Synthesis and Evaluation of Hydroxamamide-Based Tetradentate Ligands as a New Class of Thiol-Free Chelating Molecules for 99mTc Radiopharmaceuticals

ISSN 0969-8051/98/$19.00 1 0.00 PII S0969-8051(97)00208-4

Nuclear Medicine & Biology, Vol. 25, pp. 295–303, 1998 Copyright © 1998 Elsevier Science Inc.

Synthesis and Evaluation of Hydroxamamide-Based Tetradentate Ligands as a New Class of Thiol-Free Chelating Molecules for 99mTc Radiopharmaceuticals Le-Cun Xu,1 Morio Nakayama,1 Kumiko Harada,1 Hitoshi Nakayama,1 Seiji Tomiguchi,2 Akihiro Kojima,2 Mutsumasa Takahashi2 and Yasushi Arano3 1

FACULTY OF PHARMACEUTICAL SCIENCES, KUMAMOTO UNIVERSITY, 5-1 OE-HONMACHI, KUMAMOTO 862, JAPAN;

2

DEPARTMENT OF RADIOLOGY, KUMAMOTO UNIVERSITY SCHOOL OF MEDICINE, HONJO, KUMAMOTO 860, JAPAN; AND

3

DEPARTMENT OF RADIOPHARMACEUTICAL CHEMISTRY, FACULTY OF PHARMACEUTICAL SCIENCES, KYOTO UNIVERSITY, SAKYO-KU, KYOTO 606, JAPAN

ABSTRACT. Both N,N*-ethylene bis(benzohydroxamamide) [(C2(BHam)2)] and N,N*-propylene bis(benzohydroxamamide) [(C3(BHam)2)] were designed as new thiol-free chelating molecules for 99mTc radiopharmaceuticals. Synthetic procedures using oxadiazoline intermediates were developed for C2(BHam)2 and C3(BHam)2. Both C2(BHam)2 and C3(BHam)2 formed 99mTc complexes with high yields over a wide pH range (pH 3–12) at room temperature. Complexation yields of over 95% were achieved at ligand concentrations as low as 2.5 3 1026 M. Reversed-phase HPLC analyses indicated that both C2(BHam)2 and C3(BHam)2 formed 99mTc complexes as single species with stabilities much higher than those of 99m Tc-BHam. Selective complex formation of 99mTc with the two ligands was observed in the presence of human IgG. No decomposition with low protein binding were demonstrated when the two 99mTc complexes were incubated in murine plasma. Although further structural studies are required, these findings implied that the Ham-based tetradentate ligands would serve as new chelating molecules for 99mTc radiopharmaceuticals. NUCL MED BIOL 25;3:295–303, 1998. © 1998 Elsevier Science Inc. KEY WORDS. Hydroxamamide, Tetradentate ligand, N,N9-Ethylene bis(benzohydroxamamide), N,N9Trimethylene bis(benzohydroxamamide), 99mTc complex

INTRODUCTION Radiopharmaceuticals derived from low molecular weight peptides and antibody fragments have attracted a great deal of attention for imaging of the sites of tumor, infection and thrombosis (1, 15, 29). The pharmacokinetics of these classes of polypeptides and peptides are well matched with the physical half-life of the most widely used and least expensive radionuclide for imaging, technetium-99m (99mTc) (12). Since most polypeptides do not possess binding sites to form 99mTc chelates of high in vivo stability, appropriate chelating molecules are incorporated into peptide molecules to prepare 99mTc-labeled peptides for in vivo application. Tetradentate ligands with N2S2 or N3S coordination molecules are representative reagents currently being used for this purpose (2, 6, 8, 14, 32). While the thiol groups in the molecules facilitated formations of mononuclear 99mTc complexes of high stability, presence of the free thiol group may restrict the conjugation reactions with polypeptides and subsequent storage of the resulting bioconjugates. Protection of a thiol group was performed to conjugate a N3S chelating molecule to an antibody fragment by the maleimide-thiol chemistry, which necessitated a deprotection step before 99mTc complexation reaction (32). Protection of a thiol group is also performed in the kit formulation of mercaptoacetyl triglycine (MAG3) for long-term storage (30). Exchange reactions between the free thiol groups of the chelating agents and disulfide bonds in peptides may also Address all correspondence to: Morio Nakayama, Ph.D., Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862, Japan; e-mail: [email protected] Accepted 10 September 1997.

constitute a potential problem in the conjugation reactions. In addition, some chelating agents require harsh 99mTc complexation conditions to prepare 99mTc chelates with high radiochemical yields (4, 7, 13, 27). Thus, while chelating agents with thiol groups were useful to prepare 99mTc-labeled peptides and polypeptides, development of thiol-free chelating molecules that provide 99mTc complexes of high in vivo stability with high radiochemical yields under mild complexation conditions would provide further applications of 99m Tc to a variety of polypeptides of interest for diagnostic nuclear medicine. N-Hydroxy-carboximidamide (hydroxamamide, abbreviated as Ham hereafter) is well known to form chelates with metal ions in which the metal atom is linked to the oxime group and the amino group (9, 16, 26, 33). We have recently reported that bidentate Ham ligands form stable 99mTc complexes in high radiochemical yields over a wide pH range at low ligand concentrations (22–24). Previous studies also suggested formations of mononuclear 99mTc-Ham complexes at a ligand-to99m Tc molar ratio of 2:1 (25). Furthermore, alkylation of the nitrogen atom of the Ham ligands did not impair the chelating ability of unsubstituted Ham with 99mTc (M. Nakayama, unpublished data). The gathered findings strongly implied that Hambased tetradentate ligands would serve as a new class of thiol-free chelating molecules for 99mTc labeling of polypeptides. To estimate the hypothesis, we designed and synthesized N,N9ethylene bis(benzohydroxamamide) (C2(BHam)2) and N,N9propylene bis(benzohydroxamamide) (C3(BHam)2) as prototype compounds (Fig. 1). Complexation reactions of these ligands with 99mTc and stabilities of the resulting 99mTc complexes were

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Synthesis of Potassium 3-phenyl-D2-1,2,4-oxadiazolin5-one (3) To 40 mL of methanol containing 2 (15.0 g, 0.093 mol) was added 50 mL of methanol containing KOH (0.2 g, 0.093 mol). After being stirred for a few minutes, the solvent was removed in vacuo. The crude product was crystallized from ethanol. IR (KBr) 1716 cm21 (CAO). 1H-NMR (methanol-d4): d 7.45–7.80 (m, 5H, aromatic H). 13C-NMR: d 165.0, 161.2 (CAN, CAO), 131.5, 129.3, 126.4 (C6H5). Anal. calcd. for C8H5N2O2K: C 47.99, H 2.52, N 13.99. Found: C 48.27, H 2.55, N 14.11.

Synthesis of 3-Phenyl-4-(2-bromoethyl)-D2-1,2, 4-oxadiazolin-5-one (4)

FIG. 1. Chemical structures of BHam, C2(BHam)2 and C3(BHam)2.

estimated. Applicability of the new tetradentate ligands as a chelating molecule for 99mTc radiopharmaceuticals was also discussed.

Compound 3 (12.0 g, 0.060 mol) was dissolved in 50 mL N,Ndimethylformamide (DMF) and 1,2-dibromoethane (33.8 g, 0.18 mol) was added. After 3 days of being stirred at room temperature, the precipitate was filtered and the filtrate was evaporated in vacuo (30 mmHg). Chloroform (50 mL) was added to the residue and the organic phase was washed successively with 1.0 M NaOH and water (20 mL each). After being dried over anhydrous MgSO4, the solvent was removed in vacuo. The solid residue was recrystallized from petroleum ether:chloroform (1:5) to give 8.1 g of 4 (50%) as a colorless crystal. mp: 79 – 80°C. IR (KBr) 1762 cm21 (CAO). 1 H-NMR (DMF-d7): d 7.93– 8.10 (m, 5H, aromatic H), 4.47 (t, 2H, J 5 6.2, Hz, CH2Br), 3.99 (t, 2H, J 5 6.2, Hz, NCH2). 13C-NMR: d 158.9, 158.5 (CAN, CAO), 132.1, 129.3, 128.6, 122.8 (C6H5), 43.9, 28.9 (NCH2CH2Br). MS (EI) (M1): m/z 268, 270. Anal. calcd. for C10H9N2O2Br: C 44.63, H 3.37, N 10.41. Found: C 44.59, H 3.26, N 10.33.

Synthesis of 4,4*-Ethylene bis(3-phenyl-D2-1,2, 4-oxadiazolin-5-one) (5) MATERIALS AND METHODS Benzohydroxamamide (BHam) was prepared by reaction of benzonitrile with hydroxylamine (28). O-Carboethoxybenzohydroxamamide (1) and 3-phenyl-D2-1,2,4-oxadiazoline-5-one (2) were synthesized according to the procedures described by Falck (10, 11). Melting points were determined with a Yanaco micro melting point apparatus and are reported uncorrected. 1H-NMR and 13C-NMR spectra were obtained on a JNM-500 (500 MHz) instrument (JEOL Ltd.). Mass spectra were obtained with a JMS-DX303HF mass spectrometer (JEOL Ltd.). [99mTc]Pertechnetate (99mTcO42) was eluted in saline solution on a daily basis from Daiichi Radioisotope Laboratories generators. In thin-layer chromatography (TLC) analysis of 99mTc complexes, samples (10 mL each) was spotted on Merck cellulose strips (Art 5577) and methanol:water (85:15 [v/v]) was used as the developing solvent. Cellulose acetate electrophoresis (CAE) was run at an electrostatic fields of 0.8 mA/cm for 20 min in phosphate buffer (I 5 0.05, pH 7.0). High performance liquid chromatography (HPLC) analyses were carried out in an HPLC system consisting of a Hitachi L-6200 intelligent pump and a Lichrospher 100 RP-18(e) reversed-phase column (150 3 4.6 mm inner diameter, Merck). Samples (5 mL each) were injected and eluted using methanol:0.05 M NH4HCO3 (55:45 [v/v]) or acetonitrile:0.05 M NH4HCO3 (30:70 [v/v]) at a flow rate of 1 mL/min and monitored with an PS-201 well-type NaI(Tl) scintillation detector (Aloka) coupled with a TCD501 universal scalar (Aloka). Human nonspecific immunoglobulin G (IgG) was supplied by the ChemoSerotherapeutic Research Institute.

3-Phenyl-4-(2-bromoethyl)-D2-1,2,4-oxadiazolin-5-one (4) (4.8 g, 0.018 mol) and potassium 3-phenyl-D2-1,2,4-oxadiazolin-5-one (3) (4.0 g, 0.020 mol) were dissolved in 50 mL of DMF. The solution was stirred at 50°C for 2 days. After filtering the precipitate, the filtrate was evaporated in vacuo (30 mmHg). The solid residue was washed with 1.0 M NaOH, water and methanol to give 3.4 g of 5 (54%) as a solid. The crude solid was recrystallized from acetone. mp: 205–206°C. IR (KBr) 1747 cm21 (CAO). 1H-NMR (DMFd7): d 7.84 – 8.03 (m, 10H, aromatic H), 4.26 (s, 4H, CH2CH2). 13 C-NMR: d 172.7, 168.0, 158.8, 158.9 (CAN, CAO), 132.2, 132.0, 131.6, 129.4, 129.2, 129.1, 128.6, 128.3, 126.6, 126.2, 122.8 (C6H5), 70.6, 41.6 (NCH2CH2N). MS (EI) (M1): m/z 350. Anal. calcd. for C18H14N4O4: C 61.71, H 4.03, N 15.99. Found: C 61.99, H 4.12, N 15.95.

Synthesis of 3-Phenyl-4-(3-bromopropyl)-D2-1,2, 4-oxadiazolin-5-one (6) Compound 6 was synthesized by a procedure similar to that described for compound 4 in 57% yield as colorless crystals. mp: 77–78°C. IR (KBr) 1762 cm21 (CAO). 1H-NMR (CDCl3): d 7.27–7.66 (m, 5H, aromatic H), 3.85 (t, 2H, J 5 7.3, NCH2CH2CH I 2Br), 3.34 (t, 2H, J 5 6.0, NCH I 2CH2CH2Br), 2.20 –2.25 (m, 2H, NCH2CH I 2CH2Br). 13C-NMR: d 158.9, 158.8 (CAN, CAO), 131.9, 129.2, 128.6, 123.2 (C6H5), 57.8, 41.3, 30.4, 30.2 (NCH2CH2CH2Br). MS (EI) (M1): m/z 282, 284. Anal. calcd. for C11H11N2O2Br: C 46.67, H 3.92, N 9.89. Found: C 46.88, H 3.76, N 9.98.

Hydroxamamide-Based Tetradentate Ligands

Synthesis of 4,4*-Trimethylene bis(3-phenyl-D2-1,2, 4-oxadiazolin-5-one) (7)

297 solution containing both ligands (0.1 mL; 5 3 1024 M each) was added a saturated stannous tartrate (0.15 mL) and Na99mTcO4 (0.05 mL, 37 MBq/mL). After incubation for 3 h at room temperature, samples were analyzed by HPLC using methanol:0.05 M NH4HCO3 (55:45 [v/v]) or acetonitrile:0.05 M NH4HCO3 (30:70 [v/v]) as an eluent.

3-Phenyl-4-(3-bromopropyl)-D2-1,2,4-oxadiazolin-5-one (6) (5.1 g, 0.018 mol) and potassium 3-phenyl-D2-1,2,4-oxadiazolin-5-one (3) (4.0 g, 0.020 mol) were dissolved in 50 mL of DMF. After 3 days of being stirred at room temperature, the precipitate was filtered and the filtrate was evaporated in vacuo. Chloroform (30 mL) was added to the residue and the organic phase was washed successively with 1.0 M NaOH and water (20 mL each). After being dried over anhydrous MgSO4, the solvent was removed in vacuo (30 mmHg). The solid residue was recrystallized from ethanol:water (5:1) to give 4.3 g of 7 (66%) as a colorless crystal. mp: 148 –149°C. IR (KBr) 1781 cm21 (CAO); 1H-NMR (DMSO-d6): d 7.57–7.70 (m, 10H, aromatic H), 7, 3.57 (t, 4H, J 5 7.3, NCH I 2CH2CH I 2N), 1.76 (t, 2H, J 5 7.3, NCH2CH I 2CH2N). 13C-NMR: d 158.8, 158.6 (CAN, CAO), 132.0, 129.3, 128.3, 122.8 (C6H5), 40.0, 26.4 (NCH2CH2CH2N). MS (EI) (M1): m/z 364. Anal. calcd. for C19H16N4O4: C 62.63, H 4.43, N 15.38. Found: C 62.41, H 4.22, N 15.55.

Tc-BHam was prepared by adding Na99mTcO4 (37 MBq/mL, 0.05 mL) to a mixed solution of stannous tartrate (0.15 mL) and BHam (1024 M, 0.20 mL) in 0.02 M acetate buffer (pH 5). A solution of C2(BHam)2 or C3(BHam)2 (0.2 mL; 1024 M, pH 5) was then added to the solution of 99mTc-BHam. After 6 h incubation at room temperature, the reaction mixture was analyzed by HPLC using acetonitrile:0.05 M NH4HCO3 (30:70 [v/v]) as an eluent. Under similar experimental conditions, BHam was added to the solution of 99mTc-C2(BHam)2 or C3(BHam)2 and the respective reaction mixture was analyzed by HPLC.

Synthesis of C2(BHam)2

99m

4,49-Ethylene bis(3-phenyl-D2-1,2,4-oxadiazolin-5-one) (5) (2.2 g, 0.0063 mol) was added to 50 mL of 5% NaOH. The mixture was stirred and heated at 90°C for 1 h. The resulting solution was cooled to room temperature and neutralized with 1 M HCl. The white precipitate was filtered and recrystallized from ethanol:water (1:10) to give 1.3 g of C2(BHam)2 as colorless crystals (69%). mp: 207–208°C. IR (KBr) 3214 (broad, O-H) and 1645 cm21 (CAN). 1 H-NMR (DMSO-d6) d 9.77 (s, 2H, OH), 7.28 –7.41 (m, 10H, aromatic H), 5.80 (s, 2H, NH), 2.90 (s, 4H, CH2CH2), 13C-NMR: d 154.6 (CAN), 132.4, 128.8, 128.2, 128.1 (C6H5), 44.0 (CH2CH2) MS (EI) (M1). m/z 298. Anal. calcd for C16H18N4O2: C 64.41, H 6.08, N 18.78. Found: C 64.11, H 6.16, N 18.59.

A solution of C2(BHam)2 or C3(BHam)2 (0.1 mL; 1.0 3 1024 M to 1.0 3 1023 M) in 0.01 M phosphate-buffered saline (PBS) (pH 7.4) was mixed with a solution of human IgG (0.10 mL; 1.0 3 1024 M) in the same buffer. To the mixed solution was added a saturated solution of stannous tartrate (0.15 mL) and Na99mTcO4 (37 MBq/mL; 0.05 mL) and the reaction mixture was incubated at room temperature. After 1 h, the reaction mixtures were analyzed by TLC.

Synthesis of C3(BHam)2 C3(BHam)2 was synthesized by a procedure similar to that described for C2(BHam)2 in 77% yield as colorless crystals. mp 73–74°C. IR (KBr) 3203 (broad, OH) and 1635 cm21 (CAN). 1H-NMR (DMSO-d6): d 9.66 (s, 2H, OH), 7.33–7.41 (m, 10H, aromatic H), 5.68 –5.70 (t, 2H, NH), 2.87 (q, 4H, J 5 6.7, NCH I 2CH2CH I 2N), 1.38 (m, 2H, J 5 6.7, NCH2CH I 2CH2N). 13C-NMR: d 154.8 (CAN), 132.7, 128.9, 128.5, 128.1 (C6H5), 41.1, 32.6 (CH2CH2CH2). MS (EI) (M1): m/z 312. Anal. calcd. for C17H20N4O2: C 65.37, H 6.45, N 17.94. Found: C 65.51, H 6.56, N 17.76.

Preparation of

99m

Tc Complexes

C2(BHam)2 or C3(BHam)2 was dissolved in 0.02 M HCl and the solution was diluted with buffered solutions to various concentrations and pH values (1024–1026 M; pH 2–12). Na99mTcO4 (37 MBq/mL, 0.05 mL) was added to the respective ligand solution (0.2 mL), and a saturated solution of stannous tartrate in N2-purged distilled water (0.15 mL) was then added. After incubation of the reaction mixture at room temperature for 2, 5, 15, 30 and 60 min, an aliquot of samples was withdrawn and analyzed by TLC, CAE and HPLC. 99m Tc complexation reaction was also performed in the presence of equimolar amounts of C2(BHam)2 and C3(BHam)2. To a

Ligand Exchange Reaction 99m

Tc Complexation Reactions of C2(BHam)2 and C3(BHam)2 in the Presence of IgG

Stability Estimation of 99m Tc-C3(BHam)2

99m

Tc-C2(BHam)2 and

Both 99mTc-C2(BHam)2 and 99mTc-C3(BHam)2 were prepared at pH 7.0 with a ligand concentration of 1 3 1024 M. A solution of 99m Tc-C2(BHam)2 or 99mTc-C3(BHam)2 (20 mL each) was added to a microcentrifuge tube containing 200 mL of freshly prepared murine plasma. After incubation for 15, 60, 120 or 180 min at 37°C, a 400 mL solution of cold (4°C) ethanol was added to precipitate the proteins (19). The samples were then centrifuged at 3000 rpm for 10 min (4°C), and the radioactivity in the supernatant and the precipitate was measured with an auto-well g-counter (ARC-2000; Aloka). The radioactivity in the supernatant was then analyzed by HPLC using acetonitrile:0.05 M NH4HCO3 (30:70 [v/v]) as the eluent. In the control experiments, a saline solution was used in place of the murine plasma, and the stability of each 99mTc complex was monitored by HPLC. Stabilities of 99mTc-BHam, 99mTcC2(BHam)2 or 99mTc-C3(BHam)2 were also estimated in the presence of cysteine. A solution of each 99mTc complex (0.1 mL) with a ligand concentration of 1 3 1025 M was mixed with a 0.1 mL solution of cysteine to make the final concentrations of cysteine in the range of 5 3 1025 to 5 3 1022 M. The mixture was incubated at 25°C and analyzed by TLC using 0.005 M phosphate buffer as a eluent. In this TLC system, the 99mTc-cysteine showed an Rf value above 0.8, while those of 99mTc-BHam, 99mTcC2(BHam)2 and 99mTc-C3(BHam)2 were 0.3– 0.4, 0.55– 0.65 and 0.55– 0.65, respectively.

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FIG. 2. Synthesis of C2(BHam)2 and C3(BHam)2.

Biodistribution Study Animal studies were performed in compliance with generally accepted guidelines governing such work. 99mTc-C2(BHam)2 or 99m Tc-C3(BHam)2 with a ligand concentration of 5 3 1024 M was prepared at pH 7 as described above. Biodistribution of radioactivity was determined after intravenous administration of each 99mTc complex to male ddY mice (weighing 20 –25 g) (17). Groups of five mice were sacrificed at various intervals up to 2 h after injection, the organs or tissues of interest were removed and weighed, and the radioactivity was determined using an ARC-2000 auto-well g-counter system (Aloka).

reaction of NOH group of BHam with ethyl chloroformate, followed by cyclization of the resulting compound 1 in 5% NaOH solution. Treatment of 2 with a methanol solution of KOH gave a potassium salt 3, and the amine was alkylated with dibromoalkane. The cyclic precursors, compounds 5 and 7, were obtained after isolating the intermediate compounds 4 and 6, followed by the reaction with the potassium salt 3 at almost equimolar ratios. Finally, decyclization of compound 5 or 7 in 5% NaOH solution gave C2(BHam)2 and C3(BHam)2 with overall yields of 14% and 21%, respectively. The intermediates and the final products were identified by elemental analysis, IR, 1H- and 13C-NMR and mass spectroscopy.

RESULTS 99m

Synthesis of Ligands

Preparation and Characterization of the

Tc Complexes

C2(BHam)2 and C3(BHam)2 were synthesized according to the procedures outlined in Figure 2. Compound 2 was prepared by the

99m Tc complexes of C2(BHam)2 and C3(BHam)2 were prepared at room temperature by the stannous tartrate reduction. The reaction

Hydroxamamide-Based Tetradentate Ligands

TABLE 1. TLC and CAE of 99m Tc-C3(BHam)2

99m

Tc-C2(BHam)2 and

TLCb

99m

Tc Complexa

Rf Value

99m

Tc-C2(BHam)2 99m Tc-C3(BHam)2 99m TcO42

0.85–0.97 0.83–0.95 0.50–0.60

299

Electrophoresisc

Complex Yields (%)

Migration Distance from Origin (cm)

.95 .95

0 0 14–16

Each 99mTc complex was prepared at the ligand concentration of 5 3 1025 M (in 5 mM phosphate buffer, pH 7.0) by incubating 30 min at room temperature. b TLC was developed with a mixture of methanol:water (85:15 [v/v]). c CAE was run at an electrostatic fields of 0.8 mA/cm for 25 min in phosphate buffer (pH 7.0, m 5 0.05). a

mixtures were analyzed by TLC. In this system, the reducedhydrolyzed 99mTc that formed by the stannous reduction of 99m TcO42 in the absence of ligand remained at the origin. As shown in Table 1, new radioactive peaks derived from 99mTcC2(BHam)2 and 99mTc-C3(BHam)2 were clearly distinguishable from those of 99mTcO42 (0.50 – 0.60) and the reduced-hydrolyzed 99m Tc. CAE indicated that 99mTc-C2(BHam)2 and 99mTcC3(BHam)2 remained at the origin. Figure 3 shows the complexation yields of 99mTc-C2(BHam)2 and 99m Tc-C3(BHam)2 as functions of incubation time (Fig. 3A), reaction pH (Fig. 3B) and ligand concentrations (Fig. 3C) when determined by TLC. Over 95% complexation yields were obtained with a reaction time of 15 min and a ligand concentration of 5 3 1025 M at pH 7.0 for both ligands. High-yield complex formation was observed over wide pH range (3–12) at ligand concentrations as low as 2.5 3 1026 M. Typical HPLC profiles of 99mTc-BHam, 99mTc-C2(BHam)2 and 99mTc-C3(BHam)2 prepared at pH 5 and pH 7 are shown in Figure 4. When methanol:0.05 M NH4HCO3 (Fig. 4A– 4C) was used as the eluent, 99mTc-BHam showed two separated peaks with retention times (Rt) of 3.0 and 3.7 min, respectively and the ratios of the two peaks changed with reaction pH (Fig. 4A). In contrast, both 99mTc-C2(BHam)2 and 99mTc-C3(BHam)2 showed single peaks at both reaction pH (Fig. 4B and 4C). When acetonitrile:0.05 M NH4HCO3 (Fig. 4D– 4F) was used as the mobile phase, the separation efficiency of the two peaks of 99m Tc-BHam (Rt 5 3.0 and 5.7 min) was markedly improved (Fig. 4D). Under analytical conditions, both 99mTc-C2(BHam)2 and 99mTc-C3(BHam)2 still exhibited single peaks in the radiochromatograms (Fig. 4E and 4F). When 99mTc complexation reaction was performed in the presence of equimolar amounts of C2(BHam)2 and C3(BHam)2, two major radioactivity peaks, corresponding to 99mTc-C2(BHam)2 and 99m Tc-C3(BHam)2, were observed in HPLC analysis, as shown in Figure 5A. Under the experimental conditions used, radioactivity recovery from the column was over 90%. Figure 5B shows the radiochromatograms of 99mTc-BHam. When C2(BHam)2 or C3(BHam)2 was added to 99mTc-BHam, the radioactivity peaks associated with 99mTc-BHam disappeared and a new peak was observed as the major component with the Rt identical to 99m Tc-C2(BHam)2 (Fig. 5C) or 99mTc-C3(BHam)2 (Fig. 5D), respectively. However, addition of BHam to 99mTc-C2(BHam)2 or 99m Tc-C3(BHam)2 did not induce any changes in the original chromatographic profiles. No changes in radiochromatograms were

observed when C2(BHam)2 was added to 99mTc-C3(BHam)2 or vice versa. Table 2 shows the radiochemical yields of 99mTc-C2(BHam)2 or 99m Tc-C3(BHam)2 when 99mTc complexation reactions were performed in the presence of varying amounts of human IgG. Both C2(BHam)2 and C3(BHam)2 generated the 99mTc complexes over 94% radiochemical yields in the presence of equimolar amounts of human IgG. When 99mTc-C2(BHam)2 or 99mTc-C3(BHam)2 was incubated in murine plasma at 37°C, over 95% of the radioactivity was recovered in the supernatant after precipitating the plasma proteins with ethanol, as shown in Figure 6A. HPLC analyses of the supernatant indicated that more than 95% of the radioactivity was detected at the Rt identical to 99mTc-C2(BHam)2 or 99mTcC3(BHam)2, as shown in Figure 6B and Figure 6C. These HPLC profiles were very similar to those obtained in the control experiment. The percentage of the radioactivity dissociated from the three Ham-based 99mTc complexes in the presence of varying amounts of cysteine is shown in Figure 7. Although all three 99mTc complexes dissociated 99mTc with an increase in cysteine concentration, significant differences were observed among the three Ham-based 99m Tc complexes. At a cysteine concentration similar to that in the blood (5 3 1025 M) (20), both 99mTc-C2(BHam)2 and 99mTcC3(BHam)2 liberated only a small portion of 99mTc, whereas 99m Tc-BHam dissociated more than 40% of initially chelated

FIG. 3. Effect of incubation time, pH and ligand concentration on the complexation yields of 99mTc-C2(BHam)2 and 99m Tc-C3(BHam)2. 99mTc complexes were prepared at pH 7 at a ligand concentration of 5 3 1025 M (A), at a ligand concentration of 5 3 1025 M for 1 h incubation (B) and at pH 7 for 1 h incubation (C).

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FIG. 4. HPLC radiochromatograms of 99mTc-BHam (A and D), 99mTc-C2(BHam)2 (B and E) and 99mTc-C3(BHam)2 (C and F) eluted with methanol:0.05 M NH4HCO3 (A–C) and acetonitrile:0.05 M NH4HCO3 (D–E). 99m

Tc. At higher cysteine concentrations, 99mTc-C2(BHam)2 dissociated significantly higher amount of 99mTc than that observed with 99mTc-C3(BHam)2. The biodistributions of 99mTc-C2(BHam)2 and 99mTcC3(BHam)2 in normal mice were shown in Table 3. The radioactivity rapidly disappeared from the blood and high radioactivity levels were observed in the liver. Although both 99m Tc-C2(BHam)2 and 99mTc-C3(BHam)2 displayed similar radioactivity distribution profiles, 99mTc-C3(BHam)2 showed faster clearance of the radioactivity from the liver and intestine, which was reflected in the higher radioactivity levels in the intestine after administration of 99mTc-C3(BHam)2. Low radioactivity levels in the stomach were also observed following administrations of both 99mTc complexes. DISCUSSION The purpose of this study was to estimate bis(Ham) ligands as the potential thiol-free chelating molecules for 99mTc labeling of polypeptides. Among various bidentate Ham ligands, benzohydroxamamide (BHam) was selected as a model, owing to its lipophilicity, suitable to characterize by HPLC analyses. In the design of tetradentate ligand, two bis(BHam) compounds with an ethylene (C2(BHam)2) or a propylene (C3(BHam)2) carbon spacer were synthesized as prototype compounds, since some chelating reagents showed a chelate ring effect on the properties of the resulting 99mTc chelates (4, 18). In syntheses of the bis(BHam) ligands, since the NOH group of BHam is more reactive than the NH2 group, the hydroxyl group was protected by cyclization before alkylation of the amine group. We initially attempted to prepare compounds 5 and 7 by direct alkylation of compound 3 with corresponding alkylbromides. While 7 was obtained in fair yields by the reaction at room temperature, compound 5 was not obtained by this method, presumably owing to a steric interference caused by the rigid structure of compound 5. Alkylation reactions at elevated temperatures failed to produce the desired product in satisfactory yield, owing to a formation of O-alkyl side products (data not shown). The desired compound 5 was obtained after isolating the intermediate compound 4, followed by reaction with an equimo-

lar amount of compound 3. Similarly, 7 was prepared after isolation of 6, followed by the reaction with 3. Decyclization of compounds 5 and 7 under the controlled alkaline conditions provided C2(BHam)2 and C3(BHam)2 in moderate yields. Since the present synthetic procedure involved the isolation of monoalkylated Ham intermediates, this method would be applicable to prepare a variety of bis(Ham) derivatives with not only symmetrical structures, but also those with asymmetrical structures. Such synthetic procedures would allow versatile molecular design of a variety of bioconjugates with bis(Ham) chelating groups. In addition, high stability of the final compounds, C2(BHam)2 and C3(BHam)2, as well as their intermediates during long-term storage (over 3 months) at room temperature, makes these compounds easy to handle. To estimate the ability of C2(BHam)2 and C3(BHam)2 as the chelating molecules for 99mTc-labeled polypeptides, 99mTc complexation reactions were investigated with varying ligand concentrations and in the presence of IgG in which nonspecific binding of reduced 99mTc species was observed (3, 13, 21). Both C2(BHam)2 and C3(BHam)2 generated 99mTc complexes over a wide pH range under mild reaction conditions within short reaction times. High complexation yields (over 95%) were achieved at ligand concentrations as low as 2.5 3 1026 M (Figs. 3 and 4). The abilities of C2(BHam)2 and C3(BHam)2 to form 99m Tc chelates with good reaction kinetics were also demonstrated by the 99mTc complexation reactions in the presence of varying amounts of human IgG. More than 95% of the 99mTc complex of C2(BHam)2 or C3(BHam)2 were obtained when 99m Tc labeling of each ligand was performed in the presence of an equimolar amount of IgG (Table 2). To characterize the 99m Tc complexes of C2(BHam)2 and C3(BHam)2, 99mTc complexation reaction was performed in the presence of equimolar amounts of both C2(BHam)2 and C3(BHam)2. Selective formation of 99mTc-C2(BHam)2 and 99mTc-C3(BHam)2 was demonstrated without generating any additional 99mTc species composed of the two ligands (Fig. 5A). On the other hand, prior study indicated that 99mTc complexation reaction in the presence of equimolar amounts of two bidentate Ham ligands showed mixed ligand 99mTc complexes along with 99mTc complexes corresponding to each ligand (25). These findings suggested that

Hydroxamamide-Based Tetradentate Ligands

301

TABLE 2. 99mTc Labeling of C2(BHam)2 and C3(BHam)2 in the Presence of IgG

Ligand:IgGa molar ratio 10:1 5:1 1:1 a b

Complex Yieldb (%)

Ligand Concentration (M)

99m TcC2(BHam)2

99m TcC3(BHam)2

2.5 3 1024 1.25 3 1024 2.5 3 1025

97 (95–98) 97 (96–98) 95 (94–96)

97 (96–98) 96 (94–97) 96 (95–97)

Final concentration of IgG was 2.5 3 1025 M in 10 mM PBS (pH 7.4). Means of duplicate experiments (ranges in parenthesis).

implied that selective complexation of 99mTc to C2(BHam)2 or C3(BHam)2 would be achieved when these ligands are covalently attached to polypeptides at a molar ratio of 1:1. To further estimate C2(BHam)2 and C3(BHam)2 as the chelating

FIG. 5. HPLC radiochromatograms of 99mTc complexes prepared in the presence of equimolar amount of C2(BHam)2 and C3(BHam)2 (A), 99mTc-BHam (B), the reaction mixtures of 99mTc-BHam and C2(BHam)2 after 6 h incubation at room temperature (C) and the mixtures of 99mTc-BHam and C3(BHam)2 after 6 h incubation at room temperature (D). Acetonitrile:0.05 M NH4HCO3 (30:70 [v/v]) was used as the eluent. 99m

both C2(BHam)2 and C3(BHam)2 would form Tc complexes at the ligand-to-99mTc ratios of 1:1 (Fig. 5A), although further structural studies using 99Tc or rhenium are required to estimate the chemical structures of the resulting 99mTc complexes. The exchange reactions between 99mTc-BHam with C2(BHam)2 or C3(BHam)2 indicated that both C2(BHam)2 and C3(BHam)2 provided 99mTc complexes with stabilities much higher than 99m Tc-BHam (Fig. 5C and 5D). The gathered findings strongly

FIG. 6. Stability of 99mTc-C2(BHam)2 and 99mTc-C3(BHam)2 in murine plasma. (A) Percentage radioactivity remaining in the supernatant after incubation of 99mTc-C2(BHam)2 and 99mTcC3(BHam)2 in murine plasma at 37&C, followed by the precipitation of the plasma proteins by ethanol. Each bar represents the means 6 SD of three determinations. (B) HPLC radiochromatograms of 99mTc-C2(BHam)2 after incubation in murine plasma for 3 h at 37&C. (C) HPLC radiochromatograms of 99m Tc-C3(BHam)2 after incubation in murine plasma for 3 h at 37&C.

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302

FIG. 7. Percentage of 99mTc dissociated from 99mTc-BHam, 99m Tc-C2(BHam)2 and 99mTc-C3(BHam)2 after incubation in varying amount of cysteine for 1 h. molecules for polypeptides, we estimated the stability of the resulting 99mTc complexes in freshly prepared murine plasma or in the presence of varying amounts of cysteine. The unchanged radiochromatograms of both 99mTc-C2(BHam)2 and 99mTcC3(BHam)2 after incubation in freshly prepared murine plasma indicated that both C2(BHam)2 and C3(BHam)2 would form 99m Tc complexes with stabilities sufficient for in vivo applications (Fig. 6). The superiority of the tetradentate ligands, C2(BHam)2 and C3(BHam)2, to the bidentate ligand, BHam, in preparing stable 99mTc complexes was reconfirmed by incubating the three 99mTc complexes in the presence of varying amounts of cysteine (Fig. 7). These studies also indicated different stabilities between 99mTc-C2(BHam)2 and 99mTc-C3(BHam)2. While both C2(BHam)2 and C3(BHam)2 provided 99mTc complexes stable at the cysteine concentration similar to that in plasma (5 3 1025 M), the former released more 99mTc species with an increase in the cysteine concentration, indicating that C3(BHam)2 would serve as more suitable chelating molecules. Thus, C3(BHam)2 would provide more favorable geometry for 99mTc coordination than C2(BHam)2, as have been documented with tetradentate ligands of double-bond structures (4, 18). If speculation may be allowed, previous studies (16, 25) together with the present findings sug-

gested that the two secondary amine groups and the two oxygen groups in the C3(BHam)2 might participate in the 99mTcC3(BHam)2 complex. In biodistribution studies, both 99mTc-C2(BHam)2 and 99mTcC3(BHam)2 showed low radioactivity levels in the stomach and rapid disappearance of the radioactivity from the liver to intestine, indicating that in vivo reoxidation to pertechnetate or colloidal formation would be negligible. The rapid clearance of the radioactivity from the blood would reflect low binding of the two 99mTc complexes to serum proteins, as also demonstrated by the in vitro serum incubation studies. Such characteristics render bis(BHam) compounds attractive as the basic 99mTc chelating molecule of the bifunctional chelating agents with metabolizable linkages. In light of recent studies of radioiodination reagents for protein radiopharmaceuticals, radiolabeling reagents that generate radiometabolites of urinary excretion would be preferable to reduce hepatic radioactivity levels of protein radiopharmaceuticals (5, 31). In a previous study, while 99mTc-BHam was mainly excreted from the liver, 99mTc-pyridine-4-Ham was excreted by urinary excretion (23). In addition, 99mTc-C2(BHam)2 and 99m Tc-C3(BHam)2 displayed radioactivity distribution similar to that of 99mTc-BHam (22). Thus, substitutions of benzyl groups of C3(BHam)2 to hydrophilic groups such as pyridine would alter in vivo behaviors of the resulting 99mTc complexes, and such chemical modifications would provide tetradentate bis(Ham) molecules more favorable for the application to 99mTc-labeled polypeptides. In conclusion, Ham-based tetradentate ligands were designed and a standard procedure for their syntheses was developed. Both C2(BHam)2 and C3(BHam)2 demonstrated high complexation yields with 99mTc under mild reaction conditions at low ligand concentrations. Selective formation of 99mTc complexes were also observed even in the presence of an equimolar amount of IgG. High stabilities of the two 99mTc complexes with low protein binding characteristics were also observed. Although further structural studies are required, these findings indicated that the Ham-based tetradentate ligands, especially C3(BHam)2 derivatives, would serve as a new class of thiol-free chelating molecules for preparation of a variety of 99mTc radiopharmaceuticals for radioimmunoimagining and receptor imaging.

TABLE 3. Biodistribution of Radioactivity after Administration of Mice

99m

Tc-C2(BHam)2 and

99m

Tc-C3(BHam)2 in Normal

Time after Administration Organ

5 min

30 min

120 min

5 min 99m

99m

Tc-C2(BHam)2

a

Blood Spleen Pancreas Kidney Liver Stomach Intestine Lung Heart

5.72 (0.86) 0.19 (0.06) 0.14 (0.03) 2.03 (0.47) 41.35 (5.60) 0.09 (0.15) 18.02 (5.69) 0.89 (0.18) 0.21 (0.07)

0.54 (0.07) 0.18 (0.06) 0.04 (0.01) 0.72 (0.09) 16.87 (2.04) 0.34 (0.12) 51.31 (9.33) 0.16 (0.04) 0.04 (0.01)

30 min

0.29 (0.03) 0.11 (0.03) 0.02 (0.01) 0.43 (0.11) 10.21 (1.01) 0.84 (0.11) 59.08 (6.65) 0.12 (0.04) 0.02 (0.01)

3.49 (0.57) 0.11 (0.02) 0.13 (0.02) 2.16 (0.31) 44.43 (4.21) 0.61 (0.22) 17.45 (3.04) 0.18 (0.05) 0.58 (0.19)

Expressed as percent injected dose per organ. Mean (SD) of five animals for each point. a Expressed as percent injected dose per gram.

120 min

Tc-C3(BHam)2 0.41 (0.05) 0.04 (0.01) 0.02 (0.01) 0.29 (0.03) 6.73 (1.33) 0.27 (0.12) 60.59 (3.82) 0.02 (0.00) 0.12 (0.03)

0.30 (0.03) 0.04 (0.01) 0.01 (0.00) 0.20 (0.05) 4.16 (0.88) 0.49 (0.06) 80.43 (10.18) 0.02 (0.01 0.07 (0.01)

Hydroxamamide-Based Tetradentate Ligands

This study was supported in part by a Grant-in-Aid for Developing Scientific Research (numbers 08557135, 09672190 and 09672279) from the Ministry of Education, Science, Sports and Culture of Japan.

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