Synthesis of Ring- and Side-Chain-Substituted m ... - ACS Publications

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786

Bioconjugate Chem. 2001, 12, 786−797

Synthesis of Ring- and Side-Chain-Substituted m-Iodobenzylguanidine Analogues Ganesan Vaidyanathan,* Sriram Shankar, and Michael R. Zalutsky Department

of

Radiology,

Duke

University

Medical

Center,

Durham,

North

Carolina

27710.

Received March 6, 2001; Revised Manuscript Received June 22, 2001

With the goal of developing MIBG analogues with improved targeting properties especially for oncologic applications, several radioiodinated ring- and side-chain-substituted MIBG analogues were synthesized. Except for 3-[131I]iodo-4-nitrobenzylguanidine and N-hydroxy-3-[131I]iodobenzylguanidine, the radioiodinated analogues were prepared at no-carrier-added levels from their respective tin precursors. The radiochemical yields generally were in the range of 70-90% except for 3-amino-5-[131I]iodobenzylguanidine for which a radiochemical yield of about 40% was obtained. While the silicon precursor N1,N2-bis(tert-butyloxycarbonyl)-N1-(4-nitro-3-trimethylsilylbenzyl)guanidine did not yield 3-[131I]iodo-4-nitrobenzylguanidine, its deprotected derivative, N1-(4-nitro-3-trimethylsilylbenzyl)guanidine was radioiodinated in a modest yield of 20% providing 3-[131I]iodo-4-nitrobenzylguanidine. Exchange radioiodination of 3-iodo-4-nitrobenzylguanidine gave 3-[131I]iodo-4-nitrobenzylguanidine in 80% radiochemical yield. No-carrier-added [131I]NHIBG was prepared from its silicon precursor N1-hydroxy-N3-(3-trimethylsilylbenzyl)guanidine in 85% radiochemical yield.

INTRODUCTION

Despite its wide applications in oncology and cardiology, the radiopharmaceutical m-iodobenzylguanidine (MIBG1) remains a less than ideal agent (Shapiro, 1991; Wafelman et al., 1994). Although MIBG is an effective diagnostic agent for neuroblastoma and pheochromocytoma (Gefland, 1993), its therapeutic efficacy is less than desired (Sisson et al., 1990). To augment the therapeutic effect of [131I]MIBG, its use in a tandem with other modalities has been attempted (Gaze et al., 1995; Mastrangelo et al., 1995). Both higher peak uptake in tumor and prolonged tumor retention of an endoradiotherapeutic agent are important for maximizing therapeutic effects. Although high uptake of radioiodinated MIBG in a number of neuroblastoma cell lines has been demonstrated, the ability of these cells to retain the radioactivity for more than a few hours was rather poor (Lashford et al., 1991; Mairs et al., 1991; Smets et al., 1989; 1990; 1991). A short biological half-life of MIBG in tumor has been observed in clinical settings as well (Sisson and Wieland, 1986), and attempts have been made to boost MIBG retention by pharmacological intervention (Blake et al., 1988). * Correspondence to: Ganesan Vaidyanathan, Ph.D., Box 3808, Department of Radiology, Duke University Medical Center, Durham, NC 27710. Telephone: (919) 684-7811, FAX: (919) 684-7122, email: [email protected]. 1 Abbreviations: BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; CIBG, 4-chloro-3iodobenzylguanidine; DHIBG, 3,4-dihydroxy-5-iodobenzylguanidine; DIAD, diisopropyl azodicarboxylate; FIBG, 4-fluoro-3iodobenzylguanidine; HIBG, 4-hydroxy-3-iodobenzylguanidine; HIMBG, 4-hydroxy-3-iodo-5-methoxybenzylguanidine; mAIBG, 5-amino-3-iodobenzylguanidine; MIBG, m-iodobenzylguanidine; mINBG, 5-iodo-3-nitrobenzylguanidine; MOM, methoxymethyl; nca, no-carrier-added; NCS, N-chlorosuccinimide; NET, norepinephrine transporter; NHIBG, N1-hydroxy-N3-3-[131I]iodobenzylguanidine; pAIBG, 4-amino-3-iodobenzylguanidine; pINBG, 3-iodo-4-nitrobenzylguanidine; TPP, triphenylphosphine.

Maximizing tumor-to-normal tissue ratios is an objective that needs to be given important consideration in order to reduce normal tissue toxicity and to improve the therapeutic outcome. Achieving higher tumor-to-normal tissue ratios rapidly is essential for diagnostic applications as well. One of the drawbacks of MIBG-aided diagnosis is that an elapsed period of 1-2 days is necessary to provide sufficient target-to-background ratios for tumor detection (Sisson and Shulkin, 1999). We have hypothesized that improvements in the selective tumor uptake and retention probably can be made by altering the structure of the MIBG molecule. For example, in SK-N-SH cells in vitro and in mouse target tissues in vivo, 4-fluoro-3-[*I]iodobenzylguanidine (FIBG) was retained to a greater extent than MIBG itself (Vaidyanathan et al., 1997a). While tumor uptake depends in part on binding to norepinephrine transporter (Mairs et al., 1994; Glowniak et al., 1993; Hadrich et al., 1999), tumor retention to a certain degree is dictated by the stability of the compound. MIBG is known to be generally resistant to metabolism; however, tumor retention of radioactivity might be increased as well if MIBG was rendered less susceptible to deiodination by structural alterations. Indeed, one of the contributing factors for the enhanced retention of FIBG described above might be its higher resistance to deiodination. In the current study, a number of ring- and side-chainsubstituted MIBG derivatives have been synthesized with the goal of maximizing tumor-to-normal tissue ratios. Due to the encouraging results obtained with FIBG, we were interested in the properties of the corresponding chloro-analogue. Analogues with singly and multiply substituted polar groups such as nitro, amino, methoxy, and hydroxyl at different positions were also prepared. This paper deals with the syntheses of these novel analogues, and their biological evaluation is described in the accompanying paper.

10.1021/bc010031z CCC: $20.00 © 2001 American Chemical Society Published on Web 08/18/2001

MIBG Analogues MATERIALS AND METHODS

General. All chemicals were purchased from Aldrich Chemical Co. unless otherwise noted. Sodium [131/125I]iodide in 0.1 N NaOH was supplied by DuPont-New England Nuclear (North Billerica, MA). Melting points were determined on a Haake Buchler apparatus and were uncorrected. High-pressure liquid chromatography (HPLC) was performed using one of two systems: (1) a Beckman System Gold HPLC equipped with a Model 126 programable solvent module, a Model 168 diode array detector, a Model 170 radioisotope detector, and a Model 406 analog interface module; or (2) a Perkin-Elmer Series 4 Liquid Chromatograph connected to a Perkin-Elmer LC-95 UV/visible spectrophotometer detector and a Perkin-Elmer LCI-100 Laboratory Computing Integrator. Methods were programmed using a Perkin-Elmer 6312 display terminal. For reversedphase chromatography, a Waters µ Bondapak C18 (10 µm, 3.9 × 300 mm) column was used. Normal-phase HPLC was performed using a 4.6 × 250 mm (10 µm) Partisil silica column (Alltech, Deerfield, IL). Gas chromatography was performed using a SRI 8610C gas chromatograph (SRI Instruments, Torrance, CA) equipped with an FID detector. The samples were analyzed using a RESTEK 15 m MXT-1 column and using helium as the carrier gas at 5 psi; different temperature programs were used for different samples. Analytical TLC was performed on aluminum-backed sheets (Silica gel 60 F254), and normal-phase column chromatography was performed using Silica gel 60; both obtained from EM Science (Gibbstown, NJ). Column chromatographic fractions were collected either manually, by using a Gilson model 203 micro fraction collector (Middleton, WI) or by using an ISCO Foxy 200 fraction collector (Lincoln, NE), and the products were identified by TLC. In some cases, an ISCO UA-6 UV-Vis detector was placed between the column outlet and the fraction collector to identify fractions. Preparative thick layer chromatography was performed using 20 × 20 cm, 1000 µm plates (Whatman, Clifton, NJ). Before applying the sample, the plates were run in ethyl acetate to remove any adsorbed impurities. One plate per approximately 50 mg of sample was used. Radio-TLC was initially analyzed using a System 200 Imaging Scanner (BioScan, Washington, D.C.) and then cut into strips and counted using an automatic gamma counter (LKB 1282, Wallac, Finland). 1H NMR (300 MHz) and 13C NMR (75 MHz) were obtained on a GE Midfield GN-300 or on a Varian Mercury 300 spectrometer. Chemical shifts are reported in δ units; solvent peaks were referenced appropriately. Mass spectra were obtained on a Hewlett-Packard GC/MS/DS Model HP5988A instrument, or on JEOL SX-102 high-resolution mass spectrometer. IR spectra were obtained on a MB100 IR spectrometer (BOMEM, Que´bec, Canada). Elemental analyses were provided by Galbraith Laboratories (Knoxville, TN) or by Microanalysis Laboratory, University of Illinois (Urbana, IL). 4-Chloro-3-iodobenzyl Alcohol (2). Sodium borohydride (76 mg, 2 mmol) was added to a homogeneous mixture of 1 (Trans World Chemicals; 566 mg, 2 mmol), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP; Fluka; 974 mg, 2.2 mmol), and diisopropylethylamine (349 mg, 2.6 mmol) in THF (10 mL). The mixture was stirred at room temperature for 20 min. The THF was evaporated from the mixture, and 1 N HCl (5-10 mL) was added to the residue. The resultant aqueous suspension was extracted repeatedly with EtOAc. The combined organic layers were washed with 1 N NaOH and dried with Na2SO4. The mixture was

Bioconjugate Chem., Vol. 12, No. 5, 2001 787

adsorbed onto silica gel and chromatographed using 20% EtOAc/hexane to obtain 397 mg (74%) of an oil: 1H NMR (CDCl3): 1.82 (br s, 1H), 4.63 (s, 2H), 7.26 (m, 1H), 7.40 (m, 1H), 7.86 (s, 1H). GCMS m/z: 286 (M + NH4)+, 268 (M+). Anal. Calcd for C7H6ClIO: C, 31.32; H, 2.32. Found: C, 31.84; H, 2.32. 4-Chloro-3-(trimethylstannyl)benzyl Alcohol (3). A mixture of 2 (135 mg, 0.5 mmol), bis(triphenylphosphine)palladium dichloride (100 mg, 0.14 mmol), and hexamethylditin (900 mg, 2.75 mmol) in dioxane (10 mL) was refluxed under argon for 1 h. Dioxane was evaporated, and a suspension of the dark residue in hexane was passed through a plug of silica gel. Very nonpolar byproducts were removed by washing with hexane, and the more polar components of the reaction mixture including 3 were eluted from the silica gel with ethyl acetate. EtOAc was removed by rotary evaporation, and the residue was purified by preparative TLC using 20% EtOAc/hexane (four plates were used) to yield 38 mg (25%) of an oil: 1H NMR (CDCl3): 0.37 (s, 9H), 1.66 (t, 1H), 4.66 (d, 2H), 7.26-7.37 (m, 3H). GCMS m/z: cluster peaks at 307 (MH+). Anal. Calcd for C10H15ClOSn: C, 39.33; H, 4.95. Found: C, 39.41; H, 5.16. N1,N2-Bis(tert-butyloxycarbonyl)-N1-[4-chloro-3(trimethylstannyl)benzyl]guanidine (4). Diisopropyl azodicarboxylate (DIAD; 203 mg, 1 mmol) was added dropwise to a solution of 3 (153 mg, 0.5 mmol), N,N′-bis(tert-butyloxycarbonyl)guanidine (259 mg, 1 mmol), and TPP (275 mg, 1.1 mmol) in THF (ca. 5 mL), and the mixture was stirred overnight at room temperature. The THF was evaporated, and the residual oil was chromatographed using 10% EtOAc/hexane to obtain 184 mg (67%) of a thick oil: 1H NMR (CDCl3): 0.35 (s, 9H [119Sn-H, d]), 1.39 (s, 9H), 1.49 (s, 9H), 5.19 (s, 2H), 7.15-7.42 (m, 3H), 9.40 (br d, 2H). MS (FAB+) m/z: cluster peaks at 548 (MH+), 492, 436. HRMS (FAB+) calcd for C21H35ClN3O4118Sn (MH+): 546.1334. Found: 546.1352. Anal. Calcd for C21H34ClN3O4Sn: C, 46.14; H, 6.27; N, 7.69. Found: C, 46.76; H, 6.59; N, 7.27. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(4-chloro-3iodobenzyl)guanidine (5). This was prepared from 2 following a procedure similar to that used for the preparation of 4. The crude mixture was chromatographed using a stepwise gradient of 20% CHCl3/hexane to 100% CHCl3 to obtain 160 mg (63%) of a solid: mp 146-147 °C. 1H NMR (CDCl3): 1.40 (s, 9H), 1.50 (s, 9H), 5.15 (s, 2H), 7.25 (d, 1H), 7.40 (d, 1H), 7.80 (s, 1H), 9.35 (br d, 2H). MS (FAB+) m/z: 510 (MH+), 454, 398. HRMS (FAB+) calcd for C18H26ClIN3O4 (MH+): 510.0657. Found: 510.0643. Anal. Calcd for C18H25ClIN3O4: C, 42.41; H, 4.94; N, 8.24. Found: C, 42.72; H, 4.66; N, 8.43. N1-(4-Chloro-3-iodobenzyl)guanidine (6). Stannic chloride (75 mg, 0.3 mmol) was added dropwise to a solution of 5 (25 mg, 0.05 mmol) in anhydrous EtOAc (0.5 mL), and the mixture was stirred at room temperature for 3 h. EtOAc and SnCl4 were evaporated on a rotary evaporator initially using a water-aspirator and eventually with a vacuum pump. Ether (5 mL) was added to the residue with stirring, and the mixture was left stirring overnight at 4 °C. The resultant white precipitate was filtered, washed with ether, and dried to yield 14 mg (82%) of a solid: mp 262-268 °C (decomp). 1H NMR (CD3OD): 4.37 (s, 2H), 7.33 (d, 1H), 7.51 (d, 1H), 7.88 (s, 1H). MS (FAB+) m/z: 310 (MH+). 13C NMR (CD3OD): 44.4, 98.7, 129.6, 130.4, 138.1, 138.8, 140.0, 158.5. HRMS (FAB+) calcd for C8H10ClIN3 (MH+): 309.9608. Found: 309.9610 ( 0.0009 (n ) 4). 3-Chloro-4-(trimethylsilyl)toluene (8). Chlorotrimethylsilane (7.6 mL, 60 mmol) was added via a syringe

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to a stirred mixture of 7 (Trans World Chemicals; 10 g, 40 mmol) and magnesium turnings (1 g, 42 mmol) in ether (40 mL), and then the mixture was slowly heated to reflux. After the initial vigorous reaction subsided, the mixture was refluxed for an additional 1 h. Saturated NH4Cl was added to quench the reaction, and the aqueous layer was extracted twice with ether. The pooled ethereal layer was washed with brine, dried, and distilled under water-aspirator vacuum to obtain 2.7 g (34%) of an oil. Gas chromatographic analysis indicated the presence of minor impurities, but the product was used without further purification for the next step. GCMS m/z: 199 (MH+), 183, 91. 3,4-Bis(trimethylsilyl)toluene (9). Sodium (690 mg, 30 mmol) was taken up in a mixture of anhydrous toluene (3 mL) and xylene (4.7 mL) and melted by heating the mixture to reflux. To this finely divided suspension of sodium were added dropwise 8 (2.7 g, 13.6 mmol) and chlorotrimethylsilane (1.9 mL, 15 mmol) simultaneously to maintain a steady reflux. The reaction mixture was heated for an additional 1 h at reflux. The precipitate was filtered through a Celite bed, and the filtrate was concentrated. The residual oil was chromatographed using pentane to get 1.2 g (37%) of an oil: 1H NMR (CDCl3): 0.35 (s, 9H), 0.36 (s, 9H), 2.34 (s, 3H), 7.18 (d, 1H), 7.46 (s, 1H), 7.59 (d, 1H). GCMS m/z: 237 (MH+), 221, 166, 149. 4-Nitro-3-(trimethylsilyl)toluene (10). A solution of 70% nitric acid (0.56 g) in acetic anhydride (2.4 mL) was added dropwise to a solution of 9 (418 mg, 1.8 mmol) in acetic anhydride (2.9 mL), and the mixture was heated at 100 °C for 6 h, cooled, and added to 0.4 N NaOH (50 mL). The aqueous solution was extracted with ether, and the pooled ethereal extract was washed, dried, and evaporated to get an oil. The oil was chromatographed using pentane to obtain 120 mg (32%) of a pale yellow solid: mp 46-48 °C. IR (NaCl): cm-1 2926, 1522, 1345, 845. 1H NMR (CDCl3): 0.35 (s, 9H), 2.45 (s, 3H), 7.31 (d, 1H), 7.46 (s, 1H), 8.09 (d, 1H). GCMS m/z: 227 (M + NH4)+, 194. Anal. Calcd for C10H15NO2Si: C, 57.38; H, 7.22; N, 6.69. Found: C, 57.20; H, 7.36; N, 6.57. That this compound is not the isomeric 3-nitro-4-trimethylsilyltoluene is shown by the fact that chemical shifts of aromatic protons are closer to those predicted (ChemDraw software) for the 4-nitro derivative than for the 3-nitro isomer. 4-Nitro-3-(trimethylsilyl)benzyl Bromide (11). A mixture of 10 (157 mg, 0.75 mmol), NBS (133 mg, 0.75 mmol), and a grain of benzoyl peroxide in CCl4 (10 mL) was refluxed for 1 h in the presence of an incandescent light. The insolubles were filtered and washed, and the CCl4 was evaporated from the filtrate. The residue was chromatographed with pentane to obtain 97 mg (34%) of a pale yellow oil: 1H NMR (CDCl3): 0.37 (s, 9H), 4.52 (s, 2H), 7.56 (d, 1H), 7.66 (s, 1H), 8.16 (d, 1H). GCMS m/z: 305 and 307 (M + NH4)+, 272 and 274 (M-CH3)+. Anal. Calcd for C10H14BrNO2Si: C, 41.67; H, 4.90; N, 4.86. Found: C, 41.53; H, 4.87; N, 4.63. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(4-nitro-3-trimethylsilylbenzyl)guanidine (12). To a heterogeneous mixture of sodium hydride (60% dispersion; 9.6 mg, 0.24 mmol) in DMF (silylation grade, Pierce; 1 mL) was added N1,N2-bis(tert-butyloxycarbonyl)guanidine (63 mg, 0.24 mmol), and the reaction mixture was stirred at room temperature for 5 min, resulting in a homogeneous solution. A solution of 11 (72 mg, 0.25 mmol) in DMF (1 mL) was added dropwise and the progress of reaction was monitored by TLC. After 30 min, the reaction mixture was partitioned between water and EtOAc, and

Vaidyanathan et al.

the organic layer was washed with brine, dried, and concentrated. Preparative TLC of the crude product using 10% EtOAc/hexane gave 51 mg (46%) of a yellow solid: mp 104-106 °C. IR (KBr): cm-1 3386, 2973, 1717, 1609, 1519, 1293, 1244, 1147, 1121, 848. 1H NMR (CDCl3): 0.34 (s, 9H), 1.36 (s, 9H), 1.49 (s, 9H), 5.26 (s, 2H), 7.40 (d, 1H), 7.68 (s, 1H), 8.18 (d, 1H). MS (FAB+) m/z: 467 (MH+), 411, 355. Anal. Calcd for C21H34N4O6Si: C, 54.06; H, 7.34; N, 12.01. Found: C, 54.10; H, 7.60; N, 11.71. N1-(4-Nitro-3-trimethylsilylbenzyl)guanidine (13). Compound 12 (20.1 mg, 0.04 mmol) was dissolved in 10% (v/v) H2SO4/dioxane (0.5 mL), and the mixture was stirred at room temperature for 2 h. The solvents were evaporated, and the residue in a small amount of water was loaded onto an activated C18 solid-phase cartridge (tC18 ENV; Waters). The cartridge was washed with water (4 × 5 mL), and the product was eluted with MeOH (∼5 mL) in almost quantitative yield. 1H NMR(CD3OD): 4.19 (s, 2H), 7. 55 (d, 1H), 7.70 (s, 1H), 8.20 (d, 1H). MS (FAB+) m/z: 267 (MH+). HRMS (FAB+) calcd for C11H19N4O2Si (MH+): 267.1277. Found: 267.1280 ( 0.0003 (n ) 3). 3-Iodo-4-nitrotoluene (15). A solution of sodium nitrite (110 mg, 1.6 mmol) in water (0.5 mL) was added with stirring to a cold slurry of 5-methyl-2-nitroaniline (221 mg, 1.47 mmol) in concentrated HCl (0.7 mL) and ice (1 g). The mixture was stirred for 20 min at 0 °C, the resultant cold solution was added dropwise to a stirred solution of KI (2.44 g, 14.6 mmol) in water (3 mL) at room temperature, and the resultant mixture was stirred overnight. The reaction mixture was extracted with EtOAc, and the organic layer was washed with 1 N NaOH, 1 N HCl, and brine, dried, and concentrated. Column chromatography with 1% EtOAc/hexane gave 380 mg (98%) of an oil. An analytical sample was obtained by further purification by preparative TLC using the same solvent: 1H NMR (CDCl3): 2.39 (s, 3H), 7.27 (d, 1H), 7.82 (d, 1H), 7.89 (s, 1H). GCMS m/z: 281 (M + NH4)+, 264 (MH+), 155. Anal. Calcd for C7H6INO2: C, 31.96; H, 2.30; N, 5.33. Found: C, 32.14; H, 2.39; N, 5.30. 3-Iodo-4-nitrobenzyl Bromide (16). The title compound was prepared from 15 (269 mg, 1.02 mmol) by a procedure similar to that used for the preparation of 11. Chromatography using 5% EtOAc/hexane provided 45 mg (30%) of a solid: mp 61-63 °C. 1H NMR (CDCl3): 4.41 (s, 2H), 7.52 (d, 1H), 7.85 (d, 1H), 8.05 (s, 1H). Anal. Calcd for C7H5BrINO2: C, 24.59; H, 1.47; N, 4.10. Found: C, 24.76; H, 1.52; N, 4.07. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-iodo-4-nitrobenzyl)guanidine (17). The guanidination of 16 (87 mg, 0.25 mmol) was performed by a procedure similar to that used for the preparation of 12 from 11. Chromatography using a gradient of 5-10% EtOAc/hexane yielded 74 mg (90% based on the starting material consumed) of a yellow solid: mp 142-144 °C. 1H NMR (CDCl3): 1.41 (s, 9H), 1.49 (s, 9H), 5.16 (s, 2H), 7.43 (d, 1H), 7.85 (d, 1H), 8.01 (s, 1H), 9.40 (br d, 2H). MS (FAB+) m/z: 521 (MH+). HRMS (FAB+) calcd for C18H26IN4O6 (MH+): 521.0897. Found: 521.0899. Anal. Calcd for C18H25IN4O6: C, 41.55; H, 4.84; N, 10.77. Found: C, 42.25; H, 5.03; N, 10.90. N1-(3-Iodo 4-nitrobenzyl)guanidine (18). This was prepared quantitatively from 17 (52 mg, 0.1 mmol) in a similar fashion as 6 from 5: mp 258-259 °C (decomp). IR (KBr): cm-1 3443, 3355, 1660, 1518, 1352. 1H NMR (CD3OD): 4.49 (s, 2H), 7.5 (d, 1H), 7.85 (d, 1H), 8.10 (s, 1H). 13C NMR (CD3OD): 44.4, 87.3, 126.2, 128.8, 141.0,

MIBG Analogues

143.8, 154.1, 158.5. MS (FAB+) m/z: 320 (M+). HRMS (FAB+) calcd for C8H10IN4O2 (MH+): 320.9848. Found: 320.9847. 3-Iodo-5-nitrobenzoic Acid (20). A heterogeneous mixture of 3-amino-5-nitrobenzoic acid (3.64 g, 20 mmol) in 10 mL of concentrated HCl was stirred at 0 °C as a solution of NaNO2 (1.66 g, 24 mmol) in 10 mL of icewater was added. The resultant homogeneous orange solution was stirred at ca. 0 °C for 1 h, then added slowly with stirring to a solution of KI (8.3 g, 50 mmol) in 30 mL of water at 0 °C. The reaction mixture was slowly warmed to room temperature over 2 h. The aqueous solution was extracted with EtOAc, and the organic layer was washed with water, aqueous saturated sodium metabisulfite, and brine, dried, and concentrated to give 5.3 g of a dirty brown solid. Chromatography with 20% MeOH/EtOAc afforded 5.2 g (89%) of a brown solid: IR (KBr): cm-1 3481, 3389, 1704, 1632, 1584, 1532, 1480, 1355. 1H NMR (CD3OD): 8.59 (t, 1H), 8.67 (dd,1H), 8.74 (dd, 1H). 13C NMR (CD3OD): 94.1, 124.4, 135.6, 136.5, 144.91, 149.7, 166.1. HRMS (FAB-) calcd for C7H3INO4 (M - H)-: 291.9107. Found: 291.9115. 3-Iodo-5-nitrobenzyl Alcohol (21). A solution of 20 (1.5 g, 5.1 mmol) in 10 mL of anhydrous THF was stirred and refluxed for 3.5 h with 2 M BH3-THF (13 mL, 25.6 mmol) and B(OMe)3 (5.7 mL, 51.2 mmol) under a flow of argon. Excess borane was removed by addition of MeOH, and the solvents were evaporated. The resultant precipitate was reconstituted in EtOAc, washed with water and brine, dried, and concentrated by rotary evaporation to give 1.5 g of the crude product mixture. Chromatographic separation using a stepwise gradient of 50% to 75% EtOAc/hexane provided 720 mg (51%) of a pale yellow solid: IR (CCl4): cm-1 2977, 2866, 1742, 1537, 1348, 1239, 1119. 1H NMR (CDCl3): 4.79 (d, 2H), 8.04 (td, 1H), 8.19 (ddd, 1H), 8.46 (dd, 1H). 13C NMR (CDCl3): 63.1, 94.2, 120.7, 131.1, 141.1, 144.4. MS (FAB-) m/z: 278.93 (M-). HRMS (FAB-) calcd for C7H4INO3 (M - H)-: 277.9314. Found: 277.9324. Anal. Calcd for C7H6INO3: C, 30.13; H, 2.17; N, 5.02. Found: C, 30.03; H, 2.26; N, 4.92. 3-Nitro-5-tri-n-butylstannylbenzyl Alcohol (22). Compound 21 (100 mg, 0.36 mmol) in 2 mL of anhydrous toluene was refluxed with (Ph3P)2PdCl2 (60 mg, 0.09 mmol) as hexabutylditin (414 mg, 0.72 mmol) was added by syringe, and the ensuing black heterogeneous mixture was stirred and refluxed under a flow of argon for 5 h. The reaction mixture was cooled to room temperature, filtered over Celite to remove suspended particles, and washed with EtOAc. The filtrate was concentrated to provide 464 mg of a brown oil, which was purified by column chromatography using a hexanes-EtOAc gradient to give 115 mg (73%) of a brown oil: IR (NaCl): cm-1 3397, 2915, 2856, 1523, 1463, 1343. 1H NMR (CDCl3): 0.89 (t, 3H), 1.13 (m, 2H), 1.36 (m, 2H), 1.57 (m 2H), 4.81 (d, 2H), 7.74 (ddd, 1H), 8.14 (td, 1H), 8.20 (dd, 1H). 13C NMR (CDCl3): 10.0, 13.7, 27.4, 28.9, 29.1, 30.6, 63.6, 121.0, 129.1, 140.3, 144.9, 147.6. Anal. Calcd for C19H33NO3Sn: C, 51.61; H, 7.52; N, 3.17. Found: C, 51.48; H, 7.31; N, 3.21. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-nitro-5-trin-butylstannylbenzyl)guanidine (23). The benzyl alcohol 22 (111 mg, 0.25 mmol) in 2 mL of anhydrous THF was stirred at room temperature under argon with N,N′bis(tert-butyloxycarbonyl)guanidine (131 mg, 0.5 mmol) and TPP (331 mg, 1.25 mmol) as DIAD (247 mL, 1.25 mmol) was added, and the resulting yellow heterogeneous mixture was stirred for 3 h under a flow of argon. The solvent was evaporated, and the resulting solids were reconstituted in 15 mL of EtOAc, washed with 2 ×15 mL

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of water and 20 mL of brine, dried, and concentrated. Chromatographic purification of the crude product yielded 44 mg of a mixture, composed of the protected guanidine and diisopropylhydrazine dicarboxylate, which was further purified by preparative TLC using 5% EtOAc/hexane to give 18 mg (11%) of a colorless solid: IR (NaCl): cm-1 3386, 2963, 2924, 1721, 1613, 1528, 1363, 1147. 1H NMR (CDCl3): 0.78 (t, 3H), 1.02 (m, 2H), 1.21 (m, 2H), 1.33 (m, 2H), 1.30 (s, 9H), 1.40 (s, 9H), 5.13 (s, 2H), 7.66 (d, 1H), 8.00 (dd, 1H dd,), 8.07 (d, 1H), 9.3 (br d, 2H). 13C NMR (CDCl3): 10.0, 13.7, 27.3, 28.0, 28.3, 29.0, 122.2, 129.1, 141.5. MS (FAB-) m/z: cluster peaks at 683.2 (M-). HRMS (FAB-) calcd for C30H51N4O6Sn (M - H)-: 681.2831. Found: 681.2806. Anal. Calcd for C21H36N4O4Sn: C, 52.72; H, 7.67; N, 8.20. Found: C, 53.61; H, 7.63; N, 7.86. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-iodo-5-nitrobenzyl)guanidine (24). A solution of 21 (139 mg, 0.5 mmol) in 5 mL of anhydrous THF was stirred at room temperature under argon with N1,N2-bis-tert-butyloxycarbonyl-S-methylisothiourea (259 mg, 1 mmol) and TPP (656 mg, 2.5 mmol) as DIAD (492 mL, 2.5 mmol) was added, and the resulting yellow heterogeneous mixture was stirred for 5 h under a flow of argon. The solvent was evaporated, and the resulting solids were dissolved in EtOAc, washed with water and brine, dried, and concentrated to give 439 mg of crude products. Purification by column chromatography provided 156 mg (60%) of a pale yellow solid: IH NMR (CDCl3): 1.45 (s, 9H), 1.53 (s, 9H), 2.38 (s, 3H), 4.78 (s, 2H), 8.0 (t, 1H), 8.20 (dd, 1H), 8.47 (dd, 1H). HRMS (FAB+) calcd for C8H9IN4O2 (MH+): 320.9848. Found: 320.9848 ( 0.0013 (n ) 2). The above product (130 mg, 0.24 mmol) in 1.5 mL of MeOH was refluxed in liquid NH3 (ca. 2 mL) for 2.5 h. Rotary evaporation of the reaction mixture gave 125 mg (100%) of a pale yellow oil: 1H NMR (CDCl3): 1.47 (s, 9H), 1.51 (s, 9H), 5.14 (s, 2H), 8.16 (s, 1H), 8.27 (s, 1H), 8.44 (s, 1H), 9.3 (br d, 2H). MS (FAB-) m/z: 519.0 (M-). HRMS (FAB-) calcd for C18H24IN4O6 (M - H)-: 519.0741. Found: 519.0735. N1-(3-Iodo-5-nitrobenzyl)guanidine (25). A solution of 24 (125 mg, 0.24 mmol) in 4 mL of CH2Cl2 and 1 mL of TFA was stirred and heated at 65 °C in a sealed flask for 4 h. Evaporation of the solvents afforded 67 mg (64%) of the deprotected benzyl guanidine as an oil: IR (NaCl): cm-1 3347, 2503, 1673, 1616, 1533, 1352, 1203, 1141. 1H NMR (CD3OD): 4.89 (s, 2H), 8.08 (td, 1H), 8.18 (dd, 1H), 8.49 (dd, 1H). 13C NMR (CD3OD): 44.3, 94.6, 122.2, 132.4, 142.0, 142.9, 149.9, 158.2. 3-Bromo-5-methoxy-4-methoxymethoxybenzaldehyde (27). To a solution of 5-bromovanillin (1.16 g, 5 mmol) in anhydrous THF (20 mL) was added dropwise a 1M solution of potassium tert-butoxide (5 mL) followed by MOM-chloride (0.5 mL, 6.6 mmol). The reaction mixture was stirred for about 4 h at room temperature and the solvents were evaporated. The residual material was partitioned between water and EtOAc, and the combined organic layer was washed with brine, dried, and concentrated. Chromatography using 30% EtOAc/ hexane yielded 1.1 g of an oil: 1H NMR (CDCl3): 3.54 (s, 3H), 3.81 (s, 3H), 5.18 (s, 2H), 7.27 (d, 1H), 7.55 (d, 1H), 9.73 (s, 1H). GCMS m/z: 303 and 305 (M + C2H5)+, 292 and 294 (M + NH4)+, 275 and 277 (M+). Anal. Calcd for C10H11BrO4: C, 43.66; H, 4.03. Found: C, 43.95; H, 4.24. 3-Bromo-5-methoxy-4-methoxymethoxybenzyl Alcohol (28). To a stirred solution of 27 (1.05 g, 3.9 mmol) in anhydrous CH2Cl2 (15 mL) was added dropwise a 1 M solution of DIBAL in CH2Cl2 (5.4 mL), and the mixture was stirred for 1-2 h. Methanol (10 mL) and water (10

790 Bioconjugate Chem., Vol. 12, No. 5, 2001

mL) were added to the cooled reaction mixture, and the resultant gelatinous material was filtered through a bed of Celite and washed alternately with a total volume of 200 mL of CH2Cl2 and 50 mL of water. The layers of the filtrate were separated, and the CH2Cl2 layer was dried and concentrated to obtain 1.06 g (100%) of an oil: 1H NMR (CDCl3): 2.21 (br s, 1H), 3.64 (s, 3H), 3.82 (s, 3H), 4.57 (s, 2H), 5.13 (s, 2H), 6.85 (d, 1H), 7.10 (d, 1H). GCMS m/z: 294 and 296 (M + NH4)+, 278 and 280 (MH)+, 259 and 261, 215 and 217. Anal. Calcd for C10H13BrO4: C, 43.34; H, 4.73. Found: C, 43.50; H, 5.06. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-bromo-5methoxy-4-methoxymethoxybenzyl)guanidine (29). The title compound was prepared from 28 (522 mg, 1.9 mmol) by a protocol similar to that used for the preparation of 4, 5, and 23. Chromatography using a 10-30% EtOAc/hexane gradient afforded 506 mg (51%) of a solid: mp 131-133 °C. 1H NMR (CDCl3): 1.45 (s, 9H), 1.49 (s, 9H), 3.64 (s, 3H), 3.82 (s, 3H), 5.03 (s, 2H), 5.15 (s, 2H), 7.12 (m, 2H), 9.34 (br d, 2H). MS (FAB+) m/z: 518 and 520 (MH+), 462 and 464, 406 and 408. Anal. Calcd for C21H32BrN3O7: C, 48.65; H, 6.22; N, 8.11. Found: C, 48.31; H, 6.35; N, 7.94. 3-Iodo-5-methoxy-4-methoxymethoxybenzyl Alcohol (30). To a stirred solution of 28 (278 mg, 1 mmol) in THF (5 mL) under argon at -78 °C was added dropwise n-BuLi (1.6 M in hexanes; 1.45 mL, 2.32 mmol) over a period of 5 min, and the reaction mixture was stirred at -78 °C for an additional 15 min. To the resultant mixture at -78 °C was then added gradually 2-iodo-1,1,1-trifluoroethane (550 µL, 5.6 mmol) over a period of 5 min. The reaction mixture was allowed to warm gradually to room temperature and left stirring overnight. Solvents were evaporated from the reaction mixture, and the residual material was partitioned between EtOAc and water. Workup and silica gel chromatography using 40% EtOAc/hexane gave 88 mg (27%) of an oil: 1H NMR (CDCl3): 1.79 (br s, 1H), 3.67 (s, 3H), 3.84 (s, 3H), 4.60 (s, 2H), 5.15 (s, 2H), 6.92 (d, 1H), 7.36 (d, 1H). GCMS m/z: 342 (M + NH4)+, 324 (M+), 307, 263. The Rf values on an analytical TLC plate (50% EtOAc/ hexane) were the same (∼0.4) for both 28 and 30. However, their retention times on a reversed-phase HPLC column were marginally different. The tR for 28 was 22.1 min versus 23.0 min for 30 when eluted on a µ Bondapak C18 column with a 0.1% TFA in water (A) 0.1% TFA in acetonitrile (B) gradient (5% B for 10 min and then to 80% B in 20 min) at a flow rate of 1 mL/min. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-iodo-5methoxy-4-methoxymethoxybenzyl)guanidine (31). Compound 31 was prepared from 30 (88 mg, 0.28 mmol) by a protocol similar to that for 29. Chromatography with 10% EtOAc/hexane gave 113 mg (74%) of a waxy solid: mp 107-109 °C. 1H NMR (CDCl3): 1.47 (s, 9H), 1.49 (s, 9H), 3.66 (s, 3H), 3.81 (s, 3H), 5.01 (s, 2H), 5.14 (s, 2H), 7.18 (d, 1H), 7.38 (d, 1H), 9.32 (br d, 2H). HRMS (FAB+) calcd for C21H33IN3O7 (MH+): 566.1363. Found: 566.1372 ( 0.0013 (n ) 2). Anal. Calcd for C21H32IN3O7: C, 44.61; H, 5.70; N, 7.43. Found: C, 44.87; H, 5.88; N, 7.45. N1-(3,4-Dihydroxy-5-iodobenzyl)guanidine (32). A solution of BBr3 (1 M in CH2Cl2; 0.5 mL, 0.5 mmol) was added to 31 (33.8 mg, 0.06 mmol), and the mixture stirred at room temperature for 30 min. The CH2Cl2 was evaporated under a stream of argon, and residual BBr3 was removed by coevaporation with 2 × 1 mL of EtOAc. The residual mixture was taken in 1-2 mL of water and passed through two activated C18 solid-phase cartridges (tC18 ENV; Waters) connected in series. The cartridge was washed with water, and the product was eluted with

Vaidyanathan et al.

1 mL portions of EtOH. The second EtOH fraction, containing a majority of the product (HPLC), was concentrated to yield 6 mg (33%) of an oil. An analytical sample was obtained by semipreparative reversed-phase HPLC: 1H NMR (CD3OD): 4.20 (s, 2H), 6.73 (s, 1H), 7.11 (s, 1H). MS (FAB+) m/z: 330 (M + Na)+, 308 (MH)+. 13C NMR (CD3OD): 45.1, 84.5, 115.4, 129.3 (C1 and C6), 146.8, 147.0, 158.6. HRMS (FAB+) calcd for C8H11IN3O2 (MH+): 307.9896. Found: 307.9897 ( 0.0002 (n ) 3). The structure was further confirmed by heteronuclear multi bond connectivity (HMBC, Bax and Summers, 1986) and heteronuclear multiple quantum coherence (HMQC, Summers et al., 1986) NMR correlation experiments. N1-(4-Hydroxy-3-iodo-5-methoxybenzyl)guanidine (33). Stannic chloride (10 mg, 0.04 mmol) was added to a solution of 31 (24 mg, 0.04 mmol) in EtOAc (0.1 mL), and the reaction mixture was stirred at room temperature for 3 h. The EtOAc was evaporated, and ether (0.5 mL) was added with stirring resulting in a precipitate. The precipitate was washed with 2 × 0.5 mL of ether, the ether was decanted, and the precipitate was dried under vacuum for several hours to yield 33 as a crystalline solid in quantitative yield: mp, decomposes. 1 H NMR (CD3OD): 3.83 (s, 3H), 4.27 (s, 2H), 6.90 (s, 1H), 7. 23 (s, 1H). MS (FAB+) m/z: 322 (MH+). 13C NMR (CD3OD): 45.2, 56.7, 83.6, 111.9, 130.2 (C1 and C6), 130.5, 147.9, 148.6, 158.5. HRMS (FAB+) calcd for C9H13IN3O2 (MH+): 322.0052. Found: 322.0050 ( 0.0008 (n ) 2). The structure was further confirmed by HMBC/HMQC NMR experiments. The title compound can be also prepared by treatment of 31 with TFA at room temperature. 3-Methoxy-4-methoxymethoxy-5-(trimethylstannyl)benzyl Alcohol (34). A solution of n-BuLi (1.6 M in hexanes; 750 µL, 1.2 mmol) was added to a solution of 28 (142 mg, 0.52 mmol) in THF (2.5 mL), and the mixture was stirred at -78 °C for 15 min. A solution of trimethyltin chloride (1 M in hexanes; 1.2 mL, 1.2 mmol) was added, and the mixture was brought to room temperature gradually and stirred overnight. Evaporation of solvents, aqueous workup, and column chromatography with 40% EtOAc/hexane gave 51 mg (27%) of an oil: 1H NMR (CDCl3): 0.32 (s, 9H [119Sn-H, d]), 3.54 (s, 3H), 3.85 (s, 3H), 4.65 (s, 2H), 5.10 (s, 2H), 6.94 (m,2H). MS (FAB+) m/z: cluster peaks at 362 (M+) and 345. Anal. Calcd for C13H22O4Sn: C, 43.25; H, 6.14. Found: C, 43.98; H, 6.56. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-methoxy4-methoxymethoxy-5-trimethylstannylbenzyl)guanidine (35). The title compound was prepared from 34 (51 mg, 0.14 mmol) by a procedure similar to that used for 31. Chromatography using 10% EtOAc/hexane yielded 81 mg (95%) of an oil: 1H NMR (CDCl3): 0.30 (s, 9H [119Sn-H, d]), 1.45 (s, 9H), 1.49 (s, 9H), 3.53 (s, 3H), 3.82 (s, 3H), 5.08 (s, 2 × 2H), 6.95 (d, 1H), 7.22 (d, 1H), 9.40 (br d, 2H). MS(FAB+) m/z: cluster peaks at 604 (MH+); HRMS (FAB+) calcd for C24H42N3O7120Sn (MH+): 604.2045; Found: 604.2052 ( 0.0006 (n ) 2). Anal. Calcd for C24H41N3O7Sn: C, 47.86; H, 6.86; N, 6.98. Found: C, 48.25; H, 7.03; N, 7.00. N1-Hydroxy-N3-(3-iodobenzyl)guanidine (38). A solution of CNBr (220 mg, 2.1 mmol) in methanol (0.7 mL) was added dropwise with stirring to a cooled mixture of 3-iodobenzylamine (obtained by the basification of its commercially available hydrochloride salt) (414 mg,1.8 mmol) and NaOAc (323 mg, 3.9 mmol) in methanol (3.5 mL). The reaction mixture was stirred at 5 °C for 2-3 h and at ambient temperature overnight. The mixture was filtered through a frit funnel, and the filtrate was adsorbed onto silica gel and chromatographed using 3050% EtOAc/hexane to obtain 146 mg (32%) of a white

MIBG Analogues

crystalline solid: mp 49-50 °C; 1H NMR (CDCl3): 3.68 (br s, 1H), 4.21 (d, 2H), 7.13 (t, 1H), 7.32 (d, 1H), 7.69 (m, 2H). GCMS m/z: 276 (M + NH4)+, 259 (MH+), 234, 217. Since 37 was not very stable, it was carried over to the next step immediately. To a solution of NH2OH‚HCl (70 mg, 1 mmol) and 37 (146 mg, 0.57 mmol) in 1 mL of DMF was added Na2CO3 (200 mg, 1.9 mmol) in small portions. The mixture was stirred at room temperature for 2 h and partitioned between EtOAc and water. The organic phase was washed with brine, dried, and concentrated to obtain a chromatographically homogeneous oil in quantitative yield: 1H NMR (CD3OD): 4.15 (s, 2H), 7.07 (t, 1H), 7.29 (d, 1H), 7.58 (d, 1H), 7.70 (s, 1H). MS(FAB+) m/z: 292 (MH+), 276. HRMS (FAB+) calcd for C8H11IN3O (MH+): 291.9947; Found: 291.9940 ( 0.0000 (n ) 2). 3-(Trimethylsilyl)benzylamine (40). To a solution of 3-bromobenzylamine (obtained from its hydrochloride salt; 393 mg, 2.1 mmol) in THF (12 mL) at -78 °C was added dropwise n-BuLi (1.6 M in hexanes; 3.9 mL, 6.3 mmol) over a period of 5 min. The resultant pink solution was stirred for 45 min at -78 °C, and Me3SiCl (795 µL, 6.3 mmol) was added dropwise. The mixture was gradually warmed to room temperature and stirred overnight, worked up, and chromatographed using 80/20/1 CHCl3/ MeOH/HOAc yielding 144 mg (38%) of a waxy solid: mp 191-193 °C. 1H NMR(CDCl3): 0.28 (s, 9H), 1.47 (s, 2H), 3.88 (s, 2H), 7.29-7.46 (m, 4H). GCMS m/z: 180 (MH+). N 1 -Hydroxy-N 1 -(3-trimethylsilylbenzyl)guanidine (42). Compound 41 was prepared from 40 (370 mg, 2.1 mmol) by a procedure similar to that for 37. Chromatography using a 25-100% EtOAc/hexane gradient afforded 196 mg (46%) of an oil: 1H NMR (CDCl3): 0.26 (s, 9H), 3.76 (br s, 1H), 4.25 (d, 2H), 7.32-7.52 (m, 4H). GCMS m/z: 222 (M + NH4)+, 205 (MH+), 163. Compound 42 was prepared from 41 (176 mg, 0.86 mmol) in 94% yield by the same protocol used for 38: mp 128-130 °C. 1H NMR (CD3OD): 0.26 (s, 9H), 4.19 (s, 2H), 7.42-7.64 (m, 4H). MS (FAB+) m/z: 238 (MH+). Anal. Calcd for C11H19N3OSi: C, 55.66; H, 8.07; N, 17.70. Found: C, 55.82; H, 8.24; N, 17.66. 3-Amino-5-iodobenzyl Alcohol (43). A mixture of 21 (280 mg, 1 mmol), indium (836 mg, 7.3 mmol), saturated NH4Cl (1.2 mL), and EtOH (4 mL) was refluxed for 1 h. The reaction mixture was worked up as per the literature protocol (Moody and Pitts, 1998), and the resultant oil was purified by silica gel chromatography using 1:1 EtOAc:hexanes to yield 83 mg (33%) of an off-white solid: mp 65-67 °C. IR (KBr): cm-1 3417, 3296, 3193, 1598, 1568, 1262, 1099, 1026, 800. 1H NMR (CDCl3): 1.62 (br s, 1H), 3.69 (br s, 2H), 4.55 (s, 2H), 6.62 (dd, 1H), 6.96 (dd, 1H), 7.08 (d, 1H). GCMS (CI): m/z: 250 (MH+), 232, 123. HRMS (EI+) calcd for C7H8INO (M+): 248.9651; Found: 248.9653 ( 0.0011 (n ) 4). N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-amino-5iodobenzyl)guanidine (44). The title compound was prepared from 43 (198 mg, 0.8 mmol) by a Mitsunobu protocol as described earlier. Chromatography using 30% EtOAc/hexane gave a solid in quantitative yield: mp 179-180 °C (dec). 1H NMR (CDCl3): 1.37 (s, 9H), 1.49 (s, 9H), 3.64 (br s, 2H), 5.02 (s, 2H), 6.51 (m, 1H), 6.91 (m, 1H), 6.96 (m, 1H), 9.40 (br d, 2H). MS(FAB+) m/z: 491 (MH+). HRMS (FAB+) calcd for C18H28IN4O4 (MH+): 491.1155; Found: 491.1149 ( 0.0014 (n ) 4). Anal. Calcd for C18H27IN4O4: C, 44.09; H, 5.55; N, 11.43. Found: C, 45.55; H, 6.16; N, 12.04. 3-Amino-5-iodobenzylguanidine (45). Compound 44 (25 mg, 0.05 mmol) was treated with TFA (100 µL) at

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room temperature for 15 min. TFA was evaporated and the residue was triturated thrice with EtOAc and dried under high vacuum to yield 45 as an oil in quantitative yield: 1H NMR (CD3OD) 4.34 (s, 2H), 6.84 (s, 1H), 7.22 (m, 2H). MS (FAB+) m/z: 291 (MH+). HRMS (FAB+) calcd for C8H12IN4 (MH+): 291.0107; Found: 291.0111 ( 0.0005 (n ) 4). N1,N2-Bis(tert-butyloxycarbonyl)-N1-[3-amino-5(trimethylstannyl)benzyl]guanidine (46). A mixture of 44 (20 mg, 0.04 mmol), (Ph3P)2PdCl2 (25 mg, 0.04 mmol), and (Me3Sn)2 (130 mg, 0.4 mmol) in anhydrous dioxane (0.5 mL) was refluxed for 1 h. The reaction mixture was worked up as described for 3, and the crude product was purified by preparative TLC using 20% EtOAc in hexanes (the TLC plate was dried after the first run and subjected to a second development to ensure separation between starting material and product) to obtain 19 mg (83%) of a pale amber-colored oil: 1H NMR (CDCl3): 0.24 (s, 9H [119Sn-H, d]), 1.35 (s, 9H), 1.49 (s, 9H), 3.60 (br s, 2H), 5.07 (s, 2H), 6.53 (m, 1H), 6.68 (dd, 1H), 6.76 (d, 1H), 9.40 (br d, 2H). MS(FAB+) m/z: cluster peaks around 529 (MH+). HRMS (FAB+) calcd for C21H36N4O4120Sn (M+): 528.1763; Found: 528.1772 ( 0.0009 (n ) 4). Anal. Calcd for C21H36N4O4Sn: C, 47.84; H, 6.88; N, 10.09. Found: C, 47.70; H, 6.68; N, 10.09. N1-(4-Chloro-3-[131I]iodobenzyl)guanidine (6a). To 4 (0.25 mg) in a 1/2-dram vial was added 131I (∼1 mCi in 1-2 µL of 0.1 N NaOH) followed by a solution of NCS in HOAc (0.4 mg in 20 µL). The vial was capped and heated in an oil bath at 70 °C for 30 min. Trifluoroacetic acid (20 µL) was then added to the mixture, and the vial was heated for an additional 5 min. The solvents were evaporated, and the residual activity reconstituted in methanol (25 µL) and injected onto a reversed-phase HPLC column eluted with 20/80 CH3CN/water containing 0.1% TFA at a flow rate of 1 mL/min. The product activity (tR ) 15-16 min) was isolated in about 70% radiochemical yield. N1-(3-[131I]Iodo 4-nitrobenzyl)guanidine (18a). (A) Attempted Radioiododesilylation of 12. To 131I in 0.1 N NaOH (1 µL, 100-200 µCi) in a 1/2-dram vial was added a solution of NCS in TFA (0.4 mg in 10 µL) followed by 12 (0.2 mg) in TFA (10 µL). The vial was vortexed and maintained at room temperature or heated at 70 °C for 5-30 min. The contents were injected onto a reversedphase HPLC column eluted with 15/85 THF/0.2 M NH4H2PO4 at a flow rate of 1 mL/min. Although other unidentified radioactive peaks were observed, no activity peak corresponding to 17a (tR ) 26-27 min) was noticed. (B) Radioiododesilylation of 13. To 0.2 mg of 13 in a 1/ -dram vial was added about 1 µL of 131I in 0.1 N NaOH 2 (50-100 µCi) followed by NCS in TFA (0.4 mg in 10 µL). The reaction mixture was left at room temperature for 30 min, and injected onto a reversed-phase HPLC column eluted with 25/75 CH3CN/water containing 0.1% TFA at a flow rate of 1 mL/min. About 18% of the injected activity eluted with a retention time (7 min) corresponding to that of 18a. Conducting the reaction at 70 °C reduced the radiochemical yield of the product, presumably due to deiodination. (C) Exchange Radioiodination of 18. In accordance with a reported procedure (Mock and Weiner, 1988), a Reacti vial fitted with a vent tube and containing 1-5 µL of 131I in 0.1 N NaOH (∼1 mCi), (NH4)2SO4 in water (10 mg in 1 mL), and 17 (0.2 mg) was heated at 150 °C for 30 min. To ensure that radioiodination had occurred, an aliquot of the reaction mixture was injected onto a reversedphase column eluted with 15/85 CH3CN/water containing 0.1% TFA at a flow rate of 1 mL/min. HPLC analysis

792 Bioconjugate Chem., Vol. 12, No. 5, 2001

indicated that ca. 88% of the injected activity was associated with a peak coeluting with 18 (tR ) 16-17 min). The product activity from the bulk of the reaction mixture was isolated in about 75% yield using a C18 solid-phase cartridge (see below). N1-(3-[131I]Iodo-5-nitrobenzyl)guanidine (25a). A solution of 23 (0.2 mg) in 10 µL of AcOH was added to a 1 /2-dram vial containing 131I in NaOH (∼0.5 mCi in 1-2 µL) and 0.4 mg NCS in 10 µL of AcOH, capped and vortexed, and heated at 75 °C for 15 min. TFA (10 µL) was added and vortexed, and the vial was heated again at 75 °C for 5 min. The contents of the vial were then injected onto a reversed-phase HPLC column eluted with a 85:15 water/CH3CN solvent system containing 0.1% TFA at a flow rate of 1 mL/min. The principal radioactive peak (tR ) 16-17 min) corresponding to 25 was isolated in 45-50% radiochemical yield. HPLC analyses indicated that extended heating of the reaction mixture resulted in deiodination of the product. N1,N2-Bis(tert-butyloxycarbonyl)-N1-(3-[131I]iodo5-methoxy-4-methoxymethoxybenzyl)guanidine(31a). To 0.2-0.3 mg of 35 in a 1/2-dram vial was added 1 µL of 131I in 0.1 N NaOH (0.5-1.0 mCi) followed by a 3:1 (v/v) solution of HOAc:30% H2O2. The mixture was sonicated for 30 s and injected onto a silica HPLC column eluted with 5/95 EtOAc/hexanes containing 0.2% HOAc at a flow rate of 1 mL/min. The activity corresponding to 31a (tR ) 15-16 min) was isolated in about 80% radiochemical yield. N1-(3,4-Dihydroxy-5-[131I]iodobenzyl)guanidine (32a). About 100 µCi of 31a (HPLC fractions) was taken in a 1/ -dram vial, and the solvents were evaporated to dry2 ness. To the residual activity was added TFA (100 µL), and the vial was vortexed and maintained at room temperature for 15 min. TFA was evaporated, and the residue was triturated with 2 × 100 µL of EtOAc. To the residual activity was added anhydrous dichloromethane (80 µL) followed by BBr3 (25 mg), and the reaction was allowed to proceed at room temperature for 15 min. The solvent was evaporated and the residue triturated with 2 × 100 µL of EtOAc. The activity was dissolved in water (50 µL) and injected onto a reversed-phase HPLC column. The column was eluted with a solvent mixture consisting of 0.1% (w/v) TFA in each of water (A) and acetonitrile (B) at 1 mL/min. For the first 10 min, the solvent contained 3% of B which was then linearly increased to 90% over a period of 15 min. About 40% of the injected activity was associated with a peak corresponding to 32a (tR ) 12 min); another 30-40% of the injected activity was associated with a nonpolar peak (tR ) 19 min) which is presumably 33a. N1-(4-Hydroxy-3-[131I]iodo-5-methoxybenzyl)guanidine (33a). To 100 µCi of 31a in 25 µL of EtOAc in a 1/ -dram vial was added SnCl (5 µL), and the solution 2 4 was vortexed and maintained at room temperature for 10 min. The EtOAc was evaporated, and the residue was triturated with 2 × 25 µL of EtOAc. The activity was redissolved in methanol (25 µL) and injected onto a reversed-phase column eluted with a gradient method as above starting from an initial solvent composition of 5% B. About 80-90% activity eluted at a retention time (17 min) corresponding to that for 33a. N1-Hydroxy-N3-(3-[131I]iodobenzyl)guanidine (38a). To a 1/2-dram vial containing 131I (∼0.5 mCi) in 1-2 µL of 0.1 N NaOH was added NCS (0.4 mg in 10 µL of TFA) and 42 (0.2 mg in 10 µL of TFA). The vial was vortexed and maintained at room temperature for 10 min. The contents of the vial were injected onto a reversed-phase HPLC column eluted with 85/13.5/1/0.5 water/THF/

Vaidyanathan et al.

triethylamine/H3PO4 at a flow rate of 1.2 mL/min. The product activity (tR ) 16-17 min) was isolated in about 85% radiochemical yield. 3-Amino-5-[131I]iodobenzylguanidine (45a). About 1 mCi of 131I in 1 µL of 0.1 N NaOH was added to a 1/2dram vial containing 46 (0.2 mg) in CHCl3 (20 µL). A 1:3 mixture of 30% H2O2:HOAc (10 µL) was added to the above mixture, and the vial was sonicated for 30 s. Most of the solvents were evaporated under a flow of argon, and the residual mixture was treated with TFA (0.1 mL) at room temperature for 5-10 min. The product 45a was isolated in 35-40% radiochemical yield by reversedphase HPLC (tR ) 11 min). For this, a C18 column (µ Bondapak) was eluted initially with 99.9:0.1 water: acetonitrile each containing 0.1% (w/v) TFA at a flow rate of 1 mL/min; after 15 min, the acetonitrile content was linearly increased to 90% over 15 min. All the above radioiodinated guanidine derivatives except 32a and 45a were concentrated from the HPLC fractions by solidphase extraction and reconstituted in appropriate media for biological evaluations, as has been reported earlier (Vaidyanathan et al., 1997a). Concentration by solidphase extraction of 45a was performed by a slightly modified protocol (Wafelman et al., 1993). While a regular C18 cartridge was used in most cases, for very polar compounds such as 32a and 45a a bigger cartridge (tC18 ENV; Waters) was used. RESULTS AND DISCUSSION

From an extensive structure-activity study of MIBG analogues, it has been shown that ring substituents in general are better tolerated than are modifications to the guanidinomethyl functionality (Wieland, 1986). We anticipated that derivatives with polar substituents might yield higher tumor-to-normal tissue ratios, because these analogues may be expected to clear faster from normal tissues. Efforts to synthesize MIBG analogues with highly polar groups such as sulfonic and phosphonic acid are underway; however, in the current study, we were able to prepare other analogues of varying polarity. FIBG, the MIBG analogue with a fluorine at the 4-position had shown increased uptake and retention in tumor cells, as well as lower levels of deiodination (Vaidyanathan et al., 1997a). Although substituting fluorine in FIBG with a chlorine is expected to decrease its polarity, we wished to investigate whether a chlorinesubstituted analogue will have a higher tumor uptake without concomitant increases in normal tissue uptake. The synthetic approaches used in the preparation of the unlabeled 4-chloro-3-iodobenzyl guanidine (6; CIBG), and the tin precursor 4, from which 6a could be obtained at a no-carrier-added level, are presented in Scheme 1. Commercially available 4-chloro-3-iodobenzoic acid was converted to 2 by NaBH4-mediated reduction of the active ester, formed in situ by its treatment with BOP (McGeary, 1998). Compound 2 was converted to the protected guanidine 5 by using the Mitsunobu protocol (Dodd and Kozikowski, 1994), and 6 was obtained from 5 by treatment with SnCl4 (Miel and Rault, 1997). Attempts to convert 5 to 4 either by palladium-catalyzed stannylation (Wigerinck et al., 1993), or by initial Li/I exchange and subsequent stannylation were not successful. However, it was possible to convert 2 to 3, from which 4 was obtained. Radioiodination of 4 with NCS in acetic acid and subsequent in situ deprotection with TFA resulted in 6a with a radiochemical yield of about 70%. The π value, an indicator of polarity, is typically negative for a nitro group (-0.28, 0.14, and 0.71 for NO2,

MIBG Analogues Scheme 1a

Bioconjugate Chem., Vol. 12, No. 5, 2001 793 Scheme 3a

a (a) NaNO , HCl, KI; (b) NBS, benzoyl peroxide, CCl ; (c) 2 4 N,N′-bis-Boc-guanidine, NaH DMF; (d) SnCl4, EtOAc; (e) 131I, NCS, TFA.

Scheme 4a

a (a) BOP, DIPEA; NaBH , THF; (b) (Ph P) PdCl , (Me Sn) , 4 3 2 2 3 2 dioxane; (c) N,N′-bis-Boc-guanidine, TPP, DIAD; (d) SnCl4; (e) 131I, NCS, HOAc; (f) TFA.

Scheme 2a

a (a) NaNO , HCl, KI; (b) BH /THF, B(OMe) ; (c) (Ph P) PdCl , 2 3 3 3 2 2 (Bu3Sn)2, toluene; (d) N,N′-bis-Boc-guanidine,TPP, DIAD, THF; (e) N,N′-bis-Boc-S-methylisothiourea, TPP, DIAD, THF; (f) liquid NH3; (g) TFA, CH2Cl2, 65 °C; (h) 1. 131I, NCS, HOAc, 2. TFA.

a (a) Mg, Me SiCl, Et O; (b) Na, Me SiCl, toluene, xylene; (c) 3 2 3 70% HNO3, Ac2O; (d) NBS, benzoyl peroxide, CCl4; (e) N,N′-bisBoc-guanidine, NaH, DMF; (f) H2SO4, dioxane.

F, and Cl, respectively; Fujita et al., 1964; Hansch, 1995), and nitro-substituted derivatives are often more polar than their parent molecules. To investigate the effect of a 4-NO2 substituent on the resulting MIBG analogue, a

precursor for 3-[131I]iodo-4-nitrobenzyl guanidine (p[131I]INBG) was sought (Scheme 2). Radioiodination of aryl stannanes are more facile in comparison to aryl silanes. However, due to the success we have had in the preparation of n.c.a. [131I]MIBG from a corresponding silicon precursor (Vaidyanathan and Zalutsky, 1993), 12 was initially chosen as a precursor for the preparation of radioiodinated p-INBG. The intermediate 10 was prepared from 3-chloro-4-iodotoluene in three steps using literature procedures (Clark et al., 1951; Eaborn et al., 1964; 1969). Benzylic bromination of 10 yielded 11 which was converted to the protected guanidine 12 by a method reported earlier (Vaidyanathan and Zalutsky, 1997b). A

794 Bioconjugate Chem., Vol. 12, No. 5, 2001

Vaidyanathan et al.

Scheme 5a

a (a) MOMCl, potassium tert-butoxide; (b) DIBAL, CH Cl ; (c) N,N′-bis-Boc-guanidine, DIAD, TPP, THF; (d) BuLi, CF CH I, THF; 2 2 3 2 (e) BuLi, Me3SnCl,THF; (f) BBr3; (g) SnCl4, EtOAc; (h) 131I, H2O2, HOAc, sonication; (i) TFA.

different synthetic route was taken to prepare unlabeled p-INBG (Scheme 3). The key intermediate, the benzyl bromide 16, was prepared from 14 in two steps and converted to the protected guanidine 17 as above. The guanidine 18 was obtained by the SnCl4-mediated deprotection of 17. Radioiodination of 12 and subsequent TFA treatment did not show a peak on HPLC corresponding to that of 18a. Our prior unpublished results have shown that TFA sometime fails to remove both Boc groups from bis-Boc guanidines. To ensure that the absence of a peak corresponding to 18a under reversed-phase HPLC conditions did not result from this partial deprotection, 12 was deprotected to 13 before being subjected to radioiodination. Although a peak corresponding to 18a was observed by HPLC, the radiochemical yield was poor. Before undertaking the synthesis of a tin precursor to 18a, an exchange radioiodination method (Mock and Weiner, 1988) was used to prepare 18a in order to check its suitability as an MIBG analogue. One of the problems associated with pINBG was its in vivo instability (see following article). This may be due to the presence of a strong electron-withdrawing group ortho to the iodine which may facilitate the nucleophilic displacement of iodine. To investigate the effect of switching the NO2 group to the meta-position on the stability, and to examine the overall suitability of mINBG as a MIBG analogue, the isomeric (3-iodo-5-nitro)benzylguanidine (mINBG; 25; Scheme 4) and its tin precursor 23 were synthesized. Compound 21 was prepared in two

steps from 19. Although 24 could have been prepared directly from 21 by its coupling with bis-Boc-guanidine under Mitsunobu conditions, the S-methylisothiourea derivative was first prepared serendipitously and then converted quantitatively to 24 by treatment with liquid ammonia. It is often difficult to isolate products from Mitsunobu reactions by chromatography due to the coelution of starting materials and byproducts (O’Neil et al., 1998; Falconer et al., 1999; Kiankarimi et al., 1999). While this was generally the case for the purification of our diprotected guanidines, chromatographic separation of the S-methylisothiourea intermediate from the byproducts was much easier, at least in this specific instance, affording the pure product in a greater isolated yield. Treatment of 24 with TFA in CH2Cl2 yielded 25. Nocarrier-added m[131I]INBG was prepared from 23 in 50% radiochemical yield. The lower radiochemical yield may be a result of the chemical instability of 25a under these reaction conditions. Accordingly, use of protracted reaction times and higher temperatures resulted in higher amounts of radioiodide, presumably from the deiodination of 25a. The natural substrate of the NET is norepinephrine, which has two hydroxy substituents at the 3- and 4-positions of its benzene ring. The potential of a “norepinephrine-like” benzylguanidine analogue with two hydroxyl groups has been suggested previously (Wieland, 1986). We wanted to investigate whether an MIBG derivative with two ring hydroxyl groups would retain affinity for NET. A nca synthesis of 3,4-dihydroxy-5-[131I]-

MIBG Analogues Scheme 6a

a

Bioconjugate Chem., Vol. 12, No. 5, 2001 795 Scheme 7a

(a) CNBr, NaOAc, MeOH; (b) NH2OH‚HCl, Na2CO3, DMF.

iodobenzyl guanidine ([131I]DHIBG; 32a) was accomplished following an approach depicted in Scheme 5. The syntheses of unlabeled DHIBG and that of a suitable tin precursor were commenced from commercially available bromovanillin, 26. To make the chemical manipulations facile, the 4-OH group of 26 was protected with a methoxymethoxy (MOM) group. The resultant 27 was reduced to the benzyl alcohol 28 which in turn was converted to 29. Neither attempted stannylation of 29 to 35, either by the palladium-catalyzed reaction or by the lithiation pathway, nor the conversion of 28 to 34 via the modified Stille protocol, were successful. However, 34 could be prepared by the lithium/bromine exchange of 28 and subsequent stannylation of the intermediate anion. Compounds 30 and 34 were readily converted to their respective guanidine derivatives, 31 and 35, under Mitsunobu conditions. Both the methoxy and the MOM group as well as the Boc groups of 31 were removed simultaneously by treatment with BBr3 to afford 32. A radiochemical yield of about 80% was obtained for the conversion of 35 to 31a under mild reaction conditions. Compound 31a was smoothly converted to 33a by its treatment with either SnCl4 or TFA in 80-90% radiochemical yields. Treatment of 31a with BBr3 at room temperature gave 32a but in meager radiochemical yields. In situ treatment of 33a, formed by TFA treatment of 31a, with BBr3 increased the radiochemical yield of 32a to some extent (ca. 40%). The potential for enhancing the radioiodination yield of 32a by using alternative demethylating agents exists but was not investigated. As indicated earlier, structural alterations at the side chain of MIBG have been less tolerated (Wieland, 1986). Except when the radioiodine was at the ortho position, both N1-mono- and N1,N2-dialkylated MIBG derivatives had insignificant target uptake in the dog adrenal medulla. Introduction of an amino group on the guanidine function had deleterious effects as well; however, a N-hydroxy-substituted MIBG derivative has not been studied. A number of N-hydroxyguanidines have been developed as potential replacements for the anti-hypertensive guanethidine; two of these had potencies similar to that of guanethidine (Balley and DeGrazia, 1973). MIBG itself was derived by combination of the guanidine group of guanethidine and the benzyl portion of bretyllium, another neuron blocking agent. On the basis of these facts, we were interested in the development of the N-hydroxy-modified MIBG. The synthetic pathways for the preparation of N1-hydroxy-N3-(3-iodobenzyl)guanidine (NHIBG; 38) and its silicon precursor 42 are presented in Schemes 6 and 7, respectively. These were prepared from their corresponding benzylamines following a literature procedure (Balley and DeGrazia, 1973). Compound 40 (Vaidyanathan and Zalutsky, 1993) was prepared from 39 via a lithiation/silylation procedure. Radioiodination of 42 to 38a was achieved in 80-90% radiochemical yield. The MIBG derivative with an amino group at the 4-position (pAIBG) was better than the parent compound

a (a) BuLi, THF, Me SiCl; (b) CNBr, NaOAc, MeOH; (c) 3 NH2OH.HCl, Na2CO3, DMF; (d) 131I, NCS, TFA.

Scheme 8a

a (a) In, aq NH Cl; (b) N,N′-bis-Boc-guanidine, DIAD, TPP; 4 (c) (Me3Sn)2, (Ph3P)2PdCl2, dioxane; (d) TFA; (e) 131I, H2O2, HOAc, sonication; (f) TFA.

with respect to its human myocardial uptake. However, the thyroid uptake was also higher, suggesting its higher degree of dehalogenation (Shulkin et al., 1986), probably due to the presence of an amino group ortho to the carbon carrying the iodine. We wanted to investigate whether the isomeric compound, wherein the amino group has been switched to the meta position (mAIBG, 45), would exhibit less dehalogenation. The synthesis of 45 and its tin precursor were readily achieved as shown in Scheme 8. Compound 43 was prepared from the nitro derivative 21 by metallic indium-mediated reduction. Guanidinylation of 43 via the Mitzunobu protocol, followed by TFAmediated deprotection of Boc groups from the resultant guanidine derivative 44 delivered the target benzylguanidine. The tin precursor 46 was obtained by the stannylation of 44 using the modified Stille reaction. Both 44 and 46 exhibited very similar polarity, and hence it was difficult to separate [131I]44 from 46 by normal-phase HPLC. For this reason, 46 was directly converted to 45a in one-pot reaction and the latter isolated by reversedphase HPLC. Based on a few attempts, the radiochemical yield for this conversion is about 40%. In summary, this paper describes the syntheses of various MIBG analogues and their tin or silicon precursors. Methods for the preparation of radioiodinated derivatives have been described as well. In vitro and in

796 Bioconjugate Chem., Vol. 12, No. 5, 2001

vivo evaluation of these analogues is the subject of the article which follows. ACKNOWLEDGMENT

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