Evaluating a triaza-18-crown-6 picolinate chelator for [^212/203Pb]Pb²⁺ and [^212/213Bi]Bi³⁺ theranostics

Abstract

H₃tripa (H₃macrotripa), a triaza-18-crown-6 macrocycle bearing three picolinate arms, enables quantitative radiolabeling of [²⁰³Pb]Pb²⁺ and [²¹³Bi]Bi³⁺ and exhibits robust human serum stability. NMR spectroscopy, X-ray crystallography, density functional theory, and UV-potentiometry investigate its coordination geometry and thermodynamics with Bi³⁺ and Pb²⁺, highlighting H₃tripa as a promising Pb/Bi theranostic chelator.

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Targeted alpha therapy (TAT) has emerged as a promising approach in oncology, leveraging the high linear energy transfer (LET) of alpha particles to induce localized and irreparable DNA damage in cancer cells. Among the alpha-emitting radioisotopes currently under investigation, ²¹³Bi has garnered significant attention due to its relatively short half-life (45.6 min) and high LET, making it effective for targeting small tumours or micrometastases.^1,2 While the parent radionuclide, ²²⁵Ac, is better matched to large, slow-clearing biomolecules due to its extended half-life (9.92 d), the shorter half-life of ²¹³Bi aligns better with the pharmacokinetics of small targeting agents.³ In addition to its therapeutic capabilities, ²¹³Bi emits a gamma photon (440 keV), enabling SPECT imaging that facilitates real-time monitoring of the radiopharmaceutical distribution and personalized dose adjustment.^4,5 Despite these advantages, the short half-life of ²¹³Bi poses logistical challenges in production, distribution, and administration.⁶ To mitigate these issues, researchers are exploring an in vivo generator based on ²¹²Pb (10.64 h, β⁻), which decays to ²¹²Bi (60.55 min, α). The longer half-life of ²¹²Pb allows for more flexible handling and extended accumulation at tumour sites.⁶ Moreover, the combination of ²⁰³Pb (51.8 h, γ) for SPECT imaging and ²¹²Pb/²¹²Bi for therapy constitutes a powerful theranostic pair, enabling both treatment and monitoring within the same framework.

The effectiveness of Pb/Bi-based radiopharmaceuticals inherently depends on the chelators used to stably bind these radionuclides (Fig. 1). Established chelators, such as H₄DOTA and TCMC, while valuable, may not fully retain ²¹²Bi after the decay of ²¹²Pb due to internal conversion events that can lead to dissociation of the daughter nuclide. This phenomenon presents a major challenge in designing chelators for in vivo²¹²Pb/²¹²Bi generators.^7,8 New classes of chelators, including diaza-crown ethers appended with picolinic acid groups, have shown promise in improving binding affinity and radiolabeling efficiency, although further studies are needed to understand their in vivo performances.^9–12

Fig. 1 Chemical structures of discussed chelators for Pb²⁺ and Bi³⁺.

We investigated a triaza-18-crown-6 macrocycle featuring three picolinate arms (first reported¹³ by Hu et al. as H₃macrotripa while this work was just beginning) for its dual size-selectivity within the lanthanoid series, and designated it H₃tripa. This study presents the first radiopharmaceutical evaluation of H₃tripa with [²⁰³Pb]Pb²⁺ and [²¹³Bi]Bi³⁺, employing NMR spectroscopy, X-ray crystallography, and density functional theory (DFT) to investigate coordination geometries, and UV-potentiometry to determine protonation and formation constants with Pb²⁺ and Bi³⁺.

The coordination properties of H₃tripa were explored by forming binary metal complexes with Pb²⁺ and Bi³⁺. Equimolar amounts of the ligand were reacted with corresponding metal salts in D₂O and pD was adjusted to 6–7 using 0.5 M NaOD. Spectral changes were observed using NMR spectroscopy (¹H and ¹H–¹H COSY) and further confirmed using high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), in which the expected [M + H]⁺ species verified metal coordination.

The ¹H NMR spectrum of the Bi³⁺ complex (Fig. 2A) exhibits well-defined, sharp peaks, indicating a rigid conformation environment with no fluctuation around the metal. Furthermore, the complex presents the characteristic diastereotopic splitting consistent with chirality generation upon binding to a metal centre, thus confirming metal ion complexation. The spectrum of [Bi(Htripa)]⁺ shows the formation of an asymmetric complex in solution, as indicated by three inequivalent proton signals in the aromatic region that correspond to three picolinic acid arms binding in distinct environments. More specifically, two arms (A and B) give a set of overlapping signals, namely a triplet at δ 8.36 ppm (2H) and a broad multiplet at δ 8.13–8.04 ppm (4H), whereas the third arm (C) produces a separate set consisting of a triplet at δ 7.96 ppm (1H) and two doublets at δ 7.78 ppm (1H) and δ 7.49 ppm (1H). The methylene hydrogens associated with the picolinic acids give rise to three pairs of doublets as they become inequivalent upon coordination to the metal centre; these are recognisable by their large geminal J_H–H′ couplings of approximately 15–17 Hz. Although partially obscured in the ¹H NMR spectrum by the water signal, the doublets are observable in the ¹H–¹H COSY spectrum (Fig. S14†).

Fig. 2 (A) ¹H NMR spectra of H₃tripa (400 MHz, D₂O, pD 2), [Bi(Htripa)]⁺ and [Pb(Htripa)] (400 MHz, D₂O, pD 6–7). (B) Variable temperature ¹H NMR spectra of [Pb(Htripa)] (400 MHz, D₂O, pD 7).

In contrast, the ¹H NMR spectrum for [Pb(Htripa)] reveals a dynamic heterogeneous coordination environment. Multiple broad peaks appear in both the aromatic and aliphatic regions, consistent with metal–ligand species rapidly exchanging. In the aromatic region two sets of picolinate resonances can be identified, a major set (purple asterisks) and a weaker less intense group (red asterisks), suggesting either two rapidly interconverting coordination isomers, with one conformer strongly favored (purple asterisks), or alternatively a single asymmetric species in which the three arms become magnetically non-equivalent. The broad signals in the aliphatic region support the presence of fast exchange phenomena, typically associated with the flexibility of Pb²⁺ complexes attributed to the lone pair activity on Pb²⁺ and its inherent stereochemical lability.^14,15 To probe this fluxional behaviour further, variable temperature (VT) ¹H NMR measurements were performed 25–90 °C (Fig. 2B). As the temperature was raised to ∼55 °C, further broadening of the aliphatic peaks was observed, suggesting increased molecular motion or conformational exchange. Simultaneously, one set of aromatic peaks diminished, likely merging with the predominant species as exchange processes accelerated. At even higher temperatures (75–90 °C), the aromatic signals sharpened, indicating that conformational averaging led to the predominance of a single species with rapid interconversion, although the aliphatic signals remained partially broad. These observations leave open the possibility that the dynamic averaging originates either from symmetric reorientation within the coordination sphere or from rapid dissociation and recoordination of donor groups. This behaviour is especially relevant, as kinetic inertness is a criterion for radiopharmaceutical applications, and the observed fluxionality might impact the long-term stability of the Pb²⁺ complex.

An X-ray quality crystal of the Pb²⁺ complex with the H₃tripa ligand was obtained via slow evaporation of water at ambient temperature, yielding irregularly shaped crystals. The molecular structure is bimetallic (Fig. 3).²²

Fig. 3 Representation of the Cl-bridged dimer {[Pb2(tripa)Cl]}₂. Only the asymmetric unit, one [Pb2(tripa)Cl] fragment, is shown; the second half is generated by inversion symmetry. Longer interactions (Pb–O/N ≥ 3.1 Å) are represented with dashed lines, hydrogens have been omitted for clarity.

The H₃tripa ligand coordinates one Pb²⁺ ion, designated ‘Pb2’, through two bidentate picolinic acid groups in a cis arrangement – both are positioned on the same side of the metal centre. Additionally, Pb2 is coordinated by five donor atoms from the macrocycle: one oxygen and one nitrogen interact more strongly (Pb–O/N ≤ 2.7 Å), whereas the other three donors form longer interactions (3.1–3.3 Å). The third picolinic acid group coordinates the second Pb²⁺ ion, designated ‘Pb1’ here, along with two oxygen atoms, and one nitrogen atom from the macrocycle.

The coordination environment around Pb2 likely provides a representation of how H₃tripa may bind Pb²⁺, with at least two picolinic acid groups playing a key role in binding. Unfortunately, due to the presence of the second Pb²⁺ ion, further structural insights into the monometallic binary [Pb(tripa)]⁻ complex via crystallography remain limited. Unfortunately, no X-ray quality crystals were obtained for the Bi(tripa) complex.

Given the complexities observed in the NMR and crystallographic data, DFT calculations were conducted to further elucidate the coordination geometries of the [Pb(tripa)]⁻ and Bi(tripa) complexes. Optimized geometries for both were obtained (Fig. 4), and several conformations explored, including those where all three picolinate arms coordinate from one side of the coordination plane and alternate arrangements in which two arms adopt a cis configuration while the third binds more flexibly from the opposite side. In some cases, only two picolinate groups coordinate the metal while the third remains unbound.

Fig. 4 DFT-optimized structures of [Pb(tripa)]⁻ (left) and Bi(tripa) (right). Hydrogen atoms are omitted for clarity.

The lowest energy conformation for [Pb(tripa)]⁻, favoured by an energy margin of approximately 20.4 kJ mol⁻¹, displays octadentate coordination. In this geometry, two picolinic acid groups bind strongly in a cis arrangement, while the third arm extends away from the coordination plane. This arrangement distorts the macrocyclic backbone so that only four out of six donor atoms (two nitrogens and two oxygens) are directly involved in coordination. These trends are consistent with experimental observations seen in related macrocycles based on picolinic acid, where donor atoms tend to coordinate from one side of the metal centre, leading to a hemidirected geometry.^16,17 For the Bi(tripa) complex, the optimized geometry closely mirrors that of the Pb²⁺ complex. On average, the Bi³⁺ complex shows shorter metal–donor bond lengths compared to the Pb²⁺ complex. This contraction arises from both the smaller ionic radius and the higher charge density of Bi³⁺, the latter leading to the chelate having stronger interactions with Bi³⁺; Bi–O bond distances are shorter (approximately 2.28 Å) compared to Pb–O bonds, and similar trends are noted for Bi–N versus Pb–N distances.

Combined UV-potentiometric titrations between pH 2 and 11 were used to determine protonation constants for H₃tripa, with results consistent with Hu et al. (Table S2†),¹³ and formation constants for H₃tripa with Pb²⁺ and Bi³⁺ (Fig. 5 and Table 1). It was found that H₃tripa forms complexes with both metal ions at very low pH; however, the direct evaluation of formation constants under highly acidic conditions was challenging due to the limitations of the potentiometric electrode. To overcome this, in-batch UV-Vis spectrophotometric titrations were undertaken, involving incrementally acidifying preformed complexes. This approach allowed for the complete determination of formation constants for [Bi(H₂tripa)]²⁺ and [Pb(H₂tripa)]⁺.

Fig. 5 Speciation plot for H₃tripa with (A) Bi³⁺ and (B) Pb²⁺; dashed line represents pH 7.4. The gradient-filled area of the graph indicates where precipitation occurred, and speciation could not be elucidated.

Table 1 Stepwise stability constants (log K) of H₃tripa complexes with Bi³⁺ and Pb²⁺

Equilibrium reaction	Bi^3+	Pb^2+
a UV-potentiometric titration at I = 0.16 M (NaCl) and 25 °C. b Bi-H3tripa UV acidic spectrophotometric titration at 25 °C. c Pb-H3tripa UV acidic spectrophotometric titration at 25 °C. Charges are omitted for clarity.
M + L ⇆ ML	27.21(4)^a	24.75(1)^a
ML + H ⇆ MHL	7.02(4)^a	7.89(2)^a
MHL + H ⇆ MH2L	3.37(2)^a^,^b	3.07(2)^a^,^c
M(OH)L + H ⇆ ML	9.0(1)^a	10.58(4)^a

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For the Bi³⁺ complex, the speciation diagram evinces the predominant species to be the neutral Bi(tripa) at pH 7.4, with a minor population of the monoprotonated [Bi(Htripa)]⁺. As pH > 8, the hydroxide-bound species [Bi(tripa)(OH)]⁻ becomes prevalent, verifying that the Bi–ligand interactions are robust even in mildly alkaline media. Conversely, the Pb²⁺ complex exists predominantly as the neutral monoprotonated [Pb(Htripa)] species at physiological pH, with only a small fraction present as the fully deprotonated [Pb(tripa)]⁻. This behaviour suggests that proton competition plays a more significant role for the binding of Pb²⁺ than for Bi³⁺, consistent with the observed higher binding affinity of H₃tripa for Bi³⁺.

pM values are a useful parameter to compare chelators of different basicity and denticity for their metal scavenging ability, with higher pM values corresponding to stronger binding.¹⁸ In Table S3† are listed the pM values of different macrocyclic chelators relevant in TAT. The pM data for Pb²⁺ and Bi³⁺ indicate that H₃tripa exhibits the highest binding affinity for both metal ions, with pBi = 30.2 and pPb = 25.1. In comparison, H₄DOTA shows the second highest pM values for Pb²⁺, and retains high thermodynamic stability for Bi³⁺.^19,20 A dipicolinate derivative of H₄DOTA, H₂Me-do2pa, demonstrates higher Bi³⁺ affinity than H₄DOTA, confirming the strong binding preference of Bi³⁺ for picolinates.²¹ However, H₂Me-do2pa exhibits significantly lower affinity for Pb²⁺. H₂macropa, containing two picolinic acid in a diaza-18-crown-6 ether backbone, shows lower affinity for Pb²⁺ compared to H₃tripa¹² – possibly due to the different symmetry of the diaza-18-crown-6 framework and the absence of a third picolinic acid, which might allow for different arrangements around the Pb²⁺. H₂BADPA-18, a benzene derivative of H₂macropa, exhibits strong Bi³⁺ binding but reduced Pb²⁺ affinity.¹¹ The lower affinity for Pb²⁺ might be attributed to the increased conformational rigidity introduced by the benzene ring.

Lastly, we assessed the radiolabeling of H₃tripa with [²¹³Bi]Bi³⁺ and [²⁰³Pb]Pb²⁺. Given the short half-life of ²¹³Bi (45.6 min), rapid radiolabeling kinetics (5–10 min) are essential to achieve near-complete incorporation of the radionuclide under mild conditions (ambient temperature, pH 5–7). The radiolabeling efficiency of H₃tripa for [²¹³Bi]Bi³⁺ was evaluated across a range of ligand concentrations (10⁻³–10⁻⁷ M) at ambient temperature and compared to H₄DOTA at elevated temperature (80 °C) after a 10 minute incubation (Fig. 6A). H₃tripa demonstrated high efficiency, achieving quantitative incorporation of [²¹³Bi]Bi³⁺ (>95% radiochemical conversion, RCC%) at 10⁻⁶ M, with a slight decrease to approximately 85% at 10⁻⁷ M. In contrast, H₄DOTA required a significantly higher concentration (10⁻⁴ M) at 80 °C to achieve comparable radiolabeling efficiency. The performance of H₃tripa is comparable to that of H₂macropa and H₂S₂macropa¹² – all three chelators achieved quantitative chelation of [²¹³Bi]Bi³⁺ down to 10⁻⁶ M, with RCC% dropping to ∼80% at 10⁻⁷ M. H₂BADPA-18, tested with [²⁰⁷Bi]Bi³⁺ (t_1/2 = 31.2 years, EC), showed slightly lower yields compared to the other picolinic acid-based chelators.¹¹ This discrepancy may be partially attributed to the lower incubation time used for the H₂BADPA-18 radiolabeling (1–2 min).

Fig. 6 Concentration-dependent radiolabeling studies of H₃tripa and (A) DOTA with [²¹³Bi]Bi³⁺(100 kBq) in MES (1 M, pH 5.5) and (B) TCMC with [²⁰³Pb]Pb²⁺(100 kBq) in NH₄OAc (0.5 M, pH 7). Human serum stability studies of H₃tripa with (C) [²¹³Bi]Bi³⁺ (200 kBq) and (D) [²⁰³Pb]Pb²⁺ (300 kBq). All reactions were performed at 37 °C and monitored using radio-TLC.

Concentration-dependent radiolabeling studies with [²⁰³Pb]Pb²⁺ were conducted to evaluate the performance of H₃tripa compared to the gold-standard chelator for [^212/203Pb]Pb²⁺, TCMC, at ambient temperature with a 30 minute incubation. H₃tripa achieved quantitative incorporation of [²⁰³Pb]Pb²⁺ at 10⁻⁵ M, with a slight decline to 91% at 10⁻⁶ M, while the RCC% decreased further at 10⁻⁷ M. In contrast, TCMC maintained higher radiolabeling efficiency at 10⁻⁷ M. Both H₂S₂macropa and H₂macropa were also capable of incorporating [²⁰³Pb]Pb²⁺ at 10⁻⁷ M, which deviates from the expected thermodynamic stability trends (pPb: H₃tripa > H₂macropa > H₂S₂macropa). This highlights the importance of radiolabeling studies over bulk solution stability assessments in the context of radiopharmaceutical development and translation.

The observed discrepancies may also be attributed to differences in experimental conditions, particularly the residual thallium present in the [²⁰³Pb]Pb²⁺ solution and the molarity of ammonium acetate (NH₄OAc) buffer used.¹¹ H₃tripa was radiolabeled in 0.5 M NH₄OAc, while H₂S₂macropa and H₂macropa were radiolabeled in 0.1 M NH₄OAc.¹² Since ammonium acetate can compete with the radiometal for chelator binding, the higher buffer concentration may have contributed to the reduced RCC% observed for H₃tripa.

The in vitro serum stability of both complexes was evaluated in human serum (Sigma-Aldrich H4522). [²¹³Bi][Bi(Htripa)]⁺ was incubated for 2 hours (equivalent to approximately 2.6 half-lives of ²¹³Bi), while [²⁰³Pb][Pb(Htripa)] was incubated for 60 hours, with aliquots taken at specified time points (Fig. 6C and D). Both radiolabeled complexes demonstrated exceptional stability, remaining intact with over 95% radiochemical retention throughout their respective incubation periods. These results underscore the significant potential of H₃tripa as a chelator for radiopharmaceutical application.

Conclusions

This work establishes H₃tripa as a versatile chelator for Pb/Bi theranostics, delivering rapid, high-yield radiolabeling of both isotopes at micromolar concentrations, and with exceptional human serum stability. Thermodynamic solution studies demonstrated, to our knowledge the highest stability constants reported to date for both [Pb(tripa)]⁻ and Bi(tripa) complexes, underscoring the remarkable affinity of H₃tripa for these metals. NMR spectroscopy provided insights into the solution–state binding interactions, while X-ray crystallography of the Pb²⁺ complex illustrated the binding preference of H₃tripa in the solid state. Additionally, DFT-optimized geometries indicated octadentate coordination for both metals, revealing that only two picolinic acid moieties contribute to strong interactions while the third may act more dynamically. Moving forward, efforts will focus on exploring structural modifications to incorporate a bifunctional handle, facilitating targeted, kit-style radiopharmaceutical development.

Author contributions

Desiree Fiaccabrino: conceptualization, investigation, formal analysis, writing – original draft. Tinotenda Masvikeni: investigation, synthesis of precursors. Brooke McNeil: production of [²⁰³Pb]Pb²⁺. Brian O. Patrick: X-ray crystallography. María de Guadalupe Jaraquemada-Peláez: formal analysis, supervision, writing – review & editing. Paul Schaffer: funding acquisition, supervision, writing – review & editing. Chris Orvig: funding acquisition, supervision, writing – review & editing. All authors have given approval of the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the ESI.†

Acknowledgements

Funding came from the Natural Sciences and Engineering Research Council (NSERC) of Canada through NSERC Discovery Grants (PS, CO). We gratefully acknowledge all personnel involved at TRIUMF's ISAC facility for ion beam delivery for ²²⁵Ac production and Dr François Bénard at BC Cancer Research Institute for allowing the use of the Molecular Oncology laboratory to conduct experiments. TRIUMF receives federal funding via a contribution agreement with the National Research Council of Canada.

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Footnote

† Electronic supplementary information (ESI) available: Experimental section, additional data, figures and tables. See DOI: https://doi.org/10.1039/d5dt01103e

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