Iranian Journal of Pharmaceutical Research (2013), 12 (4): 729-733 Received: January 2013 Accepted: August 2013 Copyright © 2013 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services Original Article A convenient Simple Method for Synthesis of Meta-iodobenzylguanidine (MIBG) Zahra Sheikholislama, Zohreh Soleimania, Abolghasem Moghimib and Soraya Shahhosseinia* a Pharmaceutical Chemistry Department, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran, P O Box: 14155-6153. bDepartment of Chemistry, University of Imam Hossein. Abstract Radioiodinated meta-iodobenzylguanidine (MIBG) is one of the important radiopharmaceuticals in Nuclear Medicine. [123/131I] MIBG is used for imaging of Adrenal medulla, studying heart sympathetic nerves, treatment of pheochromacytoma and neuroblastoma. For clinical application, radioiodinated MIBG is prepared through isotopic exchange method, which includes replacement of radioactive iodine in a nucleophilic substitution reaction with cold iodine (127I). The unlabelled MIBG hemisulfate is synthesized by the procedure described by Wieland et al. (1980). The availability of a more practical and cost-effective procedure for MIBG preparation encouraged us to study the MIBG synthesis methods. In this study the preparation of MIBG through different methods were evaluated and a new method, which is one step, simple and cost-effective is introduced. The method has ability to be scaled up for production of unlabelled MIBG. Keywords: Meta-iodobenzylguanidine; Neuroendocrine tumors; Sympathetic neurons; Radioiodinated MIBG. Introduction Meta-iodobenzylguanidine (MIBG) a structural and functional analogue of norepinephrine (NE), is transported, stored, and released in the adrenergic neuron by the same mechanisms as NE which includes neuronal (uptake-1) and extra neuronal (uptake-2, myocyte) speciic membrane transport process and passive diffusion in and from both cellular compartments. MIBG unlike NE is a stable and non-metabolizable compound in vivo, resistant to monoamine oxidase (MAO) and catechol-omethyl transferase (COMT) enzyme systems. Studies have shown that the norepinephrine transporter (NET) is highly over expressed in * Corresponding author: E-mail: s_shahoseini@sbmu.ac.ir neuroendocrine tumors such as neuroblastoma, pheochromocytoma, paraganglioma, and carcinoid. Since MIBG is a substrate for NET, therefore, MIBG and its radioiodinated forms accumulate in neuroendocrine tumors. In clinic, radioiodinated MIBG is used for treatment and diagnosis of neuroendocrine tumors. Accumulation of radioiodinated MIBG in adrenergic neurons clinically used for noninvasive assessment of sympathetic neuronal functions (1-9). Although MIBG has been radiolabelled with radioisotopes such as 211At, 18F, 76Br none of these radiopharmaceuticals are in clinic yet (10-13). The most important radioisotopes for radiolabeling MIBG are still iodine radioisotopes (123/131I). Radioiodinated MIBG is an important diagnostic and therapeutic radiopharmaceutical in clinic. For clinical application, radioiodinated Sheikholislam Z et al. / IJPR (2013), 12 (4): 729-733 at room temperature. The crystals was iltered by vacuum, washed by cold water, and dried in vacuum oven. Re-crystallization was done in a mixture of water-ethanol (50:50) (2). M.P 166-167.5°C, IR (KBr, cm-1): 3302 (NH), 3138 (NH), 1656 and 1537 (C=N), 1057 (S=O), 785 and 686 (1, 3-disubstituted benzene). 1H-NMR (D2O/500 MHz): δ 7.1 (t, 1H, phenyl H5), 7.3 (d, 1H, phenyl H6), 7.5 (m, 2H, phenyl H2, H4), 4.3 (s, 1H, CH2 Benzyl) ppm. 13C-NMR (D2O/125 MHz): δ 40 (CH2-Benzyl), 95 (C3-phenyl), 130 (C6-phenyl), 135 (C5–phenyl), 137 (C4phenyl), 140 (C2- phenyl), 142 (C1- phenyl), 158 (C-Imine) ppm. MS (ESI): 276 (M+H+). Method 2: To a solution of 3.3 g sodium (Na) in 20 mL super dry ethanol, 2 mmole guanidine HCl was added, and reluxed for 15 min. After iltration of sodium chloride, 2 mmole metaiodobenzylbromide in anhydrous ethanol was added to iltrate and reluxed for 36 hrs. Solvent was removed by rotary evaporation. MIBG was crystallized in methanol (17-18). Yield was 43%. IR (KBr, cm-1): 3367 and 3162 (NH), 1636 and 1560 (C=N), 801 and 737 (1,3-disubstituted benzene). 1H-NMR (CDCl3/500 MHz): δ 7 (t, 1H, phenyl H5), 7.1 (d, 1H, phenyl H6), 7.3 (m, 2H, phenyl H2, H4), 4 (s, 1H, CH2Benzyl) ppm. 13C-NMR (CDCl3/125 MHz): δ 40 (CH2Benzyl), 93 (C3- phenyl), 130 (C6- phenyl), 135 (C5- phenyl), 135 (C4- phenyl), 139 (C2phenyl), 142 (C1- phenyl), 158 (C-Imine) ppm. MS (ESI): 276 (M+H+). Method 3: A mixture of 0.8 mmole cyanoguanidine with 2 mmole MIBA HCl was heated for 6 h at 200°C while purging with nitrogen gas. MIBG was crystallized upon cooling (19). Yield was 60.5%. IR (KBr, cm-1): 3298 and 3011(NH), 1685 and 1628 (C=N), 841 and 697 (1,3-disubstituted benzene). 1H-NMR (CDCl3/500 MHz): δ 7 (t, 1H, phenyl H5), 7.3 (d, 1H, phenyl H6), 7.5 (m, 2H, phenyl H2, H4), 3.9 (s, 1H, CH2 Benzyl) ppm. 13C-NMR (CDCl3/125 MHz): δ 40(CH2-Benzyl), 95 (C3phenyl), 125 (C6-phenyl), 130 (C5-phenyl), 135 (C4- phenyl), 137 (C2- phenyl), 139 (C1phenyl), 160 (C-Imine) ppm. MS (ESI): 276 (M+H+). Method 4: To a mixture of 0.7 mmole MIBA HCl in 15 mL water, 0.3 mmole S-ethylisothiouronium sulfate [NH2C(NH) MIBG is prepared through isotopic exchange method (14-16). In this method, radioactive iodine (123/131I) in a nucleophilic substitution reaction replaces cold iodine (127I) in MIBG molecule. The unlabelled MIBG hemisulfate is synthesized by the procedure described by Wieland et al. (1980). The availability of a more practical procedure for MIBG preparation encouraged us to study the MIBG synthesis methods. In this study, MIBG was synthesized through 4 different methods. In each method, the product was characterized by melting point and spectroscopic methods (IR, 1H-NMR, 13C-NMR, Mass). The purity was determined using Reverse phase HPLC and TLC. The spectra data for inhouse MIBG was in agreement with structure of MIBG. The purity of MIBG hemisulfate synthesized using meta-iodobenzylamine and S-ethylisothiouronium (method four) was found to be comparable with authentic reference MIBG (Sigma). The synthesis compare to Wieland procedure, which is used for MIBG synthesis, has fewer steps. It is simple and because of production of ethanethiol as a by-product is costeffective. The new method has the ability to be replaced by traditional methods for preparation of unlabelled MIBG. Experimental All chemicals were purchased from Merck except meta-iodobenzyamine hydrochloride (MIBA HCl) and reference MIBG which were purchased from Sigma-Aldrich. Four different methods were used for synthesizing MIBG. Method 1: MIBG hemisulfate was synthesized according to the procedure described by Wieland et al. (1980). Briely, a mixture of MIBA HCl (1 mmole) and cyanamide (1.5 mmole) was reluxed 4 hrs at 100C. The resulting oily product was dissolved in 0.5 mL water and 0.5 mL KHCO3 (100 mg/ 0.5 mL) was added slowly with stirring. The precipitated MIBG bicarbonate was iltered by vacuum, washed by cold water, and dried in vacuum oven (M.P 121-124°C, yield: 60%). To a mixture of MIBG bicarbonate (0.6 mmole) in 2 mL water, 0.3 mL 2 N H2SO4 was added slowly. The MIBG hemisulfate was dissolved in water by heat and crystallized upon cooling 730 A convenient Simple Method for Synthesis of Meta-iodobenzylguanidine NH NH CH2Br H 2N NH2 N H NH2 Figure 1. Scheme for the synthesis of MIBG from meta-iodobenzylbromide with guanidine. S-Et+]2SO42- was added gradually. The mixture was stirred 5 hrs at room temperature. Water was removed by rotary evaporation and the precipitate was crystallized in acetone. Recrystallization was done on acetone. Ethanethiol a valuable by-product of reaction was collected during the reaction. Yield was 63%. IR (KBr, cm1 ): 3339 and 3274 (NH), 1643 and 1662 (C=N), 1092 (S=O), 887 and 701 (1,3-disubstituted benzene). 13C-NMR (D2O/125 MHz): δ 40 (CH2Benzyl), 95 (C3- phenyl), 125 (C6- phenyl), 127 (C5- phenyl), 130 (C4- phenyl), 133 (C2phenyl), 135 (C1- phenyl), 165 (C-Imine) ppm. MS (ESI): 276 (M+H+). Synthesis of S-ethylisothiouronium sulfate [NH2C(NH)S-Et]2+SO42- was done based on method of Osklo-Salmani A, 2003. Briely, a reaction mixture of 0.15 mole diethylsulfate and 0.2 mole thiourea was stirred at 100°C for 2.5 hrs and then at 140°C for an additional 1.5 hrs. Upon cooling, S-ethylisothiouronium sulfate was obtained with melting point 219-222°C and 93% yield (20). IR (KBr, cm-1): 3385, 3140, 2820, 2125, 1620, 1405, 1380, 1330, 1230, 1080, 1000, 920, 870. 13C-NMR (DMSO-d6, D2O/125 MHz): δ 172.1 (C=N), 25.9 (SCH2), 13.9 (CH3) ppm. Quality control of synthesized MIBG was done through RP-HPLC and TLC based on United States Pharmacopeia USP (21, 2008). HPLC (Merck-Hitachi, L7100, L7420): C18 Chro.Monolithic 100-4.6 mm, mobile phase: 3.04 g triethylamine per 1 liter of Water: Acetonitrile (90:10), pH 4. Flow rate: 1.5 mL/min, 229 nm. TLC: Silica gel, HCCl3: CH3OH (95:5). important radiopharmaceuticals in Nuclear Medicine. [123/131I]MIBG is used for imaging of Adrenal medulla, studying heart sympathetic nerves, treatment of pheochromacytoma and neuroblastoma. For clinical application, radioiodinated MIBG is prepared by exchange of radioactive iodine with non-radioactive one using isotope exchange method. The method used for synthesis of unlabelled MIBG is based on Wieland’s procedure (2). In this method, the reaction of MIBA HCl and cyanamide in the presence of KHCO3, results in MIBG bicarbonate formation. By adding H2SO4 to MIBG bicarbonate, MIBG hemisulfate is crystallized. A few times re-crystallization in a mixture of water:ethanol (50:50) is used for puriication of MIBG hemisulfate. The reaction of alkyl halide with appropriate amine was used for preparation of MIBG in second method. Guanidine HCl in a solution of Na in ethanol makes a good base to react with m-iodobezylbromide to produce MIBG (Figure 1). Resulting MIBG was crystallized on methanol. Yield was 43%. This method does not have any advantages to Wieland procedure. The reaction of cyanoguanidine with ammonium salts, which results in guanidinium salts, was used in third method for MIBG preparation. Mixture of Cyanoguanidine and MIBA HCl was heated up 200°C for 6 hrs (Figure 2). Crystals of MIBG was formed upon cooling. The yield was 60.5%. Since no solvent is used in this reaction, there is no step to eliminate solvent which is advantageous of this method. The reaction of MIBA HCl with salt of S-ethylisothiouronium sulfate produced MIBG sulfate in one step. Yield was 63%. Ethanethiol, the by-product, was collected which is used for synthesis of other chemicals. In this method, Results and Discussion Radioiodinated MIBG is one of the 731 Sheikholislam Z et al. / IJPR (2013), 12 (4): 729-733 NH NH2 NH N N H N H NH2 NH2 Figure 2. Scheme for the synthesis of MIBG from meta-iodobenzylamine with cyanoguanidine. MIBG hemisulfate is formed directly without adding H2SO4. Comparing the methods, method 2 and 3 are similar and do not have any speciic advantages to Wieland procedure which is routinely used to synthesis MIBG sulfate. Method four is a new method which is reported for irst time. In this method, MIBG hemisulfate is prepared through one step. The by-product of reaction is easily collected and is used in industry for synthesizing other chemicals. Considering yield, cost, steps, and simplicity the new method looks promising to be replaced instead of Wieland procedure. In summary, a convenient and simple method for the synthesis of MIBG was developed. The method has ability to be scaled up for production of unlabelled MIBG. (4) Glowniak JV, Turner FE, Gray LL, Palac RT, Lagunas-Solar MC and Woodward WR. 131 I-metaiodobenzylguanidine imaging of the heart in idiopathic congestive cardiomyopathy and cardiac transplants. J. Nucl. Med. (1989) 30: 1182-1191. (5) Wafelman AR, Hoefnagel CA and Beijnen JH. Radiochemical stability during infusion of 131I-labelled meta-iodobenzylguanidine for therapeutic use. Nucl. Med Commun. (1993) 14: 819-822. (6) Hanson MW, Feldman JM, Beam CA, Leight GS and Coleman RE. Iodine-131 labeled metaiodobenzylguanidine scintigraphy and biochemical analysis in suspected pheochromocytoma. Arch. Interr. Med. (1991) 151: 1397-1402. (7) DeGrado TR, Zalutsky MR and Vaidyanathan G. Uptake mechanisms of 123I-MIBG in isolated rat heart. Nucl. Med. Biol. (1995) 22: 1-12. (8) Trendelenburg U. Extraneuronal uptake and metabolism of catecholamines as a site of loss. Life Sci. (1978) 22: 1217-1222. (9) Vaidyanathan G, Afleck DJ, Alston KL, Zhao XG, Hens M, Hunter DH, Babich J and Zalutsky MR. A kit method for the high level synthesis of [211At] MABG. Bioorganic and Medicinal Chemistry. (2007) 15: 3430-3436. (10) Berry CR, Garg PK, Zalutsky MR. Coleman RE and DeGrado TR. Uptake and retention kinetics of para luorine-18 luorobenzylguanidine in isolated rat heart. J. Nucl. Med. (1996) 37: 2011-2016. (11) Garg P. K, Garg S, Zalutsky M. R. Synthesis and preliminary evaluation of para and meta 18-luorobenzylguanidine. Nucl. Med. Biol. (1994) 21: 97-103. (12) Vaidyanathan G, Afleck DJ, Alston KL, Zhao XG, Hens M, Hunter DH, Babich J and Zalutsky M R. A kit method for high level synthesis of 211At-MABG. Bioorganic and Medicinal Chemistry. (2007) 15: 3430-3436. (13) Loc’h C, Mardon K, Valette H, Brutesco C, Merlet P, Syrota A and Maziere B. preparation and pharmacological characterization of 76Br-MBBG. Nucl. Med. Biol. (1994) 21: 49-55. (14) Mock BH and Weiner RF. Simpliied solid state labeling of 123I-MIBG. Applied Radiation and Isotopes. (1988) 39: 939-942. Acknowledgment This study was supported inancially by a grant from Vice-Chancellor’s ofice for Research and Technology affairs, Shahid Beheshti University of Medical Sciences, Tehran, Iran. References (1) Wieland DM, Swanson DP, Brown EL and Beierwaltes WH. Imaging the adrenal medulla with an 131I-labeled antiadrenergic agent. J. Nucl. Med. (1979) 20: 155158. (2) Wieland DM, W JL, Brown LE, Mangner TJ, Swanson DP and Beierwaltes WH. Radiolabeled adrenergic neuron-blocking agents: adrenomedullary imaging with 131I-iodobenzylguanidine. J. Nucl. Med. (1980) 21: 349-353. (3) Wieland DM, Mangner TJ, Inbasekaran MN, Brown LE and Wu JL. Adrenal medulla imaging agents: a structure-distribution relationship study of radiolabeled aralkylguanidines. J. Med. Chem. (1984) 27: 149-155. 732 A convenient Simple Method for Synthesis of Meta-iodobenzylguanidine synthesis. (1941) Coll. Vol. 1, P: 102-104; (1928) Coll. Vol. 8, P: 38. (18) Cymerman CJ, Yong J. 1-benzyl piperazine. Organic synthesis. (1973) Coll. Vol. 5, P: 88; (1962) 42: 19. (19) Lecher H, Graf F. 1923, Ber. 56 B, 1326-1330. C.A, 1923; 17: 3319. (20) Osklo-Salmani A. Synthesis of alkylguanidines. Master of Sciences thesis in organic chemistry. Chemistry department, Faculty of basic Sciences, Imam Hossein University. (2003): 45-60. (15) Prabhakar G, Mehra K.S, Ramamoorthy N, Chattopadhyay S, Oommen R, Narasimhan S, Nair NA and Gunasekaran S. Evaluation of radioiodination of MIBG catalysed by in situ generated Cu(I) and directly added Cu(II). Applied Radiation and Isotopes. (1999) 50: 1011-1014. (16) Donovan A C and Valliant J F. A convenient solution phase method for the preparation of MIBG in high effective speciic activity. Nuclear Medicine and Biology (2008) 35: 741-746. (17) Willson FGI, Wheeler TS. Benzly aniline. Organic This article is available online at http://www.ijpr.ir 733