99mTc-Labeled MIBG Derivatives: Novel 99mTc Complexes as

Bioconjugate Chem. 1999, 10, 159−168

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ARTICLES 99mTc-Labeled

MIBG Derivatives: Novel 99mTc Complexes as Myocardial Imaging Agents for Sympathetic Neurons

Zhi-Ping Zhuang,† Mei-Ping Kung,† Mu Mu,† Catherine Hou,† and Hank F. Kung*,†,‡ Departments of Radiology and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Received November 29, 1997; Revised Manuscript Received October 1, 1998

Radioactive-iodine-labeled meta-iodobenzylguanidine (MIBG) is currently being used as an in vivo imaging agent to evaluate neuroendocrine tumors as well as the myocardial sympathetic nervous system in patients with myocardial infarct and cardiomyopathy. It is generally accepted that MIBG is an analogue of norepinephrine and its uptake in the heart corresponds to the distribution of norepinephrine and the density of sympathetic neurons. A series of MIBG derivatives containing suitable chelating functional groups N2S2 for the formation of [TcvO]3+N2S2 complex was successfully synthesized, and the 99mTc-labeled complexes were prepared and tested in rats. One of the compounds, [99mTc]2, tested showed significant, albeit lower, heart uptakes post iv injection in rats (0.21% dose/g at 4 h) as compared to [125I]MIBG (1.7% dose/g at 4 h). The heart uptake of the 99mTc-labeled complex appears to be specific and can be reduced by co-injection with nonradioactive MIBG or by pretreatment with desipramine, a selective norepinephrine transporter inhibitor. Further evaluation of the in vitro uptake of [99mTc]2 in cultured neuroblastoma cells displayed consistently lower, but measurable uptake (approximately 10% of that for [125I]MIBG). These preliminary results suggested that the mechanisms of heart uptake of [99mTc]2 may be related to those for [125I]MIBG uptake. If suitable 99mTc-labeled MIBG derivatives can be further developed, the prevalent availability of 99mTc in nuclear medicine clinics will allow them to be readily available for widespread application.

INTRODUCTION

In the past 15 years meta-iodobenzylguanidine (MIBG,1 Figure 1) has attracted widespread acceptance as an imaging agent for studying norepinephrine neuronal function. Initially, radioiodine-labeled MIBG was developed as a norepinephrine analogue which localized in the adrenal medulla (1). The molecule was designed as a congener of an antihypertensive drug, guanethidine, whose pharmacological action is directly related to inhibition of the release of norepinephrine from adrenergic nerve endings. Using cloned cell lines (CV-1, green monkey kidney cells, and HeLa cells) expressing specific * Address correspondence to this author at the Department of Radiology, University of Pennsylvania, 3700 Market St., Room 305, Philadelphia, PA 19104. Telephone: (215) 662-3096. Fax: (215) 349-5035. E-mail: [email protected]. † Department of Radiology. ‡ Department of Pharmacology. 1 Abbreviations: MIBG, meta-iodobenzylguanidine; DMI, desipramine; ECD, ethylene cysteine dimer; TRODAT-1, [2-[[2[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]amino]ethanethiolato(3-)N2,N2′,S2,S2′]oxo-[1R-(exo-exo)]; LAH, lithium aluminum hydride; THF, tetrahydrofuran; Boc, tert-butoxycarbonyl; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; NET, norepinephrine transporter; DAT, dopamine transporter; TMS, tetramethylsilane; DCC, 1,3-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; PTLC, preparative thinlayer chromatography; MPLC, medium-performance liquid chromatography; EDTA, ethylenediaminetetraacetic acid disodium salt; PC, partition coefficient.

Figure 1. Chemical structures of norepinephrine, guanethidine, and meta-iodobenzylguanidine (MIBG).

dopamine, norepinephrine, and serotonin transporters, MIBG uptake was reported to be mediated specifically by the norepinephrine transporter with a Km value of 264 nM. The specific uptake was blocked by desipramine (DMI), a specific norepinephrine transporter inhibitor, and unaffected by dopamine and serotonin transporter inhibitors (2). As an analogue of norepinephrine, [131I]MIBG localizes in the tumors and provides a useful diagnostic tool of neuronal storage of norepinephrine (1, 3). It was later discovered that [131I]MIBG is useful as an agent for neuroendocrine tumor (neuroblastomas and pheochromocytomas) imaging and therapy (4). One other important observation of using MIBG was that it also localized in myocardium (5). It was systematically established by the research group at the University of Michigan and others that the pharmacokinetics of MIBG are indeed related to norepinephrine uptake and release (6-9). Recent studies in denervated dog heart strongly suggested that the localization of MIBG reflected the integrity of adrenergic neurons. The cardiac scan at time points later than 1 h after injection of [123I]MIBG provides a closed resemblance of the distribution of the sympathetic nerve endings (9-11). It was suggested that

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assessment of the sympathetic nervous system by MIBG imaging may be important for patients with ventricular tachycardia, cardiomyopathy, and arrhythmia (12). Thus, MIBG scan may have predictable value for patients at risk of sudden death (12-15) and patients with diabetes (16, 17). However, due to the limited supply of [123I]MIBG, large-scale clinical studies are not readily achievable (12). Due to the popularity of 99mTc routinely used in the clinical community, there is a strong need to develop 99m Tc-based compounds, which can be used on a routine basis. Our successful development of [99mTc]TRODAT-1, a SPECT imaging agent targeting dopamine transporters (DAT) in humans (18), opened up a new avenue. The similar strategy aimed at developing 99mTc-labeled agents for other specific binding sites can be applied, by which they may provide important clinical applications. The [TcvO]3+N2S2 complex was chosen for the initial attempt to prepare 99mTc-labeled MIBG derivatives because this complex has been successfully applied in the development of a brain perfusion imaging agent, [99mTc]ECD (ethylene cysteine dimer) (19-21), as well as for the DAT imaging agent, [99mTc]TRODAT-1 (22). Other 99mTc complexes may be possible, but in this initial report we are going to concentrate on the [TcvO]3+N2S2 complexes. If 99mTc-labeled MIBG derivatives can be successfully developed based on the same [TcvO]3+N2S2 complex, the prevalent availability of 99mTc in nuclear medicine clinics will permit a widespread clinical application on patients with myocardial disease or various endocrine tumors. We report herein the initial effort in preparing novel 99mTclabeled MIBG derivatives, which may potentially be valuable for routine clinical imaging study of the sympathetic nervous system of the heart. EXPERIMENTAL PROCEDURES

General. Reagents used in the syntheses were purchased from Aldrich (Milwaukee, WI) or Fluka (Ronkonkoma, NY), and were used without further purification unless otherwise indicated. Anhydrous Na2SO4 was used as a drying agent. Reaction yields are reported without attempts at optimization. 1H NMR spectra were obtained on a Bruker spectrometer (Bruker AC 200; 200 MHz). Chemical shifts are reported as δ values with chloroform or TMS as the internal reference. Coupling constants are reported in hertz. The multiplicity is defined by s (singlet), d (doublet), t (triplet), br (broad), and m (multiplet). Elemental analyses were performed by Elemental Analysis Laboratory, University of Pennsylvania. Mass spectrometry was performed by the Mass Spectrometry Center, University of Pennsylvania. N-3-Cyanophenyl-2-(4′-methoxybenzyl)mercaptoacetamide (5). A mixture of 3-aminobenzonitrile (1.18 g, 10 mmol), 2-(4-methoxybenzyl)mercaptoacetic acid (4, 2.12 g, 1 equiv), and DCC (2.18 g, 1 equiv) in CH2Cl2 (30 mL) was stirred under reflux overnight. After the mixture was cooled to room temperature, water was added and extracted with CH2Cl2. The organic extracts was dried and concentrated to give the crude product, which was purified by medium-pressure liquid chromatography (MPLC) (Hex:EtOAc ) 5:1) to give 2.17 g of product (70%). 1 H NMR: 3.31 (2H, s, Ar-CH2-S-), 3.70 (3H, s, OCH3), 3.74 (2H, CO-CH2-S-), 6.78 (2H, dot, J ) 8.6, 2.5 Hz, ArH), 7.18 (2H, d,t, J ) 8.6, 2.5 Hz, ArH), 7.35-7.40 (2H, m, ArH), 7.54-7.63 (1H, m, ArH), 7.78 (1H, m, ArH), 8.4 (1H, br, CONH). Anal. (C17H16N2O2S ): Calcd: C, 65.36; H, 5.16; N, 8.97. Found: C, 65.02; H, 5.08; N, 8.94.

Zhuang et al.

3-Aminomethylphenyl-2-(4′-methoxybenzyl)mercaptoethylamine (6). To a suspension of LAH (0.72 g, 18.9 mmol) in THF (15 mL) was added dropwise a solution of 5 (1.19 g, 3.8 mmol) in THF (15 mL) at room temperature. The mixture was stirred under reflux for 1 h and stirred at room temperature overnight. Water (0.9 mL), NaOH (1 M, 0.9 mL), and additional water (2.7 mL) were added successively with caution to decompose excess LAH. The resulting mixture was stirred at room temperature for another 10 min, and the mixture was filtered through Celite. The filtrate was concentrated and purified by preparative thin-layer chromatography (PTLC) (CH2Cl2:MeOH:NH4OH ) 9:1:0.1) to give 633 mg of oil (55%). 1 H NMR: 2.68 (2H, t, J ) 6.4 Hz, NHCH2), 3.27 (2H, t, J ) 6.4 Hz, S-CH2), 3.68 (2H, s, Ar-CH2-S), 3.77 (2H, s, Ar-CH2-NH2), 3.79 (3H, s, OCH3), 6.47 (1H, d,d, J ) 7.8, 2.0 Hz, ArH), 6.54 (1H, s, ArH), 6.65 (1H, d, J ) 7.5 Hz, ArH), 6.84 (2H, d,t, J ) 8.6, 2.8 Hz, ArH), 7.12 (1H, t, J ) 7.7 Hz, ArH), 7.22 (2H, d,t, J ) 8.6, 2.1 Hz, ArH). Anal. (C17H22N2OS‚0.25H2O ): Calcd: C, 66.52; H, 7.39; N, 9.13. Found: C, 66.70; H, 7.38; N, 8.98. 3-(N′-tert-Butoxycarbonyl)aminomethylphenyl-2(4′-methoxybenzyl)mercaptoethylamine (7). To a solution of 6 (100 mg, 0.33 mmol) and Et3N (0.1 mL) in CH2Cl2 (5 mL) was added dropwise a solution of (Boc)2O (72 mg, 1 equiv) in CH2Cl2 (2 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h. Water was added, and the reaction mixture was extracted with CH2Cl2. The combined organic extract was dried, concentrated, and purified by PTLC (Hex:EtOAc ) 3:2) to give 107 mg of product (80%). 1 H NMR: 1.47 (9H, s, Boc-H), 2.67 (2H, t, J ) 6.4 Hz, NHCH2), 3.21 (2H, t, J ) 6.4 Hz, S-CH2), 3.68 (2H, s, Ar-CH2-S), 3.79 (3H, s, OCH3), 4.22 (2H, d, J ) 5.8 Hz, Ar-CH2-NH2), 6.47 (1H, d, J ) 6.7 Hz, ArH), 6.49 (1H, s, ArH), 6.62 (1H, d, J ) 7.5 Hz, ArH), 6.85 (2H, d,t, J ) 8.6, 2.8 Hz, S-ArH), 7.08 (1H, t, J ) 7.6 Hz, ArH), 7.22 (2H, d,t, J ) 8.6, 2.0 Hz, ArH). Anal. (C22H30N2O3S ): Calcd: C, 65.64; H, 7.50; N, 6.96. Found: C, 65.46; H, 7.57; N, 6.74. N-3-(N′-tert-Butoxycarbonyl)aminomethylphenylN-2-(4′-methoxybenzyl)mercaptoethyl-2-chloroacetamide (8). To a solution of 7 (100 mg, 0.25 mmol) and Et3N (0.1 mL) in CH2Cl2 (5 mL) was added a solution of chloroacetyl chloride (34 mg, 1.2 equiv) in CH2Cl2 (1 mL) at 0 °C in an ice bath. The mixture was stirred at room temperature for 1 h. Water was added, and the reaction mixture was extracted with CH2Cl2. The combined extracts were dried, concentrated, and purified by PTLC (Hex:EtOAc ) 1:1) to give 116 mg of product (97%). 1H NMR: 1.45 (9H, s, Boc-H), 2.53 (2H, t, J ) 7.6 Hz, NHCH2), 3.69 (2H, s, Ar-CH2-S), 3.81 (3H, s, OCH3), 3.78-3.85 (4H, m, S-CH2, COCH2Cl), 4.32 (2H, d, J ) 6.2 Hz, Ar-CH2-NH2), 6.78 (2H, d,t, J ) 8.6, 2.8 Hz, ArH), 7.07-7.72 (6H, m, ArH). Anal. (C24H31ClN2O4S‚0.5H2O): Calcd: C, 59.07; H, 6.61; N, 7.26. Found: C, 59.33; H, 6.54; N, 7.13. N-3-(N′-tert-Butoxycarbonyl)aminomethylphenylN-2-(4′-methoxybenzyl)mercaptoethyl-2-(4-methoxybenzyl)mercaptoethylaminoacetamide (10). A mixture of 8 (390 mg, 0.81 mmol), 2-(4-methoxybenzyl)mercaptoethylamine 9 (320 mg, 2 equiv), KI (50 mg, 0.3 mmol), and K2CO3 (500 mg, 4.5 equiv) in DMF (10 mL) was stirred at room temperature overnight. Water was added and the reaction mixture was extracted with a mixed solvent (CH2Cl2:MeOH ) 9:1). The combined extract was dried, condensed, and purified by PTLC (CH2Cl2:MeOH ) 95:5) to give 387 mg of product (74%)

99mTc-Labeled 1H

MIBG Derivatives as Myocardial Imaging Agents

NMR: 1.45 (9H, s, Boc-H), 2.44-2.67 (6H, m, NHCH2-, SCH2-), 3.02 (2H, s, CO-CH2-NH), 3.64 (2H, s, Ar-CH2-S), 3.65 (2H, s, Ar-CH2-S), 3.77 (6H, s, OCH3), 3.70-3.83 (2H, m, CONCH2-), 4.30 (2H, d, J ) 5.9 Hz, ArCH2NHBoc), 6.74-6.86 (4H, m,ArH), 6.98-7.39 (8H, m, ArH). Anal. (C34H45N3O5S2‚2H2O): Calcd: C, 60.42; H, 7.30; N, 6.21. Found: C, 60.89; H, 7.02; N, 5.72. N-3-Aminomethylphenyl-N-2-(4′-methoxybenzyl)mercaptoethyl-2-(4-methoxybenzyl)mercaptoethylaminoacetamide (11). Compound 10 (387 mg, 0.6 mmol) was dissolved in HCl-EtOAc (10 mL, 3 M), and the mixture was stirred at room temperature for 1 h. The solvent was removed, and the residue was dissolved in MeOH (1 mL). Water (5 mL) was added, and the solution was adjusted to pH 10 with concentrated NH4OH. The resulting mixture was extracted with a mixed solvent (CH2Cl2:MeOH ) 9:1). The combined extracts were dried, evaporated, and purified by PTLC (CH2Cl2:MeOH: NH4OH ) 9:1:0.2) to give 288 mg of product (88%). 1 H NMR: 2.42-2.67 (6H, m, NHCH2-, SCH2-), 3.03 (2H, s, Ar-CH2-NH2), 3.63 (2H, s, Ar-CH2-S), 3.65 (2H, s, Ar-CH2-S), 3.75 (6H, s, OCH3), 3.79-3.83 (2H, m, CONCH2-), 3.86 (2H, s, COCH2N-), 6.76 (2H, d,t, J ) 8.6, 2.9 Hz, ArH), 6.80 (2H, d,t, J ) 8.6, 2.9 Hz, ArH-), 6.957.38 (8H, m, ArH). Anal. (C29H37N3O3S2‚1.5H2O): Calcd: C, 61.40; H, 7.11; N, 7.41. Found: C, 61.59; H, 6.96; N, 7.17. N2-3-{N-2-(4-Methoxy)benzylmercaptoethyl-N-2[N′-2-(4-methoxy)benzylmercaptoethyl]aminoethyl}aminobenzyl-N1,N3-di-tert-butoxycarbonylguanidine (12). To a suspension of LAH (28 mg, 0.73 mmol) in THF (3 mL) was added a solution of 11 (100 mg, 0.19 mmol) in THF (3 mL) dropwise at room temperature. The reaction mixture was stirred under reflux for 1 h after which one 1 drop of H2O, 1 drop of NaOH (1 M), and 3 drops of H2O were added successively to decompose the excess LAH. The resulting mixture was filtered, and the filtrate was concentrated to give 100 mg of crude product, 3-{N-2-(4-methoxy)benzylmercaptoethyl-N-2-[N′-2(4-methoxy)benzylmercaptoethyl]aminoethyl}aminobenzylamine, which is pure enough for the next reaction without further purification. 1 H NMR: 2.53-2.76 (8H, m, S-CH2-, NCH2-), 3.65 (2H, s, Ar CH2-S-), 3.68 (2H, m, Ar-CH2-NH2), 3.71 (2H, s, Ar-CH2-S-), 3.78 (3H, s, OCH3), 3.79 (3H, s, OCH3), 6.49 (1H, d,d, J ) 8.2, 2.3 Hz, ArH), 6.62 (2H, m, ArH), 6.80-6.87 (4H, m, ArH), 7.11-7.30 (5H, m, ArH). A mixture of the amine obtained above (260 mg, 0.50 mmol) and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (156 mg, 1 equiv) in THF (10 mL containing 0.2 mL of water) was stirred at 65 °C for 3 h. The solvent was removed, and the residue was purified by PTLC (CH2Cl2:CH3OH ) 93:7) to give 124 mg of product, 12 (33%). 1H NMR: 1.46 (9H, s, Boc-H), 1.51 (9H, s, Boc-H), 2.52-2.80 (8H, m, -CH2-), 3.32-3.46 (4H, m, -CH2-), 3.60 (2H, s, Ar-CH2-S-), 3.67 (2H, s, Ar-CH2-S-), 3.77 (3H, s, OCH3), 3.78 (3H, s, OCH3), 4.52 (2H, d, J ) 4.9 Hz, Ar-CH2-NH-), 6.50 (1H, d,d, J ) 8.1, 2.3 Hz, ArH), 6.586.65 (2H, m, ArH), 6.79-6.96 (4H, m, ArH), 7.04-7.32 (5H, m, ArH), 8.55 (1H, t, J ) 4.5 Hz, Ar-CH2-NH-), 11.52 (1H, s, NHBoc). Anal. (C40H57N5O6S2): Calcd: C, 62.55; H, 7.48; N, 9.32. Found: C, 62.54; H, 6.96; N, 9.12. N2-3-[N-2-Mercaptoethyl-N-2-(N′-2-mercaptoethyl)aminoethyl]aminobenzylguanidine (13). Compound 12 (124 mg, 0.16 mmol) in TFA (5 mL) was stirred at room temperature for 30 min, and the mixture was

Bioconjugate Chem., Vol. 10, No. 2, 1999 161

cooled to 0 °C in an ice bath. Anisole (2 drops) was added followed by Hg(OAc)2 (123 mg, 1.2 equiv). The mixture was stirred at 0 °C for 1 h. Solvent was removed by vacuum, and ether was added. The solid, which was produced after adding the ether, was collected by filtration and washed with ether to give 180 mg of white solid which was dissolved in a mixed solvent (20 mL, EtOAc: EtOH ) 1:1) to which H2S gas was bubbled through for 10 min. The mixture was filtered through Celite and washed with MeOH. The filtrate was condensed to give an oil, which was washed with CHCl3 3 times to give 66 mg of 13 (73%). 1 H NMR: 2.68 (2H, t, J ) 6.9 Hz, -CH2-), 2.82 (2H, t, J ) 6.1 Hz, -CH2-), 3.24 (4H, t, J ) 6.9 Hz, -CH2-), 3.57 (2H, t, J ) 7.0 Hz, -CH2-), 3.74 (2H, t, J ) 7.0 Hz, -CH2-), 4.35 (2H, s, -Ar-CH2-N-), 6.71-6.81 (3H, ArH), 7.21-7.30 (1H, m, ArH). HRMS calcd(C14H25N5S2): m/z 328.1629 (M++1); Found: m/z 328.1613 (M++1). Anal. (C14H25N5S2‚5CF3COOH): Calcd: C, 32.11; H, 3.37; N, 7.82. Found: C, 32.59; H, 3.53; N, 8.12. N-3-Chloromethylbenzylphthalimide (14). To a solution of R,R′-m-dichloroxylene (1.3 g, 7.4 mmol) in DMF (4 mL) was added potassium phthalimide (925 mg, 5 mmol) in solid form. The reaction mixture was heated to 70 °C under stirring for 2 h. The reaction mixture was cooled to room temperature, and DMF was removed under vacuum. The residue was purified by MPLC (hexane:EtOAc ) 2:1) to give 1.01 g of white solid (70%). 1H NMR: 4.55 (2H, s, ClCH -Ar), 4.84 (2H, s, Ar2 CH2-N-), 7.26-7.44 (4H, m, ArH), 7.67-7.75 (2H, m, ArH), 7.83-7.89 (2H, m, ArH). Anal. (C16H12ClNO2): Calcd: C, 67.26; H, 4.23; N, 4.90. Found: C, 67.47; H, 4.09; N, 4.91. N-3-{N′-2-(4-Methoxy)benzylmercaptoethyl-N′-2[N′′-2-(4-methoxy)benzylmercaptoethyl]aminoethyl}aminomethylbenzylphthalimide (17a). The mixture of 14 (286 mg, 1 mmol), amine 16a (420 mg, 1 equiv), K2CO3 (552 mg, 4 equiv), and KI (50 mg, 0.3 mmol) in DMF (5 mL) was stirred at room temperature overnight. Water was added, and the mixture was extracted with a mixed solvent (CH2Cl2:MeOH ) 9:1). The combined organic extract was dried over Na2SO4 and filtered. The filtrate was evaporated under vacuum to give the crude product, which was purified by PTLC (EtOAc) to give 330 mg of product, 17a (49.5%). 1H NMR: 2.33-2.74 (12H, m, -CH -), 3.52 (2H, s, 2 Ar-CH2-S), 3.53 (2H, s, Ar-CH2-S), 3.64 (2H, s, ArCH2-N-), 3.76 (6H, s, OCH3), 4.81 (2H, s, Ar-CH2-NCO-), 6.76-6.83 (4H, m, ArH), 6.99-7.32 (8H, m, ArH), 7.56-7.71 (2H, m, ArH), 7.77-7.86 (2H, m, ArH). MS: m/z 670 (M++1). Anal. (C38H43N3O4S2‚2H2O): Calcd: C, 64.65; H, 6.71; N, 5.95. Found: C, 64.90; H, 6.37; N, 5.96. N-3-{N′-2,2-Dimethyl-2-(4-methoxy)benzylmercaptoethyl-N′-2-[N′′-2,2-dimethyl-2-(4-methoxy)benzylmercaptoethyl]aminoethyl}aminomethylbenzylphthalimide (17b). The same procedure for preparation of 17a was employed using 14 (286 mg, 1 mmol), 16b (476 mg, 1 equiv), K2CO3 (552 mg, 4 equiv), and KI (100 mg, 0.6 mmol) in DMF (5 mL) to give 476 mg of product, 17b (66%). 1 H NMR: 1.29 (12H, s, -CH3), 2.50 (2H, s, -CH2-), 2.61 (4H, s, -CH2-), 2.66 (2H, s, -CH2-), 3.63 (2H, s, ArCH2S-), 3.67 (2H, s, Ar-CH2S-), 3.71 (2H, s, Ar-CH2-N-), 3.75 (3H, s, OCH3), 3.76 (3H, s, OCH3), 4.81 (2H, s, ArCH2-NCO), 6.79 (4H, d,d, J ) 8.6, 0.8 Hz, ArH), 7.19 (4H, d,d, J ) 8.6, 1.7 Hz, ArH), 7.27-7.34 (3H, m, ArH),


7.40 (1H, br, ArH), 7.68 (2H, d,d, J ) 5.4, 3.1 Hz, ArH), 7.81 (2H, d,d, J ) 5.6, 3.1 Hz, ArH). Anal. (C42H51N3O4S2‚0.5H2O): Calcd: C, 68.63; H, 7.13; N, 5.80. Found: C, 68.49; H, 6.93; N, 5.45. 3-{N-2-(4-Methoxy)benzylmercaptoethyl-N-2-[N′2-(4-methoxy)benzylmercaptoethyl]aminoethyl}aminomethylbenzylamine (18a). To a solution of 17a (510 mg, 0.76 mmol) in MeOH (10 mL) was added hydrazine anhydrate (0.3 mL) at room temperature. The mixture was stirred at room temperature overnight. The precipitate was removed by filtration, and the filtrate was concentrated to give 325 mg of white solid (79%), which was pure enough for the next reaction without further purification. 1 H NMR: 2.40-2.64 (12H, m, -CH2-), 3.52 (2H, Ar-CH2-), 3.55 (2H, Ar-CH2-), 3.62 (2H, Ar-CH2-), 3.73 (6H, s, OCH3-), 3.79 (2H, Ar-CH2-), 6.79 (4H, d,d, J ) 8.6, 3.0 Hz, ArH), 7.10-7.27 (8H, m, ArH). MS: m/z 540 (M++1). Anal. (C30H41N3O2S2‚0.5H2O): Calcd: C, 65.66; H, 7.71; N, 7.66. Found: C, 65.53; H, 7.61; N, 7.51. 3-{N-2,2-Dimethyl-2-(4-methoxy)benzylmercaptoethyl-N-2-[N′-2,2-dimethyl-2-(4-methoxy)benzylmercaptoethyl]aminoethyl}aminomethylbenzylamine (18b). The same procedure for preparation of 18a was employed with 17b (476 mg, 0.66 mmol) and hydrazine (0.2 mL) in MeOH (15 mL) to give 335 mg of product, 18b (86%). 1H NMR: 1.23 (6H, s, -CH ), 1.32 (6H, s, -CH ), 2.42 3 3 (2H, s, -CH2-), 2.51-2.59 (4H, m, -CH2-), 2.64 (2H, s, -CH2-), 3.59 (2H, Ar-CH2-), 3.68 (2H, Ar-CH2-), 3.70, 3.71 (10H, s, OCH3-, Ar-CH2-), 6.78 (4H, d,d, J ) 8.7, 2.2 Hz, ArH), 7.14-7.32 (8H, m, ArH). Anal. (C34H49N3O2S2‚0.5H2O): Calcd: C, 67.51; H, 8.33; N, 6.61. Found: C, 67.28; H, 8.22; N, 6.76. N2-3-{N-2-(4-Methoxy)benzylmercaptoethyl-N-2[N′-2-(4-methoxy)benzylmercaptoethyl]aminoethyl}aminomethylbenzyl-N 1 ,N 3 -di-tert-butoxycarbonylguanidine (19a). To a mixture of 18a (100 mg, 0.19 mmol) and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (54 mg, 1 equiv) in THF (3 mL) was added 6 drops of water, and the mixture was stirred at 65 °C for 3 h. The solvent was removed, and the residue was purified by PTLC (CH2Cl2:CH3OH ) 9:1) to give 94 mg of product, 19a (65%). 1H NMR: 1.46 (9H, s, tert-CH ), 1.51 (9H, s, tert-CH ), 3 3 2.44-2.72 (12H, m, CH2), 3.56 (2H, s, Ar-CH2-N-), 3.60 (2H, s, Ar-CH2-S-), 3.64 (2H, s, Ar-CH2-S-), 3.77 (6H, s, OCH3), 4.60 (2H, d, J ) 5.1 Hz, Ar-CH2-NH-), 6.81 (4H, d,d, J ) 8.6, 3.2 Hz, CH3O-ArH), 7.13-7.32 (8H, m, ArH), 8.54 (1H, t, J ) 4.7 Hz, Ar-CH2-NH-), 11.53 (1H, s, NHBoc). HRMS calcd(C41H59N5O6S2): m/z 782.3915 (M++1); found: m/z 782.3937 (M+ + 1). Anal. (C41H59N5O6S2‚1.5H2O): Calcd: C, 60.86; H, 7.72; N, 8.66. Found: C, 60.56; H, 6.53; N, 9.08. N2-3-{N-2,2-Dimethyl-2-(4-methoxy)benzylmercaptoethyl-N-2-[N′-2,2-dimethyl-2-(4-methoxy)benzylmercaptoethyl]aminoethyl}aminomethylbenzyl-N1,N3-di-tert-butoxycarbonylguanidine (19b). The same procedure for preparation of 19a was employed using 18b (138 mg, 0.23 mmol) and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (80 mg, 1 equiv) to give 96 mg of product, 19b (49%). 1 H NMR: 1.29 (6H, s, -CH3-), 1.32 (6H, s, -CH3-), 1.47 (9H, s, tert-CH3), 1.52 (9H, s, tert-CH3), 2.50 (2H, s, -CH2-), 2.64 (4H, s, -CH2-), 2.67 (2H, s, -CH2-), 3.63 (2H, s, Ar-CH2-), 3.70 (2H, s, Ar-CH2-), 3.73 (2H, s, Ar-CH2-), 3.76 (6H, s, OCH3-), 4.60 (2H, d, J ) 4.9 Hz, Ar-

Zhuang et al.

CH2NH-), 6.80 (4H, d,d, J ) 8.5, 1.7 Hz, ArH), 7.197.29 (8H, m, ArH). Anal. (C45H67N5O6S2‚1.5H2O): Calcd: C, 62.47; H, 8.15; N, 8.10. Found: C, 62.29; H, 7.11; N, 8.43. N2-3-[N-2-Mercaptoethyl-N-2-(N′-2-mercaptoethyl)aminoethyl]aminomethylbenzylguanidine (20a). The same procedure for preparation of 13 was employed by using 19a (350 mg, 0.45 mmol) in TFA (10 mL), Hg(OAc)2, (341 mg), and anisole (4 drops) to give 150 mg of product, 20a (37%). 1H NMR: 2.85 (4H, m, -CH -), 3.04-3.45 (8H, m, 2 -CH2-), 4.27 (2H, s, Ar-CH2-N-), 4.45 (2H, s, Ar-CH2NH-), 7.46 (4H, m, ArH). HRMS calcd(C15H27N5S2): m/z 342.1786 (M++1); found: m/z 342.1778 (M+ + 1). Anal. (C15H27N5S2‚5CF3COOH): Calcd: C, 32.94; H, 3.54; N, 7.68. Found: C, 32.52; H, 3.66; N, 7.66. N2-3-[N-2,2-Dimethyl-2-mercaptoethyl-N-2-(N′-2,2dimethyl-2-mercaptoethyl)aminoethyl]aminomethylbenzylguanidine (20b). The same procedure for preparation of 13 was employed by using 19b (84 mg, 0.1 mmol), TFA (4 mL), Hg(OAc)2 (77 mg), and anisole (2 drops) to give 50 mg of product, 20b (68%). 1 H NMR: 1.37 (6H, s, CH3-), 1.41 (6H, s, CH3-), 2.80 (2H, s, N-CH2-), 3.03 (2H, t, J ) 6.5 Hz, N-CH2-CH2NH-), 3.07 (2H, s, N-CH2-), 3.29 (2H, t, J ) 6.8 Hz, N-CH2-CH2-NH-), 3.92 (2H, s, Ar-CH2-), 4.43 (2H, s, ArCH2-NH-), 7.40 (4H, m, ArH). Anal. (C19H35N5S2‚5CF3COOH): Calcd: C, 35.99; H, 4.16; N, 7.24. Found: C, 36.27; H, 4.15; N, 8.03. Radiolabeling Procedure. Unlabeled MIBG was prepared by the same two-step method employed for the synthesis of 13 and 20 from 11 and 18. Labeling of MIBG with 125I was performed by the ammonium sulfate, heat-mediated exchange procedure according to a published method (1). A generic description for preparation of all the 99mTc-labeled complexes is described as the following: A small amount of N2S2 ligand (1-2 mg) dissolved in 100 µL of EtOH was mixed with 200 µL of HCl (1 N) and 1 mL of Sn-glucoheptonate solution (containing 12 µg of SnCl2 and 120 µg of Na-glucoheptonate, pH 6.67) and 50 µL of EDTA solution (0.1 N). [99mTc]Pertechnetate solution (100-200 µL; ranging from 1 to 3 mCi) was then added. The reaction was heated for 30 min at 100 °C, and cooled to room temperature. The reaction mixture was directly analyzed on either TLC (EtOH:concentrated NH3 ) 9:1) or ITLC SG (100% MeOH) to determine the purity of the product. For the animal study, the mixture was neutralized with phosphate buffer to pH 5.0. The ratio of the amount of ligand (a possible pseudo-carrier) to [99mTc]pertechnetate used for labeling was carefully monitored to result in a final dosing solution with less than 10 µg/Kg body weight/ dose (23). Partition Coefficients. Partition coefficients were measured by mixing 99mTc-labeled complexes or [125I]MIBG with 3 g each of 1-octanol and buffer (pH 7.0 or 7.4, 0.1 M phosphate) in a test tube. The test tube was vortexed for 3 min at room temperature, and then centrifuged for 5 min. Two weighed samples (0.5 g each) from the 1-octanol and buffer layers were counted in a well counter. The partition coefficient was determined by calculating the ratio of cpm/g of octanol to that of buffer. Samples from the octanol layer were repartitioned until consistent partition coefficient values were obtained. The measurement was repeated 3 times. Biodistribution in Rats. Male Sprague-Dawley rats (225-300 g) allowed free access to food and water were used for in vivo biodistribution studies (24, 25). While

99mTc-Labeled



Scheme 1

under ether anesthesia, 0.2 mL of a saline solution containing 99mTc-labeled agents (5-10 µCi) was injected directly into the femoral vein of rats, and the rats were sacrificed by cardiac excision at various time points postinjection. The organs of interest were removed and weighed, and the radioactivity was counted with an automatic gamma counter (Packard 5000). The percentage dose per gram of organ was calculated by a comparison of the tissue counts to suitably diluted aliquots of the injected material. For the dual-tracer experiment, [99mTc]2 (5-10 µCi) and [125I]MIBG (1-5 µCi) were coinjected together to the rats, and the biodistribution was performed as described. The samples were counted for 99mTc activity at the day of the experiment using the energy window of 99mTc (80-200 keV), and after a 3-day decay period, 125I activity in the samples was determined and used without further decay correction. Blocking studies were carried out either by pretreating rats with desipramine (10 mg/kg, ip) 30 min prior to tracer injection or by coinjecting nonradioactive MIBG (1 mg/kg) together with the tracers. Uptake Studies in Cultured Cells. The human neuroblastoma cell lines SK-N-SH (uptake-1 positive) and SKN-MC (uptake-1 negative) (26) were purchased from the American Type Culture Collection (Rockville, MD). Cell uptake study was performed similar to that reported previously for MIBG and its related derivatives (27). Briefly, bulk quantities of cells were grown in Falcon tissue culture flasks (75 cm2) at 37 °C in a 5% CO2 humidified atmosphere. The culture medium was minimal essential medium (MEM) supplemented with glutamine, Earl’s salts, sodium pyruvate, and 10% fetal bovine serum. The cells were seeded into 24-well plates

[(2-4) × 105 cells/per well in 500 µL of medium] and incubated at 24 h at 37 °C. The transporters for dopamine (DAT) or norepinephrine (NET) expressed in the pig kidney cell line (LLC-PK1) were kindly provided by Dr. Rudnick (Yale University, New Haven, CT). These cells, grown as a monolayer culture as described (28), were seeded and incubated similarly to SK-N-SH cells. On the day of the experiment, [125I]MIBG and [99mTc]2 in the medium were incubated with cells in phosphate-buffered saline (PBS) for 60 min. The cells were solubilized with 500 µL of 0.5 N NaOH and then counted for the associated activity in a gamma counter (Packard 5000). Nonspecific activity was determined either by preincubating cells with DMI (50 µM) or by measuring tracer binding to SK-N-MC cells. Both methods gave similar results. RESULTS

Synthesis of Ligands. Ligands 13, 20a, and 20b, were prepared by reactions described in Schemes 1 and 2. For the synthesis of ligand 13, a stepwise approach was used to build the N2S2 ligand system. The starting material, 3-aminobenzonitrile, was reacted with 2-(4-methoxybenzyl)mercaptoacetic acid, 4, in the presence of DCC to produce the amide 5 in 70% yield. Reduction of the amide and the nitrile groups by LAH produced the desired amine 6 (55% yield). Selective protection of 6 with (Boc)2O resulted in 7 (80% yield). The protected amine, 7, was acylated with chloroacetyl chloride by which the desired amide, 8, was produced in 97% yield. Substitution of chlorine in 8 with 2-(4-methoxybenzyl)mercaptoethylamine, 9, afforded 10 (yield 74%). Deprotection of the Boc group on 10 by acid gave amine 11 (yield 88%).


Zhuang et al.

Scheme 2

Scheme 3

Reduction of the amide moiety of 11 followed by a selective alkylation of the primary amine of the reductive product with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea in THF (29) introduced the guanidine moiety. This two-step reaction produced 12 in 33% yield. A one-pot reaction was used to remove both Boc and methoxybenzyl protection groups simultaneously. This was accomplished by treating 12 with TFA and Hg(OAc)2 followed by treatment with H2S to give the final product 13 (73%). The other two ligands, 20a and 20b, were prepared by reactions described in Scheme 2. To synthesize 20a and 20b, 2 equiv of R,R′-m-dichloroxylene was reacted with potassium phthalimide to give monophthalimide 14 in 70% yield. Monoalkylation of 16a (30) or 16b (31) by 14 produced the desired N2S2 derivatives 17a,b directly. In this selective monoalkylation reaction, when 1 equiv of 16a was used, the ratio of monoalkylation vs dialkylation was 2:1; when 2 equiv of 16a was used in the same reaction, the ratio was increased to 4:1. In the case of preparation of 17b, the reaction was much slower compared to that of 17a, and no dialkylation product was detected even when 1 equiv of 16b was used, which is

likely due to the steric hindrance of tetramethyl substitution on 16b. The yield of 17b was improved from 33% to 66% by adding KI to the same reaction mixture. Hydrolysis of phthalimide of 17a and 17b with hydrazine afforded amines 18a and 18b in 79% and 86% yield, respectively. The same procedure for introducing the guanidine moiety as that reported for 13 was used to produce 20a and 20b in 37% and 68% yield, respectively. Preparation of 99mTc-Labeled MIBG Derivatives. Preparation of 99mTc-labeled compounds from the free thiols, 13, 20a, and 20b, was achieved by using stannous(II) glucoheptonate as the reducing agent for [99mTc]pertechnetate (Scheme 3). Formations of 99mTc-labeled complexes resulted in similar Rf values (0.1-0.2) as that of [125I]MIBG by a similar TLC method [Whatman silica gel plates (PESIL G/UV) under various solvent systems (data not shown)]. Using a solvent system of ethanol/concentrated NH3 (9:1), the blank labeling reaction (without adding the ligand) showed one peak, most likely the 99mTc glucoheptonate peak with a R value of 0.8-0.9. The f radiochemical purity of the final complexes evaluated on TLC (EtOH:concentrated NH3 ) 9:1) showed greater

99mTc-Labeled


Table 1. Biodistribution in Rats after iv Injections of ( SD)

Bioconjugate Chem., Vol. 10, No. 2, 1999 165 99mTc-Labeled

[99mTc]1

Complexes 1, 2, and 3 (% Dose/g, Average of 3 Rats

[99mTc]2

[99mTc]3

organ

2 min

30 min

2 min

30 min

2 min

30 min

blood heart muscle lung kidney spleen liver skin brain heart:BL ratios

1.01 ( 0.15 0.52 ( 0.05 0.18 ( 0.01 2.92 ( 0.35 2.48 ( 0.39 0.94 ( 0.34 4.17 ( 0.24 0.13 ( 0.02 0.02 ( 0.008 0.51

0.37 ( 0.07 0.24 ( 0.04 0.07 ( 0.001 1.65 ( 0.14 1.46 ( 0.40 1.13 ( 0.05 4.08 ( 0.73 0.13 ( 0.003 0.01 ( 0.003 0.65

0.30 ( 0.03 0.67 ( 0.07 0.04 ( 0.01 1.42 ( 0.12 8.63 ( 0.71 0.51 ( 0.08 3.68 ( 0.49 0.17 ( 0.02 0.01 ( 0.00 2.23

0.07 ( 0.006 0.41 ( 0.01 0.04 ( 0.004 0.75 ( 0.03 5.70 ( 0.78 0.31 ( 0.03 2.09 ( 0.77 0.11 ( 0.002 0.006 ( 0.001 5.85

1.04 ( 0.09 0.18 ( 0.009 0.04 (0.006 0.26 ( 0.05 4.78 ( 0.05 0.40 ( 0.09 3.96 ( 0.50 0.06 ( 0.01 0.01 ( 0.002 0.17

0.03 ( 0.003 0.034 ( 0.002 0.015 ( 0.0003 0.064 ( 0.008 4.68 ( 0.64 0.08 ( 0.01 1.41 ( 0.20 0.04 ( 0.003 0.002 ( 0.000 1.13

Table 2. Dual-Tracer Biodistribution of [99mTc]2 and [125I]MIBG in Sprague-Dawley Rats (iv Injection, % Dose/g, Average of 3 Rats ( SD) organ

2 min

blood heart heart:BL ratios

0.25 ( 0.04 0.45 ( 0.03 1.86


0.50 ( 0.08 3.35 ( 0.24 6.68

1h

2h

4h

0.033( 0.004 0.22 ( 0.01 6.66

0.026 ( 0.004 0.21 ( 0.01 8.11

[99mTc]2

0.056 ( 0.01 0.26 ( 0.03 4.69 [125I]MIBG 0.17 ( 0.02 2.68 ( 0.16 15.76

than 95% purity (Rf ) 0.15). Alternately, the formed complexes were evaluated on ITLC SG (Gelman Sciences) with 100% MeOH as the solvent. Without any chelating agent present, the reduced pertechnetate (most likely chemical form is TcO2) moved slightly from the origin (Rf ) 0.15). Formation of the 99mTc-labeled MIBG derivatives gave Rf values around 0.4, in contrast to the Rf value of 1.0 in the absence of the new ligands (data not shown). These results lend support to the formation of the proposed 99mTc-labeled complexes ([99mTc]1, [99mTc]2, and [99mTc]3). These complexes were stable up to 6 h in phosphate buffer (pH 5.0) when evaluated in the same TLC system at room temperature. It is important to note that the labeling reaction may produce isomers. Attempts were made to separate the 99mTc-labeled complexes on either TLC or HPLC (normal phase or reversed phase columns) with various solvent combinations but without any success. A more systematic effort is currently being made to identify possible isomers, which may not be detected by the TLC system described above. Biodistribution Study of 99mTc-Labeled MIBG Derivatives in Rats. Lipophilicities, as measured by the partition coefficient (PC) between 1-octanol and pH 7.0 phosphate buffer, of these 99mTc-labeled complexes were comparable to that of [125I]MIBG (PC ) 1.1 for [125I]MIBG and 1.99, 6.74, and 4.54 for [99mTc]1, [99mTc]2, and [99mTc]3, respectively). Preliminary biodistribution studies of these 99mTc-labeled MIBG derivatives showed reasonably good heart uptakes in rats (Table 1). The initial heart uptake of [99mTc]3 (heart uptake 0.18 and 0.034% dose/g, at 2 and 30 min, respectively) clearly indicated that this compound is the least favorable one among all three 99m Tc complexes in terms of penetrating the cell membrane to localize in heart tissues. It is likely that, despite the advantage of adding a tetramethyl group on the molecule, which increases the lipophilicity (may facilitate diffusion process), the bulk hindrance may play an even more important role in obstructing the ability of this molecule to cross the membrane. We therefore concentrated on studying compounds [99mTc]1 and [99mTc]2, without the tetramethyl substitution groups. It appears that compounds [99mTc]1 and [99mTc]2 are both interesting, with similar heart uptakes at 2 and 30 min (0.52

0.14 ( 0.01 2.11 ( 0.09 15.07

0.13 ( 0.02 1.70 ( 0.20 13.0

and 0.24% dose/g for [99mTc]1 and 0.67 and 0.41% dose/g for [99mTc]2, respectively). However, [99mTc]2 displayed a higher heart-to-blood ratio (5.85) at 30 min as compared to that of [99mTc]1 (0.65). Therefore, compound [99mTc]2 was chosen for further studies in rats. It is interesting to note that these three 99mTc-labeled complexes displayed different uptakes in other organs such as lung and liver. [99mTc]1 appeared to be more concentrated in these two organs and was slowly washed out as compared to [99mTc]2 and [99mTc]3. In contrast, [99mTc]1 displayed lower accumulation in kidney as compared to [99mTc]2 and [99mTc]3. The dual-tracer biodistribution study provides a direct comparison of heart uptake and washout between the 99m Tc-labeled agent and [125I]MIBG. Therefore, a dualtracer experiment, the biodistribution of [99mTc]2 and [125I]MIBG, in Sprague-Dawley rats was carried out (Table 2). In these preliminary experiments using tracer doses, the loading doses for nonradioactive MIBG and also the ligands for making technetium complexes were kept below 10 µg/kg per rat per dose, so the concentrations of carrier or pseudo-carrier will not be sufficient to compete for the norepinephrine uptake sites (23). [99mTc]2 displayed the desired heart uptake and slow washout (0.45% dose/g at 2 min and 0.21% dose/g at 4 h, respectively; see Table 2). However, in a direct comparison with [125I]MIBG, the heart uptake of the 99mTc-labeled complex was approximately one-eighth of that for [125I]MIBG (3.35 and 1.71% dose/g at 2 and 4 h, respectively). Unlike [125I]MIBG, the contrast between heart vs blood at 4 h for [99mTc]2 appeared to be lower (8.11 and 13.00 for [99mTc]2 and [125I]MIBG, respectively). The preliminary data are encouraging in that it is the first time a 99mTc-labeled MIBG derivative displays a measurable heart uptake. To specifically examine whether the heart uptake is related to norepinephrine distribution, we have performed the dual-tracer experiment in rats either by coinjecting nonradioactive MIBG (1.0 mg/ kg) or by pretreating rats with desipramine (DMI, specific norepinephrine transporter blocker) prior to tracer injection. In rats with a coinjected loading dose of MIBG (1.0 mg/kg), the heart uptake of [125I]MIBG was blocked by 47.5% at 2 h, while there was about 32.0% blockade of


Zhuang et al.

Table 3. Biodistribution of [99mTc]2 and [125I]MIBG (Dual-Isotope Experiment) in Rats with or without Coinjection of Nonradioactive MIBG (% Dose/g, Average of 3 Rats ( SD)a control organ

2 min


0.28 ( 0.05 0.53 ( 0.06 1.89


0.47 ( 0.09 3.14 ( 0.20 6.59

a

treated (nonradioactive MIBG) 2h

0.046 ( 0.01 0.22 ( 0.01 4.78 0.11 ( 0.01 2.17 ( 0.07 19.37

2 min [99mTc]2 0.40 ( 0.05 0.36 ( 0.04 0.90 [125I]MIBG 0.79 ( 0.05 1.99 ( 0.03 2.51

% of blocking

2h

% of blocking

32.1b

0.043 ( 0.01 0.15 ( 0.01 3.58

32.0b

36.7b

0.13 ( 0.02 1.14 ( 0.12 8.61

47.5b

In treated rats: MIBG (1.0 mg/kg) was coinjected together with the tracers, [99mTc]2 and [125I]MIBG. b p < 0.05, Student’s t-test.

Table 4. Blocking Studies with Desipramine on Dual-Tracer Uptakes in Rats (% Dose/g, Average of 3 Rats ( SD)a control organ

2 min

blood heart adrenal gland heart:BL ratios

0.49 ( 0.03 0.46 ( 0.06 0.61 ( 0.11 0.94

blood heart adrenal gland heart:BL ratios

0.64 ( 0.06 3.04 ( 0.15 2.47 ( 0.30 4.75

treated (desipramine; DMI) 2h

0.064 ( 0.004 0.23 ( 0.03 0.23 ( 0.01 3.62 0.14 ( 0.01 2.29 ( 0.25 1.50 ( 0.19 16.20

2 min

% of blocking

2h

% of blocking

20b

0.054 ( 0.011 0.20 ( 0.02 0.21 ( 0.04 3.81

12b

[99mTc]2

0.35 ( 0.05 0.37 ( 0.09 0.52 ( 0.09 1.05 [125I]MIBG 0.65 ( 0.06 2.23 ( 0.72 0.52 ( 0.09 3.41

27b

0.091 ( 0.01 1.58 ( 0.22 1.73 ( 0.88 17.36

32b

a Control rats and rats pretreated with desipramine (DMI); 10 mg/kg, ip 30 min prior to tracer injection was used for this study. b p < 0.05, Student’s t-test.

Figure 2. Uptake of [99mTc]2 and [125I]MIBG in SK-N-SH human neuroblastoma cells in vitro as a function of tracer concentration (in cpm). The data shown above were from a set of representative experiments.

heart uptake for [99mTc]2 at the same time point (Table 3). This result suggests that the heart uptake of [99mTc]2 may be related to the MIBG uptake. To test the hypothesis that the binding of [99mTc]2 is specific for norepinephrine transporters, rats were pretreated with desipramine (DMI; 10 mg/kg) ip for 30 min prior to tracer injection, and the same biodistribution was carried out (see Table 4). Even though the specific blockade for [99mTc]2 is smaller than that of [125I]MIBG (12% vs 32% reduction, respectively), the result is consistent with the notion that the new 99mTc-labeled complex, [99mTc]2, displayed specific binding to norepinephrine transporters. Uptake Studies in Cells Expressing Monoamine Transporters. Cell uptakes of [99mTc]2 were evaluated together with [125I]MIBG in either SK-N-SH neuroblastoma cells or LLC-NET and LLC-DAT cells. [99mTc]2 uptake by SKN-SH cells, like [125I]MIBG, increased as a function of input activity (Figure 2). The specific uptake of [99mTc]2 was found, however, to be much lower than that of [125I]MIBG (about one-tenth). A higher nonspecific uptake of [99mTc]2 (approximately 40% of total uptake) was ob-

served in contrast to low nonspecific uptake for [125I]MIBG (10%). Interestingly, a similar specific uptake of [99mTc]2 was also observed in LLC-NET cells but not in LLC-DAT cells (data not shown). The data are consistent with the results for [125I]MIBG and related derivatives in the same SK-N-SH cells reported previously (27). DISCUSSION

In the last 10 years, the localization of myocardial adrenergic nerve endings in normal subjects and patients with various cardiac diseases has been reported (9, 10, 12). MIBG in its 123I-labeled form is the imaging agent of choice, and it is now commercially available in Japan and in Europe, which attests to its importance as a clinical diagnostic tool. MIBG initially was designed as an attempt to attach a more stable iodine at the meta position of benzenylguanidine as an adrenal medulla imaging agent (1, 3). It is generally accepted that the iodine substitution at the paraposition is subjected to rapid in vivo deiodination. However, at the practical level, a comparable 99mTc-labeled MIBG derivative will provide a much wider clinical acceptance. In developing 99mTc-

99mTc-Labeled


labeled MIBG derivatives, in vivo deiodination is not a subject of concern; therefore, it is likely that the [TcvO]3+N2S2 complex can be attached to benzylguanidine and both meta- and para-substituted 99mTc derivatives may be potentially useful. We made the initial attempt to replace the iodo group at the meta position of MIBG with [TcvO]3+N2S2 complex. Three complexes, [99mTc]1, -2, and -3, were prepared and tested. By substituting the iodo group with [TcvO]3+N2S2 complex, the resulting 99mTc complexes displayed comparable lipophilicities as the parent MIBG molecule (reflected by comparable partition coefficients). However, the 99mTc complexes displayed a lower heart uptake in rats. It is possible that these complexes with enlarged molecular size due to [TcvO]3+N2S2 complex prohibit their easy passage through the cell membranes. Dual-tracer distribution studies clearly delineated that the 99mTc-labeled complex [99mTc]2, similar to [125I]MIBG, was concentrated in the heart. It remained trapped in the heart for up to 4 h postinjection. Despite the lower amount of heart uptake, [99mTc]2 closely resembled [125I]MIBG, i.e., displayed significantly reduced heart uptake when coinjected with nonradioactive MIBG or pretreated with desipramine, a norepinephrine transporter inhibitor. Two components of MIBG uptake have been identified: (a) the specific neuronal uptake-1 pathway mediated by NE transporters, which is blocked by desipramine (6); and (b) the nonspecific uptake-2 pathway (possibly by a simple diffusion mechanism). Both pathways are considered to be involved when [123I]MIBG was used for myocardial imaging. It is likely that the heart uptake of 99mTc-labeled compound, [99mTc]2, derived from [125I]MIBG, also contained specific and nonspecific pathways and only the specific pathway can be blocked by pretreatment with desipramine (less than 20% for [99mTc]2). The smaller percentage of the specific uptake-1 pathway observed with [99mTc]2 could be likely due to the presence of the mixtures of diastereomers. It is also possible that only a certain structure conformation or configuration of the 99mTc complex can be preferably recognized by the specific uptake-1 pathway but not the other diastereomers. We attempted to separate different diastereomers using various HPLC columns and solvent combinations but without any success. Future efforts will be directed to the separation of individual isomers of the 99mTc complexes and the evaluation of the isolated isomer for their in vivo distribution. The specific activity of radiolabeled MIBG appears to be important for the in vivo localization (23). An initial report by Wieland et al. used an exchange reaction for preparation of radiolabeled MIBG (1). The exchange reaction requires the use of a higher quantity of nonradioactive MIBG, which leads to a lower specific activity. Biodistribution studies in rats, as predicted, showed a substantially decreased heart uptake when nonradioactive carrier was added. The saturable uptake in the heart is consistent with the proposed mechanism of uptake and retention. Consistent heart uptake was observed in rats only when the loading nonradioactive dose was below 12 µg/kg (23). Recent studies showed that the no-carrieradded preparation, using the corresponding 3-trimethylsilylbenzylguanidine as the starting material, clearly suggested that the uptake in the heart of mice is carrierdependent (32, 33). It was also reported that in humans the heart uptake appeared to be higher when the nocarrier-added preparation of MIBG was used (the carrieradded preparation specific activity was 2 mCi/µmol for 5.4 mCi dose/70 kg man ) 10.6 µg/kg). However, the image quality was not significantly improved in subjects


receiving the no-carrier-added preparation of MIBG. It was suggested that the in vivo de-iodination might be more significant at a lower “carrier” dose (higher specific activity); therefore, the background level was higher. To avoid carrier effect, the loading dose for nonradioactive MIBG or unchelated technetium ligand used for the study was kept below 10 µg/kg per rat. The uptake of MIBG into SK-N-SH cells by an active uptake-1 mechanism was reported previously (2). The substantially lower accumulation (one-tenth) of the 99m Tc-labeled compound, [99mTc]2, as compared to that of MIBG in SK-N-SH cells, was observed (Figure 2). This lower cellular uptake is consistent with the finding of lower heart uptake in rat biodistribution (the heart uptake was one-eighth of that for MIBG). Further studies will endeavor to determine the specificity of the cellular uptake of these 99mTc-labeled complexes. It is suggested that an ideal Tc-99m-labeled agent should have the following properties: (i) A heart uptake of at least 0.75% dose/organ at 2 h post iv injection into rats (this is about 50% of that reported for [125I]MIBG) and a heart vs blood ratio (percentage dose:gram ratio) g10 at 2 h postinjection. (ii) A specific heart uptake selective to norepinephrine storage sites (i.e., the uptake can be significantly reduced after pretreatment of “cold” MIBG or desipramine, DMI). The 99mTc-labeled analogue of MIBG should have a selective uptake similar to that of [125I]MIBG (at least 50% of the binding is related to selective norepinephrine distribution). In conclusion, initial results of a series of novel 99mTclabeled MIBG derivatives showed mixed results. At least in principle, it appears feasible to replace the iodine of MIBG with a [TcvO]3+N2S2 complex. The heart uptake in rats for this series of compounds is relatively low (about one-eighth of that of MIBG). The other challenge is that the ligand may form several isomers of 99mTc complexes, resulting in differential biodistribution in vivo We are currently preparing additional compounds to ameliorate the deficiencies mentioned above, and we remain optimistic that agents with desirable properties could be developed in the future for imaging myocardial sympathetic neuronal functions ACKNOWLEDGMENT

We thank Susan West and Laura Danich for their assistance in preparing the manuscript. This work was supported by the U.S. grants awarded by the U.S. Department of Energy (DOE-ER61657) and the National Institutes of Health (NS 18509). LITERATURE CITED (1) Wieland, D. M., Wu, J.-l., Brown, L. E., Mangner, T. J., Swanson, D. P., and Beierwaltes, W. H. (1980) Radiolabeled adrenergic neuron-blocking agents: adrenomedullary imaging with [131I]iodobenzylguanidine. J. Nucl. Med. 21, 349353. (2) Glowniak, J. V., Kilty, J. E., Amara, S. G., Hoffman, B. J., and Turner, F. E. (1993) Evaluation of metaiodobenzylguanidine uptake by the norepinephrine, dopamine and serotonin transporters. J. Nucl. Med. 34, 1140-1146. (3) Wieland, D. M., Mangner, T. J., Inbasekaran, M. N., Brown, L. E., and Wu, J.-l. (1984) Adrenal medulla imaging agents: a structure-distribution relationship study of radiolabeled aralkylguanidines. J. Med. Chem. 27, 149-155. (4) Shapiro, B., Sisson, J. C., Shulkin, B. L., Gross, M. D., and Zempel, S. (1995) The current status of meta-iodobenzylguanidine and related agents for the diagnosis of neuro-endocrine tumors [review; 60 refs]. Q. J. Nucl. Med. 39, 3-8. (5) Wieland, D. M., Brown, L. E., Rogers, W. L., Worthington, K. C., Wu, J.-L., Clinthorne, N. H., Otto, C. A., Swanson, D.

168 Bioconjugate Chem., Vol. 10, No. 2, 1999 P., and Beierwaltes, W. H. (1981) Myocardial imaging with a radioiodinated norepinephrine storage analogue. J. Nucl. Med. 22, 22-31. (6) Sisson, J. C., Wieland, D. M., Sherman, P., Mangner, T. J., Tobes, M. C., and Jacques, J., S. (1987) Metaiodobenzylguanidine as an index of the adrenergic nervous system integrity and function. J. Nucl. Med. 28, 1620-1624. (7) McGhie, A. I., Corbett, J. R., Akers, M. S., Kulkarni, P., Sills, M. N., Kremers, M., Buja, M. L., Durant-Reville, M., Parkey, R. W., and Willerson, J. T. (1991) Regional cardiac adrenergic function using I-123 meta-iodobenzylguanidine tomographic imaging after acute myocardial infarction. Am. J. Cardiol. 67, 236-242. (8) Kline, R. C., Swanson, D. P., Wieland, D. M., Thrall, J. H., Gross, M. D., Pitt, B., and Beierwaltes, W. H. (1981) Myocardial imaging in man with I-123 metaiodobenzylguanidine. J. Nucl. Med. 22, 129-132. (9) Dae, M. W., O′Connell, J. W., Botvinick, E. H., and Michael, C. (1995) Acute and chronic effects of transient myocardialischemia on sympathetic-nerve activity, density, and norepinephrine content. Cardiovasc. Res. 30, 270-280. (10) Demarco, T., Dae, M., Yuengreen, M. S. F., Kumar, S., Sudhir, K., Keith, F., Amidon, T. M., Rifkin, C., Lau, D., Botvinick, E. H., and Chatterjee, K. (1995) I-123 metaiodobenzylguanidine scintigraphic assessment of the transplanted human heart: evidence for late reinnervation. J. Am. Coll. Cardiol. 25, 927-931. (11) Merlet, P., Caussin, C., Poiseau, E., Piot, O., Mazie`re, B., and Syrota, A. (1996) In vivo assessment of neurotransmitter system in cardiovascular diseases. Clinical issues [review]. Q. J. Nucl. Med. 40, 108-120. (12) Dae, M. W. (1995) Scintigraphy of myocardial innervation with metaiodobenzylguanidine (MIBG): is there a clinical application? [editorial]. J. Nucl. Cardiol. 2, 151-154. (13) Fagret, D., Wolf, J.-E., and Cornet, M. (1989) Myocardial uptake of meta-[123I]-iodobenzylguanidine ([123I]-MIBG) in patients with myocardial infarct. Eur. J. Nucl. Med. 15, 624628. (14) Fagret, D., Wolf, J.-E., Vanzetto, G., and Borrel, E. (1993) Myocardial uptake of metaiodobenzylguanidine in patients with left ventricular hypertrophy secondary to valvular aortic stenosis. J. Nucl. Med. 34, 57-60. (15) Merlet, P., Valette, H., Dubois-Rande´, J. L., Moyse, D., Duboc, D., Dove, P., Bourguignon, M., Benvenuti, C., Duval, A.-M., Agostini, D., Loisance, D., Castaigne, A., and Syrota, A. (1992) Prognostic value of cardiac metaiodobenzylguanidine imaging in patients with heart failure. J. Nucl. Med. 33, 471-477. (16) Schnell, O., Muhr, D., Dresel, S., Weiss, M., Haslbeck, M., and Standl, E. (1997) Partial restoration of scintigraphically assessed cardiac sympathetic denervation in newly diagnosed patients with insulin-dependent (type 1) diabetes mellitus at one-year follow-up. Diabetic Med. 14, 57-62. (17) Schnell, O., Muhr, D., Weiss, M., Dresel, S., Haslbeck, M., and Standl, E. (1996) Reduced myocardial 123I-metaiodobenzylguanidine uptake in newly diagnosed IDDM patients. Diabetes 45, 801-805. (18) Kung, H. F., Kim, H.-J., Kung, M.-P., Meegalla, S. K., Plo¨ssl, K., and Lee, H.-K. (1996) Imaging of dopamine transporters in humans with technetium-99m TRODAT-1. Eur. J. Nucl. Med. 23, 1527-1530. (19) Walovitch, R. C., Hill, T. C., Garrity, S. T., Cheesman, E. H., Burgess, B. A., O’Leary, D. H., Watson, A. D., Ganey, M. V., Morgan, R. A., and Williams, S. J. (1989) Characterization

Zhuang et al. of technetium-99m-L,L-ECD for brain perfusion imaging, Part 1: Pharmacology of technetium-99m ECD in nonhuman primates. J. Nucl. Med. 30, 1892-1901. (20) Le´veille´, J., Demonceau, G., DeRoo, M., Rigo, P., Taillefer, R., Morgan, R. A., Kupranick, D., and Walovitch, R. C. (1989) Characterization of technetium-99m-L,L-ECD for brain perfusion imaging, Part 2: biodistribution and brain imaging in humans. J. Nucl. Med. 30, 1902-1910. (21) Walovitch, R. C., Franceschi, M., Picard, M., Cheesman, E. H., Hall, K. M., Makuch, J., Watson, M. W., Zimmerman, R. E., Watson, A. D., and Ganey, M. V. (1991) Metabolism of [99mTc]-L,L-ethyl cysteinate dimer in healthy volunteers. Neuropharmacology 30, 283-292. (22) Meegalla, S. K., Plo¨ssl, 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 Tc-99m labeled tropanes as dopamine transporter imaging agents. J. Med. Chem. 40, 9-17. (23) Mock, B. H., and Tuli, M. M. (1987) Influence of specific activity on myocardial uptake of 123I-mIBG in rats. Nucl. Med. Commun. 9, 663-667. (24) Kung, H. F., Molnar, M., Billings, J. J., Wicks, R., and Blau, M. (1984) Synthesis and biodistribution of neutral lipidsoluble Tc-99m complexes that cross the blood-brain barrier. J. Nucl. Med. 25, 326-332. (25) Kung, H. F., Yu, C. C., Billings, J. J., Molnar, M., and Blau, M. (1985) Synthesis of new bis-aminoethanethiol (BAT) derivatives: possible ligands for Tc-99m brain imaging agents. J. Med. Chem. 28, 1280-1284. (26) Biedler, J. L., Helson, L., and Sprengler, B. A. (1973) Morphology and growth, tumorigenicity and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res. 33, 2643-2652. (27) Vaidyanathan, G., Zhao, X.-G., Strickland, D. K., and Zalutsky, M. R. (1997) No-carrier-added iodine-131-FIBG: evaluation of an MIBG analogue. J. Nucl. Med. 38, 330 -334. (28) Gu, H., Wall, S. C., and Rudnick, G. (1994) Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. J. Biol. Chem. 269, 7124-7130. (29) Monache, G. D., Botta, B., Delle Monache, F., Espinal, R., De Bonnevaux, S. C., De Luca, C., Botta, M., Corelli, F., and Carmignan, M. (1993) Novel hypotensive agents from Verbesina caracasana. 2. Synthesis and pharmacology of caracasanamide. J. Med. Chem. 36, 2956-2963. (30) Oya, S., Plo¨ssl, K., Kung, M.-P., Stevenson, D. A. and Kung, H. F. (1998) Small and neutral TcO(V) BAT, bisaminoethanethiol (N2S2) complexes for developing new brain imaging agents. Nucl. Med. Biol. 25, 135-140. (31) Ohmomo, Y., Francesconi, L. C., Kung, M.-P., and Kung, H. F. (1992) New conformationally restricted [99mTc]N2S2 complexes as myocardial perfusion imaging agents. J. Med. Chem. 35, 157-162. (32) Vaidyanathan, G., and Zalutsky, M. R. (1993) No-carrieradded synthesis of meta-[131I]iodobenzylguanidine. Int. J. Radiat. Appl. Instrum., Part A, Appl. Radiat. Isot. 44, 621628. (33) Vaidyanathan, G., and Zalutsky, M. R. (1995) No-carrieradded meta-[123I]iodobenzylguanidine: synthesis and preliminary evaluation. Nucl. Med. Biol. 22, 61-64.

BC970207Q

99mTc-Labeled MIBG Derivatives: Novel 99mTc Complexes as

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