5.1.1 Characterization of metal-bispa2 complexes
In the light of potential radiopharmaceutical applications, the coordination chemistry of H2bispa2 (B2) with InIII, LuIII, and LaIII was studied. To this end, equimolar solutions of the ligand and the respective metal salts in methanol (LuIII and LaIII) or in a mixture of methanol and water (InIII) were combined. With InIII and LuIII the respective metal acetates were used, where acetate acts as a base for the deprotonation of the ligand. For the synthesis of the LaIII complex, the chloride was used instead of the acetate salt and, therefore, diluted sodium hydroxide solution was added. The formation of the metal complexes was confirmed by HR-MS as well as 1H- and 13C-NMR spectroscopy. Two-dimensional NMR correlation experiments (1H-1H and 1H-13C) were used to assign the signals unambiguously.
The picolinate and pyridine donors of the ligand can rotate freely, leading to a simple 1H-NMR spectrum (see Experimental Section and Appendix A). Upon coordination of the free ligand to the metal ions, significant changes in chemical shifts and coupling patterns of the NMR spectra are observed. The 1H-NMR spectrum of the [LaIII(B2)]+ (LaIII-B2) complex displays sharp and well-resolved diastereotopic splitting of the methylene protons associated with the picolinic acid arms. In addition, all previously equivalent sets of protons show different chemical shifts. A slightly different but altogether similar splitting pattern can be observed in the 1H-NMR spectrum of the corresponding LuIII complex. In contrast, the 1H-NMR of the [InIII(B2)]+ (InIII-B2) complex reveals changes in chemical shifts but shows no additional splitting of the signals. Furthermore, some peaks in the spectra of the complex become broad in comparison to the free ligand, suggesting some sort of fluxional behavior in solution.
Crystals of the InIII complex suitable for solid state X-ray analysis were obtained by slow evaporation from a methanolic solution. The complex cation [InIII(B2)]+ crystallizes with one trifluoroacetate (TFA) as counterion and four molecules of water in the asymmetric unit. The central InIII ion is coordinated by seven donor atoms of the doubly deprotonated N6O2-type ligand, with bond lengths between 2.200(1) Å for In-Npa3 and 2.486(1) Å for In-N3. The pyridine nitrogen Npy1 has an In-Npy1 distance of 3.1073(11) Å and might hence be described as semi-coordinating since the pyridine lone pair is oriented towards the metal ion and efficiently shields the metal center from other donor groups, such as coordinating solvents or anions. The solid state structure of the complex cation [InIII(B2)]+
is depicted in Figure 58 and the corresponding structural and crystallographic data are
presented in Table 16 and Appendix B.
Figure 58. ORTEP plot of the complex cation of [InIII(B2)](TFA). Ellipsoids are shown at
the 50 % probability level; co-crystallized solvent molecules, counterions, and hydrogen atoms are omitted for clarity.[256]
Table 16. Selected bond distances of H2bispa2 (B2) and [InIII(B2)](TFA).
H2bispa2 (B2) [InIII(B2)](TFA) distance [Å] In-N7 2.3867(10) In-N3 2.4859(10) In...Npy1 3.1073(11) In-Npy2 2.3784(10) In-Npa7 2.2201(10) In-Opa7 2.2599(9) In-Npa3 2.2000(10) In-Opa3 2.2270(9) N3...N7 2.766(3) 2.9355(14) Npy1...Npy2 4.679(3) 5.0052(15)
The elemental analysis of the InIII-B2 complex confirms the presence of water molecules in the solid sample. For the corresponding LuIII and LaIII complexes, no crystals suitable
In N3 N7 Npy1 Npy2 Npa7
for X-ray crystallography were obtained. However, NMR and HR-MS as well as the elemental analyses of the compounds confirm structures similar to the corresponding InIII complex, i.e. a 1:1 metal ion to ligand complex with TFA as a counterion and several water molecules.
5.1.2 Potentiometric titrations
The thermodynamic stabilities of the InIII-, LuIII- and LaIII-complexes based on B2 were measured by potentiometric titration (H2O, 25 °C, 0.1 M KCl), and the respective constants are listed in Table 17. While for LuIII and LaIII the stability constants were accessible by direct titration of equimolar ligand / metal ion solutions, a competing ligand was needed to determine the stability of the InIII-B2 complex. The acyclic chelator EDTA
(L9) was used for the ligand-ligand competition titrations (see Experimental Section for
details).
Table 17. Metal complex stability constants (log β) and pM7.4 values of B2 (H2O, 25 °C,
0.1 M KCl).
model (a) InIII(b) LuIII LaIII
ML 24.39(6) 8.51(3) 11.42(6)
MLH 12.60(22) 15.49(13)
MLH2 16.35(11)
MLH-1 0.50(4)
pM7.4(c) 25.0 9.1 12.0
(a) L denotes the respective ligand with completely deprotonated basic centers; the charges of the
species are omitted for clarity. (b) Formation of InL was determined by ligand-ligand competition
titrations. (c) Calculated for 10 µM total ligand and 1 µM total metal ion concentration at pH 7.4 and
25 °C.
The formation constants log βML and pM values (pM = -log [Mn+] at pH 7.4) for InIII complexes with several selected chelators are summarized in Table 18. The logarithmic stability constant of the InIII-B2 complex is 24.4 and therefore significantly larger than the respective stability constant observed for the InIII-transferrin complex (18.3). The iron-binding human serum protein is a main in vivo competitor for InIII.[406] Compared to other ligand systems relevant for nuclear medicine, InIII-B2 was found to have a higher stability constant than [InIII(DOTA)]- (23.9),[250] but to be less stable than [InIII(DTPA)]2- (29.0)[407] and [InIII(octapa)]- (26.8).[158] In this context, it is interesting to note
that InIII-DOTA is significantly more stable in vivo than InIII-DTPA, even though the log β ML and pM7.4 values for InIII-DTPA are much higher than for the corresponding DOTA complex.[48] This demonstrates that these thermodynamic parameters can provide initial insight into the stability of metal ion complexes, but cannot be correlated directly with their in vivo behavior, where inertness rather than stability are of importance (kinetics vs. thermodynamics). In addition, hydrolysis plays a major role considering the nM to pM concentrations of the complexes in comparison to the relatively high concentrations of hydroxides at physiological pH. The complex stabilities log βML of the complexes LuIII-B2 and LaIII-B2 were also determined and are relatively small (8.5 and 11.4 respectively) in comparison to the corresponding complexes with other chelators (see Appendix C). The species distribution diagrams of B2 in the presence of the investigated metal ions are
shown in Appendix C.
Table 18. Stability constants (log βML) and pM7.4 values for selected InIII complexes.
ligand log βML pM7.4(a) ref
DOTA (L5) 23.9(1) 18.8 [158,250]
DTPA (L10) 29.0 25.7 [158,407]
H4octapa (L35) 26.8(1) 26.5 [158]
transferrin 18.30 18.7 [158,406]
H2bispa2 (B2) 24.39(6) 25.0