Synthesis of hexa aza cages, SarAr-NCS and AmBaSar and a study of their metal complexation, conjugation to nanomaterials and proteins for application in radioimaging and therapy.

A novel hexa aza cage, N(1)-(4-isothiocyanatobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine (SarAr-NCS) was synthesized in good yield and characterized by (1)H NMR and electrospray mass spectrometry. A new method for the synthesis of the related N(1)-(4-carboxybenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine (AmBaSar) using the p-carboxybenzaldehyde is reported. The complexation of Cu(2+), Co(2+) and Zn(2+) by the two ligands over a range of pHs was found to be similar to the parent derivative SarAr. SarAr-NCS was conjugated to both silica particles (≈90 nm diam.) and the model B72.3 murine antibody. The SarAr-NCSN-silica particles were radiolabeled with Cu(2+) doped (64)Cu and the number of ligands conjugated was calculated to be an average of 7020 ligands per particle. Conjugation of SarAr-NCS to the B72.3 antibody was optimized over a range of conditions. The SarAr-NCSN-B72.3 conjugate was stored in buffer and as a lyophilized powder at 4 °C over 38 days. Its radiolabeling efficiency, stability and immunoreactivity were maintained. The development of a high yielding synthesis of SarAr-NCS should provide an entry point for a wide range of Cu and Zn radiometal PET imaging agents and potentially radiotherapeutic agents with (67)Cu.

An unusually flexible expanded hexaamine cage and its Cu(II) complexes: variable coordination modes and incomplete encapsulation.

The bicyclic hexaamine "cage" ligand Me(8)tricosaneN(6) (1,5,5,9,13,13,20,20-octamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosane) is capable of encapsulating octahedral metal ions, yet its expanded cavity allows the complexed metal to adopt a variety of geometries comprising either hexadentate or pentadentate coordination of the ligand. When complexed to Cu(II) the lability of the metal results in a dynamic equilibrium in solution between hexadentate- and pentadentate-coordinated complexes of Me(8)tricosaneN(6). Both [Cu(Me(8)tricosaneN(6))](ClO(4))(2) (6-coordinate) and [Cu(Me(8)tricosaneN(6))](S(2)O(6)) (5-coordinate) have been characterized structurally. In weak acid (pH 1) a singly protonated complex [Cu(HMe(8)tricosaneN(6))](3+) has been isolated that finds the ligand binding as a pentadentate with the uncoordinated amine being protonated. vis-NIR and electron paramagnetic resonance (EPR) spectroscopy show that the predominant solution structure of [Cu(Me(8)tricosaneN(6))](2+) at neutral pH comprises a five-coordinate, square pyramidal complex. Cyclic voltammetry of the square pyramidal [Cu(Me(8)tricosaneN(6))](2+) complex reveals a reversible Cu(II/I) couple. All of these structural, spectroscopic, and electrochemical features contrast with the smaller cavity and well studied "sarcophagine" (sar, 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane) Cu(II) complexes which are invariably hexadentate coordinated in neutral solution and cannot stabilize a Cu(I) form.

Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64Cu-SarAr immunoconjugates.

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 tumor-specific (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 microCi/microg (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.

Octahedral and trigonal prismatic structure preferences in a bicyclic hexaamine cage for zinc(II), cadmium(II) and mercury(II) ions.

The synthesis and characterisation of complexes of the hexaamine cage ligand facial-1,5,9,13,20-pentamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosane (fac-(Me)(5)-D(3 h)tricosaneN(6)) with Zn(II), Cd(II) and Hg(II) is reported. Single crystal X-ray structural analyses of the Cd(II) and Hg(II) complexes reveal that the coordination spheres of both cations have an unusual trigonal prismatic stereochemistry organised by the ligand substituents and cavity size. This is unprecedented for hexaamine complexes of these metal ions, and in stark contrast to the distorted octahedral stereochemistry found previously for the analogous Zn(II) complex. An X-ray structural analysis of single crystals of the diprotonated ligand [fac-(Me)(5)-D(3h)tricosaneN(6) - 2H](CF(3)SO(3))(2) shows that it also prefers to adopt a trigonal prismatic structure. The (13)C NMR spectra of the metal complexes indicate that their structures are preserved at 20 degrees C in solution. However, heating the Zn(II) complex to approximately 130 degrees C appears to convert it to the trigonal prismatic form. In contrast cooling the trigonal prismatic Hg(II) complex to -80 degrees C does not convert it to the octahedral structure. The results are also compared to the structures of various other transition metal ion complexes of the same or similar ligands. This comparison yields overall an appreciation of the factors that determine the final structures of complexes formed with such tricosaneN(6) ligands.

An expanded cavity hexaamine cage for copper(II).

The crystal structure of the bicyclic hexaamine complex [Cu(fac-Me5-tricosane-N6)](ClO4)2.H2O (fac-Me5-tricosane-N6 = facial-1,5,9,13,20-pentamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosane) at 100 K defines an apparently tetragonally compressed octahedral geometry, which is attributed to a combination of dynamic interconversion and static disorder between two tetragonally elongated structures sharing a common short axis. This structure is fluxional at 60 K and above as shown by EPR spectroscopy. Aqueous cyclic voltammetry reveals that a remarkably stable Cu(I) form of the complex is stabilised by the encapsulating nature of the expanded cage ligand.

New 64Cu PET imaging agents for personalised medicine and drug development using the hexa-aza cage, SarAr.

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.

Facile cobalt(III) template synthesis of novel branched hexadentate polyamine monocarboxylates.

New hexadentate polyamine monocarboxylate ligands, 11-amino-9-(2-aminoethyl)-3,6,9-triazaundecanoate (tren-engly-), 12-amino-10-(2-aminoethyl)-3,7,10-triazadodecanoate (tren-tngly-) and 13-amino-11-(2-aminoethyl)-3,8,11-triazatridecanoate (tren-bngly-), were synthesized by intramolecular coupling of tetradentate tris(2-aminoethyl)amine (tren) and didentate N-([small omega]-formylalkyl)glycinates, OCH(CH2)nNHCH2CO2-, in easily and stereoselectively assembled cobalt(III) templates, p-[Co(tren){(RO)2CH(CH2)nNHCH2CO2}](O3SCF3)2, n = 1-3 (R = Me or Et). The reaction sequences comprised assembly of the template from [Co(tren)(O3SCF3)2]O3SCF3 (1) and (RO)2CH(CH2)nNHCH2CO2Et, deprotection of the pendant acetal in acid, intramolecular condensation of the resulting aldehyde with a coordinated primary amine at intermediate pH to form the imine and reduction of this by NaBH4. For n= 1, imine formation occurred exclusively at the primary amine trans to the carboxylate producing the hexadentate 11-amino-9-(2-aminoethyl)-3,6,9-triazaundeca-5-enoato (tren-enimgly-) complex, i-[Co(tren-enimgly)]Cl2.3.5H2O. In all instances, subsequent imine reduction gave the s isomer complex, exclusively. Complexes p-[Co(tren){(MeO)2CHCH2gly}](O3SCF3)2 (3), i-[Co(tren-enimgly)]ZnCl4.H2O (5), s-[Co(tren-engly)]ZnCl(4)(s-6), s-[Co(tren-tngly)]ZnCl4.H2O (s-7) and s-[Co(tren-bngly)ZnCl3]2ZnCl4 (s-8) were structurally characterized by X-ray crystallography. Charcoal-catalyzed equilibration of s-[Co(tren-engly)]Cl(2).2H(2)O dissolved in water produced the s- (s-6), p- (p-6) and t-[Co(tren-engly)]2+ (t-6) isomers in comparable amounts. p-6 and t-6 were also structurally characterized as their tetrachlorozincate and chloride salts, respectively. In base-catalyzed reactions, s-6 and t-6 each also formed p-6. Reduction of s-[Co(tren-engly)]Cl2.2H2O with (NH4)2S and acidification liberated the pentaamino carboxylic acid ligand which was isolated as the hydrochloride salt.

Specificity in template syntheses of hexaaza-macrobicyclic cages: [Pt(Me5-tricosatrieneN6)]4+ and [Pt(Me5-tricosaneN6)]4+.

The racemic C3 hexadentate cage complex, [Pt(Me5-tricosatrieneN6)]Cl4 (1,5,9,13,20-pentamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosa- 3,14,18-triene)platinum(IV) tetrachloride), was synthesised stereospecifically and regiospecifically from a reaction of the bis-triamine template [Pt(tamc)2]Cl4 (bis[1,1,1-tris(aminomethyl)ethane]- platinum(IV) tetrachloride) with formaldehyde and then propanal, in acetonitrile under basic conditions. Largely, one racemic diastereoisomer was obtained in a surprisingly high yield (approximately 50%), even though the molecule has seven chiral centres. The origins of the stereoselective synthesis are addressed. The crystal structure of [Pt(Me5-tricosatrieneN6)]-(ZnCl4)1.5Cl.2H2O showed that all three imines were attached to one tame fragment with a chiral amine site ([symbol: see text] SSS, delta RRR) and a chiral methine carbon site ([symbol: see text] RRR, delta SSS) on each ligand strand. The PtN6(4+) moiety had a slightly distorted octahedral configuration with the two types of Pt-N bonds related to the imine and the amine donors, 2.050(7) and 2.072(6) A, respectively. Treatment with sodium borohydride (15 s, 20 degrees C) at pH approximately 12.5 reduced the imine groups, but not the Pt(IV) ion, producing a C3 saturated ligand complex [Pt(Me5-tricosaneN6)]Cl4 ((1,5,9,13,20- pentamethyl-3,7,11,15,18,22- hexaazabicyclo[7.7.7]tricosane)platinum(IV)tetrachloride). X-ray crystallographic analysis showed that the average Pt-N bond distance in the cation increased upon imine reduction to 2.10 (av) A. The cyclic voltammograms of the two cage complexes displayed irreversible two-electron reduction waves in aqueous media and a approximately 0.3 V shift to more positive potentials compared to that of the smaller cavity sar (3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) analogue. After reduction, net dissociation of one strand of the cage was also evident, to give unstable square planar Pt(II) macrocyclic products.

Assembly of polyamines via amino acids from three components using cobalt(III) template methodology.

A versatile and efficient template synthesis has been developed to synthesise novel polyamines [e.g. rac-N3-(3-aminopropyl)butane-1,3-diamine, isospermidine 1] via amino acids [e.g. (2R,4S/2S,4R)-N4-(3-aminopropyl)-2,4-diaminopentanoic acid] using cobalt(III) to assemble the three precursor components in a biomimetic manner.

Synthesis, Characterization, and Reactivity of Urea Derivatives Coordinated to Cobalt(III). Possible Relevance to Urease.

The syntheses of a cobalt(III) complex, 2, containing N-(2-pyridylmethyl)urea and of six complexes, 3, containing phenyl-substituted N-2-pyridylmethyl-N'-(X)phenylureas (where X = 4-H, 4-CH(3), 4-Br, 3-Cl, 4-CF(3), and 4-NO(2)), have been accomplished by reaction of [(en)(2)Co(OSO(2)CF(3))(2)](CF(3)SO(3)) with the urea ligands in tetramethylene sulfone. The complexes have been characterized by UV-vis, FTIR, (1)H NMR, and (13)C NMR spectra along with elemental analysis. Also, X-ray crystallographic analysis of 2 confirms that the urea ligand chelates as a bidentate through the pyridyl nitrogen atom and the endo deprotonated, urea nitrogen atom to form a stable five-membered ring. Crystals of the perchlorate salt of 2 were monoclinic, space group P2(1)/c with a = 9.743(1) Å, b = 13.924(3) Å, c = 15.006(4) Å, beta = 97.07(1) degrees, and Z = 4. Reflection data (3454) with I = 3sigma(I) were refined to conventional R factors of 0.037 and 0.051. In acidic solution (0.05-1.00 M HCl at 55 degrees C), the phenyl-substituted complexes undergo hydrolysis to form the bis(ethylenediamine)(2-picolylamine-N,N')cobalt(III) ion, 4, aniline, and CO(2). The hydrolysis kinetics of the phenyl-substituted complexes were studied by UV-vis spectroscopy (I = 1.00 M HCl/LiCl). At 55 degrees C the observed rate constants fit the rate law k(obsd) = kK[H(+)]/(1 + K[H(+)]). It is proposed that the protonated urea eliminates aniline to give a coordinated isocyanate intermediate that hydrolyzes rapidly to the pyridyl methylamine complex and CO(2) via the carbamate complex. Since all of the studies of this kind to date appear to involve the NCO intermediate, it raises the prospect that urease also functions by a similar path and that urease should be tested with NCO(-) as a substrate.

Cobalt Cage Complexes with N(3)S(3) Donor Sets and Differing Cavity Sizes: A Novel Macrobicyclic Cage with a Contracted Cap.

Treatment of the cobalt(III) complex of the hexadentate tripodal N(3)S(3) ligand ten (4,4',4' '-ethylidynetris(3-thiabutan-1-amine) with propanal and paraformaldehyde under basic conditions, followed by borohydride reduction and reoxidation of the metal center, leads largely to the encapsulated (red) metal complex cation [Co(Me(2)-N(3)S(3)sar)](3+) (Me(2)-N(3)S(3)sar = 1,8-dimethyl-3,13,16-trithia-6,10,19-triazabicyclo[6.6.6]icosane). Unexpectedly, significant amounts of the homologous (yellow) complex cation [Co(Me(2)-N(3)S(3)absar)](3+) (Me(2)-N(3)S(3)absar = 1,8-dimethyl-3,13,16-trithia-6,10,19-triazabicyclo[6.6.5]nonadecane) were also obtained. This macrobicyclic complex has a contracted cavity resulting from a cap containing one fewer methylene units than Me(2)-N(3)S(3)sar. The structures of both cobalt(III) complexes have been determined by X-ray crystallography. [Co(Me(2)-N(3)S(3)sar)]Cl.ZnCl(4).H(2)O crystallizes in the cubic space group P2(1)3 with Z = 4, a = 13.9683(11) Å. [Co(Me(2)-N(3)S(3)absar)](ClO(4))(3).0.5CH(3)CN.0.5H(2)O crystallizes in the triclinic space group P&onemacr; with Z = 4, a = 12.036(4) Å, b = 15.932(9) Å, c = 17.212(14) Å, alpha = 64.93(7) degrees, beta = 72.77(5) degrees, gamma = 88.91(7) degrees. The surprising structural rearrangement is examined, along with the spectral and redox properties of both cobalt complexes. The influence of the reduced cavity size in the absar type cage is reflected in a shift of the bands in the electronic spectrum of both the cobalt(II) and cobalt(III) complexes to higher energy, and a more negative value for the Co(III/II) redox potential. The demetalation of the complexes is also described.

Critical Evaluation of Metal Complex Molecular Mechanics. Part 1. Cobalt(III) Hexaamines.

The ability of available molecular mechanics programs to calculate structures and relative energies of metal complexes is examined via a comparative study of five different force fields: Molmec, Momec91(H), Momec91(C), Xnviron, and Spartan. The method used for assessing the validity of the force fields showed that four of the force fields were able to reproduce successfully the structures of various Co(III) hexaamine cations determined by X-ray analysis, even when these structures were considerably distorted. In certain cases, the calculated relative steric energies were not reliable. Small variations in force fields parameters sometimes led to large changes in the calculated steric energies, and in some instances, in the order of steric strain for different isomers. The most notable changes occurred when metal-dependent parameters were altered.

Ligand Dehydrogenation in Ruthenium-Amine Complexes: Reactivity of 1,2-Ethanediamine and 1,1,1-Tris(aminomethyl)ethane.

The mechanisms of oxidative ligand dehydrogenation in high-valent ruthenium hexaamine complexes of bidentate 1,2-ethanediamine (en) and tridentate 1,1,1-tris(aminomethyl)ethane (tame) are elucidated in detail. In basic aqueous solution, [Ru(III)(tame)(2)](3+) undergoes rapid initial deprotonation (pK(III) = 10.3). This is followed by a pH-dependent disproportionation step involving either [Ru(III)(tame)(2)-H(+)](2+) + [Ru(III)(tame)(2)](3+) (k(1d) = 8300 M(-)(1) s(-)(1)) or two singly deprotonated [Ru(III)(tame)(2)-H(+)](2+) ions (k(2d) = 3900 M(-)(1) s(-)(1)). The products are [Ru(II)(tame)(2)](2+) and either the singly deprotonated species [Ru(IV)(tame)(2)-H(+)](3+) (pK(IV) = 8.2) or the doubly deprotonated [Ru(IV)(tame)(2)-2H(+)](2+). These Ru(IV) complexes undergo spontaneous dehydrogenation to give the imine [Ru(II)(imtame)(tame)](2+) (imtame = 1,1-bis(aminomethyl)-1-(iminomethyl)ethane), with first-order rate constants of k(1im) = 320 s(-)(1) and k(2im) = 1.1 s(-)(1), respectively. In the [Ru(III)(en)(3)](3+) system, the initial deprotonation (pK(III) = 10.4) is followed by the corresponding disproportionation reactions (k(1d) = 9000 M(-)(1) s(-)(1), k(2d) = 3800 M(-)(1) s(-)(1)). The complex [Ru(IV)(en)(3)-H(+)](3+) (pK(IV) = 8.9) and its deprotonated counterpart, [Ru(IV)(en)(3)-2H(+)](2+), undergo dehydrogenation to give [Ru(II)(imen)(en)(2)](2+) (imen = 2-aminoethanimine) with first-order rate constants of k(1im) = 600 s(-)(1) and k(2im) = 1.0 s(-)(1), respectively. In the light of this analysis, the disproportionation and ligand oxidation of the [Ru(III)(sar)](3+) ion are reexamined (k(1d) = 4 x 10(7) M(-)(1) s(-)(1), k(2d) >/= 2 x 10(7) M(-)(1) s(-)(1), pK(IV) = 2.0, k(1im) = 17 s(-)(1), k(2im) = 5 x 10(-)(4) s(-)(1) at 25 degrees C). While the disproportionation to Ru(II) and Ru(IV) has been recognized in such systems, the complexity of the paths has not been realized previously; the surprising variation in the rates of the intramolecular redox reaction (from days to milliseconds) is now dissected and understood. Other facets of the intramolecular redox reaction are also analyzed.