Hydroxypyridinone Chelators: From Iron Scavenging to Radiopharmaceuticals for PET Imaging with Gallium-68

Abstract

Derivatives of 3,4-hydroxypyridinones have been extensively studied for in vivo Fe³⁺ sequestration. Deferiprone, a 1,2-dimethyl-3,4-hydroxypyridinone, is now routinely used for clinical treatment of iron overload disease. Hexadentate tris(3,4-hydroxypyridinone) ligands (THP) complex Fe³⁺ at very low iron concentrations, and their high affinities for oxophilic trivalent metal ions have led to their development for new applications as bifunctional chelators for the positron emitting radiometal, ⁶⁸Ga³⁺, which is clinically used for molecular imaging in positron emission tomography (PET). THP-peptide bioconjugates rapidly and quantitatively complex ⁶⁸Ga³⁺ at ambient temperature, neutral pH and micromolar concentrations of ligand, making them amenable to kit-based radiosynthesis of ⁶⁸Ga PET radiopharmaceuticals. ⁶⁸Ga-labelled THP-peptides accumulate at target tissue in vivo, and are excreted largely via a renal pathway, providing high quality PET images.

1. Introduction

Positron emission tomography (PET) is a whole body diagnostic three-dimensional molecular imaging modality used in nuclear medicine that detects radiation arising from the decay of unstable positron-emitting radioisotopes. The availability of the positron-emitting isotope, gallium-68 (⁶⁸Ga) from decay of ⁶⁸Ge in a bench top ⁶⁸Ge/⁶⁸Ga generator is likely to have significant clinical impact on the use of PET for molecular imaging [1]. Clinical use of ⁶⁸Ga receptor-targeting radiopharmaceuticals for neuroendocrine cancers (⁶⁸Ga-DOTA-TATE) [2] and prostate cancers (⁶⁸Ga-HBED-PSMA) [3] has changed patient management in centres that have routine access to such agents. ⁶⁸Ga molecular imaging agents typically consist of chelators tethered to a receptor-targeted peptide, protein or small molecule. As Ga³⁺ can incorporate up to six ligands in an octahedral coordination sphere, hexadentate ligands are normally utilised. The Ga³⁺ ion has a high charge density, and is categorised as a “hard” Lewis acid, and so the majority of hexadentate ligands incorporate oxygen and/or nitrogen donor atoms. Both macrocyclic and acyclic chelators have been explored for ⁶⁸Ga³⁺ complexation [1,4,5,6,7].

Clinical radiosyntheses of ⁶⁸Ga-DOTA-TATE and ⁶⁸Ga-HBED-PSMA involve heating at 80–100 °C for 5–20 min at pH 3–5 [1,4], followed by post-synthetic purification and work-up to remove impurities, unreacted ⁶⁸Ga and reaction components that are not physiologically compatible. In the case of ⁶⁸Ga-DOTA-TATE, heating is required for complexation of ⁶⁸Ga³⁺. For ⁶⁸Ga-HBED-PSMA, three geometric cis/trans isomers are possible, with each possible geometric isomer having a diastereomer. In this case, heating the reaction favours formation of the thermodynamically preferred species.

For ⁶⁸Ga to be adopted in routine clinical practice, chelators that provide efficient and reproducible kit-based radiolabelling methods—preferably a single manipulation—are required. The chelators that we have developed based on hydroxypyridinones allow one-step quantitative ⁶⁸Ga³⁺ radiolabelling at neutral pH and ambient temperature.

Hydroxypyridinone (HP) ligands have high affinities for “hard” metal ions, and originally, the class of HPs described herein was developed extensively for therapeutic in vivo Fe³⁺ chelation in iron overload disease [8,9,10]. The bidentate HP ligand, deferiprone (1, Figure 1), is used routinely for this treatment [11]. Through chemical modification of substituents, Fe³⁺ affinity, hydrophilicity, metabolic stability and functionality of HPs can be tailored [10].

Ga³⁺ is an oxophilic metal ion, with a high charge density and ionic radius and coordination preferences similar to Fe³⁺. The (crystal) ionic radius for Ga³⁺ is 76 pm, and for high spin Fe³⁺ is 78.5 pm [12]. Recently, we have investigated the use of HPs for rapid and quantitative chelation of ⁶⁸Ga³⁺ (as well as a long-lived PET isotope, ⁸⁹Zr⁴⁺) and adapted a class of HPs to enable functionalisation with peptides and proteins for targeted molecular imaging [13,14,15,16].

Here we describe our research on the development of HPs for Fe³⁺ chelation, and how the design of such chelators is suitable for direct translation to ⁶⁸Ga PET radiopharmaceuticals. It is not intended as an exhaustive review of chelators for either treatment of iron overload disease or complexation of radioisotopes of gallium used in nuclear medicine. The literature in both areas has been surveyed in depth in recent years in several excellent reviews and books [4,5,6,7,10,17,18,19].

2. Hydroxypyridinones

HPs consist of a six-membered aromatic N-heterocycle, with a hydroxyl and a ketone functionality. Varying relative positions of the hydroxyl and ketone functional groups within a bidentate HP molecule results in three types of hydroxypyridinones: 1,2-hydroxypyridinone (2); 3,2-hydroxypyridinone (3); and 3,4-hydroxypyridinone (4) (Figure 2). Neutral HPs can be protonated and deprotonated, with the first pK_a typically corresponding to protonation/deprotonation at the oxo group, and the second, at the hydroxyl group. Delocalisation of the electrons of the N¹ ring atom leads to aromaticity (Figure 1 and Figure 2).

When HPs are deprotonated at the hydroxyl group, they are capable of complexing metal ions in a bidentate O₂ mode, forming five-membered chelate rings. The relative positions of the hydroxyl and ketone groups, as well as ring substituents, influence pK_a values and metal binding affinities. In general, pK_a (the negative logarithm of the acid dissociation constant) and metal ion affinity values (both stepwise and cumulative stability constants of the metal-chelator complex) follow the order 3,4-HP > 3,2-HP and 1,2-HP (

Table 1. Protonation constants, stepwise stability constants (log K) and cumulative Fe³⁺ stability constants (log β₃) for 1,2-HP, 3,2-HP and 3,4-HP.

Compound	pKa1	pKa2	log K1	log K2	log K3	log β3 Fe3+	Ref.
1,2-HP (2)	1.2	5.86	10.6	9.5	7.1	27.2	[22]
	−0.9	5.78	10.3	9.0	7.6	26.9	[23]
3,2-HP (3)	0.1	8.66	11.7	9.8	8.1	29.6	[23]
3,4-HP (4)	3.34	9.01	14.2	11.6	9.3	35.1	[23]
	3.60	9.60	–	–	–	36.9	[20]

). This order reflects the relative decrease in delocalisation of the N¹ atom lone pairs over the ring, and a corresponding decrease in charge density on the O atoms. The measurement of pM (negative logarithm of free metal concentration) is a more useful comparative measure for chelators than log K_a or affinity constants, as pM accounts for ligand basicity, denticity and protonation. Measurements are typically obtained at pH 7.4, with [total ligand] = 10⁻⁵ M and [total metal ion] = 10⁻⁶ M. Derivatives of high affinity 3,4-HPs have been extensively studied for iron-overload disease, and generally, 3,4-HPs have higher pM values than homologous 3,2- and 1,2-HPs. Additionally, 3,4-HPs are neutral at physiological pH, and when appropriately functionalised, can cross cell membranes.

3,4-HPs have high affinity for both Fe³⁺ and Ga³⁺, and form neutral 3:1 bidentate complexes with Fe³⁺ and Ga³⁺ (Figure 3,

Table 2. Protonation constants and Fe³⁺ and Ga³⁺ stability constants for deferiprone (1) (measured at 22.5–25 °C, ionic strength 0.1–0.2 M; for pM values, pH 7.4, with [total ligand] = 10⁻⁵ M and [total metal ion] = 10⁻⁶ M).

Metal Ion	pKa1	pKa2	log K1	log K2	log K3	log β3	pM3+	Ref.
Fe3+	3.56	9.64	14.92	12.23	9.79	37.2	–	[20]
	3.68	9.77	14.56	12.19	9.69	36.4	19.4	[34]
	3.62	9.76	15.14	11.54	9.24	35.92	18.3	[35]
	–	–	15.10	11.51	9.27	35.88	–	[25]
	3.61	9.78	15.03	27.42	–	37.35	20.74	[36]
Ga3+	–	–	13.17	12.26	10.33	35.76	–	[25]
	3.70	9.86	17.07	12.19	9.16	38.42	–	[37]

) (as well as Al³⁺, In³⁺, Cr³⁺, Mn³⁺ and Gd³⁺) [24,25,26,27,28,29,30,31]. Both the [Fe(deferiprone)₃] [31] and [Ga(deferiprone)₃] [29] complexes crystallise in the same octahedral geometries with comparable bond lengths and angles. In the three coordinating deferiprone ligands in both complexes, O³ atoms are fac to each other (see

Table 3. Protonation constants and Fe³⁺ stability constants for 3,4-HP derivatives.

Compound	R1	R2	R5	R6	pKa1	pKa2	log β3 (Fe3+)	pFe3+	Ref.
1	CH3	CH3	H	H	3.68	9.77	36.4	19.4	[34]
					3.61	9.78	37.35	20.74	[36]
4	H	H	H	H	3.34	9.01	35.1	–	[20,23]
5	H	CH3	H	H	3.70	9.76	37.2	–	[20]
					3.64	9.73	36.63	20.17	[36]
6	CH3	CH2OH	H	H	2.92	9.11	35.3	20.9	[34]
7	CH2CH3	CH2CH3	H	H	3.81	9.93	36.8	19.7	[34]
7	CH2CH3	CH2OH	H	H	2.80	9.27	35.3	21.0	[34]
9	CH2CH3	CH(OH)CH3	H	H	3.03	8.77	35.1	21.4	[34]
10	H	CON(CH3)2	H	CH3	2.53	8.20	33.2	20.4	[38]
11	H	CONHCH3	H	CH3	6.66	2.32	32.5	22.8	[38]
12	CH2CH3	CH3	H	H	3.65	9.88	37.7	–	[20]
13	CH3	CH3	CH3	H	3.37	10.32	37.93	19.71	[36]
14	H	CH3	CH3	H	3.43	10.27	37.28	19.22	[36]

for atom numbering scheme). Fe–O³ bond lengths are 1.998 Å, and Fe–O⁴ bond lengths, 2.038 Å while in the Ga analogue the corresponding Ga–O bond lengths are 1.967 Å, and 1.990 Å, respectively. In both complexes, both C–O bonds are intermediate between single and double bonds, with the C⁴–O⁴ bond shorter than the C³–O³ bond. Within each deferiprone ligand, bond angles are 80.91° for O³–Fe–O⁴, and 83.22° for O³–Ga–O⁴. Other metal ions (M = Al³⁺, In³⁺, Cr³⁺, Mn³⁺) form isostructural [M(deferiprone)₃] complexes [27,28,29,30], and in all cases, fac geometries are observed in the solid state. However, ¹H NMR studies on diamagnetic Ga³⁺ and Al³⁺ complexes suggest that conversion between fac and mer geometric isomers occurs in solution [30].

In living organisms, iron plays an essential role in transport of oxygen, electron transfer, and activation and functioning of enzymes. Iron transport and metabolism are tightly controlled in living organisms. In patients with iron overload disease, a disruption to iron homeostasis results in “free” iron, largely present as a citrate-albumin complex [32]. This iron pool gives rise to Fenton redox cycling between Fe²⁺ and Fe³⁺, producing free radicals that result in toxic tissue damage [33]:

Fe²⁺ + H₂O₂ → Fe³⁺ + HO· + OH⁻

(1)

Fe³⁺ + H₂O₂ → Fe²⁺ + HOO· + H⁺

(2)

Therapeutic chelators, including 3,4-HPs, are clinically used for sequestration of Fe³⁺ in vivo [7,8,9,10]. 3,4-HPs can be functionalised at the N¹, C², C⁵ or C⁶ positions of the ring (Table 3). An understanding of the effects of substitution is critical in optimizing 3,4-HP derivatives for either therapeutic Fe³⁺ chelation, or radiopharmaceutical Ga³⁺ chelation.

3. 3,4-Hydroxypyridinones for Fe³⁺ Complexation

3.1. Tailoring 3,4-HP Properties by Ring Substitution

Substitution at the C² position results in pronounced effects on metal ion affinity. Alkylation increases pyridinone ring electron density, and increases pK_a values and log K and log β_3(Fe3+) constants (log β₃ = 35.1 when R² = H (4); log β₃ = 37.2 when R² = CH₃ (5)) [20], although pFe³⁺ values are not markedly affected [26].

Substitution of C² alkyl groups for 1′-hydroxyalkyl groups decreases pK_a and log β_3(Fe3+) constants (compare 1 with 6; and 7, 8 and 9) [34]. This decrease in affinity is a result of: (i) a decrease in ring electron density due to a negative induction effect of the C² 1′-hydroxylalkyl group; and (ii) intramolecular hydrogen bonding between the C² 1′-hydroxyalkyl group and the deprotonated C³ hydroxyl group (Figure 4). This hydrogen bonding stabilises the ionised C³ hydroxyl group. However, although Fe³⁺ affinity is decreased, the concurrent lowering of pK_a2 actually results in an increase in pFe³⁺. The decrease in proton affinity favours the negative form that coordinates to Fe³⁺. In a side-by-side comparison at pH 7.4, pFe³⁺ values of the C² 1′-hydroxyalkyl derivatives are higher than C² alkyl derivatives [34].

Similar effects are also observed upon introduction of a C² amido group (Figure 4) [38]. A combination of a negative induction effect and hydrogen bonding from the C² amido NH to the C³ hydroxyl group decreases Fe³⁺ stability constants, but the concurrent decrease in pK_a serves to increase pFe³⁺ values at physiological pH relative to C² alkyl derivatives. The C² doubly alkylated N(CH₃)₂ amido group of 10 cannot form a hydrogen bond with the C³ hydroxyl group (Figure 4), and so its pFe³⁺ value is lower when compared to 11 bearing a singly alkylated NH(CH₃) that can form a hydrogen bond (Table 3) [38].

In contrast to C², varying alkyl substituents at the N¹ position (H, methyl, ethyl) of 3,4-HP does not markedly affect the proton or metal ion affinity of 3,4-HPs (compounds 1, 5, 12 in Table 3) [20]. The exception to this is the C² amido derivatives discussed above, where N¹ alkylation sterically inhibits coplanarity of the C² amido group with the HP ring, disrupting hydrogen bonding and thus decreasing pFe³⁺ values [38]. As such, N¹ alkyl substitution can be a useful strategy for tailoring chelators’ lipophilicity, cell permeability [20], in vivo biodistribution [34], and rates of metabolism [39] without deleterious effects on metal ion affinity. Substitution at N¹ sites has also been utilised to functionalise 3,4-HPs with fluorescent tags [40] or biological vectors [41].

Alkyl substitution at the C⁵ position increases 3,4-HP affinity for Fe³⁺, however as there is a concurrent increase in pK_a2, pFe³⁺ does not increase relative to derivatives that do not contain a C⁵ alkyl group (compounds 13 and 14) [36]. There are no studies directly comparing and quantifying the effect of C⁶ substitution, but existing data of C⁶ methylated derivatives suggest that alkylation has little influence on 3,4-HP metal affinity [36].

Increasing the lipophilicity of 3,4-HPs increases their cell membrane permeability. Higher intracellular 3,4-HP accumulation results in greater intracellular Fe³⁺ scavenging, however excessive cellular uptake of 3,4-HPs results in toxicity [20]. A prodrug strategy that involves oral administration of an N¹-substituted hydrophobic ester 3,4-HP (15, Figure 5) has proved particularly successful in scavenging iron in a preclinical iron overloaded rat model [34]. In iron overload disease, excess iron is stored in the liver, and the rat model mimics this. The ester compound 15 is absorbed effectively in the gastrointestinal tract, and delivered to the liver, where it enters target iron-overloaded hepatocyte cells. The compound is proposed to undergo ester hydrolysis and metabolism intracellularly (16, 17) prior to complexing Fe³⁺ [20]. The complex is then excreted, recovering excess iron from diseased animals efficiently. In contrast, when the more hydrophilic N¹ propyl alcohol derivative 16 (Figure 5) is directly administered to rats, absorption is less effective, and Fe³⁺ recovery is lower.

3.2. Deferiprone, Desferrioxamine and Deferasirox

Deferiprone (or Ferriprox) (1), developed by Hider and colleagues, has been clinically approved (in Europe in 1999, and the USA, in 2011) for treatment of iron overload diseases, including hemochromatosis and transfusion-dependant thalassemia [42]. The advantage of deferiprone is that it is both orally active and effective at sequestering Fe³⁺ from the blood stream, heart (where iron overload toxicity can be fatal) and liver (where excess iron is stored).

Two other approved treatments for iron overload are available. The first is the hexadentate hydroxamate chelator, desferrioxamine-B (DFO, 18, Figure 6), which forms a complex with Fe³⁺ with a metal to ligand stoichiometry of 1:1 and an overall charge of +1 under physiological conditions. DFO requires parenteral infusion over 8–12 h, several times a week, as it is not orally absorbed [42]. Clinical studies have demonstrated that either deferiprone alone, or a combination of deferiprone and DFO, are more effective therapies for myocardial iron overload than DFO alone. Additionally, a combination of oral deferiprone and parenteral DFO therapies is more viable for patients than parenteral DFO therapy alone, as combination therapy results in fewer parenteral infusions.

Like deferiprone, deferasirox [43] (19, Figure 6) is an effective orally active treatment for iron overload with clinical approval (in Europe in 2006 and the USA in 2005). It is a tridentate chelator, with Fe³⁺ complexes bearing a 3-charge at physiological pH. Data from clinical comparisons of the efficacy of deferiprone and deferasirox are conflicting and inconclusive, possibly due to variations in doses of the two treatments [44,45,46,47]. Some clinical data suggest that deferiprone is more effective at reducing iron levels in cardiac tissue [46,47].

3.3. Synthesis of 3,4-HPs

The structural diversity of 3,4-HPs is a result of extensive synthetic research that spans several decades. It is beyond the scope of this review to describe all of these synthetic routes in great detail, but it is worth highlighting common starting routes, and routes that give rise to N¹- and C²-substituted 3,4-HPs that are important precursors for new, bioactive compounds.

Pyranones such as maltol (20) containing a benzyl (Bn) protecting group can simply be converted to pyridinones by reaction with primary amines (Scheme 1) [20]. This allows diverse substitution at N¹, including incorporation of reactive groups such as carboxylates or primary amines that lead to further functionalization [20,40].

Compound 22 is a pyranone containing a Bn protecting group, and a reactive alcohol. It is a key synthetic precursor enabling versatile manipulation of functionality at the C² position of 3,4-HPs (Scheme 2). It is synthesised in high yields from the commercially available and inexpensive precursor, kojic acid (21) in four steps [48,49]. As discussed above, the Bn-protected 22 can be converted to Bn-protected pyridinone 23 by reaction with methylamine [49]. Protection of the ethyl alcohol group (and subsequent deprotection) increases yields in this reaction. A Mitsunobu reaction of 23 with phthalamide gives a Bn-protected pyridinone (24) that contains a phthalamide at the C² position, that can be simply converted to a primary amine (25) [49]. Bn-protected 25 is a very useful precursor for preparation of tris(hydroxypyridinones) such as 26 (see below).

Alternatively, 22 can be converted at the alcohol to a carboxylic acid in two steps to give pyranone 27 (Scheme 3) [48]. Compound 27 can then be coupled with 2-mercaptothiazoline using appropriate reagents to give pyranone 28 that contains an active amide. Compound 28 can be reacted with primary or secondary amines, resulting in pyranone amide derivatives that can be converted to Bn-protected pyridinones by reaction with methylamine or ammonia [38,48]. For example, Bn-protected precursors to compounds 10 and 11 are prepared in this fashion.

Reactive carboxylates are synthetically accessible from Bn-protected pyridinones (Scheme 4) [50]. Bn-protected 29 can be further protected, and the methyl group substituted to ultimately yield 30. Compound 30 can be converted to a carboxylate, yielding 31. The carboxylate group of 31 can be further activated with an N-hydroxysuccinimide if required (32). Such derivatives have been used to prepare tris(hydroxypyridinones) such as 33 and 34 as well as other amide derivatives (35) [50].

Deprotection of Bn-protected hydroxyl groups proceeds via either hydrogenation reactions (catalysed by palladium on carbon) followed by acidification, or treatment with dissolved boron trichloride in an aprotic solvent, followed by addition of an alcohol (Scheme 1, Scheme 2 and Scheme 4).

4. Hexadentate Tris(hydroxypyridinone) Ligands

4.1. Topology and Fe³⁺ Affinity

Incorporation of three bidentate 3,4-HP ligands into a tripodal construct provides hexadentate tris(hydroxypyridinone) ligands (THPs) that, in a suitably designed scaffold, saturate the coordination sphere of octahedral metal ions. THPs have been designed for applications in gastrointestinal scavenging of Fe³⁺ [51], antimicrobial activity (via deprivation of microbes’ Fe³⁺ pool) [52,53], and fluorescence imaging of cellular Fe³⁺ distribution [54]. The topology of tripodal THPs is critically important to formation of octahedral complexes with 1:1 stoichiometry. The backbone of the tripod should be connected ortho to a coordinating O atom [55].

The synthesis of the first generation of THP ligands utilised 3,4-HP groups with a C² carboxylate substitution (32), allowing reaction with tripodal polyamines to yield compounds such as 33 and 34 (Scheme 4). In 33 and 34, 3,4-HP units are attached via a C² amide group, and fulfil the topology requirements outlined above for formation of hexadentate compounds with 1:1 stoichiometry. For compound 34, log K₁ = 30.7, whereas for the bidentate homologue 35, log β₃ = 31.4 [50]. In this case, the affinity of the bidentate 3,4-HP for Fe³⁺ is already optimal, and incorporation into a hexadentate form does not result in an increase in thermodynamic stability, as might normally be expected for an increase in ligand denticity and accompanying lower entropic costs. The pFe³⁺ of 34 (30.5 at pH 7.4) is higher than the pFe³⁺ of 35 (22.0) [50]. This arises because the formation constant of a hexadentate complex of 34 has only a first order dependence on ligand concentration (1:1 ligand to metal stoichiometry), whereas that of 35 necessarily has third order dependence on free ligand concentration (3:1 ligand to metal stoichiometry). In solutions of 34 where the hexadentate ligand concentration = 10 µM, the total concentration of single 3,4-HP units is three times greater than in solutions of 35 where the bidentate ligand concentration = 10 µM.

Instead of polyamine tripods, the next generation of THP ligands derivatised tripodal carboxylate groups, using 3,4-HP units with a C² aminomethyl substituent (25, Scheme 2) [51]. In the resulting compounds, for example THP-Ac (26, Scheme 2), each 3,4-HP group is tethered to the tripod via an amidomethyl linker at the C² position. The N¹ and C⁶ positions are methylated. With the exception of C⁶-methylation, THP-Ac’s single 3,4-HP unit is structurally similar to deferiprone. For THP-Ac, log K₁ = 32.52 and pFe³⁺ = 28.47 [51].

The incorporation of three 3,4-HP groups into a tripodal ligand also has implications for the lability of hexadentate complexes. For example, in [Fe(THP-Ac)], dissociation of a single 3,4-HP unit is likely to be followed by its rapid recoordination to the metal centre. During dissociation, this 3,4-HP unit will remain spatially close to the metal centre, as the other two 3,4-HP groups, to which it is covalently tethered, are likely to still be bound to Fe³⁺. On the other hand, in [Fe(deferiprone)₃], if a deferiprone ligand dissociates, it has a lower probability of recoordinating to the same metal centre, as it is not anchored to any other coordinating ligands. The activation energy barrier to dissociation of [Fe(THP-Ac)] is higher than that of [Fe(deferiprone)₃], and [Fe(THP-Ac)] is more kinetically inert than [Fe(deferiprone)₃].

4.2. Dendrimers Based on THP Units

Dendritic THP molecules, for example 36, have incorporated between three and six THP groups, allowing coordination of between three and six equivalents of coordinatively saturated Fe³⁺ per molecule [51,56]. Dendrimers have been prepared from both 25 and 32. Affinity constants as well as pFe³⁺ values of dendrimer 36 (Figure 7) (log K₁ = 32.74, pFe³⁺ = 28.69) and other derivatives do not meaningfully deviate from values for the single THP-Ac homologue 26, indicating that maximum Fe³⁺ binding efficiency is achieved using 3,4-HP motifs in a tripodal THP topology.

4.3. Derivatising THP Ligands

THP compounds of the same topology and substitution as THP-Ac (26) are synthetically accessible from the β-alanine derivative, THP-NH₂ (37, Figure 8) using similar reaction routes to that described in Scheme 2 [51]. THP derivatives such as 33 or 34 are less amenable to derivatisation in this fashion. The presence of an apical primary amine in THP-NH₂ allows attachment to dendritic scaffolds [51], biomolecules [13,14,15,16], polymer units [57], and fluorophores [54]. This ability to functionalise THP compounds makes them attractive for technological applications where trivalent metal ion complexes of high affinity and kinetic stability are required. We have explored THP derivatives for PET imaging with Ga³⁺.

5. Tris(hydroxypyridinone) Ligands for Radiolabelling with ⁶⁸Ga³⁺

5.1. Radiolabelling Peptides with ⁶⁸Ga for PET Imaging: The Case for Tris(hydroxypyridinone) Derivatives

Peptide-based molecular imaging agents can rapidly accumulate at target tissue and clear from circulation within 1–2 h. The 68 min half-life and positron emission properties of ⁶⁸Ga (β⁺ 90%, E_max = 1880 KeV) match these requirements for PET imaging of peptide receptor expression. Moreover, ⁶⁸Ga is conveniently available by elution of the ⁶⁸Ge/⁶⁸Ga generator to produce no-carrier-added solutions of ⁶⁸Ga³⁺ in hydrochloric acid [58].

Hydrated Ga³⁺ species such as [Ga(H₂O)₆]³⁺ exist in aqueous solution below pH 4. As the pH is raised above 4, the poorly soluble hydroxide species Ga(OH)₃ predominates in solution, until pH > 6.3, where tetradentate [Ga(OH)₄]⁻ predominates [59,60]. For efficient ⁶⁸Ga³⁺ radiolabelling of chelate-peptide conjugates at neutral or near neutral pH, chelate complex formation must effectively compete with unreactive ⁶⁸Ga-colloid formation. Preferably, the rate of chelation will be diffusion-controlled, so that complex formation outcompetes ⁶⁸Ga³⁺ colloid formation. Additionally, the amounts of ⁶⁸Ga eluted from clinical generators are in the range of 200–2000 MBq, approximately equivalent to 2–20 pmol of ⁶⁸Ga³⁺ in 1–5 mL of solution. Highly efficient chelators are required to quantitatively complex such low concentrations of metal ion without using excessively high chelator concentration. In clinical formulations of chelator bioconjugates, low concentrations of chelator-peptide conjugate are important. Clinical formulations do not usually separate unlabelled bioconjugate from labelled conjugate, and large amounts of unlabelled conjugate can lead to saturation of target receptors in vivo.

As THP ligands have extraordinarily high pFe³⁺ values, and Ga³⁺ has similar coordination preferences to Fe³⁺, it was reasoned that such chelators could be very efficient at quantitatively coordinating ⁶⁸Ga³⁺ at low chelator concentrations [13]. The acyclic nature of THP, and hence flexibility compared to macrocyclic ligands, results in low activation barriers to complexation, resulting in rapid rates of reaction at room temperature.

It is also critical that the ⁶⁸Ga³⁺ complex is sufficiently kinetically stable over the period of time required for imaging (1–2 h) to withstand transchelation by competing endogenous proteins, such as transferrin, and other ligands that compete for Ga³⁺ in vivo [4]. The iron transport protein transferrin is abundant in serum and its two metal binding sites have high affinity for Fe³⁺ (log β₁ = 22.8, log β₂ = 44.3) and Ga³⁺ (log β₁ = 20.3, log β₂ = 39.6) [61]. Transchelation of ⁶⁸Ga³⁺ to endogenous ligands results in increased non-target tissue uptake and lower tumour/diseased tissue uptake, decreasing PET image quality.

As 3,4-HP and THP derivatives can bind Fe³⁺ under physiological conditions, with the resulting complexes excreted, it was hypothesised that a THP Ga³⁺ complex could be sufficiently stable in vivo. Mouse biodistribution studies with the γ-emitting isotope, ⁶⁷Ga³⁺ (half-life = 78 h), comparing the biodistribution of [⁶⁷Ga(deferiprone)₃] with [⁶⁷Ga(citrate)₃] show that mice administered the deferiprone complex intravenously have lower ⁶⁷Ga blood activity one day post-administration than animals administered the citrate complex [37]. Subsequent studies demonstrate that ⁶⁷Ga: (i) clears more rapidly from animals administered the deferiprone complex compared to animals administered the citrate complex; and (ii) clears predominantly via a renal pathway in animals administered the deferiprone complex [37,62]. N¹-functionalised derivatives of 3,4-HP are also efficacious at sequestering ⁶⁷Ga³⁺ in vivo, with biodistribution of radioactivity modified by N¹ substituents [62]. Such results suggest that [⁶⁷Ga(deferiprone)₃] has appreciable stability in vivo, and that 3,4-HPs can effectively compete with endogenous protein ligands for Ga³⁺ [37,62].

5.2. Tris(hydroxypyridinone) Bioconjugates

We first reported the utility of THP-Ac (26, Figure 8) as a basis for highly efficient ⁶⁸Ga labelling under very mild conditions after undertaking side-by-side comparisons of THP-Ac with chelators already commonly used to complex ⁶⁸Ga³⁺: macrocyclic derivatives DOTA (41) (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and NOTA (42) (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the acyclic chelator HBED (43) (bis(2-hydroxybenzyl)ethylenediaminediacetic acid) (Figure 9) [13]. Each chelator was reacted with generator-produced ⁶⁸Ga³⁺ at progressively lower chelator concentrations (100 nM–1 mM, each 100 µL corresponding to amounts of 10 pmol–100 nmol) (Figure 10), with the reasoning that the most efficient, rapidly complexing chelators would maintain high labelling efficiency at the lowest ligand concentrations. Optimised reaction conditions (as reported in the radiochemical literature) for each chelator were employed. At a concentration of 10 µM, THP-Ac complexes ⁶⁸Ga³⁺ in 5 min in >98% radiochemical yield at pH 6.5 at room temperature. Radiochemical yields for DOTA are >95% at the same chelator concentration, but this requires heating at 100 °C at pH 4.4. NOTA has been reported to coordinate ⁶⁸Ga³⁺ efficiently at room temperature, but at 10 µM concentration at room temperature, pH 4.4, radiochemical yields only average 80%. At 10 µM, HBED is able to complex ⁶⁸Ga³⁺ in >96% radiochemical yield at pH 4.6. Whilst HBED radiochemical yields under acidic conditions are comparable to those of THP-Ac at near neutral pH, HBED forms geometric isomers when complexed to Ga³⁺ [4]. From a regulatory perspective, the presence of different geometric isomers is undesirable, as it is possible that the different isomers have different pharmacological profiles.

The β-alanine-derived compound, THP-NH₂ (37) [51], can be functionalised for bioconjugation via a maleimide [13,16] and isothiocyanates [14,15] (Figure 8). The bifunctional chelators THP-mal (38), THP-Ph-NCS (39) and THP-NCS (40) have all been conjugated to biomolecules. Amide conjugation of THP-NH₂ (37) to activated carboxylates of peptides and proteins is also a viable conjugation strategy.

The protein C2A, containing an engineered cysteine residue, has been conjugated to THP-mal (38), resulting in a single equivalent of THP-mal attached per protein molecule [13]. The conjugate can be radiolabelled with ⁶⁸Ga³⁺ at pH 5.5 (6 nmol of protein in 100 µL, at a concentration of 60 µM), giving quantitative labelling after 5 min. In vivo PET imaging in healthy mice demonstrates that ⁶⁸Ga-THP-mal-C2A clears to the kidneys, and does not release any ⁶⁸Ga³⁺ over a 90 min period. In contrast, in mice administered solutions of unchelated ⁶⁸Ga³⁺, radioactivity is distributed throughout the body 90 min post-injection. In competition studies where ⁶⁸Ga-THP-Ac is incubated with transferrin, [⁶⁸Ga(THP-Ac)] remains intact. On the other hand, when ⁶⁸Ga-transferrin is incubated with THP-Ac, ⁶⁸Ga is quickly transchelated to form a complex with THP-Ac [13].

THP-Ph-NCS and THP-NCS have both been attached to the cyclic pentapeptide, c(RGDfK) via lysine sidechains [14]. The “RGD” peptide motif targets α_vβ₃ integrin receptors that are expressed on the surface of many metastatic tumour cells, as well as inflamed tissue and blood vessels undergoing angiogenesis. Both THP-Ph-NCS-RGD (41, Figure 11) and THP-NCS-RGD (42, Figure 11) can be radiolabelled with generator-produced ⁶⁸Ga³⁺ eluate at conjugate concentrations of 4–5 µM, or total amounts of 10–12 nmol, giving radiochemical yields of 95%–99%. These reactions proceed in aqueous solution, pH 5.5–6.5 in less than 5 min at ambient temperature to give a single product corresponding to either [⁶⁸Ga(THP-Ph-NCS-RGD)] or [⁶⁸Ga(THP-NCS-RGD)].

Both [⁶⁸Ga(THP-Ph-NCS-RGD)] and [⁶⁸Ga(THP-NCS-RGD)] retain affinity for α_vβ₃ integrin receptors in vitro and in vivo [14]. PET imaging and biodistribution studies of mice bearing U87MG tumours demonstrate that both [⁶⁸Ga(THP-Ph-NCS-RGD)] and [⁶⁸Ga(THP-NCS-RGD)]: (i) selectively target α_vβ₃ integrin receptors; and (ii) clear from the body within 1–2 h post-injection, predominantly via a renal route (Figure 11).

The molecular imaging agent, [⁶⁸Ga(DOTA-TATE)] is routinely used for PET imaging of neuroendocrine tumours. DOTA-TATE (43, Figure 12) is a conjugate of the macrocyclic chelator DOTA (27, Figure 9) and Tyr³-octreotate, an eight amino acid cyclic peptide that targets somatostatin 2 receptors (SSTR) overexpressed on the surface of neuroendocrine tumours. Using THP-NCS, THP-TATE (44, Figure 12) has been synthesised [15]. Similar to RGD conjugates, THP-TATE can be radiolabelled at room temperature at concentrations of 5 µM (10 nmol of conjugate) at near neutral pH in less than 2 min. In contrast, DOTA-TATE requires temperatures of 80–90 °C at pH 3–5, with reaction times of 5–10 min, and in clinical radiolabelling protocols, post-synthetic purifications procedures are invariably employed [15]. PET imaging and biodistribution experiments show that [⁶⁸Ga(THP-TATE)] has similar uptake in SSTR-positive tumours to the clinical standard [⁶⁸Ga(DOTA-TATE)]. [⁶⁸Ga(THP-TATE)] clears via a renal pathway, but significantly higher kidney and liver retention of [⁶⁸Ga(THP-TATE)] is observed (Figure 12). In PET images of mice bearing SSTR2-positive tumours administered [⁶⁸Ga(THP-TATE)], tumours can be clearly delineated.

In all of these radiolabelled derivatives, the radiotracer demonstrates high serum stability and in vivo stability, with no evidence of dissociation of ⁶⁸Ga³⁺ from the THP chelator.

5.3. Preparation, Radiolabelling and In Vitro Uptake of a Trastuzumab Immunoconjugate

New protein constructs that target receptors with high affinity and specificity have similar utility to peptides in molecular imaging. For proteins with short circulation times and rapid accumulation at target tissue, ⁶⁸Ga PET imaging will be clinically viable, provided that appropriate radiolabelling protocols are available. The sensitivity of proteins’ tertiary structures to acidic pH and extremes of temperature requires mild radiolabelling conditions, raising problems when using conventional Ga³⁺ chelators. HBED, NOTA and DFO are capable of radiolabelling Ga³⁺ isotopes at room temperature, but HBED [63] and NOTA [64] require low pH for reactions to proceed quantitatively, and DFO complexes of Ga³⁺ are unstable [65].

Trastuzumab is a therapeutic monoclonal antibody used for treatment of breast cancer. It targets the human epidermal growth factor receptor 2 (HER2). In vivo, antibodies such as trastuzumab require extended periods of time (6 to 48 h) to clear circulation and accumulate at HER2-positive target tissue, and given the short half-life of ⁶⁸Ga, it is likely impractical to use ⁶⁸Ga-labelled trastuzumab to image HER2 expression. Nonetheless, it is instructive to radiolabel THP-PhNCS-trastuzumab to assess whether a THP protein conjugate that is sensitive to acidic pH (less than pH 5) can be radiolabelled rapidly under mild conditions (pH 6–7) to provide a formulation suitable for injection without further purification.

The bifunctional chelator THP-PhNCS has been conjugated to the monoclonal antibody (mAb), trastuzumab by incubating a HEPES buffered solution containing both reagents under mild conditions [66,67]. The immunoconjugate, THP-PhNCS-trastuzumab has been isolated using solid phase size exclusion chromatography [66,67]. Addition of generator-produced ⁶⁸Ga³⁺ (~10 MBq, 50 µL, aqueous 0.1 M HCl) to solutions of THP-PhNCS-trastuzumab (50 µL, 0.65 mg·mL⁻¹, 0.2 M ammonium acetate, pH 6–7) results in formation of [⁶⁸Ga(THP-PhNCS-trastuzumab)], with specific activities of up to 50 MBq·nmol⁻¹ and radiochemical yields of 99% as measured by size exclusion HPLC (Figure 13a, red trace). The major signal at 7.98 min in the radiochromatogram matches the retention time of native trastuzumab, and the minor signal at 6.83 min is typical of formation of radiolabelled immunoconjugate aggregates [66]. Addition of ⁶⁸Ga³⁺ solutions to samples containing native, unconjugated trastuzumab do not provide labelled antibody (Figure 13a, black trace)—the mobile phase in these experiments contains ethylenediaminetetraacetate (EDTA), and the single signal in the radiochromatogram with a retention time of 12.08 min corresponds to [⁶⁸Ga(EDTA)]⁻.

To establish that the labelled immunoconjugate, [⁶⁸Ga(THP-PhNCS-trastuzumab)], retains affinity for HER2 receptors, [⁶⁸Ga(THP-PhNCS-trastuzumab)] has been incubated with HER2-positive HCC1954 cells and HER2-negative A375 cells. Additionally, a blockade experiment where [⁶⁸Ga(THP-PhNCS-trastuzumab)] is incubated with HCC1954 cells in the presence of a large excess of native trastuzumab has been undertaken. Uptake of [⁶⁸Ga(THP-PhNCS-trastuzumab)] in HCC1954 cells measures 35.12 ± 0.53 percentage of added radioactivity per one million cells (%AR/million cells), whereas uptake in A375 cells measures 1.98% ± 0.02 %AR/million cells and uptake in the trastuzumab blockade measures 0.30% ± 0.03 %AR/million, indicating that uptake of [⁶⁸Ga(THP-PhNCS-trastuzumab)] is receptor-mediated, and that in vitro [⁶⁸Ga(THP-PhNCS-trastuzumab)] retains affinity for HER2-expressing cells (Figure 13b). Addition of a solution containing ⁶⁸Ga³⁺ to HCC1954 cells does not result in significant uptake of activity (1.09% ± 0.05 %AR/million cells).

Thus, THP enables efficient and rapid ⁶⁸Ga³⁺ radiolabelling of proteins under mild, aqueous conditions. This radiolabelling strategy will have utility for ⁶⁸Ga PET imaging of proteins such as fusion proteins and antibody fragments that have shorter clearance times than full length antibodies.

5.4. Other THP Derivatives

Alternative THP chelators such as NTP(PrHP)₃ (45) have been reported for complexation of the SPECT isotope, ⁶⁷Ga [68,69]. In these derivatives (Figure 14), the 3,4-HP groups are attached to the tripodal scaffold via the N¹ ring atoms, and the chelating O atoms are meta and para to the linker. Such a topology can lead to formation of either dinuclear structures or structures where one 3,4-HP unit has dissociated [55]. Both species are generally more kinetically labile than species of a 1:1 stoichiometry and are not ideal for in vivo applications. The presence of more than one complex structure is also undesirable, as these species can have different biological behaviours. NTP(PrHP)₃, with its tripodal topology centred on a tertiary amine rather than carbon, is not readily adapted to use as a bifunctional chelator. SPECT imaging and biodistribution studies with ⁶⁷Ga³⁺ indicate that most [⁶⁷Ga-NTP(PrHP)₃] rapidly clears circulation within 1 h, and remaining [⁶⁷Ga-NTP(PrHP)₃] does not release ⁶⁷Ga³⁺ over 24 h [69].

6. Hydroxypyridinones for Radiolabelling ⁸⁹Zr⁴⁺

We have also investigated THP-Ac and a THP-mal-trastuzumab conjugate for radiolabelling with the long-lived PET isotope, zirconium-89 (⁸⁹Zr⁴⁺) (half-life = 78 h) [16]. The Zr⁴⁺ ion is oxophilic, and bifunctional hexadentate DFO derivatives are most commonly utilised for incorporating ⁸⁹Zr⁴⁺ into antibodies. There is evidence that [⁸⁹Zr(DFO)]⁺ derivatives are unstable in vivo, with observations of ⁸⁹Zr accumulation in the skeleton over the course of a week [16]. Here, skeletal uptake is assumed to be indicative of dissociation of ⁸⁹Zr⁴⁺ from a chelator-conjugate.

Both THP-Ac and THP-mal-trastuzumab can be radiolabelled with ⁸⁹Zr⁴⁺ at room temperature and near neutral pH in quantitative yield. For THP-Ac, this was achieved at 1 mM concentration and 10 µL volume, corresponding to 10 nmol of ligand. Competition experiments indicated a thermodynamic preference for Zr⁴⁺ coordination to THP-Ac over DFO. This is consistent with higher metal ion affinities of 3,4-HPs compared to hydroxamates. However, experiments in which ⁸⁹Zr-THP-mal-trastuzumab was administered to healthy animals indicate that in vivo, ⁸⁹Zr⁴⁺ dissociates from THP-mal-trastuzumab within 48 h, with activity accumulating in the skeleton (Figure 15). In vivo stability of the [⁸⁹Zr(THP)]⁺ complex is inferior to that of the [⁸⁹Zr(DFO)]⁺ complex, possibly a result of the different chelator topologies.

Zr⁴⁺ can accommodate up to eight donor atoms in its coordination sphere, and these coordination requirements are likely to be a factor in the instability of [⁸⁹Zr(THP)]⁺ in vivo. The presence of two coordination sites unoccupied by THP chelator allows coordination of endogenous ligands, and provides pathways to transmetallation/ligand exchange.

It is possible that a tetrakis(3,4-hydroxypyridinone) ligand could impart greater in vivo stability, but there are no reports of such ligands to date. Other researchers have studied octadentate tetrakis(hydroxypyridinone) derivatives for ⁸⁹Zr⁴⁺ incorporating 1,2-HPs and 3,2-HPs. The bifunctional open chain (1,2-HP)₄ chelator (46, Figure 16) can: (i) coordinate Zr⁴⁺ in an octadentate environment; and (ii) be conjugated to trastuzumab. Immunoconjugate (1,2-HP)₄-trastuzumab can be radiolabelled with ⁸⁹Zr⁴⁺ at room temperature, and PET imaging and biodistribution studies demonstrate high in vivo stability of the complex, with concomitant high tumour uptake [70].

Bifunctional macrobicyclic octadentate (3,2-HP)₄ (47, Figure 16) has also been derivatised for conjugation to trastuzumab [71]. The coordination environment of the Zr⁴⁺ complex is not well defined, and chromatographic analysis indicates that multiple Zr-bound (3,2-HP)₄ species form under the mild reaction conditions described (room temperature, 15 min incubation). PET imaging and biodistribution studies in mice using ⁸⁹Zr-labelled (3,2-HP)₄-antibody conjugates suggest that at least one form of ⁸⁹Zr⁴⁺-(3,2-HP)₄ is unstable in vivo, as significantly higher bone uptake is observed for (3,2-HP)₄ conjugates compared to DFO conjugates. This highlights the difficulty in interpreting in vivo results based on radiolabelled compounds that contain metal complexes in more than one conformation.

7. Concluding Remarks

Derivatisation of 3,4-HPs via substitution of ring protons allows for tailoring of chelator properties including the proton and Fe³⁺ affinities, and in vivo distribution and reactivity towards endogenous enzymes. 3,4-HPs can be further functionalised with fluorescent tags and biologically active motifs, and can be incorporated into chelators of higher denticity. Hexadentate THP chelators based on 3,4-HPs possess extraordinarily high pFe³⁺ values, and are very potent Fe³⁺ scavengers. As Fe³⁺ and Ga³⁺ have similar coordination preferences, THP chelators are ideal candidates for development of PET radiopharmaceuticals based on ⁶⁸Ga³⁺, and direct translation of this chemistry has enabled rapid development of bioconjugates of THP chelators for ⁶⁸Ga PET imaging.

Simplicity of radiolabelling with minimal need for complex equipment and radiochemical expertise is likely to be a key to the wider availability of ⁶⁸Ga PET, and this is afforded by appropriate design of a ⁶⁸Ga chelator. THP derivatives fulfil these requirements whereas other established chelator designs do not. Current clinical radiosynthetic protocols based on DOTA and HBED derivatives require heating (>80 °C), low pH (3–5) and post-synthetic purification/formulation. In contrast, THP compounds can be radiolabelled and formulated by treatment with generator-produced ⁶⁸Ga³⁺ in over 95% radiochemical yield under ambient conditions in less than 5 min, at low chelator concentrations, neutral pH and in aqueous solution. There is no requirement for post-synthetic purification or reformulation, as reactions are quantitative and components (solvents and buffers) are physiologically compatible. Bifunctional THP derivatives enable a means of attachment to peptides and proteins, and biological studies have demonstrated that the peptide radiotracers are stable in vivo with respect to dissociation of the ⁶⁸Ga-THP complex, retain affinity for target receptors and clear rapidly from circulation. All of these characteristics make THP superlative for one-step kit-based radiosynthesis.