Bioconjugate Chem. 2004, 15, 923−926
923
TECHNICAL NOTES Carbohydrate Conjugates for Molecular Imaging and Radiotherapy: 99mTc(I) and 186Re(I) Tricarbonyl Complexes of N-(2′-Hydroxybenzyl)-2-amino-2-deoxy-D-glucose Simon R. Bayly,†,‡ Cara L. Fisher,†,‡ Tim Storr,† Michael J. Adam,*,‡ and Chris Orvig*,† Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada, and TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia, V6T 2A3, Canada. Received January 26, 2004; Revised Manuscript Received April 28, 2004
An approach to a new class of potential radiopharmaceuticals is demonstrated by the labeling of a glucosamine derivative with the tricarbonyls of 99mTc and 186Re. The proligand HL2 (N-(2′-hydroxybenzyl)-2-amino-2-deoxy-d-glucose) was produced by hydrogenation of the corresponding Schiff base and reacted with [NEt4]2[Re(CO)3Br3] to form the neutral complex [(L2)Re(CO)3] in 40% yield. 1H and 13 C NMR spectra indicate that the {Re(CO)3} core is bound in a tridentate fashion via the amino N, phenolato O, and C-3 hydroxyl O atoms of the ligand. At the tracer-level, labeling of HL2 with [99mTc(CO)3(H2O)3]+ and [186Re(CO)3(H2O)3]+ was achieved in aqueous conditions in 95 ( 2% and 94 ( 3% average radiochemical yields, respectively.
INTRODUCTION
2-[18F]-Fluoro-2-deoxy-D-glucose (FDG) is the preeminent radiotracer used in positron emission tomography (1). Because the production of 18F requires a cyclotron and the isotope has a short (110 min) half-life, its utility is somewhat limited compared to that of single photon emitters in nuclear medicine. By comparison, 99mTc, the isotope most commonly used in single photon emission computed tomography (SPECT), is produced in the form of Na99mTcO4 from a 99Mo generator; hence, it is widely available and relatively inexpensive (2). The third row transition metal analogue of technetium, rhenium, has similar chemistry to that of technetium and has particleemitting radioisotopes with physical properties applicable to therapeutic nuclear medicine (3). For these reasons, we seek a 99mTc SPECT tracer that will mimic the biodistribution of FDG and the therapeutic potential of the analogous rhenium compounds. Our approach is to attach to glucose a chelating ligand that, in a subsequent reaction, will bind the radioisotope 99mTc or 186/188Re. A metal-chelate could be preformed and then attached to glucose. To mimic the properties of FDG it is imperative that the effects of the tracer group on the properties of the glucose molecule are minimized. Existing 99mTc labeled glucose derivatives fail this criterion: they are either ionic or have relatively high molecular weight (i.e. carry two glucose moieties) (4). We elected to use the versatile low valent fac-{M(CO)3} core (M ) 99mTcI or 186ReI), whose recent chemistry has been pioneered by Alberto and co-workers (5). The facially * Corresponding authors. E-mail:
[email protected] and
[email protected]. † University of British Columbia. ‡ TRIUMF.
coordinated carbonyl ligands stabilize the Tc +1 oxidation state, obviating the elaborate, often macrocyclic, polydentate structures required to stabilize other intermediate oxidation states of Tc and Re. In neutral complexes with simple N,O donors the fac-{M(CO)3} core possesses intermediate lipophilicity, an advantage in living systems (6). Glucosamine (2-amino-2-deoxy-D-glucose) is a highly attractive scaffold for a glucosyl ligand, because the amine acts both as a potential coordination site and as a useful target for further functionalization. Furthermore, there is much evidence in the literature to suggest that N-functionalized glucosamines show activity with GLUTs (glucose transporters) and hexokinasesthe enzymes that are most closely associated with the metabolism of FDGs even when the functional group is large (7). EXPERIMENTAL PROCEDURES
All solvents and chemicals (Fisher, Aldrich) were reagent grade and used without further purification unless otherwise specified. HL1 (8) and [NEt4]2[Re(CO)3Br3] (9) were prepared according to previously published procedures. 1H and 13C NMR spectra were recorded on a Bruker AV-400 instrument at 400.132 and 100.623 MHz, respectively. Assigned chemical shifts for the compounds prepared are recorded in Table 1. Mass spectra (+ ion) were obtained on dilute methanol solutions using a Macromass LCT (electrospray ionization, ESI). Elemental analyses were performed at the University of British Columbia Chemistry Department by Mr. M. Lakha (Carlo Erba analytical instrumentation). HPLC analyses were performed on Knauer Wellchrom K-1001 HPLC equipped with a K-2501 absorption detector, a Kapintek radiometric well counter, and a Synergi 4 µm C-18 Hydro-RP analytical column with dimensions 250 × 4.6 mm. The
10.1021/bc0499681 CCC: $27.50 © 2004 American Chemical Society Published on Web 06/10/2004
924 Bioconjugate Chem., Vol. 15, No. 4, 2004 Table 1.
1H
and
13C{1H} 1H
HL2 C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13
Technical Notes
NMR Data (DMSO-d6) (δ in ppm) for the r-Anomers of HL2 and [(L2)Re(CO)3] 13C{1H}
NMR (δ in ppm) [(L2)Re(CO)
5.11 2.34 3.52 3.06 3.39 3.4, 3.6 3.80
5.22 2.37 3.66 3.20 3.43 3.4, 3.6 3.85, 4.30
6.7 7.05 6.7 7.05
6.35 6.80 6.45 6.95
3]
δcomplex - δligand 0.11 0.03 0.14 0.14 0.04
-0.35 -0.25 -0.25 -0.10
HPLC solvent consisted of 0.1% trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B). Samples were analyzed with a linear gradient method (100% solvent A to 100% solvent B over 30 min). Synthesis. N-(2′-Hydroxybenzyl)-2-amino-2-deoxy-Dglucose (HL2). HL1 (1.00 g, 3.53 mmol) was dissolved in MeOH (60 mL), and 10% Pd/C w/w (50 mg) was added. The mixture was stirred under a pressurized H2 atmosphere (50 bar) for 24 h. The reaction mixture was clarified by filtration and the solvent evaporated to give HL2 (0.98 g, 98%). ESI-MS: 286 ([M + H]+). Anal. Calcd for C13H19NO6‚H2O: C, 51.48; H, 6.98; N, 4.62. Found: C, 51.50; H, 6.81; N, 4.60. Tricarbonyl(N-(2′-Hydroxybenzyl)-2-amino-2-deoxy-Dglucose)rhenium(I) (ReL2(CO)3). [NEt4]2[Re(CO)3Br3] (200 mg, 0.26 mmol), HL2 (74 mg, 0.26 mmol), and sodium acetate trihydrate (40 mg, 0.32 mmol) were dissolved in H2O (7 mL) and heated with stirring to 50 °C for 2 h. The solvent was then removed in vacuo and the residue dissolved in CH2Cl2 (10 mL) for 30 min. On standing, a brown residue was recovered by decanting off the solvent. This was purified to an off-white powder (58 mg, 0.10 mmol, 40%) by column chromatography (silica, 5:1CH2Cl2:CH3OH). ESI-MS: 556, 554 ([M + H]+), 578, 576 ([M + Na]+). Anal. Calcd for C16H18NO9Re‚H2O: C, 33.57; H, 3.52; N, 2.45. Found: C, 33.55; H, 3.53; N, 2.75. Radiolabeling. [99mTc(CO)3(H2O)3]+ was prepared from a saline solution of Na[99mTcO4] (1 mL, 100 MBq) using an ”Isolink” boranocarbonate kit generously donated by Mallinckrodt Inc. Due to the increased chemical inertness and lower redox potential of rhenium, [186Re(CO)3(H2O)3]+ was not accessible by the kit preparation used for technetium. [186Re(CO)3(H2O)3]+ was prepared by addition 4.5 µL of 85% H3PO4 to a saline solution of Na[186ReO4] (0.5 mL, 100 MBq), followed by addition of this solution to 3 mg of borane ammonia complex that had been flushed with CO for 10 min. The mixture was heated at 60 °C for 15 min and then cooled to room temperature. Labeling was achieved by mixing an aliquot of one of the above final solutions (0.5 mL) with a 1 mM solution of HL2 in PBS (pH 7.4, 1 mL) and incubating at 75 °C for 30 min. Stability Evaluation. [(L2)99mTc(CO)3(H2O)] (100 µL, 10 MBq, 1 mM in HL2) was added to 900 µL of either 1 mM histidine or 1 mM cysteine in PBS. The solutions were incubated at 37 °C and aliquots were removed at 1, 4, and 24 h, at which time HPLC analysis was run. Histidine labeling was achieved by adding a solution containing [99mTc(CO)3(H2O)3]+ to a 1 mM solution of histidine in PBS (pH 7.4, 1 mL) and incubating at 75 °C for 30 min. HPLC analysis confirmed the formation of a single radiolabeled product.
NMR (δ in ppm)
HL2
[(L2)Re(CO)3]
δcomplex - δligand
90.4 61.3 72.4 71.0 72.4 61.5 48.7 124.8 157.5 119.6 128.9 116.1 129.6
87.5 58.0 79.8 70.6 71.8 59.8 51.1 119.4 163.2 120.3 129.1 114.1 130.6
-2.9 -3.3 7.4 -0.4 -0.6 -1.7 2.4 -5.4 5.7 0.7 0.2 -2.0 1.0
Scheme 1
RESULTS AND DISCUSSION
The Schiff base formed by condensation of glucosamine with salicylaldehyde HL1 (Scheme 1) has been previously investigated as a ligand for transition metals, including 99Tc(V) (8). Using the starting material [NEt ] [Re(CO) 4 2 3 Br3] (9) as a “cold” surrogate for [M(CO)3(H2O)3]+ (M ) 99m Tc or 186Re), we synthesized the complex [(L1)Re(CO)3] (as observed by ESIMS (+)); however, both the imine and the complex are unstable to hydrolysis and proved to be unsuitable for aqueous radiolabeling chemistry. To circumvent the hydrolysis problem, we reduced HL1 to the more hydrolytically robust amine phenol HL2 (N(2′-hydroxybenzyl)-2-amino-2-deoxy-D-glucose, Scheme 1). Catalytic hydrogenation of HL1 provided HL2 in 98% yield, with sufficient purity for subsequent radiolabeling studies. The reaction of HL2 with [NEt4]2[Re(CO)3Br3] and NaOAc in H2O produced the compound [(L2)Re(CO)3] in 40% yield after column chromatographic purification. The molecular ion was identified as [((L2)Re(CO)3) + H]+ by ESIMS, and the formulation of the bulk sample was confirmed by elemental analysis. Comparison of the anomeric ratio (R/β) observed in the 1H NMR spectrum (CD3OD) showed a change from 1.9 for HL2 to 1.1 for the complex, indicating that complexation has decreased the difference in thermodynamic stability between the two anomers. For solubility reasons full NMR studies were carried out in DMSO-d6 solution (Table 1). The 1H NMR spectrum (DMSO-d6) of the complex is highly convoluted, but the shifting and broadening out of the aromatic resonances compared to those of HL2 signify that the phenol “arm” participates, as desired, in the binding of the {ReI(CO)3} moiety. The splitting of the methine
Technical Notes
Bioconjugate Chem., Vol. 15, No. 4, 2004 925 Table 2. Percentage of [(L2)99mTc(CO)3] Remaining after Incubation at 37 °C in 1 mM Cysteine or Histidine for 1, 4, and 24 h % of [(L2)99mTc(CO)3] remaining incubation in cysteine incubation in histidine
Figure 1. Radiation traces and retention times from HPLC analysis using a C18 column eluting with the gradient 100% 0.1% trifluoroacetic acid buffer to 100% acetonitrile over 30 min.
proton signals into two doublets for each anomer indicates the methine proton inequivalence on formation of the complex. Binding of the ligand N and O donor atoms incorporates the methine in a ring, rigidly holding the two protons in diastereotopic chemical environments. Signals due to the sugar C1 protons were shifted downfield in both anomers compared to those of HL2. Peaks due to the sugar C2 protons are also well-resolved and compared to those of HL2 are also shifted slightly downfield in both anomers. Small extraneous peaks in the spectrum also indicate that at least one other minor species is present. When kept overnight in CD3OD or DMSO-d6 solution, samples of the complex become visibly brown and the relative intensities of these peaks increase, indicating that they arise from decomposition products. The signals do not correlate with the chemical shifts of uncomplexed HL2. Minor species are also detected by UV/ visible spectroscopy in the HPLC of the complex and become more significant over time. The 13C{1H} NMR spectrum (d6-DMSO) of the complex was fully assigned for the R-anomer, and partially assigned for the β-anomer (Table 1). The Re carbonyls show three sharp resonances at 196-198 ppm as expected due to the lack of symmetry. In both anomers, peaks due to the phenol CO and the CH2 linker are shifted significantly downfield from their values of HL2, giving a clear indication that the Re is bound both by the phenol O and glucosamine N (10). The C1 and C2 signals of both anomers are shifted upfield on complexation, presumably reflecting some slight conformational change in the hexose skeleton. The result of this could be destabilization of the R-anomer and hence the changed anomeric ratio compared to that of HL2 itself. In the R-anomer the C3 signal has shifted downfield 7.4 ppm, suggesting that the C3 glucosamine hydroxyl is binding to the Re center in place of the predicted solvent molecule (11). Unfortunately, C3 for the β-anomer could not be assigned, due to the lower concentration of the anomer in DMSO solution. Less polar than water or methanol, DMSO is unable to stabilize the unfavorable dipole moments present in the β-anomer (12). It is unlikely that the stereochemistry at C1 can have any effect on the geometry-dependent propensity of the C3 hydroxyl to coordinate to Re, thus both anomers are predicted to bind Re in a similar tridentate manner. Labeling HL2 with [99mTc(CO)3(H2O)3]+ and [186Re(CO)3(H2O)3]+ was achieved in 95 ( 2% and 94 ( 3% average
1h
4h
88 50
28 24
24 h not detected 4
radiochemical yields, respectively, as measured by HPLC (Figure 1). The identities of the radiolabeled complexes were confirmed to be [(L2)99mTc(CO)3] (tR ) 17.9 min) and [(L2)186Re(CO)3] (tR ) 18.2 min) by coinjection of the radiolabeled product with the authentic “cold” Re complex (tR ) 17.9 min.). To make a preliminary assessment of the potential in vivo stability of the 99mTc complex, cysteine/histidine challenge experiments were performed. In a typical test, the radiolabeled complex was incubated at 37 °C in aqueous phosphate buffer solution (pH 7.4) containing either 1 mM cysteine or 1 mM histidine, and aliquots were removed at 1, 4, and 24 h (Table 2). HPLC analysis showed the complex to be stable in either histidine or cysteine solution but only in the short term; by 4 h, less than 30% of the complex remained intact. Histidine-labeled [99mTc(CO)3(H2O)3]+ was determined to be the major decomposition product of the histidine challenge experiments. The complex instability may be due to the relatively weak binding ability of the donor atoms, especially the secondary amino group and the carbohydrate hydroxyl. When considering modifications to increase complex stability, the fortuitous tridentate binding has directed us to investigate purposely tridentate ligands, and those containing binding groups with higher affinities for the soft {M(CO)3} center. In summary, neutral, low molecular weight 99mTc- and 186Re-labeled carbohydrate complexes were produced in high radiochemical yield from a simple functionalized glucosamine. HL2 is in trials as a ligand for 62/64Cu and 67/68Ga, and other carbohydrate-containing ligands for 99m Tc and 186/188Re are under study. ACKNOWLEDGMENT
We acknowledge NSERC for a Strategic Grant and a Postgraduate Scholarship (T.S.), Mallinckrodt Inc. for the Isolink kits, the UBC Hospital Department of Nuclear Medicine for Na99mTcO4, and MDS Nordion Inc. for Na186ReO4. LITERATURE CITED (1) Fowler, J. S., and Wolf, A. P. (1997) Working Against Time: Rapid Radiotracer Synthesis and Imaging the Human Brain. Acc. Chem. Res. 30, 181-188. (2) Jurisson, S. S., and Lydon, J. D. (1999) Potential Technetium Small Molecule Radiopharmaceuticals. Chem. Rev. 99, 22052218. (3) Volkert, W. A., and Hoffman, T. J. (1999) Therapeutic Radiopharmaceuticals. Chem. Rev. 99, 2269-2292. (4) (a) Petrig, J., Schibli, R., Dumas, C., Alberto R., and Schubiger, P. A. (2001) Derivatization of Glucose and 2-Deoxyglucose for Transition Metal Complexation: Substitution Reactions with Organometallic 99mTc and Re Precursors and Fundamental NMR Investigations. Chem. Eur. J. 7, 18681873. (b) Yang, D. J., Kim, C. G., Schechter, N. R., Azhdarinia, A., Yu, D. F., Oh, C. S., Bryant, J. L., Won, J. J., Kim, E. E., and Podoloff, D. A. (2003) Imaging with 99mTc ECDG Targeted at the Multifunctional Glucose Transport System: Feasibility Study with Rodents. Radiology 226, 465-473. (5) (a) Alberto, R., Schibli, R., Waibel, R., Abram, U., and Schubiger, P. A. (1999) Basic Aqueous Chemistry of [M(OH2)3(CO)3]+ (M ) Re, Tc) Directed Towards Radiopharmaceutical
926 Bioconjugate Chem., Vol. 15, No. 4, 2004 Application. Coord. Chem. Rev. 190-192, 901-919. (b) Schibli, R., Schwarzbach, R., Alberto, R., Ortner, K., Schmalle, H., Dumas, C., Egli, A., and Schubiger, P. A. (2002) Steps Toward High Specific Activity Labeling Biomolecules for Therapeutic Application: Preparation of Precursor [188Re(H2O)3(CO)3]+ and Synthesis of Tailor-Made Bifunctional Ligand Systems. Bioconjugate Chem. 13, 750-756. (6) Zhang, M., Zhang, Z., Blessington, D., Li, H., Busch, T. M., Madrak, V., Miles, J., Chance, B., Glickson, J. D., and Zheng, G. (2003) Pyropheophorbide 2-Deoxyglcoseamide: a New Photosensitizer Targeting Glucose Transporters. Bioconjugate Chem. 14, 709-714, and references within. (7) Schibli, R., La Bella, R., Alberto, R., Garcia-Garayoa, E., Ortner, K., Abram, U., and Schubiger, P. A. (2000) Influence of the Denticity of Ligand Systems on the In Vitro and In Vivo Behavior of 99mTc(I)-Tricarbonyl Complexes: A Hint for the Future Functionalization of Biomolecules. Bioconjugate Chem. 11, 345-351. (8) (a) Adam, M. J., and Hall, L. D. (1982) Synthesis of MetalChelates of Amino Sugars: Schiff’s Base Complexes. Can. J. Chem. 60, 2229-2237. (b) Duatti, A., Marchi, A., Magon, L., Deutsch, E., Bertolasi, V., and Gilli, G. (1987) Synthesis and Structure of an Amino Sugar Schiff Base Complex of Technetium(V) Containing Salicylaldehyde in an Unusual Coordination Mode. Inorg. Chem. 26, 2182-2186.
Technical Notes (9) Alberto, R., Egli, A., Abram, U., Hegetschweiler, K., Gramlich, V., and Schubiger, P. A. (1994) Synthesis and reactivity of [NEt4][ReBr3(CO)3]. Formation and structural characterization of the clusters [NEt4][Re3(µ3-OH)(µ-OH)3(CO)9] and [NEt4][Re2(µ-OH)3(CO)6] by alkaline titration. J. Chem. Soc., Dalton Trans. 2815-2820. (10) Klufers, P., and Kunte, T. (2001) A Transition Metal Complex of D-glucose. Angew. Chem., Int. Ed. 40, 4210-4212. (11) (a) Andrews, M. A., Voss, E. J., Gould, G. L., Klooster, W. T., and Koetze, T. F. (1994) Regioselective Complexation of Unprotected Carbohydrates by Platnium(II): Synthesis, Structure, Complexation Equilibria and Hydrogen-Bonding in a Carbonate Derived Bis(phosphine) Platnium(II) Diolate and Alditolate Complexes. J. Am. Chem. Soc. 116, 5730-5740. (b) Klufers, P., and Kunte, T. (2003) Palladium(II) Complexes of the Reducing Sugars d-Aribinose, D-Ribose, rac-Mannose, and D-Galactose. Chem. Eur. J. 9, 2013-2018. (c) Klufers, P., Oswald, K., and Ossberger, M. (2002) Oxorhenium(V) Complexes of Carbohydrate Ligands. Eur. J. Inorg. Chem. 2002, 1919-1923. (12) Prayly, J. P., and Lemieux, R. U. (1987) The Influence of Solvent on the Anomeric Effect. Can. J. Chem. 65, 213-223.
BC0499681
