Pyridine-Containing 6-Hydrazinonicotinamide Derivatives as Potential

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Bioconjugate Chem. 2004, 15, 728−737

Pyridine-Containing 6-Hydrazinonicotinamide Derivatives as Potential Bifunctional Chelators for 99mTc-Labeling of Small Biomolecules Ajay Purohit, Shuang Liu,* Charlie E. Ellars, Dave Casebier, Stephen B. Haber, and D. Scott Edwards Discovery Chemistry, Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received August 14, 2003; Revised Manuscript Received April 30, 2004

As a continuation of our interest in novel 99mTc chelating systems, several pyridine-containing HYNIC (6-hydrazinonicotinamide) derivatives (L1-L5) have been synthesized and characterized by NMR (1H and 13C) and LC-MS. 99mTc complexes of L1-L5 were prepared by the reaction of the HYNIC derivative with 99mTcO4- in the presence of excess tricine and stannous chloride. Results from this study show that the attachment site of the linker is critical for the formation of macrocyclic 99mTc complexes. For example, the pyridine-N in L3 is not able to bond to the Tc, because the lysine linker is attached to the 4-position. When the linker is at the 2-position, L1 forms the macrocyclic complex [99mTc(L1)(tricine)], but the radiochemical purity is relatively low. If the linker is attached to the 3-position of the pyridine ring, the HYNIC derivatives form macrocyclic complexes [99mTc(L)(tricine)] (L2, L4, and L5) in high yield (>95%). The HPLC data suggest that the macrocyclic complex [99mTc(L2)(tricine)] exists in solution as four isomers: two diastereomers and two conformational isomers. Diastereomers are due to a combination of the chirality of the lysine linker and of the Tc chelate. Replacing lysine with a pentamethylenediamine linker results in the macrocyclic complex [99mTc(L4)(tricine)] with two conformational isomers, which interconvert rapidly at room temperature. Changing the linker from pentamethylenediamine to hexamethylenediamine did not eliminate the minor isomer; but the percentage of the minor isomer was reduced from ∼10% for [99mTc(L4)(tricine)] to only 6% for [99mTc(L5)(tricine)]. The linker length is an important parameter to minimize the minor isomer. LC-MS data of complexes [99mTc(L)(tricine)] (L2, L4, and L5) are completely consistent with their proposed compositions. On the basis of these data, it is concluded that pyridine-containing HYNIC derivatives have the potential as bifunctional chelators for 99mTc-labeling of small biomolecules if the linker is attached to the 3-position of the pyridine ring.

INTRODUCTION

For the last several years, we have been successful in using a ternary ligand system (HYNIC, tricine, and TPPTS) (HYNIC ) 6-hydrazinonicotinamide, TPPTS ) trisodium triphenylphosphine-3,3′,3′′-trisulfonate) for 99m Tc-labeling of small biomolecules (BM), including chemotactic peptides (1) and LTB4 receptor antagonists (2, 3) for imaging infection and inflammation, integrin Rvβ3 receptor antagonists for tumor imaging (4), and a GPIIb/IIIa receptor antagonist for imaging thrombus (510). The combination of HYNIC-BM, tricine, and TPPTS results in a unique and versatile ternary ligand system that forms 99mTc complexes [99mTc(HYNIC-BM)(tricine)(TPPTS)] (BM ) peptide or nonpeptide receptor ligands) with extremely high specific activity. These ternary ligand-99mTc complexes are stable in solution for at least 6 h and are formed as equal mixtures of two diastereomers, if the biomolecule has a chiral center. The composition of ternary ligand-99mTc complexes has been determined to be 1:1:1:1 for Tc:HYNIC-BM:tricine:TPPTS, through a series of mixed ligand experiments, and confirmed by LC-MS at both the tracer (99mTc) and * To whom correspondence should be addressed at the Department of Industrial and Physical Pharmacy, School of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907-2051. Phone: 765-494-0236. Fax 765-4963367. E-mail: [email protected].

macroscopic (99Tc) levels (11, 12). The use of HYNIC for Tc-labeling of small biomolecules has recently been reviewed (13-16). Previously, we reported two prototype phosphinecontaining HYNIC chelators (Figure 1), HYNIC-KpDPPB and HYNIC-Ko-DPPB (K ) lysine, DPPB ) diphenylphosphinobenzoic acid), and their macrocyclic 99mTc complexes (17). We found that both HYNIC-KpDPPB and HYNIC-Ko-DPPB form highly stable 99mTc complexes, [99mTc(HYNIC-Ko-DPPB)(tricine)] and [99mTc(HYNIC-Kp-DPPB)(tricine)]. We also found that [99mTc(HYNIC-Kp-DPPB)(tricine)] exists as only one detectable isomer in solution while [99mTc(HYNIC-Ko-DPPB)(tricine)] has three isomers, which interconvert at elevated temperatures. Unfortunately, HYNIC-Kp-DPPB is very prone to oxidation during radiolabeling. This presents a significant challenge, since the oxidized phosphinecontaining HYNIC chelator (HYNIC-Kp-ODPPB) will form a binary ligand complex, [99mTc(HYNIC-Kp-ODPPB)(tricine)2], as a radioimpurity. Like phosphines, pyridine analogues have also been used as coligands for 99mTc-labeling of small biomolecules (10). Compared to phosphines, pyridine analogues are airstable and are more amendable for further derivatization. Therefore, as a continuation of our interest in new 99mTc chelating systems, we now present the synthesis of several pyridine-containing HYNIC derivatives (Figure 99m

10.1021/bc034141c CCC: $27.50 © 2004 American Chemical Society Published on Web 07/01/2004

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Figure 1. Phosphine- and pyridine-containing HYNIC derivatives and their macrocyclic Tc complexes.

1, L1-L5). 1 The main objective of this study is to explore the potential of L1-L5 as bifunctional chelators (BFCs) for 99mTc-labeling of small biomolecules. EXPERIMENTAL SECTION

N--(tert-Butoxycarbonyl)lysine methyl ester hydrochloride was purchased from Calbiochem Inc. 1,6-Diaminohexane, 1,5-diaminopentane, 1-hydroxyazabenzotriazole, 4 M HCl in dioxane, isonicotinic acid, nicotinic acid, picolinic acid, 1-O-(N,N′,N′′,N′′′-tetramethyluronium)azabenzotriazoloxy hexafluorophosphate, and tricine were purchased from Sigma-Aldrich (St. Louis, MO). Na99mTcO4 was obtained from a Technelite 99Mo/99mTc generator at Bristol-Myers Squibb Medical Imaging Inc., N. Billerica, MA. Sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-NHS) was prepared according to a literature procedure (18). Synthesis of N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester was described in our previous communication (17). 1 1Abbreviations: L1 ) N--(isonicotinyl)-N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl )lysine methyl ester; L2 ) N--(nicotinyl)-N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester; L3 ) N--(picolinyl)-N-R-(6-(2-(2sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester; L4 ) 1-(N-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl))6-(nicotinoyl)pentanediamine; and L5 ) 1-(N-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl))-6-(nicotinoyl)hexanediamine.

Instruments. NMR spectral data (1H and 13C) were recorded on a 600 MHz Bruker DRX FT NMR spectrometer and are reported as δ (ppm) relative to TMS. Electrospray MS analyses were performed using an IonSpec Ultima FT mass spectrometer in the positive ion mode. LC-MS spectral data were collected using a HP1100 LC/MSD system with an API-electrospray interface. The LC-MS method used a Zorbax C18 column (4.6 mm × 150 mm, 3.5 µm particle size). The flow rate was 1 mL/min with a gradient mobile phase starting at 80% solvent A (25 mM ammonium acetate buffer, pH 6.8) and 20% solvent B (acetonitrile) and going to 75% A and 25% solvent B at 20 min. The preparative HPLC method used a Varian Prep Star system equipped with an UV/visible detector (λ ) 220 nm) and a Jupiter C18 prep column (15 µm, 300Å, 21.2 × 250 mm). The flow rate was 80 mL/min with the mobile phase starting with 100% solvent A (0.1%TFA in water) and going to 80% solvent B (0.1%TFA in 90% acetonitrile) and 20% solvent A at 40 min. The radioHPLC methods used a HP-1100 system with an IN-US radio-detector and a Zorbax C18 column (4.6 mm × 250 mm, 300 Å). The flow rate was 1 mL/min with a gradient mobile phase starting at 80% solvent A (25 mM ammonium acetate buffer, pH 6.8) and 20% solvent B (acetonitrile) and going to 75% A and 25% solvent B at 20 min. The ITLC method used GelmanSciences silica gel paper strips and a 1:1 mixture of acetone and saline as eluant. By this method, 99mTc complexes migrate to

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the solvent front while 99mTcO4- and [99mTc]colloid remain at the origin. Synthesis of N-E-(Isonicotinyl)-N-r-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine Methyl Ester (L1). To a 25 mL round-bottom flask was added isonicotinoyl chloride hydrochloride (16.0 mg, 0.90 mmol) and 3 mL of DMF. To the solution above was added N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester hydrochloride (50 mg, 0.10 mmol), followed by addition of DIEA (0.0695 mL, 51.62 mg) in DMF (1 mL). The reaction mixture was stirred for 20 min. DMF was removed in vacuo and the crude oil subjected to HPLC purification. The collected fractions were combined and lyophilized to give a white powder. The yield was 35.5 mg (∼70%). MS (ESI, +ve): m/z ) 463.15 for C20H25N5O6S ([M + H]+). 1H NMR (DMSO-d6 + D2O): 9.38 (s, 1H), 9.06 (d, 2H, J ) 6.6 Hz), 8.632 (s, 1H), 8.49-8.36 (m, 4H), 7.81 (dd, 1H, J ) 8.4, 7 Hz), 7.4 (m, 2H), 7.22 (br, 1H), 4.4 (m, 1H), 3.65 (s, 3H), 3.3 (m, 2H), 1.86 (m, 2H), 1.59-1.43 (m, 4H). Synthesis of N-E-(Nicotinyl)-N-r-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine Methyl Ester (L2). To a 25 mL round-bottom flask was added nicotinic acid hydrochloride (14.32 mg, 0.09 mmol), followed by 5 mL of DMF and 1-O-(N,N′,N′′,N′′′-tetramethyluronium)azabenzotriazoloxy hexafluorophosphate (41 mg, 0.1 mmol), 1-hydroxyazabenzotriazole (14.6 mg, 0.1 mmol) and DIEA (0.104 mL, 0.774 g). After stirring for 5 min, N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester hydrochloride (50 mg, 0.10 mmol) was added. The resulting solution was stirred for 30 min. DMF was removed under vacuum and the crude oil subjected to HPLC purification. The collected fractions were combined and lyophilized to give a fluffy white solid. The yield was 42 mg (82%). MS (ESI, +ve): m/z ) 569.1815 for C26H28N6O7S ([M + H]+). 1H NMR (DMSO-d6 + DCl): 9.3 (d, 2H, J ) 16.2 Hz), 9.0 (d, 1H, J ) 4.8 Hz), 8.9 (d, 1H, J ) 8.4 Hz), 8.6 (s, 1H), 8.49 (d of d, 1H, J ) 2.4, 7.2 Hz), 8.43 (d, 1H), 8.1 (m, 1H), 7.8 (dd, 1H, J ) 2.4, 1.2 Hz), 7.4(m, 2H). 7.28(d, 1H), 4.4 (m, 1H), 3.65 (s, 3H), 3.29 (m, 2H), 1.8 (m, 2H), 1.59-1.44 (m, 4H). Synthesis of N-E-(Picolinyl)-N-r-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)-nicotinyl)lysine Methyl Ester (L3). To a round-bottom flask were added picolinic acid (10 mg, 0.08 mmol), 5 mL of DMF, 1-O-(N,N′,N′′,N′′′tetramethyluronium)azabenzotriazoloxy hexafluorophosphate (38 mg, 0.1 mmol), 1-hydroxyazabenzotriazole (14.6 mg, 0.1 mmol), and DIEA (0.104 mL, 0.077 gm). After the solution was stirred for 7 min, N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester hydrochloride (50 mg, 0.10 mmol) was added. The reaction mixture was stirred for another 30 min. DMF was removed under vacuum and the crude oil subjected to HPLC purification. The collected fractions were combined and lyophilized to give a fluffy white solid. The yield was 34 mg (73%). MS (ESI, +ve): m/z ) 569.1815 for C26H28N6O7S ([M + H]+). 1H NMR (DMSO-d6 + DCl): 9.32 (br s, 1H), 8.62 (m, 1H), 8.52 (s, 1H), 8.3 and 8.24 (bs, 2H), 8.0-7.9 (m, 2H), 7.8 (dd, 1H, J ) 1.2 Hz, J ) 6 Hz), 7.5 (m, 1H), 7.4 (m, 2H), 7.19 (d, 1H, 9.6 Hz), 4.4 (m, 1H), 3.64 (s, 3H), 3.3 (m, 2H), 1.9-1.8 (m, 2H), 1.61.54 (m, 2H), 1.45-1.25 (m, 2H). Synthesis of 1-(N-(6-(2-(2-Sulfonatobenzaldehyde)hydrazono)nicotinyl))-6-(nicotinoyl)pentanediamine (L4). To a 15 mL round-bottom flask was added sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (50 mg, 0.113 mmol) followed by DMF (3 mL) and by pentamethylenediamine (13.8 mg, 0.135

Purohit et al.

mmol). The mixture was stirred for 30 min. A precipitate begins to form a few minutes into the reaction. The precipitate was filtered and was confirmed as the intermediate product according to LC-MS. The intermediate was used directly without any further purification for the next step. 1-(N-(6-(2-(2-Sulfonatobenzaldehyde)hydrazono)nicotinyl))pentanediamine (23 mg, 0.056 mmol) was added to a flask containing nicotinic acid (7.5 mg, 0.061 mmol), 1-O-(N,N′,N′′,N′′′-tetramethyluronium)azabenzotriazoloxy hexafluorophosphate (24 mg, 0.063 mmol), and diisopropylethylamine (58 mg, 0.45 mmol) in DMF (3 mL). The reaction mixture stirred at room temperature for 4 h. DMF was removed in vacuo and the crude product was purified by HPLC. The collected fractions were combined and lyophilized to give a fluffy white solid. The yield was 15 mg (52%). MS (ESI, +ve; m/z ) 511.1754 for C24H26N6O5S ([M + H]+). 1H NMR (600 MHz, DMSOd6 + DCl): 9.35 (s, 1H), 9.33 (s, 1H), 9.0 (m, 2H), 8.58 (s, 1H), 8.4 (d of d, 1H, J ) 2.0, 7.2 Hz), 8.1 (m, 1 H), 7.8 (m, 1H), 7.4 (m, 2H), 7.23 (br, 1H), 3.3 (t, 2H), 3.2 (t, 2H), 1.5 (m, 4H), 1.3 (m, 2H). Synthesis of 1-(N-(6-(2-(2-Sulfonatobenzaldehyde)hydrazono)nicotinyl))hexanediamine. To a 15 mL round-bottom flask was added sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (100 mg, 0.226 mmol) followed by DMF (3 mL) followed by hexamethylenediamine (26.3 mg, 0.226 mmol). The mixture was stirred for 10 min. DMF was removed in vacuo and the crude mixture subjected to purification by HPLC. The collected fractions were combined and lyophilized to give a white solid. The yield was 46 mg (∼49%). MS (ESI, +ve): m/z ) 406.15 for C18H24N5O4S ([M + H]+). 1H NMR (DMSO-d6 + DCl): 9.34 (s, 1H), 8.59 (s, 1H), 8.50 (dd, 2H, J ) 2.0, 7.2 Hz), 8.09 (t, 1 H), 7.8 (m, 1H), 7.4 (m, 2H), 7.23 (br, 1H), 3.25 (t, 2H), 2.73 (m, 2H), 1.5 (m, 4H), 1.31 (m, 4H). Synthesis of 1-(N-(6-(2-(2-Sulfonatobenzaldehyde)hydrazono)nicotinyl))-6-(nicotinoyl)hexanediamine (L5). To a 15 mL round-bottom flask was added nicotinic acid (5.34 mg, 0.043 mmol) and 1O-(N,N′,N′′,N′′′-tetramethyluronium)azabenzotriazoloxy hexafluorophosphate(18.14 mg, 0.047 mmol). To the above mixture were added DMF (3 mL) and diisopropylethylamine (16.78 mg, 0.130 mmol, 0.022 mL). The solution was stirred for 5 min, after which 1-(N-(6-(2-(2sulfonatobenzaldehyde)hydrazono)nicotinyl))hexanediamine (20 mg, 0.047 mmol) was added in one lot. The reaction mixture was stirred for another 30 min at room temperature. After removal of DMF, the crude oil was subjected to HPLC purification. The collected fractions were combined and lyophilized to give a white solid. The yield was 18 mg (80%). MS (ESI, +ve): m/z ) 525.1915 for C25H28N6O5S ([M + H]+). 1H NMR (600 MHz, DMSOd6 +DCl): 9.3 (d, 2H, J ) 7.2 Hz), 9.04 (d, 1H, J ) 5.4 Hz), 9.02 (d, 1H, J ) 8.4 Hz), 8.58 (s, 1H), 8.48 (dd, 2H, J ) 1.8, 7.8 Hz), 8.17 (m, 1H), 7.79 (m, 1H), 7.4 (m, 2H), 7.23 (br, 1H), 3.3 (t, 2H, J ) 7.2 Hz), 3.2 (t, 2H, J ) 7.2 Hz), 1.54 (m, 4H), 1.35 (m, 4H). General Procedure for Preparation of 99mTc Complexes. To a 5.0 mL vial were added the HYNIC chelator (1.0-40 µg), 0.4 mL of 0.25 M succinate buffer (pH ) 5.0), and 0.4 mL of tricine solution (25-100 mg/mL in 0.25 M succinate buffer, pH ) 5.0). After addition of 0.5 mL of 99mTcO - solution (40-80 mCi/mL in saline) and 25 µL 4 of SnCl2‚2H2O solution (1.0 mg/mL in 0.1 N HCl), the mixture was heated in a water bath at 95-100 °C for 10-15 min. After cooling to room temperature, a sample

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Scheme 1. Synthesis of L1-L3

of the resulting solution was analyzed by radio-HPLC and ITLC. In all cases, formation of [99mTc]colloid was minimal. Solution Stability Studies. For solution stability in the kit matrix, the macrocyclic complex [99mTc(L2)(tricine)] was prepared, and samples of the resulting reaction were analyzed by radio-HPLC at t ) 0, 1, 2, 3, and 4 h postlabeling. For solution stability of HPLCpurified [99mTc(L2)(tricine)], peaks A and C were collected into a 25 mL round-bottom flask. Solvents were removed using a rotary evaporator. The residue was dissolved in saline, and samples of the resulting solution were analyzed by radio-HPLC over 4 h postpurification. For TPPTS and cysteine challenge experiments, the complex [99mTc(L2)(tricine)] was prepared first and diluted with an equal volume of saline containing TPPTS (1 mg/mL) or cysteine (1 mg/mL). Samples of the resulting solution were analyzed by radio-HPLC over 6 h. Radiolabeling Efficiency Experiment. To a 5.0 mL vial were added 0.05-0.5 mL of the L2 solution (20 µg/ mL) in 0.25 M succinate buffer (pH ) 5.0) and 0.2 mL of tricine solution (100 mg/mL in 0.25 M succinate buffer, pH ) 5.0). The total volume in each vial was adjusted to ∼1.2 mL using 0.25 M succinate buffer, pH ) 5.0). After addition of 0.3 mL of 99mTcO4- solution (∼30 mCi) and 25 µL of SnCl2‚2H2O solution (1.0 mg/mL in 0.1 N HCl), the mixture was heated at 100 °C for 10 min. After cooling to room temperature, the resulting solution was analyzed by radio-HPLC. Each condition was carried out in duplicate and the radiolabeling yield was calculated as the average of the two vials. General Procedure for Preparation of Macrocyclic 99Tc Complexes. To a 5.0 mL vial were added the pyridine-containing HYNIC chelator (500-1000 µg), 0.5 mL of 0.25 M succinate buffer (pH ) 5.0), and 0.5 mL of tricine solution (100 mg/ mL in 0.25 M succinate buffer, pH ) 5.0). After addition of 0.5 mL of 99TcO4- solution (1 mg/mL in water) and 25 µL of SnCl2‚2H2O solution (10 mg/mL in 0.1 N HCl), the mixture was heated at 100 °C for ∼15 min. After cooling to room temperature, a sample of the resulting solution was analyzed by LCMS.

RESULTS AND DISCUSSION

Synthesis of Pyridine-Containing HYNIC Derivatives. L1-L5 were prepared according to Schemes 1 and 2. For L1-L3, HYNIC-NHS was allowed to react with ω-Boc-lysine methyl ester at room temperature in DMF to give the intermediate HYNIC-(ω-Boc)K as its methyl ester. The Boc-protecting group was readily removed in a 4 M HCl dioxane solution to afford HYNIC-K(OMe) as the hydrochloride salt. Conjugation of HYNICK(OMe) with picolinic acid, nicotinic acid, and isonicotinyl chloride produced L1-L3, respectively, as their methyl esters. For L4 and L5, the synthetic procedure was similar to that for the synthesis of L1-L3, except that pentamethylenediamine and hexamethylenediamine were used as the linker instead of lysine methyl ester. L1-L5 were purified by HPLC with the purity being >98% before radiolabeling. NMR (1H and 13C) and mass spectral data are completely consistent with the proposed structures. Synthesis of 99mTc Complexes. 99mTc complexes of L1-L5 were prepared (Scheme 3) using conditions similar to those described in our previous communication (17). The HYNIC derivative was allowed to react directly with 99m TcO4- in the presence of stannous chloride and excess tricine. Radiolabeling was achieved by heating the reaction mixture at 100 °C for 10-15 min. Since the pyridinecontaining HYNIC derivatives are air-stable, there was no need for exclusion of air before addition of 99mTcO4-. After radiolabeling, a sample of the resulting solution was analyzed by radio-HPLC and TLC. The 99mTc-labeling efficiency is extremely high for L2, L4, and L5. The minimum amount of L2 to achieve high-yield 99mTclabeling (>95%) was about 1 µg for 30 mCi of 99mTcO4-, corresponding to a molar ratio of ∼8:1 for L2:Tc. We also tried to use other polydentate aminocarboxylates, such as nitrilotriacetic acid (NTA), N-(2-hydroxyethyl)iminodiacetic acid (HIDA), N-(2-hydroxyethyl)glycine (monocine), ethylenediamine-N,N′-diacetic acid (EDDA), and N-(hydroxyethyl)ethylenediamine triacetic acid (HEDTA), as coligands to replace tricine. The yields for these 99mTc complexes were always low (50 °C). Compared to the triphenylphosphine moiety, the pyridine group is much smaller. The energy barrier between different conformational isomers is much lower than that in [99mTc(HYNICKo-TPPB)(tricine)]. It is not surprising that A is able to convert to D (or C to B) and to reach equilibrium within 3 h even at room temperature. To further support these hypothesis, we used LC-MS to determine the composition of each isomer of complexes [99mTc(L)(tricine)] (L ) L2, L4, and L5) at the tracer level. The LC-MS data are listed in Table 1. Due to the extremely low concentration, determination of the composition for the smaller peaks {B and D of [99mTc(L2)(tricine)] and B of [99mTc(L4)(tricine)] and [99mTc(L5)(tricine)]} was not possible at the tracer level. Therefore, we prepared 99Tc complexes [99Tc(L)(tricine)] (L ) L2, L4, and L5) at the milligram level. The composition of all the isomers in the macrocyclic complexes [99Tc(L)(tricine)] (L2, L4, and L5) has been determined. The LC-MS data for macrocyclic 99Tc complexes are listed in Table 2 and are completely consistent with the proposed composition for macrocyclic 99mTc complexes [99mTc(L)(tricine)]. All four isomers of [99Tc(L2)(tricine)] have the same molecular weight at m/z ) 674 for [M + H]+. Both isomers of [99Tc(L)(tricine)] (L ) L4 and L5) also have the same molecular weight at m/z ) 616 for [99Tc(L4)(tricine)] and m/z ) 630 for [99Tc(L5)(tricine)]. These results clearly show that the presence of four peaks in the radio-HPLC chromatogram of [99mTc(L2)(tricine)] is indeed due to resolution of four different isomers. The formation of

these four isomers is in part due to the presence of chirality in the lysine linker and in part due to the restricted rotation of the macrocyclic Tc chelate ring. Linker Length and Isomerism in Macrocyclic 99mTc Complexes. In ternary ligand 99mTc complexes, [99mTc(HYNIC-BM)(tricine)(L)] (L ) pyridine analogues), HYNIC is most likely monodentate due to the presence of the tricine and monodentate pyridine coligands (10). However, the HYNIC group in [Tc(L)(tricine)] (L ) L2, L4, and L5) may be forced to become bidentate (Figure 6) due to the bonding of pyridine-N. The tridentate tricine may result in formation of many different coordination isomers due to its asymmetric nature in bonding to the Tc. If that were true, the coordinated tricine would have been much more fluxional and there would have been a rapid exchange among all four isomers (A, B, C, and D). The fact that there is no conversion from A to C or C to A is not consistent with this explanation. Therefore, HYNIC is most likely to be monodentate in [Tc(L)(tricine)] (L ) L2, L4, and L5). There are several ways to minimize the number of conformational isomers. These include (1) increasing the linker length so that the macrocycle can rotate freely and (2) reducing the linker length in such a way that only one conformational isomer is possible. We prepared HYNIC-en-NIC to see if the use of the 1,2-ethylenediamine linker would result in the reduction of isomerism. Unfortunately, the ethylenediamine linker is too short for the pyridine-N of the nicotinyl group to bond to the Tc (unpublished data). The minimum linker length seems to be C4-C5 or an equivalent. In this study, we prepared L5 using a hexamethylenediamine linker. In doing so, we were able to reduce the percentage of the minor isomer B from ∼10% for [99mTc(L4)(tricine)] to only ∼6% for [Tc(L5)(tricine)]. However, we were not able to totally eliminate the minor conformational isomer at ∼16 min (Figure 4). Solution Stability of Macrocyclic 99mTc Complexes. We examined the solution stability of the macrocyclic complex [99mTc(L2)(tricine)] in the kit matrix. The stability was assessed by radio-HPLC by performing six injections over 6 h. Figure 7 shows the plot of RCP versus time for [99mTc(L2)(tricine)]. There is no significant RCP change over 6 h, suggesting that [99mTc(L2)(tricine)] is stable in the kit matrix for at least 6 h. We also examined the solution stability of the HPLC-purified [99mTc(L2)(tricine)]. Peaks A and C were collected into separate flasks. After removal of volatiles under vacuum, the residue was dissolved in saline. The solution was then monitored by radio-HPLC. It was found that [99mTc(L2)(tricine)] remains stable for at least 4 h at room temperature after HPLC purification (Figure 5). There is no indication of any significant decomposition during this period of time, suggesting that [99mTc(L2)(tricine)] is

Figure 6. Possible structures for macrocyclic complexes [99mTc(L)(tricine)] (L ) L2, L4 and L5).

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Figure 7. Solution stability of the complex [99mTc(L2)(tricine)] in the matrix and in the presence of TPPTS (1 mg/mL) or cysteine (1 mg/mL). Table 3. Radiolabeling Yield for the Macrocyclic Complex [99mTc(L2)(tricine)] L2 level (µg)

99mTcO - (mCi) 4

amount of

average % yield

1 2 4 5 10

30 30 30 30 30

94.8 95.6 96.8 98.8 98.8

highly stable, since the decomposition of the 99mTc complex often leads to formation of 99mTcO4-. For the TPPTS and cysteine challenge experiments, we added TPPTS and cysteine (1 mg/mL: 2-5 × 10-3 M) into solutions containing [99mTc(L2)(tricine)]. It was found that the addition of TPPTS or cysteine caused minimal changes in the radiochemical purity over 6 h at room temperature (Figure 7). The fact that [99mTc(L2)(tricine)] remains intact in the presence of a large excess (107-fold) of TPPTS is significant, because TPPTS is a much stronger ligand than pyridine analogues (10). This suggests that the ligand exchange reaction is not dependent on the concentration of the entering ligand, and dissociation of pyridine-N is most likely the rate-limiting step. Therefore, the high solution stability of [99mTc(L2)(tricine)] is probably due to its kinetic inertness. Similar results were also obtained for ternary ligand 99mTc complexes [99mTc(HYNIC-BM)(tricine)(L)] (L ) pyridine analogues) (10) and the macrocyclic 99mTc complex [99mTc(HYNIC-Ko-DPPB)(tricine)] (17). Radiolabeling Efficiency. We examined the radiolabeling efficiency of L2 by varying the L2 concentration in the formulation matrix from 1 to 10 µg/mL for 30 mCi of 99mTcO4-. The tricine concentration remained the same in all cases. Each condition was carried out in duplicate and the yield was reported as the average of two vials. The radiolabeling yield was calculated as the total percentage of all four peaks (A, B, C, and D), and is summarized in Table 3. It is clear that [99mTc(L2)(tricine)] could be prepared in high yield (>95%) using 1 µg of L2 for 30 mCi of 99mTcO4-, corresponding to the molar ratio of 8:1 for L2:99mTc. The fact that the pyridine-N atoms in L2, L4, and L5 are able to bond to the Tc and form the macrocyclic Tc chelate is truly amazing. Traditionally, it is often believed that for the chelator to form a stable metal chelate, the number of atoms in each chelate ring is ether five or six. Chelate rings containing more than six atoms often result in less stability. The number of atoms between the β-N of HYNIC and pyridine-N of the nicotinyl group is more than 18 in macrocyclic complexes [99mTc(L)(tricine)] (L ) L2, L4, and L5), but these macrocyclic complexes are formed with high stability and extremely high radiolabeling efficiency.

There are several factors contributing to the high solution stability of macrocyclic complexes [99mTc(L)(tricine)] (L ) L2, L4, and L5). The pyridine-containing HYNIC chelators (L2, L4, and L5) are designed in such a way that when HYNIC binds to the Tc center, the “effective concentration” of the attached pyridine-N donor in the vicinity of Tc will be increased dramatically. This makes it much easier for the pyridine-N donor to bond to the Tc and form a macrocyclic 99mTc chelate. Of course, the linker length and the attachment site on the pyridine ring have to be appropriate for metal binding, as demonstrated in this study. Once the pyridine-N donor binds to the Tc and completes the octahedral coordination sphere, it is hard for the pyridine-N to become dissociated, since six-coordinated Tc(III) complexes are often kinetically inert (11). This is supported by the fact that [99mTc(L2)(tricine)] remains intact in the presence of a large excess (107-fold) of TPPTS while the corresponding 99Tc analogue, [99Tc(L2)(tricine)], remains stable in aqueous solution for 2-3 weeks without any significant decomposition. Since Abrams and co-workers first reported the use of organic hydrazines as BFCs for the 99mTc-labeling of polyclonal IgG (19, 20), HYNIC has been used for 99mTclabeling of a variety of biomolecules for the development of target-specific radiopharmaceuticals. These include antibodies (19-22), chemotactic peptides (23-26), somatostatin analogues (27-32), liposomes (33), antisense oligonucleotides (34, 35), and a folate receptor ligand (36). It is well-documented that the 99mTc-labeled HYNICprotein conjugates have very high solution stability (1922), while the 99mTc-labeled small biomolecule-HYNIC conjugates are not stable and exist in solution as a mixture of multiple species if tricine or glucoheptonate is used as coligand (1, 5, 14, 27, 31). We are also puzzled by the high level of blood activity for 99mTc-labeled HYNIC conjugates of small biomolecules, including somatostatin analogues (27, 31) and oligonucleotides (34, 35). Results from this study provide a reasonable explanation of these observations. Proteins, including antibodies and antibody fragments, contain histidine residues. When the HYNIC group bonds to the Tc, the imidazole-N from a nearby histidine residue may be able to coordinate to the Tc and form an “intramolecular” macrocycle, which is kinetically inert toward further ligand exchange with other chelators. As a result, the 99mTc-labeled HYNIC-protein conjugates often show high in vitro and in vivo stability. On the other hand, small biomolecules often contain no histidine residues and are not able to form the “intramolecular” macrocycle. When tricine is used as the coligand, the HYNIC-BM conjugates will form binary ligand complexes [99mTc(HYNIC-BM)(tricine)2], which are not stable and exist as multiple species in solution. Once the binary ligand complex [99mTc(HYNIC-BM)(tricine)2] is injected into the biological system, it may react with the imidazole group of the histidine residue from circulating blood proteins such as albumin. The serum protein binding capability is most likely responsible for the high blood activity levels of 99mTc-labeled HYNIC-BM conjugates. CONCLUSIONS

In this study, we report the synthesis of several pyridine-containing derivatives (L1-L5) and their 99mTc complexes. Preliminary results have demonstrated that the attachment site of the linker on the pyridine ring is critical for the formation of macrocyclic 99mTc complexes. For example, the pyridine-N in L3 is not able to bond to

736 Bioconjugate Chem., Vol. 15, No. 4, 2004

the Tc because the lysine linker is at the 4-position. If the lysine linker is at the 2-position, L1 is able to form the macrocyclic complex [99mTc(L1)(tricine)], but the radiochemical purity is low. When the lysine linker is attached to the 3-position of the pyridine-N, L2 is able to form the macrocyclic complex [99mTc(L2)(tricine)] in high yield (>95%). The radio-HPLC data suggest that [99mTc(L2)(tricine)] exists in solution as four isomers: two diastereomers and two conformational isomers. Diastereomers are formed due to the chirality of the lysine linker and the Tc chelate. Replacing lysine with a pentamethylenediamine linker results in the macrocyclic complex [99mTc(L4)(tricine)] with only two conformational isomers, which interconvert rapidly at room temperature. Changing the linker from pentamethylenediamine to hexamethylenediamine did not completely eliminate the minor isomer, but the percentage of the minor isomer was reduced from ∼10% for [99mTc(L4)(tricine)] to only 6% for [99mTc(L5)(tricine)]. The LC-MS data for complexes [99mTc(L)(tricine)] (L2, L4, and L5) are completely consistent with the proposed composition. On the basis of these results, it is concluded that pyridine-containing HYNIC chelators have the potential as bifunctional chelators for 99mTc-labeling of small biomolecules, if the linker is attached to the 3-position of the pyridine ring. Although L2 is able to form the macrocyclic 99mTc complex in high yield (>95%) and high radiolabeling efficiency, it is not ideal as a BFC for the 99m Tc-labeling of small biomolecules, mainly due to the presence of two diastereomers and two conformational isomers. Our ultimate goal is to develop a chelating system that forms the macrocyclic 99mTc complex with only diastereomers or more preferably without isomerism. LITERATURE CITED (1) Edwards, D. S., Liu, S., Ziegler, M. C., Harris, A. R., Crocker, A. C., Heminway, S. J., Barrett, J. A., Bridger, G. J., Abrams, M. J., and Higgins, J. D. (1999) RP463: A stabilized technetium-99m complex of a hydrazino nicotinamide conjugated chemotactic peptide for infection imaging. Bioconjuate Chem. 10, 884-891. (2) Brouwers, A. H., Laverman, P., Boerman, O. C., Oyen, W. J. G., Barrett, J. A., Harris, T. D., Edwards, D. S., and Corstens, F. H. M. A (2000) 99Tcm-labeled leukotriene B4 receptor antagonist for scintigraphic detection of infection in rabbits. Nucl. Med. Commun. 21, 1043-1051. (3) Liu, S., Edwards, D. S., Ziegler, M. C., and Harris, A. R. (2002) 99mTc-labeling of a hydrazinonictotinamide-conjugated LTB4 receptor antagonist useful for imaging infection. Bioconjugate Chem. 13, 881-886. (4) Liu, S., Edwards, D. S., Ziegler, M. C., Harris, A. R., Hemingway, S. J., and Barrett, J. A. (2001) 99mTc-Labeling of a hydrazinonictotinamide-conjugated vitronectin receptor antagonist. Bioconjugate Chem. 12, 624-629. (5) Liu, S., Edwards, D. S., Looby, R. J., Harris, A. R., Poirier, M. J., Barrett, J. A., Heminway, S. J., and Carroll, T. R. (1996) Labeling a hydrazinonicotinamide-modified cyclic IIb/IIIa receptor antagonist with 99mTc using aminocarboxylates as co-ligands. Bioconjugate Chem. 7, 63-70. (6) Edwards, D. S., Liu, S., Barrett, J. A., Harris, A. R., Looby, R. J., Ziegler, M. C., Heminway, S. J., and Carroll, T. R. (1997) A new and versatile ternary ligand system for technetium radiopharmaceuticals: Water soluble phosphines and tricine as coligands in labeling a hydrazino nicotinamide-modified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc. Bioconjugate Chem. 8, 146-154. (7) Edwards, D. S., Liu, S., Harris, A. R., and Ewels, B. A. (1999) 99mTc-labeling hydrazones of a hydrazinonicotinamide conjugated cyclic peptide. Bioconjuate Chem. 10, 803-807. (8) Liu, S., Edwards, D. S., Harris, A. R., Ziegler, M. C., Poirier, M. J., Ewels, B. A., DiLuzio, W. R., and Hui, P. (2001)

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