Petr Hermann

Complexes of tetraazacycles bearing methylphosphinic/phosphonic acid pendant arms with copper(II), zinc(II) and lanthanides(III). A comparison with their acetic acid analogues

Abstract A comparison of complexing properties of cyclen and cyclam derivatives containing acetic acid pendant arms on one hand and their methylphosphonic or methylphosphinic acid analogues on the other is made mainly with complexes of copper and lanthanides due to their applications in medicine. The stability constant values of Cu(II) complexes are determined mainly by the basicity of amine groups. The influence of other possible effects, such as the macrocyclic effect in tetraazacycle in contrast to linear amines or the number of additional donor atoms in or outside of pendant arms and their basicity, seems to be very small. The stability constants for Gd(III), in addition to the basicity of amines, are also influenced by the basicity of the pendants and their number. The values of cyclam derivatives are lower than those of cyclen and it corresponds to the ring size effect as was found for zinc(II) complexes. In contrast to Cu(II), the Gd(III) stability constants of the phosphonic acid ligands are also lower than those with H4dota derivatives. A comparison of co-ordination polyhedra of the carboxylic and phosphonic or phosphinic derivatives shows similar motifs that are more determined by the macrocyclic effect than by the kind of donor groups in pendant arms. The differences between the polyhedra observed result from longer C–P(O) bond in the phosphorus derivatives than that C–C(O) in the acetate pendants. Consequently, the lanthanide(III) complexes with phosphorus acid derivatives are more sterically hindered; oxygen atoms in the O4 plane are close to one another and there is insufficient room for co-ordination of a water molecule, which is crucial in MRI applications.

TRAP, a Powerful and Versatile Framework for Gallium‐68 Radiopharmaceuticals

The remarkable success of Ga-labeled peptides in neuroendocrine tumor diagnostics gave a major boost to the development of Ga-labeled tracers for positron emission tomography (PET). In contrast to common PET isotopes, Ga (t1=2 =68 min; Eb+ ,max =1.89 MeV) is not produced by on-site cyclotrons but, similarly to Tc production for scintigraphy, is obtained from Ge/Ga radioisotope generators (t1=2 ACHTUNGTRENNUNG(68Ge)=270.8 days). Conjugation of biologically active molecules to bifunctional chelate ligands and subsequent Ga complexation delivers radiopharmaceuticals that bind to pathologically overexpressed target structures with high affinity and thus allow for non-invasive molecular imaging in patients. Until now, the design of Ga tracers mainly relied on DOTA (Scheme 1), which is ideally suited for larger metal ions (particularly lanthanides) but less so for the smaller Ga ion. However, the utility of the smaller NOTA system (Scheme 1) for Ga complexation has been shown two decades ago. As it has been found that it forms Ga complexes with higher kinetic stability and allows higher Ga labeling yields, conjugates of some of its bifunctional derivatives (mainly NODAGA, Scheme 1) are increasingly utilized in the development of Ga radiopharmaceuticals in recent times. Inspired by these results, we report here the synthesis, properties, and preclinical evaluation of Ga radiopharmaceuticals derived from NOTA-analogous Tri-Azacyclononane-Phosphinic acids (abbreviated TRAP, see Scheme 1 and Table 1) and reveals their enormous utility for nuclear medicine and molecular imaging. The Ga complexation behavior was compared for TRAP ligands (Table 1), DOTA, and NOTA. Figure 1 shows that TRAP ligands are able to incorporate Ga nearly quantitatively (>95 %) at much lower ligand concentrations (cL) and, due to the low pKa of phosphinic acid moieties (ca. 0.7–1.5), also in strongly acidic media; in the case of TRAP-OH and TRAP-Pr even at a pH as low as 0.5. As this allows Ga labeling by directly using the neat eluate (0.1 m HCl) of the very popular Obninsk generator, greatly facilitated kit production of Ga tracers could be achieved. Figure 2 illustrates that particularly TRAP-Pr rapidly incorporates Ga at 25 8C and cL = 3 mm, whereas neither DOTA nor NOTA can be labeled under these conditions. However, in line with previous reports we found that unlike DOTA, NOTA can be readily labeled at room temperature when using a fairly high ligand concentration of cL = 100 mm (see the Supporting Information for details). [a] Dr. J. Notni, J. Šimeček, Prof. Dr. H.-J. Wester Lehrstuhl f r Pharmazeutische Radiochemie Technische Universit t M nchen Walther-Meissner-Strasse 3, 85748 Garching (Germany) and Klinik f r Nuklearmedizin Technische Universit t M nchen Ismaninger Strasse 22, 81675 M nchen (Germany) E-mail : johannes.notni@tum.de [b] J. Šimeček, Prof. Dr. P. Hermann Department of Inorganic Chemistry, Faculty of Science Charles University in Prague (Czech Republic) [**] TRAP =Triazacyclononane-phosphinic acid. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201103503. Scheme 1. Macrocyclic chelators discussed in the text (see also Table 1).

Gallium(III) complexes of DOTA and DOTA-monoamide: kinetic and thermodynamic studies.

Given the practical advantages of the (68)Ga isotope in positron emission tomography applications, gallium complexes are gaining increasing importance in biomedical imaging. However, the strong tendency of Ga(3+) to hydrolyze and the slow formation and very high stability of macrocyclic complexes altogether render Ga(3+) coordination chemistry difficult and explain why stability and kinetic data on Ga(3+) complexes are rather scarce. Here we report solution and solid-state studies of Ga(3+) complexes formed with the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, (DOTA)(4-), and its mono(n-butylamide) derivative, (DO3AM(Bu))(3-). Thermodynamic stability constants, log K(GaDOTA) = 26.05 and log K(GaDO3AM(Bu)) = 24.64, were determined by out-of-cell pH-potentiometric titrations. Due to the very slow formation and dissociation of the complexes, equilibration times of up to ∼4 weeks were necessary. The kinetics of complex dissociation were followed by (71)Ga NMR under both acidic and alkaline conditions. The GaDOTA complex is significantly more inert (τ(1/2) ∼12.2 d at pH = 0 and τ(1/2) ∼6.2 h at pH = 10) than the GaDO3AM(Bu) analogue (τ(1/2) ∼2.7 d at pH = 0 and τ(1/2) ∼0.7 h at pH = 10). Nevertheless, the kinetic inertness of both chelates is extremely high and approves the application of Ga(3+) complexes of such DOTA-like ligands in molecular imaging. The solid-state structure of the GaDOTA complex, crystallized from a strongly acidic solution (pH < 1), evidenced a diprotonated form with protons localized on the free carboxylate pendants.

PET/CT imaging of osteoblastic bone metastases with 68Ga-bisphosphonates: first human study

Bisphosphonates are well established ligands for Tc for planar and SPECT/CT imaging of osteoblastic metastases. A novelDOTA-based bisphosphonate, (4-{[bis-(phosphonomethyl)) carbamoyl]methyl}-7,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid (BPAMD) [1, 2], was labelled using the Ge/Ga generator-derived positron emitter Ga [3], resulting in the PET tracer [Ga]BPAMD. [Ga]BPAMD was injected i.v. into a patient with known extensive bone metastases of prostate cancer. [Ga]BPAMD [maximum intensity projection (MIP) 50 min post-injection (p.i.), 462 MBq] revealed intense accumulation in multiple osteoblastic lesions in the central skeleton, ribs and proximal extremities: a = coronal PET, b = sagittal PET/CT. For comparison, c shows F-fluoride PET (sagittal, MIP 90 min p.i., 270 MBq). Metastases were detected in the whole skeleton with a maximal standardized uptake value (SUVmax) of 77.1 and 62.1 in the 10th thoracic and L2 vertebra vs 39.1 and 39.2 for F-fluoride, respectively. The advantages of this new bone imaging PET tracer are the very high target to soft tissue ratios and fast clearance, its ease of use and the generator-based availability of Ga which becomes especially important in these days of Tc shortage. While F-fluoride is adsorbed on bone surface and is related to blood flow, the bisphosphonate [Ga]BPAMD is taken up also by osteoclasts reflecting the farnesyl diphosphate synthase enzyme dynamics in the HMG-CoA reductase pathway. Since this pathway is mainly responsible for the osteoclastic bone destruction, [Ga]BPAMD may be superior in osteoclastic lesions. Finally, [Ga]BPAMD seems to be an ideal PET/CT tracer to plan and monitor bisphosphonate therapy in several bone disorders like osteoporosis, osteitis deformans, bone metastases, multiple myeloma, osteogenesis imperfecta, etc. and also to monitor radionuclide therapy for palliation of bone pain.

Mn2+ Complexes with Pyridine-Containing 15-Membered Macrocycles: Thermodynamic, Kinetic, Crystallographic, and 1H/17O Relaxation Studies

Given its five unpaired d-electrons, long electronic relaxation time, and fast water exchange, Mn(2+) is a potential candidate for contrast agent application in medical magnetic resonance imaging. Nevertheless, the design of chelators that ensure stable Mn(2+) complexation and optimal relaxation properties remains a coordination chemistry challenge. Here, we report the synthesis of two pyridine-containing ligands L1 and L2, with 15-membered triaza-dioxa-crown and pentaaza-crown ether macrocycles, respectively, and the characterization of their Mn(2+) complexes. Protonation constants of the ligands and stability constants of various metal complexes were determined by potentiometry. The presence of the pyridine in the macrocyclic ring induces rigidity of the complexes which results in a greater thermodynamic stability with respect to the nonpyridine analogues. Solid-state structures of MnL1 and MnL2 confirmed seven-coordination of Mn(2+) with Cl(-) and H(2)O in axial positions. The dissociation kinetics of MnL2 in the presence of Zn(2+) were followed by relaxometric measurements. They proved the prime importance of the proton-assisted dissociation while the zinc(II)-assisted pathway is not important at physiological pH. For MnL1, the dissociation was too fast to be studied by conventional relaxivity measurements under pH 6. A combined (17)O NMR and (1)H NMRD study on MnL1 and MnL2 yielded the parameters that govern the relaxivity of these complexes. The water exchange rate for MnL1, k(ex)(298) = 0.38 x 10(7) s(-1), is the lowest value ever reported for a Mn(2+) complex, while a considerably higher value was obtained for MnL2 (k(ex)(298) = 6.9 x 10(7) s(-1)). Anion binding was studied by relaxometric titrations. They revealed weak interactions between MnL2 and phosphate or citrate, leading to the formation of monohydrated species. Overall, the incorporation of a pyridine into a polyaza macrocycle scaffold has several beneficial effects on the Mn(2+) chelates with respect to potential MRI contrast agent applications: (i) The thermodynamic and the kinetic stability of the complexes is increased. (ii) The rigidified ligand backbone results in higher coordination numbers of the metal ion, allowing for two inner-sphere water molecules in aqueous solution.

Thermodynamic study of lanthanide(III) complexes with bifunctional monophosphinic acid analogues of H4dota and comparative kinetic study of yttrium(III) complexes.

New bifunctional H(4)dota-like ligands with three acetic acid and one phosphinic acid pendant arms and propionate (H(5)do3ap(PrA)) or 4-aminobenzyl (H(4)do3ap(ABn)) reactive groups bound to the phosphorus atom were investigated. Potentiometric studies showed that the ligands have a similar basicity to the parent H(4)dota and the stability constants of their complexes with sodium(i) and selected lanthanide(III) ions are also similar. Formation and acid-assisted decomplexation kinetics of yttrium(III) complexes with a series of H(4)dota-like ligands (H(4)dota and its phosphinic/phosphonic acid analogues) were studied and the reactions are sensitive to a slight modification of the ligand structure. The (2-carboxyethyl)phosphinic acid derivative H(5)do3ap(PrA) and the phosphonic acid ligand H(5)do3ap form complexes faster than H(4)dota. The most kinetically inert complex is that with H(4)do3ap(ABn). Rates of complexation and decomplexation can depend on the ability to transfer proton(s) outside/inside the complex cavity and, therefore, on the hydrophobicity of the ligands. The results demonstrate that the new bifunctional ligands are suitable for labelling biomolecules with yttrium(iii) radioisotopes for utilization in nuclear medicine.

Lanthanide(III) Complexes of Bis(phosphonate) Monoamide Analogues of DOTA : Bone-Seeking Agents for Imaging and Therapy

Lanthanide complexes of DOTA derivatives 2a (BPAMD) and 2b (BPAPD), having a monoamide pendant arm with a bis(phosphonate) moiety, were comparatively tested for application in MRI, radiotherapy, and bone pain palliation. (1)H, (31)P, and (17)O NMR spectroscopy show that they are nine-coordinated, with one water molecule in the first coordination sphere of the Ln(III) ion. The bis(phosphonate) moieties are not coordinated to the lanthanide and predominantly mono- and diprotonated at physiological pH. The parameters governing the longitudinal relaxivities of the Gd complexes are similar to those of other monoamides of DOTA reported in the literature. Upon adsorption on hydroxyapatite, the relaxivities at 20 MHz and 25 degrees C of Gd-2a and Gd-2b were 22.1 and 11 s(-1) mM(-1), respectively. An in vivo gamma-ray imaging study showed that the (177)Lu complexes of 2a and 2b have a high affinity for bones, particularly for growth plates and teeth with a prolonged retention.

Dissociation kinetics of Mn2+ complexes of NOTA and DOTA

The kinetics of transmetallation of [Mn(nota)](-) and [Mn(dota)](2-) was investigated in the presence of Zn(2+) (5-50-fold excess) at variable pH (3.5-5.6) by (1)H relaxometry. The dissociation is much faster for [Mn(nota)](-) than for [Mn(dota)](2-) under both experimental and physiologically relevant conditions (t(½) = 74 h and 1037 h for [Mn(nota)](-) and [Mn(dota)](2-), respectively, at pH 7.4, c(Zn(2+)) = 10(-5) M, 25 °C). The dissociation of the complexes proceeds mainly via spontaneous ([Mn(nota)](-)k(0) = (2.6 ± 0.5) × 10(-6) s(-1); [Mn(dota)](2-)k(0) = (1.8 ± 0.6) × 10(-7) s(-1)) and proton-assisted pathways ([Mn(nota)](-)k(1) = (7.8 ± 0.1) × 10(-1) M(-1) s(-1); [Mn(dota)](2-)k(1) = (4.0 ± 0.6) × 10(-2) M(-1) s(-1), k(2) = (1.6 ± 0.1) × 10(3) M(-2) s(-1)). The observed suppression of the reaction rates with increasing Zn(2+) concentration is explained by the formation of a dinuclear Mn(2+)-L-Zn(2+) complex which is about 20-times more stable for [Mn(dota)](2-) than for [Mn(nota)](-) (K(MnLZn) = 68 and 3.6, respectively), and which dissociates very slowly (k(3)∼10(-5) M(-1) s(-1)). These data provide the first experimental proof that not all Mn(2+) complexes are kinetically labile. The absence of coordinated water makes both [Mn(nota)](-) and [Mn(dota)](2-) complexes inefficient for MRI applications. Nevertheless, the higher kinetic inertness of [Mn(dota)](2-) indicates a promising direction in designing ligands for Mn(2+) complexation.

How is 68Ga Labeling of Macrocyclic Chelators Influenced by Metal Ion Contaminants in 68Ge/68Ga Generator Eluates?

To assess the influence of Zn2+, Cu2+, Fe3+, Al3+, TiIV, and SnIV on incorporation of 68Ga3+ into pendant‐arm macrocyclic chelators, the 68Ga labeling of 1,4,7‐triazacyclononane‐1,4,7‐triacetic acid (NOTA), 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA), 1,4,7‐triazacyclononane‐1,4,7‐tris[methyl(2‐carboxyethyl)phosphinic acid]) (TRAP), and 1,4,7‐triazacyclononane‐1‐[methyl(2‐carboxyethyl)phosphinic acid]‐4,7‐bis[methyl(2‐hydroxymethyl)phosphinic acid] (NOPO), as well as their peptide conjugates, was investigated in the presence of varying concentrations of these metal ions. The 68Ga labeling yield for carboxylate‐type chelators NOTA and DOTA is decreased at lower metal ion contaminant concentrations compared with phosphinate‐type chelators TRAP and NOPO. The latter are able to rapidly exchange coordinated ZnII with 68Ga3+, as confirmed by mass spectrometry and 31P NMR spectroscopy. 68Ga labeling of ZnII complexes of TRAP and NOPO proceeds as efficient as labeling of neat NOTA; this applies also to the corresponding peptide conjugates of these chelators. This behavior results in substantially improved selectivity for Ga3+ and, therefore, in more robust and reliable 68Ga labeling procedures. In addition, none of the investigated chelators binds 68Ge, rendering post‐labeling purification protocols, for example, solid‐phase extraction, a reliable means of removal of 68Ge contamination from 68Ga radiopharmaceuticals.

68Ga-BPAMD: PET-imaging of bone metastases with a generator based positron emitter

PURPOSE Bone metastases are a serious aggravation for patients suffering from cancer. Therefore, early recognition of bone metastases is of great interest for further treatment of patients. Bisphosphonates are widely used for scintigraphy of bone lesions with (99m)Tc. Using the (68)Ge/(68)Ga generator together with a macroyclic bisphosphonate a comparable PET-tracer comes into focus. PROCEDURES The bisphosphonate DOTA-conjugated ligand BPAMD was labelled with (68)Ga. [(68)Ga]BPAMD was evaluated in vitro concerning binding to hydroxyapatite and stability. The tracers in vivo accumulation was determined on healthy rats and bone metastases bearing animals by μ-PET. RESULTS BPAMD was labelled efficiently with (68)Ga after 10 min at 100°C. [(68)Ga]BPAMD showed high in vitro stability within 3h and high binding to hydroxyapatite. Consequently, μ-PET experiments revealed high accumulation of [(68)Ga]BPAMD in regions of pronounced remodelling activity like bone metastases. CONCLUSIONS (68)Ga BPAMD reveals great potential for diagnosis of bone metastases via PET/CT. The straight forward (68)Ga-labelling could be transferred to a kit-preparation of a cyclotron-independent PET tracer instantaneously available in many clinical sites using the (68)Ge/(68)Ga generator.