Chelating Rare-Earth Metals (Ln³⁺) and ²²⁵Ac³⁺ with the Dual-Size-Selective Macrocyclic Ligand Py₂-Macrodipa

Abstract

Radioisotopes of metallic elements, or radiometals, are widely employed in both therapeutic and diagnostic nuclear medicine. For this application, chelators that efficiently bind the radiometal of interest and form a stable metal−ligand complex with it are required. Towards the development of new chelators for nuclear medicine, we recently reported a novel class of 18-membered macrocyclic chelators that is characterized by their ability to form stable complexes with both large and small rare-earth metals (Ln³⁺), a property referred to as dual size selectivity. A specific chelator in this class called py-macrodipa, which contains one pyridyl group within its macrocyclic core, was established as a promising candidate for ¹³⁵La³⁺, ²¹³Bi³⁺, and ⁴⁴Sc³⁺ chelation. Building upon this prior work, here we report the synthesis and characterization of a new chelator called py₂-macrodipa with two pyridyl units fused into the macrocyclic backbone. Its coordination chemistry with the Ln³⁺ series was investigated by NMR spectroscopy, X-ray crystallography, DFT calculations, analytical titrations, and transchelation assays. These studies reveal that py₂-macrodipa retains the expected dual size selectivity and possesses an enhanced thermodynamic affinity for all Ln³⁺ compared to py-macrodipa. By contrast, the kinetic stability of Ln³⁺ complexes with py₂-macrodipa is only improved for the light, large Ln³⁺ ions. Based upon these observations, we further assessed the suitability of py₂-macrodipa for use with ²²⁵Ac³⁺, a large radiometal with valuable properties for targeted alpha therapy. Radiolabeling and stability studies revealed py₂-macrodipa to efficiently incorporate ²²⁵Ac³⁺, and to form a complex that is inert in human serum over 3 weeks. Although py₂-macrodipa does not surpass the state-of-the-art chelator macropa for ²²⁵Ac³⁺ chelation, it does provide another effective ²²⁵Ac³⁺ chelator. These studies shed light on the fundamental coordination chemistry of the Ln³⁺ and may inspire future chelator design efforts.

Graphical Abstract

INTRODUCTION

Radiopharmaceutical agents, which leverage radionuclides for medicinal therapy and diagnosis, have been receiving increasing attention as new modalities progress to the clinic.^1–5 Across the entire periodic table, many metallic elements have been identified to possess radioisotopes suitable for this application.^6–8 In order to harness these radiometals for clinical use, a chelator that can rapidly form thermodynamically and kinetically stable complexes is usually required. Facile complex formation minimizes radioactivity loss during handling before in vivo administration, and high complex stability prevents in vivo release of the potentially harmful radiometal. A significant body of research has been devoted to chelator design for radiometals by addressing their distinct chemical properties and coordination chemistry preferences.^7–10

As a recent contribution to this area, we developed a class of ligands that exhibit “dual size selectivity”, a property characterized by their ability to preferentially bind both the large and small rare-earth metal ions (Ln³⁺). This dual-size-selective property is attributed to a significant conformational toggle that occurs in this ligand class as they bind metal ions of different sizes (Scheme 1) to optimize complex stability. For large Ln³⁺, a 10-coordinate, nearly C₂-symmetric complex is formed (Conformation A), whereas for small Ln³⁺, an 8-coordinate asymmetric complex arises (Conformation B).^10–12 The first member of this ligand class identified was the macrocyclic chelator macrodipa (Chart 1).¹¹ Despite the unique selectivity profile of macrodipa, the kinetic lability of the corresponding Ln³⁺−macrodipa complexes limited its use for biomedical applications.¹²

Scheme 1.

Depiction of the Conformational Toggle Present in Dual-Size-Selective Chelators when Binding Ln³⁺ Ions with Different Sizes.^a

^aReproduced from J. Am. Chem. Soc. 2020, 142, 13500. Copyright 2020 American Chemical Society.

Chart 1.

Structures of Ligands Discussed in This Work.

To overcome this limitation, we modified the structure of macrodipa by incorporating a stronger pyridyl donor in place of an ethereal oxygen within the macrocycle to afford the second-generation ligand py-macrodipa (Chart 1). This change led to a significant enhancement of both the thermodynamic and kinetic stability of its Ln³⁺ complexes, while maintaining its dual size selectivity. Based on these improvements of macrodipa, we showed that py-macrodipa efficiently formed stable complexes with ¹³⁵La³⁺ and ⁴⁴Sc³⁺, two valuable radionuclides for nuclear medicine with significantly disparate ionic radii.¹² To further validate the potential of py-macrodipa for nuclear medicine applications, we assessed its suitability for chelating ²²⁵Ac³⁺ and ²¹³Bi³⁺, two therapeutically valuable α-emitters.^13,14 These studies showed that py-macrodipa is a promising candidate for ²¹³Bi³⁺ chelation, but does not form a complex of sufficient kinetic stability for ²²⁵Ac³⁺ nuclear medicine applications.¹⁵

The inefficacy of py-macrodipa for the stable chelation of ²²⁵Ac³⁺ motivated us to target a third-generation dual-size-selective chelator that forms complexes with even greater thermodynamic and kinetic stability. Based on the improvements that were observed upon the incorporation of a single pyridyl group into the macrocycle of macrodipa, we targeted the novel chelator py₂-macrodipa (Chart 1), which contains an additional pyridyl donor. In this work, we discuss the synthesis of this new ligand, its coordination chemistry with Ln³⁺ ions, and its potential application for ²²⁵Ac³⁺ radiotherapy.

RESULTS AND DISCUSSION

The chelator py₂-macrodipa was synthesized via the five-step procedure shown in Scheme 2, with an overall yield of 20%. The benzyl-protected macrocyclic backbone 2 was constructed in two steps commencing from commercially available 2,6-bis(bromomethyl)pyridine and 2-benzylaminoethanol. The benzyl groups were then removed by reduction with H₂ over a Pd/C catalyst to yield the desired dipyridyl macrocycle 3. Subsequent installation of the two picolinate pendent arms via alkylation and acidic deprotection afforded py₂-macrodipa. The identity and purity of the intermediates and final product were verified by NMR spectroscopy, mass spectrometry, and analytical HPLC (Figures S1−S13).

Scheme 2.

Synthetic Route to py₂-macrodipa.

To understand the influence of the second pyridyl unit on the coordination chemistry of py₂-macrodipa, the crystal structures of its complexes with two representative Ln³⁺ ions, La³⁺ and Sc³⁺, were determined by X-ray crystallography (Figure 1). La³⁺, the largest Ln³⁺, forms a complex with py₂-macrodipa that attains the 10-coordinate, distorted C₂-symmetric Conformation A, with all nitrogen and oxygen donor atoms on the ligand engaged in metal binding. By contrast, the py₂-macrodipa complex of Sc³⁺, the smallest Ln³⁺, adopts the 8-coordinate, asymmetric Conformation B. In this crystal structure, three of the macrocycle-based ligand donor atoms (O1, O2, N4) do not directly interact with the Sc³⁺ center, whose coordination sphere is completed by an inner-sphere water molecule. The conformations observed for the La³⁺ and Sc³⁺ crystal structures are in line with our expectations, based on the well characterized conformational toggle (Scheme 1) observed for the earlier generation analogues macrodipa and py-macrodipa upon binding to Ln³⁺ of different sizes.

Figure 1.

Crystal structures of (a) [La(py₂-macrodipa)]⁺ and (b) [Sc(py₂-macrodipa)(OH₂)]⁺ complexes. Thermal ellipsoids are drawn at the 50% probability level. Solvent, counterions, and nonacidic hydrogen atoms are omitted for clarity.

Having verified that this conformational change occurs in the solid state, we next leveraged NMR spectroscopy to characterize the solution structures of Ln³⁺−py₂-macrodipa complexes with the diamagnetic La³⁺, Y³⁺, Lu³⁺, and Sc³⁺ ions, for which the ionic radius decreases from 103.2 pm to 74.5 pm.¹⁶ The ¹H and ¹³C{¹H} NMR spectra of these four complexes were acquired in D₂O at pD 7 (Figures 2 and S17−S24). Figure 2 shows a stacked arrangement of the ¹H NMR spectra of these four complexes for comparison. The ¹H NMR spectra of the complexes of La³⁺ and Y³⁺ show equivalency in their resonances, indicating that the symmetric Conformation A predominates for the py₂-macrodipa complexes of these larger ions. By contrast, Sc³⁺, the smallest Ln³⁺, yields an NMR spectrum of lower symmetry, revealing it to attain the asymmetric Conformation B in aqueous solution. The ¹³C{¹H} NMR spectra of these complexes are also consistent with these conformational assignments (Figures S18, S20, and S24). These NMR observations indicate that the solid-state crystallographic studies of the La³⁺ and Sc³⁺ complexes (Figure 1) are a good representation of their solution structures.

Figure 2.

¹H NMR spectra of La³⁺−, Y³⁺−, Lu³⁺−, and Sc³⁺−py₂-macrodipa complexes (600 MHz, D₂O, pD 7, 25 °C). The yellow asterisks in the spectra of the La³⁺ and Y³⁺ complexes highlight the decrease in symmetry of Conformation A in moving from larger to smaller ions. The purple (Conformation A) and red (Conformation B) asterisks in the spectrum of the Lu³⁺ complex indicate the two conformers present in solution.

The ¹H NMR spectrum of the py₂-macrodipa complex of the intermediately-sized Lu³⁺ is more complicated than those of La³⁺, Y³⁺, and Sc³⁺. Two different species, in a molar ratio of 4:1, are observed. The ¹³C{¹H} NMR spectrum of this complex likewise reveals the presence of two species (Figure S22). These two species are assigned to be a mixture of Conformation A and B in equilibrium. Close inspection of the aromatic region of the ¹H NMR spectrum (Figures 2 and S21), reveals an asymmetry in both conformers, despite our prior observations that Conformation A typically gives rise to spectra with symmetry. Our tentative explanation for this observation is that the Conformation A gradually descends in symmetry as the Ln³⁺ gets smaller, and the size-matching becomes poorer. This argument is supported by comparing the ¹H NMR spectra of the large La³⁺ and smaller Y³⁺ complexes (Figure 2), where a decrease in symmetry of Conformation A begins to appear. Most apparent are the two triplets in the aromatic region (labeled with orange asterisks), which can be assigned as the geminal hydrogens on the carbon atoms labeled with orange asterisks in Chart 1. For the large La³⁺, the chemical shifts of these two triplets are nearly identical, whereas for the smaller Y³⁺, their chemical shifts begin to diverge more substantially and indicate different chemical environments for these two H atoms, presumably due to a decrease in symmetry of Conformation A.

To further understand the conformational switch of py₂-macrodipa across the Ln³⁺ series, we carried out density functional theory (DFT) calculations. All Ln³⁺−py₂-macrodipa complexes in both Conformations A and B were optimized, using the same level of theory (ωB97XD/6–31G(d,p)/LCRECP) that we previously applied to study Ln³⁺−macrodipa and Ln³⁺−py-macrodipa.^{11,12,17–24} From these geometry optimizations and thermochemical calculations, the standard free energy difference (ΔG°) between Conformations A and B for each Ln³⁺ complex was determined (eq 1), as plotted in Figure 3.

Figure 3.

DFT-computed standard free energy differences (ΔG°) between Conformations A and B for Ln³⁺−py₂-macrodipa complexes, plotted versus ionic radii.

$${\left[\text{Ln}\left({\text{py}}_2-\text{macrodipa}\right)\right]}^{+}\left(\text{Conformation}\text{A},aq\right)+{\text{H}}_2\text{O}\left(l\right)\rightleftharpoons {\left[\text{Ln}\left({\text{py}}_2-\text{macrodipa}\right)\left({\text{OH}}_2\right)\right]}^{+}\left(\text{Conformation}\text{B},aq\right)$$ (1)

As the Ln³⁺ series is traversed, ΔG° gradually shifts from positive to negative values, consistent with our experimental observations that Conformation A is favored for large Ln³⁺, whereas Conformation B is preferred for small Ln³⁺. A similar computational analysis was applied in our studies of macrodipa and py-macrodipa, which also showed that Conformation A was lower in free energy for large Ln³⁺ and vice versa. The crossover point where ΔG° switches its sign occurs roughly at Tm³⁺, Er³⁺, and Tb³⁺ for py₂-macrodipa, py-macrodipa,¹² and macrodipa¹¹ systems, respectively. Thus, modifications of the macrocycle can alter the conformational dynamics and preferences of this ligand class.

Having confirmed by X-ray crystallography and NMR spectroscopy that Ln³⁺ complexes of py₂-macrodipa undergo a Ln³⁺-size-dependent conformational toggle like its earlier analogues macrodipa and py-macrodipa, we next set out to verify our hypothesis that the additional macrocyclic pyridyl moiety would lead to enhanced complex stability. In this regard, we first evaluated the thermodynamic stability of Ln³⁺−py₂-macrodipa complexes by determining their stability constants. The magnitude of stability constant measures the thermodynamic affinity of a ligand for a metal ion and is valuable for assessing its potential in different applications.^10,25,26 The protonation constants (K_i) of py₂-macrodipa, as well as the stability constants (K_LnL) of its Ln³⁺ complexes, measured via either potentiometric titrations or UV−Vis spectrophotometric titrations,^27–29 are collected in Table 1. These quantities are defined in eqs 2–3, where the concentration terms represent those at chemical equilibrium, and L signifies the uncomplexed ligand in its fully deprotonated state.

Table 1.

Protonation Constants of py₂-macrodipa, py-macrodipa, macrodipa, macropa, and Stability Constants of Their Ln³⁺ Complexes.

	py2-macrodipaa	py-macrodipa	macrodipa	macropa
log K1	7.58(4)	7.20b	7.79c	7.41d, 7.41e
log K2	6.48(1)	6.54b	7.04c	6.85d, 6.90e
log K3	3.52(3)	3.17b	3.18c	3.32d, 3.23e
log K4	2.60(5)	2.31b	2.41c	2.36d, 2.45e
log K5	2.10(11)			1.69d
log KLaL	16.68(8)	14.31b	12.19c	14.99d
log KCeL	17.13(7)	14.65b	12.50c	15.11d
log KPrL	17.28(6)	14.81b	12.41c	14.70d
log KNdL	17.11(3)	14.51b	12.25c	14.36d
log KSmL	16.56(4)	13.66b	11.52c	13.80d
log KEuL	15.93(4)	13.29b	10.93c	13.01d
log KGdL	15.25(7)	12.63b	10.23c	13.02d
log KTbL	14.76(6)	11.95b	9.68c	11.79d
log KDyL	14.04(2)	11.47b	9.36c	11.72d
log KHoL	12.68(5)	10.69b	9.36c	10.59d
log KErL	12.17(3)	10.60b	9.71c	10.10d
log KTmL	11.98(2)	10.92b	10.13c	9.59d
log KYbL	11.82(5)	11.31b	10.48c	8.89d
log KLuL	11.90(3)	11.54b	10.64c	8.25c
log KScL	16.28(4)	15.83b	14.37b

^a0.1 M KCl, this work. The values in the parentheses are one standard deviation of the last significant figure.

^b0.1 M KCl, ref 12.

^c0.1 M KCl, ref 11.

^d0.1 M KCl, ref³⁷.

^e0.1 M KCl, ref³⁸.

$$K_i=\left[{\mathrm{H}}_i\mathrm{L}\right]/\left[{\mathrm{H}}^{+}\right]\left[{\mathrm{H}}_{i-1}\mathrm{L}\right]$$ (2)

$$K_{\text{LnL}}=\left[\mathrm{L}\mathrm{n}\mathrm{L}\right]/\left[\mathrm{L}{\mathrm{n}}^{3+}\right]\left[\mathrm{L}\right]$$ (3)

The log K_LnL values of py₂-macrodipa, and those of the related structures macrodipa, py-macrodipa, and macropa, are plotted against Ln³⁺ 6-coordinate ionic radius¹⁶ in Figure 4. Like macrodipa and py-macrodipa, py₂-macrodipa exhibits a dual-size-selective pattern that is not altered via the introduction of an additional pyridyl group in the macrocycle. Importantly, the log K_LnL values of py₂-macrodipa are systematically greater compared to those of py-macrodipa, indicating that the additional macrocyclic pyridyl group of py₂-macrodipa has a pronounced effect on enhancing the overall Ln³⁺-binding affinity. The log K_LnL values of py₂-macrodipa are also significantly higher across the entire Ln³⁺ series than those of macropa, a chelator established as a promising candidate for several large radiometals including ²²⁵Ac³⁺, ^132/135La³⁺, ¹³¹Ba²⁺, ²²³Ra²⁺, and ²¹³Bi³⁺.^30–34 This observation highlights the potential of py₂-macrodipa for large radiometal chelation. Although these log K_LnL values are smaller than those observed for the Ln³⁺ complexes of the commonly applied chelator DOTA (Chart 1), for which log K_LnL spans 22.9−25.4 from La³⁺ to Lu³⁺, the established efficacy of macropa, which gives rise to substantially lower log K_LnL values, for these radiometals demonstrate that such high K_LnL values are not strictly needed for practical use in nuclear medicine. However, future ligand design efforts within this ligand class to further increase these stability constants may further improve value for use in nuclear medicine.

Figure 4.

Stability constants of Ln³⁺ complexes formed with py₂-macrodipa, py-macrodipa, macrodipa, and macropa plotted versus ionic radii.

Notably, the increase in log K_LnL in moving from py-macrodipa to py₂-macrodipa is much greater for the large Ln³⁺ than for the small Ln³⁺. This observation can be rationalized by the crystal structures. The newly introduced second pyridyl unit directly interacts and stabilizes the metal center in Conformation A (Figure 1a), whereas it is not bound to the metal in Conformation B (Figure 1b). As such, Conformation A benefits more significantly from the presence of the additional macrocyclic pyridyl donor present in py₂-macrodipa. Thus, for large Ln³⁺ complexes, for which Conformation A prevails, a substantial log K_LnL enhancement is observed, but diminished impact is found for small Ln³⁺ complexes, where Conformation B is dominant. Another noteworthy characteristic discerned from our potentiometric titrations is the identification of the protonated complex species (LnHL) for late Ln³⁺−py₂-macrodipa systems (Figures S28−S32), which was not observed for any Ln³⁺ complexes of py-macrodipa.¹² Based on this information, the most likely protonation site to form the LnHL species is the second pyridyl group (N4, Chart 1), which is substantially basic and not engaged with the Ln³⁺ center (Figure 1b) in Conformation B.

After demonstrating an enhancement of Ln³⁺ complex thermodynamic stability afforded by the second pyridyl group of py₂-macrodipa, we next evaluated its impact on complex kinetic stability, a property of critical importance for chelators applied in radiopharmaceutical settings.^1,9 For comparison to our previous studies, an identical DTPA transchelation challenge^12,39 was applied to assess the Ln³⁺−py₂-macrodipa complex kinetic stability. Specifically, the Ln³⁺−py₂-macrodipa complexes were treated with 100 equiv of DTPA at pH 7.4 and RT (22 °C), a condition that thermodynamically favors the formation of the Ln³⁺–DTPA complexes.^40–42 This transchelation process, which follows pseudo-first-order kinetics with the large excess of DTPA, was monitored by UV−Vis spectroscopy. The half-lives (t_1/2) afforded from these pseudo-first-order processes (Table 2) provide a quantitative comparative measure of the complex kinetic stability.

Table 2.

Half-lives of Ln³⁺−py₂-macrodipa, Ln³⁺−py-macrodipa, and Ln³⁺−macrodipa Complexes when Challenged with 100 Equivalents of DTPA.^a

	Ln3+–py2-macrodipa	Ln3+–py-macrodipab	Ln3+–macrodipab
La3+	≫ 5 weeks	6.3 d	1678 s
Gd3+	4.5 ± 0.2 d	5524 s	54 s
Lu3+	253 ± 6 s	853 s	65 s
Sc3+	5.9 ± 0.4 h	16.6 h	782 s

^a[LnL] = 100 μM, pH 7.4 in MOPS, 22 °C.

^bRef 12.

The trend in kinetic stability of the Ln³⁺−py₂-macrodipa complexes follows closely with that observed for their thermodynamic stability, with the light and heavy Ln³⁺ complexes undergoing slower transchelation than those in the middle of the series. In comparison to those of macrodipa and py-macrodipa, the early Ln³⁺ complexes of py₂-macrodipa exhibit a remarkable kinetic stability, with <10% dissociation of the La³⁺ complex observed after 5 weeks. However, for the smallest Sc³⁺, the kinetic stability of its py₂-macrodipa complex is significantly lower than that of py-macrodipa, indicating that the second macrocyclic pyridyl is detrimental in this regard. The enhancement of kinetic stability of the La³⁺ complex is easily rationalized from the X-ray crystallographic data (Figure 1a), where both pyridyl groups strongly engage the La³⁺ center. By contrast, the non-coordinated pyridyl group present in the Sc³⁺−py₂-macrodipa complex is positioned closely to the inner-sphere water molecule (N4–O7 distance of 2.91 Å), within a range that is consistent with hydrogen bonding, for which the N···O distances typically span from 2.8−3.0 Å.⁴³ Thus, the pendent pyridyl group is oriented appropriately to facilitate proton-assisted metal-ion dissociation, which will substantially labilize the complex in aqueous solution. The increasing kinetic lability by protonation was also illustrated in other metal complex systems.^44–46 Collectively, the inclusion of this second pyridyl unit of py₂-macrodipa enhances the thermodynamic stability for all Ln³⁺ complexes, but only improves the kinetic stability of complexes with the large Ln³⁺. Thus, py₂-macrodipa is a promising candidate for large Ln³⁺ chelation.

On account of the remarkable thermodynamic and kinetic stability of the La³⁺−py₂-macrodipa complex, we next sought to investigate the capability of py₂-macrodipa to chelate the largest trivalent cation Ac³⁺, as both ions possess similar coordination chemistry.⁴⁷ Furthermore, the radioisotope ²²⁵Ac³⁺ (t_1/2 = 9.9 d)⁴⁸ emits four α particles through its decay chain, making it valuable for use in targeted internal radiotherapy.^49–51 To assess the suitability of py₂-macrodipa as a chelator for this radiometal, we carried out ²²⁵Ac³⁺ radiolabeling studies with py₂-macrodipa and benchmarked the results to macropa and DOTA (Chart 1), two macrocyclic chelators that have established precedence for ²²⁵Ac³⁺ chelation.^30,52

Concentration-dependent radiolabeling studies were carried out by incubating different concentrations of py₂-macrodipa, macropa, and DOTA with 9.5–11.1 kBq of ²²⁵Ac³⁺ at pH 6.0 and 25 °C. The radiochemical yields (RCYs), determined by radio-TLC, are summarized in Figure 5 and Table S3. Notably, py₂-macrodipa quantitatively incorporates ²²⁵Ac³⁺ at a low concentration of 10⁻⁵ M within 5 min, revealing it to be an efficient chelator for ²²⁵Ac³⁺. This property is nearly on par with that of macropa, the current gold standard, which quantitatively radiolabels ²²⁵Ac³⁺ at micromolar concentration, an observation consistent with previously reported results.^15,30 By contrast, DOTA did not significantly undergo ²²⁵Ac³⁺ radiolabeling even at a millimolar concentration. Increasing the reaction time to 60 min had only a slight effect on the RCYs for py₂-macrodipa and macropa, indicating that the radiolabeling process is nearly complete within 5 min under these mild conditions for both chelators. In addition, the radiolabeling efficiency of py₂-macrodipa is comparable to that of py-macrodipa and significantly better than macrodipa.¹⁵

Figure 5.

Radiochemical yields (RCYs) of ²²⁵Ac³⁺ radiolabeling with py₂-macrodipa, macropa, and DOTA at different ligand concentrations (pH 6.0, 25 °C). (a) 5-min reaction time; (b) 60-min reaction time.

After establishing effective radiolabeling of py₂-macrodipa with ²²⁵Ac³⁺, the kinetic stability of its ²²⁵Ac³⁺ complex was assessed. Specifically, the ²²⁵Ac³⁺ complexes of py₂-macrodipa and macropa were incubated in human serum at 37 °C over 3 weeks to model the conditions that would be encountered when a radiopharmaceutical agent is administered in vivo. Figure 6 shows the percentage of intact complex remaining throughout the course of this experiment. Both radiocomplexes are sufficiently stable in human serum, as reflected by the fact that they remain >90% intact after 3 weeks. The resistance to human serum for [²²⁵Ac]Ac³⁺−macropa observed here is also consistent with prior studies.^15,30,53 Although the radiolabeling efficiency of py₂-macrodipa is comparable to that of py-macrodipa, the kinetic stability of [²²⁵Ac]Ac³⁺−py₂-macrodipa is substantially enhanced relative to that of [²²⁵Ac]Ac³⁺−py-macrodipa, which showed a ~90% complex dissociation in human serum at 37 °C after 24 h.¹⁵ This observation is also consistent with the DTPA transchelation challenge results (Table 2), which also revealed an overall higher Ln³⁺ complex kinetic stability for py₂-macrodipa. Collectively, py₂-macrodipa shows promise for use with ²²⁵Ac³⁺ based on its efficient radiolabeling and high complex stability. It exhibits the best performance for ²²⁵Ac³⁺ chelation among the dual-size-selective chelators.

Figure 6.

The stability of [²²⁵Ac]Ac³⁺−py₂-macrodipa and [²²⁵Ac]Ac³⁺−macropa in human serum at 37 °C.

CONCLUSION

Building upon macrodipa and py-macrodipa, we designed and prepared the third-generation dual-size-selective chelator py₂-macrodipa. Its coordination chemistry with Ln³⁺ ions was characterized by multiple techniques including X-ray crystallography, NMR spectroscopy, DFT calculations, analytical titrations, and transchelation assays. These three chelators all exhibit dual-size selective properties and are based on the same 18-membered macrocyclic core structures. They differ by the presence of 0, 1, or 2 pyridyl groups within the macrocyclic core. Without an integrated pyridyl group, macrodipa is a relatively poor chelator with respect to both the thermodynamic and kinetic stability of its Ln³⁺ complexes. The single pyridyl group in py-macrodipa serves to enhance both properties. In this study, we showed that the introduction of a second pyridyl group in py₂-macrodipa further enhanced the thermodynamic stability for all Ln³⁺ complexes, but the kinetic stability was only improved for the large Ln³⁺ complexes. As a general conclusion, it appears that the inclusion of pyridyl groups in place of ethereal donors does benefit complex stability, but the conformations of the resulting complexes also need to be considered when trying to anticipate the magnitudes of these effects. Despite the weaker performance of py₂-macrodipa for small Ln³⁺, its promising chelation properties for large Ln³⁺ prompted us to investigate its use with the therapeutic radiometal ²²⁵Ac³⁺. Radiolabeling and serum stability studies with ²²⁵Ac³⁺ revealed py₂-macrodipa to perform comparably to the state-of-the-art chelator macropa. Although the dual-size-selective properties were demonstrated both thermodynamically and kinetically, efforts need to be undertaken to improve the stability of its complexes with small metal ions for practical nuclear medicine applications. Additionally, ongoing work is also directed toward preparation of a bifunctional analogue of py₂-macrodipa that can be used to conjugate with biomolecules. Both the macrocyclic backbone and the picolinate pendant arms provide potential opportunities for this functionalization. In any case, this work affords a new potential candidate for ²²⁵Ac³⁺ chelation as well as provides new insights on the fundamental Ln³⁺ coordination chemistry of and ²²⁵Ac³⁺ radiochemistry for future chelator development endeavors.

Synopsis.

A novel macrocyclic chelator called py₂-macrodipa is reported, and its coordination chemistry with the rare-earth metals is described. These studies show it to have promising properties for the chelation of large radiometal ions for nuclear medicine. Thus, its ability to chelate the therapeutic radionuclide ²²⁵Ac³⁺ was assessed, demonstrating efficient formation of inert ²²⁵Ac³⁺ complexes.