Exploring the use of rigid 18-membered macrocycles with amide pendant arms for Pb(II)-based radiopharmaceuticals

Abstract

We report a detailed investigation on the use of chelators PYTAM, H₄PYTAMGly, CHX-PYTAM and H₄CHX-PYTAMGly for the complexation of Pb(II)-radioisotopes with potential use in the preparation of radiopharmaceuticals. The macrocyclic backbones from which the chelators are prepared, PYAN (3,6,10,13-tetraaza-1,8(2,6)-dipyridinacyclotetradecaphane) or CHX-PYAN ((4¹R,4²R,10¹R,10²R)-3,5,9,11-tetraaza-1,7(2,6)-dipyridina-4,10(1,2)-dicyclohexanacyclododecaphane), are readily synthesized in high yields using a Ba(II) template synthesis. The single difference between the two backbones is that while PYAN contains ethylene spacers, CHX-PYAN incorporates more rigid cyclohexyl spacers. The pendant arms incorporated into the backbone were strategically chosen, selecting amide pendants of different nature, compatible with the borderline Lewis acid character of Pb(II). To study the complexation of Pb(II), first, a detailed characterization of the non-radioactive complexes was carried out, including X-ray crystallography, NMR, and the determination of chelator protonation and stability constants of the complexes. Once the complexes were fully characterized and showed favourable properties towards Pb(II), a radiochemical study using the SPECT compatible radioisotope lead-203 was performed. Although all tested chelators sufficiently complexed lead-203 and presented excellent complex stability in human serum, it was found that novel chelator CHX-PYTAM was superior over the others due to its high kinetic inertness. This effect is due to increased rigidity of the complex with cyclohexyl spacers in the backbone and use of primary amides, over secondary amides, as pendant arms and thus this chelator is a promising candidate for future in vivo studies.

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Introduction

There are a wide variety of radioactive metals that have potential use in radiopharmaceutical compounds, both for diagnostic and therapeutic applications.¹ Within the family of diagnostic isotopes we can find positron (β⁺) emitters that can be used for positron emission tomography (PET) imaging, such as commonly used gallium-68 (⁶⁸Ga, t_1/2 = 67.7 min), or isotopes that directly emit gamma (γ-) rays for SPECT (single photon emission computed tomography) imaging, such as technetium-99m (^99mTc, t_1/2 = 6.00 h).² Therapeutic isotopes, on the other hand, release cytotoxic radiation and can be divided into three categories depending on the type of radiation that is produced: alpha (α)-emitters, beta (β⁻)-emitters and Auger electron emitters.³

Nowadays, the emerging field of theranostics combines both applications of radioisotopes in order to achieve a more personalized treatment.⁴ First, a diagnostic agent is administered to evaluate the uptake of the radiopharmaceutical and if the uptake is high, the therapeutic is administered subsequently. There are different combinations of isotopes that can be used in this procedure, “true theranostics”, same-element isotope pairs, and different-element pairs.^5,6 True theranostic radionuclides emit both imaging compatible radiation and therapeutic particles, such as the β⁻-emitter lutetium-177 (¹⁷⁷Lu, t_1/2 = 6.64 days) which emits an accompanying γ-ray compatible with SPECT imaging. Although a true theranostic may be convenient, one of the drawbacks of the use of these isotopes, is the potential for off target effects if there is not significant uptake shown in the image.⁷ This is why, when evaluating the use of ¹⁷⁷Lu for patient treatment, ⁶⁸Ga is often used for pre-therapy imaging and uptake evaluation.⁸ However, when using a different-element pair, the pharmacokinetics of both radiopharmaceuticals may not be identical, which could be problematic if the update of the imaging tracer does not accurately reflect the biodistribution of the therapeutic pharmaceutical.⁵ This can be avoided by using a same element isotope pair, where two different isotopes of the same element are appropriate for either diagnosis or therapy, such as the lead-212 (²¹²Pb, t_1/2 = 10.6 h)/lead-203 (²⁰³Pb, t_1/2 = 51.9 h) theranostic pair.^9,10

The ²¹²Pb/²⁰³Pb theranostic pair has emerged in recent years as a promising candidate for use in personalized theranostics, which has triggered efforts focused on finding chelators that are adequate to take advantage of the potential of this pair.^10–15 The ²⁰³Pb-radionuclide emits a 279 keV (81%) γ-photon, and can be produced through proton bombardment of Tl on medical cyclotrons via either the ²⁰³Tl (p,n) ²⁰³Pb reaction or the ²⁰⁵Tl (p,3n) ²⁰³Pb reaction.^10,16 The ²¹²Pb-radionuclide emits β⁻-particles and, indirectly, an α-particle through the decay of its daughters bismuth-212 (²¹²Bi, t_1/2 = 60.6 min) or polonium-212 (²¹²Po, t_1/2 = 294.3 ns) and can be obtained via different generators based on isotopes from the thorium-228 (²²⁸Th, t_1/2 = 1.9 years) decay series.¹⁷ The half-life of this isotope pairs nicely with the biological half-life of small biomolecules, like peptides, that can bind to targets such as prostate-specific membrane antigen (PSMA) and somatostatin receptors (SSTRs) which are overexpressed in some tumours.^18–20 It should be noted that phase 1 clinical trial results using [²¹²Pb]Pb-DOTAMTATE (bifunctional chelator [DOTAM] coupled to SSTR-targeting peptide [TATE]) have recently been published, in which SSTR-expressing neuroendocrine tumours are targeted, with very promising results, showing an objective radiologic response (ORR) of 80% compared to an ORR of 13% obtained with [¹⁷⁷Lu]Lu-DOTATATE (bifunctional chelator [DOTA] coupled to SSTR-targeting peptide [TATE]).²⁰

One of the most commonly used strategies to harness the decay properties of these metals in vivo is through the use of bifunctional chelators (BFCs), in which the radiometal of interest is coordinated by a chelator and attached, through a linker, to a biological targeting vector such as a peptide or an antibody.^21,22 This allows for the radioactivity to be delivered selectively to the diseased tissue, while minimally damaging surrounding healthy tissue.²³ Coordination chemistry plays a key role in the design of these BFCs, as it is necessary to tailor the properties of the chelator to the properties of each specific metal to obtain thermodynamically stable and kinetically inert complexes, to avoid the release of the metals in vivo and possible toxicity.²⁴

When designing a new chelator, Pearson's hard–soft acid–base (HSAB) principle should be considered, so that donor atoms matching the metal ion are incorporated into the structure of the chelator.²⁵ Stable complexation of lead(II), a borderline acid, is generally achieved using amide donor groups, borderline bases, with the CYCLEN (1,4,7,10-tetraazacyclododecane)-based macrocyclic derivative DOTAM being the leading chelator for this metal ion (Scheme 1).^26,27 Macrocyclic chelators are often preferred as they are pre-organized in a way such that the entropic penalty associated with coordination is reduced, resulting in thermodynamically stable and kinetically inert complexes.^28–30 In recent years, the effect of incorporating rigid spacers into the backbone of different acyclic ligands has been studied, in most occasions resulting in higher kinetic inertness.^31–34 However, when studying the effect of the incorporation of a cyclohexyl spacer into the backbone of the 18-membered macrocycle H₂MACROPA, the kinetic lability of the resulting lanthanide complexes actually increased.³⁵

Scheme 1 Chelators discussed in this work and numbering scheme used for NMR spectral assignment.

Concerning the use of macrocyclic chelators for biomedical applications, DOTA (2,2′,2′′,2′′′-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid) is the most commonly used system.³⁶ This platform is based on the azamacrocycle CYCLEN, although macrocycles such as TACN (1,4,7-triazacyclononane) and CYCLAM (1,4,8,11-tetraazacyclotetradecane) have also been extensively investigated.³⁷ The popularity of these systems is mainly due to the presence of amine nitrogen atoms that can be used to functionalise the macrocycle with additional pendant arms to increase the denticity of the systems. Apart from these systems, larger 18-membered macrocycles incorporating ether or pyridine donors, have been studied in recent years for the complexation of large radiometals.^38–44 Although not yet reported for radiopharmaceutical uses, PYTAM (Scheme 1) is a decadentate macrocyclic chelator with four acetamide pendant arms, belonging to the PYAN family, first used for the complexation of lanthanides, resulting in very inert complexes.^45,46

Herein, we present a study on the coordination ability towards [^nat/203Pb]Pb(II) of PYTAM and another three new decadentate hexaazamacrocylic chelators. Additional rigidity was introduced into the PYTAM framework by incorporating two cyclohexyl units, giving rise to the new chelator CHX-PYTAM. To improve solubility of these chelators at more basic pH values, the acetamide groups were substituted with glycinate arms giving chelators H₄PYTAMGly and H₄CHX-PYTAMGly. The intermediate HSAB character of the primary and secondary amides, along with the pyridyl groups present in the structure of these chelators, is expected to pair nicely with coordination preference of Pb(II). The [^natPb]Pb(II) complexes were prepared in situ and studied in solution through a combination of multinuclear (¹H, ¹³C, ²⁰⁷Pb) NMR spectroscopy and DFT calculations. We also report the X-ray crystal structures of two of the complexes. Protonation constants of two of the chelators and the stability constants of the resulting Pb(II) complexes were determined through a combined spectrophotometric and potentiometric approach. Subsequently, radiolabelling and stability studies were performed with [²⁰³Pb]Pb(II) produced using a 13 MeV cyclotron. It should be noted that asymmetric functionalization of 3,6,10,13-tetraaza-1,8(2,6)-dipyridinacyclotetradecaphane, the backbone on which the chelators are based, has been recently described,⁴² paving the way for bifunctional systems based on this platform.

Results and discussion

Synthesis of the chelators

CHX-PYTAM was synthesized following a procedure similar to the one reported previously for PYTAM,⁴⁵ through alkylation of macrocycle CHX-PYAN with 2-bromoacetamide (Scheme 2), yielding CHX-PYTAM as a white solid (40% yield). The CHX-PYTAM precursor was obtained by a Ba(II)-templated Schiff-base condensation reaction, following a slight modification of the method reported by Jackels.⁴⁷ H₄PYTAMGly and H₄CHX-PYTAMGly were prepared through alkylation of the corresponding macrocyclic backbone with tert-butyl (2-chloroacetyl)glycinate followed by deprotection of the tert-butyl groups using TFA, affording the final chelators in 13% and 44% yields, respectively, over the two steps (see Experimental section for details).

Scheme 2 General procedure for the synthesis of the chelators. (a) Bromoacetamide and Na₂CO₃ in acetonitrile. (b) tert-butyl (2-chloroacetyl)glycinate K₂CO₃ in acetonitrile, followed by 1 : 1 TFA : CH₂Cl₂.

X-ray crystal structures of [Pb(PYTAM)](PF₆)₂ and [Pb(H₄PYTAMGly)](NO₃)₂·5H₂O

Addition of excess KPF₆ to an aqueous solution of the [Pb(PYTAM)]²⁺ complex and slow evaporation of an acidic solution of the [Pb(H₄PYTAMGly)]²⁺ complex provided single crystals suitable for X-ray analysis (Fig. 1 and Fig. S1, S2, ESI†). Bond distances of the Pb(II) coordination environments are listed in Table 1. Compound [Pb(PYTAM)](PF₆)₂ crystallizes in the centrosymmetric orthorhombic Fddd space group, with the asymmetric unit showing only one quadrant of the molecule due to its crystallographically-imposed D₂ symmetry. Crystals contain two non-coordinated PF₆⁻ anions involved in hydrogen-bonding interactions with the chelator amide N–H groups. On the other hand, [Pb(H₄PYTAMGly)](NO₃)₂·5H₂O crystallizes in the monoclinic P2₁/c space group and contains a network of water molecules interacting via hydrogen bonds with nitrate anions the protonated terminal carboxylate groups of the glycinate arms.

Fig. 1 ORTEP views (30%) of the cations present in the crystal structures of [Pb(PYTAM)](PF₆)₂ [(a)–(c)] and [Pb(H₄PYTAMGly)](NO₃)₂·5H₂O [(d)–(f)] with atom labelling. Hydrogen atoms bonded to C atoms, solvent molecules and anions are omitted for simplicity.

Table 1 Interatomic distances (Å) of the metal coordination environment in crystals of [Pb(PYTAM)](PF₆)₂ and [Pb(H₄PYTAMGly)](NO₃)₂·5H₂O

[Pb(PYTAM)](PF6)2		[Pb(H4PYTAMGly)](NO3)2·5H2O
Pb(1)–N(1)	2.646(6)	Pb(1)–N(1)	2.650(2)
		Pb(1)–N(4)	2.623(2)
Pb(1)–N(2)	2.732(5)	Pb(1)–N(2)	2.733(2)
		Pb(1)–N(3)	2.7109(19)
		Pb(1)–N(5)	2.7663(19)
		Pb(1)–N(6)	2.753(2)
Pb(1)–O(1)	2.791(4)	Pb(1)–O(1)	2.8648(19)
		Pb(1)–O(4)	2.7828(17)
		Pb(1)–O(7)	2.6935(18)
		Pb(1)–O(10)	2.9217(17)

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The Pb(II) ion in [Pb(PYTAM)](PF₆)₂ is ten-coordinated by the chelator, which binds through the six N donor atoms of the macrocycle and the four amide O atoms. The Pb–N distances involving pyridyl N atoms (2.646(6) Å) are ∼0.09 Å shorter than those to amine N atoms (2.732(5) Å), a trend that is consistent with X-ray structures reported in the literature for eight-coordinate Pb(II) complexes.^15,48 The distances between the metal ion and the O donor atoms (2.791(4) Å) are longer than those involving N donor atoms, a situation observed previously for complexes containing amide groups such as [Pb(DOTAM)]²⁺,⁴⁹ or even negatively charged carboxylate groups (i.e. [Pb(DOTA)]²⁻).⁵⁰

The Pb(II)-donor distances in [Pb(H₄PYTAMGly)]²⁺ point to a slight distortion of the metal coordination environment compared to [Pb(PYTAM)](PF₆)₂ (Table 1). Indeed, one of the Pb–O distances is particularly short [Pb(1)–O(7) = 2.6935(18) Å], while the remaining three Pb–O distances are in the range of 2.78 to 2.92 Å. Similarly, the distances to amine N atoms differ by up to ∼0.06 Å. The deformation of the coordination sphere appears to be related to steric interactions involving methylene protons of neighbouring terminal acetic acid groups, which display H⋯H distances as short as 2.77 Å. The distortion of the metal coordination sphere is also reflected in the angle involving the two pyridyl N atoms and the metal ion, which is linear in [Pb(PYTAM)]²⁺ takes a value of 177.99(6)° in [Pb(H₄PYTAMGly)]²⁺, with the pyridine rings being slightly bent towards the hemisphere of the metal coordination environment where O(7) and O(1) are situated. The slight alteration of the Pb(II) coordination sphere in [Pb(H₄PYTAMGly)]²⁺ does not appear to be related to a significant activity of the 6s² lone pair. In fact, previous works associated the stereochemical activity of the lone pair with a polarization of the 6s² lone pair with a significant 6p contribution.^51,52 However, the analysis of the natural bond orbitals using DFT in both [Pb(PYTAM)]²⁺ and [Pb(H₄PYTAMGly)]²⁺ indicates that the Pb(II) lone pair has a 100% 6s character (Table S1, ESI†). This is in line with the general trend that holodirected structures are favoured by the presence of neutral donor atoms and high coordination numbers.⁵¹

The conformation of the macrocycles in these complexes is similar to those observed for lanthanide complexes based on the same macrocycle and functionalised with identical pendant arms.^53–57 Two pendant arms attached to opposite N atoms on the macrocyclic structure are placed above the mean plane defined by the pyridine rings, while the remaining two pendant arms are placed below that plane. This conformation, together with the symmetrical coordination environment imposes a D₂ symmetry for the [Pb(PYTAM)]²⁺ complex. Fig. 1 shows views of the structure of this complex along the three perpendicular C₂ axes characterising the D₂ point group. The five-membered chelates generated upon coordination of the ethylenediamine spacers adopt the same conformations, which can be represented as (λλ) or (δδ) (Fig. S3, ESI†).^58,59 Furthermore, the four amine N atoms adopt identical configurations. The two structures are centrosymmetric, with both (λλ)-RRRR and (δδ)-SSSS enantiomers being present in the crystal.

Solution structure of the Pb(II) complexes

The Pb(II) complexes of the four chelators were prepared in situ by adding Pb(NO₃)₂ to a solution of the chelator in D₂O. Upon addition of the metal salt, the pD dropped to low values, between 1 and 2. While ¹H-NMR of the complexes with ethylene spacers show one species in solution, the complexes with cyclohexyl spacers do not show one unique species, presenting more complex spectra, at this pD (Fig. S4, ESI†). The pD was then raised to ∼7 using NaOD to record the NMR spectra of the complexes (Fig. 2).

Fig. 2 ¹H NMR spectra of [Pb(PYTAM)]²⁺ (pD = 6.5), PYTAM (pD = 1.4) [Pb(CHX-PYTAM)]²⁺ (pD = 7.4), CHX-PYTAM (pD = 1.7), [Pb(PYTAMGly)]²⁻ (pD = 6.8), H₄PYTAMGly (pD = 2.0), [Pb(CHX-PYTAMGly)]²⁺ (pD = 6.3) and H₄CHX-PYTAMGly (pD = 2.0), recorded in D₂O solution (300 MHz, 298 K).

The ¹H NMR spectra of the four complexes are characteristic of highly rigid structures, as evidenced by the well-resolved signals that can be observed in the aliphatic region, where doublets corresponding to AB systems associated with the geminal coupling of the protons of the CH₂ groups appear (Fig. 2). These characteristic doublets are easily identified through their large ²J values (∼15 Hz) and roof effect. This is due to the rigidity of the system, which locks the protons of these groups into specific positions, making them no longer chemically equivalent. The NMR data in solution indicate the presence of a single complex species in each case. The number of ¹H and ¹³C NMR signals point to an effective D₂ symmetry of the complexes in solution. A full assignment of the ¹H and ¹³C NMR spectra, achieved with the aid of 2D COSY, HSQC and HMBC experiments, along with J coupling values, is provided in the ESI (Tables S2–S4†). Noteworthy, the ¹H NMR spectra of the free PYTAM and H₄PYTAMGly chelators are characteristic of flexible molecules, with the signals due to CH₂ groups being observed as singlets. The ¹H NMR spectra of the corresponding cyclohexyl derivatives display broad signals, which reflects a certain degree of rigidity of the free chelator in solution.

Additional information on the structures of the complexes in solution was obtained from 2D ¹H–²⁰⁷Pb heteronuclear multiple-quantum correlation (HMQC) spectra (Fig. 3).⁶⁰ These spectra provide two strong cross-peaks relating the ²⁰⁷Pb resonance and the equatorial protons of the methylenic groups of the molecule (H4 and H5, see Scheme 1). Additionally, another strong cross peak is observed for one of the signals of the CH₂ protons of the ethylene spacer (H7) in [Pb(PYTAM)]²⁺ and [Pb(PYTAMGly)]²⁻. The set of three geminal (axial) CH₂ signals provide however weak cross peaks. This indicates that axial and equatorial protons provide rather different ³J_Pb–H coupling constants. Furthermore, the axial C–H protons (H7) of the cyclohexyl group in [Pb(CHX-PYTAM)]²⁺ and [Pb(CHX-PYTAMGly)]²⁻ do not provide cross peaks with the ²⁰⁷Pb nucleus. Thus, we conclude that equatorial protons give much stronger cross peaks in the HMQC spectra than the axial counterparts. Equatorial CH₂ protons are characterised by H–C–N–Pb dihedral angles of ∼160–170°, while axial protons give dihedral angles of ∼70–80°. This suggests that the ³J_Pb–H coupling constants involving methylenic protons follow a Karplus-like relationship with the dihedral H–C–N–Pb angle,⁶¹ as observed previously for properties originated by through-bond effects (i.e. spin densities).⁶² The strong ³J_Pb–H coupling involving equatorial protons is also evident by the presence of satellites in the ¹H NMR spectra (Fig. 3) due to coupling with ²⁰⁷Pb (I = ½), which has a 22.6% natural abundance. These satellites provide ³J(¹H–²⁰⁷Pb) couplings of ∼8–9 Hz, which are in the low range reported for Pb(II) complexes containing H–C–N(sp³) bonds (ca. 8–20 Hz).^60,63 Larger coupling constants of up to ∼43 Hz were reported for ³J(¹H–²⁰⁷Pb) couplings involving sp² N donor atoms.^64–66 Nevertheless, the presence of the ³J(¹H–²⁰⁷Pb) coupling constants confirms the coordination of the macrocyclic unit to the Pb(II) ion and reveals a certain degree of covalence of the Pb–N_amine bonds. We also notice that the pyridyl protons H2 give a sizeable ⁴J(¹H–²⁰⁷Pb) coupling of ∼3 Hz, pointing to a rather strong coordination of the pyridyl N atom to the metal ion.

Fig. 3 ¹H–²⁰⁷Pb HMQC spectrum of [Pb(PYTAM)]²⁺ (pD = 6.5) recorded in D₂O solution (400 MHz, 298 K). The inset shows an expansion of the aliphatic region where satellites due to ³J(¹H–²⁰⁷Pb) coupling are evident.

The ²⁰⁷Pb NMR spectra of the complexes display signals in the range −2178 to −2255 ppm, with the cyclohexyl derivatives providing slightly more negative chemical shifts and the glycinate derivatives slightly more positive shifts (Fig. 4). The similar δ values observed for the four complexes point to comparable Pb(II) coordination environments. The ²⁰⁷Pb NMR signals observed here are shielded by ∼4000 ppm with respect to those reported for EDTA derivatives^60,67 and [Pb(DOTAM)]²⁺.⁶⁸ Positive chemical shift values were also reported for complexes with crown ether derivatives⁶⁴ (the chemical shift of neat Pb(CH₃)₄ used as a reference corresponds to +2961 ppm versus the 1 M aqueous Pb(NO₃)₂ reference used here).⁶⁷ The negative δ values found for the ten-coordinated Pb(II) ions reported here follow the general trend of an increasing shielding with higher coordination numbers.⁶⁹ A similar situation was observed for Y(III) complexes, as ten-coordinated derivatives displayed unusually shielded ⁸⁹Y resonances.⁷⁰ In the latter case the experimental ⁸⁹Y shifts could be approximated by the sum of the shielding contributions of the chelator donor atoms, and thus an increased coordination number results in a more important shielding effect. A similar behaviour may affect the ²⁰⁷Pb NMR shifts, though investigation of a wider range of complexes is required to find correlations between the number and type of donor atoms and the observed shifts.

Fig. 4 ²⁰⁷Pb NMR spectra of [Pb(PYTAM)]²⁺ (pD = 6.5), [Pb(CHX-PYTAM)]²⁺ (pD = 7.4), [Pb(PYTAMGly)]²⁻ (pD = 6.8), [Pb(CHX-PYTAMGly)]²⁺ (pD = 6.3) recorded in D₂O solution (84 MHz, 298 K).

Protonation constants and thermodynamic stability of the Pb(II) complexes of the glycinate derivatives

The protonation constants of the chelators and the stability constants of the lead(II) complexes were determined for the glycinate derivatives, as the chelators containing acetamide pendants presented solubility issues in basic media. Chelator protonation constants were measured using potentiometric and spectrophotometric titrations, following the absorption of the pyridyl groups at ca. 265 nm. Potentiometric titrations afforded three protonation constants for PYTAMGly⁴⁻ above pH 5.0 and up to four protonation constants with log K values in the range ∼2.4 to 4.1. The assignment of the protonation constants was aided by spectrophotometric titrations (Fig. 5). Indeed, the absorption spectrum recorded at pH 1.30 displays a maximum at 263 nm and a shoulder at 267 nm. Increasing the pH above ∼4.0 provokes a slight shift of the absorption maximum to 265 nm as the band becomes more rounded and its intensity decreases. These spectral changes are compatible with the protonation of a pyridyl unit.³⁴ The log K value obtained from spectrophotometric titrations (4.98) is in very good agreement with that determined by potentiometry (log K₃ = 5.11(1)). The spectrum experiences further changes above pH ∼ 5.6, with the absorption maximum experiencing a slight shift to lower energies (267 nm) as its intensity increases. Spectrophotometric titrations afforded a single protonation constant of log K = 6.29(1), which coincides with the average of log K₁ (6.70(1)) and log K₂ (5.88(1)) obtained by potentiometry. We therefore assign these protonation constants to the amine groups of the macrocycle, though the significant spectral changes suggest that the pyridine units are also involved in these protonation processes. A comparison of the protonation constants log K₁ and log K₂ determined for PYTAMGly⁴⁻ and DOTAMGly⁴⁻ indicate that the amine groups of the 12-membered CYCLEN ring are considerably more basic than those of the 18-membered ring of PYTAMGly⁴⁻. The 18-membered macrocycle crown-4Py displays an intermediate behaviour.¹⁵ The protonation constants determined for PYAN using 0.1 M KNO₃ and 0.1 M KCl as background electrolytes evidence that the incorporation of the amide pendants in PYTAMGly⁴⁻ considerably reduces the basicity of the N atoms of the macrocycle (Table 2).^71–73

Fig. 5 (a) UV-Vis absorption spectra recorded for a solution of PYTAMGly⁴⁻ (10⁻⁴ M, I = 0.15 M NaClO₄, 25 °C) at different pH values. The spectra highlighted in red, violet, and blue correspond to pH values of 1.30, 5.57 and 8.01, respectively. The inset shows the spectral changes at selected wavelengths and the fits of the data. (b) UV-Vis absorption spectra recorded to monitor the dissociation of the pre-formed Pb(II) complex with PYTAMGly⁴⁻ (10⁻⁴ M, I = 0.15 M NaClO₄, 25 °C) at different pH values. The spectra highlighted in red, and blue correspond to pH values of 0.82 and 3.58, respectively. The inset shows the spectral changes at 261 nm and the fit of the data.

Table 2 Chelator protonation constants and stability constants of Pb(II) complexes (I = 0.15 M NaClO₄, 25 °C)

	PYTAMGly^4−	CHX-PYTAMGly^4−	MACROPA^2− ^a	Cyclen-4Py^b	Crown-4Py^b	DOTAMGly^4−^c	PYAN^d
a Data from ref. 78, 0.1 M KNO3, 25 °C. b Data from ref. 15, 0.1 M KCl, 25 °C. c Data from ref. 74, 0.1 M KCl, 25 °C. d Data from ref. 71, 0.1 M KNO3, 25 °C.
log K1	6.70(1)	8.69(5)	7.52	10.05	7.91	9.19	8.99
log K2	5.88(1)	7.80(7)	7.03	7.53	7.33	6.25	8.22
log K3	5.11(1)	4.38(9)	3.58	3.54	4.64	4.08	6.03
log K4	4.08(1)	3.66(9)	2.47	2.23	4.00	3.45	5.34
log K5	3.46(1)	3.31(9)				3.20	2.11
log K6	3.13(1)	2.62(9)				1.40
log K7	2.46(1)	2.05(9)
log KPbL	17.03(1)	17.18(1)	16.23	19.95	13.29
log KPbHL	4.58(2)	4.691(2)	2.86	2.06	5.22
log KPbH2L	3.57(4)	3.66(7)			3.50
log KPbH3L	3.49(4)	3.52(8)
log KPbH4L	2.87(3)	3.09(6)
pPb	17.9	16.3	16.7	17.88	13.40

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The protonation constants log K₄−log K₇ determined by potentiometry are not associated to noticeable spectral changes in the absorption spectra, and therefore can be safely assigned to the remote glycinate groups. Similar protonation constants were reported for the glycinate groups of DOTAMGly⁴⁻.⁷⁴ The protonation of four non-interacting carboxylates is expected to have statistical separations of Δlog K = 0.42, 0.36 and 0.42. The experimental protonation constants yield Δlog K_4,5 = 0.62, log K_5,6 = 0.33 and log K_6,7 = 0.67, in reasonable agreement with the values expected according to the statistical factor.^75,76

The same combined methodology was used to determine the protonation constants of CHX-PYTAMGly⁴⁻. The constants involving the protonation of amine groups, log K₁ and log K₂, are about 2 log K units higher than those determined for PYTAMGly⁴⁻ (Table 2). This is expected, as in general the substitution of ethyl for cyclohexyl groups significantly increases chelator basicity.^31,77 The protonation constants log K₃−log K₆ are attributed to the protonation of the glycinate groups on the basis of the minor spectral changes observed in the absorption spectra (Fig. S6, ESI†). These protonation constants follow again reasonably well the trend expected according to the statistical factor. Interestingly, spectrophotometric experiments demonstrate that the protonation of the pyridine ring occurs at low pH, with a protonation constant of log K₇ = 2.05(9). The rigidity of the macrocycle introduced by the presence of cyclohexyl rings appears to be responsible for the sharp decrease in the protonation constant of the pyridyl unit.

The stability constants of the Pb(II) complexes with PYTAMGly⁴⁻ and CHX-PYTAMGly⁴⁻ were determined using a combination of potentiometric and spectrophotometric titrations. Potentiometry was used to obtain the protonation constants of the complexes, while complex dissociation was investigated spectrophotometrically following the changes in the absorption band of the pyridyl groups. Of note, the amount of free Pb(II) at pH ∼2 is only 1.5 and 6.5% for PYTAMGly⁴⁻ and CHX-PYTAMGly⁴⁻, respectively, preventing the determination of stability constants by direct potentiometry. The absorption band of the pyridyl rings of PYTAMGly⁴⁻ at 263 nm undergoes a slight blue shift to 260 nm as its intensity increases as a result of complex formation (Fig. 5b). Similar spectral variations are observed for CHX-PYTAMGly⁴⁻ (Fig. S6, ESI†). The fits of the data afforded the stability and protonation constants shown in Table 2. These studies showed that the incorporation of the cyclohexyl spacer did not have a large effect on the value of log K_PbL affording values of 17.03 and 17.17 for [Pb(H₄PYTAMGly)]²⁻ and [Pb(H₄CHX-PYTAMGly)]²⁻, respectively. These values are higher than those reported for the complexes with other potentially decadentate 18-membered macrocycles such as MACROPA²⁻ and crown-4Py.^15,78 Higher stability constants were reported for CYCLEN derivatives containing pyridyl rings such as CYCLEN-4Py.¹⁵ However, the low basicity of PYTAMGly⁴⁻ and especially CHX-PYTAMGly⁴⁻ renders rather high conditional stability at physiological pH. This can be conveniently assessed by calculating pPb values, defined as −log[Pb(II)]_free for a metal ion concentration of 1 μM and a total chelator concentration of 10 μM.⁷⁹ The pPb values obtained for PYTAMGly⁴⁻ and CYCLEN-4py are virtually identical in spite of the three orders of magnitude difference in stability constants. The pPb value calculated for CHX-PYTAMGly⁴⁻ is nearly two units lower than that of PYTAMGly⁴⁻ as a result of the lower basicity of the latter, though comparable to that of MACROPA²⁻.

The speciation diagrams calculated from equilibrium constants (Fig. 6) indicate that complex protonation takes place below pH ∼ 7, with the non-protonated complex species being at nearly 100% abundance around neutral pH. Complex dissociation occurs below pH ∼ 2 and pH ∼ 3 for PYTAMGly⁴⁻ and CHX-PYTAMGly⁴⁻, respectively. The two complexes show a very similar protonation behaviour, with four protonation constants associated to the glycinate groups. The Δlog K_PbHL,PbH2L values of 1.01 and 1.03 for the complexes with PYTAMGly⁴⁻ and CHX-PYTAMGly⁴⁻, respectively, are higher than that expected according to the statistical effect (0.42), while the values of Δlog K_PbH2L,PbH3L < 0.15 are lower than the statistical value of 0.36.⁷⁶ This suggests a certain negative cooperativity for the formation of the deprotonated complex species. We also note that the glycinate groups are characterized by higher protonation constants in the complexes compared with the free ligands. Complex formation forces the negatively charged glycinate groups to be in rather close proximity (Fig. 1), which likely favours their protonation.

Fig. 6 Speciation diagrams of the H₄PYTAMGly:Pb(II) (a) and H₄CHX-PYTAMGly⁴⁻:Pb(II) (b) systems calculated for [Pb(II)]_tot = [L]_tot = 1 mM (25 °C, I = 0.15 M NaClO₄).

Radiolabelling studies

Once the non-radioactive studies showed that these chelators presented appropriate properties for Pb(II) complexation, lead-203 radiolabelling studies were performed. [²⁰³Pb]Pb(II) was produced and purified according to the procedure reported by McNeil et al., without any modifications.⁸⁰ Concentration dependent radiolabelling for all four chelators and DOTAM, the gold-standard for radiolabelling with lead(II) isotopes, was performed in NH₄OAc buffer (0.1 M, pH 7) at five different chelator concentrations (10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ M). The reactions were followed at two time points (30 and 60 minutes) and two temperatures (room temperature: ∼25 °C, and 80 °C). The radiochemical yields (RCYs) were determined by spotting an aliquot of the reaction onto iTLC-SA plates and developed using 50 mM EDTA (pH 5) as the mobile phase.

The RCYs observed for both temperatures at 60 min (Fig. 7 and Tables S4, S5, ESI†) improve slightly with respect to those recorded at 30 min (Fig. S8 and Tables S6, S7 ESI†). At room temperature (Fig. 7a and Table S5, ESI†), CHX-PYTAM is the only chelator that does not reach quantitative yields at 10⁻⁴ M, although the RCY is still high with a value of 97.2 ± 1.2% at this concentration. This effect is most likely due to the rigidity introduced by the cyclohexyl spacers, since at higher temperatures the yields are superior. At 80 °C (Fig. 7b and Table S6, ESI†), the RCYs of PYTAM and CHX-PYTAM are comparable to the values observed for DOTAM. The glycinate derivatives, however, behave differently, and their RCYs do not improve significantly as the labelling efficiency of these chelators is nearly identical at both room temperature and 80 °C.

Fig. 7 Radiolabelling studies using ²⁰³Pb (∼150 kBq, 0.1 M NH₄OAc buffer, pH 7) (n = 3 for each data point). (a) Concentration dependent radiolabelling at room temperature after 60 min. (b) Concentration dependent radiolabelling at 80 °C after 60 min.

Since ²⁰³Pb-radiolabelling of CHX-PYTAM at room temperature was not fully quantitative at the highest chelator concentration, labelling of this chelator was also performed at 37 °C (Table S9, ESI†), in addition to room temperature (∼25 °C) and 80 °C (Fig. 8). The radiochemical yields improve significantly at this temperature for all concentrations, with yields of nearly 100% for both 10⁻⁴ and 10⁻⁵ M (98.4 ± 0.4 and 97.3 ± 0.5%, respectively) after 30 minutes and even higher yields after 60 min (100% and 99.6 ± 0.6%, respectively). This result indicates that although labelling of 100% was not achieved at room temperature, the system is still quite promising for the design of bifunctional systems, as heating to 37 °C is still compatible with most biological targeting vectors.

Fig. 8 Temperature dependent ²⁰³Pb-radiolabelling (150 kBq, 0.1 M NH₄OAc buffer, pH 7, 30 min) of CHX-PYTAM at different concentrations (n = 3 for each data point).

Once the ²⁰³Pb-labelled complexes were prepared, challenge experiments were performed to assess the kinetic inertness of the ²⁰³Pb complexes. The complexes were formed at either room temperature and/or 80 °C to be representative of the tested radiolabelling conditions (Fig. 9 and Fig. S9, Tables S10–S14, ESI†). The purpose of this experiment was to evaluate the influence of complexation temperature on stability. For the stable Pb(II) and EDTA challenges, 20 equivalents of the respective chemical was added to the radiolabelling solution. DOTAM forms incredibly stable complexes in the studied competitive media, remaining 100% intact in all the conditions investigated. The complexes formed with PYTAM, H₄PYTAMGly, and CHX-PYTAM presented excellent stability in presence of 20 equiv. of EDTA (Fig. 9b) over the course of over one half-life, showing no indications of transchelation. However, H₄CHX-PYTAMGly did not remain intact, neither when prepared at room temperature or 80 °C. It may be possible that the steric hindrance introduced by the simultaneous presence of cyclohexyl spacer and acetylglycine pendant arms facilitates transchelation.

Fig. 9 Challenging studies of the ²⁰³Pb-labelled complexes (150 kBq, 0.1 M NH₄OAc buffer, pH 7) (n = 3 for each data point). (a) Human serum stability challenge. (b) Excess EDTA challenge (20 equiv.). (c) Excess stable Pb(II) challenge (20 equiv.).

When incubated with excess (20 equiv.) of stable Pb(II) (Fig. 9c), CHX-PYTAM is the only chelator that forms a complex that is stable under these conditions. The complex is 100% intact after 24 hours and after 72 hours the majority (93.2 ± 0.4%) of the radiocomplex remains intact. H₄PYTAMGly is the least stable as within 2 hours, only 13.4 ± 2.0% of the complex remained intact at this time point. PYTAM and H₄CHX-PYTAMGly, while not as stable as CHX-PYTAM, performed better than H₄PYTAMGly, with similar results, as 37.0 ± 2.0% and 54.3 ± 1.5% of the complex is stable after 72 hours, respectively. It is surprising, after seeing the results of the competition with H₄EDTA, that H₄CHX-PYTAMGly performed better than H₄PYTAMGly under these conditions, though transchelation and transmetallation occur through different mechanisms. The presence of glycinate groups likely favours the formation of the dinuclear intermediate responsible for the Pb(II)-assisted dissociation mechanism. The high stability of CHX-PYTAM is most likely due to the increase in rigidity introduced by the two cyclohexyl groups in the backbone and the use of acetamide pendant arms, which appears to be the ideal combination for complex stability. It should be noted though that the complex formed with DOTAM does, in fact, remain 100% stable. However, the 93.2 ± 0.4% of intact complex, observed for CHX-PYTAM at 72 hours, is the highest of all of the chelators presented in this work, and it should not present any issues in vivo as high concentrations of Pb(II) are not expected to be found in the body.

Finally, the kinetic inertness of the complexes was evaluated when incubated in human serum at 37 °C (Fig. 9a). CHX-PYTAM showed no dissociation over 72 hours while the other three chelators did drop slightly but remained above 95% in all cases. These results point towards all four complexes possibly being stable under in vivo conditions, although further studies would be necessary to confirm this hypothesis.

Conclusions

We have shown that macrocyclic chelators based on the 18-membered platform PYAN show promising properties for the development of Pb-based radiopharmaceuticals. Stable Pb(II) complexation was achieved by incorporating four amide pendant arms, leading to the formation of stable and rigid complexes with ten-coordinated Pb(II) ions. The inertness of the complexes is improved by incorporating rigid cyclohexyl spacers into the macrocyclic ring. The presence of bulky acetylglycine pendant arms appears to introduce some steric hindrance, while the chelator CHX-PYTAM displays excellent properties for the development of Pb-based radiopharmaceuticals. We are currently developing bifunctional derivatives of this platform to be able to further study the potential of these chelates.

Experimental section

General considerations

Solvents and reagents were purchased from commercial sources and were used as supplied without further purification. Medium performance liquid chromatography (MPLC) was performed in a Puriflash XS 420 InterChim Chromatographer equipped with a UV-DAD detector and a 20 g BGB Aquarius C18AQ reversed-phase column (100 Å, spherical, 15 μm) using H₂O and CH₃CN with 0.1% TFA as the mobile phases. Semi-preparative high performance liquid chromatography (HPLC) was performed using a Jasco LC-4000 instrument equipped with a UV-4075 detector, in manual injection and collection mode, using a Fortis C18 column (5μm, 250 × 10 mm) and H₂O and CH₃CN with 0.1% TFA as the mobile phases, operating at a flow rate of 4.0 mL min⁻¹. High-resolution electrospray-ionization time-of-flight ESI-TOF mass spectra were recorded in positive mode using a LTQ-Orbitrap Discovery Mass Spectrometer coupled to a Thermo Accela HPLC.¹H and ¹³C NMR spectra of the chelators and their complexes were recorded on Bruker AVANCE III 300, Bruker AVANCE 400 or Bruker AVANCE 500 spectrometers. PYAN (3,6,10,13-tetraaza-1,8(2,6)-dipyridinacyclotetradecaphane), tert-butyl (2-chloroacetyl)glycinate and PYTAM (2,2′,2′′,2′′′-(3,6,10,13-tetraaza-1,8(2,6)-dipyridinacyclotetradecaphane-3,6,10,13-tetrayl)tetraacetamide) were prepared according to previously reported procedures.^45,81,82

2,2′,2′′,2′′′-((2,2′,2′′,2′′′-(3,6,10,13-Tetraaza-1,8(2,6)-dipyridinacyclotetradecaphane-3,6,10,13-tetrayl)tetrakis(acetyl))tetrakis(azanediyl))tetraacetic acid (H₄PYTAMGly)

PYAN (0.1869 g, 0.57 mmol), K₂CO₃ (0.4734 g, 3.43 mmol) and KI (0.0095 g, 0.057 mmol) were added to CH₃CN (40 mL) while the mixture was heated to 50 °C. tert-Butyl (2-chloroacetyl)glycinate (0.5920 g, 2.85 mmol) was dissolved in CH₃CN (15 mL) and added dropwise over the course of 2 hours after which the reaction was left stirring at 50 °C for 6 days after which the solvent was eliminated in the rotary evaporator. The product was isolated by suspending the oil in H₂O (50 mL) and performing extractions with CHCl₃ (3 × 50 mL). The organic phase was separated, dried using Na₂SO₄, and concentrated in the rotary evaporator. A yellow oil was obtained after drying in the vacuum line, which is dissolved in 20 mL of a 1 : 1 TFA : CH₂Cl₂ mixture and left stirring overnight. The solvent was then eliminated under a nitrogen flow and was washed with water (3 × 15 mL), obtaining a brown oil. This oil was dissolved in water (1.5 mL) and was purified by reverse phase using method A (Table S15†). The desired compound eluted at 36% CH₃CN (retention time 8.6 min, 6.3 CV). The fraction of interest was lyophilized and an off-white solid was obtained (0.0887 g, 13% yield). ¹H NMR (300 MHz, D₂O, pD 2.0) δ (ppm) 8.31 (s, br, 2H), 7.80 (d, J = 7.4 Hz, 4H), 4.59 (s, 8H), 3.92 (s, 8 H), 3.85 (s, br, 8H), 3.48 (s, 8H).¹³C NMR (75 MHz, D₂O, pD 2.0) δ (ppm) 172.96, 169.33, 150.57, 126.03, 55.12, 52.00, 41.15. Elemental analysis calcd (%) for C₃₄H₄₆N₁₀ O₁₂·4TFA: C 40.59, H 4.06, N 11.27; found: C 40.68, H 4.35, N 11.35. Experimental MS (ESI⁺, MeOH/H₂O): m/z 413.1459, 424.1364, 825.2847; calculated for [C₃₄H₄₇N₁₀ O₁₂K]²⁺ 413.1501, calculated for [C₃₄H₄₇N₁₀ O₁₂NaK]²⁺ 424.1410, calculated for [C₃₄H₄₆N₁₀ O₁₂K]⁺ 825.2928.

(4¹R,4²R,10¹R,10²R)-3,5,9,11-Tetraaza-1,7(2,6)-dipyridina-4,10(1,2)-dicyclohexanacyclododecaphane (CHX-PYAN)

Synthesis adapted from a previously reported procedure.⁴⁷ Pyridine-2,6-dicarbaldehyde (0.6250 g, 4.63 mmol) was dissolved in MeOH (150 mL) along with barium chloride dihydrate (0.4830 g, 2.32 mmol). (1R,2R)-Cyclohexane-1,2-diamine (0.5281 g, 4.62 mmol) was dissolved in MeOH (10 mL) and added dropwise to the refluxing reaction mixture over the course of 2 hours. The reaction was then left heating for an additional 3 hours after which it was cooled to 0 °C in an ice bath and NaBH₄ (0.2122 g, 6.07 mmol) was added in small increments. The reaction mixture was allowed to reach room temperature, a second addition of NaBH₄ (0.1062 g, 3.02 mmol) was made and the reaction was left stirring for 3 hours. The solvent was then eliminated in the rotary evaporator and water (50 mL, pH adjusted to 9 using NaOH) was added. Extractions with CHCl₃ (3 × 50 mL) (50 mL) were performed and the combined organic phases were dried using NaSO₄, filtered and concentrated in the rotary evaporator, obtaining a yellow solid (1.2429 g, yield 87%). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.55 (s, 2H), 7.04 (d, ³J = 7.6 Hz, 4H), 4.10 (d, ²J = 15.4 Hz, 4H), 3.80 (d, ²J = 15.7 Hz, 5H), 2.17–1.95 (m, 8H), 1.63 (d, ²J = 7.3 Hz, 4H), 1.18–0.96 (m, 8H). ¹³C NMR (126 MHz, CDCl₃) δ 137.19, 121.63, 59.56, 32.97, 24.99. Elemental analysis calcd (%) for C₂₆H₃₈N₆·1.65CHCl₃: C 52.64, H 6.23, N 12.97; found: C 52.58, H 6.33, N 13.31. Experimental MS (ESI⁺, MeOH/H₂O): m/z 435.3206, 457.3022; calculated for [C₂₆H₃₉N₆]⁺ 435.3231, calculated for [C₂₆H₃₉N₆Na]⁺ 457.3050.

2,2′,2′′,2′′′-((4¹R,4²R,10¹R,10²R)-3,5,9,11-Tetraaza-1,7(2,6)-dipyridina-4,10(1,2)-dicyclohexanacyclododecaphane-3,5,9,11-tetrayl)tetraacetamide (CHX-PYTAM)

CHX-PYAN·1.65CHCl₃ (0.3008 g, 0.484 mmol) was dissolved in CH₃CN (20 mL) and Na₂CO₃ (0.3204 g, 3.02 mmol) was added. 2-Bromoacetamide (0.2689 g, 1.95 mmol) was dissolved in CH₃CN (10 mL) and added dropwise over the course of an hour. After stirring for three days, the solvent was evaporated, and the resulting yellow solid was then prepared for purification by MPLC. The solid was dissolved in water (1 mL) by lowering the pH to pH = 3 using HCl. This solution was purified by reverse phase using method B (Table S16†), and the desired compound eluted at 27% CH₃CN (retention time 12.2 min, 8.9 CV). The fraction of interest was lyophilized and an off-white solid was obtained (180.0 mg, 40% yield). ¹H NMR (400 MHz, D₂O, pD = 1.74, 343.15 K) δ (ppm) 8.26 (s, 2H), 7.99 (d, ³J = 5.9 Hz, 2H), 7.60 (s, 2H), 5.19–4.85 (m, 3H), 4.69 (d, ²J = 15.3 Hz, 2H), 4.47 (d, ²J = 14.6 Hz, 2H), 4.17–3.75 (m, 5H), 3.60 (d, ²J = 13.6 Hz, 4H), 2.97 (d, ²J = 10.6 Hz, 2H), 2.83–2.56 (m, 2H), 2.53–2.29 (m, 4H), 2.09 (d, ²J = 10.1 Hz, 2H), 1.98–1.70 (m, 6H).¹³C NMR (101 MHz, D₂O, pD = 1.74, 343.15 K) δ (ppm) 174.07, 169.61, 157.17, 151.74, 140.35, 126.54, 126.51, 123.26 (br), 62.93 (br), 61.57 (br), 58.33, 54.46 (br), 52.97, 52.28, 24.39, 24.14. Elemental analysis calcd (%) for C₃₄H₅₀N₁₀O₄·1.5TFA·1.5HBr: C 46.52, H 5.59, N 14.66; found: C 46.46, H 5.61, N 14.72. Experimental MS (ESI⁺, MeOH/H₂O): m/z 332.2083, 663.4094, 685.3912; calculated for [C₃₄H₅₂N₁₀O₄]²⁺ 332.2081, calculated for [C₃₄H₅₁N₁₀O₄]⁺ 663.4089, calculated for [C₃₄H₅₀N₁₀O₄K]⁺ 685.3909.

2,2′,2′′,2′′′-((2,2′,2′′,2′′′-((4¹R,4²R,10¹R,10²R)-3,5,9,11-Tetraaza-1,7(2,6)-dipyridina-4,10(1,2)-dicyclohexanacyclododecaphane-3,5,9,11-tetrayl)tetrakis(acetyl))tetrakis(azanediyl))tetraacetic acid (H₄CHX-PYTAMGly)

CHX-PYAN·(CHCl₃)_1.65 (0.2000 g, 0.32 mmol), K₂CO₃ (0.3890 g, 2.81 mmol) and tert-butyl (2-chloroacetyl)glycinate (0.3920 g, 1.89 mmol) were added to CH₃CN (30 mL) and left stirring at room temperature for 30 days, following the progress of the reaction using mass spectrometry. After this time, since the reaction was not evolving to form the tetraalkylated derivative, more tert-butyl (2-chloroacetyl)glycinate (0.0294 g, 0.14 mmol) was added, and the reaction was left stirring for another 30 days after which the solvent was concentrated in the rotary evaporator and the product was isolated by adding CHCl₃ (30 mL) and H₂O with the pH adjusted to 9 using NaOH (30 mL) and performing an additional two extractions with CHCl₃ (30 mL). The organic phase was separated, dried using Na₂SO₄, and concentrated in the rotary evaporator. A yellow oil was obtained after drying in the vacuum line, which was dissolved in 10 mL of a 1 : 1 TFA : CH₂Cl₂ mixture and left stirring overnight. The solvent was then eliminated under a nitrogen flow and was washed with water (3 × 15 mL), obtaining a brown oil. This oil was dissolved in water (1.5 mL) and was purified by reverse phase using method C (Table S17†). The desired compound eluted at 41% CH₃CN (retention time 12.4 min, 9.1 CV). The fraction of interest was lyophilized, and a white solid was obtained (185.6 mg, 44% yield). ¹H NMR (400 MHz, D₂O, pD = 10.2, 343.15 K) δ (ppm) 8.02 (t, ³J = 7.7 Hz, 2H), 7.64 (d, ³J = 7.5 Hz, 4H), 4.33–3.43 (m, 16H), 3.22 (s, 4H), 2.51 (d, ²J = 10.0 Hz, 4H), 2.28–1.99 (m, 5H), 1.76–1.37 (m, 6H).¹³C NMR (101 MHz, D₂O, pD = 10.2, 343.15 K) δ (ppm) 176.34, 161.69, 158.74, 139.19, 123.26, 43.60, 25.67, 25.01. Elemental analysis calcd (%) for C₄₂H₅₈N₁₀O₁₂·3.5TFA·2H₂O: C 44.25, H 4.96, N 10.53; found: C 44.02, H 4.71, N 10.76. Experimental MS (ESI⁺, MeOH/H₂O): m/z 448.2192, 895.4324, 917.4142; calculated for [C₄₂H₆₀N₁₀O₁₂]²⁺ 448.2191, calculated for [C₄₂H₅₉N₁₀O₁₂]⁺ 895.4308, calculated for [C₄₂H₅₈N₁₀O₁₂K]⁺ 917.4128.

General procedure for the synthesis of the lead complexes

The Pb(II) complexes were prepared in situ by adding Pb(NO₃)₂ to a solution of each chelator in deuterated water (0.5 mL) in a molar ratio of 1 : 1.1 (H₄L: Pb(NO₃)₂). At this point, the mixture was quite acidic so the pD is adjusted above pD = 6 using a diluted solution of NaOD while the formation of the complex was monitored using NMR.

Crystal structure determination

Compounds [Pb(PYTAM)](PF₆)₂ and [Pb(H₄PYTAMGly)](NO₃)₂ were analysed by X-ray diffraction. Table S19† shows the crystallographic data and structure refinement parameters. Crystallographic data were collected on a Bruker D8 Venture diffractometer with a Photon 100 CMOS detector at 100 K with Mo-Kα radiation (λ = 0.71073 Å) generated by an Incoatec high brilliance microfocus source equipped with Incoatec Helios multilayer optics. The APEX4⁸³ software was used for collecting frames of data, indexing reflections, and the determination of lattice parameters, while SAINT⁸⁴ was used for integration of intensity of reflections, and SADABS⁸⁵ for scaling and empirical absorption correction. The structure was solved by dual-space methods using the program SHELXT.⁸⁶ All non-hydrogen atoms were refined with anisotropic thermal parameters by full-matrix least-squares calculations on F² using the program SHELXL-2014.⁸⁷ Hydrogen atoms were inserted at calculated positions and constrained with isotropic thermal parameters. The crystal structure of [Pb(H₄PYTAMGly)](NO₃)₂ shows a B level Alert in the checkcif due a short distance between two hydrogen atoms: one belonging to a water molecule (H5WA) and the other to a protonated glycinate group (H2A). The protonated glycinate group shows hydrogen bond interaction with the water molecule. Also, H5WA shows a hydrogen bond interaction with one nitrate group. These interactions determine the position of the hydrogens. CCDC 2305213 and 2305214 contain the supplementary crystallographic data.†

Potentiometric and spectrophotometric measurements

Stability constants and chelator protonation constants were determined through potentiometric and spectrophotometric titrations using HYPERQUAD.⁸⁸ All data were collected at 25 °C and NaClO₄ was used as an inert electrolyte which is added to all solutions to maintain the ionic strength at a constant value of I = 0.15 M.

Potentiometric titrations were carried out in dual-wall thermostated cell, using recirculating water. Nitrogen was bubbled on the surface to avoid CO₂ absorption and the solutions were homogenised using magnetic stirring. The titrant was added using a Crison microBu 2030 automatic burette and the electromotive force (emf) was recorded using a Crison micropH 2000 pH meter connected to a Radiometer pHG211 glass electrode and a Radiometer REF201 reference electrode. A 1 mM solution of the chelator (10 mL) was added to the cell, and the pH of the solution was adjusted to 11 using NaOH. This solution was then titrated using a standard HClO₄ solution to determine all pK_a values in the same experiment. Once the solution reached an acidic pH, an equimolar amount of Pb(II) was added and the mixture was left stirring for one hour to ensure complex formation. The solution was titrated with a solution of NaOH to determine the protonation constants of the complex. At this point it became clear that the stability constant of the complex must be determined by spectrophotometry, as the dissociation occurs at pH values that are too low to be determined using the glass electrode.

To determine equilibrium constants, HYPERQUAD calculates the proton concentration at each point of the titration using emf values, E, and using the electrode calibration equation, E = E°′ + s log[H⁺], with E°′ representing the formal potential and s, the slope of the electrode. Both parameters are determined with a calibration of the electrode system, conducted by titrating HClO₄ with NaOH, both with known concentrations and adjusted ionic strength, as [H⁺] is known for each calibration point and E is measured, with a lineal regression giving the values of E°′ and s.

Spectrophotometric titrations were performed using a Uvikon-XS (Bio-Tek Instruments) double-beam spectrophotometer and 1 cm path length quartz cuvettes, recording spectra between 220 and 300 nm. The spectra of each chelator at concentrations between 6–10 × 10⁻⁵ M at different pH values were used to obtain the pK_a values of the chelators. The desired pH was obtained using different buffers (acetate, phosphate, borax) and for very low pH values, HClO₄ was used directly (i.e. stability constant determination). Dissociation of the complexes was achieved at low pH values, and although equilibrium is reached quite quickly, the samples were equilibrated for a few hours before each measurement.

The ligand protonation constants reported in this work are defined as in eqn (1), where L represents a general ligand:

The stability constants of the Pb(II) complexes and their protonation constants are defined as in eqn (2) and (3), respectively.

DFT calculations

DFT calculations were performed with the Gaussian 16 program package,⁸⁹ with the TPSSh functional and the Def2-TZVPP basis set for chelator atoms. For Pb, we selected the small-core relativistic effective core potential of Dolg et al. (ECP60MDF),⁹⁰ which incorporates 60 electrons in the core, together with the cc-pVTZ valence basis set, which has a (12s11p8d1f)/[5s4p3d1f] contraction scheme.⁹¹ Calculations were performed using crystallographic structures in which only the positions of H atoms were optimised. The integration grid was set with the integral = ultrafinegrid keyword. Natural bond orbital analysis was carried out with the NBO program (version 3.1)⁹² included in Gaussian.

Radiolabelling

Caution: ²⁰³Pb emits ionizing radiation and should only be used in a facility in accordance with appropriate safety controls.

All chelators were purified by HPLC before radiolabelling experiments to ensure high purity (Table S18, ESI†).

Stock solutions, with a concentration of 10⁻³ M of DOTAM, PYTAM, H₄PYTAMGly, CHX-PYTAM and H₄CHX-PYTAMGly were prepared in deionized water. Serial dilution was then used to prepare chelator solutions with concentrations between 10⁻⁴ M and 10⁻⁷ M. For the ²⁰³Pb labelling studies, a 10 μL aliquot of chelator (or deionized water for the negative control) was added to 78 μL of deionized water and 10 μL of 1 M NH₄OAc (pH 7) and 2 μL of [²⁰³Pb]Pb(Cl)₂ (150 kBq) were added and mixed to begin the reactions, which were performed in triplicate and left at either room temperature or 80 °C until measurement at 30 and 60 minutes. iTLC was used to determine radiochemical yields (RCY) of the reactions, using iTLC-SA (iTLC paper impregnated with silicic acid) plates (1.5 × 10 cm, baseline at 1.5 cm; Agilent Technologies), developed using 50 mM EDTA (pH 5). With this system, complexed ²⁰³Pb(II) remained at the baseline (R_f = 0) while uncomplexed ²⁰³Pb(II) migrated at the solvent front (R_f = 1) (representative iTLCs are included in ESI, Fig. S10–S16†). Aliquots of the reactions were taken at 30 and 60 min, spotted onto the plates, and developed. RCYs were then measured using a BioScan System 200 Scanner (Washington, DC) and quantified with WinScan software.

Human serum stability studies

The stability of the five radiocomplexes was determined by adding 90 μL of human serum to previously radiolabelled solutions (10⁻⁴ M chelator concentration, 90 μL) and the resulting mixture was incubated at 37 °C. Aliquots (10 μL) were taken at 24, 48 and 72 hours and spotted onto the iTLC-SA plates, developed, and then analysed using the previously reported procedure, to determine the amount of intact complex.

EDTA and Pb(II) challenging studies

Stock solutions of EDTA (pH = 7) and Pb(OAc)₂ were prepared with a concentration of 20 mM. Preformed ²⁰³Pb-complexes (10⁻⁴ M chelator concentration, 100 μL volume), prepared both at room temperature and 80 °C, were then challenged with a 20-fold excess of either EDTA or Pb(II) as 10 μL of the 20 mM solution was added to the radiolabelling reaction. Aliquots (10 μL) were taken at 0.083, 0.5, 1, 2, 24, 48 and 72 hours and spotted onto the iTLC-SA plates, which were then analysed using the previously reported procedure, to evaluate the inertness of the complexes.

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

D. E.-G. and C. P.-I. thank Ministerio de Ciencia e Innovación (Grants PID2019-104626GB-I00 and PID2022-138335NB-I00) and Xunta de Galicia (ED431C 2023/33) for generous financial support. C. H. thanks Ministerio de Ciencia e Innovación (Grant PRE2020-092888) for funding her PhD contract and a short-term research visit to SFU and TRIUMF. C. P.-I. and C. H. thank Centro de Supercomputación de Galicia (CESGA) for providing supercomputer facilities. L. V. is indebted to CACTI (Universidade de Vigo) for X-ray measurements. C. F. R and B. L. M thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for funding through the Discovery grants (RGPIN-2019-07207) and Canada Graduate Scholarships – Doctoral (CGS-D) programs, respectively. B. L. M thanks the TRIUMF cyclotron operators for their continued support with ²⁰³Pb production at TRIUMF. Funding for open access provided by Universidade da Coruña/CISUG.

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Footnote

† Electronic supplementary information (ESI) available: ¹H, ¹³C, high-resolution crystal data and structure refinement details, thermodynamic constant determination and radiolabeling experiments. CCDC 2305213 and 2305214. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d3qi02354k

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