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A new hexaazamacrobicyclic cage ligand, 1-N-(4-aminobenzyl)- 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (SarAr) has been designed for conjugation to proteins. SarAr was synthesised and characterised by microanalyses, 1 H NMR and electrospray mass spectrometry. The complexation of selected transition metal ions (Cu(), Ni() and Co() at 10 Ϫ6 M) by SarAr was complete within 30 min over pH 6 to 8. The [ 64 Cu(SarAr)] 2ϩ complex was investigated with a view to applications in radioimaging. The [ 64 Cu(sar)] 2ϩ complex was found to be stable in human plasma for at least 174 h and biodistribution studies in mice, showed that the [ 64 Cu(SarAr)] 2ϩ complex was rapidly excreted through the renal system unlike the free 64 Cu 2ϩ . Overall, the simple synthesis, ready complexation behaviour of SarAr, the kinetic inertness of the [Cu(SarAr)] 2ϩ complex to dissociation of 64 Cu and its facile elimination from mice make it an attractive prospect for use in nuclear medicine.

Ascorbate (Asc) reductions of the oral anticancer platinum() prodrugs cis,trans,cis-[PtCl 2 (OAc) 2 (cha)(NH 3 )] (JM216) and cis,trans,cis-[PtCl 2 (OCOC 3 H 7 ) 2 (cha)(NH 3 )] (JM221) and of the isomers of JM216, viz. trans,cis,cis-[PtCl 2 (OAc) 2 (cha)(NH 3 )] (JM394) and trans,trans,trans-[PtCl 2 (OAc) 2 (cha)(NH 3 )] (JM576) (OAc = acetate, cha = cyclohexylamine) have been investigated in a 1.0 M aqueous perchlorate medium using stopped-flow and conventional UV/VIS spectrophotometry as a function of temperature and pH. JM216 and 221 are reduced to cis-[PtCl 2 (cha)(NH 3 )] (JM118) and JM394 and 576 to cis-and trans-[Pt(OAc) 2 (cha)(NH 3 )], respectively. The redox reactions follow the second-order rate law:where k is a pH dependent second-order overall rate constant andReduction of JM216 and JM221 is slow (overall rate constants k 298 = 5.08 ± 10 Ϫ2 and 3.25 × 10 Ϫ2 mol Ϫ1 dm 3 s Ϫ1 at pH 7.12, respectively) and is suggested to take place via an outer-sphere mechanism. Reductions of JM394 and JM576 are more than three orders of magnitude faster (k 298 = 230 ± 6 mol Ϫ1 dm 3 s Ϫ1 at pH 7.0 for JM394). They are suggested to take place by a mechanism involving a reductive attack on one of the mutually trans chloride ligands by Asc 2Ϫ and less efficiently by HAsc Ϫ leading to the formation of a chloride-bridged activated complex. The second-order rate constants for reduction of JM394 by HAsc Ϫ and Asc 2Ϫ at 25 ЊC are 0.548 ± 0.004 and (4.46 ± 0.01) × 10 6 mol Ϫ1 dm 3 s Ϫ1 , respectively. The rate constants for reduction of JM216 and JM221 by Asc 2Ϫ at 25 ЊC are calculated to be 672 ± 15 and 428 ± 10 mol Ϫ1 dm 3 s Ϫ1 , respectively and reduction by HAsc Ϫ was not observed under these conditions. Thus, Asc 2Ϫ is up to 7 orders of magnitude more efficient as a reductant than HAsc Ϫ . H 2 Asc is virtually inactive. The activation parameters ∆H ‡ and ∆S ‡ for reduction of JM216, JM221, JM394, and JM576 by Asc 2Ϫ are 52 ± 1, 46 ± 1, 56.2 ± 0.5, and 63 ± 2 kJ mol Ϫ1 and Ϫ97 ± 4, Ϫ120 ± 4, Ϫ24 ± 2, and Ϫ8 ± 5 J K Ϫ1 mol Ϫ1 , respectively. An isokinetic relationship gives further support to the mechanistic assignments.

The advancement of positron emission tomography (PET) depends on the development of new radiotracers that will complement 18 F-FDG. Copper-64 ( 64 Cu) is a promising PET radionuclide, particularly for antibody-targeted imaging, but the high in vivo lability of conventional chelates has limited its clinical application. The objective of this work was to evaluate the novel chelating agent SarAr (1-N-(4-aminobenzyl)-3, 6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) for use in developing a new class of tumorspecific 64 Cu radiopharmaceuticals for imaging neuroblastoma and melanoma. The anti-GD2 monoclonal antibody (mAb) 14.G2a, and its chimeric derivative, ch14.18, target disialogangliosides that are overexpressed on neuroblastoma and melanoma. Both mAbs were conjugated to SarAr using carbodiimide coupling. Radiolabeling with 64 Cu resulted in >95% of the 64 Cu being chelated by the immunoconjugate. Specific activities of at least 10 Ci/ g (1 Ci ‫؍‬ 37 GBq) were routinely achieved, and no additional purification was required after 64 Cu labeling. Solid-phase radioimmunoassays and intact cell-binding assays confirmed retention of bioactivity. Biodistribution studies in athymic nude mice bearing s.c. neuroblastoma (IMR-6, NMB-7) and melanoma (M21) xenografts showed that 15-20% of the injected dose per gram accumulated in the tumor at 24 hours after injection, and only 5-10% of the injected dose accumulated in the liver, a lower value than typically seen with other chelators. Uptake by a GD2-negative tumor xenograft was significantly lower (<5% injected dose per gram). MicroPET imaging confirmed significant uptake of the tracer in GD-2-positive tumors, with minimal uptake in GD-2-negative tumors and nontarget tissues such as liver. The 64 Cu-SarAr-mAb system described here is potentially applicable to 64 Cu-PET imaging with a broad range of antibody or peptide-based imaging agents.

Template syntheses based on tris (ethane-1,2-diamine)cobalt(III) lead to cobalt(III) complexes of cage hexamines of the ' sarcophagine ' type ( sarcophagine = sar = 3,6,10,13,16,19- hexaazabicyclo [6.6.6] icosane ) rapidly and in high yield. Reduction of these species to their cobalt(II) forms enables the ligands to be removed in concentrated acids at elevated temperatures, and in hot aqueous solutions containing excess cyanide ion. The free sarcophagine and 1,8-diaminosarcophagine [(NH2)2sar or diamsar] ligands are strong bases, accepting up to four and five protons, respectively, in aqueous solution. In chloride medium, I = 1.0, at 298 K, pK1 = 11.95, pK2 = 10.33, pK3 = 7.17, pK4 ≈ 0 for sarcophagine , and pK1 = 11.44, pK2 = 9.64, pK3 = 6.49, pK4 = 5.48, pK5 ≈ 0 for diaminosarcophagine , with very similar values being found for triflate medium. Crystal structure determinations for both free bases, the chloride, sulfate, perchlorate and nitrate salts of diamsar , the complex of zinc chloride with sar, and the magnesium nitrate complex with diamsar show remarkably small variations in the cavity defined by the bicyclic ligands, though relatively subtle bond length and bond angle changes can be rationalized in terms of the effects of proton and metal ion binding. Exhaustive methylation of sarcophagine produces the highly lipophilic, hexatertiary base hexamethylsarcophagine , which, in the solid state, adopts quite different conformations and nitrogen-atom configurations to those of sar itself. All the ligands rapidly form metal ion complexes of generally exceptional kinetic and thermodynamic stability.

The success of positron emission tomography (PET) in personalised medicine and drug development requires radioisotopes that provide high quality images and flexible chemistry for a broad application. 64Cu is arguably one of the most suitable PET isotopes for imaging with the evolving target agents, but there are not many appropriate chelating agents for 64Cu and this has limited its wider application. The bi-functional chelator, SarAr is known to bind 64Cu2+ quantitatively (i.e. one metal per ligand present) and rapidly (<2 min) at 10(-6) M over a range of pH (4-9). In this paper the conjugation of SarAr to the whole and fragmented antibody is described. Conjugation of the SarAr to the protein does not impair its coordination of the 64Cu. It complexes the 64Cu2+ rapidly, quantitatively and essentially irreversibly at pH 5. Animal studies show that the 64Cu-SarAr-immunoconjugates maintain their specificity for the target and are stable in vivo. Also, SarAr is a platform technology, is easy to use in a kit formulation and is readily adaptable for the wider application in 64Cu PET imaging.

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