Hydroxyquinoline-functionalised aza-crown macrocycles for lanthanide coordination

Abstract

Emerging therapeutic radiolanthanides have utility for systemic molecular radiotherapy in nuclear medicine, provided that suitable chemical technology is available to incorporate them into receptor-targeted radiopharmaceuticals. In this work, N,N′-bis(8-hydroxyquinoline-2-ylmethyl)-4,13-diaza-18-crown-6 (H₂KHQ) was synthesised, and its binding ability, thermodynamic stability and selectivity for Ln³⁺ ions (Ln³⁺ = La, Tb, and Lu) investigated. The design of H₂KHQ involves pendant arms featuring 8-hydroxyquinoline units, known to possess metal-chelating properties and desirable activity in other therapeutic molecules. H₂KHQ exhibited selectivity for the larger Ln³⁺ ions, confirmed by experimentally measured stability constants as well as DFT calculations. H₂KHQ was able to bind the larger, non-radioactive La³⁺ and Tb³⁺ ions within 30 minutes at room temperature, forming a single, 2-fold symmetric species in solution. The structure of [La-HKHQ]²⁺, as determined by single crystal XRD, emphasized the need for high denticity chelators to satisfy the coordination sphere of the Ln³⁺, showing a 10-coordinate La³⁺ metal centre. H₂KHQ was radiolabelled with [¹⁶¹Tb]TbCl₃ under mild conditions in 92% radiochemical yield in promising proof-of-concept measurements.

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Introduction

Radioactive nuclides of the lanthanides have significant utility in nuclear medicine for both diagnostic imaging and systemic radiotherapy. In particular, radiolanthanides that emit cytotoxic particles including beta (β⁻), alpha (α) and Auger Electrons (AE), have demonstrated efficacy in theranostic radiopharmaceuticals.^1–3 Diagnostic and therapeutic (“theranostic”) pairs of radiopharmaceuticals typically utilise the same biologically active receptor-targeted vector to deliver either an imaging radionuclide or a cytotoxic therapeutic radionuclide to diseased tissue. This “look and treat” approach uses the radiotracer, in combination with either Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) imaging to provide diagnostic information that guides decisions on suitability of the companion therapeutic radiotracer.^1,4 For example, the PET radionuclide gallium-68 (⁶⁸Ga, t_1/2 = 68 min, β⁺) is commonly used in tandem with therapeutic lutetium-177 (¹⁷⁷Lu, t_1/2 = 6.64 days, β⁻) for the treatment of somatostatin receptor (SSTR)-positive neuroendocrine tumours, using an “octreotate” peptide attached to a macrocyclic chelator.^3,5,6 While the use of the ⁶⁸Ga/¹⁷⁷Lu radionuclide pair is effective, “true theranostic” agents consisting of a pair of imaging and therapeutic radionuclides of the same element could provide significant advantages. In “true theranostic” pairs, the chemically identical imaging radiotracer and radiotherapeutic agent exhibit equivalent biodistributions: the imaging radiotracer can be used to determine accurate dosimetry of the radiotherapeutic agent. Examples include copper-64/copper-67 (⁶⁴Cu, β⁺/⁶⁷Cu, β⁻), terbium-155/terbium-161 (¹⁵⁵Tb, γ/¹⁶¹Tb, β⁻, AE, γ) and scandium-44/scandium-47 (⁴⁴Sc, β⁺/⁴⁷Sc, β⁻).^3,7–9 Terbium radioisotopes have potential for receptor-targeted theranostic radiopharmaceuticals. There are four clinically relevant radioisotopes: ¹⁴⁹Tb (t_1/2 = 4.12 hours, E_α = 3.97 MeV, I_α = 16.7%) for targeted α therapy, ¹⁵²Tb (t_1/2 = 17.5 hours, E_β+,av = 1.14 MeV, I = 20.3%) for PET and ¹⁵⁵Tb (t_1/2 = 5.3 days, E_γ = 86.6, 105.3 and 180.1 keV) for SPECT. Finally, ¹⁶¹Tb (t_1/2 = 6.95 days) is a β⁻-emitter (154 keV) that undergoes low-energy internal conversion (IC), co-emitting high-energy AE (∼12.12 e⁻, <40 keV per decay) and γ-rays.^3,7,10 In side-by-side preclinical studies, ¹⁶¹Tb has showcased superior in vitro and in vivo efficacy compared to ¹⁷⁷Lu, alongside an ability to deliver higher absorbed doses.^10–14 The co-emission of both long-range β⁻ and shorter-range AE emissions from ¹⁶¹Tb in comparison to ¹⁷⁷Lu (β⁻ emitter only) is hypothesized to increase its efficacy and radiotherapeutic effect.^7,15,16 Phase I/II clinical trials with ¹⁶¹Tb radiopharmaceuticals are currently evaluating the efficacy of ¹⁶¹Tb-based radiopharmaceuticals.¹⁷

A suitable chelating agent is required to coordinate radiolanthanides such as ¹⁶¹Tb and subsequently attach them to biologically active motifs that target surface receptors of diseased cells. Over the years, a series of aza-crown macrocyclic chelators have been developed, for coordination of rare-earth metals available for theranostic applications (Fig. 1). The coordination chemistry of macropa, macrodipa and next-generation analogues have been extensively studied with regards to their ability to bind clinically relevant radionuclides, including actinium-225 (²²⁵Ac) and lanthanum-135 (¹³⁵La).^18–27 Macropa displayed preferential binding with radionuclides of larger ionic radius, over smaller radionuclides whereas macrodipa, py-macrodipa and py₂-macrodipa complexes with both large and small radionuclides demonstrated increased thermodynamic and kinetic stability.^21–23 Blei et al. have further highlighted the abilities of macropa to bind radiometals with larger ionic radii (lead-212 (²¹²Pb) and lanthanum-133 (¹³³La)) in 100% radiochemical conversion (RCC), comparable to labelling with ²²⁵Ac.²⁷ Macropa was also able to complex the smaller radionuclide, ¹⁷⁷Lu, however, the complex's kinetic stability was lower and higher ligand concentrations were required to achieve satisfactory RCC.²⁷ Still, macropa is one of the two state-of-the-art chelators for ²²⁵Ac, alongside 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and is currently being used in clinical trials.^28,29

Fig. 1 Examples of azamacrocyclic chelators.

In this work, we explore a diaza-18-crown-6 derivative, H₂KHQ, containing two 8-hydroxyquinoline pendant arms. This chelator is related to macropa, in that the picolinic acid groups of the latter are replaced by 8-hydroxyquinoline motifs. H₂KHQ has highly similar topology to macropa, with increased rigidity in the pendant arms. H₂KHQ has been previously studied for the complexation of divalent metals ions of zinc, barium, copper, as well as monovalent potassium and sodium.³⁰ However, it has not been reported for the complexation of trivalent lanthanides or for use in nuclear medicine. In general, 8-hydroxyquinoline units and derivatives are widely used in medicinal chemistry and drug discovery.^31–36 They are well-known to possess metal chelating properties and have been assessed as ionophores,^37,38 antiseptic,³⁹ antioxidant,^40,41 and anticancer agents.^42,43 In addition, they exhibit photophysical properties enabling them to act as sensitizers for Ln³⁺ in a wide variety of applications including biological imaging and materials chemistry.^44–49

Results

Ligand synthesis

H₂KHQ (N,N′-bis(8-hydroxyquinoline-2-ylmethyl)-4,13-diaza-18-crown-6) was synthesised in a three-step reaction as shown in Scheme 1. 8-Hydroxyquinoline-2-carbaldehyde was reduced and brominated following a previous procedure.⁵⁰ Subsequent attachment of intermediate 2 onto a Kryptofix®22 backbone was carried out via nucleophilic substitution in the presence of sodium carbonate, adapted from previous procedures.^24,26 The product was isolated as a pure, white solid in 27% yield post purification (Fig. S1–S6, SI).

Scheme 1 Synthesis of H₂KHQ.

Characterisation of lanthanide complexes

To investigate the metal chelating abilities of H₂KHQ, the non-radioactive [Tb-KHQ]⁺ complex was synthesised by mixing H₂KHQ and TbCl₃·6H₂O (1.2 eq.) in H₂O (pH 4–5) at room temperature for 30 min. The pure complex was isolated by C18 reverse-phase chromatography as a yellow solid in 65% yield. The ¹H NMR spectrum shows no evidence of unchelated H₂KHQ ligand and contains characteristic paramagnetic NMR shifts of Tb³⁺, with signals observed between −200 and +250 ppm (Fig. 2A).⁵¹ LC-MS (Fig. S26) and HR-ESI-MS (Fig. S27) detected a molecular ion peak corresponding to the stoichiometry of [Tb-KHQ]⁺ at m/z 733.2076 (for [C₃₂H₃₈N₄TbO₆]⁺ calcd = 733.2039). HPLC confirmed the presence of a single [Tb-KHQ]⁺ species in solution, with a retention time distinct to that of unchelated H₂KHQ ligand (Fig. 2B).

Fig. 2 (A) ¹H NMR spectrum of [Tb-KHQ]⁺ (D₂O, 400 MHz, 298 K). (B) HPLC chromatogram of H₂KHQ and [Tb-KHQ]⁺ (1 mM in NH₄OAc, pH 6.5).

The diamagnetic [La-KHQ]⁺ complex was also prepared: H₂KHQ and LaCl₃·7H₂O (1.2 eq.) were mixed in D₂O (pD 6.5) at room temperature for 30 min. The ¹H NMR spectrum (Fig. 3) indicated a single symmetric species present: only five resonances in the aromatic region were observed, corresponding to the five distinct proton environments on the 8-hydroxyquinoline arms. Notably, large geminal ¹H–¹H coupling constants (²J_HH > 10 Hz) were observed for all macrocyclic CH₂ protons. This is consistent with metal binding, leading to chemically inequivalent geminal proton environments. In the ¹³C{¹H} NMR spectrum (Fig. S12), sixteen ¹³C signals were detected, further supporting the presence of a 2-fold symmetric species. Upon La³⁺ binding, an increase in the chemical shift of aromatic 8-hydroxyquinoline ¹³C resonances was observed, relative to the unchelated H₂KHQ ligand. The chemical equivalence of ¹H and ¹³C atoms of both 8-hydroxyquinoline motifs is similar to analagous complexes (La³⁺-macrodipa, La³⁺-macropa), in which 2-fold symmetry was also observed in solution by ¹H and ¹³C NMR spectroscopy.^20,21,52

Fig. 3 Expanded ¹H NMR spectrum of H₂KHQ (top) and [La-KHQ]⁺ (bottom). (D₂O, pD 6.5, 400 MHz, 298 K). The macrocyclic protons are labelled as H_macro.

To further probe the solution-state chemistry of [La-KHQ]⁺, a mixture of H₂KHQ and LaCl₃·7H₂O (1.2 eq.) in D₂O was monitored via ¹H NMR spectroscopy from pD 2–13 (Fig. S15). The ¹H NMR spectrum of H₂KHQ ligand did not change significantly with pD. Binding of La³⁺ to H₂KHQ was not observed at low pD but was seen at pD 6.5, with only a single species noted. With increasing pD, the chemical shifts of [La-KHQ]⁺ did not change significantly.

Lastly, the formation of [Lu-KHQ]⁺ complex was also explored via ¹H NMR studies (Fig. S18–S20). H₂KHQ and LuCl₃·6H₂O (1.2 eq.) were reacted in D₂O, and ¹H NMR spectra were obtained over pD 2–9. At low pD, no complex formation was detected whereas at pD 6, H₂KHQ ligand and a [Lu-KHQ]⁺ species were observed in a 1 : 2 ratio. At pD 9, full conversion to a [Lu-KHQ]⁺ metal complex was observed. Similarly to [La-KHQ]⁺, the Lu³⁺ complex showed 2-fold symmetry, as evidenced by a single set of aromatic protons.

To explore the solid-state structure of the [La-KHQ]⁺ complex, it was re-synthesised by mixing H₂KHQ with La(ClO₄)₃ (1.2 eq.) in MeOH at room temperature for 30 min. Single crystals of [La-HKHQ](ClO₄)₂ were obtained by vapour diffusion of pentane into a solution of [La-HKHQ](ClO₄)₂ in EtOH. Single crystal X-ray diffraction analysis revealed the crystalline material to be [La-HKHQ](ClO₄)₂ (Fig. 4). The La³⁺ metal centre sits in a 10-coordinate environment, with all nitrogen and oxygen donor atoms bound to the metal centre. The complex adopted a syn-conformation, where the 8-hydroxyquinoline arms were both coordinated to the metal centre on the same face relative to the macrocyclic ring. The syn-conformation of the ligand implies the presence of two helices, one for the pendant arms (absolute configuration Δ or Λ) and one for the six five-membered chelate rings formed (absolute configuration δ or λ).^53,54 The crystal structure reveals the most stable isomer to be Δ(δλλ)(δλδ), present as part of a racemate. One of the hydroxyl groups remained protonated, forming a hydrogen bond to a second perchlorate counter-anion, and a long La–O8 bond (2.673(3) Å). The non-protonated oxygen atom in the 8-hydroxyquinoline arms formed a strong La–O18 bond (2.365(3) Å). In [La-HKHQ]²⁺, La–N12 (2.658(4) Å), La–N2 (2.655(3) Å), La–N23 (2.847(4) Å) were all relatively shorter than the La–N bonds in [La(Hmacropa)(H₂O)], suggesting that the 8-hydroxyquinoline arms provide a basis for stronger binding in comparison to the picolinate motif.²⁰ The solid-state structure was consistent with the ¹H NMR solution-state data.

Fig. 4 The structure of [La-HKHQ](ClO₄)₂ (50% probability ellipsoids). H atoms and second perchlorate anion omitted for clarity. H-bonding contacts in teal and interatomic distances in green.

Lanthanide complexes thermodynamic stability

Potentiometric titrations were employed to obtain ligand protonation constants (K_a) and complex stability constants (K_LnL) (defined in eqn (S4) and (S5) respectively).

As shown in Table 1, the first and second H₂KHQ protonation constants (log K_H1 and log K_H2) correspond to the hydroxyl groups of the two 8-hydroxyquinoline arms. In free 8-hydroxyquinoline, the hydroxyl groups have a pK_a of 9.9.⁵⁵ The differences in protonation constants between the two 8-hydroxyquinoline substituents on H₂KHQ is likely a result of complex intramolecular hydrogen bonding patterns in the unchelated ligand, such as those between the O–H and the macrocyclic nitrogen atoms (Fig. S9).⁵⁶ The third and fourth protonation constants (log K_H3 and log K_H4) correspond to the macrocyclic tertiary amine atoms, while the fifth and sixth protonation constants (log K_H5 and log K_H6) correspond to the nitrogen atoms of the 8-hydroxyquinoline motifs.

Table 1 Protonation constants (log K_a) and stability constants (log K_LnL) obtained by potentiometric titrations

	H2KHQ^a	Macropa^24,57	Py2-macrodipa^23	Py-macrodipa^22	Macrodipa^21
a This work: 0.1 M NaCl, 25 °C. Ligand concentration 0.018 mmol. pH range used 3–10.5. Three repeats. The values in the parentheses are one standard deviation of the last significant figure. pM is the negative logarithm of the free metal concentration in equilibrium with complexed and free ligand, at a fixed pH 7.4.
Log Ka1	10.40(2)	7.41	7.58(4)	7.20	7.79
Log Ka2	7.70(2)	6.85	6.48(1)	6.54	7.04
Log Ka3	4.03(4)	3.32	3.52(3)	3.17	3.18
Log Ka4	3.74(3)	2.36	2.60(5)	2.31	2.14
Log Ka5	3.10(5)	1.69	2.10(11)	—	—
Log Ka6	3.08(4)	—	—	—	—
Log KLaL	12.10(8)	14.99	16.68(8)	14.31(6)	12.19(2)
Log KTbL	11.05(2)	11.79	14.76(6)	11.95(3)	9.68(1)
Log KLuL	8.58(5)	8.25	11.90(3)	11.54(2)	10.64(4)
Log KLaHL	—	2.28	—	—	—
Log KLuLH−1	2.46(1)	—	—	—	—
pLa	9.47	15.58	17.20	15.03	12.49
pTb	8.54	12.38	15.28	12.62	9.98
pLu	7.50	8.84	12.42	12.26	10.94

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The log K_LnL values for H₂KHQ and similar macrocyclic chelators such as macropa and py₂-macrodipa were plotted against the ionic radii of key Ln³⁺ ions (Fig. 5).^{21–23,57,58} The affinity of H₂KHQ for trivalent cationic lanthanide metal ions decreases as the ionic radius decreases. Importantly, the log K_LnL (L = KHQ²⁻, Ln = Tb³⁺, Lu³⁺) values were comparable to those of macropa, highlighting that substituting picolinic acid groups for hydroxyquinoline groups does not have a marked effect on the binding affinity of this class of chelators. Indeed, the log K_LnL values for Ln³⁺ complexes of H₂KHQ are lower than those of many newer chelators (such as py₂-macrodipa).

Fig. 5 Stability constants of [Ln-KHQ]⁺ (Ln = La, Tb, Lu) plotted versus Ln³⁺ ionic radii.

However, to compare ligands with different basicities, the relevant parameter pM, is used as an indicator of affinity (Table 1). The pM value is the negative logarithm of the free metal concentration in equilibrium with complexed and free ligand, at a fixed pH 7.4. Analysis of the pM(Ln(III)) values emphasizes that with H₂KHQ at pH 7.4, more Ln(III) ions are present in solution, in comparison to other chelators.

Speciation plots (Fig. 6) indicate that under the conditions studied here, all available H₂KHQ ligand molecules were bound to La³⁺ at pH ∼6, Tb³⁺ at pH ∼6.5 and Lu³⁺ at pH ∼7.5. This was also consistent with ¹H NMR studies which indicated that a higher pH was needed to achieve quantitative coordination of Lu³⁺ by H₂KHQ, as compared to the analogous La³⁺ complex. The distribution of both La³⁺ and Tb³⁺ species supports the formation of one major species at ambient pH, consistent with ¹H NMR spectroscopic studies. Significantly, the Lu³⁺ speciation plot indicates that the major species forming between Lu³⁺ and H₂KHQ at above pH 6.5 is LuL_H−1. This most likely corresponds to a species where a water molecule is interacting with the complex. Attempts to elucidate the solid-state structure of [Lu-KHQ]⁺ via crystals were unsuccessful.

Fig. 6 Representative species distribution for [Ln-KHQ]⁺ (Ln = La, Tb, Lu) modelled in HySS. H₂KHQ c = 0.018 mmol, Ln³⁺ c = 0.018 mmol. Initial volume V = 30 mL. Data fitting and speciation distribution over the pH range shown. L represents fully deprotonated ligand, KHQ²⁻.

DFT calculations

DFT is a useful tool for exploring the coordination chemistry of Ln³⁺ with macrocyclic chelators and has been used to better understand the origin of size selectivity in macropa and second-generation analogues.^21–23 Herein, we explored the binding of H₂KHQ across the lanthanide series (La³⁺–Lu³⁺) to gain insight into the size selectivity of H₂KHQ complexation. Initially, geometries were taken from the crystal structure of syn [La-HKHQ]²⁺ with removal of H8 to model the overall ‘+1’ species. Different conformational arrangements were modelled with the 8-hydroxyquinoline arms in either a syn or anti arrangement around the metal centre (whilst retaining symmetry). Across the lanthanide series, the syn conformation was favoured over the anti-conformation (Fig. S30). The relative Gibbs free energy (ΔΔG) for the transmetallation reaction ([LaKHQ]⁺ + Ln³⁺ → [LnKHQ]⁺ + La³⁺) was calculated. The results show a thermodynamic preference for the larger Ln³⁺ ions (Fig. 7). This calculated trend is in good qualitative agreement with experimental data (ΔΔG_exp = −2.303RT log(K_LnL − K_LaL), ΔΔG_Tb–La = +1.43 kcal mol⁻¹, ΔΔG_Tb–La = +4.80 kcal mol⁻¹) which shows decreasing thermodynamic stability across the series. The quantitative discrepancy is attributed to the over estimation of gas-phase Ln³⁺ ion energies in our model, which does not include explicit solvation effects. The destabilisation evidently results from a greater degree of ligand strain in the macrocyclic framework to facilitate the coordination of the oxygen atoms to the smaller Ln³⁺ ions (Fig. S34). Furthermore, H₂KHQ did not exhibit the conformational toggle that is predominant in macrodipa and analogues.^21–23

Fig. 7 DFT-computed relative Gibbs free energies (ΔΔG) for the transmetallation reaction: values are calculated as: .

Structural analysis revealed a coordination number of 10 for all Ln³⁺, involving four nitrogen atoms and six oxygen atoms in coordination to the metal centre (Fig. S31). Across the series, the hapticity of the KHQ ligand was η¹⁰, with the metal ion located above the plane of the macrocyclic ring system, enclosed by both 8-hydroxyquinoline units. Additionally, the presence of a water molecule in the first coordination sphere was modelled, however the resulting coordination number of 11 was less favoured across the series (Table S2, ).

Analysis of the structure of [La-HKHQ]²⁺ reveals that there are 16 possible conformations (8 enantiomeric pairs of diastereoisomers) with C₂ symmetry.²⁴ To better explore the in-solution behaviour, an in-depth conformational screen of 8 different diastereomers was carried out for La³⁺, Tb³⁺ and Lu³⁺. As shown in Fig. S32, our calculations predict that the Δ(δλδ)(δλδ) conformation is the lowest energy form in aqueous solution across the three Ln³⁺ ions tested, and is lower in energy by ca. 1.9 kcal mol⁻¹ than the solid-state conformation observed for [La-HKHQ]²⁺. The relative energies of the different conformations in aqueous solution are given in Table S3 and highlight the multiple C₂-symmetric conformational modes that likely exist in equilibrium in solution. This result is consistent with the NMR solution-state studies on [La-KHQ]⁺ where a complex with C₂ symmetry was observed.

To explore the effects of the nature of the pendant arm on the stability of these complexes a parallel conformational screen of macropa complexes (for La³⁺, Tb³⁺ and Lu³⁺) was carried out, where our model also favoured the Δ(δλδ)(δλδ) conformation (see Fig. S33). Analysis of gas-phase Gibbs free binding energies and ligand strain was performed to compare the influence of the pendant arms of macropa and KHQ on ligand preorganisation and metal binding. The results show that whilst ligand strain generally increases with decreasing ionic radius for both ligands, the effect is much less pronounced for macropa (Fig. S34). Additionally, our calculations suggest that macropa provides a consistently greater, albeit modest, metal–ligand stabilisation for La³⁺, Tb³⁺ and Lu³⁺ (Table S4). Whilst experiments show the reverse trend for Lu³⁺, the differences are small enough that calculated values are likely within error. Together with experimental results, it appears that the greater rigidity of the pendant arms in KHQ forces a higher degree of overall ligand strain in the metal-bound conformation of the macrocycle.

Phosphorescence spectroscopy

The pendant 8-hydroxyquinoline motifs possess useful photophysical properties and can potentially act as antennae to exploit the luminescence emission properties of Tb³⁺. Luminescence studies were carried out to ascertain whether the [Tb-KHQ]⁺ complex exhibited characteristic Tb phosphorescent emissions. Indeed, sharp emission peaks at 518, 565, 583 and 621 nm were observed for [Tb-KHQ]⁺ via the antenna effect, following excitation of the ligand at 242 nm (Fig. 8A). These are characteristic of the ⁵D₄ → ⁷F_n transitions (n = 6, 5, 4, 3). This preliminary experiment highlights the potential of 8-hydroxyquinoline based ligands to be used for optical imaging, with further studies needed to optimise ligand design for efficient sensitization.

Fig. 8 (A) Phosphorescence spectrum for [Tb-KHQ]⁺ (H₂O, 20 μM, 20 nm slits, 0.1 ms delay). (B) RadioHPLC of H₂KHQ (1 mM) with [¹⁶¹Tb]Tb³⁺ (2.5 MBq, 1 mM HCl) after 60 min at 90 °C. RCY = 92%. [¹⁶¹Tb][Tb-KHQ]⁺ retention time = 18.3 min.

Radiolabelling experiments

Finally, [¹⁶¹Tb]Tb³⁺ radiolabelling experiments were undertaken, with [¹⁶¹Tb]TbCl₃ provided by the Paul Scherrer Institute. Solutions of H₂KHQ (1 mM) and [¹⁶¹Tb]Tb³⁺ (2.5 MBq, 1 mM HCl) in NH₄OAc (20 mM) at pH 8.5 were reacted at 25 °C. After 20 min and 180 min, the reactions were analysed by reverse-phase radio-HPLC (Fig. S41–43), which indicated formation of [¹⁶¹Tb][Tb-KHQ]⁺ with a retention time (R_t) of 18.33 min. We observed that unreacted [¹⁶¹Tb]Tb³⁺ was initially retained on the column, presumably as a colloidal species, similar to prior radiochemical observations.⁵⁹ However, upon switching to an aqueous mobile phase at the end of analysis, free [¹⁶¹Tb]Tb³⁺ eluted, appearing at >29 min, enabling quantification of radiochemical yield (RCY). Preliminary radiolabelling studies show that at 25 °C, H₂KHQ was radiolabelled in 33% RCY after 20 min, which increased to 68% after 180 min. Upon heating to 90 °C for 60 min, a high RCY of 92% was achieved (Fig. 8B). In comparison, macropa radiolabelling with [¹⁶¹Tb]Tb³⁺ (5 MBq, 2 mM HCl) was achieved at 62 and 55% RCY respectively at room temperature and 90 °C after 30 minutes (Fig. S44 and S45).

Conclusions

The coordination of H₂KHQ with Ln³⁺ ions has been interrogated via X-ray crystallography, NMR spectroscopy, DFT calculations, analytical chromatography and potentiometric titrations. H₂KHQ exhibited a higher binding affinity towards larger Ln³⁺ ions compared to the smaller ions. DFT calculations corroborate the experimental evidence: [Ln-KHQ]⁺ complexes demonstrated decreasing thermodynamic stability as ionic radii decrease across the lanthanide series. In solid-state studies, XRD analysis of [La-KHQ]⁺ showed that the La³⁺ metal ion adopts a 10-coordinate conformation with the 8-hydroxyquinoline arms binding in a syn orientation relative to the macrocycle. This was consistent with solution-state NMR studies, that suggest a 2-fold symmetric complex present. The optical properties of [Tb-KHQ]⁺ were investigated by ligand sensitization at 242 nm, and characteristic phosphorescence emission peaks were observed. It has been noted that in order to effectively exploit the capabilities of 8-HQ moieties as antennae, optimised ligand analogues can be designed for increased sensitization and signal enhancement. The promising chelation properties seen with La³⁺ and Tb³⁺ prompted us to explore the chelation of KHQ with [¹⁶¹Tb]Tb³⁺. Preliminary radiolabelling corroborated the ability of H₂KHQ to coordinate [¹⁶¹Tb]Tb³⁺ in high radiochemical yields (92%). Further in-depth radiolabelling studies are required to explore concentration, time, pH and temperature dependencies as well as maximum molar activity. Complex stability and inertness for in vivo applications shall be adequately explored in future work. Still, the combination of radioactive properties with photophysical properties could enable the development of a dual-modal Tb³⁺ probe for future medical applications.^60,61

Author contributions

C. S. synthesised the compounds and performed the analyses. The radiolabelling was performed with the help of B. E.O. and M. T. M. DFT and X-ray crystallography was carried out by R. K. B. Potentiometric titrations were performed by C. S. and C. R. M. T. M. and N. J. L. supervised the project, and all the authors contributed to the writing 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 supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5dt03015c.

CCDC 2435038 and 2435039 contain the supplementary crystallographic data for this paper.^62a,b

Acknowledgements

The research was supported by the EPSRC programme for Next Generation Molecular Imaging and Therapy with Radionuclides (EP/S019901/1, “MITHRAS”), the EPSRC Centre for Doctoral Training in Smart Medical Imaging (EP/S022104/1), and as part of the PRISMAP project supported by the European Union's Horizon 2020 research and innovation program under grant agreement no. 101008571. CR acknowledges funding from the Marie Sklodowska-Curie grant agreement no. [101032337].

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