Biotin Reagents in Antibody Pretargeting. 6 ... - ACS Publications

Bioconjugate Chem. 2004, 15, 601−616

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Biotin Reagents in Antibody Pretargeting. 6. Synthesis and in Vivo Evaluation of Astatinated and Radioiodinated Aryl- and nido-Carboranyl-biotin Derivatives D. Scott Wilbur,*,† Donald K. Hamlin,† Ming-Kuan Chyan,† Brian B. Kegley,† Janna Quinn,‡ and Robert L. Vessella‡ Departments of Radiation Oncology and Urology, University of Washington, Seattle, Washington 98195. Received December 18, 2003; Revised Manuscript Received February 21, 2004

An investigation has been conducted to prepare and evaluate several radiohalogenated biotin derivatives as part of our studies to develop reagents for carrying 211At in cancer pretargeting protocols. The primary goal of the investigation was to determine the in vivo stability and distribution properties of astatinated biotin derivatives. In addition to astatination, the biotin derivatives were radioiodinated for in vitro and in vivo comparison. Biodistributions were conducted in athymic mice, with sacrifice times of 1, 4, and 24 h to correspond to 9%, 32%, and 90% of 211At decay (t1/2 ) 7.21 h). In the investigation, two biotin derivatives, 1a and 2a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, an ether linker moiety, and an aryl stannane moiety for radiohalogenation. Biotin derivatives 1a and 2a were radiolabeled with 125/131I to give [125/131I]1b or [125I]2b and with 211At to give [211At]1c or [211At]2c. In vivo studies demonstrated that co-injected [125I]2b and [131I]1b had very similar tissue distributions in athymic mice. Co-injection of [211At]2c and [125I]2b provided data that indicated that rapid deastatination occurred in vivo. A second set of biotin derivatives, 3a, 4a, and 5a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, and an anionic nido-carborane moiety for radiohalogenation. The biotin derivatives 4a and 5a contained an aryl moiety not present in 3a, and 5a had a trialkylamine functionality not present in 3a or 4a. Biotin derivative 3a was radioiodinated, but was not further investigated. Biotin derivatives 4a and 5a were radiolabeled with 211At and 125I to produce [125I]4b/ [211At]4c and [125I]5b/[211At]5c. Comparison of [125I]4b and (separately) [125I]5b with [131I]1b showed that the nido-carborane containing biotin derivatives were retained in blood and tissue more than the aryl iodide derivative. In vivo evaluations of [211At]4c/[125I]4b and (separately) [211At]5c/[125I]5b indicated that some deastatination occurred in these compounds, but it was much less than observed for the aryl derivative [211At]2c. While the nido-carborane containing biotin derivatives provide a significant improvement in astatine stability over biotin derivatives previously studied, additional derivatives need to be prepared and studied to further improve the in vivo stability and blood/tissue clearance of these compounds.

INTRODUCTION

We are investigating the use of the R-particle-emitting radionuclide astatine-211 (211At) in “targeted radionuclide therapy” 1 (TRT2) of metastatic cancer. R-Particle-emitting radionuclides have properties that make them favorable for use in the therapy of small groups of cancer cells (micrometastatic disease) and single cancer cells (15). Those favorable properties include very short path lengths (50-85 µm) of the R-particle, allowing irradiation of only a few cell diameters, and very high cellular toxicity due to a large energy deposition (e.g. 100 keV/ µm) over that short path length (6). Among the few * Address correspondence to D. Scott Wilbur, Ph.D., Department of Radiation Oncology, University of Washington, 2121 N. 35th Street, Seattle, WA 98103-9103. Phone: 206-685-3085. Fax: 206-685-9630. E-mail: [email protected]. † Department of Radiation Oncology. ‡ Department of Urology. 1 “Targeted radionuclide therapy”, “targeted radiopharmaceutical therapy”, “targeted radiotherapy”, and “endoradiotherapy” are all terms used to describe an approach to radiotherapy that involves administration (injection) of a diseasetargeting radiopharmaceutical that carries a therapeutic radionuclide.

R-emitting radionuclides that have favorable decay properties for in vivo use (7), 211At is particularly attractive, as it is readily prepared in quantities sufficient for clinical application (8). Importantly, it has a half-life that is short enough (t1/2 ) 7.2 h) for use in an outpatient approach but long enough to allow adequate targeting of cancer cells in vivo. This latter point is only true if the cancertargeting carrier molecule used in combination with 211At has appropriate pharmacokinetics. A number of cancer-targeting intact monoclonal antibodies (mAb) and their F(ab′)2 fragments have been labeled with 211At (9). However, except in special applications, these carrier molecules do not have appropriate cancer targeting and blood clearance properties for use with 211At due to its short half-life (10). Smaller mAb 2 Abreviations: ChT, chloramine-T.; cpm, counts per minute; DCC, 1,3-dicyclohexylcarbodiimide; DTDC.; di-tert-butyl dicarbonate; mAb, monoclonal antibody; NCS, N-chlorosuccinimide; %ID/g, percent injected dose/gram; PBS, phosphate-buffered saline; pi, postinjection; rt, room temperature, Sav, streptavidin; tBoc, tert-butoxycarbonyl; TFA, trifluoroacetic acid; TFP, 2,3,5,6tetrafluorophenyl; TFP-OH, 2,3,5,6-tetrafluorophenol; TFPOTFA, 2,3,5,6-tetrafluorophenyl trifluoroacetate; TRT, targeted radionuclide therapy.

10.1021/bc034229q CCC: $27.50 © 2004 American Chemical Society Published on Web 04/30/2004

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Chart 1. Biotin Derivatives Prepared in the Study

fragments, such as Fab′ or single chain Fv fragments, have more appropriate pharmacokinetics, but they have a propensity to localize to kidney making their use unfavorable. To improve the pharmacokinetics of the astatine carrier molecule, we are investigating an alternative approach, termed “pretargeting” (11-14), which employs radiolabeled biotin derivatives to deliver 211At to cancer cells in vivo. In the pretargeting approach, a monoclonal antibody-streptavidin conjugate (mAb-SAv) is injected and allowed to bind antigens on cancer cells. After a period of time for adequate tumor targeting (e.g. 24 h), a clearing agent is used to remove excess mAbSAv conjugate from blood. After an appropriate time (e.g. 1-3 h), the 211At-labeled biotin derivative is administered such that it can bind with the prebound (pretargeted) mAb-SAv on cancer cells. Importantly, this approach has been shown to provide very good tumor targeting, with cures for human tumor implants (xenografts) in athymic mice using other therapeutic radionuclides (15, 16), and has progressed to clinical trials (17). While a number of radiometal-chelated biotin derivatives have been used successfully in pretargeting protocols, there have been no reports of successful applications of radiohalogen-labeled biotin derivatives. Thus, our initial studies were focused on improving the in vivo characteristics of radiohalogenated biotin derivatives. Importantly, those studies provided information on structural requirements for blocking the serum enzyme biotinidase (18, 19) while retaining the desired high binding affinity of the biotin derivatives (20). In some of the studies, the required structural features were combined with stannylbenzoates to prepare radioiodinated biotin derivatives for evaluation. We knew from previous studies with aryl stannanes that they could be used for astatination (21, 22), leading us to believe that an astatinated biotin derivative for in vivo use could be readily obtained. However, as the studies described

herein point out, astatinated aryl-biotin derivatives can be quite unstable to in vivo deastatination. Therefore, additional biotin derivatives were prepared which incorporated an alternate 211At-binding moiety, a nido-carborane.3 Previous studies by Hawthorne et al. demonstrated that a nido-carborane conjugate could be used to radioiodinate antibodies (23). We also investigated nidocarboranes as hydrophilic pendant groups for coupling radionuclides of iodine to biomolecules (24, 25). Those investigations demonstrated that nido-carborane-containing molecules radioiodinated rapidly ( 250 °C (dec); 1H NMR (CD3OD, 500 MHz) δ 1.411.53 (m, 4H), 1.57-1.78 (m, 6H), 1.88-1.95 (m, 1H), 2.23-2.34 (m, 2H), 2.68-2.71 (m, 1H), 2.91-2.94 (m, 3H), 3.19-3.23 (m, 1H), 3.30-3.31 (m, 1H), 4.31 (dd, J ) 4.3, 8.0 Hz, 1H), 4.39 (dd, J ) 4.3, 8.0 Hz, 1H), 4.50 (dd, J ) 5.0, 8.0 Hz, 1H). HRMS (ES+) calcd for C16H29N4O4S (M + H)+ 373.1910, found 373.1915; HPLC: tR ) 2.7 min. 1-N-(Biotinyl)-5-N-(3′-(closo-carboranyl)propionyl)L-lysine, 18. A 750 mg quantity of 17b (2.02 mmol) and Et3N (285 µL, 2.02 mmol) were dissolved into 4 mL of anhydrous DMF. To the solution was added 15 (743 mg, 2.20 mmol), and the reaction mixture was stirred for 1.5 h at room temperature. The reaction solution was diluted with 10 mL of H2O and then washed with EtOAc (2 × 25 mL). The H2O and DMF were removed under vacuum (Kugelrohr). The residue was further dried under high vacuum to yield 966 mg (84%) of 18 as a colorless solid: mp 176-179 °C; 1H NMR (CD3OD, 200 MHz) δ 1.361.85 (m, 17H), 2.23-2.45 (m, 5H), 2.54-2.73 (m, 4H), 2.93 (dd, J ) 5.1, 12.8 Hz, 1H), 3.12-3.26 (m, 4H), 3.29-3.32 (m, 1H), 4.27-4.38 (m, 2H), 4.49 (dd, J ) 5.0, 8.0 Hz, 1H), 4.55 (bs, 1H); 11B NMR (CD3OD, decoupled, 64 MHz) δ -8.33 (4B), -10.49 (6B). HRMS (ES+) calcd for C21H43B10N4O5S (M + H)+ 573.3885, found 573.3914; HPLC: tR) 13.2 min. 1-N-(Biotinyl)-5-N-(3′-(nido-carboranyl)propionyl)L-lysine, 3a. A 50 mg quantity (0.088 mmol) of 18 was dissolved in pyrrolidine (1 mL) cooled in an ice/H2O bath. This mixture was allowed to warm to room temperature over 30 min. H2O (5 mL) and diethyl ether (5 mL) were added to the mixture. The ether was separated, and the H2O layer was washed with ether (4 × 5 mL). The H2O was removed under vacuum, and the crude residue was dissolved in a 5:4:1 mixture of H2O:CH3CN:MeOH (1 mL). This solution was loaded onto an ion exchange column, which was prepared as follows. The ion exchange column was prepared by connecting two Alltech IC-H ion exchange columns in series and washing them with 10 mL of 1 N NaOH. Following this, the columns were washed with H2O until column reached neutral pH. The columns were then washed with 20 mL of 5:4:1 H2O:CH3CN: MeOH solution. Crude product was eluted in 1 mL fractions (20×). Fractions containing product were combined, and solvents were removed under vacuum to yield 42 mg (83%) of 3a as a tacky solid; 1H NMR (CD3OD, 300 MHz) δ 1.33-1.91 (m, 16H), 1.98-2.02 (m, 5H), 2.25-2.31 (m, 4H), 2.69 (d, J ) 12.5 Hz, 1H), 2.96 (dd, J ) 5.2, 12.5 Hz, 1H), 3.15 (t, J ) 6.7 Hz, 2H), 3.223.32 (m, 5H), 4.22 (dd, J ) 4.3, 8.0 Hz, 1H), 4.33 (dd, J ) 4.3, 8.0 Hz, 1H), 4.50 (dd, J ) 5.0, 8.0 Hz, 1H); 11B NMR (CD3OD, decoupled, 96 MHz) δ -10.52 (2B), -13.11 (1B), -16.05 (1B), -17.82 (1B), -20.91 (1B), -32.73 (2B),

Radiohalogenated Biotin Derivatives

-37.04 (1B). HRMS (ES-) calcd for C21H41B9N4O5S (M)560.3635, found 560.3663; HPLC: tR) 7.9 min. 3-(3′-(closo-Carboranyl)propionyl)amino-1-aminobenzene, 20. A 1.0 g quantity (2.72 mmol) of 15 was dissolved in 20 mL of anhydrous DMF. In a separate flask, 19 (0.59 g, 5.43 mmol) was dissolved in 20 mL of anhydrous DMF. The solution containing 15 was added dropwise to the solution containing 19 over a 1 h period. After the addition was complete, the reaction mixture was stirred at room temperature for 2.5 h. DMF was removed under vacuum at 50 °C (Kugelrohr). The residue was dissolved in 50 mL of diethyl ether and was washed with 1% HOAc/H2O (2 × 25 mL) and then H2O (25 mL). The ether layer was removed under vacuum, and the colorless solid was dried under vacuum to yield 0.790 g (95%) of 20: mp 136-137 °C; 1H NMR (CDCl3, 500 MHz) δ 1.57-2.51 (m, 12H), 2.57 (t, J ) 7.1 Hz, 2H), 2.68 (t, J ) 7.1 Hz, 2H), 3.85 (s, 1H), 6.47 (d, J ) 8.0 Hz, 1H), 6.65 (d, J ) 8.0 Hz, 1H), 7.06 (s, 1H), 7.09 (t, J ) 8.0 Hz, 1H), 7.14 (s, 1H); 11B NMR (CDCl3, decoupled, 160 MHz) δ -8.66 (2B), -9.63 (2B), -10.76 (1B), -12.02 (3B), -12.98 (2B). HRMS (ES+) calcd for C11H23B10N2O (M + H)+ 309.2741, found 309.2734; HPLC: tR ) 13.5 min. 3-(3′-(closo-Carboranyl)propionyl)amino-1-isothiocyanatobenzene, 21. A 81 mg quantity (0.263 mmol) of 20 was dissolved into 3 mL of DMF. To this solution was added 1,1-thiocarbonyldiimidazole (47 mg, 2.63 mmol), and the reaction mixture was stirred at room temperature for 30 min. DMF was removed under vacuum (Kugelrohr). The residue was dissolved in MeOH (0.5 mL), and 3 mL of H2O was added. The mixture was stirred for 30 min, and the H2O was decanted. The solid was washed with another 3 mL of H2O and dried under vacuum to yield 50 mg (54%) of 21 as a tacky solid. 1H NMR (CD3OD, 300 MHz) δ 1.27-2.56 (m, 10H), 2.612.73 (m, 4H), 4.59 (bs, 1H), 6.98-7.02 (m, 1H), 7.34 (t, J ) 8.0 Hz, 1H), 7.39-7.43 (m, 1H), 7.64 (t, J ) 2.1 Hz, 1H); 11B NMR (CD3OD, decoupled, 96 MHz) δ -1.60 (1B), -4.89 (1B), -8.72 (2B), -10.85 (6B). HRMS (ES+) calcd for C12H20B10N2ONaS (M + Na)+ 373.2124, found 373.2125; HPLC: tR ) 15.7 min. 3-(3′-(closo-Carboranyl)propionyl)amino-1-N-(1′(N-biotinyl)-5′-lysyl-thiourea)benzene, 22. A 50 mg quantity (0.14 mmol) of 21, 17b (58 mg, 0.16 mmol), and Et3N (100 µL, 0.71 mmol) were dissolved in 5 mL of DMF. The reaction mixture was stirred for 30 min, and the DMF was removed under vacuum. The residue was dissolved in minimum amount of MeOH, loaded onto a silica gel (20 g) column (1.5 cm × 26 cm), and eluted with an EtOAc/MeOH mixture (50:50). The solvents were removed from the fractions containing the product, and the colorless solid was dried under vacuum to yield 67 mg (65%) of 22: mp 178-181 °C; 1H NMR (CD3OD, 500 MHz) δ 1.29 (s, 1H), 1.40-1.46 (m, 4H), 1.55-1.78 (m, 12H), 1.85-1.92 (m, 2H), 2.26-2.29 (m, 4H), 2.67-2.69 (m, 1H), 2.92 (dd, J ) 5.0, 13.0 Hz, 1H), 3.18-3.22 (m, 2H), 3.30-3.31 (m, 5H), 3.57 (bs, 1H), 4.28-4.33 (m, 2H), 4.47 (dd, J ) 5.0, 8.0 Hz, 1H), 4.61 (bs, 1H), 7.02 (d, J ) 8.0 Hz, 1H), 7.29 (t, J ) 8.0 Hz, 1H), 7.39 (d, J ) 8.0 Hz, 1H), 7.59 (s, 1H); 11B NMR (CD3OD, decoupled, 160 MHz) δ -1.00 (2B), -8.08 (2B), -10.10 (4B), -11.40 (2B). HRMS (ES+) calcd for C28H47N6O5S2B10Na2 (M - H + 2Na)+ 767.3775, found 767.3776; HPLC: tR ) 13.3 min. 3-(3′-(nido-Carboranyl)propionyl)amino-1-N-(1′(N-biotinyl)-5′-lysyl-thiourea)benzene, 4a. A 44 mg quantity (0.06 mmol) of 22 was dissolved in pyrrolidine (1 mL), and the resultant mixture was stirred at room temperature for 1 h. Pyrrolidine was evaporated from the reaction mixture in a fume hood under gentle stream of

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air. The residue was rinsed with ether (2 × 10 mL), and the ether was decanted. The crude product was dissolved in a 5:4:1 mixture of H2O:CH3CN:MeOH (1 mL) and was loaded on an ion exchange column (prepared as described in the synthesis of 3a). The column was eluted with 1 mL fractions (20×). Fractions containing 4a were combined, and solvents were removed under vacuum to yield 28 mg (67%) of 4a as a pale yellow solid: mp 161-163 °C; 1H NMR (CD3OD, 300 MHz) δ 1.33-1.45 (m, 3H), 1.53-1.71 (m, 6H), 1.78-1.93 (m, 11H), 2.24 (t, J ) 7.3 Hz, 2H), 2.47 (t, J ) 7.8 Hz, 2H), 2.64 (d, J ) 12.5 Hz, 1H), 2.90 (dd, J ) 5.0, 13.0 Hz, 1H), 3.06-3.11 (m, 5H), 3.24-3.29 (m, 2H), 3.49-3.57 (m, 1H), 4.20 (dd, J ) 4.7, 8.3 Hz, 1H), 4.27 (dd, J ) 4.3, 8.0 Hz, 1H), 4.46 (dd, J ) 4.3, 8.0 Hz, 1H), 7.03 (d, J ) 8.0 Hz, 1H), 7.27 (t, J ) 8.0 Hz, 1H), 7.38 (d, J ) 8.0 Hz, 1H), 7.57 (t, J ) 2.1 Hz, 1H); 11B NMR (CD3OD, decoupled, 96 MHz) δ -10.42 (2B), -12.68 (1B), -15.92 (1B), -17.72 (1B), -32.63 (2B), -36.78 (2B). HRMS (ES-) calcd for C28H47N6O5S2B9 (M)710.3887, found 710.3895; HPLC: tR ) 10.0 min. 3-(3′-(closo-Carboranyl)propionyl)amino-5-aminobenzoic acid, 24. 3,5-Diaminobenzoic acid dihydrochloride (23) (3.7 g, 16.3 mmol) and Et3N (3.41 mL, 24.5 mmol) were dissolved in 20 mL of DMF under argon atmosphere. A 3 g quantity (8.1 mmol) of TFP ester 15 was dissolved in 20 mL of DMF and was added dropwise over 30 min. The reaction was stirred at room temperature for 1 h. the DMF was removed under vacuum. The crude product was dissolved in hexane/ethyl acetate (4: 1), loaded onto a silica column (100 g), and eluted with gradient of starting with hexane/ethyl acetate (4:1). The product eluted with 100% EtOAc. Fractions containing 24 were collected, and solvent was removed under vacuum. The isolated product was dried under vacuum to yield 2.6 g (91%) of 24 as a colorless solid: mp 123125 °C; 1H NMR (CD3OD, 500 MHz) δ 1.56-2.55 (m, 10H), 2.58-2.61 (m, 2H), 2.65-2.68 (m, 2H), 4.56 (bs, 1H), 7.11 (s, 1H), 7.24 (s, 1H), 7.41 (s, 1H); 11B NMR (CD3OD, decoupled, 160 MHz) δ -1.11 (1B), -4.32 (1B), -8.06 (2B), -10.15 (4B), -11.37 (2B). HRMS (ES+) calcd for C12H22B10N2O3Na (M + Na)+ 375.2459, found 375.2454; HPLC: tR ) 13.4 min. 3-(3′-(closo-Carboranyl)propionyl)amino-5-(N-tertbutyloxycarbonyl)aminobenzoic Acid, 25. A 1.0 g quantity (2.84 mmol) of 24 and NaOH (142 mg, 3.55 mmol) were dissolved in 20 mL of DMF/H2O (1:1). Ditert-butyl dicarbonate (776 mg, 3.55 mmol) was added to the reaction flask, and the reaction mixture was stirred for 16 h at room temperature. The solvent was removed under vacuum. Crude product was dissolved into 10 mL of water and was acidified with 4 mL of 1 N HCl. The precipitate was collected by filtration and was dried under vacuum to yield 920 mg (72%) 25 as a colorless solid: mp 185-186 °C; 1H NMR (CD3OD, 500 MHz) δ 1.52 (s, 9H), 1.58-2.54 (m, 10H), 2.61-2.64 (m, 2H), 2.66-2.70 (m, 2H), 4.58 (bs, 1H), 7.78 (s, 1H), 7.84 (s, 1H), 7.99 (s, 1H); 11B NMR (CD3OD, decoupled, 160 MHz) δ -1.11 (1B), -4.41 (1B), -8.11 (2B), -10.21 (4B), -11.41 (2B). HRMS (ES+) calcd for C17H30B10N2O5Na (M + Na)+ 475.2983, found 475.2995; HPLC: tR ) 14.5 min. 3-(3′-(closo-Carboranyl)propionyl)amino-5-(N-tertbutyloxycarbonyl)aminobenzoic Acid Tetrafluorophenyl Ester, 26. A100 mg quantity (0.22 mmol) of 25 and Et3N (276 µL, 1.3 mmol) were dissolved into 3 mL of anhydrous DMF. The solution was cooled to 4 °C in an ice/H2O bath. TFP-OTFA (129 µL, 0.67 mmol) was added, the reaction mixture was stirred at 4 °C for 30 min, and then 30 mL of H2O was added. The precipitate was filtered, washed with water (4 × 10 mL), and dried


under vacuum to yield 115 mg (87%) of 26 as a colorless solid: mp 122-125 °C; 1H NMR (CD3OD, 500 MHz) δ 1.57 (s, 9H), 1.66-2.61 (m, 10H), 2.67-2.75 (m, 4H), 4.64 (bs, 1H), 7.48-7.55 (m, 1H), 8.05 (s, 1H), 8.12 (s, 1H), 8.14 (s, 1H); 11B NMR (CD3OD, decoupled, 160 MHz) δ -1.17 (1B), -4.23 (1B), -7.94 (2B), -10.04 (4B), -11.27 (2B). HRMS (ES+) calcd for C23H30B10N2O5F4Na (M + Na)+ 623.2919, found 623.2929; HPLC: tR ) 15.4 min. 3-(3′-(closo-Carboranyl)propionyl)amino-5-(N-tertbutyloxycarbonyl)amino-1-(3′-dimethyl-aminopropyl)benzamide, 27. 3-(Dimethylamino)propylamine (49 µL, 3.84 mmol) and Et3N (33 µL, 0.23 mmol) were dissolved in 4 mL of anhydrous DMF. To that solution was added 26 (115 mg, 0.19 mmol). The reaction mixture was stirred for 1 h at room temperature, 50 mL of water was added, and this mixture was stirred for an additional 1 h. The precipitate was filtered, washed with water (4 × 20 mL), and dried under vacuum to yield 90 mg (88%) of 27 as a colorless solid: mp 111-112 °C. 1H NMR (CD3OD, 500 MHz) δ 1.24-1.45 (m, 4H), 1.52 (s, 9H), 1.64-1.72 (m, 2H), 1.79-1.84 (m, 2H), 1.87-2.25 (m, 4H), 2.32 (s, 6H), 2.49 (t, J ) 7.4 Hz, 2H), 2.61-2.64 (m, 2H), 2.66-2.70 (m, 2H), 3.39 (t, J ) 7.4 Hz, 2H), 4.59 (bs, 1H), 7.54 (s, 1H), 7.62 (s, 1H), 7.81 (s, 1H); 11B NMR (CD3OD, decoupled, 160 MHz) δ -1.25 (1B), -4.09 (1B), -7.32 (1B), -8.26 (1B), -9.37 (1B), -10.45 (3B), -11.62 (2B). HRMS (ES+) calcd for C22H43B10N4O4 (M + H)+ 537.4215, found 537.4220; HPLC: tR ) 16.4 min. 3-(3′-(closo-Carboranyl)propionyl)amino-5-amino1-(3′-dimethylaminopropyl)benzamide, 28. A 73 mg quantity (0.14 mmol) of 27 was dissolved in TFA (2 mL). The solution was stirred for 30 min at room temperature, and the excess TFA was removed using a gentle stream of air in a fume hood. The crude residue was washed 2× with 3 mL of ether, and then the residue was dried under high vacuum to yield the TFA salt of 28 as a colorless tacky solid, which was used directly in the next reaction step. 1H NMR (CD3OD, 200 MHz) δ 0.74-1.47 (m, 8H), 1.61-1.76 (m, 2H), 2.17 (s, 6H), 2.28-2.35 (m, 3H), 2.452.61 (m, 5H), 3.27 (t, J ) 7.4 Hz, 2H), 4.48 (bs, 1H), 6.71 (s, 1H), 6.98 (s, 1H), 7.08 (s, 1H); 11B NMR (CD3OD, decoupled, 96 MHz) δ -1.77 (1B), -5.30 (1B), -8.72 (2B), -10.80 (4B), -11.86 (2B). HRMS (ES+) calcd for C17H35B10N4O2 (M + H)+ 437.3691, found 437.3698; HPLC: tR ) 12.5 min. 3-(3′-(closo-Carboranyl)propionyl)amino-5-(1′-(Nbiotinyl)-5′-lysylthiourea)(3′-dimethylaminopropyl)benzamide, 29. The crude 28 (0.14 mmol) was dissolved in a minimum amount of MeOH, made basic with Et3N (2 mL), and stirred for 30 min at room temperature. The MeOH and excess Et3N were removed under a stream of air and then under vacuum to yield a colorless oil. The oily residue and 1,1-thiocarbonyldiimidazole (22 mg, 0.14 mmol) were dissolved in 3 mL of anhydrous DMF. Reaction progress for formation of the phenyl isothiocyanate was followed by HPLC (1 h). Upon complete formation of the isothiocyanate, the biotin-lysine adduct 17b (56 mg, 0.15 mmol) and Et3N (57 µL, 0.41 mmol) were added, and then the reaction mixture was stirred for 3 h at room temperature. DMF was removed under vacuum, and the residue was dissolved in a minimum amount of MeOH. The crude reaction mixture was loaded onto a silica gel column (20 g, 2.5 cm × 35 cm) and was eluted using 1% Et3N/MeOH. The purified product was dried under vacuum to yield 85 mg (74%) of 29 as a colorless oil: 1H NMR (CD3OD, 500 MHz) δ 1.24-1.46 (m, 4H), 1.53-1.74 (m, 6H), 1.79-1.90 (m, 4H), 2.252.31 (m, 3H), 2.34 (s, 6H), 2.79-2.84 (m, 4H), 2.91 (dd, J ) 5.0, 13.0 Hz, 1H), 3.16-3.21 (m, 1H), 3.30-3.32

Wilbur et al.

(m, 2H), 3.34-3.43 (m, 3H), 3.58 (bs, 1H), 4.25-4.30 (m, 2H), 4.45-4.49 (m, 1H), 4.62 (bs, 1H), 7.05 (s, 1H), 7.437.90 (m, 2H); 11B NMR (CD3OD, decoupled, 160 MHz) δ -1.08 (1B), -4.41 (1B), -8.09 (2B), -10.16 (4B), -11.40 (2B). HRMS (ES+) calcd for C34H61B10N8O6S2 (M + H)+ 851.5086, found 851.5083; HPLC: tR ) 12.6 min. 3-(3′-(nido-Carboranyl)propionyl)amino-5-(1′-(Nbiotinyl)-5′-lysylthiourea)(3′-dimethylaminopropyl)benzamide, 5a. A 60 mg quantity (0.07 mmol) of 29 was dissolved in pyrrolidine (1 mL) and was stirred for 30 min at room temperature. Excess pyrrolidine was removed under a gentle stream of air in a fume hood. The residue was rinsed with ether (2 × 10 mL), and the ether was decanted. The crude product was dissolved in a 5:4:1 mixture of H2O:CH3CN:MeOH (1 mL) and was loaded on an ion exchange column (prepared as described in the synthesis of 3a). The column was eluted with 1 mL fractions (20×). Fractions containing 5a were combined, and solvents were removed under vacuum to yield 40 mg (68%) of 5a as a tacky light-yellow solid: 1H NMR (CD3OD/DMSO-d6, 500 MHz) δ 1.11-1.27 (m, 3H), 1.51-1.60 (m, 3H), 1.65-1.74 (m, 2H), 1.80-1.84 (m, 2H), 1.872.01 (m, 2H), 2.08-2.22 (m, 2H), 2.25 (s, 6H), 2.26-2.32 (m, 1H), 2.53 (t, J ) 7.4 Hz, 2H), 2.77 (t, J ) 7.8 Hz, 2H), 2.88-2.97 (m, 2H), 3.41-3.49 (m, 2H), 3.55-3.56 (m, 2H), 3.69-3.89 (m, 4H), 4.54-4.75 (m, 4H), 7.928.27 (m, 3H); 11B NMR (CD3OD, decoupled, 160 MHz) δ -10.04 (2B), -12.29 (1B), -15.14 (1B), -17.13 (2B), -20.02 (1B), -31.63 (1B), -35.76 (1B). HRMS (ES-) calcd for C34H59B9N8O6S2 (M - H)- 838.4836, found 838.4869; HPLC: tR ) 9.8 min. General Procedure for Iodination of nido-Carborane Derivatives, 3a, 4a, and 5a. To a 500 µL quantity of a 2 mg/mL solution of nido-carborane derivative in MeOH/1% HOAc was added an appropriate microliter quantity of a 22 mg/mL solution of NaI in H2O to introduce 0.5 mol equiv of iodide. Immediately following this, an appropriate microliter quantity of a 22 mg/ mL solution of N-chlorosuccinimide (NCS) in MeOH was added to introduce 0.5 mol equiv. The reaction mixture generally turned light brown as the NCS was added but returned to colorless almost immediately. After 30 s, the reaction was quenched by the addition of 10 µL of a 22 mg/mL solution of sodium metabisulfite in H2O. Iodinated nido-carboranyl species were used for radioHPLC peak identification. HPLC analysis of the reaction mixtures showed both starting material and an iodinated product. To obtain pure samples, the HPLC peak corresponding to the iodinated product was collected from HPLC effluent. Identity of the collected material was obtained from high-resolution mass spectral analysis. 1-N-(Biotinyl)-5-N-(3′-(iodo-nido-Carboranyl)propionyl)-L-lysine, 3b. HRMS calcd for C21H40B9N4O5SI (M-) 686.2594, found 686.2602; HPLC: tR ) 9.8 min. 3-(3′-(Iodo-nido-Carboranyl)propionyl)amino1-N-(1′-(N-biotinyl)-5′-lysylthiourea)-benzene, 4b. HRMS calcd for C28H46N6O5S2B9I (M-) 836.2889, found 836.2853; HPLC: tR ) 10.7 min. 3-(3′-(Iodo-nido-carboranyl)propionyl)amino-5(1′-(N-biotinyl)-5′-lysylthiourea)(3′dimethylaminopropyl)benzamide, 5b. HRMS calcd for C34H58B9IN8O6S2 (M-) 964.3791, found 964.3803; HPLC: tR ) 11.0 min. General Procedure for Radiohalogenation of Arylstannanes, 1a and 2a. To a 50-100 µL aliquot of a 1 mg/mL of the aryl stannanes (1a or 2a) in MeOH/5% HOAc was added 3-10 µL of Na[125I]I, Na[131I] in 0.1 N NaOH or Na[211At] in 0.05 N NaOH. Following this, 10 µL of a 1 mg/mL solution of NCS in MeOH was added.


The reaction was allowed to proceed for 1-10 min, and then 10 µL of 1 mg/mL solution of Na2S2O5 in H2O was added to quench the reaction. The radiohalogenated aryl halide derivatives were isolated from reversed-phase HPLC effluent. After isolation, the MeOH and HOAc was removed under a stream of nitrogen (vented through charcoal containing syringe). The radiohalogenation reactions can also be conducted using chloramine-T (ChT) in H2O as described below for 2c. N′-(13-(4′′-[131I]iodobenzamido)-4,7,10-trioxatridecanyl)-N-methylglycylbiotinamide, [131I]1b. Reaction was conducted with 3 µL of Na[131I] (2.14 mCi) in 0.1 N NaOH. [131I]1b was isolated from the HPLC effluent and radio-HPLC analysis indicated that the reaction provided 95% yield, and 69% was isolated from the HPLC: tR ) 16.7 min. N′-(13-(4′′-[211At]astatobenzamido)-4,7,10-trioxatridecanyl)-N-methylglycylbiotinamide, [211At]1c. Reaction was conducted with 10 µL of Na[211At] (547 µCi) in 0.05 N NaOH with a 10 min reaction time. The reaction mixture containing [211At]1c was purified by radio-HPLC to give 64% isolated yield: tR ) 12.6 min (Note: this was a different HPLC column from that used for the radioiodinated product listed above). 2-(N-Biotinyl)-4-(N′-(13′-(4′′-[125I]iodobenzoyl)amino)-4′,7′,10′-trioxatridecaneamino)-L-aspartate,[125I]2b. Reaction was conducted with 5 µL of Na[125I] (2.76 mCi) in 0.1 N NaOH. [125I]2b was isolated from the HPLC effluent. Radio-HPLC analysis indicated that the reaction provided 90% yield, but only 22% was isolated from the HPLC: tR ) 15.2 min. In four other experiments, 37%, 43%, 45%, and 49% (isolated) radiochemical yields were obtained. 2-(N-Biotinyl)-4-(N′-(13′-(4′′-[211At]astatobenzoyl)amino)-4′,7′,10′-trioxatridecaneamino)- L -aspartate, [211At]2c. Reaction was conducted with 100 µL of Na[211At] (1.39 mCi) in 0.1 N NaOH. [211At]2c was isolated from the HPLC effluent. Radio-HPLC analysis indicated that the reaction provided ∼100% yield, and 41% was isolated from the HPLC: tR ) 14.6 min. In another experiment, 100 µL of 2a in H2O (1 mg/ mL) and 20 µL of ChT (1 mg/mL) were reacted in Na phosphate buffer, pH 7.4 (100 µL), with 50 µL of (295 µCi) Na[211At]At in 1:1 0.05 N NaOH/MeOH for 5 min at room temperature to give 23% isolated yield of [211At]2c. General Procedure for Radiohalogenation of nido-Carborane Derivatives, 3a, 4a, and 5a. To a solution containing 50-100 µL of 1 mg/mL solution of nido-carborane derivative (3a, 4a, or 5a) in MeOH/ 5%HOAc was added 1-5 µL of Na[125I]I, Na[131I]I, or 20100 µL of Na[211At]At in 0.05N NaOH. To the resultant solution was added 10-20 µL of a 1 mg/mL solution of NCS in MeOH or ChT in H2O. The reaction was quenched after 30 s with 10-20 µL of a 1 mg/mL solution of Na2S2O5 in H2O. Alternatively, 100 µL of a 1 mg/mL solution of nidocarborane derivative, 5b, was reacted with 15-20 µg of a 1 mg/mL solution of ChT in H2O and 5-15 µL (1.51.9 mCi) of Na[125I]I in 0.05 N NaOH. After 1 min reaction time, the reaction was quenched with 15-20 µL of a 1 mg/mL solution of Na2S2O5 in H2O. The radiohalogenated nido-carborane derivatives were isolated from reversed-phase HPLC effluent. After isolation, the MeOH and HOAc was removed under a stream of nitrogen (vented through charcoal containing syringe). Due to the propensity of the nido-carborane compounds to adhere to glass surfaces, plastic vials are routinely used. The radiohalogenated derivatives used in animal


studies were diluted with a solution of 1% BSA in PBS after the MeOH/HOAc (or H2O) was removed. This was necessary to keep the labeled material from adhering to the surfaces in the vial and syringe. 1-N-(Biotinyl)-5-N-(3′-([125I]iodo-nido-carboranyl)propionyl)-L-lysine, [125I]3b. Reaction was conducted with 3 µL of Na[125I]I (853 µCi) in 0.1 N NaOH. [125I]3b was isolated from the HPLC effluent. Radio-HPLC indicated that the reaction provided >75% yield, and 67% was isolated from the HPLC: tR ) 14.0 min. 3-(3′-([125I]Iodo-nido-carboranyl)propionyl)amino1-N-(1′-(N-biotinyl)-5′-lysylthiourea)-benzene, [125I]4b. Reaction was conducted with 3 µL of Na[125I]I (766 µCi) in 0.1 N NaOH. [125I]4b was isolated from the HPLC effluent. Radio-HPLC indicated that the reaction provided 61% yield. HPLC: tR ) 16.7 min. In a separate reaction, a 59% yield was obtained. 3-(3′-([125I]Iodo-nido-carboranyl)propionyl)amino5-(1′-(N-biotinyl)-5′-lysylthiourea)(3′dimethylaminopropyl)benzamide, [125I]5b. Reaction was conducted with 5 µL of Na[125I]I (2.38 mCi) in 0.1 N NaOH. RadioHPLC indicated that the reaction provided ∼61% [125I]5b, and 50% was isolated from the HPLC: tR ) 14.9 min. In another reaction employing NCS as the oxidant, an 83% (HPLC) yield was obtained. In two other reactions employing ChT in H2O, 72% and 86% (HPLC) yields were obtained. 3-(3′-([211At]Astato-nido-carboranyl)propionyl)amino-1-N-(1′-(N-biotinyl)-5′-lysylthiourea)benzene, [211At]4c. Reaction of 20 µL (160 µCi) of Na[211At]At with 4a provided 60% radiochemical yield. The product was isolated from the HPLC effluent: tR) 16.7 min. In a separate reaction, 40 µL (210 µCi) of Na[211At]At in a 1:1 mixture of 0.05 N NaOH/MeOH provided a 36% isolated yield. 3-(3′-([211At]Astato-nido-carboranyl)propionyl)amino-5-(1′-(N-biotinyl)-5′-lysylthiourea)(3′dimethylaminopropyl)benzamide, [211At]5c. Reaction of 3 µL of Na[211At]At (853 µCi) in 0.1 N NaOH provided 23% radiochemical yield of [211At]5c. The product was isolated from the HPLC effluent. tR) 14.0 min. In a separate reaction, 25 µL (125 µCi) of Na[211At]At in a 1:1 mixture of 0.05 N NaOH/MeOH was reacted with NCS to provide a 57% isolated yield. Biodistribution Studies. The animal studies were approved by the University of Washington’s Institutional Animal Care and Use Committee prior to being conducted. Animal care and use was conducted in accordance with the NIH guidelines.4 Mice were placed on a biotin deficient diet for 5-7 days prior to the start of each investigation. This was done to mimic the conditions used in the pretargeting studies. Male athymic mice (BALB/c nu/nu), 4-5 weeks of age were obtained from Simonsen Laboratories (Gilroy, CA). Mice were housed for 1 week in the animal facility prior to beginning the study. All injections of reagents were administered to mice in a total volume of approximately 100 µL (injectate weighed) via the lateral tail vein. Animals were sacrificed at 1, 4, and 24 h postinjection of the radiolabeled materials. These times correspond to 9%, 32%, and 90% decay of 211At. The data for tissues excised are shown in the Tables S1-S6 in the Supporting Information, and the average animal weight in a biodistribution, and average tumor weights for groups involving mice bearing tumor xenografts are provided in the table legends. Blood samples were obtained by cardiac 4 NIH guidelines are described in NIH Publication 86-23: “Guide for the Care and Use of Laboratory Animals”.


Wilbur et al.

Scheme 1. Synthesis and Radiohalogenation of 2-(N-Biotinyl)-4-(N′-(13′-p-(tri-n-butylstannyl)benzoylamino)4′,7′,10′-trioxatridecaneamino-L-aspartate, 2a

a Reagents and conditions: (a) H O, acetone, NaHCO , rt, 6 h; (b) TFP-OTFA, Et N, rt, 20 min; (c) Et N, DMF, 4 °C-rt, 1 h; (d) 2 3 3 3 TFA, rt, 30 min, ∼100%; (e) Et3N, DMF, 12a or 12b, rt, 30 min to 1 h, 70% (2a), 80% (2b); (f) MeOH/5% HOAc, NaX (X ) 125I or 211At), NCS, rt, 1-10 min.

puncture immediately before sacrifice. Urine samples were obtained by syringe bladder tap at the time the tissues were excised. Excised tissues were blotted free of blood, weighed, and counted. Calculation of the percent injected dose (%ID) and percent injected dose per gram (% ID/g) in the tissues was based on internal standards containing 1 µL of the injected dose. The standards were prepared by mixing 10 µL of the injectate with 990 µL of saline. After mixing thoroughly, four 100 µL aliquots were placed in tubes and counted as standards. Standards were counted with tissues obtained at each time point, and an average value for the four standards were used in the calculations. The 211At counts were compensated for decay, and where dual radionuclide counting was required, the 125I counts were compensated (13.5%) for spillover counts from the 131I. Total blood volume was estimated to be 8% of body weight for the calculations (10). Statistical analysis of the data was conducted using the paired student’s t-test. Differences were considered statistically significant when the p < 0.05. RESULTS

Synthesis of Biotin Compounds Containing Arylstannanes, 1a/2a, and Aryl Iodides, 1b/2b. Syntheses of 1a and 1b were conducted as previously described (18). Syntheses of 2a and 2b were conducted as shown in Scheme 1. Both syntheses were conducted through the common intermediate, biotin derivative 11. The synthesis of compound 11 has been previously reported (19). Briefly, the synthesis started with reaction of biotin TFP ester, 6, with aspartate R-tert-butyl ester, 7, to form an adduct containing a free carboxylate. The carboxylate was subsequently esterified using TFP-OTFA to provide the TFP ester 8. Reaction of 8 with the mono-Boc protected 4,7,10-trioxatridecane-1,13-diamine 9 gave the adduct 10. In the previous studies, 10 was deprotected with neat trifluoroacetic acid (TFA) to provide 11, which was then reacted with p-[125I]iodobenzoate N-hydroxysuccinimide ester to form [125I]2b. In these studies, the aryl stannanes 2a was sought as an intermediate to prepare both [125I]2b and [211At]2c. To prepare 2a, biotin derivative 11 was isolated as the TFA salt after treatment of 10 with neat TFA. Reaction of the TFA salt of 11 with p-(tri-n-butylstannyl)benzoate TFP ester 12a and Et3N provided 2a in 70% yield. Similarly, reaction the

TFA salt of 11 with p-iodobenzoate TFP ester 12b and Et3N provided 2b in 80% yield. Synthesis of Biotin Compounds 3a, 4a, and 5a, Containing nido-Carborane Moieties. The reactions employed to obtain the biotin-nido-carborane derivative 3a are shown in Scheme 2. The synthesis of the 3-(closocarboranyl)propionate TFP ester, 15, was conducted as previously described (27). Briefly, the TFP ester of pentynoic acid 14 was prepared by reaction of 4-pentynoic acid with dicyclohexylcarbodiimide (DCC) and tetrafluorophenol (TFP-OH). Formation of the closo-carborane moiety to produce 15 was accomplished by reaction of the acetylenic group with decaborane (B10H14). It should be noted that the TFP ester was used in the reaction as free carboxylates are problematic in reactions with decaborane. Preparation of the biotin-lysine adduct 17a was accomplished in 87% yield by reaction of biotin TFP ester 6 with -N-(tert-butoxycarbonyl)-L-lysine, 16, and sodium bicarbonate in aqueous acetone at room temperature. Removal of the t-Boc protecting group from 17a was facile in neat TFA, providing 17b in 98% yield. Reaction of biotin-lysine adduct 17b with 15 and Et3N in anhydrous DMF provided the biotin derivative containing a closocarborane moiety, 18, in 84% yield. The closo-carborane moiety in 18 was readily converted into the nidocarborane by boron abstraction reaction with pyrrolidine to provide the 3-(nido-carboranyl)propionic acid, 3a in 83% yield. The synthesis employed to obtain 4a is shown in Scheme 3. The synthesis is similar to that employed to prepare 3a, except it incorporates 1,3-diaminobenzene, 19, into the structure. Reaction of an excess of 19 with the 3-(closo-carboranyl)propionate TFP ester, 15, in anhydrous DMF provided the closo-carborane adduct 20 in 95% yield. The aniline amine in 20 was converted to an isothiocyanate derivative 21 in 54% (isolated) yield by reaction with thiocarbonyldiimidazole in DMF at room temperature. The isothiocyanate 21 was reacted with the biotin-lysine adduct 17b and Et3N in anhydrous DMF at room temperature to provide the biotin derivative 22, containing a closo-carborane moiety, in 65% yield. The targeted nido-carboranyl-biotin derivative 4a was prepared in 67% yield by reaction of 22 with pyrrolidine. Synthesis of the biotin derivative 5a, containing a trialkylamine moiety, is shown in Scheme 4. 3,5-Diami-



Scheme 2. Synthesis and Radioiodination of 1-N-(Biotinyl)-5-N-(3′-(nido-carboranyl)propionyl)-L-lysine, 3a

a Reagents and conditions: (In carborane structures, open circles represent boron or B-H atoms and closed circles represent carbon atoms) (a) THF, DCC, TFP-OH, rt, 16-24 h; (b) B10H14, CH3CN, ∆, 18-24 h; (c) H2O, acetone, NaHCO3, rt, 16 h, 87%; (d) TFA, rt, 30 min, 98%; (e) DMF, Et3N, rt, 1.5 h, 84%; (f) pyrrolidine, 0 °C - rt, 30 min, 83%; (g) MeOH/5% HOAc, NaX (X ) 125I or 211At), NCS, rt, 1 min.

Scheme 3. Synthesis and Radiohalogenation of 3-(3′-(nido-Carboranyl)propionyl)amino-1-N-(1′(N-biotinyl)-5′-lysyl-thiourea)benzene, 4a

a Reagents and conditions: (In carborane structures, open circles represent boron or B-H atoms and closed circles represent carbon atoms) (a) DMF, rt, 3.5 h, 95%; (b) DMF, TCDI, rt, 30 min, 54%; (c) DMF, Et3N, rt, 30 min, 65%; (d) pyrrolidine, rt, 1 h, 67%; (e) MeOH/5% HOAc, NaX (X ) 125I or 211At), NCS, rt, 1 min.

nobenzoic acid, 23, was used to provide a trifunctional aryl moiety for introducing a trialkylamine moiety. Reaction of an excess of 23 and Et3N with 15 in DMF at room temperature provided the closo-carborane adduct 24 in 91% yield. Protection of the aniline amine of 24 to give 25 was accomplished in 72% yield by reaction with di-tert-butyl dicarbonate in a mixture of DMF and H2O at room temperature. Subsequent formation of the TFP ester derivative 26 was accomplished in 87% yield by reaction of 25 with TFP-OTFA in DMF at 4 °C. Reaction of 26 with 3-(dimethylamine)propylamine and Et3N in DMF at room temperature provided 27 in 88% yield. Removal of the t-Boc group from 27 with neat TFA gave

the aniline 28 in nearly quantitative yield. Conversion of the aniline amine in 28 to an isothiocyanate functionality using thiocarbonyldiimidazole in anhydrous DMF was followed by reaction with the biotin-lysine adduct 17b to provide the closo-carboranyl-biotin derivative 29 in 74% overall yield. Reaction of the closo-carborane in 29 with pyrrolidine provided the nido-carborane derivative 5a in 95% yield. Radiohalogenation Reactions of Biotin Derivatives 1a, 2a, 3a, 4a, and 5a. The product mixtures for the radiohalogenation reactions were assessed by reversedphase HPLC using nonradioactive iodinated derivatives (1b, 2b, 3b, 4b, 5b) as HPLC standards. All radiolabeled


Wilbur et al.

Scheme 4. Synthesis and Radiohalogenation of 3-(3′-(nido-Carboranyl)propionyl)amino-5(1′-(N-biotinyl)-5′-lysylthiourea)-(3′dimethylaminopropyl)benzamide, 5a

a Reagents and conditions: (In carborane structures, open circles represent boron or B-H atoms and closed circles represent carbon atoms) (a) DMF, Et3N, rt, 1.5 h, 91%; (b) NaOH, DMF, H2O, DTDC, rt, 16 h, 72%; (c) DMF, Et3N, TFP-OTFA, 4 °C, 30 min, 87%; (d) 3-(dimethylamino)propylamine, DMF, Et3N, rt, 1 h, 88%; (e) TFA, rt, 30 min, ∼100%; (f) DMF, TCDI, 1 h (not isolated); (g) 17b, DMF, Et3N, rt, 3 h, 74%; (h) pyrrolidine, rt, 30 min, 95%; (i) MeOH/5% HOAc, NaX (X ) 125I or 211At), NCS, rt, 1 min.

samples were obtained pure by collection of radioactivity from the HPLC effluent. Astatinated compounds had retention times slightly longer than that of the iodinated standard. Under the elution conditions employed, biotin derivatives containing aryl moieties had narrow HPLC peaks (sometimes as doublets), whereas, biotin derivatives containing nido-carboranyl moieties had much broader HPLC peaks. In most cases, radioiodination reactions were conducted multiple times. Due to the cost and quantity of 211At, astatinations were (generally) conducted only at the time of the labeling for the animal study. No optimization studies of the radiohalogen labeling conditions were conducted. Radioiodination and astatination of biotin derivatives containing aryl stannane moieties, 1a and 2a (50-100 µg), were conducted by reaction of N-chlorosuccinimide (NCS) (10 µg) and Na[125I]I or Na[211At]At in MeOH containing 1-5% HOAc at room temperature for 1-10 min. The reaction was then stopped by the addition of (10 µg) sodium metabisulfite. Radioiodination of 1a has been conducted several times as the resultant compound, [125/131I]1b, is used as a control in studies of biotinidase stability. As an example, isolated yields of 34%, 35%, 39%, and 69% were obtained in four radioiodination experiments. Astatination of 1a was conducted using the same reaction conditions, giving yields of 22% after 3 min reaction time, and in a second reaction 64% after 10 min. Radioiodination of 2a has also been conducted a number of times under the same reaction conditions. In four radioiodination experiments with 2a, isolated yields of 37%, 43%, 45%, and 49% for [125I]2b were obtained. Astatination of 2a was conducted twice. One experiment was conducted using NCS as the oxidant, and the other experiment used ChT as the oxidant. The NCS reaction provided 41% isolated yield of [211At]2c, and the ChT reaction provided 23% isolated yield. Radioiodination was conducted on all three biotin derivatives 3a, 4a and 5a, containing a nido-carborane moiety, whereas astatination was only conducted on 4a

and 5a. Radioiodination of 3a, 4a, and 5a were conducted by reaction of NCS and Na[125I]I in MeOH containing 1-5% HOAc at room temperature for 30 s to 5 min. Following that, sodium metabisulfite was added to quench the reaction. The radiochemical yields obtained under these reaction conditions were [125I]3b, 67%; [125I]4b, 82% and 59% (isolated); and [125I]5b, 83%. Radioiodination of 5a was also conducted by reaction of ChT and Na[125I]I in H2O at room temperature for 1 min. The radiochemical yields (HPLC) of [125I]5b obtained in those reactions were 72% and 86%. Astatinations of 4a and 5a were conducted using NCS as the oxidant to give 36% and 23% isolated radiochemical yields of [211At]4c and [211At]5c, respectively. Astatination of 5a was also conducted using ChT in H2O and that reaction gave [211At]5c in 57% isolated yield. Biodistributions of Radiohalogenated Biotinlysine-nido-carborane Derivatives [125I]1b, [125I]2b, [211At]2c, [125I]3b, [125I]4b, [211At]4c, [125I]5b, and [211At]5c. A total of six biodistributions of radiohalogenated biotin derivatives were conducted in the investigation. Due to the propensity of the nido-carboranes to adhere to glass and metal surfaces, 1% BSA was added to the radiolabeled compounds when diluted from the (rotoevaporator) concentrated HPLC effluent. All studies evaluated the tissue distribution of radiolabeled compounds at 1, 4, and 24 h postinjection in athymic mice. The complete tissue distribution data are provided in the Supporting Information as Tables S1-S6. Three biodistribution studies utilized coinjected, duallabeled (125I and 131I) compounds to compare the in vivo tissue distributions of biotin derivatives that have different structures. In an initial animal study, the distribution of radioiodinated biotin derivatives [125I]2b and [131I]1b, containing aryl labeling moieties, were evaluated. The biodistribution data is provided as Table S1 in Supporting Information. Although the data has a fairly large spread, evaluation of concentrations of the two radionuclides in individual tissues by the Student’s t-test



Figure 1. Concentrations of 125I and 131I in selected tissues at 4 h pi for the radiolabeled compounds listed (top of panels A and B). Note that the Y-axis range for the percent injected dose/gram has been set at 0-15 to correlate with graphs in Figure 2. Complete data sets are provided as (A) Table S3 and (B) Table S5 in the Supporting Information.

Figure 2. Concentrations of 211At and 125I in selected tissues at 4 h pi for the radiolabeled compounds listed (top of panels A thru D). Data for the graph in panel A was obtained from the literature (27) (with permission). Complete data sets are provided as (B) Table S2, (C) Table S4, and (D) Table S6 in the Supporting Information.

indicates that there is little difference between the two in vivo. The two other studies involving radioiodinated biotin derivatives compared the in vivo distribution of a biotin derivative containing an aryl moiety [131I]1b with the biotin derivatives [125I]4b or [125I]5b. Both biotin derivatives 4b and 5b contain an aryl group and a nidocarborane moiety, and 5b also contains a dimethylamine moiety. From the data obtained, it is readily apparent that the nido-carborane containing biotin derivative, [125I]4b, is cleared from blood, lung, kidney, and liver much slower than [131I]1b. These differences are observed at

all time points (Table S3, Supporting Information) and can be readily seen in the concentration of radionuclides in selected tissues at 4 h postinjection, which are plotted in Figure 1, panel A. Similar results were obtained in the comparison of [125I]5b and [131I]1b. The data obtained in that study are provided as Table S5 and tissue concentrations in selected tissues at 4 h postinjection are plotted in Figure 1, panel B. Three studies were conducted to evaluate the distribution of radionuclides in mice for biotin derivatives (2b/c, 4b/c, and 5b/c), which were dual labeled with 211At and

612 Bioconjugate Chem., Vol. 15, No. 3, 2004 125

I (Figure 2, panels B-D). To use as a reference, a plot of data from a previous evaluation of free Na[211At]At and Na[125I]I in selected tissues of mice27 is provided as Figure 2, panel A. The results of [211At]2c vs [125I]2b are provided as Table S2 in Supporting Information, and data obtained at 4 h postinjection in selected tissues are plotted in Figure 2, panel B. Unfortunately, the high concentrations of 211At and large differences between 211At and 125I in lung, spleen, and neck indicate that [211At]2c undergoes considerable deastatination. Indeed, comparison of the pattern of tissue concentrations in graphs provided in panels A and B of Figure 2 provides strong evidence that large amounts of free astatide are present in the animals injected with [211At]2c. Biodistribution data for biotin derivatives [211At]4c and [125I]4b, that contain a radiohalogenated nido-carborane moiety, are provided in Table S4 in Supporting Information, and data obtained at 4 h postinjection for selected tissues are plotted in Figure 2, panel C. Much smaller differences in the lung and spleen concentrations of the two radionuclides is observed at the three time points, indicating that the deastatination is much lower than observed for [211At]2c. Unfortunately, while the nidocarborane appears to provide a more stable attachment for astatine, it also prolongs the blood residence time. The biodistribution data for the biotin derivatives [211At]5c and [125I]5b, containing a nido-carborane and a trialkylammonium moiety, are provided in Table S6 in Supporting Information, and the 4 h data for selected tissues are plotted in Figure 2, panel D. It is apparent that some deastatination also occurs with this compound because of the differences observed in the concentrations of the two radionuclides in lung, spleen, and neck. DISCUSSION

The high cytotoxicity of radionuclides that emit R-particles makes them attractive for developing new radiopharmaceuticals for targeted radionuclide therapy (TRT) of cancer. Further, the short path length (e.g. 50-80 µm) of the emitted R-particles makes these radionuclides particularly well suited for therapy of micrometastatic disease, disease in compartmental spaces (e.g. ovarian carcinoma), and cancer cells remaining in surgical tumor resection areas. One of the most promising R-emitting radionuclides is 211At. It is particularly attractive because it can be readily prepared in high radionuclidic purity and does not produce R-emitting daughter radionuclides. While its short half-life (t1/2 ) 7.21 h) is problematic for use with intact monoclonal antibodies, smaller tumor targeting agents, such as biotin derivatives or peptides, may provide good carrier molecules for this radionuclide. There are many reagents and approaches that may be taken in TRT of cancer. One approach that utilizes the proven tumor targeting of monoclonal antibodies (mAb), is termed “pretargeting” (13, 14, 33-35). The pretargeting approach is particularly attractive as it employs a two- or three-step procedure to separate the pharmacokinetics of the molecule carrying the radionuclide (e.g. mAb) from the pharmacokinetics of the tumor targeting monoclonal antibody. This fact makes it attractive for use with short half-lived radionuclides such as 211At. Although several different pretargeting approaches have been described, we have chosen to investigate the use of antibody-streptavidin (mAb-SAv) conjugates in combination with radiolabeled biotin derivatives. This combination of reagents using other radionuclides has been successfully applied to treatment of human tumor xenografts in athymic mice (15, 16, 36) and has undergone

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clinical study (14, 17, 37). The success of the approach is due, in part, to the extremely high binding affinity of biotin with streptavidin (38), which provides high tumor targeting efficiency and long retention at the tumor site. Further, the small size of biotin derivatives provides rapid renal clearance of radiolabeled biotin derivatives, eliminating much of the irradiation of nontarget tissues. While a variety of biotin derivatives containing metal radionuclides have been successfully targeted to tumors in mouse models (34, 36, 39, 40), no examples of successful cancer targeting of radiohalogenated biotin derivatives have appeared in the literature. We hypothesized that the lack of examples of pretargeting with radiohalogens was due to the structural and lipophilic differences between biotin derivatives that bind metal radionuclides and those that bind radiohalogens. On the basis of this hypothesis, we have conducted a large number of studies to optimize the structural features of biotin derivatives so the in vivo characteristics of radiohalogenated biotin derivatives are improved. Unlike radiometal-containing biotin derivatives, radiohalogenated biotin derivatives can be quite insoluble in aqueous medium, so many of the compounds that have been investigated include ether containing linkers between the biotin moiety and the radiohalogen bonding moiety. Because all biotin derivatives are subject to in vivo degradation by the enzyme biotinidase (41-43), we (19, 44) and other investigators (45) have conducted studies to determine which structural features will block the biotinamide hydrolysis reaction of that enzyme. In addition to determining whether biotinidase cleaved biotin from radiohalogenated biotin derivatives, we also investigated the effect of structural features on biotin binding with avidin and streptavidin (46). We believe that it is important to retain the high binding affinity of biotin with these proteins for optimal in vivo tumor targeting. An outcome of those studies was identification of biotin derivatives which have a carboxylate functionality R to the biotinamide bond as an optimal structural feature. The R-carboxylate is optimal because it blocks biotinidase cleavage while retaining high binding affinity. Thus, all of the biotin derivatives investigated in vivo contain a carboxylate functionality R to the biotinamide bond. The design of the biotin derivatives for carrying the radioiodine and astatine must include a moiety for radiohalogens to react with. It is very important that radiohalogenation reactions are rapid and efficient and that the radiohalogenated moiety be stable to in vivo dehalogenation. Due to the presence of deiodinase enzymes (47), radioiodinations of small molecules for in vivo applications are generally conducted on deactivated aryl moieties or vinyl moieties. To facilitate rapid incorporation of radiohalogens, organometallic intermediates are generally used (48, 49). In these studies, stannylbenzamides and nido-carboranyl moieties were included in the biotin derivatives as functional moieties for reacting with radiohalogens. Aryl stannanes are very reactive toward electrophilic radiohalogens, producing site-specific incorporation. Aryl stannanes have been used extensively as intermediates for radioiodination and for astatination of molecules. Similarly, high reactivity of nido-carboranes with electrophilic radioiodine and astatine has been demonstrated. Pioneering work of Hawthorne and coworkers (50-52) and our previous studies (24, 25, 53) have shown radiohalogenation of nido-carboranes can occur in excellent radiolabeling yields under mild reaction conditions. Unlike the aryl stannanes, halogenation of nido-carboranes is not site-specific, with substitution potentially occurring on any boron atom of the nido-


carborane moiety. However, it seems most likely that when only one halogen is substituted, halogenation occurs on a boron atom in the open rim. Hawthorne et al. have reported crystal structures of two iodo-nidocarboranes, wherein the iodine atom substituted on the open rim R to a carbon atom (50, 54). Thus, while the structures shown in Schemes 2-4 depict only one of the potential halogenated regioisomers, it is likely to be a major one. Significantly, the presence of halogenated regioisomers is not an important factor in the compounds studied, since it is very unlikely that biotin binding with streptavidin will be affected by substitution on the nidocarborane. Biotin binding can be affected if any of the three contiguous, all-cis, chiral centers on the thiophane ring are altered. Importantly, the all-cis (+) configuration of biotin stereocenters is retained under the reaction conditions used. A fourth chiral center is present in the aspartyl group of compound 2 and the lysine groups of compounds 3-5. The presence of this chiral center does not appear to affect biotin binding (18-20). The presence of chiral centers and iodinated carboranyl regioisomers can, however, result in diasteriomeric pairs of compounds that complicate the NMR spectra and are observable as doublets in HPLC chromatograms. In vivo stability of the 211At bond to the carrier molecule is absolutely critical to its successful application in TRT. On the basis of our previous experiences, we anticipated some instability of 211At in the biotin derivatives prepared. Fortunately, the release of 211At from carrier molecules in vivo is easily monitored, as free 211At (astatide) localizes in lung, spleen, stomach, and thyroid. Localization of 211At in those tissues can be estimated by comparing its concentration against that of free radioiodine (or a radioiodinated compound). This comparison was first reported by Hamilton et al. (55) in an extensive study of the localization of 211At (vs 131I) in rats and monkeys. We have used the comparison of tissue concentrations to demonstrate that 211At-labeled mAb Fab′ fragments are unstable to in vivo deastatination (56) and more recently to show stability of astatinated benzamides, and nido-carboranyl derivatives in vivo (27). The graphs provided as panels in Figures 1 and 2 include the 211 At-localizing tissues: lung, spleen, and neck (thyroid). The graphs also include concentration data from blood and the excretory tissues, kidney, and liver. Only the 4 h data for the tissues is provided in the graphs, as that data is adequate to point out the important differences between the biotin derivatives or between 211At and 125I. For comparison purposes, a graph of the tissue concentrations of [211At]astatide and [125I]iodide in the selected tissues, from data previously published, has been included as Figure 2, panel A. The primary objective of the investigation was to determine the in vivo stability of the astatinated biotin derivatives. The biotin derivative 1a was the first prepared for astatination and evaluation. Astatination of 1a was fairly efficient, providing 64% isolated radiochemical yield after reaction for 10 min at room temperature. However, the astatinated compound, [211At]1c was not evaluated in vivo, as it was subsequently shown that the biotinidase-blocking N-methyl group dramatically decreases binding with avidin and streptavidin (20). Therefore, the biotin derivative 2b, which has a carboxylate group R to the biotinamide bond, was prepared and evaluated. In an vivo experiment, [125I]2b and [131I]1b were co-injected and tissue concentrations were evaluated at 1, 4, and 24 h postinjection. The results from the experiment (Table S1) indicated that there was little difference between the two compounds in vivo. Therefore,


2a was chosen for astatination and in vivo study. In the experiment, [211At]2c and [125I]2b were co-injected, and their biodistribution was evaluated. The data obtained (Table S2) clearly show that [211At]2c is unstable to in vivo deastatination based on the observed differences in 211 At and 125I concentrations in lung, spleen, and neck (thyroid) (compare Figure 2, panels A and B). The magnitude of difference between the concentrations of 211 At and 125I in those tissues suggests that the deastatination is very rapid. The rationale for evaluating nido-carboranes as radiohalogen-binding moieties in biotin derivatives was that these moieties may provide higher in vivo stability of 211 At. Because boron-halogen bonds are generally stronger than aryl-halogen bonds (57), it seemed possible that boron-halogen bonding would be more stable in vivo. Further, our studies with structurally simple benzamide derivatives and nido-carboranyl derivatives appeared to indicate some in vivo stability could be gained by using astatinated nido-carborane derivatives (27). Our initial study of an astatinated biotin derivative with a nidocarborane moiety demonstrated that it could be rapidly astatinated; however, an in vivo evaluation was not conducted as the compound was not stable to biotinidase. The biotin derivative employed contained a methyl group R to the biotinamide moiety (26). In vitro studies later demonstrated that this structural feature slowed the biotinidase cleavage rate (to ∼25% of an unaltered biotinamide) but did not block it (18). Once it was established that an R carboxylate could be used to completely block biotinidase, a biotin-lysine adduct, 3b, containing the nido-carborane moiety, was prepared and radioiodinated. However, preparation of 3b was also premature, as other studies demonstrated that inclusion of an aryl moiety decreased the in vivo release of 211At over simpler nido-carboranyl compounds (27). Therefore, a biotin derivative, 4a, that contains a phenyl group and a nido-carborane, was prepared and evaluated in vivo. In an initial study, [125I]4b was co-injected with [131I]1b to compare the radioiodinated nido-carboranyl-biotin derivative with a biotin derivative containing an aryl halide. The results show that [125I]4b is cleared much slower from blood, lung, and kidney than [131I]1b. This was consistent with the data previously obtained with similar derivatives that did not include biotin moieties (27). Following that experiment, [211At]4c and [125I]4b were co-injected and evaluated in vivo. The differences in concentration of 211At and 125I in lung, spleen, and neck are much smaller, indicating that less free 211At was present. This is very encouraging, but the high concentrations of astatinated and radioiodinated biotin derivatives remaining in blood and tissues makes this biotin derivative not optimal. The cause of the longer blood residence time of the nido-carborane-containing compounds is not known, but it may be due to their binding with serum proteins. In our studies, it has been noted that the concentration of radiolabeled biotin derivative in aqueous solution could decrease dramatically over a short period of time, with the radioactivity being bound to the surfaces of the container. Additionally, injection or transfer of the solutions via syringes were complicated by adsorption to the metal syringe needle. We found that preparations containing nido-carboranes were easier to handle and quantify when a 1% BSA solution was added prior to isolation. These observations led to a conclusion that the adsorption may be due to the nido-carborane moiety’s interaction with cationic ions on surfaces and on BSA. If this were true, it seemed that one way to potentially improve the


in vivo characteristics of biotin derivatives containing nido-carborane moieties would be to incorporate a trialkylamine that, when protonated, could act as an intramolecular counterion. Thus, a nido-carboranyl-biotin derivative which contained a trialkylamine functionality, 5a, was prepared and tested in vivo. An initial in vivo study compared [125I]5b with [131I]1b. Interestingly, the tissue concentrations of [125I]5b did not appear to be very different from that of [125I]4b, with the exception of the kidney, which appeared to have a considerably lower concentration at the 1 and 4 h time points (compare Tables S3 and S5). The biodistribution of [211At]5c is very similar to that of [211At]4c (compare Figure 2, panels C and D; Tables S4 and S6). The rationale for preparing 5a was that this compound might have decreased blood concentration due to a lower propensity to bind with serum proteins. In the study comparing co-injected [211At]5c and [125I]5b (Table S6) there appeared to be a large decrease in blood concentration for [125I]5b but not for [211At]5c. However, the biodistribution data for [125I]5b in the initial study (Table S5) did not show a low blood concentration. Therefore, the question of whether the intermolecular trialkylammonium counterion actually decreased the propensity to bind with serum proteins has not been answered from our data. Some differences in tissue concentrations of [211At]5c and [125I]5b are most likely due to having free 211At present (i.e. deastatination occurred). Importantly, it appears that the deastatination is of the same magnitude as that observed for [211At]4c, so the addition of the trialkylammonium counterion had little effect on the deastatination. In conclusion, we have investigated the synthesis, radiohalogenation, and in vivo tissue distribution of some biotin derivatives containing aryl stannanes or nidocarboranes for radiohalogenation. The studies have shown that biotin derivatives containing nido-carboranyl moieties can be readily prepared using standard synthetic organic chemistry methods. The studies have provided data that indicate the biotin derivatives containing astatinated nido-carborane moieties are more stable to in vivo deastatination than aryl astatine derivatives. This is a significant improvement over the use of astatinated aryl moieties; however, the astatinated nido-carboranyl derivatives do undergo some deastatination. The fact that the nido-carboranyl biotin derivatives are not completely stable toward in vivo deastatination, along with their long retention in blood, makes these derivatives nonoptimal. Continuing studies will investigate biotin derivatives with other borane cage molecules, and different charged functionalities, in an attempt to further improve them for use in pretargeting protocols. ACKNOWLEDGMENT

We thank Dr. Ruedi Risler for his efforts in the preparation of 211At, and Kent Buhler for his participation in animal studies. We are grateful for the financial support provided by the Department of Energy, Medical Applications and Biophysical Research Division, Office of Health and Environmental Research, under grant number DE-FG06-95ER62029. Supporting Information Available: Tables containing biodistribution data for compounds studied in vivo are included as Tables S1-S6. HPLC chromatograms, 1H NMR spectra, and mass spectra are provided for new compounds (11, 2a, 17a, 17b, 18, 3a, 20, 21, 22, 4a, 24, 25, 26, 27, 28, 29, 5a) prepared in the research described

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