A Novel AmineâDioxime Chelator for Technetium-99m - American

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Bioconjugate Chem. 2000, 11, 652−663

A Novel Amine-Dioxime Chelator for Technetium-99m: Synthesis and Evaluation of 2-Nitroimidazole-Containing Analogues as Markers for Hypoxic Cells Zi-Fen Su,†,‡ James R. Ballinger,‡,§,| A. M. Rauth,*,†,‡ Douglas N. Abrams,⊥ and Mervyn W. Billinghurst⊥ Departments of Medical Biophysics and Pharmaceutical Sciences, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada, Division of Experimental Therapeutics, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada, and Department of Radiopharmacy, Health Sciences Centre, 820 Sherbrooke Street, Winnipeg, Manitoba R3A 1R9, Canada. Received December 7, 1999; Revised Manuscript Received May 10, 2000

A novel amine-dioxime chelator for 99mTc has been developed. It offers the advantages of ease of synthesis and flexibility in alteration of lipophilicity. Labeling by stannous reduction of pertechnetate takes place rapidly and efficiently at room temperature and is stable for 24 h. The 99mTc:ligand ratio is believed to be 1:2. Seven different alkyl moieties were used to achieve a range of lipophilicities. Three series of compounds were prepared: 2-nitroimidazoles as potential hypoxia-targeting agents, 4-nitroimidazoles as a less easily reduced isomer, and untargeted anilines. In an in vitro model of cellular hypoxia, the 2-nitroimidazole compounds all showed selective accumulation whereas 4-nitroimidazoles showed variable selectivity and aniline showed no selectivity. These experiments demonstrate the potential utility of the 2-nitroimidazole derivatives of the amine-dioxime class of chelator as hypoxia-targeting agents.

INTRODUCTION 99m

Technetium-99m ( Tc) is the most widely used radionuclide in nuclear medicine because of its ready availability on-site from a 99Mo/99mTc generator, short half-life of 6 h, reasonable cost, suitable γ energy of 140 keV, and flexibility of application (1). Being a metal, 99m Tc does not form covalent bonds with targeting molecules, but rather must be attached via a chelating group. In the last 15 years, several new classes of metal chelators have been developed and exploited in the design of radiopharmaceuticals; these vary in their ease of synthesis, ease of incorporation of 99mTc, strength of chelation, and flexibility of application. These include diamino-dithiol (2-4), bis amine-oxime (5-7), and boronic acid adducts of technetium oxime (BATO) complexes (8), all of which form neutral, lipophilic complexes with 99mTc, “hiding” the metal within the structure and allowing the physicochemical properties of the complex to be determined by pendant groups rather than the metal. Although these chelators are used in a variety of radiopharmaceuticals, they can suffer from certain limitations: Because of the structural requirements for chelation, they may be limited in flexibility in terms of what parts of the chemical structure can be altered to achieve targeting. The lipophilicity of derivatives may not * To whom correspondence should be addressed. Phone: (416) 946-2977. Fax: (416) 946-2984. E-mail: [email protected]. † Departments of Medical Biophysics. ‡ Ontario Cancer Institute. § Pharmaceutical Sciences. | Present address: Department of Nuclear Medicine, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom. ⊥ Department of Radiopharmacy.

vary over a wide range and may be difficult to adjust; for example, the bis amine-oxime and BATO chelators tend to be extremely lipophilic, and this has led to unsatisfactory results with targeting groups such as somatostatin on PnAO (9) and 2-nitroimidazole on BATO (10). Many are asymmetrical and form two or more isomers with TcdO; these isomers can be difficult to separate and may have different properties (11). Finally, some of these chelates, especially the bis amine-oximes, have limited stability in vitro and in vivo (12, 13). Two classes of oxime-containing chelators have been used as radiopharmaceuticals. The bis amine-oxime or PnAO class is used in the brain perfusion agent exametazime or HMPAO (5) and the hypoxia agents BMS181321 and BRU59-21 (6, 7). These are neutral, lipophilic complexes, which form at room temperature under stannous reduction, although the first two compounds have only limited stability postlabeling. In contrast, the BATO chelates formed by template synthesis from vicinal dioximes (e.g., dimethylglyoxime, cyclohexanedione dioxime) and a boronic acid (e.g., methyl boronic acid, boric acid) (8) to form neutral, lipophilic complexes are extremely stable. These are also prepared by stannous reduction but require heating for the desired complex to form. Two 99mTc BATO complexes formed from vicinal dioximes have been reported by Su et al. (14). These formed lipophilic, cationic complexes with 99mTc upon reduction with sodium borohydride but did not show sufficient myocardial localization to be useful for imaging (14). In the present series of compounds, stannous reduction of pertechnetate to TcO3+ was used to prepare neutral complexes with amine-dioximes. One area in which lipophilicity is believed to play an important role is hypoxia imaging (15). Cellular hypoxia is believed to be an important factor in resistance of tumors to radiation and other forms of therapy. It is also

10.1021/bc9901705 CCC: $19.00 © 2000 American Chemical Society Published on Web 08/22/2000

Amine−Dioxime Chelator for Technetium-99m

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Figure 1. Synthetic scheme of three classes of chelating agents, 3a-g, 4a-g, and 5a,c-g, 6a,c-g, 7a, and 7e.

important to detect hypoxic but viable tissue following myocardial or cerebral infarction. 2-Nitroimidazoles are a class of compounds, which are reduced enzymatically and selectively retained in hypoxic tissue. For both therapeutic and diagnostic applications of 2-nitroimidazoles, optimization of lipophilicity has been important (15, 16). It has recently been suggested that the optimum octanol/water partition coefficient (PC) for a hypoxia imaging agent is ∼1, although the compounds which have been evaluated clinically have PC values which range from 0.4 for [18F]fluoromisonidazole to 40 for BMS181321 (15). In the present report, the flexibility of the aminedioxime has been used to design 99mTc complexes with a wide range of PC values. The lead series contained 2-nitroimidazole as a hypoxia-targeting group. Two series of control compounds, which contained 4-nitroimidazole and aniline groups, were compared with the 2-nitroimidazoles. 4-Nitroimidazoles, which have lower electron affinity than 2-nitroimidazoles, would not be as efficiently reduced and bound. Aniline analogues were also prepared and have no hypoxia-targeting group. These three series of compounds have been synthesized, 99mTc complexes prepared and analyzed, and the behavior of the complexes in an in vitro model of cellular hypoxia was evaluated. EXPERIMENTAL PROCEDURES

Materials and Instruments. Acetone, 2-butanone, 2-pentanone, 2-methyl-3-pentanone, tert-butyl methyl ketone, acetophenone, benzylacetone, 2-nitroimidazole, 4-nitroimidazole, 2-bromoethylphthalimide, hydrazine hydrate, phthalic acid dinonyl ester, and dibutyl phthalate were purchased from Aldrich Chemical Co. Hydroxylamine hydrochloride, aniline, N,N-dimethylformamide (DMF), and silica gel (120-200 mesh) were bought from VWR Canlab. Imidazole, diethylamine, triethylamine, diethylenetriaminepentaacetic acid (DTPA), and bovine serum albumin (BSA) were purchased from Sigma-

Aldrich Canada Ltd. Stannous chloride dihydrate was obtained from BDH Chemicals. Na99mTcO4 was obtained from a 99Mo/99mTc generator (DuPont Pharma). Analysis and purification of 99mTc-labeled compounds was carried out on a Beckman model 125 System Gold (Fullerton, CA) reversed-phase high-pressure liquid chromatography (HPLC) system with Zorbax SB or Beckman ODS 4.6 × 250 mm 5 µm C18 columns. UV and radiometric detectors were connected in series. H2O/MeOH and 0.02 M, pH 4.6, NH4OAc/MeOH were used as mobile phases (flow rate, 1.0 mL/min). Tests of the binding of the 99mTc tracers to BSA were performed using a size exclusion column, TSK-Gel 7.8 × 300 mm from TosoHaas (Japan), with 0.05 M, pH 6.7, phosphate buffer (either 0.7 or 1.0 mL/min) as mobile phase. 1H NMR spectra were recorded on a 300 MHz Bruker or a 500 MHz Varian UNITYPlus-500 spectrometer with tetramethylsilane as reference. High-resolution mass spectra were recorded on a Micromass 70-250S (double focusing) spectrometer. General Procedure for Preparing 1-Chloro-1,2dione-1-oxime (1a-g, Figure 1). A flask containing 2.5 equiv of NOCl (17) in 200-300 mL of carbon tetrachloride or diethyl ether was set in a -10 °C ice-salt bath. To the solution was added dropwise 1 equiv of the R-methyl ketone. The ice-salt bath was removed after the completion of the addition, and the mixture was stirred at room temperature. The reaction took place slowly with release of gas during the first few hours and then become more vigorous. The temperature of the reaction mixture was maintained at 20-27 °C by means of the ice bath. The mixture was stirred at room temperature for 2 h further after the completion of the reaction. The crystalline precipitate was collected by filtration, washed with a minimum amount of carbon tetrachloride, and dried in a desiccator under vacuum. Yields: 1-chloro-1-oximeacetone-2 (1a), 83%; 1-chloro-1-oxime-butanone-2 (1b), 72%; 1-chloro-1-oxime-pentanone-2 (1c), 84%; 1-chloro-

654 Bioconjugate Chem., Vol. 11, No. 5, 2000

1-oxime-4-methylpentanone-2 (1d), 79%; 1-chloro-1-oximepinaclone-2 (1e), 84%; 1-chloro-1-oxime-acetophenone-2 (1f), 94%; 1-chloro-1-oxime-phenylacetone-2 (1g), 79%. General Procedure for Preparing 1-Chloro-1,2dione Dioxime (2a-g, Figure 1). 1-chloro-1,2-dione-1oxime (1a-g) was dissolved in 2-3-fold by weight of water and chilled in an ice bath. To the solution was added 4 equiv of hydroxylamine hydrochloride dissolved in a minimum amount of water (18, 19). The ice bath was removed after the completion of addition. The reaction was stirred at room temperature for 3 h, at 50-60 °C for 30 min, and then stored in a fume hood at room temperature. The crystalline product was collected by filtration, washed with water, dried in a desiccator, and purified by recrystallization from ethanol/water. 1-Chloro-1,2-propanedione Dioxime (2a). Yield 53%. 1H NMR (DMSO-d6): δ 12.53 (s, 1H, dNOH), 11.98 (s, 1H, dNOH), 2.03 (s, 3H, -CH3). 1-Chloro-1,2-butanedione Dioxime (2b). Yield 39%. 1H NMR (DMSO-d ): δ 12.51 (s, 1H, dNOH), 11.95 (s, 6 1H, dNOH), 2.57 (q, 2H, CH3CH2CdNOH), 1.0 (t, 3H, CH3CH2-). 1-Chloro-1,2-pentanedione Dioxime (2c). Yield 44%. NMR (DMSO-d6): δ 12.51 (s, 1H, dNOH), 11.94 (s, 1H, dNOH), 2.57 (q, 2H, C2H5CH2-), 1.48 (m, 2H, CH3CH2CH2-), 0.874 (t, 3H, CH3C2H4-). 1-Chloro-4-methyl-pentanedione(1,2) Dioxime (2d). Yield 27%. 1H NMR (DMSO-d6): δ 12.50 (s, 1H, dNOH), 11.94 (s, 1H, dNOH), 2.0 [m, 1H, (CH3)2CHCH2-], 0.857 [d, 6H, (CH3)2-]. 1-Chloro-3,3-dimethyl-butanedione(1,2) Dioxime (2e). Yield 68%. 1H NMR (DMSO-d6): δ 12.23 (s, 1H, dNOH), 11.32 (s, 1H, dNOH), 1.14 [s, 9H, (CH3)3C-]. 1-Chloro-2-phenyl-ethanedione(1,2) Dioxime (2f). Yield 81%. 1H NMR (DMSO-d6): δ 12.55 (s, 1H, dNOH), 12.09 (s, 1H, dNOH), 7.29-7.39 (m, 5H, C6H5-). 1-Chloro-4-phenyl-butanedione(1,2) Dioxime (2g). Yield 38%. 1H NMR (DMSO-d6): δ 12.57 (s, 1H, dNOH), 12.08 (s, 1H, dNOH), 7.19-7.27 (m, 5H, C6H5-), 2.76-2.84 (m, 4H, phenyl-CH2CH2-). 2-(2-Nitro-1H-imidazolyl)ethylamine. The title compounds were prepared as reported by Hay et al. (20) with some modifications. 2-Nitroimidazole (2.0 g, 17.7 mmol) and 2-bromoethylphthalimide (4.72 g, 18.6 mmol) were added to a suspension of K2CO3 (2.44 g, 18.6 mmol) in 60 mL of DMF. The mixture was stirred at 110 °C for 2 h and then cooled to room temperature. After removing the solvents by rotary evaporation under vacuum, 150 mL of water was added to the residue. The precipitate was collected, washed by water, and dried in a desiccator under vacuum to give 3.19 g (63%) of 2-(2-nitro1H-imidazolyl)ethylphthalimide. 1H NMR (300 MHz, DMSO-d6): δ 7.83 (s, 4H, ArH), 7.59 (d, J ) 1.0 Hz, 1H, imidazole), 7.06 (d, J ) 1.1 Hz, 1H, imidazole), 4.61 (broad, 2H, -CH2CH2-imidazole), 4.06 (broad, 2H, -CH2CH2-imidazole). The phthalimide (3.19 g) was dissolved in 60 mL of ethanol and heated to reflux. To the solution was added 1.12 g (22 mmol) of hydrazine hydrate in 10 mL of ethanol. The mixture was stirred under reflux for 2 h. After cooling to room temperature, the reaction mixture was set in an ice bath. The precipitate was removed by filtration, and the filtrate was brought to dryness by rotary evaporation. The compound was purified by flash chromatography on a silica gel column (4 cm × 80 cm) with petroleum ether and diethyl ether as eluents to yield 1.18 g (72%) of the title compound. 1H NMR (DMSO-d6): δ 7.61 (s, 1H, imidazole), 7.15 (s, 1H, imidazole), 4.35

Su et al.

(t, J ) 6.1 Hz, 2H, -CH2CH2-imidazole), 2.90 (t, 2H, J ) 6.1 Hz, -CH2CH2-imidazole). 2-(4-nitro-1H-imidazolyl)ethylamine. This compound was prepared in a similar manner. Yield of 2-(4nitro-1H-imidazolyl)ethylphthalimide, 3.0 g (39%), mp 237-8 °C. Yield of 2-(4-nitro-1H-imidazolyl)ethylamine, 1.4 g (90%). 1H NMR (300 MHz, DMSO-d6): δ 8.38 (s, 1H, imidazole), 7.82 (s, 1H, imidazole), 4.02 (t, J ) 6.0, 2H, -CH2CH2-imidazole), 2.89 (t, J ) 6.0, 2H, -CH2CH2-imidazole). General Procedures for Preparing 1-N-[2-(2nitro-1H-imidazolyl)ethylamine]-1,2-alkanedione Dioxime (3a-g, Figure 1). On a 0.2-1.0-g scale, a mixture of 2-(2-nitro-1H-imidazolyl)ethylamine with 2 equiv of triethylamine in 10 mL of anhydrous methanol was set in an ice bath. To the solution was added 1 equiv of 1-chloro-1,2-alkanedione dioxime (2a-g). After the completion of addition, the mixture was stirred at room temperature for 3 h and then at 50-60 °C for another 3 h. The solvent was removed by rotary evaporation under vacuum. The residue was placed on a silica gel column and eluted first with 200 mL of dichloromethane and then with 500 mL of diethyl ether which removed most of the product. The ether was removed by rotary evaporation under vacuum. The residue was collected, washed with hot isopropyl ether, and dried in a desiccator under vacuum. 3b was further purified by HPLC on a Beckman ODS C18 4.6 × 250 mm column with H2O/MeOH as eluent. 3f was washed with a minimum amount of 50/50 MeOH/H2O to remove the dark brown impurities. 1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-1,2-propanedione Dioxime (3a). Yield 92.6%, mp 168-9 °C (dec). 1H NMR (300 MHz, DMSO-d6): δ 11.22 (s, 1H, dNOH), 9.98 (s, 1H, dNOH), 7.41 (d, J ) 0.86 Hz, 1H, imidazole ring), 7.14 (d, J ) 0.93 Hz, 1H, imidazole ring), 5.68 (t, 1H, -NHC2H4-), 4.46 (t, J ) 5.62 Hz, 2H, -CH2-imidazole), 3.72 (m, 2H, -NHCH2CH2-), 1.80 (s, 3H, CH3CdNOH). High-resolution MS (electron impact, EI) found for C8H12N6O4 (M+): 256.0919. Calcd: 256.0920. 1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-1,2-butanedione Dioxime (3b). Yield 47%, mp 169-170 °C (dec). 1H NMR (DMSO-d6): δ 11.17 (s, 1H, dNOH), 9.98 (s, 1H, dNOH), 7.42 (d, J ) 0.98 Hz, 1H, imidazole ring), 7.14 (d, J ) 0.97 Hz, 1H, imidazole ring), 5.68 (t, J ) 6.84 Hz, 1H, -NHC2H4-), 4.46 (t, J ) 5.62 Hz, 2H, -CH2imidazole), 3.68 (m, 2H, -NHCH2CH2-), 2.36 (q, J ) 7.49 Hz, CH3CH2CdNOH, 0.92 (t, J ) 7.46 Hz, CH3CH2-). High-resolution MS (EI) found for C9H14N6O4 (M+): 270.1064. Calcd: 270.1077. 1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-1,2-pentanedione Dioxime (3c). Yield 49%, mp 128-9 °C (dec). 1H NMR (300 MHz, DMSO-d6): δ 11.15 (s, 1H, dNOH), 9.96 (s, 1H, dNOH), 7.42 (s, 1H, imidazole ring), 7.14 (s, 1H, imidazole ring), 5.67 (t, J ) 6.70 Hz, 1H, -NHC2H4-), 4.46 (t, J ) 5.55 Hz, 2H, -CH2-imidazole), 3.68 (m, 2H, -NHCH2CH2-), 2.34 (t, 2H, CH3CH2CdNOH), 1.36 (m 2H, CH3CH2CH2-), 0.824 (t, 3H, CH3C2H4-). High-resolution MS (EI) found for C10H16N6O4 (M+): 284.1228. Calcd: 284.1233. 1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-4-methylpentanedione(1,2) Dioxime (3d). Yield 43%, mp 156-7 °C (dec). 1H NMR (300 MHz, DMSO-d6): δ 11.13 (s, 1H, dNOH), 9.94 (s, 1H, dNOH), 7.43 (d, J ) 0.92 Hz, 1H, imidazole ring), 7.13 (d, J ) 1.0 Hz, 1H, imidazole ring), 5.66 (t, J ) 6.69 Hz, 1H, -NHC2H4-), 4.17 (t, J ) 5.35 Hz, 2H, -CH2-imidazole), 3.66 (m, 2H, -NHCH2CH2-), 2.30 [d, 2H, (CH3)2CHCH2-], 1.82 [m, 1H, (CH3)2CH2CH2-], 0.80 [d, 6H, (CH3)2CH-]. High-resolution MS (EI) found for C11H18N6O4 (M+): 298.1394. Calcd: 298.1390.


1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-3,3-dimethylbutanedione(1,2) Dioxime (3e). Yield 41%, mp 164-5 °C (dec). 1H NMR (300 MHz, DMSO-d6): δ 10.90 (s, 1H, dNOH), 9.40 (s, 1H, dNOH), 7.53 (d, J ) 0.93 Hz, 1H, imidazole ring), 7.13 (d, J ) 0.93 Hz, 1H, imidazole ring), 5.98 (t, J ) 6.43 Hz, 1H, -NHC2H4-), 4.49 [t, J ) 5.71 Hz, 2H, -CH2(imidazole)], 3.23 (m, 2H, -NHCH2CH2-), 0.96 [s, 9H, (CH3)3C-]. High-resolution MS (EI) found for C11H18N6O4 (M+): 298.1386. Calcd: 298.1390. 1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-2-phenylethanedione(1,2) Dioxime (3f). Yield 83%, mp 132-133 °C (dec). 1H NMR (300 MHz, DMSO-d6): δ 11.05 (s, 1H, dNOH), 9.86 (s, 1H, dNOH), 7.42-7.31 (m, 5H, phenyl ring), 7.38 (d, J ) 0.97 Hz, imidazole ring), 7.14 (d, J ) 1.22 Hz, 1H, imidazole ring), 6.02 (t, J ) 6.60 Hz, 1H, -NHC2H4-), 4.39 (t, J ) 5.74 Hz, 2H, -CH2-imidazole), 3.50 (m, 2H, -NHCH2CH2-). High-resolution MS (EI) found for C13H14N6O4 (M+): 318.1081. Calcd: 318.1077. 1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-4-phenylbutanedione(1,2) Dioxime (3g). Yield 80%, mp 173-4 °C (dec). 1H NMR (DMSO-d6): δ 11.30 (s, 1H, dNOH), 10.05 (s, 1H, dNOH), 7.39 (d, J ) 0.78 Hz, 1H, imidazole ring), 7.26-7.13 (m, 6H, imidazole and phenyl rings), 5.68 (t, J ) 6.82 Hz, 1H, -NHC2H4-), 4.45 (t, J ) 5.71 Hz, 2H, -CH2CH2-imidazole), 3.68 (m, 2H, -CH2CH2imidazole). 2.65 (broad, 4H, phenyl-CH2CH2-). Highresolution MS (EI) found for C15H18N6O4 (M+): 346.1392. Calcd: 346.1390. General Procedures for Preparing 1-N-[2-(4Nitro-1H-imidazolyl)ethylamine]-1,2-alkanedione Dioxime (4a-g, Figure 1). The preparation of these compounds was similar to that of compounds 3a-g. 1-N-[2-(4-Nitro-1H-imidazolyl)ethylamine]-1,2-propanedione Dioxime (4a). Yield 32%, mp 192-3 °C (dec). 1 H NMR (DMSO-d6): δ 11.29 (s, 1H, dNOH), 10.09 (s, 1H, dNOH), 8.26 (d, J ) 1.41 Hz, 1H, imidazole ring,), 7.72 (d, J ) 1.35 Hz, 1H, imidazole ring), 5.68 (t, J ) 6.77 Hz, 1H, -NHC2H4-), 4.14 (t, J ) 5.81 Hz, 2H, -CH2-imidazole), 3.64 (m, 2H, -NHCH2CH2-), 1.82 (s, 3H, CH3CdNOH). High-resolution MS (EI) found for C8H12N6O4 (M+): 256.0914. Calcd: 256.0920. 1-N-[2-(4-Nitro-1H-imidazolyl)ethylamine]-1,2-butanedione Dioxime (4b). Yield 21%, mp 180-1 °C (dec). 1H NMR (DMSO-d ): δ 11.21 (s, 1H, dNOH), 10.07 (s, 6 1H, dNOH), 8.26 (d, J ) 1.33 Hz, 1H, imidazole ring), 7.72 (d, J ) 1.33 Hz, 1H, imidazole ring), 5.68 (t, J ) 6.71 Hz, 1H, -NHC2H4-), 4.15 (t, J ) 5.78 Hz, 2H, -CH2-imidazole), 3.61 (m, 2H, -NHCH2CH2-), 2.38 (q, 2H, CH3CH2CdNOH), 0.91 (t, 3H, CH3CH2-). Highresolution MS (EI) found for C9H14N6O4 (M+): 270.1084. Calcd: 270.1078. 1-N-[2-(4-Nitro-1H-imidazolyl)ethylamine]-1,2-pentanedione Dioxime (4c). Yield 34%, mp 131-2 °C (dec). 1H NMR (DMSO-d6): δ 11.21 (s, 1H, dNOH), 10.07 (s, 1H, dNOH), 8.27 (d, J ) 1.44 Hz, 1H, imidazole ring), 7.72 (d, J ) 1.35, 1H, imidazole ring), 5.60 (t, J ) 6.65 Hz, 1H, -NHC2H4-), 4.15 (t, J ) 5.76 Hz, 2H, -CH2imidazole), 3.61 (m, 2H, -NHCH2CH2-), 2.33 (t, 2H, CH3CH2CH2-), 1.35 (q, 2H, CH3CH2CH2-), 0.81 (t, 3H, CH3C2H4-). High-resolution MS (EI) found for C10H16N6O4 (M+): 284.1230. Calcd: 284.1233. 1-N-[2-(4-Nitro-1H-imidazolyl)ethylamine]-4-methylpentanedione(1,2) Dioxime (4d). Yield 81%, mp 173-4 °C (dec). 1H NMR (DMSO-d6): δ 11.19 (s, 1H, dNOH), 10.06 (s, 1H, dNOH), 8.28 (d, J ) 1.35 Hz, 1H, imidazole ring), 7.73 (d, J ) 1.36 Hz, 1H, imidazole ring), 5.67 (t, J ) 6.61 Hz, 1H, -NHC2H4-), 4.17 (t, J ) 5.75 Hz, 2H, -CH2-imidazole), 3.60 (m, 2H, -NHCH2CH2-), 2.30 [d, 2H, (CH3)2CHCH2-], 1.72 [m 1H, (CH3)2CH2CH2-],


0.78 [d, 6H, (CH3)2CH-]. High-resolution MS (EI) found for C11H18N6O4 (M+): 298.1381. Calcd: 298.1390. 1-N-[2-(5-Nitro-1H-imidazolyl)ethylamine]-3,3-dimethylbutanedione(1,2) Dioxime (4e). Yield 19%, mp 163-4 °C. 1H NMR (DMSO-d ): δ 10.91 (s, 1H, dNOH), 9.46 (s, 1H, 6 dNOH), 8.33 (d, J ) 1.32 Hz, 1H, imidazole ring), 7.78 (d, J ) 1.38 Hz, 1H, imidazole ring), 6.03 (t, J ) 6.26 Hz, 1H, -NHC2H4-), 4.21 (t, J ) 5.63 Hz, 2H, -CH2imidazole), 3.15 (m, 2H, -NHCH2CH2-), 0.98 [s, 9H, (CH3)3C-]. High-resolution MS (EI) found for C11H18N6O4 (M+): 298.1396. Calcd: 298.1390. 1-N-[2-(4-Nitro-1H-imidazolyl)ethylamine]-2-phenylethanedione(1,2) Dioxime (4f). Yield 86%, mp 203-4 °C (dec). 1H NMR (DMSO-d6): δ 11.61 (s, 1H, dNOH), 9.96 (s, 1H, dNOH), 8.17 (d, J ) 1.35, 1H, imidazole ring), 7.67 (d, J ) 1.32 Hz, imidazole ring), 7.39-7.30 (m, 5H, phenyl ring), 6.03 (t, J ) 6.57 Hz, 1H, -NHC2H4-), 4.10 (t, J ) 5.64 Hz, 2H, -CH2-imidazole), 3.45 (m, 2H, -NHCH2CH2-). High-resolution MS (EI) found for C13H14N6O4 (M+): 318.1092. Calcd: 318.1077. 1-N-[2-(2-Nitro-1H-imidazolyl)ethylamine]-4-phenylbutanedione(1,2) Dioxime (4g). Yield 20%, mp 185-6 °C (dec). 1H NMR (DMSO-d6): δ 11.35 (s, 1H, dNOH), 10.16 (s, 1H, dNOH), 8.25 (d, J ) 1.28 Hz, 1H, imidazole ring), 7.75 (d, J ) 1.28 Hz, 1H, imidazole ring), 7.28-7.12 (m, 5H, phenyl rings), 5.70 (t, J ) 6.82 Hz, 1H, -NHC2H4-), 4.12 (t, J ) 5.72 Hz, 2H, -CH2-imidazole), 3.62 (m, 2H, -NHCH2CH2-). 2.66 (broad, 4H, -CH2CH2-). Highresolution MS (EI) found for C15H18N6O4 (M+): 346.1393. Calcd: 346.1390. General Procedures for Preparing 1-N-Aniline1,2-alkanedione Dioxime (5a,c-g, Figure 1). Three equivalents of aniline was diluted in 7-15 mL of isopropyl ether and placed in an ice bath. To the solution was added 1 equiv of 2a-g (0.5-8 g scale) dissolved in a minimum amount of methanol. The mixture was stirred at 0 °C for 1 h, at room temperature for 1 h, and then at 50-60 °C for 1 h. After cooling to room temperature, the crystalline precipitate (C6H5NH2‚HCl) was removed by filtration. The filtrate was brought to dryness. The residue was dissolved in a minimum amount of methanol and placed on a silica gel column. The column was eluted first with 200 mL of dichloromethane and then with 200 mL of diethyl ether. The ether was removed and the residue was collected and dried in desiccator under vacuum. 1-N-Aniline-1,2-propanedione Dioxime (5a). Yield 29%. 1H NMR (DMSO-d ): δ 11.31 (s, 1H, dNOH); 10.65 (s, 6 1H, dNOH); 7.86 (s, 1H, -NHC6H5); 7.17-6.77 (m, 5H, -C6H5); 1.90 (s, 3H, -CH3). High-resolution MS (EI) found for C9H11N3O2 (M+): 193.0854. Calcd: 193.0851. 1-N-Aniline-1,2-pentanedione Dioxime (5c). Yield 49%, mp 60-62 °C (dec). 1H NMR (DMSO-d6): δ 11.25 (s, 1H, dNOH); 10.61 (s, 1H, dNOH); 7.80 (s, 1H, -NHC6H5); 7.16-6.78 (m, 5H, -C6H5); 2.50 (t, 2H, -CH2CH2CH3); 1.48 (m, 2H, -CH2CH2CH3); 0.88 (t, 3H, -CH2CH2CH3). High-resolution MS (EI) found for C11H15N3O2 (M+): 221.1163. Calcd: 221.1164. 1-N-Aniline-4-methyl-pentanedione(1,2) Dioxime (5d). Yield 33%. 1H NMR (DMSO-d6): δ 11.27 (s, 1H, dNOH); 10.61 (s, 1H, dNOH); 7.80 (s, 1H, -NHC6H5); 7.18-6.79 (m, 5H, -C6H5); 2.29 [d, 2H, -CH2CH(CH3)2]; 1.95 [m, 1H, -CH2CH(CH3)2]; 0.87 [d, 6H, -CH2CH(CH3)2]. Highresolution MS (EI) found for C12H17N3O2 (M+): 235.1317. Calcd: 235.1321. 1-N-Aniline-3,3-dimethyl-butanedione(1,2) Dioxime (5e). Yield 37%, mp 161-2 °C (dec). 1H NMR (DMSO-d6): δ 11.15 (s, 1H, dNOH); 10.04 (s, 1H, dNOH); 8.15 (s, 1H, -NHC6H5); 7.15 (t, 2H, phenyl); 7.05 (d, 2H, phenyl); 6.88


(t, 1H, phenyl); 0.97 [s, 9H, (CH3)3C-]. High-resolution MS (EI) found for C12H17N3O2 (M+): 235.1314. Calcd: 235.1321. 1-N-Aniline-2-phenyl-ethanedione(1,2) Dioxime (5f). Yield 41%, mp 57-8 °C (dec). 1H NMR (DMSO-d6): δ 11.63 (s, 1H, dNOH); 10.55 (s, 1H, dNOH); 8.16 (s, 1H, -NHC6H5); 7.45-6.88 (m, 10H, -C6H5, -NHC6H5). High-resolution MS (EI) found for C14H13N3O2 (M+): 255.1009. Calcd: 255.1008. 1-N-Aniline-4-phenyl-butanedione(1,2) Dioxime (5g). Yield 20%, mp 126-7 °C (dec). 1H NMR (DMSO-d6): δ 11.41 (s, 1H, dNOH); 10.71 (s, 1H, dNOH); 7.85 (s, 1H, -NHC6H5); 7.28-6.75 (m, 10H, -C6H5, -NHC6H5). High-resolution MS (EI) found for C16H17N3O2 (M+): 283.1323. Calcd: 283.1321. General Procedures for Preparing 1-N-Imidazole-1,2-alkanedione Dioxime (6a,c-g, Figure 1). To a 2:1 methanol/diethyl ether solution of compounds 1a-g, which was set in a water bath, were added dropwise 1 equiv of imidazole and triethylamine dissolved in 2:1 methanol/diethyl ether. The temperature was maintained at room temperature during the addition. After stirring at room temperature for 2 h, the mixture was heated to 60 °C for 10 min. The crystalline precipitate was filtered, washed with water, and used for the next reaction. The raw 1-N-imidazole-1,2-alkanedione monoxime was dissolved in a minimum amount of methanol and placed in a water bath. To the solution was added 1.2 equiv of NH2OH, which was prepared by neutralizing NH2OH‚ HCl with NaOH. The reaction mixture became cloudy after the addition of NH2OH. A minimum amount of methanol was added to make the cloudy mixture clear. The mixture was stirred at 60 °C for 15 min, then placed in a fume hood overnight. The precipitate was removed by filtration. The filtrate was brought to dryness and the residue was dissolved in hot methanol/water. Precipitation occurred on cooling. The precipitate was collected, washed with water, and dried in a desiccator under vacuum. 1-N-Imidazole-1,2-propanedione Dioxime (6a). Yield 11%, mp 214-5 °C (dec). 1H NMR (DMSO-d6): δ 13.05 (s, 1H, dNOH); 12.13 (s, 1H, dNOH); 9.24 (s, 1H, imidazole); 7.80 (s, 1H, imidazole); 7.46 (1H, imidazole); 2.12 (s, 3H, -CH3). 1-N-Imidazole-1,2-pentanedione Dioxime (6c). Yield 6.3%, mp 174-5 °C (dec). 1H NMR (DMSO-d6): δ 12.20 (s, 1H, dNOH); 11.82 (s, 1H, dNOH); 7.70 (s, 1H, imidazole); 7.20 (s, 1H, imidazole); 6.96 (s, 1H, imidazole); 2.62 (t, 2H, CH3CH2CH2-). 1-N-Imidazole-4-methyl-pentanedione(1,2) Dioxime (6d). Yield 10%, mp 149-150 °C (dec). 1H NMR (DMSO-d6): δ 12.22 (s, 1H, dNOH); 11.83 (s, 1H, dNOH); 7.70 (s, 1H, imidazole); 7.20 (s, 1H, imidazole); 6.96 (s, 1H, imidazole); 2.56 [s, 1H, (CH3)2CHCH2-]; 2.10 [m, 1H, (CH3)2CHCH2-]; 0.95 [d, 6H, (CH3)2CHCH2-]. 1-N-Imidazole-3,3-dimethyl-butanedione(1,2) Dioxime (6e). Yield 13%, mp 181-2 °C (dec). 1H NMR (DMSOd6): δ 11.91 (s, 1H, dNOH); 11.41 (s, 1H, dNOH); 8.14 (s, 1H, imidazole); 7.37 (s, 1H, imidazole); 6.99 (s, 1H, imidazole); 1.12 [s, 9H, (CH3)3C-]. 1-N-Imidazole-2-phenyl-ethanedione(1,2) Dioxime (6f). Yield 11%, mp 153-4 °C (dec). 1H NMR (DMSO-d6): δ 12.21 (s, 1H, dNOH); 11.98 (s, 1H, dNOH); 7.91 (s, 1H, imidazole); 7.38-7.43 (m, 6H, C6H5-, imidazole); 6.99 (s, 1H, imidazole). 1-N-Imidazole-4-phenyl-butanedione(1,2) Dioxime (6g). Yield 13.6%, mp 166-7 °C (dec). 1H NMR (DMSO-d6): δ 12.23 (s, 1H, dNOH); 11.94 (s, 1H, dNOH); 7.61 (s, 1H, imidazole); 7.13-7.30 (m, 6H, C6H5-, imidazole);

Su et al.

6.93 (d, 1H, J ) 0.93, imidazole), 2.80-2.96 (m, 4H, phenyl-C2H4-). 1-N-Diethyl-1,2-propanedione Dioxime (7a). To a solution of diethylamine (1.27 g, 17.4 mmol) in 7 mL of aectonitrile, which was set in an ice bath, was added dropwise 1.0 g (5.8 mmol) of 2a dissolved in 10 mL of methanol. The temperature of the reaction mixture was kept below 15 °C during the addition. The reaction mixture was stirred at room temperature for 4 h after the completion of addition. The crystalline precipitate (diethylamine HCl) was removed by filtration. The filtrate was brought to dryness, the residue was dissolved in a minimum amount of methanol and loaded on a silica gel column. The column was eluted first with 100 mL of dichloromethane and then with 250 mL of diethyl ether. The ether eluate was brought to dryness and the waxlike residue was dried in a desiccator under vacuum to give 1.0 g (99%) product. 1H NMR (DMSO-d6): δ 11.04 (s, 1H, dNOH); 8.82 (s, 1H, dNOH); 3.06 [q, 4H, -N(CH2CH3)2]; 1.87 [s, 3H, CH3C(dNOH)-]; 1.01 [t, 6H, -N(CH2CH3)2]. 1-N-Diethyl-1,2-propanedione Dioxime (7e). This compound was prepared in a way similar to that of 7a. Yield 1.0 g (66%). 1H NMR (DMSO-d6): δ 10.54 (s, 1H, dNOH); 8.68 (s, 1H, dNOH); 2.96 [m, 4H, -N(CH2CH3)2]; 1.13 [s, 9H, (CH3)3C-]; 1.04 [t, 6H, -N(CH2CH3)2]. 99m Tc Labeling of Compounds 3a-g, 4a-g, and 5a,c-g. The 99mTc labeling of these three classes of chelating agents was performed by two methods. Method I. To a 3 mL tube were added 40-100 µg of chelating agent (in 10-25 µL of methanol), 100 µL of 0.1 M, pH 4.6, NH4OAc buffer, 100 µL of saline, 92 µL of 0.1 N NaOH, 2-1000 MBq of Na99mTcO4 (in 20-200 µL saline), and 10 µg of SnCl2 (in 10 µL of 1 N HCl). The chelation took a few minutes to complete. The radiochemical yield and purity of the 99mTc-labeled compounds was tested by HPLC on a C18 reversed-phase column, with either 0.02 M, pH 4.6, NH4OAc/MeOH, or H2O/ MeOH as mobile phase, gradient 50% H2O to 10% H2O over 20 min, flow rate 1.0 mL/min. Method II. To a 3-mL tube were added 40-100 µg of chelating agent (in 10-25 µL of methanol), 200 µL of 10 mg/mL of pentasodium DTPA, 2-1000 MBq of Na99mTcO4 (in 20-200 µL of saline), and 50 µL of Sn-DTPA, which was prepared by mixing 10 µL of 20 mg/mL SnCl2 in 1 N HCl with 500 µL of 10 mg/mL pentasodium DTPA. The chelation took a few minutes to complete. The radiochemical yield and purity of the 99mTc-labeled compounds were analyzed by HPLC under the conditions stated above. 99mTc Labeling of Compounds 6 a,c-g, 7a, and 7e. Attempts were made to label compounds 6a,c-g, 7a, and 7e with 99mTc under the conditions described in Methods I and II. The labeling efficiency was tested by HPLC as above. 99m Tc Labeling of Mixed Ligands. Two chelating agents (either 3a-g plus 4a-g or 3a-g plus 5a,c-g) were mixed together and labeled with 99mTc by either Method I or Method II. The labeling efficiency was determined by HPLC under the conditions stated above. Stability Test of the 99mTc Tracers in Phosphate Buffer and in BSA. The 99mTc tracers were mixed with an equal volume of either PBS or 0.5 M, pH 7.4, phosphate buffer at room temperature for a period up to 24 h and were analyzed by reversed-phase HPLC. The stability of one volume of 99mTc complexes in 5 vol of 50 mg/mL BSA in PBS were incubated at 37 °C for a period up to 24 h and tested by size-exclusion HPLC.


Partition Coefficient (PC) Measurement. 99mTclabeled compounds were isolated by HPLC with H2O/ MeOH as eluent. After removing the solvent by rotary evaporation under vacuum, the residue was dissolved in 1.0 mL of saline plus 0.5 mL of ethanol. Aliquots of 10 µL were placed in a tube containing 1.0 mL of n-octanol and 1.0 mL of PBS, and vortexed for 1 min. Aliquots of 0.6 mL of each phase were transferred to 1.5-mL microcentrifuge tubes and centrifuged at 12000g for 2 min. The radioactivity in triplicate 100 µL samples from each phase was counted in a Picker Pace-1 γ well counter. The PC values were calculated by dividing the mean counts in the n-octanol phase by the counts in the PBS phase. Studies of Cellular Accumulation of 99mTc Complexes. The in vitro model for the evaluation of hypoxia markers has been described elsewhere (21). This model was used to test the cellular accumulation of the 20 99mTc complexes of 3 a-g, 4a-g, and 5a,c-g under aerobic and hypoxic conditions. Chinese hamster ovary (CHO) cells grown in suspension culture in R minimal essential medium (R-MEM) containing 10% (v/v) of fetal bovine serum were resuspended in fresh R-MEM at a concentration of (1-2) × 106 cells/mL and incubated at 37 °C. The aerobic and hypoxic conditions were generated by stirring the cell suspension under an atmosphere of air or nitrogen, respectively (both containing 5% CO2). The 99mTc tracer (2-10 MBq) was added to the suspension and aliquots were removed at different time intervals over the course of 6 h. Triplicate aliquots of 100 µL were transferred to 1.5 mL microcentrifuge tubes containing 1 mL of an oil mixture (phthalic acid dinonyl ester:dibutyl phthalate, 2:3) and centrifuged at 12000g for 2 min. Aliquots of 30 µL of the supernatant were removed for radioactivity counting. The remaining supernatant and most of the oil were removed by aspiration. The tip of the tube containing the cell pellet was clipped into a counting tube and the radioactivity in the cell pellet and supernatant sample was measured in a γ well counter. Curves of Cin/Cout under aerobic and hypoxic conditions, where Cin and Cout represented the radioactivities in the cell pellet and in the equivalent volume of supernatant, respectively, as a function of time were plotted as described previously (21). RESULTS

Synthesis of the Chelating Agents. The intermediate 1,2-alkanedione monoximes (1a-g) were prepared by bubbling NOCl through a solution of the corresponding R-methyl ketone with dry diethyl ether or carbon tetrachloride (Figure 1). NOCl reacted with acetone, 2butanone, 2-pentanone, 2-methyl-3-pentanone, and tertbutyl-methyl ketone vigorously at room temperature. However, the reaction of NOCl with acetophenone and benzylacetone proceeded rather slowly and took several days to complete. The yields of the seven dione monoxime compounds ranged from 72 to 94%. Care has to be taken in the preparation of 1b because significant amounts of byproduct, which was shown to be 2,3-butanedione monoxime, could be produced during the reaction of NOCl with 2-butanone. This byproduct could be diminished by carefully maintaining the temperature of reaction mixture at 20-25 °C. The compounds 1a-g were hygroscopic and it is advisable to convert 1a-g into the more stable 2a-g as soon as possible. The oxo group of 1a-g could be converted into an oxime by hydroxylamine hydrochloride (Figure 1). Compounds 2a-g separated as crystals from the reaction mixture on standing in a fume hood at room temperature. They were


soluble in common organic solvents, such as alcohol, acetone and acetonitrile, and were more stable than 1a-g on storage. The byproduct, 2,3-butandione dioxime, was less soluble in ethanol, acetonitrile, and cold methanol than the desired product 2b; thus, 2,3-butandione dioxime could be separated from 2b by extracting the latter into acetonitrile. The chlorine of 2a-g was active and could be substituted by either primary or secondary amines (Figure 1). This nucleophilic substitution was performed with 2-(2-nitro1H-imidazolyl)ethylamine (3a-g), 2-(4-nitro-1H-imidazolyl)ethylamine (4a-g), aniline (5a,c-g), imidazole (6a,c-g), and diethylamine (7a, 7e). The rate of substitution depended on the activity of the proton of the amine or imine, or the substituents on the imidazole ring. For example, imidazole was able to replace the chlorine of either 1a-g or 2a-g, while 2-(2-nitro-1H-imidazolyl)ethylamine and 2-(4-nitro-1H-imidazolyl)ethylamine were not. No product could be isolated from the reaction of 2-nitroimidazole or 4-nitroimidazole with 1a-g or 2a-g. The solubility of the three classes of compounds in ethanol increased in the order: 4a-g < 3a-g < 5a,c-g. The NMR data demonstrated that the chemical shifts of the two protons on the oxime groups were dependent on the R2 groups (Figure 1). In comparison with those of 2a-g (12.0 ppm and 12.2-13.0 ppm for the two protons of the dNOH groups), the oxime protons of 3a-g, 4a-g, and 5a,c-g were shifted to higher field (10.0-10.7 ppm and 11.0-11.7 ppm), while those of 6a,c-g (11.4-12.1 ppm and 11.9-13.1 ppm) were little affected. The imidazole group of 6a,c-g might play a role as an electron-withdrawing group that is similar to the chlorine atom of 1a-g. However, the two oxime protons of 7a and 7e were significantly shifted to higher field (8.7-8.8 ppm and 10.5-11.0 ppm). It is interesting to note that two of the three protons of a free imidazole ring are chemically equivalent as two single lines were seen on 1H NMR: 7.0 ppm (s, 2H) and 7.6 ppm (s, 1H). They became chemically nonequivalent when they combined with 2a-g to form compounds 6a,c-g. Three single lines (7.0, 7.2, and 7.7 ppm) on the 1H NMR of 6c-g were observed and assigned to the three protons on the imidazole ring. It should be mentioned that the three imidazole protons of 6a appeared at 7.6, 7.8, and 9.2 ppm, which oriented at lower field than those of 6c-g. The configuration of 6a might be different from its analogues in the series. Unlike 6a,c-g, the protons of the imidazole ring of 3a-g and 4a-g exhibited weak coupling (0.8-0.9 Hz and 1.3-1.4 Hz). The proton attached to the 1-N position (1-N proton) could be detected by 1H NMR. The 1-N protons of 3a-g and 4a-g appeared at 6.0 ppm, while that of 5a,c-g was at 7.8 ppm and fell in a region for protons of aromatic compounds. All 20 chelating agents, 3a-g, 4a-g, and 5a,c-g, have been analyzed by high-resolution mass spectrometry. The detected molecular ions have displayed good agreement (error limits within 5 ppm) with the calculated molecular weights of the compounds. 99mTc Labeling of Compounds 3 a-g, 4a-g, 5a,c-g, 6a,c-g, 7a, and 7e. The 99mTc labeling of these chelating agents was carried out using either SnCl2 or Sn-DTPA as reducing agent for Na99mTcO4. The chelation of 3a-g, 4a-g, and 5a,c-g to 99mTc was complete within a few minutes at room temperature, as shown by HPLC analysis with two solvent systems. 99mTc chelates of 3a-g and 4a-g exhibited a single radioactive peak on the chromatogram, while 5a,c-g showed a minor peak (5-10%) on the tail of the main peak. The retention time (tR) of a 99mTc chelate depended on the nature of the mobile


Su et al.

Table 1. Retention Time, Radiochemical Purity, and Yield of 20 99mTc Complexes Containing 2-Nitroimidazole (3a-g), 4-Nitroimidazole (4a-g), or Aniline (5a-g) with R1 Defined in Figure 1a 3a-g

4a-g

5a,c-g

R1

tR (min)

purity (%)

yield (%)

tR (min)

purity (%)

yield (%)

tR (min)

purity (%)

yield (%)

CH3C2H5C3H7isoC4H9(CH3)3CC6H5C6H5C2H4-

11.03 11.74 11.93 12.45 12.97 11.81 13.40

100 100 100 100 98 100 97

94 84 92 89 90 88 90

11.22 11.28 11.56 12.04 11.73 11.36 12.83

99 98 99 99 99 99 96

97 97 98 95 96 95 91

13.19

92

80

15.54 17.67 19.47 15.52 19.17

87 86 95 93 98

80 77 91 82 88

a The complexes were prepared by Method II, and radiochemical purity was analyzed by HPLC on a C 18 column with H2O/MeOH as mobile phase; gradient, 50 to 10% H2O over 20 min; flow rate, 1.0 mL/min. Yield was calculated as percent recovery of injected activity in the collected HPLC peak.

phases used for HPLC, but the radiochemical yield and purity remained similar. Compounds 3a-g and 4a-g are similar in structure, as are the 99mTc complexes of each pair of isomers. For example, 3a and 4a are one pair of isomers, and their 99mTc complexes are another pair of isomers. Each pair of isomers showed similar retention time on HPLC. The tR of the 99mTc complexes of 3a-g increased in the order of 3a < 3b < 3f < 3c < 3e < 3d < 3g, which is in accordance with the tR of the chelating agents themselves. The 99mTc complexes of 4a-g presented the same order of tR as well. Figure 2 presents the HPLC analysis of 99mTc complexes of 3f and 4f, representative of the remaining 12 complexes containing 2- or 4-nitroimidazole groups. The 20 99mTc complexes of 3a-g, 4a-g, and 5a,c-g were prepared using either SnCl2 or Sn-DTPA as reducing agent, and analyzed by HPLC. The retention times, purities, and radiochemical yields are summarized in Table 1. The purity of a 99mTc complex was measured by HPLC, while its radiochemical yield was calculated by comparing the radioactivity injected with that collected in the major peak. It is noted that 5a,c-g exhibited longer retention times than 3a-g and 4a-g, which suggests that 5a,c-g are more lipophilic than 3a-g and 4a-g, though the latter have higher molecular weights. Similarly, the 99m Tc complexes of 5a,c-g showed longer tR than 3a-g and 4a-g. The attempted labeling of 6a,c-g, 7a, and 7e with 99mTc by using either SnCl2 or Sn-DTPA as reducing agent was unsuccessful. No labeled compound was detected by HPLC and only 99mTc colloids resulted from these labelings. Stability of the 99mTc Complexes of 3a-g, 4a-g, and 5a,c-g in PBS/PB and BSA. All the 99mTc complexes were stable in PBS and 0.5 M PB at room temperature for a period up to 24 h (data not shown). When incubated in BSA PBS solution at 37 °C for a period up to 24 h (data not shown), most of the 99mTc complexes of 2- and 4-nitroimidazole showed less than 20% binding to BSA. The exceptions were 3e, and 4d, which showed significant BSA binding, 43 and 34% respectively. In addition, 3g and 4g exhibited approximately 65% breakdown in BSA. In contrast, the 99mTc complexes of 5a,c-g showed 95100% binding to BSA but no evidence of breakdown. The degree of BSA binding of these 99mTc tracers appeared to be independent of the incubation time. Structural identification of 99mTc Chelates of 3a-g, 4a-g, and 5a,c-g. The chemical structure of a 99mTc chelate could theoretically be determined by elemental analysis, NMR, and MS, but it is not feasible to prepare weighable amounts of a 99mTc chelate for this purpose because even as little as 1 mg of 99mTc can emit 1 × 105 GBq γ radiation (22). Alternatively, the chemical structure could be determined by using 99Tc, but its long half-life (t1/2 ) 2.1 × 105 years) and emissions (294 keV β-) restrict its use

a

Figure 2. HPLC of 99mTcO(3f)2 and 99mTcO(4f)2. The complexes were prepared by Method II. The analysis was carried out on Beckman ODS C18 4.6 × 250 mm column, with H2O/MeOH as mobile phase. Flow rate 1.0 mL/min, gradient 50 to 10% H2O over 20 min.

in chemical and radiopharmacy laboratories. Nevertheless, the structural identification of 99mTc chelates of 3a-g, 4a-g, and 5a,c-g has been attempted as described below, with the particular goal of determining the 99mTc:ligand ratio. The labeling of any one of 3a-g and 4a-g with 99mTc in the presence of either SnCl2 or Sn-DTPA was complete and efficient, with only one single peak detected by HPLC (Table 1 and Figure 2). Interestingly, three peaks were observed on HPLC after labeling a mixture of two chelating agents, one from the 3a-g series and another from either 4a-g or 5a,c-g series, for example, with 99mTc in the presence of the same reducing agent. Two examples of the HPLC analysis of the mixed-ligand (3b + 4g and 3g + 4b) labeling with 99mTc are shown in Figure 3. Three peaks were detected by HPLC upon labeling a mixture of either 3b (60 µg, 0.22 µmol) and 4g (80 µg, 0.23 µmol) or 3g (80 µg, 0.23 µmol) and 4b (60 µg,



Figure 4. Proposed structures of 99mTc complexes of 3a-g, 4a-g, and 5a,c-g.

Figure 3. HPLC of the mixed-ligands labeling with 99mTc. Upper panel: 0.22 µmol of 4b mixed with 0.23 µmol 3g. Lower panel: 0.22 µmol of 3b mixed with 0.23 µmol of 4g. The labelings were performed by Method I. The analysis was carried out on Beckman ODS C18 4.6 × 250 mm column, with 0.02 M, pH 4.6, NH4OAc/MeOH as mobile phase. Flow rate 1.0 mL/min, gradient 50 to 10% NH4OAc buffer over 20 min.

0.22 µmol) with 99mTc in the presence of SnCl2. Following isolation of the three peaks by HPLC, removal of the solvents by rotary evaporation under vacuum, redissolution of the residue in a mixture of saline and ethanol, and reinjection into HPLC, three peaks in the same pattern and with the same retention times were exhibited in Figure 3. The peaks of 99mTc(3b)2, 99mTc(4g)2, 99mTc(4b)2, and 99mTc(3g)2 in Figure 3 were identified because their retention times were similar to the four corresponding 99mTc complexes analyzed under the same conditions. The middle peaks on the upper and lower chromatograms of Figure 3 were assigned as the mixed-ligand 99mTc chelates 99mTc(3b + 4g) and 99mTc(3g + 4b), respectively. This assignment was further confirmed by coinjecting the 99mTc-labeled mixed-ligands with the four single-ligand complexes separately. That is, upon mixing 99mTc(3b)2 with 99mTc-labeled mixed-ligand (3b + 4g) products and injecting into HPLC, the peak height of 99mTc(3b)2 and 99m Tc(3b + 4g) in Figure 3 increased accordingly. The same result was observed upon the co-injection of the other three single 99mTc tracers mixed with the corresponding 99mTc-labeled mixed-ligand products. In addition, the peak height of 99mTc(3b)2 in Figure 3 varied in accordance with the change of the molar ratio of 3b in the mixture. A similar result was also observed with the other three chelating agents, 3g, 4b, and 4g. Therefore, the middle of the three peaks in the upper and lower chromatograms of Figure 3 must be the 99mTc mixedligand complexes 99mTc(4b + 3g) and 99mTc(3b + 4g),

respectively. All this evidence supported the assumption that the ratio of 99mTc:ligand is 1:2. However, it was not certain whether it was the four oxime groups of the two chelating agents that chelate 99m Tc. As stated earlier, the 99mTc labeling of 6a,c-g under similar conditions was not successful. The imidazole group of 6a,c-g, directly linked to the R-carbon without any spacer, might hinder the R-oxime group to chelate 99mTc. However, steric hindrance by the imidazole group of 6a,c-g is not likely the explanation because 99mTc labeling of 7a and 7e was unsuccessful as well, yielding mostly 99mTc colloids that could not be detected by HPLC. Compounds 7a and 7e contain a diethylamine group attached to the R-carbon, which is not considered a more significant hindrant for the R-oxime group than the aniline group of 5a,c-g, which chelated 99mTc well. Therefore, it appears that the proton on the nitrogen linked to the R-carbon is important for chelation of 99mTc and the R-oxime group of 3a-g, 4a-g, and 5a,c-g might not play a role in chelating 99mTc under these labeling conditions. The chelation of 99mTc might be through the β-oxime group and the nitrogen atom linked to the R-carbon of the chelating agents. 6a,c-g and 7a and 7g could not chelate 99mTc because there is no proton on the nitrogen attached to the R-carbon of these molecules. The structure of 99mTc(3a-g)2, 99mTc(4a-g)2, or 99mTc(5a,c-g)2 is consequently proposed in Figure 4. Partition Coefficient (PC) Determination of 99mTc(3 99mTc(3 a-g)2. The PC values of the seven a-g)2 complexes, before and after HPLC purification, were determined (Table 2). The HPLC-purified 99mTc complexes, after removing the solvents and redissolving in a mixture of saline and ethanol, and reinjecting into HPLC under the same condition, revealed the same retention times as those without HPLC purification. This suggests that purification did not change the composition of the 99mTc(3a-g)2 complexes. The PC values of the seven 99mTc complexes purified by HPLC increased in the order as: 99mTc(3a)2 < 99mTc(3b)2 < 99mTc(3f)2 < 99mTc(3c)2 < 99mTc(3 ) < 99mTc(3 ) < 99mTc(3 ) , which is similar to e 2 d 2 g 2 that of their retention times on HPLC (Table 1). The data in Table 1 demonstrated that a chelating agent which


Su et al.

Table 2. Octanol/PBS Partition Coefficients of Seven 99mTc Complexes Containing the 2-Nitroimidazole Group (3a-g) with R1 Defined in Figure 1a partition coefficient R1

unpurified

purified

CH3C2H5C3H7isoC4H9(CH3)3CC6H5C6H5C2H4-

0.11 ( 0.01 0.49 ( 0.05 4.76 ( 0.35 23.7 ( 1.70 2.63 ( 0.16 5.70 ( 0.52 107 ( 8

0.11 ( 0.01 0.51 ( 0.02 5.70 ( 0.58 26.2 ( 1.91 2.82 ( 0.18 4.92 ( 0.14 108 ( 17

a The complexes were prepared by Method II and purified by HPLC on a C18 column with H2O/MeOH as mobile phase. Ten microliters of each purified 99mTc complex or labeling mixture (unpurified) were partitioned between 1.0 mL of n-octanol and PBS. Each value is mean of three determinations ( SEM.

,c

Figure 5. Accumulation of 99mTcO(3f)2 in Chinese hamster ovary cells under aerobic and hypoxic conditions as a function of time. Cin stands for the radioactivity concentration inside cells, while Cout stands for radioactivity in supernatant. Each point is mean of three measurements and the error bars are SEM.

exhibited a longer retention time on reversed-phase HPLC usually formed a 99mTc complex with a longer retention time. However, 99mTc(3e)2 seemed to be an exception in the series as it showed an unexpectedly lower PC than its analogues. This result with 99mTc(3e)2 was also noted in the PC determination of the samples without HPLC purification (Table 2). In fact, the data in Table 2 suggest that it is the alkyl chain length of a chelating agent, rather than molecular weight, which plays an important role in determining the lipophilicity of the corresponding 99mTc complex. For example, the PC of 99mTc(3f)2 is almost equal to 99mTc(3c)2 even though the former has a higher molecular weight. Cellular Accumulation of the 99mTc Complexes of 3a-g, 4a-g, and 5a,c-g under Hypoxic and Aerobic Conditions. Samples of each 99mTc complex of 3a-g, 4a-g, or 5a,c-g were added to CHO cells incubated under hypoxic or aerobic conditions. Aliquots were removed from the cell suspension as a function of time, spun through oil to separate the cells from the supernatant medium, and the radioactivity in the cell pellet (Cin) and supernatant (Cout) were measured in a well counter. The Cin/Cout ratio under hypoxic and aerobic conditions was plotted as a function of time. Figure 5 is a plot of the data obtained with 99mTc(3f)2 and is representative of the remaining six 99mTc complexes which contain the 2-nitroimidazole group. The accumulation of 99mTc(3f)2 increased with time in both aerobic and hypoxic cells, but the increase in hypoxic cells was greater than in aerobic cells. For example, the Cin/Cout values at 5 min for aerobic and hypoxic cells were 0.47 and 0.46, respectively, while at 6 h the Cin/Cout for aerobic cells was 0.74 and for

Figure 6. The accumulation of 99mTcO(3a-g)2 (panel a), 99mTcO(4 99mTc(5 a-g)2 (panel b), and a,c-g)2 (panel c) in Chinese hamster ovary cells under aerobic and hypoxic conditions at 6 h time point. The complexes were prepared by Method I, analyzed by HPLC on Beckman ODS C18 4.6 × 250 mm column, with 0.02 M, pH 4.6, NH4OAc/MeOH as mobile phase. Flow rate 1.0 mL, gradient 30 to 10% NH4OAc buffer over 20 min. (Circle) Under aerobic condition. (Square) Under hypoxic condition. Solid symbols stand for 99mTc complexes of 3a, 4a, and 5a; open symbols stand for 99mTc complexes of 3b and 4b; dot-centered symbols stand for 99mTc complexes of 3f, 4f, and 5f; plus-centered (+) symbols stand for 99mTc complexes of 3c, 4c, and 5c; crosscentered (×) symbols stand for 99mTc complexes of 3e, 4e, and 5e; horizontal-line-centered (-) symbols stand for 99mTc complexes of 3d, 4d, and 5d; vertical-line-centered (|) symbols stand for 3g, 4g, and 5g.

hypoxic cells was 2.25, representing a 3-fold hypoxic/ aerobic differential. The remaining six 99mTc tracers of this series also demonstrated higher accumulation in hypoxic cells than in aerobic cells at 6 h. The hypoxic/ aerobic differentials at 6 h were 2.6, 3.1, 2.2, 1.7, 1.4, and 2.4 for 99mTc(3a)2, 99mTc(3b)2, 99mTc(3c)2, 99mTc(3d)2, 99mTc(3 ) , and 99mTc(3 ) , respectively. These data are e 2 g 2 illustrated in the upper panel of Figure 6, where the Cin/Cout values in hypoxic and aerobic cells at 6 h are plotted against the HPLC retention time of the complex. The 4-nitroimidazole group of 99mTc(4a-g)2 is more difficult to reduce than 2-nitroimidazole (10). This is reflected in the lower hypoxic/aerobic differentials at 6 h for 99mTc(4a-g)2 than for their 99mTc(3a-g)2 counterparts. The results are plotted in the middle panel of Figure 6. Among the seven compounds, only 99mTc(4a)2


and 99mTc(4b)2 showed significant hypoxic/aerobic differentials of 1.9 and 2.6, while 99mTc(4f)2 and 99mTc(4d)2 showed differentials of only 1.4 and 1.3, respectively. The remaining three 99mTc complexes, 99mTc(4c)2, 99mTc(4e)2, and 99mTc(4g)2 exhibited no differential in cellular accumulation under hypoxic and aerobic conditions. It was anticipated that 99mTc(5a,c-g)2 would show equal accumulation in hypoxic and aerobic cells because they do not contain a hypoxia-targeting group, such as the 2-nitroimidazole or 4-nitroimidazole groups of 99mTc(3a-g)2 and 99mTc(4a-g)2. The lower panel of Figure 6 confirms this assumption; the Cin/Cout values of aerobic and hypoxic cells for the six complexes of 99mTc(5a,c-g) were almost equal at 6 h. DISCUSSION

Propyleneamine oxime (PnAO) is a well-known chelating agent for 99mTc, forming a stable and lipophilic complex (23-25). PnAO chelates 99mTc via two amine nitrogen atoms and two oxime nitrogen atoms. The three positive charges of the 99mTcO3+ core are neutralized during complexation with the release of three protons: two amine protons and one hydroxyl proton. The hydroxyl proton is released by the formation of an intramolecular hydrogen bond between the two hydroxyl groups of PnAO. The hydrogen bond significantly increases the stability of 99mTc-PnAO class of complexes. One example of this class of complexes is 99mTc-(hexamethyl propyleneamine oxime) or HMPAO, which is used as a brain imaging agent (5). 99mTc-[PnAO-1-(2-nitroimidazole)] or BMS181321 is another PnAO-based 99mTc complex, which contains a 2-nitroimidazole group and has shown selective accumulation in hypoxic cells (6). However, in vivo studies indicated that the clearance of BMS181321 from the blood and background tissues is slow, due to its high lipophilicity (PC ) 40), which limited its usage as a hypoxia imaging agent (21). Analogues with lower lipophilicity are under investigation (7). Lipophilicity is an important factor in the biological behavior of a 99mTc complex. Complexes of inadequate lipophilicity have difficulty crossing biological membranes by passive diffusion, have a low volume of distribution, and tend to be cleared rapidly from the body by renal excretion. To develop a 99mTc based complex for imaging tumor hypoxia, 99mTc complexes which possess specific functional groups for targeting, such as 2-nitroimidazole, and appropriate lipophilicity are required. However, it is difficult to modify the lipophilicity of 99mTc-PnAO type complexes or to prepare a 99mTc-PnAO complex that bears two homo- or hetero-functional groups, such as two 2-nitroimidazole groups or one 2-nitroimidazole and one 4-nitroimidazole group. However, this can be readily achieved using the dioxime class of ligand reported in this paper. All 20 ligands, 3a-g, 4a-g, and 5a,c-g, can efficiently chelate 99mTc in the presence of either SnCl2 or Sn-DTPA in a pH range 4.6-9 at room temperature. The chemical yields and purities of the chelation were shown to be high by HPLC analysis (Table 1). The true radiochemical yields of the 99mTc complexes could be even higher than those presented in Table 1 because some highly lipophilic 99mTc complexes (3 , 4 , and 5 g g a,c-g, for example) were retained to some extent on the column which decreased their recovery from the HPLC system. The 99mTc complexes were stable for 24 h in saline at room temperature without the need for an antioxidant. The ability of the dioxime to chelate 99mTc in the presence of an excess of the strong chelating agent DTPA (Method II)


further attests to the strength of the 99mTc-dioxime complex. 99mTc labeling of 3a-g and 4a-g showed a single peak on HPLC and no isomers were evident. The 99mTc complexes of 5a,c-g could be different from those of 3a-g and 4a-g in structure. On HPLC a minor peak (about 5-10% in radiochemical yield) appeared on the tail of the main peak of 99mTc complexes of 5a,c-g. Whether or not the minor peak is an isomer of 99mTc(5a,c-g)2 remains to be confirmed. Aside from the 99mTc complexes of 3e, 4e, and 5e, which exhibited comparatively broad peaks, the 99mTc complexes of the remaining 17 ligands produced symmetrical peaks on HPLC. Judging from the results in Table 1, the tertiary butyl group of 3e, 4e, and 5e did not act as a significant steric hindrant in chelation of 99mTc. The ratio of 99mTc to chelating agent in these 20 complexes is believed to be 1:2 based on analysis of the 99mTc complexes of mixed ligands, and there is no evidence to indicate the formation of 1:3 complexes (Figure 3). It is also believed that the 99mTc is chelated by the nitrogen atom of the oxime group on the β-carbon and the nitrogen atom linked to the R-carbon, not by the two nitrogen atoms of the two oxime groups, to form a complex with a square pyramidal configuration with the oxo group at the apex. If this is the case, there are two dπ-pπ bonds formed between 99mTc and the nitrogen of the β-dioximes, and two σ bonds formed between 99mTc and nitrogen at the R-carbon. In addition to the σ bonds, there could be dπ-pπ conjugations between 99mTc, the R-carbon, the nitrogen of the R-oxime, and the nitrogen at the R-carbon. If this is the case, the two 99mTc-N bonds would be shorter than usual ones and stronger. The proton on the nitrogen that is attached to the R-carbon of 3a-g, 4a-g, and 5a,c-g is vitally important for the chelation of 99mTc. Without it, no chelation of 99mTc took place, as was seen with 6a,c-g, 7a, and 7e, despite their two oxime groups and one nitrogen atom attached to the R-carbon. This evidence further supports the proposed structure of the 99mTc complexes in Figure 4. This proposed structure suggests there might exist an intermolecular hydrogen bond between the two hydroxyl groups on the same side, which could enhance the stability of the 99mTc chelate. The three positive charges of the TcO3+ core are therefore neutralized upon chelation. This proposed structure is different from the vicinal dioxime complexes of transition metal ions, such as Ni2+, Co2+, Pd2+, and Cu2+, where the metal ions are chelated at pH 1-7 by the two nitrogen atoms of the oxime groups or by the two oxygen atoms of the oxime groups to form complexes in square planar geometry (26). Dioximes are also able to chelate 99mTc in the presence of NaBH4 to form a boronic acid adduct of 99mTcCl(dioxime)3 or BATO complex (8, 10, 14). The dioxime ligands presented in this report offer two potential advantages over PnAO. First, a 99mTc complex containing two different functional groups, by means of mixed-ligand labeling techniques, can be conveniently prepared. Two examples, 99mTc(3b + 4g) and 99mTc(3 + 4 ), are illustrated in Figure 3. The second g b advantage is that it is possible to prepare a series of 99m Tc complexes, by means of combinations of ligands, with various lipophilicities. The lipophilicity of a 99mTc complex is described by its octanol/water partition coefficient (PC). The PC values of the seven complexes of 99m Tc(3a-g)2, shown in Table 2, range from 0.11 for 99mTc(3 ) to 107 for 99mTc(3 ) , a 3 orders of magnitude a 2 g 2 difference over the series of complexes. The PC values of the 99mTc complexes appeared to be proportional to the length of the carbon chain rather than the molecular


weight. In a plot of log(PC) values (Table 2) as a function of length of carbon chain (number of the carbons of a n-alkyl chain, n), a straight line is evident (figure not shown) described by the equation log PC ) 0.86n - 1.88. Consequently, a 99mTc complex with a longer carbon chain, n-butyl for example, would have an even higher PC value (36.3) than that of 99mTc(3d)2 (PC ) 26.2). A similar result has been observed in the study of 99mTcPnAO complexes (27), where a relationship of the lipophilicity and length of carbon chain on the backbone of the chelating agents was described as log PC ) 0.88n + 0.322. Accordingly, the PC of a 99mTc-PnAO complex with two n-propyl groups on the backbone would be 916, which is 160-fold greater than that of 99mTc(3c)2. This implies that altering the alkyl groups on the backbone cannot finely modify the PC of a 99mTc-PnAO. Ten of the 14 2- and 4-nitroimidazole 99mTc complexes showed low binding (0-17%) to high concentration of BSA. Two complexes, 99mTc(3e)2 (43%) and 99mTc(4d)2 (34%), showed intermediate levels of BSA binding. Two complexes, 99mTc(3g)2 and 99mTc(4g)2, exhibited 65% breakdown in BSA and of the remaining activity 57-88% bound to BSA. In contrast, the 99mTc complexes of 5a,c-g showed no breakdown but high levels of binding to BSA (95-100%). This high level of binding might be the main cause of the low Cin/Cout values observed for this series (Figure 6c). The cellular uptake of the 20 99mTc complexes under aerobic and hypoxic conditions was investigated in an in vitro model as described above. The radioactivity associated with the CHO cells was measured as a function of incubation time. The cellular uptake of the 99mTc complexes under aerobic and hypoxic conditions at the 5 min and 6 h time points, and the hypoxic/aerobic differential at 6 h, in relation to the functional groups and the PC values will be the focus of this discussion. At 5 min, there is equal uptake of a 99mTc complex by aerobic and hypoxic CHO cells. This is the case for all the 20 99mTc complexes. However, the initial uptake value of each series of 99mTc complexes, such as 99mTc(3a-g)2, 99mTc(4 99mTc(5 a-g)2 and a,c-g)2, differed from each other and mainly depended on the functional groups and the lipophilicity. The accumulation of 99mTc(3f)2, representative of the series, in aerobic and hypoxic CHO cells as a function of time is plotted in Figure 5. The uptake of the complex in aerobic and hypoxic cells was equal at 5 min. At 6 h, the ratio of radioactivity in the aerobic cells to that in the medium (Cin/Cout) was 1.6-fold greater than that at 5 min, while the Cin/Cout under hypoxic conditions revealed a much greater increase of 4.9-fold, resulting in a hypoxic/aerobic differential of 3-fold. In general, the initial cellular uptakes under both aerobic and hypoxic conditions of the 99mTc complexes of 3a-g increased in sequence with their retention times on HPLC. The upper panel of Figure 6 shows the accumulation of radioactivity in CHO cells after 6 h incubation with the complexes of 99m Tc(3a-g)2 under either aerobic or hypoxic conditions. The Cin/Cout ratio of the seven 99mTc tracers exhibited a trend which followed the order of their retention times on HPLC, or the PC values. All showed hypoxic/aerobic differentials significantly greater than 1. The in vitro results obtained with the 4-nitroimidazole series 4a-g are presented in the middle panel of Figure 6. Contrary to what was seen in the 3a-g series, there is not as good a relationship between Cin/Cout at 6 h and HPLC retention time or PC value. In addition, only two of the 4a-g complexes show hypoxic/aerobic differentials significantly greater than 1. This was attributed to the

Su et al.

inefficient enzymatic reduction of the 4-nitroimidazole moiety in 4a-g. Finally, in the untargeted aniline series 5a,c-g presented in the lower panel of Figure 6, there is a relationship between Cin/Cout at 6 h and HPLC retention time, but no hypoxic/aerobic differentials were observed due to the lack of a hypoxia-specific targeting mechanism. Thus, only the 3a-g series of complexes containing 2-nitroimidazole all showed selective accumulation in hypoxic cells and are potentially useful for imaging hypoxic cells in vivo. However, it must be noted that the hypoxic/aerobic differentials observed in this series of compounds are modest in comparison with other reports of hypoxia imaging agents (6, 7, 21). The present series of compounds represent a proof of principle and a first step toward a hypoxia imaging agent. CONCLUSIONS

A novel amine-dioxime chelator for 99mTc has been reported which forms stable 1:2 99mTc:ligand complexes in high yield on stannous reduction at room temperature. Lengthening the alkyl chain allowed tailoring of the lipophilicity of the 99mTc complex. Complexes containing a 2-nitroimidazole moiety showed selective accumulation in hypoxic cells in vitro whereas those with 4-nitroimidazole or aniline did not. This work has shown the preparation and utility of the novel amine-dioxime chelator. ACKNOWLEDGMENT

The ligands were synthesized under grants from the Heart and Stroke Foundation, Squibb Radiology Research Fund, and Winnipeg Health Sciences Centre Research Foundation. Radiolabeling and evaluation of the complexes was performed under operating grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. LITERATURE CITED (1) Dewanjee, M. K. (1990) The chemistry of 99mTc-labeled radiopharmaceuticals. Semin. Nucl. Med. 20, 5-27. (2) Vallabhajosula, S., Zimmerman, R. E., Picard, M., Stritzke, P., Mena, I., Hellman, R. S., Tikofsky, R. S., Stabin, M. G., Morgan, R. A., and Goldsmith, S. J. (1989) Technetium-99m ECD: A new brain imaging agent: In vivo kinetics and biodistribution studies in normal human subjects. J. Nucl. Med. 30, 599-604. (3) Meegalla, S. K., Plossl, K., Kung, M. P., Chumpradit, S., Stevenson, D. A., Kushner, S. A., McElgin, W. T., Mozley, P. D., and Kung, H. F. (1997) Synthesis and characterization of technetium-99m-labeled tropanes as dopamine transporterimaging agents. J. Med. Chem. 40, 9-17. (4) Meltzer, P. C., Blundell, P., Jones, A. G., Mahmood, A., Garada, B., Zimmerman, R. E., Davison, A., Holman, B. L., and Madras, B. K. (1997) A technetium-99m SPECT imaging agent which targets the dopamine transporter in primate brain. J. Med. Chem. 40, 1835-1844. (5) Nowotnik, D. P., Canning, L. R., Cumming, S. A., Harrison, R. C., Higley, B., Nechvatal, G., Pickett, R. D., Piper, I. M., Bayne, V. J., and Forster, A. M. (1985) Development of a 99Tcm-labeled radiopharmaceutical for cerebral blood flow imaging. Nucl. Med. Commun. 6, 499-506. (6) Linder, K. E., Chan, Y. W., Cyr, J. E., Malley, M. F., Nowotnik, D. P., and Nunn, A. D. (1994) TcO(PnAO-1-(2nitroimidazole)) [BMS-181321], a new technetium-containing nitroimidazole complex for imaging hypoxia: synthesis, characterization, and xanthine oxidase-catalyzed reduction. J. Med. Chem. 37, 9-17. (7) Johnson, L. L., Schofield, L., Mastrofrancesco, P., Donahay, T., and Nott, L. (1998) Technetium-99m-nitroimidazole uptake in a swine model of demand ischemia. J. Nucl. Med. 39, 1468-1475.

Amine−Dioxime Chelator for Technetium-99m (8) Treher, E. N., Francesconi, L. C., Gougoutas, J. Z., Malley, M. F., and Nunn, A. D. (1989) Mono-capped tris dioxime complexes of technetium(III): Synthesis and structural characterization of TcX(dioxime)3B-R(X ) Cl, Br; dioxime ) dimethylglyoxime, cyclohexanedionedioxime; R ) CH3, C4H9). Inorg. Chem. 28, 3411-3416. (9) Maina, T., Stolz, B., Albert, R., Bruns, C., Koch, P., and Macke, H. (1994) Synthesis, radiochemistry and biological evaluation of a new somatostatin analogue (SDZ 219-387) labelled with technetium-99m. Eur. J. Nucl. Med. 21, 437444. (10) Linder, K. E., Chan, Y. W., Cyr, J. E., Nowotnik, D. P., Eckelman, W. C., and Nunn, A. D. (1993) Synthesis, characterization, and in vitro evaluation of nitroimidazole-BATO complexes: new technetium compounds designed for imaging hypoxic tissue. Bioconjugate Chem. 4, 326-333. (11) Meegalla, S. K., Plossl, K., Kung, M. P., Stevenson, D. A., Mu, M., Kushner, S., Liable-Sands, L. M., Rheingold, A. L., and Kung, H. F. (1998) Specificity of diastereomers of [99mTc]TRODAT-1 as dopamine transporter imaging agents. J. Med. Chem. 41, 428-436. (12) Billinghurst, M. W., Abrams, D. N., and Lawson, M. S. (1991) Stabilization of [99mTc]HMPAO - 1. Ethanolic preparation. Appl. Radiat. Isot. 42, 607-612. (13) Ballinger, J. R., and Gulenchyn, K. Y. (1991) Alternative formulations of technetium-99m HMPAO. Appl. Radiat. Isot. 42, 315-316. (14) Su, Z. F., Linse, K. H., Steinmetz, H. J., and Schwochau, K. (1992) Preparation and evaluation of cationic 99Tc/99mTc dimethoxy- and diethoxy-hexanedione dioxime complexes. J. Labelled Compd. Radiopharm. 31, 61-70. (15) Chapman, J. D., Engelhardt, E. L., Stobbe, C. C., Schneider, R. F., and Hanks, G. E. (1998) Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays. Radiother. Oncol. 46, 229-237. (16) Brown, J. M., and Workman, P. (1980) Partition coefficient as a guide to the development of radiosensitizers which are less toxic than misonidazole. Radiat. Res. 82, 171-190. (17) Morton, J. R., and Wilcox, H. W. (1953) Inorganic Syntheses, vol. IV, p 48, McGraw-Hill Book Co., New York.

Bioconjugate Chem., Vol. 11, No. 5, 2000 663 (18) Rheinboldt, H., and Schmitz-Dumont, O. (1925) Justus Liebigs Ann. Chem. 444, 113-135. (19) Behrend, R., and Schmitz, J. (1893) Justus Liebigs Ann. Chem. 277, 320. (20) Hay, M. P., Wilson, W. R., Moselen, J. W., Palmer, B. D., and Denny, W. A. (1994) Hypoxia-selective antitumor agents. 8. Bis(nitroimidazolyl)alkanecarboxamides: a new class of hypoxia-selective cytotoxins and hypoxic cell radiosensitizers. J. Med. Chem. 37, 381-391. (21) Ballinger, J. R., Wan Min Kee, J., and Rauth, A. M. (1996) In vitro and in vivo evaluation of a technetium-99m-labeled 2-nitroimidazole (BMS181321) as a marker of tumor hypoxia. J. Nucl. Med. 37, 1023-1031. (22) Schwochau, K. (1994) Technetium radiopharmaceuticals fundamentals, synthesis, structure, and development. Angew. Chem., Int. Ed. Engl. 33, 2258-2267. (23) Troutner, D. E., Volkert, W. A., Hoffman, T. J., and Holmes, R. A. (1983) A tetradentate amine oxime complex of Tc-99m. J. Nucl. Med. 24, P10. (24) Troutner, D. E., Volkert, W. A., Hoffman, T. J., and Holmes, R. A. (1984) A neutral lipophilic complex of 99mTc with a multidentate amine oxime. Int. J. Appl. Radiat. Isot. 35, 467-470. (25) Jurisson, S., Schlemper, E. O., Troutner, D. E., Canning, L. R., Nowotnik, D. P., and Neirinckx, R. D. (1986) Synthesis, characterization, and X-ray structural determinations of technetium(V)-oxo-tetradentate amine oxime complexes. Inorg. Chem. 25, 543-549. (26) Singh, R. B., Garg, B. S., and Singh, R. P. (1979) Oximes as spectrophotometric reagents - a review. Talanta 26, 425444. (27) Neirinckx, R. D., Nowotnik, D. P., Pickett, R. D., Harrison R. C., Ell, P. J. (1986) Development of a lipophilic Tc-99m complex useful for brain perfusion evaluation with conventional imaging equipment. In Amphetamines and pH Shift Agents: Basic Research and Clinical Results (H. Biersack and C. Winkler, Eds.) pp 59-70, Walter de Gruyter.

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A Novel AmineâDioxime Chelator for Technetium-99m - American

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A Novel AmineâDioxime Chelator for Technetium-99m - American