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Microwave-assisted (radio)halogenation of nitroimidazole-based Hypoxia markers
Applied Radiation and Isotopes 57 (2002) 697–703
Microwave-assisted (radio)halogenation of nitroimidazolebased Hypoxia markers P. Kumara, L.I. Wiebea,*, M. Asikoglua,1, M. Tandona, A.J.B. McEwanb a
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 3118 Dent-Pharm Bldg, Edmonton Alta, Canada T6G 2N8 b Cross Cancer Institute, Edmonton Alta, Canada T6G 2N8 Received 1 October 2001; accepted 12 June 2002
Abstract Microwave-assisted radiohalogenation for the production of short-lived radiopharmaceuticals has now been applied to the synthesis and radiolabelling of azomycin nucleosides. (Radio)halogens were incorporated either by nucleophilic substitution of a leaving group or by halogen–halogen exchange, in the synthesis of IAZA, IAZP and FAZA. A comparison of conventional labelling and microwave-assisted labelling procedures reflects a clear advantage of the microwave technique. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Microwave; (radio)halogenation; a-azomycin nucleosides; Hypoxia markers
1. Introduction The production of radiopharmaceuticals labelled with short-lived radionuclides requires procedures that afford the labelled product in a short reaction time. The use of Abbreviations: IAZA, 1-a-d-[50 -deoxy-50 -iodoarabinofuranosyl]-2-nitroimidazole; Ts-AZA, 1-a-d-[50 -O-toluenesulfonylarabinofuranosyl]-2-nitroimidazole; DAcTs-AZA, 1-a-d-[50 -Otoluenesulfonyl-20 ,30 -di-O-acetylarabinofuranosyl]-2-nitroimidazole; DAc-IAZA, 1-a-d-[50 -deoxy-50 -iodoarabinofuranosyl]2-nitroimidazole; AZP, 1-a-d-[arabinopyranosyl]-2-nitroimidazole; Ts-AZP, 1-a-d-[40 -O-toluenesulfonyl-arabinopyranosyl]-2nitroimidazole; 40 -DAcTs-AZP, 1-a-d-[40 -O-toluenesulfonylDAc20 ,30 -di-O-acetylarabinopyranosyl]-2-nitroimidazole; IAZP, 1-a-l-[40 -deoxy-40 -iodo-20 ,30 -di-O-acetylxylopyranosyl]2-nitroimidazole; IAZP, 1-a-l[-40 -deoxy-40 -iodo-xylopyranosyl]-2-nitroimidazole; FAZA, 1-a-d-[50 -deoxy-50 -fluoroarabinofuranosyl]-2-nitroimidazole *Corresponding author. Tel.: +1-780-492-5783; fax: +1780-435-0636. E-mail address: [email protected] (L.I. Wiebe). 1 Current address: Faculty of Pharmacy, Ege University, Bornova, Izmir 35100, Turkey.
microwave-assisted labelling has come into common usage because it enhances radiochemical and chemical yields by reducing reaction times without causing major degradation or introducing undesired reactions (Hwang et al., 1989; Lemaire et al., 1989). A number of azomycin derivatives have been evaluated as radiopharmaceuticals for the detection of regional tissue hypoxia. Their selectivity is attributed to initial single electron reduction of the nitro group to form an oxygen sensitive radical anion, which, upon further reduction in absence of oxygen, forms reactive intermediates that bind covalently to tissue macromolecules (Biaglow et al., 1986). [123I]IAZA, an azomycinbased nucleoside, has been studied in the patients with a variety of hypoxic disorders that include solid and malignant tumors (Parliament et al., 1992; Grosher et al., 1993; Urtasun et al., 1996), peripheral vascular disease (Al-Arafaj et al., 1994) and rheumatoid arthritis (McEwan et al., 1997). [18F]FAZA, the fluoro analog of IAZA, has shown similar hypoxia selective uptake in tumor-bearing mice (Kumar et al., 1999) and rat hypoxic liver (Patt et al., 1999; Kumar et al., 2000). IAZA and IAZP have traditionally been radiolabelled
0969-8043/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 4 3 ( 0 2 ) 0 0 1 8 5 - 9
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P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
by radioiodine/iodine exchange under conventional heating (Mannan, 1991; Mannan et al., 1992), a process that requires relatively long reaction times.
(399.374) calc. C, 45.11, H, 4.26, N, 10.53; found C, 44.98, H, 4.22 and N, 10.37.
2.2. Syntheses
2.2.2. 1-a-D-[50 -O-Toluenesulfonyl-20 ,30 -Di-Oacetylarabinofuranosyl]-2-nitroimidazole (DAcTs-AZA; 2) Acetic anhydride (0.75 ml) was added to a solution of 5 (400 mg, 1 mmol) and the mixture was stirred overnight at room temperature. The reaction was quenched by addition of water (1 ml) to the reaction mixture. The solvent was removed by evaporation on a rotavapor and the impure mixture was purified on a silica gel column using CH2Cl2:MeOH (97:3, v/v) as eluent to afford 450 mg (93%) of 2; Rf 0.55 (CH2Cl2:MeOH, 90:10, v/v); mp 1351C; 1H NMR (CDCl3)-d 1.97 and 2.16 (two s, 6 H, two CH3), 2.45 (s, 3H, toluyl CH3), 4.28 (d, Jgem ¼ 16 Hz, 2H, H-50 and H-500 ), 4.64 (m, 1H, H-30 ), 5.10 (m, 1H, H-40 ), 5.38 (m, 1H, H-20 ), 6.52 (d, J20 ;10 ¼ 1:5 Hz, 1H, H-10 ), 7.18 (d, J4;5 ¼ 1:0 Hz, 1H, H5), 7.31 (d, J5;4 ¼ 1:0 Hz, 1H, H-4), 7.37 (d, J2;3 ¼ J6;5 ¼ 8:8 Hz, 2H, phenyl H-3 and H-5) and 7.82 (d, J3;2 ¼ J5;6 ¼ 8:8 Hz, 2H, phenyl H-2 and H-6) ppm; 13 C NMR (CDCl3)-d 17.33 (COCH3), 21.60 (toluyl CH3), 67.61 (C-50 ), 76.31 (C-40 ), 80.96 (C-30 ), 85.03 (C20 ), 93.09 (C-100 ), 121.89 (C-5), 128.01 (aromatic C-3, C-5), 128.50 (C-4), 129.97 (aromatic C-2, C-4), 132.31 (aromatic C-1), 145.45 (C-2 nitroimidazole), 168.79 and 169.2263 (two C=O); analysis for C19H21N3O10S (483.44) calc. C, 47.20, H, 4.48, N, 8.69; found C, 47.33, H, 4.66 and N, 8.44.
2.2.1. 1-a-d-[50 -O-Toluenesulfonyl-arabinofuranosyl-2nitroimidazole (Ts-AZA; 5) 4-Tosyl chloride (284 mg; 1 mmol) was dissolved in anhydrous pyridine (1 ml) and added dropwise to a precooled (101C) solution of 1, (365 mg; 1.4 mmol) in anhydrous pyridine (3 ml). The reaction mixture was stirred at that temperature for 4 h and then quenched by adding few pieces of ice. The solvent was removed and the residual viscous material was purified on a silica gel column using MeOH/CH2Cl2 (3:97, v/v) as eluent to afford 310 mg (77%) of pure 5; Rf 0.43 (CH2Cl2:MeOH, 90:10); mp 621C; 1H NMR (CDCl3)-d 2.43 (s, 3H, CH3), 4.16–4.30 (m, 3H, H-40 , H-50 and H-500 ), 4.38 (d, J10 ;20 ¼ 1:5 Hz, 1H, H-20 ), 4.65 (d, J20 ;30 ¼ 1:5 Hz, of d, J40 ;30 ¼ 6:4 Hz, 1H, H-30 ), 6.29 (d, J20 ;10 ¼ 1:5 Hz, 1H, H10 ), 6.99 (d, J5;4 ¼ 1:0 Hz, 1H, H-4), 7.37 (d, J2;3 ¼ J6;5 ¼ 8:0 Hz, 2H, phenyl H-3 and H-5), 7.41 (d, J4;5 ¼ 1:0 Hz, 1H, H-5) and 7.78 (d, J3;2 ¼ J5;6 ¼ 8:0 Hz, 2H, phenyl H-2 and H-6) ppm; 13C NMR (CDCl3+CD3OD)-d 21.58 (toluyl CH3), 70.24 (C-50 ), 76.97 (C-40 ), 81.66 (C-30 ), 82.65 (C-20 ), 87.51 (C-100 ), 121.89 (C-5), 125.96 (aromatic C-3, C-5), 128.45 (C-4), 130.43 (aromatic C-2, C-6), 130.49 (aromatic C-1), 145.45 (C-2 nitroimidazole); analysis for C15H17N3O8S
2.2.3. 1-a-D-[50 -Deoxy-50 -Iodo-20 ,30 -Di-Oacetylarabinofuranosyl]-2-nitroimidazole (DAc-IAZA; 3) Sodium iodide (75 mg; 0.5 mmol) and 2, (50 mg; 0.1 mmol) were heated under reflux in anhydrous 2pentanone (5 ml) for 18 h. At this point, tlc showed no evidence of residual tosyl precursor. The solvent was removed and the impure product was purified over a silica gel column using hexanes/EtOAc (2:1, v/v) and to afford pure 3 as a viscous foam (43 mg; 93%); Rf 0.68 (CH2Cl2:MeOH, 97:3); 1H NMR (CDCl3)-d 2.02 and 2.23 (two s, each 3H, two CH3), 3.43 (d, Jgem ¼ 15:8 Hz, of d, J40 ;500 ¼ 4:0 Hz, 1H, H-500 ), 3.49 (d, Jgem ¼ 15:8 Hz of d, J40 ;50 ¼ 4:0 Hz, 1H, H-50 ), 4.66 (d, J30 ;40 ¼ 1:5 Hz of t, J50 ;40 ¼ J500 ;40 ¼ 4:0 Hz, 1H, H-40 ), 5.20 (d, J30 ;20 ¼ 2:0 Hz of d, J40 ;30 ¼ 1:5 Hz, 1H, H-30 ), 5.46 (d, J30 ;20 ¼ 2:0 Hz of d, J10 ;20 ¼ 0:9 Hz, 1H, H-20 ), 6.66 (d, J20 ;10 ¼ 0:9 Hz, 1H, H-10 ), 7.20 (d, J5;4 ¼ 0:9 Hz, 1H, H4) and 7.37 (d, J4;5 ¼ 0:9 Hz, 1H, H-5) ppm; 13C NMR (CDCl3)-d 2.56 (C-50 ), 20.53 (CH3), 78.13 (C-40 ), 81.21 (C-30 ), 87.11 (C-20 ), 92.92 (C-100 ), 121.99 (C-5), 128.48 (C-4), 168.45 (C=O at C-30 ) and 168.94 (C=O at C-20 ); +ve FAB for C12H14IN3O7 (439.15), M+ (100%).
2. Experimental 2.1. Materials All reagents were purchased from Aldrich-Sigma Chemical Co. and were used without further purification. The solvents were distilled over appropriate drying agents and collected fresh at the time of reaction. Sodium [125I]iodide and sodium [123I]iodide were purchased from Amersham Pharmacia Inc., and Nordion Canada, respectively. (Radio)halogenated products were purified either on a Waters HPLC system using a C-18 reverse phase column (300 4.5 mm2 or 300 9.0 mm2) attached to dual detectors (UV and radiometric), or by Waters Sep-Pak C-18 cartridge-assisted separation followed by HPLC analysis and/or silica gel thin layer chromatography (tlc) to determine compound purity. Microwave-assisted reactions were done in a 1 ml Reactivialt that was placed inside a Teflon (20 ml) containment vial to avoid contamination in the event of accidental vessel rupture during microwave irradiation. A 720 W, 60 Hz, 0.9 ft3 domestic microwave oven was used.
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
2.2.4. 1-a-D-[40 -O-Toluenesulfonylarabinopyranosyl]-2nitroimidazole (40 -Ts-AZP; 8) Azomycin pyranoside, 7, (0.1 g, 0.416 mmol) (Mannan et al, 1992) was dissolved in anhydrous methanol (15 ml) and dibutyltin oxide (0.12 g, 0.5 mmol) was added to this. The reaction mixture was heated under reflux for 1 h, after which, the solvent was removed by evaporation, the residue was dissolved in anhydrous toluene (10 ml) and re-evaporated. The contents were dried under hi-vacuum and re-dissolved in anhydrous toluene (20 ml). The reaction flask was attached to a Dean-Stark attachment, to remove water formed during the reaction, and pulverized molecular sieves (100 mg) were added to the reaction mixture. This was followed by the addition of Bu4NBr (0.2 g, 0.6 mmol) and toluene sulfonyl chloride (0.12 g, 0.6 mmol) and the mixture was refluxed under an inert atmosphere at 1101C. The tlc examination of the reaction mixture showed complete conversion of 7 to its toluenesulfonyl derivative 8 after 16 h. Ice was added to this mixture and the solvent was evaporated. This impure residue was purified on a silica gel column to obtain 123 mg (76%) of pure 8 as a gummy mass; 1H NMR (CDCl3)-d 2.34 (s, 3H, CH3), 3.91 (d, Jgem ¼ 12:6 Hz, 1H, H-500 ), 4.08 (d, Jgem ¼ 12:6 Hz, of d, J40 ;50 ¼ 2:0 Hz, 1H, H-50 ), 4.134.19 (m, br, 2H, H-20 , H-30 ), 4.59 (d, J30 ;04 ¼ 9:1 Hz, of d, J50 ;40 ¼ 2:0 Hz, 1H, H-40 ), 6.06 (d, J20 ;10 ¼ 8:8 Hz, 1H, H10 ), 6.95 (s, 1H, H-4), 7.25 (d, J=8.3 Hz, phenyl H-3 and H-5), 7.46 (s, 1H, H-5) and 7.7 (d, J=8.3 Hz, phenyl H-2 and H-6) ppm; 13C NMR (CDCl3)-d 21.58 (CH3), 58.34 (C-50 ), 67.59 (C-30 ), 68.98 (C-20 ), 82.85 (C-40 ), 86.04 (C10 ), 123.99 (C-10 ), 128.01 (C-5), 130.00 (C-4), 132.00 (C-4 phenyl), 136.70 (C-3 and C-5 phenyl), 144.54 (C-1 phenyl), 145.68 (C-2), 149.04 (C-2 and C-6 phenyl); +ve FAB for C15H17N3O8S (399.374), M+1 (100%). 2.2.5. 1-a-D-[40 -O-Toluenesulfonyl-20 ,30 -di-Oacetylarabinopyranosyl]-2-nitroimidazole (DAcTs-AZP, 9) Acetic anhydride (0.5 ml; 0.5 mmol) was added to a solution of 8 (0.1 g; 0.25 mmol) in anhydrous pyridine (2 ml), and the mixture was stirred at 251C until there was no evidence of 8 on tlc. The solvent was removed and the product was purified on a silica gel column using toluene/ethyl acetate (85:15; v/v) to afford 0.1 g (82%) of 9; m.p. 1261C; 1H NMR (CDCl3)-d 1.73 and 2.13 (two s, each for 3H, two COCH3), 2.46 (s, 3H, CH3), 3.88 (d, Jgem ¼ 13:6 Hz, 1H, H-500 ), 4.18 (d, Jgem ¼ 13:6 Hz, of d, J40 ;50 ¼ 1:7 Hz, 1H, H-50 ), 4.89 (d, J20 ;03 ¼ 9:4 Hz, of d, J40 ;30 ¼ 1:8 Hz, 1H, H-30 ), 5.32 (d, J50 ;40 ¼ 1:7 Hz of d, J40 ;30 ¼ 1:8 Hz, 1H, H-40 ), 5.47 (d, J30 ;02 ¼ 9:4 Hz, of d, J10 ;200 ¼ 9:1 Hz, 1H, H-20 ), 6.28 (d, J20 ;10 ¼ 9:1 Hz, 1H, H10 ), 7.17 (d, J5;4 ¼ 0:9 Hz, 1H, H-4), 7.35 (d, J=8.3 Hz, phenyl H-3 and H-5), 7.40 (d, J4;5 ¼ 0:9 Hz, 1H, H-5) and 7.76 (d, J=8.3 Hz, phenyl H-2 and H-6) ppm; 13C NMR (CDCl3)-d 20.11 and 20.79 (two COCH3), 21.72
699
(toluyl CH3), 76.57 (C-50 ), 76.78 (C-30 ), 77.00 (C-20 ), 77.54 (C-40 ), 83.89 (C-10 ), 122.02 (C-5), 127.77 (C-3 and C-5 phenyl), 128.96 (C-4), 129.98 (C-2 and C-6 phenyl), 133.05 (C-4 phenyl), 144.14 (C-1 phenyl), 145.68 (C-2), 168.87 and 169.42 (two C=O); +veFAB for C19H21N3O10S (483.444), M+1 (100%); calc. C, 47.20; H, 4.48, N, 8.69; found C, 47.33, H, 4.66 and N, 8.44.
2.2.6. 1-a-L-[40 -Iodo-40 -deoxy-20 ,30 -di-Oacetylxylopyranosyl]-2-nitroimidazole (DAc-a-IAZP, 11) A solution of 9 (0.1 g; 0.21 mmol) in anhydrous 2pentanone (5 ml) was heated at 601C with sodium iodide (0.16 g1.05 mmol) for 6 h. The solvent was removed after the reaction was complete. The impure residue was purified on a silica gel column using toluene/ethyl acetate (90:10, v/v) to give 59 mg (65%) of pure 11 as a foam; mp. 651C (softened); 1H NMR (CDCl3)-d 1.80 and 2.09 (two s, each for 3H, two COCH3), 3.88-4.09 (m, 3H,1H of H-40 and 2H of H-50 ), 4.84 (d, J20 ;03 ¼ 9:0 Hz, of d, J40 ;30 ¼ 4:8 Hz, 1H, H-30 ), 5.42 (d, J30 ;02 ¼ 9:0 Hz, of d, J10 ;200 ¼ 9:0 Hz, 1H, H-20 ), 6.12 (d, J20 ;10 ¼ 9:0 Hz, 1H, H-10 ), 7.14 (d, J5;4 ¼ 1:0 Hz, 1H, H4) and 7.66 (d, J4;5 ¼ 1:0 Hz, 1H, H-5) ppm; 13C NMR (CDCl3)-d 4.55 (C-40 ), 20.69 (two COCH3), 76.96 (C-50 ), 77.35 (C-30 ), 77.49 (C-20 ), 86.13 (C-10 ), 121.88 (C-5), 128.46 (C-4), 145.42 (C-2), 168.66 and 169.42 (two C=O); anal. for C12H14IN3O7 (439.15); calc. C, 32.82; H, 3.21, N, 9.57; found C, 32.44; H, 3.11, N, 9.55. AZA 1, IAZA 3a, AZP 8, IAZP 10 (Mannan et al., 1991; Kumar et al, 2000) and FAZA (4a; Kumar et al, 1999) were synthesized by literature methods.
2.3. Radioiodination by microwave-assisted isotopic halogen exchange A typical procedure involved evaporating the radioiodine solution (0.1 M aqueous NaOH in a Reactivialt) to dryness under a stream of nitrogen at 301C, followed by the addition of a solution of a neutralizing amount of organic acid (acetic acid or pivalic acid). The solvent was evaporated again, and substrate (haloazomycin nucleoside) was added as a solution in methanol. After evaporation to dryness, the vial was evacuated by a hivacuum pump, sealed, packaged inside the Teflon safety container, and placed in the center of the microwave oven cavity for irradiation. After microwave irradiation the mixture was cooled to room temperature, and then analyzed either by HPLC or by co-spotting with pure standard on tlc. HPLC eluant was collected 1 min intervals to determine reaction yield and product purity.
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700
2.4. Microwave-assisted nucleophilic halogen-substitution of tosylated nucleosides Radioiodinations: Aqueous sodium [125I]iodide (in 0.1 M NaOH in a Reactivialt) was evaporated to dryness and a solution of the tosylate precursor was added to the reaction vial. The vial, packaged inside the Teflon safety container, was irradiated with or without solvent, as desired, for a preset time, and then cooled to room temperature. This reaction mixture was analyzed by HPLC and/or tlc. Fluorination: Ammonium fluoride (4 mg) and Kryptofixt 2.2.2 (18.5 mg, 0.048 mmol) were added into a 1 ml Reactivialt, together with 2-pentanone (100 ml), and finally, the tosylate 2 (10.8 mg). The vial, packaged inside the Teflon safety container, was then irradiated in the microwave for 8 min. The mixture was cooled to room temperature, and the solvent was evaporated. The residue was treated with 2 M ammonia in methanol to remove the protecting groups, then analyzed by HPLC. This product was isolated and its structure confirmed by 19 F NMR.
2.5. Nucleophilic substitution of tosylate by conventional heating Iodination: The desired tosylate (50 mg; 0.1 mmol) was dissolved in 2-pentanone (2 ml) in a Reactivialt. Pulverized sodium iodide (74 mg; 0.5 mmol) was added, and the reaction mixture was heated at 1101C. Progress of the reaction was monitored by tlc until the reaction was complete (nearly 18 h), after which the solvent was evaporated.
Fluorination: The tosylated precursor 2 (10 mg; 0.02 mmol), ammonium fluoride (4 mg; 0.11 mmol) and Kryptofixt 2.2.2 (19.5 mg; 0.051 mmol) were dissolved in anhydrous 2-pentanone (1 ml) in a Reactivialt. The solution was heated at 601C for 18 h and the reaction was worked up as described (above). The formation of FAZA was confirmed by co-injecting the standard FAZA with the reaction mixture on HPLC. Radiofluorination. The tosylate precursor 2 was converted to [18F]FAZA by conventional heating technique as described in the literature (Patt et al, 1999).
3. Results and discussion Microwaves are sinusoidally reversing magnetic fields, which set electrons of molecules in the field into motion in a direction opposite to the magnetic field. This generates rapid reversal of polarity, and induces varying charge distributions in the molecules. An optimal distribution of charge changes the polarity of the environment (solvent), thereby increasing the reactivity of the molecules. The nature of the solvent, intramolecular hydrogen bonding, the sample’s polarity, dielectric constant, and related factors play important roles in microwave-assisted reactions. The domestic microwave oven operates at a microwave frequency of 2450 MHz, to exploit O–H and C–H rotational frequencies (Gedye et al, 1986). The data in Tables 1 and 2 compare radioiodination reactions and yields for IAZA 3a, IAZP 10 and FAZA 4a by conventional heating and microwave-assisted (radio)halogenation techniques. Since, both IAZA and IAZP are susceptible to alkaline hydrolysis, acetic acid
Table 1 Radioiodination of IAZA 6, DAc-IAZA 3a, IAZP 10 and DAc-IAZP 11 by the exchange method using conventional heating (C) and microwave assisted (M) synthesis techniques Reaction no. 1 2 3 4 5 6 7 8 9 10 11 12 13
Substrate 6 6 6 6 6 6 3 3 6 10 10 10 11
1 10 20 20 100 100 100 100 100 1a 3.5a 100 3.5a 10
Solvent None None None P P P P P None None MeOH None P
Neutralizer (mg) None None None None A (176) A (176) None A (176) PA (3.5)a PA (2.7)a PA (1.0)a PA (2.7)a PA (1.0)a
Time (min) 8 6 12 5 5 8 8 8 80 120 8 120 8
CY (%) 100 100 100 100 99 100 100 100 90 45 95 65 100
RCY (%)
Product
Technique
38 27 29 17 33 42 87 74 90 36 33 55 23
125
M M M M M M M M C, 901C C, 1301C M C, 1151C M
a Amount in mg. P: 2-Pentanone, A: glacial acetic acid (mg), PA: pivalic acid, CY: chemical yield, RCY: radiochemical yield.
I-3a I-3a 125 I-3a 125 I-3a 125 I-3a 125 I-3a 125 I-3 125 I-3 125 I-3a 125 I-10 125 I-10 123 I-10 125 I-11 125
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
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Table 2 (Radio)iodination and fluorination of 5, 2, and 9 by nucleophilic substitution using conventional heating (C) and microwave-assisted (M) labelling techniques Reaction no.
Substrate
Amount (mg)
Solvent
Time (min)
CY (%)
RCY (%)
14 15 16 17 18 19 20
2 5 2 2 2 2 9
50a 10 10 10 10a 10a 10
P P None None P/NH4F P/NH4 P
18b 5 8 13 18b 8 8
93 Co-elutedc Co-eluted Co-eluted 25 50 100
NA 90 90 33.5 NA NA 88
Product
Technique
3
C, 1101C M M M C, 601C M M
125
I-3a 125 I-3 125 I-3 4a 4a 125 I-11
a
Amount in mg. Time in h. c The radiolabelled product, obtained by nucleophilic substitution, was measured by both radiometry and UV (by adding calculated amounts of cold product). P: 2-pentanone, NA: not applicable since it is not a radiohalogenated product, CY: chemical yield, RCY: radiochemical yield. b
HO
TsO a,b
O HO
X
O AcO
c or d M/C Heating 2-NI
2-NI OH 1
2-NI OAc 3; X = I, 125I, 123I 4; X = F, 18F
OAc 2
g I
O AcO
O HO
a
2-NI
e or f
OH 6 TsO
O HO
I* c M/C Heating 2-NI
X
O HO
2-NI
2-NI OH
OH
OH
3a; I* = 125I
5
O HO
3a; X = I, 125I, 123I 4a; X = F, 18F
N where, 2-NI = N
NO2
where, a = toluenesulfonyl chloride/pyridine; b = Ac2O/pyridine; c =NaI*/2-pentanone; d = NH4F/KryptofixTM 2.2.2 or 18F and 2-pentanone; e = NH3/MeOH and f = 1N.NaOH
Scheme 1. Synthetic route to the 50 -(radio)halogenated azomycin arabinosides IAZA and FAZA.
or pivalic acid (trimethyl acetic acid) was added to neutralize the NaOH in the radioiodide solution. Several reactions were conducted in the absence of neutralizing acid in order to compare the chemical and radiochemical yields of labelled products in neutral and in alkaline solution. Syntheses of [125I]IAZA and [125I]IAZP require large amounts of precursor and long reaction times (e.g. 1.3–2 h) at elevated temperatures (90–1301C) under conventional heating conditions (Table 1, reactions 9, 10 and 12). The syntheses of FAZA and 3 took even
longer (18 h), with substantial chemical decomposition of the product and concomitant lower chemical yields (Table 2, reactions 18 and 14, respectively). When these (radio)syntheses were performed in the microwave, the corresponding products were obtained in very short reaction times (8 min; Schemes 1 and 2), which reduced chemical decomposition of the precursor/product, and consequently contributed to improved (radio)chemical yields. Surprisingly, the synthesis of [125I]IAZA by conventional heating procedures using exchange of radioiodine
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
702
O HO TsO OH
O AcO
c TsO
2-NI
I*
d
O AcO
M/C Heating OAc 9
2-NI
2-NI OAc 125 11; I* = I, I
a,b I
O HO
e
2-NI
HO
I*
O HO
OH
OH
7
10
d 2-NI
O HO
M/C Heating OH 10; I* =
2-NI 125 123
I,
I
N where 2-NI = N
NO2
Reaction conditions: a = Sn2O/MeOH; b = toluenesulfonyl chloride/toluene; c = Ac2O/pyridine; d = NaI* and e = Ph3P/I2
Scheme 2. Synthetic route to (radio)iodinated IAZP.
afforded the product in 90% radiochemical and 90% chemical yields (reaction 9, Table 1), whereas microwave-assisted radioiodine exchange with IAZA afforded only a 42% radiochemical yield. The microwave-assisted reaction was complete in 8 min, and there was no evidence of chemical decomposition (100% chemical yield, reaction 6, Table 1). Low radiochemical yields for microwave-assisted exchange of radioiodine in IAZA are related to the presence of free hydroxyl groups in the sugar moiety, since similar exchange radioiodination reactions with the corresponding acetylated product 3 provided much higher radiochemical yields (87%), with minimal chemical degradation (chemical yield 100%, reaction 7, Table 1). Similar, but less dramatic, effects are seen during the exchange labelling of IAZP and its diacetylated analogue 11 (reactions 10–13, Table 1).
4. Summary The microwave technique has been applied to nucleophilic substitution of a toluenesulfonyl (tosyl) group at C-50 of AZA to afford IAZA or FAZA, using the respective nucleophile. Conventional heating procedures to prepare IAZA and FAZA from 2 took longer (up to 18 h) and provided lower chemical yields (e.g. 25% vs. 50% for FAZA) than the microwave-assisted synthesis in each case. The microwave technique afforded IAZA, DAc-IAZA 3a and DAc-IAZP 11 without chemical degradation, in nearly 90% radiochemical yields within 5–8 min. The amount of precursor required for microwave labelling was only 1–10% of that used routinely for conventional exchange reactions. The most important features of microwave-assisted synthesis of IAZA, IAZP and FAZA are high yield,
reduced reaction time and the reduced amounts of precursors required. This leads to the syntheses of labelled products with higher chemical and radiochemical yields, with higher specific activities, in relatively short reaction times.
Acknowledgements The authors are grateful to Alberta Cancer Board for providing financial support (Grant RI-14) to this project.
References Al-Arafaj, A., Ryan, E.A., Hutchinson, K., Mannan, R.H., Mercer, J.R., Wiebe, L.I., McEwan, A.J.B., 1994. An evaluation of iodine-123 iodoazomycin arabinoside as a marker of localized tissue hypoxia in patients diabetes mellitus. Eur. J. Nucl. Med. 21, 1338–1342. Biaglow, J.E., Varnes, M.E., Rizen-Towle, L., Clark, E.P., Epp, F.R., Astor, M.B., Hall, E.J., 1986. Biochemistry of reduction of nitroheterocycles. Biochem. Pharmacol. 35, 77. Gedye, R., Smith, F., Westaway, K., Ali, H., Baldisera, L., Laberge, L., Rousell, J., 1986. The use of microwave ovens for rapid organic synthesis. Tetrahedron Lett. 27, 279. Grosher, D., McEwan, A.J.B., Parliament, M.B., Urtasun, R.C., Goldberg, L.E., Hoskinson, M., Mercer, J.R., Mannan, R.H., Wiebe, L.I., Chapman, J.D., 1993. Imaging tumor Hypoxia and tumor perfusion. J. Nucl. Med. 34, 885. Hwang, D.R., Moerlein, S.M., Welch, M.J., 1989. Microwavefacilitated synthesis of [18F]-Spiperone. J. Lab. Compd. Radiopharm. 26, 391 (Abstract). Kumar, P., Stypinski, D., Xia, H., McEwan, A.J.B., Machulla, H-J., Wiebe, L.I., 1999. Fluoroazomycin arabinoside (FAZA): synthesis, 2H and 3H-labelling and preliminary
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703 biological evaluation of a novel 2-nitroimidazole marker of tissue hypoxia. J. Labelled Compd. Radiopharm. 42, 3–16. Kumar, P., Patt, M., Machulla, H-J., Mc Ewan, A.J.B., Wiebe, L.I., 2000. [18F]-FAZA: A putative PET radiotracer for hypoxia. In: Ulrich Pleiss, Rolf Voges (Eds.), Synthesis and Applications of Isotopically Labelled Compounds. Wiley, UK. Lemaire, C., Cantineau, R., Christiaens, L., Guillaume, M., 1989. Radiofluorination of Altanserine a potential serotonin receptor binding radiopharmaceutical for positron emission tomography. J. Lab. Compd. Radiopharm. 36, 336 (Abstract). Mannan, R.H., 1991. Novel non-invasive markers of tumor hypoxia. Ph.D. Thesis, University of Alberta, Edmonton, Canada, pp. 53–58. Mannan, R.H., Somayaji, V.V., Lee, J., Mercer, J.R., Chapman, J.D., Wiebe, L.I., 1991. Radioiodinated 1-(5-Iodo-5deoxy-b-D-arabinofuranosyl)-2-nitroimidazole (Iodoazomycin Arabinoside: IAZA), a Novel Marker of Tissue Hypoxia. J. Nucl. Med. 32, 1764–1770. Mannan, R.H., Mercer, J.R., Wiebe, L.I., Kumar, P., Somayaji, V.V., Chapman, J.D., 1992. Radioiodinated
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azomycin pyranoside (IAZP): a novel non-invasive marker for the assessment of tumor hypoxia. J. Nucl. Biol. Med. 36, 60–67. McEwan, A.J.B., Skeith, K.J., Mannan, R.H., Davies, N., Jamali, F., Schmidt, R., Golberg, K., Wiebe, L.I., 1997. I123 Iodoazomycin arabinoside (IAZA) may have a role in imaging rheumatoid arthritis. J. Nucl. Med. 38, 300. Parliament, M.B., Chapman, J.D., Urtasun, R.C., McEwan, A.J., Goldberg, L.E., Mercer, J.R., Mannan, R.H., Wiebe, L.I., 1992. Non-invasive assessment of human tumor hypoxia with 123I-iodoazomycin arabinoside: preliminary report of a clinical study. Br. J. Cancer. 65, 90–95. Patt, M., Kumar, P., Wiebe, L.I.,Machulla, H.-J., 1999. [18F]Fluoroazomycin-arabinosid (FAZA), ein neuer Radiotracer zur Bestimmung von Gewebe-Hypoxien. International Annual Conference, Deut. G. Nuklearmed. Ulm, Germany. Urtasun, R.C., McEwan, A.J., Parliament, M.B., Mercer, J.R., Mannan, R.H., Wiebe, L.I., Morin, C., Chapman, J.D., 1996. Measurement of hypoxia in human tumours by noninvasive spect imaging of iodoazomycin arabinoside. Br. J. Cancer. 74, S209.
Microwave-assisted (radio)halogenation of nitroimidazolebased Hypoxia markers P. Kumara, L.I. Wiebea,*, M. Asikoglua,1, M. Tandona, A.J.B. McEwanb a
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 3118 Dent-Pharm Bldg, Edmonton Alta, Canada T6G 2N8 b Cross Cancer Institute, Edmonton Alta, Canada T6G 2N8 Received 1 October 2001; accepted 12 June 2002
Abstract Microwave-assisted radiohalogenation for the production of short-lived radiopharmaceuticals has now been applied to the synthesis and radiolabelling of azomycin nucleosides. (Radio)halogens were incorporated either by nucleophilic substitution of a leaving group or by halogen–halogen exchange, in the synthesis of IAZA, IAZP and FAZA. A comparison of conventional labelling and microwave-assisted labelling procedures reflects a clear advantage of the microwave technique. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Microwave; (radio)halogenation; a-azomycin nucleosides; Hypoxia markers
1. Introduction The production of radiopharmaceuticals labelled with short-lived radionuclides requires procedures that afford the labelled product in a short reaction time. The use of Abbreviations: IAZA, 1-a-d-[50 -deoxy-50 -iodoarabinofuranosyl]-2-nitroimidazole; Ts-AZA, 1-a-d-[50 -O-toluenesulfonylarabinofuranosyl]-2-nitroimidazole; DAcTs-AZA, 1-a-d-[50 -Otoluenesulfonyl-20 ,30 -di-O-acetylarabinofuranosyl]-2-nitroimidazole; DAc-IAZA, 1-a-d-[50 -deoxy-50 -iodoarabinofuranosyl]2-nitroimidazole; AZP, 1-a-d-[arabinopyranosyl]-2-nitroimidazole; Ts-AZP, 1-a-d-[40 -O-toluenesulfonyl-arabinopyranosyl]-2nitroimidazole; 40 -DAcTs-AZP, 1-a-d-[40 -O-toluenesulfonylDAc20 ,30 -di-O-acetylarabinopyranosyl]-2-nitroimidazole; IAZP, 1-a-l-[40 -deoxy-40 -iodo-20 ,30 -di-O-acetylxylopyranosyl]2-nitroimidazole; IAZP, 1-a-l[-40 -deoxy-40 -iodo-xylopyranosyl]-2-nitroimidazole; FAZA, 1-a-d-[50 -deoxy-50 -fluoroarabinofuranosyl]-2-nitroimidazole *Corresponding author. Tel.: +1-780-492-5783; fax: +1780-435-0636. E-mail address: [email protected] (L.I. Wiebe). 1 Current address: Faculty of Pharmacy, Ege University, Bornova, Izmir 35100, Turkey.
microwave-assisted labelling has come into common usage because it enhances radiochemical and chemical yields by reducing reaction times without causing major degradation or introducing undesired reactions (Hwang et al., 1989; Lemaire et al., 1989). A number of azomycin derivatives have been evaluated as radiopharmaceuticals for the detection of regional tissue hypoxia. Their selectivity is attributed to initial single electron reduction of the nitro group to form an oxygen sensitive radical anion, which, upon further reduction in absence of oxygen, forms reactive intermediates that bind covalently to tissue macromolecules (Biaglow et al., 1986). [123I]IAZA, an azomycinbased nucleoside, has been studied in the patients with a variety of hypoxic disorders that include solid and malignant tumors (Parliament et al., 1992; Grosher et al., 1993; Urtasun et al., 1996), peripheral vascular disease (Al-Arafaj et al., 1994) and rheumatoid arthritis (McEwan et al., 1997). [18F]FAZA, the fluoro analog of IAZA, has shown similar hypoxia selective uptake in tumor-bearing mice (Kumar et al., 1999) and rat hypoxic liver (Patt et al., 1999; Kumar et al., 2000). IAZA and IAZP have traditionally been radiolabelled
0969-8043/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 4 3 ( 0 2 ) 0 0 1 8 5 - 9
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by radioiodine/iodine exchange under conventional heating (Mannan, 1991; Mannan et al., 1992), a process that requires relatively long reaction times.
(399.374) calc. C, 45.11, H, 4.26, N, 10.53; found C, 44.98, H, 4.22 and N, 10.37.
2.2. Syntheses
2.2.2. 1-a-D-[50 -O-Toluenesulfonyl-20 ,30 -Di-Oacetylarabinofuranosyl]-2-nitroimidazole (DAcTs-AZA; 2) Acetic anhydride (0.75 ml) was added to a solution of 5 (400 mg, 1 mmol) and the mixture was stirred overnight at room temperature. The reaction was quenched by addition of water (1 ml) to the reaction mixture. The solvent was removed by evaporation on a rotavapor and the impure mixture was purified on a silica gel column using CH2Cl2:MeOH (97:3, v/v) as eluent to afford 450 mg (93%) of 2; Rf 0.55 (CH2Cl2:MeOH, 90:10, v/v); mp 1351C; 1H NMR (CDCl3)-d 1.97 and 2.16 (two s, 6 H, two CH3), 2.45 (s, 3H, toluyl CH3), 4.28 (d, Jgem ¼ 16 Hz, 2H, H-50 and H-500 ), 4.64 (m, 1H, H-30 ), 5.10 (m, 1H, H-40 ), 5.38 (m, 1H, H-20 ), 6.52 (d, J20 ;10 ¼ 1:5 Hz, 1H, H-10 ), 7.18 (d, J4;5 ¼ 1:0 Hz, 1H, H5), 7.31 (d, J5;4 ¼ 1:0 Hz, 1H, H-4), 7.37 (d, J2;3 ¼ J6;5 ¼ 8:8 Hz, 2H, phenyl H-3 and H-5) and 7.82 (d, J3;2 ¼ J5;6 ¼ 8:8 Hz, 2H, phenyl H-2 and H-6) ppm; 13 C NMR (CDCl3)-d 17.33 (COCH3), 21.60 (toluyl CH3), 67.61 (C-50 ), 76.31 (C-40 ), 80.96 (C-30 ), 85.03 (C20 ), 93.09 (C-100 ), 121.89 (C-5), 128.01 (aromatic C-3, C-5), 128.50 (C-4), 129.97 (aromatic C-2, C-4), 132.31 (aromatic C-1), 145.45 (C-2 nitroimidazole), 168.79 and 169.2263 (two C=O); analysis for C19H21N3O10S (483.44) calc. C, 47.20, H, 4.48, N, 8.69; found C, 47.33, H, 4.66 and N, 8.44.
2.2.1. 1-a-d-[50 -O-Toluenesulfonyl-arabinofuranosyl-2nitroimidazole (Ts-AZA; 5) 4-Tosyl chloride (284 mg; 1 mmol) was dissolved in anhydrous pyridine (1 ml) and added dropwise to a precooled (101C) solution of 1, (365 mg; 1.4 mmol) in anhydrous pyridine (3 ml). The reaction mixture was stirred at that temperature for 4 h and then quenched by adding few pieces of ice. The solvent was removed and the residual viscous material was purified on a silica gel column using MeOH/CH2Cl2 (3:97, v/v) as eluent to afford 310 mg (77%) of pure 5; Rf 0.43 (CH2Cl2:MeOH, 90:10); mp 621C; 1H NMR (CDCl3)-d 2.43 (s, 3H, CH3), 4.16–4.30 (m, 3H, H-40 , H-50 and H-500 ), 4.38 (d, J10 ;20 ¼ 1:5 Hz, 1H, H-20 ), 4.65 (d, J20 ;30 ¼ 1:5 Hz, of d, J40 ;30 ¼ 6:4 Hz, 1H, H-30 ), 6.29 (d, J20 ;10 ¼ 1:5 Hz, 1H, H10 ), 6.99 (d, J5;4 ¼ 1:0 Hz, 1H, H-4), 7.37 (d, J2;3 ¼ J6;5 ¼ 8:0 Hz, 2H, phenyl H-3 and H-5), 7.41 (d, J4;5 ¼ 1:0 Hz, 1H, H-5) and 7.78 (d, J3;2 ¼ J5;6 ¼ 8:0 Hz, 2H, phenyl H-2 and H-6) ppm; 13C NMR (CDCl3+CD3OD)-d 21.58 (toluyl CH3), 70.24 (C-50 ), 76.97 (C-40 ), 81.66 (C-30 ), 82.65 (C-20 ), 87.51 (C-100 ), 121.89 (C-5), 125.96 (aromatic C-3, C-5), 128.45 (C-4), 130.43 (aromatic C-2, C-6), 130.49 (aromatic C-1), 145.45 (C-2 nitroimidazole); analysis for C15H17N3O8S
2.2.3. 1-a-D-[50 -Deoxy-50 -Iodo-20 ,30 -Di-Oacetylarabinofuranosyl]-2-nitroimidazole (DAc-IAZA; 3) Sodium iodide (75 mg; 0.5 mmol) and 2, (50 mg; 0.1 mmol) were heated under reflux in anhydrous 2pentanone (5 ml) for 18 h. At this point, tlc showed no evidence of residual tosyl precursor. The solvent was removed and the impure product was purified over a silica gel column using hexanes/EtOAc (2:1, v/v) and to afford pure 3 as a viscous foam (43 mg; 93%); Rf 0.68 (CH2Cl2:MeOH, 97:3); 1H NMR (CDCl3)-d 2.02 and 2.23 (two s, each 3H, two CH3), 3.43 (d, Jgem ¼ 15:8 Hz, of d, J40 ;500 ¼ 4:0 Hz, 1H, H-500 ), 3.49 (d, Jgem ¼ 15:8 Hz of d, J40 ;50 ¼ 4:0 Hz, 1H, H-50 ), 4.66 (d, J30 ;40 ¼ 1:5 Hz of t, J50 ;40 ¼ J500 ;40 ¼ 4:0 Hz, 1H, H-40 ), 5.20 (d, J30 ;20 ¼ 2:0 Hz of d, J40 ;30 ¼ 1:5 Hz, 1H, H-30 ), 5.46 (d, J30 ;20 ¼ 2:0 Hz of d, J10 ;20 ¼ 0:9 Hz, 1H, H-20 ), 6.66 (d, J20 ;10 ¼ 0:9 Hz, 1H, H-10 ), 7.20 (d, J5;4 ¼ 0:9 Hz, 1H, H4) and 7.37 (d, J4;5 ¼ 0:9 Hz, 1H, H-5) ppm; 13C NMR (CDCl3)-d 2.56 (C-50 ), 20.53 (CH3), 78.13 (C-40 ), 81.21 (C-30 ), 87.11 (C-20 ), 92.92 (C-100 ), 121.99 (C-5), 128.48 (C-4), 168.45 (C=O at C-30 ) and 168.94 (C=O at C-20 ); +ve FAB for C12H14IN3O7 (439.15), M+ (100%).
2. Experimental 2.1. Materials All reagents were purchased from Aldrich-Sigma Chemical Co. and were used without further purification. The solvents were distilled over appropriate drying agents and collected fresh at the time of reaction. Sodium [125I]iodide and sodium [123I]iodide were purchased from Amersham Pharmacia Inc., and Nordion Canada, respectively. (Radio)halogenated products were purified either on a Waters HPLC system using a C-18 reverse phase column (300 4.5 mm2 or 300 9.0 mm2) attached to dual detectors (UV and radiometric), or by Waters Sep-Pak C-18 cartridge-assisted separation followed by HPLC analysis and/or silica gel thin layer chromatography (tlc) to determine compound purity. Microwave-assisted reactions were done in a 1 ml Reactivialt that was placed inside a Teflon (20 ml) containment vial to avoid contamination in the event of accidental vessel rupture during microwave irradiation. A 720 W, 60 Hz, 0.9 ft3 domestic microwave oven was used.
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
2.2.4. 1-a-D-[40 -O-Toluenesulfonylarabinopyranosyl]-2nitroimidazole (40 -Ts-AZP; 8) Azomycin pyranoside, 7, (0.1 g, 0.416 mmol) (Mannan et al, 1992) was dissolved in anhydrous methanol (15 ml) and dibutyltin oxide (0.12 g, 0.5 mmol) was added to this. The reaction mixture was heated under reflux for 1 h, after which, the solvent was removed by evaporation, the residue was dissolved in anhydrous toluene (10 ml) and re-evaporated. The contents were dried under hi-vacuum and re-dissolved in anhydrous toluene (20 ml). The reaction flask was attached to a Dean-Stark attachment, to remove water formed during the reaction, and pulverized molecular sieves (100 mg) were added to the reaction mixture. This was followed by the addition of Bu4NBr (0.2 g, 0.6 mmol) and toluene sulfonyl chloride (0.12 g, 0.6 mmol) and the mixture was refluxed under an inert atmosphere at 1101C. The tlc examination of the reaction mixture showed complete conversion of 7 to its toluenesulfonyl derivative 8 after 16 h. Ice was added to this mixture and the solvent was evaporated. This impure residue was purified on a silica gel column to obtain 123 mg (76%) of pure 8 as a gummy mass; 1H NMR (CDCl3)-d 2.34 (s, 3H, CH3), 3.91 (d, Jgem ¼ 12:6 Hz, 1H, H-500 ), 4.08 (d, Jgem ¼ 12:6 Hz, of d, J40 ;50 ¼ 2:0 Hz, 1H, H-50 ), 4.134.19 (m, br, 2H, H-20 , H-30 ), 4.59 (d, J30 ;04 ¼ 9:1 Hz, of d, J50 ;40 ¼ 2:0 Hz, 1H, H-40 ), 6.06 (d, J20 ;10 ¼ 8:8 Hz, 1H, H10 ), 6.95 (s, 1H, H-4), 7.25 (d, J=8.3 Hz, phenyl H-3 and H-5), 7.46 (s, 1H, H-5) and 7.7 (d, J=8.3 Hz, phenyl H-2 and H-6) ppm; 13C NMR (CDCl3)-d 21.58 (CH3), 58.34 (C-50 ), 67.59 (C-30 ), 68.98 (C-20 ), 82.85 (C-40 ), 86.04 (C10 ), 123.99 (C-10 ), 128.01 (C-5), 130.00 (C-4), 132.00 (C-4 phenyl), 136.70 (C-3 and C-5 phenyl), 144.54 (C-1 phenyl), 145.68 (C-2), 149.04 (C-2 and C-6 phenyl); +ve FAB for C15H17N3O8S (399.374), M+1 (100%). 2.2.5. 1-a-D-[40 -O-Toluenesulfonyl-20 ,30 -di-Oacetylarabinopyranosyl]-2-nitroimidazole (DAcTs-AZP, 9) Acetic anhydride (0.5 ml; 0.5 mmol) was added to a solution of 8 (0.1 g; 0.25 mmol) in anhydrous pyridine (2 ml), and the mixture was stirred at 251C until there was no evidence of 8 on tlc. The solvent was removed and the product was purified on a silica gel column using toluene/ethyl acetate (85:15; v/v) to afford 0.1 g (82%) of 9; m.p. 1261C; 1H NMR (CDCl3)-d 1.73 and 2.13 (two s, each for 3H, two COCH3), 2.46 (s, 3H, CH3), 3.88 (d, Jgem ¼ 13:6 Hz, 1H, H-500 ), 4.18 (d, Jgem ¼ 13:6 Hz, of d, J40 ;50 ¼ 1:7 Hz, 1H, H-50 ), 4.89 (d, J20 ;03 ¼ 9:4 Hz, of d, J40 ;30 ¼ 1:8 Hz, 1H, H-30 ), 5.32 (d, J50 ;40 ¼ 1:7 Hz of d, J40 ;30 ¼ 1:8 Hz, 1H, H-40 ), 5.47 (d, J30 ;02 ¼ 9:4 Hz, of d, J10 ;200 ¼ 9:1 Hz, 1H, H-20 ), 6.28 (d, J20 ;10 ¼ 9:1 Hz, 1H, H10 ), 7.17 (d, J5;4 ¼ 0:9 Hz, 1H, H-4), 7.35 (d, J=8.3 Hz, phenyl H-3 and H-5), 7.40 (d, J4;5 ¼ 0:9 Hz, 1H, H-5) and 7.76 (d, J=8.3 Hz, phenyl H-2 and H-6) ppm; 13C NMR (CDCl3)-d 20.11 and 20.79 (two COCH3), 21.72
699
(toluyl CH3), 76.57 (C-50 ), 76.78 (C-30 ), 77.00 (C-20 ), 77.54 (C-40 ), 83.89 (C-10 ), 122.02 (C-5), 127.77 (C-3 and C-5 phenyl), 128.96 (C-4), 129.98 (C-2 and C-6 phenyl), 133.05 (C-4 phenyl), 144.14 (C-1 phenyl), 145.68 (C-2), 168.87 and 169.42 (two C=O); +veFAB for C19H21N3O10S (483.444), M+1 (100%); calc. C, 47.20; H, 4.48, N, 8.69; found C, 47.33, H, 4.66 and N, 8.44.
2.2.6. 1-a-L-[40 -Iodo-40 -deoxy-20 ,30 -di-Oacetylxylopyranosyl]-2-nitroimidazole (DAc-a-IAZP, 11) A solution of 9 (0.1 g; 0.21 mmol) in anhydrous 2pentanone (5 ml) was heated at 601C with sodium iodide (0.16 g1.05 mmol) for 6 h. The solvent was removed after the reaction was complete. The impure residue was purified on a silica gel column using toluene/ethyl acetate (90:10, v/v) to give 59 mg (65%) of pure 11 as a foam; mp. 651C (softened); 1H NMR (CDCl3)-d 1.80 and 2.09 (two s, each for 3H, two COCH3), 3.88-4.09 (m, 3H,1H of H-40 and 2H of H-50 ), 4.84 (d, J20 ;03 ¼ 9:0 Hz, of d, J40 ;30 ¼ 4:8 Hz, 1H, H-30 ), 5.42 (d, J30 ;02 ¼ 9:0 Hz, of d, J10 ;200 ¼ 9:0 Hz, 1H, H-20 ), 6.12 (d, J20 ;10 ¼ 9:0 Hz, 1H, H-10 ), 7.14 (d, J5;4 ¼ 1:0 Hz, 1H, H4) and 7.66 (d, J4;5 ¼ 1:0 Hz, 1H, H-5) ppm; 13C NMR (CDCl3)-d 4.55 (C-40 ), 20.69 (two COCH3), 76.96 (C-50 ), 77.35 (C-30 ), 77.49 (C-20 ), 86.13 (C-10 ), 121.88 (C-5), 128.46 (C-4), 145.42 (C-2), 168.66 and 169.42 (two C=O); anal. for C12H14IN3O7 (439.15); calc. C, 32.82; H, 3.21, N, 9.57; found C, 32.44; H, 3.11, N, 9.55. AZA 1, IAZA 3a, AZP 8, IAZP 10 (Mannan et al., 1991; Kumar et al, 2000) and FAZA (4a; Kumar et al, 1999) were synthesized by literature methods.
2.3. Radioiodination by microwave-assisted isotopic halogen exchange A typical procedure involved evaporating the radioiodine solution (0.1 M aqueous NaOH in a Reactivialt) to dryness under a stream of nitrogen at 301C, followed by the addition of a solution of a neutralizing amount of organic acid (acetic acid or pivalic acid). The solvent was evaporated again, and substrate (haloazomycin nucleoside) was added as a solution in methanol. After evaporation to dryness, the vial was evacuated by a hivacuum pump, sealed, packaged inside the Teflon safety container, and placed in the center of the microwave oven cavity for irradiation. After microwave irradiation the mixture was cooled to room temperature, and then analyzed either by HPLC or by co-spotting with pure standard on tlc. HPLC eluant was collected 1 min intervals to determine reaction yield and product purity.
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
700
2.4. Microwave-assisted nucleophilic halogen-substitution of tosylated nucleosides Radioiodinations: Aqueous sodium [125I]iodide (in 0.1 M NaOH in a Reactivialt) was evaporated to dryness and a solution of the tosylate precursor was added to the reaction vial. The vial, packaged inside the Teflon safety container, was irradiated with or without solvent, as desired, for a preset time, and then cooled to room temperature. This reaction mixture was analyzed by HPLC and/or tlc. Fluorination: Ammonium fluoride (4 mg) and Kryptofixt 2.2.2 (18.5 mg, 0.048 mmol) were added into a 1 ml Reactivialt, together with 2-pentanone (100 ml), and finally, the tosylate 2 (10.8 mg). The vial, packaged inside the Teflon safety container, was then irradiated in the microwave for 8 min. The mixture was cooled to room temperature, and the solvent was evaporated. The residue was treated with 2 M ammonia in methanol to remove the protecting groups, then analyzed by HPLC. This product was isolated and its structure confirmed by 19 F NMR.
2.5. Nucleophilic substitution of tosylate by conventional heating Iodination: The desired tosylate (50 mg; 0.1 mmol) was dissolved in 2-pentanone (2 ml) in a Reactivialt. Pulverized sodium iodide (74 mg; 0.5 mmol) was added, and the reaction mixture was heated at 1101C. Progress of the reaction was monitored by tlc until the reaction was complete (nearly 18 h), after which the solvent was evaporated.
Fluorination: The tosylated precursor 2 (10 mg; 0.02 mmol), ammonium fluoride (4 mg; 0.11 mmol) and Kryptofixt 2.2.2 (19.5 mg; 0.051 mmol) were dissolved in anhydrous 2-pentanone (1 ml) in a Reactivialt. The solution was heated at 601C for 18 h and the reaction was worked up as described (above). The formation of FAZA was confirmed by co-injecting the standard FAZA with the reaction mixture on HPLC. Radiofluorination. The tosylate precursor 2 was converted to [18F]FAZA by conventional heating technique as described in the literature (Patt et al, 1999).
3. Results and discussion Microwaves are sinusoidally reversing magnetic fields, which set electrons of molecules in the field into motion in a direction opposite to the magnetic field. This generates rapid reversal of polarity, and induces varying charge distributions in the molecules. An optimal distribution of charge changes the polarity of the environment (solvent), thereby increasing the reactivity of the molecules. The nature of the solvent, intramolecular hydrogen bonding, the sample’s polarity, dielectric constant, and related factors play important roles in microwave-assisted reactions. The domestic microwave oven operates at a microwave frequency of 2450 MHz, to exploit O–H and C–H rotational frequencies (Gedye et al, 1986). The data in Tables 1 and 2 compare radioiodination reactions and yields for IAZA 3a, IAZP 10 and FAZA 4a by conventional heating and microwave-assisted (radio)halogenation techniques. Since, both IAZA and IAZP are susceptible to alkaline hydrolysis, acetic acid
Table 1 Radioiodination of IAZA 6, DAc-IAZA 3a, IAZP 10 and DAc-IAZP 11 by the exchange method using conventional heating (C) and microwave assisted (M) synthesis techniques Reaction no. 1 2 3 4 5 6 7 8 9 10 11 12 13
Substrate 6 6 6 6 6 6 3 3 6 10 10 10 11
1 10 20 20 100 100 100 100 100 1a 3.5a 100 3.5a 10
Solvent None None None P P P P P None None MeOH None P
Neutralizer (mg) None None None None A (176) A (176) None A (176) PA (3.5)a PA (2.7)a PA (1.0)a PA (2.7)a PA (1.0)a
Time (min) 8 6 12 5 5 8 8 8 80 120 8 120 8
CY (%) 100 100 100 100 99 100 100 100 90 45 95 65 100
RCY (%)
Product
Technique
38 27 29 17 33 42 87 74 90 36 33 55 23
125
M M M M M M M M C, 901C C, 1301C M C, 1151C M
a Amount in mg. P: 2-Pentanone, A: glacial acetic acid (mg), PA: pivalic acid, CY: chemical yield, RCY: radiochemical yield.
I-3a I-3a 125 I-3a 125 I-3a 125 I-3a 125 I-3a 125 I-3 125 I-3 125 I-3a 125 I-10 125 I-10 123 I-10 125 I-11 125
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
701
Table 2 (Radio)iodination and fluorination of 5, 2, and 9 by nucleophilic substitution using conventional heating (C) and microwave-assisted (M) labelling techniques Reaction no.
Substrate
Amount (mg)
Solvent
Time (min)
CY (%)
RCY (%)
14 15 16 17 18 19 20
2 5 2 2 2 2 9
50a 10 10 10 10a 10a 10
P P None None P/NH4F P/NH4 P
18b 5 8 13 18b 8 8
93 Co-elutedc Co-eluted Co-eluted 25 50 100
NA 90 90 33.5 NA NA 88
Product
Technique
3
C, 1101C M M M C, 601C M M
125
I-3a 125 I-3 125 I-3 4a 4a 125 I-11
a
Amount in mg. Time in h. c The radiolabelled product, obtained by nucleophilic substitution, was measured by both radiometry and UV (by adding calculated amounts of cold product). P: 2-pentanone, NA: not applicable since it is not a radiohalogenated product, CY: chemical yield, RCY: radiochemical yield. b
HO
TsO a,b
O HO
X
O AcO
c or d M/C Heating 2-NI
2-NI OH 1
2-NI OAc 3; X = I, 125I, 123I 4; X = F, 18F
OAc 2
g I
O AcO
O HO
a
2-NI
e or f
OH 6 TsO
O HO
I* c M/C Heating 2-NI
X
O HO
2-NI
2-NI OH
OH
OH
3a; I* = 125I
5
O HO
3a; X = I, 125I, 123I 4a; X = F, 18F
N where, 2-NI = N
NO2
where, a = toluenesulfonyl chloride/pyridine; b = Ac2O/pyridine; c =NaI*/2-pentanone; d = NH4F/KryptofixTM 2.2.2 or 18F and 2-pentanone; e = NH3/MeOH and f = 1N.NaOH
Scheme 1. Synthetic route to the 50 -(radio)halogenated azomycin arabinosides IAZA and FAZA.
or pivalic acid (trimethyl acetic acid) was added to neutralize the NaOH in the radioiodide solution. Several reactions were conducted in the absence of neutralizing acid in order to compare the chemical and radiochemical yields of labelled products in neutral and in alkaline solution. Syntheses of [125I]IAZA and [125I]IAZP require large amounts of precursor and long reaction times (e.g. 1.3–2 h) at elevated temperatures (90–1301C) under conventional heating conditions (Table 1, reactions 9, 10 and 12). The syntheses of FAZA and 3 took even
longer (18 h), with substantial chemical decomposition of the product and concomitant lower chemical yields (Table 2, reactions 18 and 14, respectively). When these (radio)syntheses were performed in the microwave, the corresponding products were obtained in very short reaction times (8 min; Schemes 1 and 2), which reduced chemical decomposition of the precursor/product, and consequently contributed to improved (radio)chemical yields. Surprisingly, the synthesis of [125I]IAZA by conventional heating procedures using exchange of radioiodine
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703
702
O HO TsO OH
O AcO
c TsO
2-NI
I*
d
O AcO
M/C Heating OAc 9
2-NI
2-NI OAc 125 11; I* = I, I
a,b I
O HO
e
2-NI
HO
I*
O HO
OH
OH
7
10
d 2-NI
O HO
M/C Heating OH 10; I* =
2-NI 125 123
I,
I
N where 2-NI = N
NO2
Reaction conditions: a = Sn2O/MeOH; b = toluenesulfonyl chloride/toluene; c = Ac2O/pyridine; d = NaI* and e = Ph3P/I2
Scheme 2. Synthetic route to (radio)iodinated IAZP.
afforded the product in 90% radiochemical and 90% chemical yields (reaction 9, Table 1), whereas microwave-assisted radioiodine exchange with IAZA afforded only a 42% radiochemical yield. The microwave-assisted reaction was complete in 8 min, and there was no evidence of chemical decomposition (100% chemical yield, reaction 6, Table 1). Low radiochemical yields for microwave-assisted exchange of radioiodine in IAZA are related to the presence of free hydroxyl groups in the sugar moiety, since similar exchange radioiodination reactions with the corresponding acetylated product 3 provided much higher radiochemical yields (87%), with minimal chemical degradation (chemical yield 100%, reaction 7, Table 1). Similar, but less dramatic, effects are seen during the exchange labelling of IAZP and its diacetylated analogue 11 (reactions 10–13, Table 1).
4. Summary The microwave technique has been applied to nucleophilic substitution of a toluenesulfonyl (tosyl) group at C-50 of AZA to afford IAZA or FAZA, using the respective nucleophile. Conventional heating procedures to prepare IAZA and FAZA from 2 took longer (up to 18 h) and provided lower chemical yields (e.g. 25% vs. 50% for FAZA) than the microwave-assisted synthesis in each case. The microwave technique afforded IAZA, DAc-IAZA 3a and DAc-IAZP 11 without chemical degradation, in nearly 90% radiochemical yields within 5–8 min. The amount of precursor required for microwave labelling was only 1–10% of that used routinely for conventional exchange reactions. The most important features of microwave-assisted synthesis of IAZA, IAZP and FAZA are high yield,
reduced reaction time and the reduced amounts of precursors required. This leads to the syntheses of labelled products with higher chemical and radiochemical yields, with higher specific activities, in relatively short reaction times.
Acknowledgements The authors are grateful to Alberta Cancer Board for providing financial support (Grant RI-14) to this project.
References Al-Arafaj, A., Ryan, E.A., Hutchinson, K., Mannan, R.H., Mercer, J.R., Wiebe, L.I., McEwan, A.J.B., 1994. An evaluation of iodine-123 iodoazomycin arabinoside as a marker of localized tissue hypoxia in patients diabetes mellitus. Eur. J. Nucl. Med. 21, 1338–1342. Biaglow, J.E., Varnes, M.E., Rizen-Towle, L., Clark, E.P., Epp, F.R., Astor, M.B., Hall, E.J., 1986. Biochemistry of reduction of nitroheterocycles. Biochem. Pharmacol. 35, 77. Gedye, R., Smith, F., Westaway, K., Ali, H., Baldisera, L., Laberge, L., Rousell, J., 1986. The use of microwave ovens for rapid organic synthesis. Tetrahedron Lett. 27, 279. Grosher, D., McEwan, A.J.B., Parliament, M.B., Urtasun, R.C., Goldberg, L.E., Hoskinson, M., Mercer, J.R., Mannan, R.H., Wiebe, L.I., Chapman, J.D., 1993. Imaging tumor Hypoxia and tumor perfusion. J. Nucl. Med. 34, 885. Hwang, D.R., Moerlein, S.M., Welch, M.J., 1989. Microwavefacilitated synthesis of [18F]-Spiperone. J. Lab. Compd. Radiopharm. 26, 391 (Abstract). Kumar, P., Stypinski, D., Xia, H., McEwan, A.J.B., Machulla, H-J., Wiebe, L.I., 1999. Fluoroazomycin arabinoside (FAZA): synthesis, 2H and 3H-labelling and preliminary
P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 697–703 biological evaluation of a novel 2-nitroimidazole marker of tissue hypoxia. J. Labelled Compd. Radiopharm. 42, 3–16. Kumar, P., Patt, M., Machulla, H-J., Mc Ewan, A.J.B., Wiebe, L.I., 2000. [18F]-FAZA: A putative PET radiotracer for hypoxia. In: Ulrich Pleiss, Rolf Voges (Eds.), Synthesis and Applications of Isotopically Labelled Compounds. Wiley, UK. Lemaire, C., Cantineau, R., Christiaens, L., Guillaume, M., 1989. Radiofluorination of Altanserine a potential serotonin receptor binding radiopharmaceutical for positron emission tomography. J. Lab. Compd. Radiopharm. 36, 336 (Abstract). Mannan, R.H., 1991. Novel non-invasive markers of tumor hypoxia. Ph.D. Thesis, University of Alberta, Edmonton, Canada, pp. 53–58. Mannan, R.H., Somayaji, V.V., Lee, J., Mercer, J.R., Chapman, J.D., Wiebe, L.I., 1991. Radioiodinated 1-(5-Iodo-5deoxy-b-D-arabinofuranosyl)-2-nitroimidazole (Iodoazomycin Arabinoside: IAZA), a Novel Marker of Tissue Hypoxia. J. Nucl. Med. 32, 1764–1770. Mannan, R.H., Mercer, J.R., Wiebe, L.I., Kumar, P., Somayaji, V.V., Chapman, J.D., 1992. Radioiodinated
703
azomycin pyranoside (IAZP): a novel non-invasive marker for the assessment of tumor hypoxia. J. Nucl. Biol. Med. 36, 60–67. McEwan, A.J.B., Skeith, K.J., Mannan, R.H., Davies, N., Jamali, F., Schmidt, R., Golberg, K., Wiebe, L.I., 1997. I123 Iodoazomycin arabinoside (IAZA) may have a role in imaging rheumatoid arthritis. J. Nucl. Med. 38, 300. Parliament, M.B., Chapman, J.D., Urtasun, R.C., McEwan, A.J., Goldberg, L.E., Mercer, J.R., Mannan, R.H., Wiebe, L.I., 1992. Non-invasive assessment of human tumor hypoxia with 123I-iodoazomycin arabinoside: preliminary report of a clinical study. Br. J. Cancer. 65, 90–95. Patt, M., Kumar, P., Wiebe, L.I.,Machulla, H.-J., 1999. [18F]Fluoroazomycin-arabinosid (FAZA), ein neuer Radiotracer zur Bestimmung von Gewebe-Hypoxien. International Annual Conference, Deut. G. Nuklearmed. Ulm, Germany. Urtasun, R.C., McEwan, A.J., Parliament, M.B., Mercer, J.R., Mannan, R.H., Wiebe, L.I., Morin, C., Chapman, J.D., 1996. Measurement of hypoxia in human tumours by noninvasive spect imaging of iodoazomycin arabinoside. Br. J. Cancer. 74, S209.