- Email: [email protected]
Facile synthesis of bis(hydroxamamide)-based tetradentate ligands for 99mTc-radiopharmaceutical
ARTICLE IN PRESS
Applied Radiation and Isotopes 62 (2005) 919–922 www.elsevier.com/locate/apradiso
Facile synthesis of bis(hydroxamamide)-based tetradentate ligands for 99mTc-radiopharmaceutical Hong Xu, Taiwei Chu, Xiangyun Wang, Xinqi Liu Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 2 December 2004; received in revised form 22 December 2004; accepted 31 December 2004
Abstract A facile, two-step synthesis of the bis(hydroxamamide)-based tetradentate ligands for 99mTc-radiopharmaceuticals is described. Firstly, the hydroxamamide was converted to hydroximic acid chloride by reaction with sodium nitrite in hydrochloric acid at 0 1C. Secondly, treating the halide with the ethylenediamine or 1,3-propylenediamine in absolute ethanol formed the desired products, N,N0 -ethylene bis(1-(4-nitroimidazole-1-yl)–propan-hydroxyiminoamide) (I) and N,N0 -propylene bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide) (II). The corresponding 99mTc complexes showed high yields and were found by paper electrophoresis to be electrically neutral under physiological conditions. The partition coefficients indicated a distinct difference between the two complexes. r 2005 Elsevier Ltd. All rights reserved. Keywords: Tetradentate ligands; 99mTc complex; Labeling; N,N0 -ethylene bis(1-(4-nitroimidazole-1-yl)–propanhydroxy-iminoamide); N,N0 -propylene-bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide)
1. Introduction Hydroxyiminoamides form colored chelates with some metal ions in which the metal atom is linked to the oxime group and the amino group (Eloy and Lenares, 1962). Nakayama et al. (1992, 1994, 1997) found that the bidentate ligand hydroxyiminoamides could form complexes with 99mTc. They also synthesized the tetradentate ligands, N,N0 -ethylene bis(benzohydroxamamide) [(C2(BHam)2)] and N,N0 -propylene bis(benzohydroxamamide) [(C3(BHam)2)], and found that their 99mTc complexes were more stable than those of the bidentate ligands, and also that the bis(hydroxamamide) derivative might be a useful new chelating moiety for the design of 99mTc radiopharmaceuticals Corresponding author. Tel.: +86 10 6275 5409; +86 10 6275 5409. E-mail address: [email protected] (T. Chu).
fax:
(Xu et al., 1998, 1999). However, their synthetic procedure contained five to six steps, including some protection and deprotection reactions, with overall yields of only 14% and 21%. All of these findings prompted us to design a more convenient method to synthesize bis(hydroxamamide) derivatives as chelating molecules for 99mTc-radiopharmaceuticals. Recently, we synthesized two new hydroxyiminoamide ligands, 1-(2-nitroimidazole-1-yl)-propanhydroxyiminoamide (N2IPA) and 1-(4-nitro -imidazole-1-yl)propanhydroxyiminoamide (N4IPA), with a bio-reducible moiety and the corresponding 99mTc complexes were evaluated as tumor hypoxia markers (Chu et al., 2004a, b). This paper describes an extension of this work. Two tetradentate ligands, N,N0 -ethylene bis(1-(4nitroimidazole-1-yl)-propanhydroxy-iminoamide) (I) and N,N0 -propylene bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide) (II) were synthesized in a two-step reaction and labeled with 99mTc; the
0969-8043/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.12.008
ARTICLE IN PRESS H. Xu et al. / Applied Radiation and Isotopes 62 (2005) 919–922
920
2.1. Synthesis of hydroximoyl chlorides (Scheme 1)
partition coefficients of these complexes were also determined.
1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide (III) (0.6 g, 3.0 mmol) was dissolved in 5 mL of water and 8 mL of concentrated hydrochloric acid at 0 1C. Sodium nitrite (NaNO2) (0.34 g, 4.9 mmol) in 3 mL of water was added dropwise to a stirred solution of amide oxime. After stirring for 1.5 h at 0–5 1C, NaHCO3 was slowly added until pH 2 was reached. The precipitate was filtered off and washed with ice-cold water to give 0.50 g of the hydroximoyl chlorides (IV), m.p. 158–159 1C. Yield: 76%. 1H NMR [DMSO-d6]: 11.80 (s, 1H, OH); 4.36 (t, 2H, CH2); 3.09 (t, 2H, CH2); 8.46 (s, 1H, imi-H); 7.87 (s, 1H, imi-H).
2. Materials and methods 1,3-Propylenediamine (99+%) was purchased from ACROS Organics Company (Geel, Belgium). 1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide (III) was prepared according to the procedure published previously (Chu et al., 2004a). All other agents were of A.R. Grade. 99mTcO 4 was obtained by elution with saline from a 99Mo-99mTc generator (China Institute of Atom Energy, Beijing). NMR spectra were recorded on a Bruker ARX-400 (400 MHz) spectrometer (Falladen, Switzerland) with deuterated dimethyl sulfoxide (DMSO-d6) as a solvent, and with tetramethylsilane (TMS) as an internal standard. Elemental analyses were carried out on the Elementar Vario EL (Hanau, Germany).
2.2. Synthesis of N,N0 -ethylene bis(1-(4-nitroimidazole1-yl)-propanhydroxy-iminoamide) (I) Compound IV, 0.28 g (1.3 mmol), was dissolved in 20 mL of absolute ethanol, and the solution was cooled
N OH
N OH O2N
O2N
NaNO 2
NH2
N
o
N
N
Cl
N
HCl, 0 C
III
IV NH2
H2N IV
OH
HO
N O2N
N NH
N
NO2
N
HN
N
N
I
NH2 NH2 OH HO
IV
N
N O2N
N
NH
HN
NO2
N N
N
II Scheme 1. Synthesis of tetradentate ligands.
ARTICLE IN PRESS H. Xu et al. / Applied Radiation and Isotopes 62 (2005) 919–922
to 0 1C. Then a solution of ethylenediamine (0.7 mmol) and triethylamine (2.2 mmol) in 10 mL of absolute ethanol was added dropwise under nitrogen atmosphere with continuous stirring. The mixture was stirred for about 1 h and the temperature was raised to 5 1C. The resulting precipitate was filtered off and recrystallized from methanol to give 0.19 g of N,N0 -ethylene bis(1-(4nitroimidazole-1-yl)-propanhydroxy-iminoamide) (I), m.p. 244–246 1C. Yield: 70%. 1H NMR [DMSO-d6]: 9.20 (s, 1H, OH); 8.43 (s, 1H, imi-H); 7.85 (s, 1H, imiH); 5.83 (t, 1H, NH); 4.22 (t, 2H, CH2); 3.09 (q, 2H, CH2); 2.70 (t, 2H, CH2). Analytical data calculated for C14H20N10O6: C, 39.62; H, 4.72; N, 33.02. Found: C, 39.34; H, 4.92; N, 33.08. 2.3. Synthesis of N,N0 -propylene bis(1-(4nitroimidazole-1-yl)–propanhydroxy-iminoamide) (II) Compound II was synthesized by a procedure similar to that described for compound I in 47% yield, m.p. 212–213 1C. 1H NMR [DMSO-d6]: 9.18 (s, 2H, OH); 8.43 (s, 2H, imi-H); 7.85 (s, 2H, imi-H); 5.62 (t, 2H, NH); 4.26 (t, 4H, CH2); 3.05 (q, 4H, CH2); 2.69 (t, 4H, CH2); 1.61 (m, 2H, CH2). Analytical data calculated for C15H22N10O6: C, 41.10; H, 5.02; N, 31.96. Found: C, 40.97; H, 5.03; N, 31.98. 2.4. Preparation of
99m
Tc-complexes
The ligand solution, 100 mL (1.0 mg/mL), was mixed with 25 mL of sodium tartrate solution (1.0 mg/mL), and 1 mL of phosphate buffer solution (pH 7.4, 0.1 mol/L) in a 10 ml glass vial and 100 mL (3.7 107 Bq) of 99m TcO 4 effluent was added. Then 5 mL of freshly prepared SnCl2 2H2O solution (1.0 mg/mL) was added to reducte the 99mTcO 4 . The mixture was then heated at 75 1C for 15 min. The yield of the 99mTc complex was determined by TLC using a polyamide strip as the fixed phase and water:acetone ¼ 1:1 (or 2:1 v/v) as the developer. The strip was cut to 0.5 cm sections and counted with a well-type gamma counting system. 2.5. Partition coefficient Five mL of 1-octanol and 5 mL of aqueous 99mTccomplex solution was mixed in a centrifuge tube. The tube was shaken for 30 min at room temperature and then centrifuged for 5 min. Aliquots were taken from each phase and counted for radioactivity. The partition coefficient was calculated by dividing the radioactivity of the octanol layer with that of the water layer. Samples of the water layer were repartitioned until consistent partition coefficient values were obtained. This measurement was repeated three times.
921
2.6. Electrophoresis Filter paper strips pre-soaked in phosphate buffer (pH ¼ 7.4, m ¼ 0:05) were placed in an electrophoresis chamber containing the phosphate buffer and the samples were spotted. Each strip was run at a constant voltage of 220 V for 25 min. After drying, the strips were cut into 1 cm sections and counted in a well-type gamma counter.
3. Results and discussion 3.1. Synthesis Unsubstituted hydroximic acid chlorides generally are prepared by direct chlorination of aldoximes. Introduction of a halogen atom in place of the amino group have been mentioned before for some of o-alkylated benzamide oximes (Eloy and Lenares, 1962). Kocevar et al. (1988) reported that pyridinecarbohydroximoyl chlorides could be synthesized by treating the amide oximes with sodium nitrite under acidic conditions, and stored at room temperature for weeks (Kocevar et al., 1988). This provides a convenient method for the preparation of hydroximic acid halides; Yarovenko et al. (1994) also prepared the halides by this method. According to these findings, we successfully synthesized hydroximoyl chlorides (IV). At the first stage, the amidoxime (III) was treated with sodium nitrite in hydrochloric acid at 0 1C to give the product (IV). A large excess hydrochloric acid was required in this procedure. Then sodium hydrogen carbonate was added to regulate the pH of the solution and to give higher yield. When less hydrochloric acid was added, we failed to get the desired product. The action of an amine on hydroximic acid halides is the usual way of preparing N-substituted amidoximes. Ethylenediamine and 1,3-propylenediamine were used in this study. The reaction was carried out in absolute ethanol under a nitrogen atmosphere. Low temperature was needed to avoid some unwanted reactions. An excess of triethylamine was used to neutralize the hydrochloric acid formed. The synthetic procedure for the bis(hydroxamamide)based tetradentate ligands reported by Xu et al. (1998) contained five to six steps, with overall yields of less than 14% and 21% . In contrast, our facile synthetic method needs only two steps, with overall yields of 53% and 36% from similar reactants. 3.2. Formation of
99m
Tc-complexes
Technetium-99 m complexes of I and II were prepared at 75 1C as described above. The reaction mixtures were analyzed by TLC. Labeling compound I with 99mTc
ARTICLE IN PRESS H. Xu et al. / Applied Radiation and Isotopes 62 (2005) 919–922
922 Table 1 Properties of complexes
References
Complexes
Complex yields (%)
P value
Migration distance from origin (cm)
99m
90 88
0.026 0.008
0 0
Tc-I Tc-II
99m
generated a spot at Rf 0.5–0.6 by using acetone–water (1:2, v/v) as the developing solvent. This spot was distinguishable as a single peak clearly separated from 99m those of free 99mTcO Tc, 4 or the reduced hydrolyzed 99m and indicated the formation of Tc-I. The complex of II with 99mTc, produced a single peak with TLC using water–acetone (1:1, v/v) with an Rf value 0.6–0.7. In this 99m system, 99mTcO Tc 4 and the reduced hydrolyzed remained at the origin. The dependence of the radiolabeling yield on pH, temperature, ligand concentration, the amount of added stannous chloride, etc. was also screened by normal procedures. Under optimized labeling conditions the two 99mTc-complexes showed good radiochemical purity (Table 1). 3.3. Other properties of the
99m
Tc complexes
The results of the electrophoresis and the partition coefficients of the 99mTc-complexes are given in Table 1. Paper electrophoresis showed that the complexes were electrically neutral under physiological conditions. The partition coefficients indicated that there were distinct differences between the two complexes. Obviously, the ethylene or propylene carbon spacer of two bis(hydroxamamide)-based tetradentate ligands showed a chelate ring effect on the properties of the resulting 99mTc complexes (Jurisson et al., 1987).
4. Conclusion The bis(hydroxamamide)-based tetradentate ligands can be conveniently prepared by a two-step reaction, and labeled with 99mTc. The complexes showed high radiochemical yields and were electrically neutral under physiological conditions. These results would render bis(hydroxamamide) derivatives useful as chelating molecules for the preparation of 99mTc-radiopharmaceuticals.
Acknowledgments Financial support was provided by the National Natural Science Foundation of China (No. 20301001).
Chu, T.W., Hu, S.W., Wei, B., Wang, Y., Liu, X.Q., Wang, X.Y., 2004a. Synthesis and biological results of technetium99m-labeled 4-nitroimidazole for imaging tumor hypoxia. Bioorg. Med. Chem. Lett. 14, 747–749. Chu, T.W., Li, R.J., Hu, S.W., Liu, X.Q., Wang, X.Y., 2004b. Preparation and Biodistribution of Technetium-99m-labeled 1-(2-nitroimidazole-1-yl)-propanhydroxyiminoamide (N2IPA) as a tumor hypoxia marker. Nucl. Med. Biol. 31, 199–203. Eloy, F., Lenares, R., 1962. The chemistry of amidoximes and related compounds. Chem. Rev. 62, 155–183. Jurisson, S., Aston, K., Fair, C.K., Schlemper, E.O., Sharp, P.R., Troutner, D.E., 1987. Effect of ring size on properties of technetium amine oxime complexes: X-ray structure of TcO2Pent(AO)2, which contains an unusual eight-membered chelate ring and of TcOEn(AO)2. Inorg. Chem. 26, 3576–3582. Kocevar, M., Polanc, S., Sollner, M., Tisler, M., Vercek, B., 1988. Simple procedure for the synthesis of pyridinecarbohydroximoyl chlorides and bromides. Synth. Commun. 18, 1427–1432. Nakayama, M., Saigo, H., Kai, E., Koda, A., Ozaki, H., Harada, K., Sugii, A., Tomiguchi, S., Kojima, A., Hara, M., 1992. Hydroxamamide as a chelating moiety for the preparation of 99mTc radiopharmaceuticals (I). Nucl. Med. Commun. 13, 445–449. Nakayama, M., Saigo, H., Koda, A., Ozeki, K., Harada, K., Sugii, A., Tomiguchi, S., Kojima, A., Hara, M., Nakashima, R., Ohyama, Y., Takahashi, M., Takata, J., Karube, Y., 1994. Hydroxamamide as a chelating moiety for the preparation of 99mTc radiopharmaceuticals-II. The 99mTc complexes of hydroxamamide derivatives. Appl. Radiat. Isot. 45, 735–740. Nakayama, M., Xu, L.C., Koga, Y., Harada, K., Sugii, A., Nakayama, H., Tomiguchi, S., Kojima, A., Ohyama, Y., Takahashi, M., Okabayashi, I., 1997. Hydroxamamide as a chelating moiety for the preparation of 99mTc radiopharmaceuticals-III. Characterization of various Tc-99m-hydroxamamides. Appl. Radiat. Isot. 48, 571–577. Xu, L.C., Nakayama, M., Harada, K., Nakayama, H., Tomiguchi, S., Kojima, A., Takahashi, M., Arano, Y., 1998. Synthesis and Evaluation of hydroxamamide-based tetradentate ligands as a new class of thiol-free chelating molecules for 99mTc radiopharmaceuticals. Nucl. Med. Biol. 25, 295–303. Xu, L.C., Nakayama, M., Harada, K., Kuniyasu, A., Nakayama, H., Tomiguchi, S., Kojima, A., Takahashi, M., Ono, M., Arano, Y., Saji, H., Yao, Z.S., Sakahara, H., Konishi, J., Imagawa, Y., 1999. Bis(Hydroxamamide)based bifunctional chelating agent for 99mTc labeling of polypeptides. Bioconjugate Chem. 10, 9–17. Yarovenko, V.N., Krayushkin, M.M., Lysenko, O.V., Kustov, L.M., Zavarzin, I.V., 1994. A new method for the synthesis of nitriles enriched with the 15N isotope. Russian Chem. Bull. 43, 402–404.
Applied Radiation and Isotopes 62 (2005) 919–922 www.elsevier.com/locate/apradiso
Facile synthesis of bis(hydroxamamide)-based tetradentate ligands for 99mTc-radiopharmaceutical Hong Xu, Taiwei Chu, Xiangyun Wang, Xinqi Liu Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 2 December 2004; received in revised form 22 December 2004; accepted 31 December 2004
Abstract A facile, two-step synthesis of the bis(hydroxamamide)-based tetradentate ligands for 99mTc-radiopharmaceuticals is described. Firstly, the hydroxamamide was converted to hydroximic acid chloride by reaction with sodium nitrite in hydrochloric acid at 0 1C. Secondly, treating the halide with the ethylenediamine or 1,3-propylenediamine in absolute ethanol formed the desired products, N,N0 -ethylene bis(1-(4-nitroimidazole-1-yl)–propan-hydroxyiminoamide) (I) and N,N0 -propylene bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide) (II). The corresponding 99mTc complexes showed high yields and were found by paper electrophoresis to be electrically neutral under physiological conditions. The partition coefficients indicated a distinct difference between the two complexes. r 2005 Elsevier Ltd. All rights reserved. Keywords: Tetradentate ligands; 99mTc complex; Labeling; N,N0 -ethylene bis(1-(4-nitroimidazole-1-yl)–propanhydroxy-iminoamide); N,N0 -propylene-bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide)
1. Introduction Hydroxyiminoamides form colored chelates with some metal ions in which the metal atom is linked to the oxime group and the amino group (Eloy and Lenares, 1962). Nakayama et al. (1992, 1994, 1997) found that the bidentate ligand hydroxyiminoamides could form complexes with 99mTc. They also synthesized the tetradentate ligands, N,N0 -ethylene bis(benzohydroxamamide) [(C2(BHam)2)] and N,N0 -propylene bis(benzohydroxamamide) [(C3(BHam)2)], and found that their 99mTc complexes were more stable than those of the bidentate ligands, and also that the bis(hydroxamamide) derivative might be a useful new chelating moiety for the design of 99mTc radiopharmaceuticals Corresponding author. Tel.: +86 10 6275 5409; +86 10 6275 5409. E-mail address: [email protected] (T. Chu).
fax:
(Xu et al., 1998, 1999). However, their synthetic procedure contained five to six steps, including some protection and deprotection reactions, with overall yields of only 14% and 21%. All of these findings prompted us to design a more convenient method to synthesize bis(hydroxamamide) derivatives as chelating molecules for 99mTc-radiopharmaceuticals. Recently, we synthesized two new hydroxyiminoamide ligands, 1-(2-nitroimidazole-1-yl)-propanhydroxyiminoamide (N2IPA) and 1-(4-nitro -imidazole-1-yl)propanhydroxyiminoamide (N4IPA), with a bio-reducible moiety and the corresponding 99mTc complexes were evaluated as tumor hypoxia markers (Chu et al., 2004a, b). This paper describes an extension of this work. Two tetradentate ligands, N,N0 -ethylene bis(1-(4nitroimidazole-1-yl)-propanhydroxy-iminoamide) (I) and N,N0 -propylene bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide) (II) were synthesized in a two-step reaction and labeled with 99mTc; the
0969-8043/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.12.008
ARTICLE IN PRESS H. Xu et al. / Applied Radiation and Isotopes 62 (2005) 919–922
920
2.1. Synthesis of hydroximoyl chlorides (Scheme 1)
partition coefficients of these complexes were also determined.
1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide (III) (0.6 g, 3.0 mmol) was dissolved in 5 mL of water and 8 mL of concentrated hydrochloric acid at 0 1C. Sodium nitrite (NaNO2) (0.34 g, 4.9 mmol) in 3 mL of water was added dropwise to a stirred solution of amide oxime. After stirring for 1.5 h at 0–5 1C, NaHCO3 was slowly added until pH 2 was reached. The precipitate was filtered off and washed with ice-cold water to give 0.50 g of the hydroximoyl chlorides (IV), m.p. 158–159 1C. Yield: 76%. 1H NMR [DMSO-d6]: 11.80 (s, 1H, OH); 4.36 (t, 2H, CH2); 3.09 (t, 2H, CH2); 8.46 (s, 1H, imi-H); 7.87 (s, 1H, imi-H).
2. Materials and methods 1,3-Propylenediamine (99+%) was purchased from ACROS Organics Company (Geel, Belgium). 1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide (III) was prepared according to the procedure published previously (Chu et al., 2004a). All other agents were of A.R. Grade. 99mTcO 4 was obtained by elution with saline from a 99Mo-99mTc generator (China Institute of Atom Energy, Beijing). NMR spectra were recorded on a Bruker ARX-400 (400 MHz) spectrometer (Falladen, Switzerland) with deuterated dimethyl sulfoxide (DMSO-d6) as a solvent, and with tetramethylsilane (TMS) as an internal standard. Elemental analyses were carried out on the Elementar Vario EL (Hanau, Germany).
2.2. Synthesis of N,N0 -ethylene bis(1-(4-nitroimidazole1-yl)-propanhydroxy-iminoamide) (I) Compound IV, 0.28 g (1.3 mmol), was dissolved in 20 mL of absolute ethanol, and the solution was cooled
N OH
N OH O2N
O2N
NaNO 2
NH2
N
o
N
N
Cl
N
HCl, 0 C
III
IV NH2
H2N IV
OH
HO
N O2N
N NH
N
NO2
N
HN
N
N
I
NH2 NH2 OH HO
IV
N
N O2N
N
NH
HN
NO2
N N
N
II Scheme 1. Synthesis of tetradentate ligands.
ARTICLE IN PRESS H. Xu et al. / Applied Radiation and Isotopes 62 (2005) 919–922
to 0 1C. Then a solution of ethylenediamine (0.7 mmol) and triethylamine (2.2 mmol) in 10 mL of absolute ethanol was added dropwise under nitrogen atmosphere with continuous stirring. The mixture was stirred for about 1 h and the temperature was raised to 5 1C. The resulting precipitate was filtered off and recrystallized from methanol to give 0.19 g of N,N0 -ethylene bis(1-(4nitroimidazole-1-yl)-propanhydroxy-iminoamide) (I), m.p. 244–246 1C. Yield: 70%. 1H NMR [DMSO-d6]: 9.20 (s, 1H, OH); 8.43 (s, 1H, imi-H); 7.85 (s, 1H, imiH); 5.83 (t, 1H, NH); 4.22 (t, 2H, CH2); 3.09 (q, 2H, CH2); 2.70 (t, 2H, CH2). Analytical data calculated for C14H20N10O6: C, 39.62; H, 4.72; N, 33.02. Found: C, 39.34; H, 4.92; N, 33.08. 2.3. Synthesis of N,N0 -propylene bis(1-(4nitroimidazole-1-yl)–propanhydroxy-iminoamide) (II) Compound II was synthesized by a procedure similar to that described for compound I in 47% yield, m.p. 212–213 1C. 1H NMR [DMSO-d6]: 9.18 (s, 2H, OH); 8.43 (s, 2H, imi-H); 7.85 (s, 2H, imi-H); 5.62 (t, 2H, NH); 4.26 (t, 4H, CH2); 3.05 (q, 4H, CH2); 2.69 (t, 4H, CH2); 1.61 (m, 2H, CH2). Analytical data calculated for C15H22N10O6: C, 41.10; H, 5.02; N, 31.96. Found: C, 40.97; H, 5.03; N, 31.98. 2.4. Preparation of
99m
Tc-complexes
The ligand solution, 100 mL (1.0 mg/mL), was mixed with 25 mL of sodium tartrate solution (1.0 mg/mL), and 1 mL of phosphate buffer solution (pH 7.4, 0.1 mol/L) in a 10 ml glass vial and 100 mL (3.7 107 Bq) of 99m TcO 4 effluent was added. Then 5 mL of freshly prepared SnCl2 2H2O solution (1.0 mg/mL) was added to reducte the 99mTcO 4 . The mixture was then heated at 75 1C for 15 min. The yield of the 99mTc complex was determined by TLC using a polyamide strip as the fixed phase and water:acetone ¼ 1:1 (or 2:1 v/v) as the developer. The strip was cut to 0.5 cm sections and counted with a well-type gamma counting system. 2.5. Partition coefficient Five mL of 1-octanol and 5 mL of aqueous 99mTccomplex solution was mixed in a centrifuge tube. The tube was shaken for 30 min at room temperature and then centrifuged for 5 min. Aliquots were taken from each phase and counted for radioactivity. The partition coefficient was calculated by dividing the radioactivity of the octanol layer with that of the water layer. Samples of the water layer were repartitioned until consistent partition coefficient values were obtained. This measurement was repeated three times.
921
2.6. Electrophoresis Filter paper strips pre-soaked in phosphate buffer (pH ¼ 7.4, m ¼ 0:05) were placed in an electrophoresis chamber containing the phosphate buffer and the samples were spotted. Each strip was run at a constant voltage of 220 V for 25 min. After drying, the strips were cut into 1 cm sections and counted in a well-type gamma counter.
3. Results and discussion 3.1. Synthesis Unsubstituted hydroximic acid chlorides generally are prepared by direct chlorination of aldoximes. Introduction of a halogen atom in place of the amino group have been mentioned before for some of o-alkylated benzamide oximes (Eloy and Lenares, 1962). Kocevar et al. (1988) reported that pyridinecarbohydroximoyl chlorides could be synthesized by treating the amide oximes with sodium nitrite under acidic conditions, and stored at room temperature for weeks (Kocevar et al., 1988). This provides a convenient method for the preparation of hydroximic acid halides; Yarovenko et al. (1994) also prepared the halides by this method. According to these findings, we successfully synthesized hydroximoyl chlorides (IV). At the first stage, the amidoxime (III) was treated with sodium nitrite in hydrochloric acid at 0 1C to give the product (IV). A large excess hydrochloric acid was required in this procedure. Then sodium hydrogen carbonate was added to regulate the pH of the solution and to give higher yield. When less hydrochloric acid was added, we failed to get the desired product. The action of an amine on hydroximic acid halides is the usual way of preparing N-substituted amidoximes. Ethylenediamine and 1,3-propylenediamine were used in this study. The reaction was carried out in absolute ethanol under a nitrogen atmosphere. Low temperature was needed to avoid some unwanted reactions. An excess of triethylamine was used to neutralize the hydrochloric acid formed. The synthetic procedure for the bis(hydroxamamide)based tetradentate ligands reported by Xu et al. (1998) contained five to six steps, with overall yields of less than 14% and 21% . In contrast, our facile synthetic method needs only two steps, with overall yields of 53% and 36% from similar reactants. 3.2. Formation of
99m
Tc-complexes
Technetium-99 m complexes of I and II were prepared at 75 1C as described above. The reaction mixtures were analyzed by TLC. Labeling compound I with 99mTc
ARTICLE IN PRESS H. Xu et al. / Applied Radiation and Isotopes 62 (2005) 919–922
922 Table 1 Properties of complexes
References
Complexes
Complex yields (%)
P value
Migration distance from origin (cm)
99m
90 88
0.026 0.008
0 0
Tc-I Tc-II
99m
generated a spot at Rf 0.5–0.6 by using acetone–water (1:2, v/v) as the developing solvent. This spot was distinguishable as a single peak clearly separated from 99m those of free 99mTcO Tc, 4 or the reduced hydrolyzed 99m and indicated the formation of Tc-I. The complex of II with 99mTc, produced a single peak with TLC using water–acetone (1:1, v/v) with an Rf value 0.6–0.7. In this 99m system, 99mTcO Tc 4 and the reduced hydrolyzed remained at the origin. The dependence of the radiolabeling yield on pH, temperature, ligand concentration, the amount of added stannous chloride, etc. was also screened by normal procedures. Under optimized labeling conditions the two 99mTc-complexes showed good radiochemical purity (Table 1). 3.3. Other properties of the
99m
Tc complexes
The results of the electrophoresis and the partition coefficients of the 99mTc-complexes are given in Table 1. Paper electrophoresis showed that the complexes were electrically neutral under physiological conditions. The partition coefficients indicated that there were distinct differences between the two complexes. Obviously, the ethylene or propylene carbon spacer of two bis(hydroxamamide)-based tetradentate ligands showed a chelate ring effect on the properties of the resulting 99mTc complexes (Jurisson et al., 1987).
4. Conclusion The bis(hydroxamamide)-based tetradentate ligands can be conveniently prepared by a two-step reaction, and labeled with 99mTc. The complexes showed high radiochemical yields and were electrically neutral under physiological conditions. These results would render bis(hydroxamamide) derivatives useful as chelating molecules for the preparation of 99mTc-radiopharmaceuticals.
Acknowledgments Financial support was provided by the National Natural Science Foundation of China (No. 20301001).
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