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Synthesis and Characterization of the Actinium Aquo Ion ... Actinium-225 for Targeted α Therapy: Coordination Chemistry and Current Chelation Approac...
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Bioconjugate Chem. 2000, 11, 510−519

Synthesis, Conjugation, and Radiolabeling of a Novel Bifunctional Chelating Agent for 225Ac Radioimmunotherapy Applications Lara L. Chappell,† Kim A. Deal,‡ Ekaterina Dadachova, and Martin W. Brechbiel* Radioimmune & Inorganic Chemistry Section, DCS, NCI, NIH, Bethesda, MD, 20892. Received November 8, 1999; Revised Manuscript Received April 13, 2000

(t1/2 ) 10 days) is an alternative R-emitter that has been proposed for radioimmunotherapy (RIT) due to its many favorable properties, such as half-life and mode of decay. The factor limiting use of 225Ac in RIT is the lack of an acceptably stable chelate for in vivo applications. Herein is described the first reported bifunctional chelate for 225Ac that has been evaluated for stability for in vivo applications. The detailed synthesis of a bifunctional chelating agent 2-(4-isothiocyanatobenzyl)-1,4,7,10,13,16hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (HEHA-NCS) is reported. This ligand was conjugated to three monoclonal antibodies, CC49, T101, and BL-3 with chelate-to-protein ratios between 1.4 and 2. The three conjugates were radiolabeled with 225Ac, and serum stability study of the [225Ac]BL-3-HEHA conjugate was performed. 225Ac

INTRODUCTION

The treatment of cancer by the use of radiolabeled monoclonal antibodies continues to be attractive and supported by recent clinical reports (1). One variable that influences the effectiveness of radioimmunotherapy (RIT) in the treatment of cancer is the choice of the radionuclide and its associated emission characteristics (2). Solid tumors have been treated with β-emitters such as 131I (t1/2 ) 8.04 days) (1a) and 90Y (t1/2 ) 64.1 h) (1b). The range of the β- particle is several millimeters, and the majority of energy deposition does not occur immediately along the emission track (3). For example, the cytotoxicity attributed to the 90Y radioimmunoconjugate of monoclonal antibody anti-Tac has been attributed to be a result of cross-fire and not a direct effect from the emission occurring at the cell surface (4, 5). Therefore, these emissions have been proposed to be less useful for micrometastatic disease and lymphomas and also contribute to normal tissue toxicity (6). In this case, an R-emitting radionuclide might be a better choice due to very high cytotoxcity, a short pathlength emission, and immediate energy deposition that should minimize collateral cytotoxicity. Examples that have been studied are 213Bi (t1/2 ) 46 min) (7, 8) and 211At (t1/2 ) 7.2 h) (9), and clinical trials employing these two radionuclides are ongoing (10, 11). Challenges to the use of the former radionuclide is a short half-life potentially limiting tumor uptake and thus effectiveness, in addition to short supply and high cost. The general utility of the latter radionuclide, 211At, in addition to possessing similar problems, albeit to a lesser degree, seems limited to being produced at facilities possessing an appropriate cyclotron. An alternative R-emitter that has been proposed for RIT is 225Ac (t1/2 ) 10 days) which is a parent of 213Bi (12). The decay process of 225Ac includes four R-emissions and * To whom correspondence should be addressed. E-mail: [email protected]. † Current address: Department of Chemistry, SUNY at Oswego, Oswego, NY, 13126. ‡ Current address: The CNA Corporation, 4401 Ford Avenue, Alexandria, VA, 22302-8268.

two β--emissions to a stable 209Bi daughter (Chart 1). As a very large amount of energy (∼28 MeV) is released during this process, much lower amounts of activity of the radionuclide are actually required to produce the desired effects and thus, this isotope demonstrates extreme cytotoxicity (13). For RIT applications, 225Ac must be linked as the metal complex to a monoclonal antibody (mAb) or immunoprotein via a suitable bifunctional chelating agent (14). The 225 Ac complex must be adequately thermodynamically and kinetically stable to minimize release of the isotope due to the extreme toxicity in vivo (13). Derivatives of acyclic polyaminocarboxylate ligands, such as ethylenediamine tetraacetic acid and diethylenetriamine pentaacetic acid (EDTA and DTPA), which form complexes with 225Ac, have been demonstrated as far too labile in vivo (15). The use of calix[4]arene ligands has also been proposed for sequestration of 225Ac in RIT applications (16). The first report demonstrated selectivity and extraction characteristics of the calix[4]arene derivative for 225Ac, but presented no actual stability data (16a). The second report presented detailed results on the conjugation of a bifunctional calix[4]arene to a mAb and protein, immunoreactivity of the product, and studies on potential immunogenicity (16b). However, no data pertaining to actual stability, either in vitro or in vivo for the 225Ac complex was presented, nor were any radiolabeling data included. Previously, our laboratory has reported on the screening of chelating agents and potential development of more stable chelating agents for 225Ac (17). A promising candidate that emerged from this study, 1,4,7,10,13,16hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid (HEHA), was found to form an 225Ac complex that was eliminated from the blood exceptionally rapidly, while also not permitting accumulation of 225Ac in the target organs, i.e., liver and bone (18). Also, a substantial reduction in observable toxicity was noted during this comparison with other screened 225Ac complexes (19). Upon the basis of this lead, an efficient synthesis and in vitro evaluation of a bifunctional version of this ligand was deemed to be the next logical step in the development

10.1021/bc990153f CCC: $19.00 © 2000 American Chemical Society Published on Web 06/28/2000

Bifunctional Chelate for

225Ac

Chart 1. Decay Characteristics of

Bioconjugate Chem., Vol. 11, No. 4, 2000 511 225Ac

of a suitable chelating agent to test the hypothesis of using 225Ac for RIT. Herein, we report the synthesis and characterization of the bifunctional C-functionalized HEHA ligand. This chelator has been conjugated to three mAbs, radiolabeled with 225Ac, and the serum stability of one of the [225Ac]HEHA-mAb conjugates evaluated. EXPERIMENTAL SECTION

Materials and Methods. All materials were of reagent grade and used without further purification. Chromatography was performed on silica gel 60, 220-440 mesh ASTM (Fluka). Thin-layer chromatography (TLC) was performed on silica gel 60 F-254 plates (EM Reagents). All glassware used in the synthesis of the ligand after ester hydrolysis was acid-washed and rinsed with distilled deionized water. 1H and 13C NMR spectra were recorded on a Varian Gemini 300 instrument at ambient temperature. Chemical shifts are reported downfield relative to TMS (CDCl3) or TSP (D2O). Chemical ionization mass spectra (CI-MS) were measured using a Finnegan 3000 instrument. Fast atom bombardment mass spectra (FAB-MS) were measured using a Extrel 400 instrument. High-resolution FAB (HR-FAB) mass spectra were obtained on a JEOL SX102 mass spectrometer operated at an accelerating voltage of 10 kV. Samples were desorbed from a glycerol matrix using 6 keV xenon atoms. Mass measurements in HR-FAB are performed at 10 000 resolution. Analytical HPLC was performed on a Beckman gradient system equipped with model 114 M pumps controlled by System Gold software and a model 165 dual wavelength detector set at 254 and 280 nm. An Altex ODS C18 column (4.6 mm × 15 cm) with a 25 min gradient of 100% aqueous 0.05 M triethylammonium acetate to 100% methanol at a flow rate of 1 mL/min was used. Infrared spectra were obtained using a Bio-Rad FTS 45 spectrophotometer with the samples prepared as Nujol mulls.

HPLC purification of radiolabeled with 225Ac conjugates, as well as HPLC analysis of samples from serum stability studies, were performed employing a Dionex system equipped with a model GPM-2 gradient pump, a Gilson model 112 UV monitor operating at 280 nm, an IN/US γ-RAM flow-through radioactivity detector, and a Gilson model 203 fraction collector. The size-exclusion HPLC column (TSK-Gel G3000SW, TosoHaas, Japan) eluted with PBS, pH 7.2, at 1 mL/min was used for these separations. Radioactive samples were counted in Minaxi γ-counter (Packard Instrument Company). Synthesis. 4-Nitrophenylalaninylglycylglycine, methyl ester (11). N-tert-Butoxycarbonyl-(D,L)-4-nitrophenylalanine (20) (20 g, 64.5 mmol), glycylglycine methyl ester hydrochloride (14.1 g, 77.2 mmol), Et3N (11 mL, 77.2 mmol), and EDC (14.8 g, 77.2 mmol) were combined in anhydrous DMF (500 mL) and stirred at room-temperature overnight. The solvent was removed under vacuum, and EtOAc (500 mL) was added. The solution was extracted with H2O (2 × 200 mL) and 5% NaHCO3 (3 × 200 mL). The aqueous layers were back-extracted with EtOAc (2 × 150 mL), and this EtOAc portion was washed as above. The EtOAc layers were combined and washed with saturated NaCl solution (1 × 200 mL). The combined EtOAc solution was dried over anhydrous Na2SO4, filtered, and the filtrate reduced to dryness. The residue was taken up in boiling EtOAc (200 mL), and hexanes (300 mL) was added. The material initially separated as an oil which then turned to a white precipitate. This precipitate was chromatographed on silica gel with 10% MeOH in CHCl3 elution. The first major band contained the product. Fractions containing product were combined and reduced to dryness to yield the product (13.16 g, 47%). 1H NMR (DMSO-d ) δ 1.27 (s, 9 H, tBu), 2.88 (m, 1 H), 6 3.17 (m, 1 H), 3.64 (s, 3 H, -OCH3), 3.79 (d, 2 H, J ) 5.7), 3.88 (m, 2 H), 4.28 (m, 1 H), 7.09 (d, 1 H, J ) 8.7, NH), 7.56 (d, 2 H, J ) 9, Ar), 8.17 (d, 2 H, J ) 8.7, Ar), 8.27 (t, 1 H, J ) 5.9, NH), 8.33 (m, 2 H, J ) 5.9, NH); 13C NMR (DMSO-d6) δ 28.04 (tBu), 37.39, 40.54, 41.88, 51.77, 55.30, (-OC(CH3)3), 78.37 (-OCH3), 123.30, 130.82, 146.43, 147.04 (Ar), 155.60, 169.62, 170.47, 171.81 (C(O)); MS (CI/NH3) m/e 456 (M + NH4+). Anal. Calcd for C19H26N4O8: C, 52.05; H, 5.98; N, 12.78. Found: C, 51.79; H, 5.93; N, 12.60. N-tert-Butoxycarbonyl-4-nitro-phenylalaninylglycylglycine N-(2-Aminoethyl)-amide (12). Methyl ester 11 (16.55 g, 37.8 mmol) in MeOH (100 mL) was added as a slurry by pipet to stirring ethylenediamine (250 mL, 3.7 mol), that had been freshly distilled from 3A sieves. The reaction mixture changed from colorless to pink, to deep red, and finally to brown and was left stirring for 3 days. The ethylenediamine was removed by vacuum rotary evaporation and the residue purified by column chromatography on silica gel with CHCl3/MeOH/NH4OH (12:4: 1) elution. 1H NMR (DMSO-d ) δ 1.27 (s, 9 H, tBu), 2.57 (t, 2 H, 6 J ) 6.3, -CH2NH2), 2.88 (m, 1 H), 3.06 (q, 2 H, J ) 6.2), 3.17 (m, 1 H), 3.3 (br, 2 H), 3.69 (d, 2 H, J ) 5.7), 3.76 (d, 2 H, J ) 4.8), 4.24 (9 m, 1 H), 7.13 (d, 1 H, J ) 8.7, NH), 7.56 (d, 2 H, J ) 9.0, Ar), 7.74 (t, 1 H, J ) 5.4, NH), 8.10 (t, 1 H, J ) 5.9, NH), 8.16 (d, 2 H, J ) 7.8, Ar), 8.35 (t, 1 H, J ) 5.9, NH); 13C NMR (DMSO-d6) δ 27.97 (tBu), 37.26, 41.21, 42.12, 42.24, 55.24 (-CH2Ar), 78.37 (-OC(CH3)3)), 123.30, 130.82, 146.43, 146.97 (Ar), 155.60, 168.89, 169.32, 171.99 (C(O)); MS (CI/NH3) m/e 467 (M+ + 1). Anal. Calcd for C20H30N6O7: C, 51.49; H, 6.48; N, 18.01. Found: C, 50.88; H, 6.46; N, 17.83.

512 Bioconjugate Chem., Vol. 11, No. 4, 2000

N-(2-Aminoethyl)-4-nitro-phenylalaninylglycylglycine amide hydrochloride (13). Dioxane (300 mL) was chilled in an ice bath and saturated with HCl (g) for 2 h. Carbamate 12 (8.95 g, 19.2 mmol) was added, and HCl (g) bubbled for an additional 30 min. The reaction mixture was stirred overnight, after which diethyl ether was added (200 mL) and the mixture chilled for 7 h. The solid was collected on a Bu¨chner funnel, washed with ether, and dried under vacuum (10.6 g). 1 H NMR (DMSO-d6) δ 2.87 (m, 2 H), 3.2-3.4 (m, 4 H), 3.5-3.9 (m, 4 H), 4.22 (m, 1 H), 7.62 (d, 2 H, J ) 9, Ar), 8.13 (br, 2 H, NH), 8.20 (d, 2 H, J ) 8.7, Ar), 8.40 (m, 1 H, NH), 8.48 (m, 1 H, NH), 9.11 (m, 1 H, NH); 13 C NMR (DMSO-d6) δ 34.47, 36.60, 38078, 42.55, 53.60, 124.02, 131.67, 144.83, 147.34, 169.13, 169.98; MS (CI/NH3) m/e 367 (M + H+). Anal. Calcd for C15H22N5O7‚2HCl‚1.2dioxane‚H2O: C, 42.24; H, 6.37; N, 14.93. Found: C, 43.28; H, 6.50; N,14.99. 1-(tert-Butoxycarbonyl)-5-(4-nitrobenzyl)-3,6,9,12,17pentaoxo-1,4,7,10,13,16-hexaazacyclohexadecane (14). Dioxane (3.7 L) was heated to 95 °C in a 5 L three-necked Morton flask. Hydrochloride 13 (4.39 g, 10 mmol) was dissolved in DMSO (35 mL), and Et3N (4.6 mL, 33 mmol) was added. The mixture was stirred for 30 min and the Et3N‚HCl was removed by filtration. The filtrate was taken up into a gastight syringe and DMSO added to bring the final volume to 50 mL. N-tert-Butoxycarbonyliminodiacetic acid dihydroxysuccinnimyl ester (20) (4.27 g, 10 mmol) was dissolved in DMF, taken up into a gastight syringe and additional DMF was added to adjust the final volume to 50 mL. The syringes were loaded onto a syringe pump and added to the dioxane such that the addition was complete after 24 h. One more addition of 10 mmol in each reactant were added over a 24 h period. The reaction was heated for an additional 18 h and cooled to room temperature. The reaction was concentrated to thick oil under vacuum and the brown residue dissolved in CHCl3 (500-600 mL). The CHCl3 layer was washed with water (2 × 200 mL), saturated NaCl solution (1 × 100 mL), 5% HCO3- (5 × 150 mL), saturated NaCl solution (1 × 100 mL), 1 M HCl (2 × 100 mL), and saturated NaCl solution (1 × 100 mL). The CHCl3 layer was dried over Na2SO4 and filtered, and the filtrate was reduced to dryness by rotary evaporation (crude yield 2.5 g). However, when purification by column chromatography on silica gel was performed, no product was isolated as determined by mass spectrometry and 1H NMR spectroscopy. N-tert-Butyloxycarbonyliminodiacetyldiglycine Diethyl Ester (1). N-tert-Butyloxycarbonyl-iminodiacetic acid (21)(20 g, 85.8 mmol), glycine ethyl ester hydrochloride (24 g, 171.9 mmol), and triethylamine (24 mL, 172 mmol) were dissolved in anhydrous DMF (700 mL). EDC (34 g, 88.7 mmol) was added and the solution stirred 18 h at room temperature. The DMF was removed under vacuum, and the residue dissolved in ethyl acetate (600 mL). The ethyl acetate was extracted with water (1 × 200 mL), 5% NaHCO3 (3 × 200 mL), brine (1 × 200 mL), 1 M HCl (1 × 200 mL), and brine (2 × 200 mL). The organic layer was dried over Na2SO4, filtered and concentrated to yield an oil (23 g, 66%). 1H NMR (DMSO-d ) δ 1.19 (t, 6 H, CH ); 1,35 (s, 9 H, 6 3 C(CH3)3); 3.30 (s, 2 H, CH2); 3.32 (s, 2 H, CH2); 3.88 (s, 2 H, CH2); 3.91 (s, 2 H, CH2); 4.08 (q, 4 H, CH2CH3); 8.80 (t, 2 H, NH); 13C NMR (DMSO-d6) δ 14.01, 27.73, 51.29, 21.77, 60.46, 79.76, 154.87, 169.86, 170.29, 170.41; MS (CI/NH3) m/e 404 (M+ + 1). Anal. Calcd for C17H29N3O8: C, 50.62; H, 7.24; N, 10.42. Found: C, 50.09; H, 7.10; N, 10.13.

Chappell et al.

N-tert-Butyloxycarbonyliminodiacetyldiglycine (2). To a stirred solution of NaOH (5.2 g, 131 mmol) in ethanol (200 mL) and H2O (100 mL) was added dropwise diester 1 (23 g, 57 mmol) in ethanol (200 mL). The solution was stirred 60 h at room temperature followed by removal of ethanol under vacuum. The residual aqueous layer was extracted with ethyl acetate (1 × 200 mL). The aqueous layer was chilled to 0 °C and acidified to pH 2 with 3 M HCl (∼50 mL). To facilitate extraction, 20 g of NaCl was added and the aqueous layer was extracted with ethyl acetate repeatedly. The product began to precipitate in the organic layer and the organic layer was heated to maintain a clear solution before drying with sodium sulfate. After drying and filtration, concentration of the organic layer yielded the pure product as a white solid (14.59 g, 74%). The product was dried under vacuum at 70 °C for 24 h. 1H NMR (DMSO-d ) δ 1.34 (s, 9 H, C(CH ) ); 3.79 (s, 2 6 3 3 H, CH2); 3.80 (s, 2 H, CH2); 3.84 (s, 2 H, CH2); 3.90 (s, 2 H, CH2); 8.75 (m, 2 H, NH); 13C NMR (DMSO-d6) δ 27.79, 51.47, 51.96. 79.76, 154.99, 170.29, 171.26; MS (CI/NH3) m/e 348 (M+ + 1). Anal. Calcd for C13H21N3O8‚HCl: C, 40.69; H, 5.77; N, 10.95. Found: C, 41.17; H, 5.61; N, 11.03. N-tert-Butyloxycarbonyliminodiacetyldiglycine Disuccinimidyl Ester (3). Thoroughly dried diacid 2 (14 g, 40.3 mmol) and N-hydroxysuccinimide (10.2 g, 88.7 mmol) were stirred in DMF (250 mL) under argon. EDC (17 g, 88.7 mmol) was added and the solution stirred 18 h. The DMF was removed under vacuum, and the residue dissolved in ethyl acetate (600 mL). The organic layer was extracted with water (1 × 150 mL), 5% NaHCO3 (3 × 200 mL), brine (1 × 200 mL), and brine (2 × 200 mL). The organic layer was dried with Na2SO4, filtered, and concentrated to yield a white solid (15.86 g, 73%). The product was thoroughly dried under vacuum at 70 °C prior to use in cyclization. 1H NMR (DMSO-d ) δ 1.34 (s, 9 H, C(CH ) ); 2.82 (s, 8 6 3 3 H, succinimide); 3.88 (s, 2 H, CH2); 3.94 (s, 2 H, CH2); 4.31 (s, 2 H, CH2); 4.33 (s, 2 H, CH2); 8.95 (t, 2 H, NH); 13C NMR (DMSO-d ) δ 25.19, 25.43, 27.74, 51.05, 79.89, 6 154.87. 166.53, 170.23, 170.54; MS (CI/NH3) m/e 542 (M+ + 1). Anal. Calcd for C21H27N5O12‚1.5H2O: C, 44.37; H, 5.31; N, 12.32. Found: C, 44.43; H, 5.54; N, 11.48. 1-(tert-Butoxycarbonyl)-8-(4-nitrobenzyl)-3,6,9,14,17pentaoxo-1,4,7,10,13,16-hexaazacyclohexadecane (4). In a 5 L three-necked Morton flask, anhydrous dioxane (3.5 L) was heated to ∼90 °C. Disuccinimidyl ester 3 (5.42 g, 10 mmol) in DMF (50 mL) and N-(2-aminoethyl)-4nitrophenylalaninamide (19) (2.52 g, 10 mmol) in DMF (50 mL) were each loaded in 50 mL gastight syringes and added to the dioxane so that the addition was complete after 24 h. Second and third additions were 10 and 5.5 mmol in each reactant, respectively. After the third addition, the reaction was heated for an additional 18 h and then cooled to room temperature. The reaction was concentrated to a thick brown oil under vacuum and the residue dissolved in chloroform (500 mL). The chloroform was washed with water (1 × 200 mL), 5% NaHCO3, (2 × 200 mL), brine (1 × 200 mL), 1 M HCl (2 × 200 mL), brine (1 × 200 mL), and water (1 × 100 mL). The CHCl3 layer was kept separate and reduced to dryness. A sticky, brown residue stuck to the inside of the separatory funnel was dissolved in MeOH and reduced to dryness. The residues isolated from the CHCl3 and MeOH were each chromatographed on short silica gel columns with 1020% MeOH in CHCl3. Fractions that contained the product were combined (Rf ) 0.55, silica gel, 20% MeOH in CHCl3). This product was purified by column chroma-

Bifunctional Chelate for

225Ac

tography on silica gel eluted with a 10%-20% MeOH in CHCl3 gradient yielded the pure macrocycle as a tan solid (2.55 g, 19%). 1H NMR (DMSO-d ) δ 1.33 (s, 9 H, C(CH ) ), 2.8-4.0 6 3 3 (overlapping multiplets, 14 H); 4.5 (m, 1 H, CH); 7.47 (s, 2 H, Ar); 7.58 (t, 0.5 H, NH); 7.74 (d, 1.5 H, NH); 7.96 (d, 1 H, NH); 8.12 (d, 2 H, Ar); 9.14 (t, 0.5 H, NH); 9.21 (m, 1 H, NH); 9.36 (t, 0.5 H, NH)); 13C NMR (DMSO-d6) δ 27.74, 52.15, 53.36, 53.54, 79.22, 80.13, 123.36, 130.65, 146.37, 146.56, 154.69, 169.20, 170.66, 171.08, 171.27, 171.69; MS (CI/NH3) m/e 564; (M+ + 1) HPLC tR ) 18.7 min. Anal. Calcd for C24H33N7O9‚2 H2O: C, 48.08; H, 6.21; N, 16.35. Found: C, 48.28; H, 5.73; N, 15.87. 2-(4-Nitrobenzyl)-3,8,11,15,18-pentaoxo-1,4,7,10,13,16hexaazacyclohexadecane Hydrochloride (5). Dioxane (50 mL) in a 250 mL three-necked round-bottom flask was chilled in an ice bath and saturated with HCl (g) for 2 h. Carbamate 4 (1.66 g, 2.9 mmol) was added and HCl (g) was bubbled through the reaction mixture for 1 h. The reaction stirred overnight (18 h) at room temperature. Diethyl ether (200 mL) was added and the reaction mixture chilled in the freezer for 6 h. The yellow precipitate was collected on a Bu¨chner funnel, washed with diethyl ether, and vacuum-dried overnight. The product was isolated as a pale, yellow solid (1.9 g, >100%). 1 H NMR (DMSO-d6) δ 3.0-3.3 (m, 7 H), 3.4 (m, 2 H), 3.70 (m, 3 H), 3.89 (m, 3 H), 4.44 (m, 1 H -CH-Ar), 7.54 (d, 2 H, Ar), 7.80 (t, 1 H, NH), 8.05 (t, 1 H, NH), 8.14 (d, 2 H, Ar) 8.20 (d, 1 H, NH), 8.96 (d of t, 2 H, NH); MS (CI/NH3) m/e 464 (M+ + 1); HPLC tR ) 15.3 min. Anal. Calcd for C19H25N7O7‚1.5 H2O‚HCl‚dioxane: C, 44.92; H, 6.06; N, 15.94. Found: C, 44.23; H, 5.90; N, 15.97. 2-(4-Nitrobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane Hexahydrochloride (6). The above product 5 (1.8 g, 3.0 mmol) and anhydrous THF (50 mL) was combined in a 100 mL two-necked round-bottom flask and chilled in an ice bath. To this suspension was added 1 M borane in THF (5-6 mL). The mixture was allowed to warm to room temperature and then heated at 50 °C. Over 3 days, additional 1 M borane in THF was added in 15, 5, and 10 mL portions, respectively, for a total of 36 mmol. The reaction mixture was evaporated to dryness and the yellow solid (1.55 g) dried under vacuum. This residue was transferred to a 250 mL three-necked flask and absolute EtOH (80 mL) was added. HCl(g) was bubbled through the reaction mixture for 2 h and then the reaction was heated at reflux for 17 h. After which, the reaction mixture was cooled to room temperature, Et2O (150 mL) was added and the suspension was placed in the freezer overnight. The pale yellow precipitate was collected on a Bu¨chner funnel, washed with diethyl ether and vacuum-dried (1.01 g, 54%). 1H NMR (D O pH 1-2) δ 3.0-4.0 (m, 25 H, macrocycle, 2 -CH2Ar), 7.5 (m, 2 H, Ar), 8.25 (m, 2 H, Ar); MS (CI/ NH3) m/e 394 (M+ + 1); HPLC tR ) 17.8 min. Anal. Calcd for C19H35N7O2‚6HCl‚H2O: C, 36.21; H, 6.87; N, 15.56. Found: C, 36.06; H, 6.80; N, 14.59. Hexa-tert-butyl 2-(4-nitrobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetate (7). Hexahydrochloride 6 (1.15 g, 1.83 mmol) was taken up in water (4 mL), and the pH raised to 13 by the addition of solid NaOH. The aqueous solution was extracted with CHCl3 (4 × 100 mL). The combined CHCl3 layers were dried over anhydrous Na2SO4 and the drying agent filtered. The free base was isolated as a yellow solid (593 mg, 80% recovery) after evaporation of the filtrate. 1 H NMR (CDCl3) δ 1.6 (br, 6 H, NH); 2.2-3.4 (m, 25 H, macrocycle, CH2Ar), 7.3 (m, 2 H, Ar), 8.05 (m, 2 H,

Bioconjugate Chem., Vol. 11, No. 4, 2000 513

Ar); 13C NMR (CDCl3) δ 38.39, 38.81, 46.34, 48.34, 48.53, 48.77, 49.07, 49.38, 52.41, 52.78, 58.67, 123.32, 123.57, 129.94, 146.34, 147.55. The above free base (593 mg, 1.51 mmol) was dissolved in anhydrous DMF (10 mL), and tert-butyl bromoacetate (1.78 g, 9.1 mmol) was added. The reaction was allowed to stir in an ice bath and then warmed to room temperature over 1 h. An aqueous solution of Na2CO3 (965 mg in 20 mL of H2O) was added, and the mixture was stirred for 2.5 h. Toluene (10 mL) was added, and the reaction mixture was stirred overnight (17 h). The reaction mixture was poured into a separatory funnel, and the aqueous layer drained. The toluene layer was saved. The aqueous layer was extracted with CHCl3 (2 × 100 mL), and the combined CHCl3 and toluene layers were reduced to dryness. The residue was determined to contain hexatert-butyl ester product by mass spectrometry. This residue was purified on two consecutive silica gel columns (2 × 10 cm) with a gradient from 5 to 10% MeOH in CHCl3 and finally 10% NH3(aq) in MeOH. Early fractions contained the hexa-tert-butyl ester product with some impurity. Later fractions contained only the hexa-tertbutyl ester product. These were combined, reduced to dryness, and vacuum-dried (462 mg, 29%). 1 H NMR (CDCl3) δ 1.45 (overlapping s, 54 H, tBu); 2.83.4 (m, 37 H, macrocycle, CH2Ar), 7.46 (d, 2 H, Ar), 8.13 (d, 2 H, Ar); 13C NMR (CDCl3) δ 27.94, 52.05, 53.32, 55.33, 55.63, 55.99, 80.58, 123.20, 130.25, 146.21, 149.21, 149.37, 170.93, 171.65; MS (CI/NH3) m/e 1078.6 (M+ + 1), 1100.6 (M+ + Na). Anal. Calcd for C55H94N7O14‚0.5 H2O: C, 60.81; H, 8.81; N, 9.03. Found: C, 58.97; H, 8.44; N, 9.01. 2-(4-Nitro-benzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic Acid (8). To a 10 mL round-bottom flask, hexa-tert-butyl 2-(4-nitrobenzyl)1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16hexaacetate (166 mg, 0.15 mmol) was combined with concentrated HCl (5 mL) and heated at reflux for 6 h. The HCl(aq) was removed by rotary evaporation, and the residue taken up in water (1-2 mL) and was lyophilized to give the hexa-acid as a pale yellow solid (100 mg, 70%). 1 H NMR (D2O, pH 1-2) δ 2.8-4.2 (m, 37 H), 7.48 (br, 2 H, Ar), 8.14 (br, 2 H, Ar); MS (CI/NH3) m/e 742 (M+ + 1); HPLC tR ) 10.5 min. Anal. Calcd for C31H47N7O14‚6 HCl: C, 38.95; H, 5.59; N, 10.26. Found: C, 39.12; H, 5.66; N, 10.49. 2-(4-Aminobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13, 16-hexaacetic acid (9). A Schlenk flask was charged with 10% Pd/C (23.5 mg) and H2O (3 mL) and fitted onto an atmospheric hydrogenator. The apparatus was flushed with H2(g) two times to fully saturate the catalyst. A solution of hexaacetic acid 8 (76.6 mg, 0.080 mmol) in H2O (3 mL) was injected via syringe into the flask. The hydrogenation was allowed to proceed until the uptake of H2(g) ceased. The reaction mixture was filtered through a bed of Celite 577 packed in a medium glass fritted funnel. The filtrate was reduced to dryness by rotary evaporation and the residue taken up in water (1-2 mL). This was then lyophilized to give the aniline as a pale yellow solid (56 mg, 75%). 1H NMR (D O, pH 1-2) δ 2.6-4.2 (m, 37 H), 7.46 (m, 2 4 H, Ar); 1H NMR (D2O, pH 13) δ 2.0-4.6 (m, 37 H), 6.83 (m, 2 H, Ar), 7.09 (m, 2 H, Ar); MS (CI/NH3) m/e 712 (M+ + 1); HPLC tR ) 8.6 min. M - H+ calcd for C31H48N7O12: 710.3371. Found [HRFAB]: m/e ) 710.3397, error ) +5.0 ppm. 2-(4-Isothiocyanatobenzyl)-1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13, 16-hexaacetic acid (10). A 1 M solution of SCCl2 in CHCl3 (50 mL) was added to aniline

514 Bioconjugate Chem., Vol. 11, No. 4, 2000

9 (30.5 mg, 0.033 mmol) dissolved in H2O (0.5 mL) in a vial. The mixture was stirred rapidly for 2 h at room temperature. The aqueous layer was decanted with a pipet into a round-bottom flask and the CHCl3 layer washed with H2O (3 × 0.5 mL). The combined aqueous portions were reduced to approximately 1 mL by rotary evaporation. The solution was then lyophilized to give the isothiocyanate as a pale yellow solid (30.9 mg, 96%). 1H NMR (DMSO-d , pH 1-2) δ 3.0-4.0 (m, 37 H), 7.33 6 (br, 4 H, Ar); 1H NMR (D2O, pH 13) δ 2.2-3.4 (m, 37 H), 7.3 (m, 4 H, Ar); MS (FAB/glycerol) m/e 754 (M+ + 1); IR (Nujol) 2034.1 cm-1; HPLC tR ) 14.95 min. M - H+ calcd for C32H46N7O12S. Found [HRFAB]: m/e ) 752.2971, error ) +6.1 ppm. Antibody Conjugation. Purified monoclonal antibody CC49 (22a), which reacts with the human pancarcinoma antigen TAG-72 and its isotype-match BL-3 (22b), were supplied by Dr. J. Schlom, (Laboratory of Tumor Immunology and Biology, NCI, NIH); T101 antibody (23) was furnished by Dr. J. Carrasquillo (Nuclear Medicine Department, Clinical Center, NIH). Antibody solutions were initially transferred to dialysis tubing (Spectra/Por CE DispoDialyers, MWCO 10 K, 5 mL) and were dialyzed against conjugation buffer, (1 L, 0.05 M CO3-2/HCO3-1, 0.15 M NaCl, 5 mM EDTA, pH 8.6) for 6 h at 4 °C. After dialysis, the protein concentration was determined spectrophotomerically. Extinction coefficients of 1.4, 0.65, and 1.3 mL/mg‚cm were determined for mAbs BL-3, CC49, and T101, respectively, based on a mAb concentration from a protein determination using the Lowry method (24) with a bovine serum albumin standard. A solution of 10 in water was added to the antibody solution, such that initial molar ratio of ligand to antibody was 50-fold. The reaction mixture was allowed to stand at room-temperature overnight (18 h). The reaction mixture was transferred to Centricon (MWCO 30 K or 50 K) (Amicon) filtration units. Ammonium acetate buffer (0.15 M NH4OAc, pH 7.0) was added to the filtration unit to a total volume of 2-2.5 mL, and the filtration units were centrifuged until the remaining volume was approximately 0.5 mL. Additional buffer was added, and this process was repeated for a total of six times. The final antibody concentration was measured spectrophotomerically and the ligand-toprotein ratio (L/P) was determined as previously described (25). In brief, the Pb(II) arsenazo III complex provided a colorometric assay that can be easily monitored. Pb(II) is efficiently and reproducibly transchelated from the dye to the mAb macrocycle conjugate thereby eliminating a radiolabeling assay. Radiolabeling of HEHA-mAb Conjugates with 225 Ac. 225Ac(NO3)3 (2.2-4.0 mCi) was obtained from Oak Ridge National Laboratories (ORNL) as a solid. The solid was taken up in 0.1 M HCl (1 mL), such that the specific activity became 0.22-0.40 mCi/0.1 mL. For radiolabeling the conjugates, an aliquot of 225Ac solution (0.05-0.2 mCi) was added to the vials containing 0.15 M NH4OAc (0.30.4 mL) at pH 4.0, 5.0 or 7.0. The HEHA-mAb conjugate (0.3-0.4 mg) in 0.15 M NH4OAc buffer, pH 7.0, was then added. The reaction mixture was incubated for 0.5 h at 37 °C. Nonspecific protein binding was investigated by incubating 0.08 mCi of 225Ac solution with 0.5 mg of bovine serum albumin (BSA) at pH 7.0 for 0.5 h at 37 °C and the assaying for labeling by HPLC. Preliminary labeling yields were assessed using 10 cm ITLC-SG strips (Gelman Sciences) spotted with 1 µL of the reaction and developed in 0.15 M NH4OAc buffer, pH 4.0. In this system, the labeled proteins are retained at

Chappell et al.

the origin while 225Ac acetate moves with the solvent front. The strips were dried, cut into 1-cm segments, and counted in the γ-counter. They were recounted 3 h later in order to obtain corrected labeling yields for 225Ac after all of the 213Bi that had been previously generated decayed. The reaction was quenched with 0.1 M EDTA, pH 6.0 (4 µL), and [225Ac]HEHA-mAb conjugate was purified on size exclusion HPLC column as described above. The protein fraction was collected and counted in a Capintec Radioisotope Calibrator and recounted 3 h later to calculate the specific activity of the product. Serum Stability Studies. Purified [225Ac]HEHA-mAb conjugate (200 µL) was incubated with fetal bovine serum (2 mL) for 3 days at 37 °C. Aliquots were withdrawn at times 0, 0.25, 1, 3, 5, 24, 48, and 72 h and analyzed by HPLC on size exclusion column as described above. The radioactive fractions containing high and low molecular weight components were collected and counted on a γ-counter immediately after elution and 3 h thereafter in order to determine if they were due to free 225Ac or 213 Bi. If a particular fraction contained 225Ac/213Bi in equilibrium, the results obtained after 3 h counts were very close to the initial value. In the case of mostly 225Ac being present, the counts increased 2-fold as the 213Bi daughter grew in and 225Ac/213Bi reached equilibrium. Finally, the counts decreased 16-fold if mostly 213Bi was present initially as 3 h are equal to ∼4 half-lives of 213Bi. As a control, free 225Ac was incubated in serum under the same conditions and aliquots checked by HPLC at the above time points. RESULTS

Ligand Synthesis. An initial attempt at the synthesis of bifunctional HEHA was using the route outlined in Scheme 1, which has been denoted as the “5 + 1” cyclization. All attempts at cyclization using compound 13 and the BOC-iminodiacetate bis-active ester failed to yield any detectable, isolable amounts of desired macrocycle. Prompted by this result, a “3 + 3” cyclization was devised (Scheme 2). tert-Butyloxycarbonyl-iminodoacetic acid was extended in both directions through diimide coupling with ethyl glycine to produce diester 1. Saponification and formation of the corresponding di-succinimidyl ester 3 prepared one-half of the targeted hexaaza18-membered. The remaining component for the macrocyclization reaction, N-(2-aminoethyl)-4-nitrophenylalanine was readily available (20). Applying methods and conditions analogous to those previously reported (21) resulted in macrocycle 4 being isolated after chromatography in relatively good yield (20%). The carbamate was then removed and the amides reduced with diborane, which after treatment with acid, yielded adequately pure cyclic polyamine as the presumed hexa-hydrochloride. Alkylation was found to be best accomplished by generation of the free base 6, which was then reacted with tert-butyl bromoacetate adapting a procedure reported by Schumann, et al (26). The fully alkylated macrocycle was isolated by chromatography, the ester cleaved, and the nitro group then hydrogenated to produce aniline 9. Treatment of the aniline with thiophosgene produced isothiocyanate 10, which was isolated as a stable, yellow powder. Conjugation. Monoclonal antibodies BL-3, T101, and CC49 were conjugated with 10 when using either a 10or 50-fold molar ratio of ligand to mAb at pH 8.6. While an initial 10-fold molar ratio yielded conjugates with L/P final ratios ranging from 0.4 to 0.6, with 50-fold initial

Bifunctional Chelate for

225Ac

Bioconjugate Chem., Vol. 11, No. 4, 2000 515

Scheme 1. Attempted “5 + 1” Macrocyclization Route to Bifunctional HEHAa

a (a) Et N, EDC, DMF, 47%. (b) Ethylenediamine, MeOH. (c) 1,4-Dioxane saturated with HCl(g). (d) 1,4-Dioxane, N-BOC3 iminodiacetic acid di-OSu ester, 90 °C. BOC ) tert-butyloxycarbonyl; EDC ) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; OSu ) succinimidyl.

Scheme 2. Successful “3 + 3” Macrocyclization Route to Bifunctional HEHAa

a Reagents and conditions: (a) NEt , EDC, DMF, 66%. (b) EtOH, H O, NaOH, 74%. (c) N-Hydroxysuccinimide, EDC, DMF, 73%. 3 2 (d) N-(2-Aminoethyl)-4-nitrophenylalaninamide, 1,4-dioxane, 90 °C, 19%. (e) 1,4-Dioxane/HCl(g). (f) 1 M borane in THF, 50 °C. (g) EtOH/HCl(g). (h) NaOH(aq), extraction with CHCl3. (i) BrCH2COOtBu, DMF, Na2CO3, H2O. (j) HCl(aq) reflux. (k) 10% Pd/C, H2O. (l) SCCl2, CHCl3, H2O. BOC ) tert-butyloxycarbonyl; EDC ) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; OSu ) succinimidyl.

excess of ligand to protein, final product L/P ratios of 2, 1.5, and 1.4 were obtained for the above mAbs, respectively. Radiolabeling. The results of radiolabeling of HEHAmAb conjugates are given in Table 1. During preliminary experiments, an incubation time of 30 min at 37 °C was found to produce the highest labeling yields while minimizing radiation damage to the antibody. No unspecific binding of 225Ac to a free thiol group in BSA known to be responsible for unspecific binding of radiometals was detected. The labeling yields for 225Ac were found to be dependent upon the pH of the reaction mixture with optimal results being obtained at pH 7.0. Labeling yields for 213Bi, the 225Ac daughter also present in the reaction mixture in equilibrium with 225Ac, showed

much less dependence on pH below 7.0. Preliminary experiments demonstrated that HEHA-mAb conjugates with a L/P < 1.0 resulted in decreased yields, generally lower than 30%. The data in Table 1 also shows that, by maintaining a L/P g 1.0, HEHA-mAb conjugates can be labeled with 225Ac in 60-85% yield with specific activity of 200-400 µCi/mg protein, which should be easily sufficient to conduct therapy studies in animals. Serum Stability. The results of serum stability studies of [225Ac]HEHA-BL-3 conjugate in fetal bovine serum at 37 °C were performed in parallel with comparison to serum binding of free 225Ac (Table 2 and Supporting Information). Free 225Ac bound instantly, and essentially quantitatively, to serum proteins with a distinctive pattern of two peaks eluted at ∼5.5 and 8.25 min. A

516 Bioconjugate Chem., Vol. 11, No. 4, 2000

Chappell et al.

Table 1. Dependence of HEHA-MAb Labeling Yields with 225Ac and 213Bi on Conjugate Concentration, pH of Reaction Mixture, and Initial Activity of 225Aca HEHA-MAb conjugate HEHA-BL-3 L/P ) 2.0

HEHA-T101 L/P ) 1.5

amt of conjugate (µg) 300 300 300 400 400 400 400

pH

225Ac activity (µCi)

213Bi labeling (% yield)

225Ac labeling (% yield)

7.0 5.0 4.0 7.0 7.0 7.0 7.0

50 50 50 50 100 200 100

80 ( 1 75 ( 3 67 ( 3 78 ( 2 82 ( 4 80 ( 4 86 ( 5

60 ( 3 43 ( 5 22 ( 2 75 ( 2 72 ( 5 85 ( 4 90 ( 3

a Volume of reaction mixture was 0.4-0.6 mL; all yields are mean values of three experiments with SD; 213Bi was present in reaction mixture in equilibrium with its parent 225Ac.

Table 2. Results of Serum Stability Studies of [225Ac]HEHA-BL-3 Conjugate in Fetal Bovine Serum at 37 °Ca

time point (h)

225Ac activity, associated with [225Ac]HEHA-BL-3 conjugate (%)

213Bi activity, associated with [225Ac]HEHA-BL-3 conjugate (%)

0 0.4 1 3 5 24 48

100 ( 1 99 ( 3 77 ( 7 73 ( 5 69 ( 5