Bioconjugate Chem. 2005, 16, 1597−1609
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Carbohydrate-Bearing 3-Hydroxy-4-pyridinonato Complexes of Gallium(III) and Indium(III) David E. Green,†,‡,§ Cara L. Ferreira,†,‡ Robert V. Stick,⊥ Brian O. Patrick,† Michael J. Adam,‡ and Chris Orvig*,† Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada, TRIUMF, 4004 Westbrook Mall, Vancouver, B.C., V6T 2A3, Canada, and Chemistry M313, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley WA 6009, Australia. Received June 23, 2005; Revised Manuscript Received August 23, 2005
Gallium and indium complexes with pendant carbohydrates have been prepared and examined for their potential as radiopharmaceuticals. Carbohydrate-bearing 3-hydroxy-4-pyridinone ligand precursors and their tris(ligand)gallium(III) and -indium(III) complexes were synthesized and characterized by mass spectrometry, elemental analysis, and 1H and 13C NMR spectroscopy, and in the case of one intermediate, by X-ray crystallography. With three equivalents of ligand, neutral complexes formed with the bidentate hydroxypyridinone moiety complexing the gallium(III) and indium(III) metal centers.
INTRODUCTION
Isotopes of gallium and indium have been used in diagnostic nuclear medicine for decades (1-5). The gamma-emitting isotopes 67Ga (t1/2 ) 78 h, γ ) 93, 185, 300 keV) and 111In (t1/2 ) 68 h, γ ) 245, 172 keV) are the most common radionuclides of gallium and indium used in nuclear medicine. Another promising gallium radionuclide with the ideal characteristics for generator technology is the positron-emitting 68Ga (t1/2 ) 68 min) produced from a 68Ge/68Ga couple. Advantages of this radionuclide are that the long half-life of 68Ge (t1/2 ) 280 d) gives the generator a life of at least one year and 68Ga has a short half-life and can also be used for quantitative PET imaging; an on-site cyclotron is not required for production. Currently, gallium citrate is the most used gallium radiopharmaceutical for diagnostic imaging; however, the coordinating citrate ligands are readily displaced by transferrin which complexes the gallium(III) ions, thus facilitating the biodistribution of the isotope 67Ga (6, 7). For a metal complex to remain intact it must be kinetically and thermodynamically stable in comparison to other biological chelators. A high stability constant for a metal complex indicates its thermodynamic stability and is one criterion considered for radiopharmaceutical evaluation. Chelators such as 8-hydroxyquinoline (Chart 1) are used to form gallium and indium complexes with high stability constants that are used in radiopharmaceutical applications (6). More recent efforts include the use of a pendant peptide linked to DTPA (pentetreotide, Chart 1). This is a bifunctional chelate approach: the DTPA portion of the molecule chelates the gallium or indium metal center, and the peptide portion helps to direct the metal complex to somatostatin receptors in vivo (8). * Corresponding author. E-mail:
[email protected]. † University of British Columbia. ‡ TRIUMF. ⊥ University of Western Australia. § Present Address: Medical Biophysics, B.C. Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, V5Z 4E6, Canada
Chart 1. Selected Relevant Compounds That Form Gallium and Indium Complexes
Recently FDA-approved, this 111In radiopharmaceutical (OctreoScan) is being used for diagnostic imaging. To expand further the types of gallium and indium radiopharmaceuticals, metal complexes stable under in vivo conditions are needed. 3-Hydroxy-4-pyridinones also have high stability constants with gallium(III) and indium(III) ions (9-11). These ligands form neutral tris(ligand)gallium(III) and indium(III) complexes that predominate from pH 4.5 to 9 and have high thermodynamic stabilities (log β3 ∼36-38 for Ga(III) and ∼31-33 for In(III)). The pyridinone moiety also has a number of sites where it can be functionalized with the addition of biomolecules such as carbohydrates. In this work, we report carbohydrate-bearing pyridinone complexes of gallium and indium. Several novel
10.1021/bc0501808 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/04/2005
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Scheme 1. Synthesis of HOG6GPa
a (a) BnCl, NaOH, MeOH/H O, 71%; (b) sodium glycinate, MeOH/H O, 48%; (c) TsCl, pyridine, 30%; (d) NaN , acetone, 97%; (e) 2 2 3 Pd black, 60 atm. H2, MeOH, 73%; (f) DCC/NHS, DMF, 76%; (g) 10% Pd/C, 1 atm H2, MeOH/H2O, 53%.
Scheme 2. Synthesis of HOGBAPa
a (a) 4-Nitrophenol, Ag CO , MeCN, 68%; (b) NaOMe, MeOH/dichloromethane, 99%; (c) 10% Pd/C, H , MeOH/H O, 87%; (d) DCC/ 2 3 2 2 NHS, DMF, 71%; (e) 10% Pd/C, 1 atm H2, MeOH/H2O, 59%.
3-hydroxy-4-pyridinone ligands bearing pendant glucose derivatives are described as well as an improved synthesis of the previously published Feralex-G (12). The glucose-bearing ligands will likely direct the metal complexes in vivo, as carbohydrates have extensive transport and metabolic pathways. The corresponding radioactive gallium or indium complexes may have applications in diagnostic nuclear medicine (67Ga, 68Ga, 111 In) and radiotherapy (111In) related to metabolic glucose imaging such as tumor, myocardial, or brain imaging or perhaps in imaging of glucose transport receptors. Several studies of carbohydrate pendant metal complexes using the SPECT radioisotope 99mTc have been reported (13-16), but none with gallium or indium radioisotopes. RESULTS AND DISCUSSION
Ligand Preparations. The conditions for pyridinone synthesis from the corresponding pyranone often require heating for extended periods of time in slightly acidic or rather basic conditions. Larger amino substituents typically hinder pyridinone formation resulting in very low or no yields. For these reasons the strategy was to form the pyridinone-linker system first using relatively smaller amines and then couple this linker to the glucose moiety. The linkers include the carboxylic acid substituent as in 3 and a phenolic substituent as in 17. The point of glucose attachment was chosen to give some variety in structure that was relatively straightforward to synthesize. All six ligand precursors HOG6GP, HOG2GP, HOGBAP, HOGBPP, HAG6GP, and HAGBAP were
prepared by various routes from maltol (1). The hydroxyl group of maltol was benzyl-protected using benzyl chloride to produce 2. Glycine was reacted with 2 under basic conditions to produce the corresponding pyridinone 3. DCC was used to activate the carboxylic acid group of 3 as the NHS ester, which was then reacted in situ with amines 7, 13, and glucosamine to produce the amidecoupled compounds 8 (Scheme 1), 14 (Scheme 2), and 19, respectively. Debenzylation of 8, 14, and 19 was accomplished by hydrogenolysis using 10% Pd/C to produce HOG6GP, HOGBAP, and HOG2GP (Feralex-G), respectively. Amine 7 (Scheme 1) was prepared starting from methyl R-D-glucopyranoside (4) that was tosylated to produce 5 and further reacted with sodium azide, according to literature preparations (17), to afford 6. Hydrogenation of the azide was accomplished at 60 atm using palladium black as a catalyst producing amine 7. Amine 13 (Scheme 2) was prepared from 2,3,4,6-tetraO-acetyl-R-D-glucopyranosyl bromide (10); KoenigsKnorr conditions (18) with silver carbonate were used to convert 10 to 4-nitrophenyl 2,3,4,6-tetra-O-acetyl-β-Dglucopyranoside (11). Deacetylation with sodium methoxide produced 4-nitrophenyl β-D-glucopyranoside (12), and the nitro group was hydrogenated with 10% Pd/C as a catalyst to produce the amine 13 that was recrystallized as the hydrochloride. The synthesis of HOGBPP is shown in Scheme 3. 4-Aminophenol was reacted with benzyl-protected maltol 2 under slightly acidic conditions to produce 3-benzyloxy-
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Scheme 3. Synthesis of HOGBPPa
a
(a) 4-Aminophenol, H2O/MeOH, 29%; (b) ADDP, Bu3P, dichloromethane, 66%; (c) 10% Pd/C, 1 atm H2, MeOH/H2O, 86%.
Scheme 4. Synthesis of HAG6GPa
a
(a) Ac2O, pyridine, 77%; (b); 10% Pd/C, 1 atm H2, MeOH/ H2O, 71%.
Scheme 5. Synthesis of HAGBAPa
a (a) Ac O, pyridine, 74%; (b); 10% Pd/C, 1 atm H , MeOH/ 2 2 H2O, 71%.
1-(4-hydroxyphenyl)-2-methyl-4(1H)-pyridinone(17). ADDP and tributylphosphine were used to couple the phenolic moiety of 17 to 2,3,4,6-tetra-O-benzyl-D-glucopyranose (16) under Mitsunobu conditions (19) which produced the β-glucoside (18) after column chromatography. Debenzylation of 18 with H2 and 10% Pd/C afforded HOGBPP. The syntheses of HAG6GP and HAGBAP are shown in Schemes 4 and 5, respectively. Compounds 8 and 14 were acetylated using acetic anhydride in pyridine to produce 9 and 15, respectively. Debenzylation of 9 and 15 with H2 and 10% Pd/C afforded HAG6GP and HAGBAP, respectively. A synthesis of HOG2GP, also known as Feralex-G, has been previously reported. In the literature preparation, the nonbenzylated analogue of 3, 1-carboxymethyl-2methyl-4(1H)-pyridinone, is coupled to glucosamine (12). Although the reported synthesis gives good yields, purification of the product posed difficulties. Easier purifica-
Scheme 6. Synthesis of HOG2GPa
a (a) Glucosamine hydrochloride, DCC, NHS, DMF, 80 °C, 54% (b) 10% Pd/C, 1 atm H2, MeOH/H2O, 100%.
tion was afforded by preparing the benzyloxy-protected HOG2GP (19). The greater lipophilicity of 19 facilitated its silica gel purification, and a protected pyridinone also prevented adventitious metal chelation from the silica. Debenzylation of 19 with H2 and 10% Pd/C afforded high purity HOG2GP (Scheme 6). All ligands were thoroughly characterized by elemental analysis and mass spectrometry, and complete 1H and 13 C NMR spectral assignments were made using 2D techniques. The 1H NMR spectrum of HOG2GP has been previously reported, but only for the R anomer (12). As a hemiacetal, HOG2GP can mutarotate and is present as a 7:3 R:β ratio in water (at RT). The other five ligands were designed as single anomers. Tris(3-oxy-4-pyridinato)gallium(III) and -indium(III) Complexes. The neutral gallium(III) and indium(III) complexes were prepared by addition of gallium or indium nitrate solutions to solutions containing the precursor ligands. The acidic solutions containing the reaction mixtures were adjusted to neutral pH with sodium hydroxide, and subsequent column purification (G10) or precipitate collection yielded the metal complexes (Scheme 7). Elemental analyses of all the metal complexes were consistent with the calculated values. All metal complexes included 1 to 5 waters of hydration that were not eliminated after drying for at least 48 h in a vacuum desiccator at RT. These waters are consistent with other gallium and indium pyridinone complexes with 0.5-5.5 waters of hydration often being reported (9), although up to 12 waters of hydration have been reported in some solid-state structures (20, 21). Drying some gallium and indium pyridinone complexes at 85 °C in vacuo for 24 h failed to remove all waters of hydration from these complexes (9). The mass spectra (+ESI) for the metal
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Scheme 7. Synthesis of ML3 Complexesa
a Ga(OG6GP) (95%)b, In(OG6GP) (89%)c, Ga(AG6GP) (83%)c, In(AG6GP) (86%)d, Ga(OGBAP) (95%)b, In(OGBAP) (91%)c, 3 3 3 3 3 3 Ga(AGBAP)3 (90%)c, In(AGBAP)3 (93%)d, Ga(OGBPP)3 (88%)b, In(OGBPP)3 (91%)c, Ga(OG2GP)3 (91%)b, and In(OG2GP)3 (70%)c. bGa(NO ) ‚9H O, H O; cIn(NO ) ‚3H O, H O; dGa(NO ) ‚9H O, H O:EtOH; eIn(NO ) ‚3H O, H O:EtOH. 3 3 2 2 3 3 2 2 3 3 2 2 3 3 2 2
Scheme 8. Synthesis of Ga(hpp)3a
a
(a) Abg, sodium phosphate buffer; (b) Ga(NO3)3‚9H2O, DMF, 86%; (c) 10% Pd/C, 1 atm H2, MeOH/DMF/H2O, 35%.
complexes revealed protonated, sodiated, or sometimes potassiated parent molecules. These parent ions had the expected fragmentation patterns and isotopic distributions. 1 H and 13C NMR Spectra. 1H NMR spectra indicated that the pyridinone ring protons had the most prominent changes in chemical shift upon metal complexation. The largest coordination induced shift (CIS) was observed for H5, (see Table 1 for pyridinone ring numbering) where metal complexation resulted in downfield shifts of 0.11-0.19 ppm for the complexes studied. These are close to the 0.19-0.32 ppm downfield shifts observed for
other (crystallographically characterized) gallium and indium pyridinone complexes in CDCl3 and (CD3)2SO (9). Significant shifts were also observed in this work for H6 with upfield shifts from 0.04 to 0.11 ppm. Gallium(III) and indium(III) ions have the potential to coordinate alkoxy groups from citric acid (22, 23) or gluconic acid (24). The overlap of the glucose protons in some of the 1H NMR spectra in this work (3.4-3.7 ppm) made it difficult to ascertain whether there were any interactions between the hydroxyl groups of glucose and the gallium or indium metal centers. 13C NMR spectra were obtained and assigned using 2D techniques to aid
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Table 1. 13C NMR Coordination Induced Shifts (∆δ) for Selected Tris(3-oxy-4-pyridinato)gallium and -indium Complexes versus the Free Ligandsa
a Only the pyridinone and glucose moieties are considered. A positive/negative value indicates a downfield/upfield shift for the metal complexes compared to the free ligand.
in determining whether any glucose-metal direct interactions take place. Also, a comparison of the pyridinone 13C NMR region upon complexation to gallium and indium has not been previously undertaken (to our knowledge). The 13C NMR coordination induced shifts for HOG6GP, HOGBAP, and HOGBPP are listed in Table 1. There are very small chemical shift differences for the glucose carbon atoms (all less than 0.1 ppm shifts), indicating clearly that the hydroxyl groups of glucose do not coordinate to the gallium or indium metal centers; one would expect more dramatic shifts if the glucose hydroxyl groups coordinated gallium or indium. When gluconic acid complexes gallium(III), the C2-C4 hydroxyl groups deprotonate/coordinate to gallium and the resulting 13C NMR shifts for C2-C4 range from -0.3 ppm (upfield shift) to +0.6 ppm (downfield shift) in 9:1 H2O:D2O (23-25). Citrate complexation of gallium at neutral pH results in a 1.1 ppm upfield 13C CIS for the tertiary hydroxyl group that deprotonates/coordinates gallium(III). While there are small changes in the glucose 13C NMR chemical shifts, all of the pyridinone ring carbon atoms shift by -5 ppm to +8 ppm, indicating that the pyridinone moiety is complexing gallium and indium. A representative 13C NMR spectrum is shown in Figure 1. The largest CIS occurs for the pyridinone C3 atom, where a downfield shift of 6.5-8.3 ppm occurs upon formation of the gallium or indium complex. Each indium complex has a slightly larger downfield CIS for C3 (∼0.3-0.5 ppm) compared to that for the corresponding gallium complex. In contrast, upfield shifts are observed for the C4 pyridinone carbonyl ranging from ∼0.4 to 2.3 ppm. Each gallium complex has a slightly larger CIS for C4 (∼0.50.7 ppm) compared to the corresponding indium complex. The C5 and C6 atoms have upfield shifts from 2.5 to 4.9 ppm after metal complexation, while only the indium complexes show a consistent downfield CIS of 1.3-2.3 ppm for the C2 atom. So far, attempts to recrystallize the metal complexes or the precursor ligands have failed to produce X-ray quality crystals. Vapor diffusion of alcohols into water
or slow evaporation of water or MeCN were the most common methods employed. Sugars are notoriously difficult to crystallize; however, X-ray quality crystals of a benzylated pyridinone-glucose compound (8) were grown by slow evaporation of a 3:1 ethyl acetate:methanol solution (Figure 2). The bond lengths and angles (Table 2) are typical for pyridinones. The torsional angles indicate the near-planarity of the pyridinone ring, and the first carbon atom of the N2-substituent (C9) is also close to the plane of the pyridinone ring, both characteristics of other pyridinone structures. The R-anomer of glucose is also displayed in the structure of 8 which concurs with the coupling constant and chemical shift in the 1H NMR spectrum (Supporting Information). Intermolecular hydrogen bonds form a tight net in the unit cell (Supporting Information). The amide H-atom (H26) is 1.931(19) Å from O2 of the glucose ring, the amide O-atom (O6) is 1.83(2) Å from H23, and the pyridinone carbonyl O-atom (O7) is 1.81(3) Å from H24. Typically, aromatic- or alkyl-substituted pyridinone ligands and their complexes have low water solubility. The solubility of the complexes is usually less of an issue in radiopharmaceutical chemistry due to the low concentrations of the tracers; however, the ligand solubility may influence the efficiency of radiolabeling. Interestingly, the presence of the attached carbohydrates increases the solubility of both the ligands and the complexes compared to those of other pyridinonebased ligands. The water solubilities of Ga(OG6GP)3, In(OG6GP)3,Ga(OGBPP)3,In(OGBPP)3,Ga(OG2GP)3, In(OG2GP)3, Ga(AG6GP)3, and In(AG6GP)3 were all greater than 10 mM at RT. Ga(OGBAP)3 and In(OGBAP)3 have water solubilities of ∼2.5-3 mM at RT while Ga(AGBAP)3 and In(AGBAP)3 have quite low water solubilities (
