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

International Journal of

Molecular Sciences Review

Hydroxypyridinone Chelators: From Iron Scavenging to Radiopharmaceuticals for PET Imaging with Gallium-68 Ruslan Cusnir 1,† , Cinzia Imberti 1,† , Robert C. Hider 2 , Philip J. Blower 1 and Michelle T. Ma 1, * 1

2

* †

Division of Imaging Sciences and Biomedical Engineering, King’s College London, Fourth Floor Lambeth Wing, St. Thomas’ Hospital, London SE1 7EH, UK; [email protected] (R.C.); [email protected] (C.I.); [email protected] (P.J.B.) Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK; [email protected] Correspondence: [email protected] These authors contributed equally to this work.

Academic Editors: Jamal Zweit and Sundaresan Gobalakrishnan Received: 2 November 2016; Accepted: 21 December 2016; Published: 8 January 2017

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, 68 Ga3+ , which is clinically used for molecular imaging in positron emission tomography (PET). THP-peptide bioconjugates rapidly and quantitatively complex 68 Ga3+ at ambient temperature, neutral pH and micromolar concentrations of ligand, making them amenable to kit-based radiosynthesis of 68 Ga PET radiopharmaceuticals. 68 Ga-labelled THP-peptides accumulate at target tissue in vivo, and are excreted largely via a renal pathway, providing high quality PET images. Keywords: hydroxypyridinone; deferiprone; gallium; iron overload; positron emission tomography; molecular imaging; bifunctional chelators

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 (68 Ga) from decay of 68 Ge in a bench top 68 Ge/68 Ga generator is likely to have significant clinical impact on the use of PET for molecular imaging [1]. Clinical use of 68 Ga receptor-targeting radiopharmaceuticals for neuroendocrine cancers (68 Ga-DOTA-TATE) [2] and prostate cancers (68 Ga-HBED-PSMA) [3] has changed patient management in centres that have routine access to such agents. 68 Ga 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 68 Ga3+ complexation [1,4–7]. Clinical radiosyntheses of 68 Ga-DOTA-TATE and 68 Ga-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 68 Ga and reaction components that are not physiologically compatible. In the case of Int. J. Mol. Sci. 2017, 18, 116; doi:10.3390/ijms18010116

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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 3+ For 68 Ga-HBED-PSMA, three 68 68Ga-HBED-PSMA, Ga-DOTA-TATE, three heating is required for isomers complexation of 68 Ga geometric cis/trans are possible, with. each possible geometric isomer geometric cis/trans isomers are possible, with each possible geometric isomer having a diastereomer. having a diastereomer. In this case, heating the reaction favours formation of the thermodynamically In this case, heating the reaction favours formation of the thermodynamically preferred species. preferred species. For 6868Ga adopted in routine clinicalclinical practice,practice, chelatorschelators that provide andefficient reproducible Gatotobe be adopted in routine thatefficient provide and kit-based radiolabelling methods—preferably a single manipulation—are required. The chelators that reproducible kit-based radiolabelling methods—preferably a single manipulation—are required. 68 Ga3+ radiolabelling at we developed based on hydroxypyridinones one-step quantitative Thehave chelators that we have developed based onallow hydroxypyridinones allow one-step quantitative 68Ga3+ radiolabelling neutral pH and ambient temperature. at neutral pH and ambient temperature. Hydroxypyridinone (HP) ligands have high affinities for “hard” metal ions, and originally, the 3+ class of HPs described herein herein was was developed developed extensively extensivelyfor fortherapeutic therapeuticin invivo vivoFe Fe3+ chelation in iron overload disease [8–10]. The bidentate HP ligand, deferiprone (1, Figure 1), is used routinely for this treatment [11]. chemical modification of substituents, Fe3+ affinity, metabolic [11].Through Through chemical modification of substituents, Fe3+ hydrophilicity, affinity, hydrophilicity, stability and functionality of HPs can be tailored [10]. metabolic stability and functionality of HPs can be tailored [10]. 3+ is an oxophilic metal ion, with a high charge density and ionic radius and coordination Ga3+ is an oxophilic metal ion, with a 3+ is 76 pm, and for high spin3+ Fe3+ is 3+ is preferences similar to to Fe Fe3+3+. .The The(crystal) (crystal) ionic radius ionic radius forfor GaGa 76 pm, and for high spin Fe is 78.5 78.5 pm [12]. Recently, we have investigated the use of HPs for rapid and quantitative pm [12]. Recently, we have investigated the use of HPs for rapid and quantitative chelationchelation of 68Ga3+ 68 3+ 89 4+ 89 4+ of Ga as (as well as aPET long-lived Zr )a and a enable class offunctionalisation HPs to enable (as well a long-lived isotope,PET Zr isotope, ) and adapted classadapted of HPs to functionalisation peptides and proteins for targeted molecular with peptides andwith proteins for targeted molecular imaging [13–16]. imaging [13–16]. 3+ Here we on on the the development of HPs and how design wedescribe describeour ourresearch research development of for HPsFefor chelation, Fe3+ chelation, andthe how the 68 of such of chelators is suitable for directfor translation to Ga PET It is not intended design such chelators is suitable direct translation to 68radiopharmaceuticals. Ga PET radiopharmaceuticals. It is not as an exhaustive review of chelators for chelators either treatment of iron overload or complexation of intended as an exhaustive review of for either treatment ofdisease iron overload disease or radioisotopes used in The literature in both areas has been surveyed in complexation of gallium radioisotopes ofnuclear galliummedicine. used in nuclear medicine. The literature in both areas has depth in recent in years in several excellent booksreviews [4–7,10,17–19]. been surveyed depth in recent years inreviews several and excellent and books [4–7,10,17–19].

Figure protonation states states [20]. [20]. Figure 1. 1. Deferiprone Deferiprone (1): (1): Resonance Resonance structures structures at at different different protonation

2. Hydroxypyridinones 2. Hydroxypyridinones HPs consistof of a six-membered aromatic N-heterocycle, with a and hydroxyl and a ketone HPs consist a six-membered aromatic N-heterocycle, with a hydroxyl a ketone functionality. functionality. Varying relative positions of the hydroxyl and ketone functional groups within a Varying relative positions of the hydroxyl and ketone functional groups within a bidentate HP molecule bidentate HP molecule results in three types of hydroxypyridinones: 1,2-hydroxypyridinone results in three types of hydroxypyridinones: 1,2-hydroxypyridinone (2); 3,2-hydroxypyridinone (2); (3); 3,2-hydroxypyridinone (3);(4)and 3,4-hydroxypyridinone (Figure and 2). deprotonated, Neutral HPs with can the be and 3,4-hydroxypyridinone (Figure 2). Neutral HPs can be(4) protonated protonated and deprotonated, with the first pK a typically corresponding to first pKa typically corresponding to protonation/deprotonation at the oxo group, and the second, at the protonation/deprotonation at theofoxo andofthe the hydroxyl group. Delocalisation hydroxyl group. Delocalisation thegroup, electrons thesecond, N1 ringatatom leads to aromaticity (Figures 1 1 ring atom leads to aromaticity (Figures 1 and 2). of the electrons of the N and 2). When HPs are are deprotonated deprotonated at the hydroxyl hydroxyl group, group, they they are are capable capable of of complexing complexing metal metal ions ions When HPs at the in aa bidentate bidentate O O2 mode, forming five-membered chelate rings. The relative positions of the hydroxyl in 2 mode, forming five-membered chelate rings. The relative positions of the hydroxyl and ketone groups, as well a values and metal binding affinities. In and ketone groups, as wellas asring ringsubstituents, substituents,influence influencepK pK a values and metal binding affinities. general, pK a (the negative logarithm of the acid dissociation constant) and metal ion affinity values In general, pKa (the negative logarithm of the acid dissociation constant) and metal ion affinity values (both stepwise stepwise and and cumulative cumulative stability stability constants constants of the metal-chelator metal-chelator complex) complex) follow order (both of the follow the the order 3,4-HP > 3,2-HP and 1,2-HP (Table 1). This order reflects the relative decrease in delocalisation of the 3,4-HP > 3,2-HP and 1,2-HP (Table 1). This order reflects the relative decrease in delocalisation of the N11 atom lone pairs over the ring, and a corresponding decrease in charge density on the O atoms. N atom lone pairs over the ring, and a corresponding decrease in charge density on the O atoms.

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The measurement measurementof of (negative logarithm freeconcentration) metal concentration) is a more useful The pMpM (negative logarithm of freeof metal is a more useful comparative comparative measure than for chelators than logconstants, Ka or affinity as ligand pM accounts ligand measure for chelators log Ka or affinity as pMconstants, accounts for basicity,for denticity − 5 basicity, denticity and protonation. Measurements are typically obtained at pH 7.4, with and protonation. Measurements are typically obtained at pH 7.4, with [total ligand] = 10 M[total and −5 M and−[total −6 ligand] = 10ion] metal ion] of = 10 Derivatives highbeen affinity 3,4-HPs studied have been [total metal = 10 6 M. Derivatives highM. affinity 3,4-HPsofhave extensively for extensively studied for and iron-overload 3,4-HPs pM values iron-overload disease, generally, disease, 3,4-HPs and havegenerally, higher pM valueshave thanhigher homologous 3,2- than and homologous 3,2- and 3,4-HPs 1,2-HPs.areAdditionally, 3,4-HPs are neutral at physiological and when 1,2-HPs. Additionally, neutral at physiological pH, and when appropriatelypH, functionalised, appropriately functionalised, can cross cell membranes. can cross cell membranes.

Figure 3,2-HP and and 3,4-HP 3,4-HP [21]. [21]. Figure 2. 2. Resonance Resonance structures structures of of 1,2-HP, 1,2-HP, 3,2-HP 3+ stability Table 1.1. Protonation Protonation constants, constants, stepwise stepwise stability stability constants constants (log (log K) K) and and cumulative cumulative Fe Fe3+ stability Table constants (log (log β β3)) for 1,2-HP, 3,2-HP and 3,4-HP. 3,4-HP. constants for 1,2-HP, 3,2-HP and 3

Compound

Compound

1,2-HP (2) 1,2-HP (2)

3,2-HP (3) 3,2-HP (3)

3,4-HP (4) 3,4-HP (4)

pKa1 1.2 1.2−0.9 −0.9 0.1 0.13.34 3.343.60

pKa2 5.86 5.865.78 5.78 8.66 8.669.01 9.019.60

log K1 10.6 10.6 10.3 10.3 11.7 11.7 14.2 14.2 –

log K2 9.5 9.59.0 9.0 9.8 9.8 11.6 11.6–

log K3 log 3 Fe3+ log β3 Fe3+ 7.1 27.2 7.17.6 27.2 26.9 7.6 26.9 8.1 29.6 8.19.3 29.6 35.1 9.3– 35.1 36.9

pKa1

pKa2

log K1

log K2

log K3

3.60

9.60

–

–

–

36.9

Ref. [22] [22] [23] [23] [23] [23] [23] [23] [20]

Ref.

[20]

3,4-HPs have high affinity for both Fe3+ and Ga3+, and form neutral 3:1 bidentate complexes with 3+ and 3+, In3+, Cr3+, Mn3+ and Gd3+) [24–31]. Both the Fe 3,4-HPs and Ga3+have (Figure 3, Table (as Fe well as Ga Al3+ high affinity for2)both , and form neutral 3:1 bidentate complexes with 3+ 3+ 3+ 3+ , Cr3+ , Mn 3+ and 3+ ) [24–31].geometries [Fe(deferiprone) 3] and [Ga(deferiprone) 3] complexes in the same Fe and Ga (Figure 3, Table 2) (as well as Al , Incrystallise Gdoctahedral Both the with comparable bond lengths and angles. In the three coordinating deferiprone ligands in both [Fe(deferiprone)3 ] [31] and [Ga(deferiprone)3 ] [29] complexes crystallise in the same octahedral 3 3 bond complexes, with O atoms are facbond to each other (see TableIn3 the forthree atomcoordinating numbering deferiprone scheme). Fe–O geometries comparable lengths and angles. ligands in 3 3 4 lengths are 1.998OÅ,atoms and Fe–O lengths, 2.038Table Å while thenumbering Ga analogue the corresponding both complexes, are facbond to each other (see 3 for in atom scheme). Fe–O bond 4 bond Ga–O bond lengths and 1.990 Å, respectively. bonds are lengths are 1.998 Å, are and1.967 Fe–OÅ, lengths, 2.038 Å whileIninboth the complexes, Ga analogueboth the C–O corresponding 4 4 3 3 bond. intermediate between double with the CIn –Oboth bond shorter than C –O Ga–O bond lengths aresingle 1.967and Å, and 1.990bonds, Å, respectively. complexes, boththe C–O bonds are 4 4 3 4, and 83.22° for O3–Ga–O43 Within each deferiprone ligand, angles are 80.91° forCO3–O –Fe–O intermediate between single andbond double bonds, with the bond shorter than the C –O . Other bond. 4. 3+) form isostructural [M(deferiprone) metal ions (Mdeferiprone = Al3+, In3+, ligand, Cr3+, Mnbond complexes and in Within each angles are 80.91◦ for O3 –Fe–O4 3,] and 83.22◦ [27–30], for O3 –Ga–O 3+ 3+ 3+ 3+ 1 all cases, fac ions geometries observed However, H NMR studies on diamagnetic Other metal (M = Alare, In , Cr , in Mnthe) solid form state. isostructural [M(deferiprone) [27–30], 3 ] complexes 1 3+ and Al3+ complexes suggest that conversion between fac and mer geometric Ga isomers occurs in and in all cases, fac geometries are observed in the solid state. However, H NMR studies on 3+ 3+ solution [30].Ga and Al complexes suggest that conversion between fac and mer geometric isomers diamagnetic occurs in solution [30]. 3+


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Figure 3.3.Representations of of thethe molecular structures of [Fe(deferiprone) Representations molecular structures of [Fe(deferiprone) 3] [31] number (CCDC 1183332) number 3 ] [31] (CCDC (left) and (left) [Ga(deferiprone) (right).[29] Only one stereoisomer is depicted 1183332) and [Ga(deferiprone) ] (CCDC1164388) number[29] 1164388) (right). Only one stereoisomer 3 ] (CCDC 3number for each complex. Orange—iron, trogen, grey—carbon, red—oxygen, and is depicted for each complex. pink—gallium, Orange—iron, blue—ni pink—gallium, blue—ni trogen, grey—carbon, white—hydrogen. These diagrams (generated in Mercury softwareincourtesy the Cambridge red—oxygen, and white—hydrogen. These diagrams (generated Mercuryofsoftware courtesyCrystal of the Database) crystallographically structures. Hydrogenstructures. atoms of methyl groups are Cambridgerepresent Crystal Database) represent determined crystallographically determined Hydrogen atoms omitted forgroups clarity.are omitted for clarity. of methyl

In living living organisms, organisms, iron iron plays plays an an essential essential role role in in transport transport of of oxygen, oxygen, electron electron transfer, transfer, and and In activation and and functioning functioning of of enzymes. enzymes. Iron tightly controlled in living living activation Iron transport transport and and metabolism metabolism are are tightly controlled in organisms. In patients with iron overload disease, a disruption to iron homeostasis results in “free” organisms. In patients with iron overload disease, a disruption to iron homeostasis results in “free” iron, largely largelypresent presentasas a citrate-albumin complex iron gives pool gives to Fenton redox iron, a citrate-albumin complex [32].[32]. ThisThis iron pool rise torise Fenton redox cycling 2+ 3+ 2+ 3+ cycling between Fe , producing free radicals thatin result toxicdamage tissue damage between Fe andFeFe and , producing free radicals that result toxicintissue [33]: [33]: Fe2+ + H2O2  Fe3+ + HO· + OH− (1) Fe2+ + H2 O2 → Fe3+ + HO· + OH− (1) Fe3+ + H2O2  Fe2+ + HOO· + H+ (2) Fe3+ + H2 O2 → Fe2+ + HOO· + H+ (2) Therapeutic chelators, including 3,4-HPs, are clinically used for sequestration of Fe3+ in vivo 3+ 2, Cclinically 5 or C6 positions [7–10]. 3,4-HPs can be functionalised the N1, Care of the ring (Tableof3).Fe An Therapeutic chelators, includingat3,4-HPs, used for sequestration 1 2 5 6 understanding of the effects substitution is 3,4-HP derivatives either in vivo [7–10]. 3,4-HPs can beoffunctionalised at critical the N , in C optimizing , C or C positions of the ring for (Table 3). 3+ 3+ therapeutic Fe chelation, or radiopharmaceutical Ga chelation. An understanding of the effects of substitution is critical in optimizing 3,4-HP derivatives for either therapeutic Fe3+ chelation, or radiopharmaceutical Ga3+ chelation. Table 2. Protonation constants and Fe3+ and Ga3+ stability constants for deferiprone (1) (measured at 3+ stability −5 (measured 22.5–25 ionic strength 0.1–0.2 forand pMGa values, pH 7.4, with [total ligand] = 10 M and [total Table 2. °C, Protonation constants andM; Fe3+ constants for deferiprone (1) at ◦ C,= ionic metal ion] 10−6 M). 22.5–25 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 Metal Ion

Fe3+ Fe3+

Ga3+ Ga3+

pKa1 3.56 pK a1 3.68 3.56 3.62 3.68 – 3.62 – 3.61 3.61 – – 3.70 3.70

pKa2 9.64 pK a2 9.77 9.64 9.76 9.77 – 9.76 – 9.78 9.78 – – 9.86 9.86

log K1 log K2 log K3 log β3 14.92 log K1 log12.23 K2 log K9.79 log β37.2 3 3 14.56 12.19 9.69 36.4 14.92 12.23 9.79 37.2 15.14 11.54 9.699.24 36.435.92 14.56 12.19 15.10 11.51 9.249.27 35.92 35.88 15.14 11.54 15.10 11.51 35.88 15.03 27.42 9.27 – 37.35 15.03 27.42 – 37.35 13.17 12.26 10.33 35.76 13.17 12.26 17.07 12.19 10.33 9.16 35.76 38.42 17.07

12.19

9.16

38.42

pM3+ pM3+– 19.4 – 18.3 19.4 18.3 – –20.74 20.74– – – –

Ref. [20] Ref. [34] [20] [35] [34] [25] [35] [25] [36] [36] [25] [25] [37] [37]

3. 3,4-Hydroxypyridinones for Fe Complexation 3. 3,4-Hydroxypyridinones for Fe3+ Complexation 3.1. Tailoring 3,4-HP Properties by Ring Substitution 3.1. Tailoring 3,4-HP Properties by Ring Substitution Substitution at the C2 position results in pronounced effects on metal ion affinity. Alkylation Substitution at thering C2 position pronounced on metal affinity. increases pyridinone electron results density,in and increaseseffects pKa values andion log K andAlkylation log β3(Fe3+) increases pyridinone ring electron density, and increases pK values and log K and log β constants 2 2 3+ a 3(Fe3+) constants (log β3 = 35.1 when R = H (4); log β3 = 37.2 when R = CH3 (5)), although pFe values are not 2 = H (4); log β = 37.2 when R2 = CH (5)) [20], although pFe3+ values are not (log β = 35.1 when R 3 3 3 markedly affected [26]. markedly affected of [26]. Substitution C2 alkyl groups for 1′-hydroxyalkyl groups decreases pKa and log β3(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) 3+

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Substitution of C2 alkyl groups for 10 -hydroxyalkyl groups decreases pKa and log β3(Fe3+) constants (compare with18,6;116and 7, 8 and 9) [34]. This decrease in affinity is a result of: (i) a decrease Int. J. Mol. Sci.1 2017, 5 of 23 in ring electron density due to a negative induction effect of the C2 10 -hydroxylalkyl group; and (ii) intramolecular hydrogen bonding between C2 10 -hydroxyalkyl group deprotonated intramolecular hydrogen bonding between the the C2 1′-hydroxyalkyl group andand thethe deprotonated C3 3 hydroxyl group (Figure 4). This hydrogen bonding stabilises the ionised C3 3 hydroxyl group. C hydroxyl group (Figure 4). This hydrogen bonding stabilises the ionised C hydroxyl group. 3+ affinity affinity is is decreased, decreased, the concurrent lowering of pKa2 actually results results in an However, although Fe3+ a2 actually 3+ pFe3+. .The Thedecrease decreaseininproton protonaffinity affinityfavours favoursthe the negative form that coordinates Fe. 3+ increase in pFe negative form that coordinates to to Fe3+ In. 3+ 2 0 3+ 2 a side-by-side comparison pH7.4, 7.4,pFe pFe values valuesofofthe theCC 1′-hydroxyalkyl 1 -hydroxyalkylderivatives derivatives are are higher aInside-by-side comparison atatpH alkyl derivatives derivatives [34]. [34]. than C22 alkyl

Figure groups of of 3,4-HPs. 3,4-HPs. Figure 4. 4. Intramolecular Intramolecular hydrogen hydrogen bonding bonding can can stabilise stabilise ionised ionised hydroxyl hydroxyl groups

Similar effects are also observed upon introduction of a C2 amido group (Figure 4) [38]. A Similar effects are also observed upon introduction of a C2 amido group (Figure 4) [38].3 combination of a negative induction effect and hydrogen bonding from the C2 amido NH to the C A combination of a negative 3+induction effect and hydrogen bonding from the C2 amido NH to hydroxyl group decreases Fe stability constants, but the concurrent decrease in pKa serves to the C3 hydroxyl group decreases Fe3+ stability constants, but the concurrent decrease in pK serves to 3+ values at physiological pH relative to C2 alkyl derivatives. The C2 doublyaalkylated increase pFe3+ increase pFe 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 the3 C3 hydroxyl group (Figure 4), N(CH )2 amido group of 10 cannot form a hydrogen bond with the C hydroxyl group (Figure 4), and and so3 its pFe3+ value is lower when compared to 11 bearing a singly alkylated NH(CH3) that can so its pFe3+ value is lower when compared to 11 bearing a singly alkylated NH(CH3 ) that can form form a hydrogen bond (Table 3) [38]. a hydrogen bond (Table 3) [38]. In contrast to C22, varying alkyl substituents at the N11 position (H, methyl, ethyl) of 3,4-HP does In contrast to C , varying alkyl substituents at the N 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]. 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 The exception to this is the 2C2 amido derivatives discussed above, where N1 alkylation sterically inhibits coplanarity of the C2 amido group with the HP ring, disrupting hydrogen bonding and thus inhibits coplanarity of the C amido group1 with the HP ring, disrupting hydrogen bonding and thus decreasing pFe3+ values [38]. As such, N alkyl substitution can be a useful strategy for tailoring 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 chelators’ lipophilicity, cell permeability [20], in vivo biodistribution [34], and rates of metabolism [39] [39] without deleterious effects on metal ion affinity. Substitution at N1 sites has also been utilised to 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]. functionalise 3,4-HPs with fluorescent tags [40] or biological vectors [41]. Alkyl substitution at the C55 position increases 3,4-HP affinity for Fe3+3+ , however as there is a Alkyl substitution at the C3+ position increases 3,4-HP affinity for Fe , however as there is5 concurrent increase in pKa2, pFe 3+ does not increase relative to derivatives that do not contain a C a concurrent increase in pK , pFe does not increase relative to derivatives that do not contain a C5 alkyl group (compounds 13a2and 14) [36]. There are no studies directly comparing and quantifying alkyl group (compounds 13 and 14) [36]. There are no studies directly comparing and quantifying the the effect of C6 substitution, but existing data of C6 methylated derivatives suggest that alkylation effect of C6 substitution, but existing data of C6 methylated derivatives suggest that alkylation has has little influence on 3,4-HP metal affinity [36]. little influence on 3,4-HP metal affinity [36]. Increasing the lipophilicity of 3,4-HPs increases their cell membrane permeability. Higher Increasing the lipophilicity of 3,4-HPs increases their cell membrane permeability. intracellular 3,4-HP accumulation results in greater intracellular Fe3+ scavenging, however excessive Higher intracellular 3,4-HP accumulation results in greater intracellular Fe3+ scavenging, however cellular uptake of 3,4-HPs results in toxicity [20]. A prodrug strategy that involves oral excessive cellular uptake of 3,4-HPs results in toxicity [20]. A prodrug strategy that involves oral administration of an N11-substituted hydrophobic ester 3,4-HP (15, Figure 5) has proved particularly administration of an N -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 successful in scavenging iron in a preclinical iron overloaded rat model [34]. In iron overload disease, disease, excess iron is stored in the liver, and the rat model mimics this. The ester compound 15 is excess iron is stored in the liver, and the rat model mimics this. The ester compound 15 is absorbed absorbed effectively in the gastrointestinal tract, and delivered to the liver, where it enters target effectively in the gastrointestinal tract, and delivered to the liver, where it enters target iron-overloaded iron-overloaded hepatocyte cells. The compound is proposed to undergo ester hydrolysis and hepatocyte cells. The compound is proposed to undergo ester hydrolysis and metabolism intracellularly metabolism intracellularly (16, 17) prior to complexing Fe3+ [20]. The complex is then excreted, (16, 17) prior to complexing Fe3+ [20]. The complex is then excreted, recovering excess iron from1 recovering excess iron from diseased animals efficiently. In contrast, when the more hydrophilic N diseased animals efficiently. In contrast, when the more hydrophilic N1 propyl alcohol derivative 16 propyl alcohol derivative 16 (Figure 5) is directly administered to rats, absorption is less effective, (Figure3+5) is directly administered to rats, absorption is less effective, and Fe3+ recovery is lower. and Fe recovery is lower.

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Figure 5. Schematic representation of the use of hydrophobic esters to enhance both the absorption Figure 5. representation of use of esters enhance both absorption Figurethe 5. Schematic Schematic representation of the theand use the of hydrophobic hydrophobic esters to toof enhance both the theSubsequent absorption from gastrointestinal tract (GIT) hepatic extraction the chelator. from the gastrointestinal tract (GIT) and the hepatic extraction of the chelator. Subsequent intracellular from the gastrointestinal tract (GIT) and the hepatic extraction of the chelator. Subsequent intracellular hydrolysis occurs yielding a hydrophilic chelator which can undergo further hydrolysis occurs yielding a hydrophilic chelator which can undergo further metabolism to the intracellular tohydrolysis occurs yieldingnegatively a hydrophilic which metabolite can undergo metabolism the extremely hydrophilic chargedchelator 1-carboxyalkyl in thefurther liver. extremely hydrophilic negatively charged 1-carboxyalkyl metabolite in the liver. Reprinted with metabolism to the extremely hydrophilic negatively charged 1-carboxyalkyl metabolite in the liver. Reprinted with permission from Liu, Z.D.; et al. Synthesis, physicochemical characterisation, and permission from Liu, Z.D.; et al. Synthesis, physicochemical characterisation, and biological evaluation Reprinted with permission from Liu, Z.D.; et al. Synthesis, physicochemical characterisation, and biological evaluation of 2-(1′-hydroxyalkyl)-3-hydroxypyridin-4-ones: Novel iron chelators with 0 3+ of 2-(1 -hydroxyalkyl)-3-hydroxypyridin-4-ones: Novel iron chelators with enhanced pFe values. biological evaluation of 2-(1′-hydroxyalkyl)-3-hydroxypyridin-4-ones: Novel iron chelators with 3+ values. J. Med. Chem. 1999, 42, 4814–4823. Copyright American Chemical Society. enhanced pFe1999, J. Med. Chem. 42, 4814–4823. Copyright American Chemical Society. enhanced pFe3+ values. J. Med. Chem. 1999, 42, 4814–4823. Copyright American Chemical Society. Table 3. Protonation constants and Fe3+ stability constants for 3,4-HP derivatives. Table 3. Protonation constants and Fe3+ stability constants for 3,4-HP derivatives. 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 2 5 6 3+) 3+ 3+ 1 2 5 6 Compound R R R R pK a1 pK a2 log β 3 (Fe Ref. Compound pKa1 9.77pKa2 Ref. R R R R log β3 (Fe ) pFe pFe3+ 3.68 36.4 19.4 [34] 1 CH3 H H CH3 3.68 9.779.77 36.4 36.4 19.4 [34] 3.68 9.78 19.4 [34] 3.61 37.35 20.74 [36] 11 3 H CH3 3 CH CHCH H H H 3 3.61 9.789.78 37.35 20.74 [36] 3.61 37.35 20.74 [36] 44 H H H H 3.34 9.01 35.1 – [20,23] H H H H 3.34 9.01 35.1 – [20,23] 4 H H H H 3.34 9.019.76 35.1 37.2 [20,23] 3.70 9.76 [20] 3.70 37.2 –– – [20] CH 5 H H H 3 5 H H H CH3 3.64 9.769.73 [36] 3.70 37.236.63 –20.17 [20] 3.64 9.73 36.63 20.17 [36] 56 H H3 CH3 CH CH2 OH H H H 2.92 9.11 35.3 20.9 [34] 3.64 9.73 36.63 20.17 [36] CH CH CH CH H H 3.81 9.93 36.8 19.7 [34] 67 3 CH 2 OH H H 2.92 9.11 35.3 20.9 [34] CH 2 3 2 3 CH CHCH H H H 2.80 9.119.27 21.0 [34] 2 CH 2 OH2OH 33 3 H 2.92 35.3 35.3 20.9 [34] CH 7679 2CH CH 2CH3 H H 3.81 9.93 8.77 36.8 19.7 [34] CH CH2 CH3 CH(OH)CH3 H H 3.03 35.1 21.4 [34] CH33 CON(CH CH 3 H H 3.81 9.93 36.8 19.7 [34] CHH22CH ) H CH 2.53 8.20 33.2 20.4 [38] 7710 CH22CH OH H H 2.80 9.27 35.3 21.0 [34] CH 3 2 3 H H CH 6.66 9.272.32 22.8 [38] 3 CH33 CONHCH CH2OH H3 2.80 35.3 32.5 21.0 [34] CHH22CH 9711 CH(OH)CH 3 H H 3.03 8.77 35.1 21.4 [34] CH 12 CH2 CH3 CH3 H H 3.65 9.88 37.7 – [20] 913 2CH3 CH(OH)CH H H 3 2.53 3.03 8.7710.32 35.137.93 21.4 [34] CH CH CH3 H 3.37 8.20 19.71 [36] 10 3)23CH3 H CH 33.2 20.4 [38] H CON(CH 3 14 H CH3 CH3 H H 3 3.43 8.2010.27 37.28 19.22 [36] 10 3 ) 2 CH 2.53 33.2 20.4 [38] H CON(CH 11 H CH3 6.66 2.32 32.5 22.8 [38] H CONHCH3 11 H CH 6.66 2.32 32.5 22.8 [38] CONHCH 12 2CH3 CH3 3 H H 3 3.65 9.88 37.7 – [20] CHH 12 2 CH 3 CH 3 H H 3.65 9.88 37.7 – [20] CH 3.2. Deferiprone, Desferrioxamine Deferasirox 13 CHand 3 CH3 H 3.37 10.32 37.93 19.71 [36] CH3 13 CH33 CH33 H 3.37 10.27 10.32 37.93 19.71 [36] CH 14 CH H 3.43 37.28 19.22 [36] H3 CH Deferiprone (or (1), developed colleagues, has been clinically approved 14 3 CH3 by H Hider 3.43and10.27 37.28 19.22 [36] H Ferriprox) CH

(in Europe in 1999, and the USA, in 2011) for treatment of iron overload diseases, including 3.2. Deferiprone, Desferrioxamine and Deferasirox hemochromatosis and transfusion-dependant 3.2. Deferiprone, Desferrioxamine and Deferasirox thalassemia [42]. The advantage of deferiprone is 3+ from Deferiprone (or Ferriprox) (1), developed by HiderFe and colleagues, hasstream, been clinically approved that it is both orally active and effective at sequestering the blood heart (where iron Deferiprone (or Ferriprox) (1), developed by Hider and colleagues, has been diseases, clinically approved (in Europe in 1999, and the USA, in 2011) for treatment of iron overload including overload toxicity can be fatal) and liver (where excess iron is stored). (in Europe in 1999, the USA, in 2011) for treatment ofThe iron overloadofdiseases, including hemochromatosis andand transfusion-dependant [42]. advantage deferiprone is that Two other approved treatments for iron thalassemia overload are available. The first is the hexadentate hemochromatosis and transfusion-dependant thalassemia [42]. The advantage of deferiprone is iron that 3+ from the blood stream, heart (where 3+ it is both orally active and effective at sequestering Fe hydroxamate chelator, desferrioxamine-B (DFO, 18, Figure 6), which forms a complex with Fe with 3+ it is both toxicity orally active effective at (where sequestering the blood stream, heart (where iron overload can beand fatal) and liver excessFe ironfrom is stored). 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 Two other approved treatments for (DFO, iron overload are Theafirst is thewith hexadentate hydroxamate chelator, desferrioxamine-B 18, Figure 6),available. which forms complex Fe3+ with 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 Int. J. Mol. Sci. 2017, 18, 116 7 of 23 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. Int. J. Mol. Sci. 2017, 18, 116 7 of 23 a metal ligand stoichiometry of [43] 1:1 and overall of +1 under conditions. Liketodeferiprone, deferasirox (19, an Figure 6) ischarge an effective orally physiological active treatment for iron DFO requires parenteral infusion(inover 8–12in h, 2006 several a week, as it isItnot absorbed [42]. overload clinical approval Europe andtimes the USA in under 2005). is aorally tridentate chelator, a metal towith ligand stoichiometry of 1:1 and an overall charge of +1 physiological conditions. 3+ Clinical studies havebearing demonstrated thatateither deferiprone or a combination of deferiprone with complexes a 3-charge physiological pH.aalone, Data comparisons the DFO Fe requires parenteral infusion over 8–12 h, several times week,from as it clinical is not orally absorbedof[42]. and DFO, are more effective therapies are for conflicting myocardialand ironinconclusive, overload than DFO alone. Additionally, efficacy of deferiprone and deferasirox possibly due to variations in Clinical studies have demonstrated that either deferiprone alone, or a combination of deferiprone adoses combination of oral deferiprone and parenteral DFO therapies is more viable for patients than of the treatments Some clinical data suggest thatthan deferiprone is more effective ata and DFO, aretwo more effective [44–47]. therapies for myocardial iron overload DFO alone. Additionally, parenteral DFO therapy alone, tissue as combination therapy results in fewer parenteral infusions. reducing iron levels in cardiac [46,47]. 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 [44–47]. Some clinical data suggest that deferiprone is more effective at reducing iron levels in cardiac tissue [46,47]. Figure 6. Structure B (DFO) (DFO) and and deferasirox. deferasirox. Figure 6. Structure of of desferrioxamine desferrioxamine B

3.3. Synthesis of 3,4-HPs Like deferiprone, deferasirox [43] (19, Figure 6) is an effective orally active treatment for iron The structural diversity of 3,4-HPs is a in result extensive synthetic that spanschelator, several overload with clinical approval (in Europe 2006ofand the USA in 2005).research It is a tridentate decades. is beyond bearing the scope of this review to describe pH. all ofData thesefrom synthetic routes in great detail, with Fe3+Itcomplexes a 3-charge at physiological clinical comparisons of the 1 but it isof worth highlighting common routes, and routes possibly that give rise to N - and efficacy deferiprone and deferasirox arestarting conflicting and inconclusive, due to variations in C2-substituted 3,4-HPs that are important forsuggest new, bioactive compounds. doses of the two treatments [44–47]. Someprecursors clinical data that deferiprone is more effective at Pyranones suchinFigure as maltol (20)[46,47]. containing a benzyl (Bn)and protecting group can simply be 6. Structure of desferrioxamine B (DFO) deferasirox. reducing iron levels cardiac tissue converted to pyridinones by reaction with primary amines (Scheme 1) [20]. This allows diverse 3.3. Synthesis at of 3,4-HPs substitution N1, including incorporation of reactive groups such as carboxylates or primary amines lead to further functionalization [20,40]. Thethat structural diversity of 3,4-HPs is a result of extensive 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, 1 - and C2 -substituted but ititis is worth highlighting common starting routes, and routes that give rise to N worth highlighting common starting routes, and routes that give rise to N1- and 2 3,4-HPs that are important precursors for new, bioactive compounds. C -substituted 3,4-HPs that are important precursors for new, bioactive compounds. Pyranones maltol (20)(20) containing a benzyl (Bn) protecting group can simply converted Pyranones such suchasas maltol containing a benzyl (Bn) protecting group canbesimply be to pyridinones by reaction with primarywith amines (Scheme 1) [20]. This allows diverse substitution at converted to pyridinones by reaction primary amines (Scheme 1) [20]. This allows diverse 1 1 1 N , includingatincorporation of incorporation reactive groups as -substituted carboxylates or primary amines that lead to substitution N , including ofsuch reactive groups such as carboxylates or primary Scheme 1. Synthetic route for N 3,4-HPs. further [20,40]. amines functionalization 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) increases1 yields in this reaction. A Mitsunobu reaction Scheme 1. 1. Synthetic Synthetic route route for for N N1-substituted 3,4-HPs. Scheme -substituted of 23 with phthalamide gives a Bn-protected pyridinone (24) that 3,4-HPs. contains a phthalamide at the C2 position, that can22 beissimply converted to a primary amine (25) [49]. and Bn-protected is a very Compound a pyranone containing a Bn protecting group, a reactive 25 alcohol. It isuseful a key Compound 22 is a pyranone containing a Bn protecting group, and a reactive alcohol. It is a key precursor for preparation of tris(hydroxypyridinones) such as 26 (see below). synthetic precursor enabling versatile manipulation of functionality at the C22 position of 3,4-HPs synthetic precursor enabling versatile manipulation of functionality at the C position of 3,4-HPs (Scheme 2). It is synthesised in high yields from the commercially available and inexpensive (Scheme 2). It is synthesised in high yields from the commercially available and inexpensive precursor, precursor, kojic acid (21) in four steps [48,49]. As discussed above, the Bn-protected 22 can be kojic acid (21) in four steps [48,49]. As discussed above, the Bn-protected 22 can be converted to converted to Bn-protected pyridinone 23 by reaction with methylamine [49]. Protection of the ethyl Bn-protected pyridinone 23 by reaction with methylamine [49]. Protection of the ethyl alcohol group alcohol group (and subsequent deprotection) increases yields in this reaction. A Mitsunobu reaction (and subsequent deprotection) increases yields in this reaction. A Mitsunobu reaction of 23 with2 of 23 with phthalamide gives a Bn-protected pyridinone (24) that contains a phthalamide at the C phthalamide gives a Bn-protected pyridinone (24) that contains a phthalamide at the C2 position, that position, that can be simply converted to a primary amine (25) [49]. Bn-protected 25 is a very useful can be simply converted to a primary amine (25) [49]. Bn-protected 25 is a very useful precursor for precursor for preparation of tris(hydroxypyridinones) such as 26 (see below). preparation of tris(hydroxypyridinones) such as 26 (see below).


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Scheme 2. Synthesis of a useful 3,4-HP with a reactive amine (25) that is used for synthesis of Scheme 2. Synthesis of a useful 3,4-HP with a reactive amine (25) that is used for synthesis of THP-Ac (26). THP-Ac (26).

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

primary and and secondary secondary amides. amides. Scheme 3. Synthesis of 3,4-HP precursors containing primary

Reactive carboxylates are synthetically accessible from Bn-protected pyridinones (Scheme 4) Reactive carboxylates are synthetically accessible from Bn-protected pyridinones (Scheme 4) [50]. [50]. Bn-protected 29 can be further protected, and the methyl group substituted to ultimately yield Bn-protected 29 can be further protected, and the methyl group substituted to ultimately yield 30. 30. Compound 30 can be converted to a carboxylate, yielding 31. The carboxylate group of 31 can be 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 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]. 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 (Schemes 1, 2 and 4).


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Scheme 4. Synthesis of a 3,4-HP containing a reactive carboxylate that allows synthesis of THPs 33 and 34. Scheme 4. Synthesis of a 3,4-HP containing a reactive carboxylate that allows synthesis of THPs 33 and 34.

Deprotection of Bn-protected hydroxyl groups proceeds via either hydrogenation reactions (catalysed by palladium on carbon) followed by acidification, or treatment with dissolved boron 4. Hexadentate Tris(hydroxypyridinone) Ligands trichloride in an aprotic solvent, followed by addition of an alcohol (Schemes 1, 2 and 4). 3+ Affinity 4.1.4.Topology and Fe Hexadentate Tris(hydroxypyridinone) Ligands

Incorporation of three bidentate 3,4-HP ligands into a tripodal construct provides hexadentate 4.1. Topology and Fe3+ Affinity tris(hydroxypyridinone) ligands (THPs) that, in a suitably designed scaffold, saturate the coordination sphere Incorporation of octahedralofmetal ions. THPs have been into designed for construct applications in gastrointestinal three bidentate 3,4-HP ligands a tripodal provides hexadentate tris(hydroxypyridinone) ligands (THPs) that, a suitably ofdesigned saturate scavenging of Fe3+ [51], antimicrobial activity (viaindeprivation microbes’scaffold, Fe3+ pool) [52,53],the and 3+ coordination sphere of octahedral metal ions. THPs have been designed for applications in fluorescence imaging of cellular Fe distribution [54]. The topology of tripodal THPs is critically 3+ pool) gastrointestinal scavenging of Fe3+ [51], antimicrobial (via deprivation microbes’ofFethe important to formation of octahedral complexes withactivity 1:1 stoichiometry. Theof backbone tripod 3+ [52,53], fluorescence imaging of cellularOFe distribution [54]. The topology of tripodal THPs is should be and connected ortho to a coordinating atom [55]. 2 carboxylate critically important to formation of octahedral with 1:1 stoichiometry. The backbone of The synthesis of the first generation of THP complexes ligands utilised 3,4-HP groups with aC the tripod should be connected ortho to a coordinating O atom [55]. substitution (32), allowing reaction with tripodal polyamines to yield compounds such as 33 and 2 The synthesis the 34, first3,4-HP generation ligands 3,4-HPgroup, groupsand withfulfil a Cthe 34 (Scheme 4). In 33ofand units ofareTHP attached viautilised a C2 amide carboxylate substitution (32), allowing reaction with tripodal polyamines to yield compounds such topology requirements outlined above for formation of hexadentate compounds with 1:1 stoichiometry. as 33 and 34 (Scheme 4). In 33 and 34, 3,4-HP units are attached via a C2 amide group, and fulfil the For compound 34, log K1 = 30.7, whereas for the bidentate homologue 35, log β3 = 31.4 [50]. In this case, topology requirements outlined above for formation of hexadentate compounds with 1:1 the affinity of the bidentate 3,4-HP for Fe3+ is already optimal, and incorporation into a hexadentate stoichiometry. For compound 34, log K1 = 30.7, whereas for the bidentate homologue 35, log β3 = 31.4 form does not result in an increase in thermodynamic stability, as might normally be expected for [50]. In this case, the affinity of the bidentate 3,4-HP for Fe3+ is already optimal, and incorporation an increase in ligand denticity and accompanying lower entropic costs. The pFe3+ of 34 (30.5 at into a hexadentate form does not result in an increase in thermodynamic stability, as might normally 3+ of 35 (22.0) [50]. This arises because the formation constant of a pHbe7.4) is higher than the pFe expected for an increase in ligand denticity and accompanying lower entropic costs. The pFe3+ of hexadentate complex of 34 has only a dependence on ligand concentration ligand 3+ of order 34 (30.5 at pH 7.4) is higher than the pFefirst 35 (22.0).[50]. This arises because the formation (1:1 constant to of metal stoichiometry), whereas that of 35 necessarily has third order dependence on free ligand a hexadentate complex of 34 has only a first order dependence on ligand concentration (1:1 ligand concentration (3:1 ligand to metal that stoichiometry). In solutions 34 where the hexadentate ligand to metal stoichiometry), whereas of 35 necessarily has thirdoforder dependence on free ligand concentration = 10 µM, the total concentration of single 3,4-HP units is three times greater than concentration (3:1 ligand to metal stoichiometry). In solutions of 34 where the hexadentate ligand in solutions of 35 where the bidentate ligand concentration 10 µM. concentration = 10 µM, the total concentration of single =3,4-HP units is three times greater than in Insteadofof35 polyamine theligand next generation of THP ligands derivatised tripodal carboxylate solutions where thetripods, bidentate concentration = 10 µM. of polyamine tripods, the next generation of THP ligands 2)derivatised groups,Instead using 3,4-HP units with a C2 aminomethyl substituent (25, Scheme [51]. In thetripodal resulting 2 aminomethyl substituent (25, Scheme 2) [51]. In the carboxylate groups, using 3,4-HP units with a C compounds, for example THP-Ac (26, Scheme 2), each 3,4-HP group is tethered to the tripod via 1 and compounds, THP-Ac (26, Scheme 2), each are 3,4-HP group isWith tethered to the an resulting amidomethyl linker atfor theexample C2 position. The N C6 positions methylated. the exception


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tripod via an amidomethyl linker at the C2 position. The N1 and C6 positions are methylated. With 6 -methylation, theCexception of C6THP-Ac’s -methylation, THP-Ac’s single 3,4-HP unitsimilar is structurally similarFor to deferiprone. of single 3,4-HP unit is structurally to deferiprone. THP-Ac, log 3+ 3+ For=THP-Ac, log K1 = = 32.52 and pFe = 28.47 [51]. K 32.52 and pFe 28.47 [51]. 1 The incorporation incorporation of 3,4-HP groups into aa tripodal tripodal ligand ligand also also has has implications implications for for the the The of three three 3,4-HP groups into lability of hexadentate complexes. For example, in [Fe(THP-Ac)], dissociation of a single 3,4-HP unit lability of hexadentate complexes. For example, in [Fe(THP-Ac)], dissociation of a single 3,4-HP unit is is likely to followed be followed byrapid its rapid recoordination the metal dissociation, this likely to be by its recoordination to theto metal centre.centre. DuringDuring dissociation, this 3,4-HP 3,4-HP unit will remain spatially close to the metal centre, as the other two 3,4-HP groups, to which it unit will remain spatially close to the metal centre, as the other two 3,4-HP groups, to which it is 3+. On the other hand, in [Fe(deferiprone)3], if a 3+ is covalently tethered, are likely to still be bound to Fe covalently tethered, are likely to still be bound to Fe . On the other hand, in [Fe(deferiprone)3 ], if a deferiprone ligand ligand dissociates, dissociates,itithas hasaalower lowerprobability probabilityofofrecoordinating recoordinating same metal centre, deferiprone toto thethe same metal centre, as as it is not anchored to any other coordinating ligands. The activation energy barrier to dissociation it is not anchored to any other coordinating ligands. The activation energy barrier to dissociation of of [Fe(THP-Ac)] is higher than of [Fe(deferiprone) 3], and [Fe(THP-Ac)] is more kinetically inert [Fe(THP-Ac)] is higher than thatthat of [Fe(deferiprone) 3 ], and [Fe(THP-Ac)] is more kinetically inert than than [Fe(deferiprone) [Fe(deferiprone) ]. 3]. 3

4.2. Dendrimers Based on THP Units THPmolecules, molecules,forfor example incorporated between threesixand THP Dendritic THP example 36, 36, havehave incorporated between three and THPsix groups, 3+ 3+ groups, allowing coordination of between three and six equivalents of coordinatively saturated allowing coordination of between three and six equivalents of coordinatively saturated Fe Fe per per molecule [51,56]. Dendrimers have been prepared from both 25 and Affinity constants as well molecule [51,56]. Dendrimers have been prepared from both 25 and 32. 32. Affinity constants as well as 3+ values of dendrimer 36 (Figure 7) (log K1 = 32.74, pFe 3+ = 28.69) and other derivatives do not 3+ 3+ as pFe pFe values of dendrimer 36 (Figure 7) (log K1 = 32.74, pFe = 28.69) and other meaningfully deviate deviatefrom fromvalues valuesfor forthe the single THP-Ac homologue 26, indicating maximum meaningfully single THP-Ac homologue 26, indicating that that maximum Fe3+ 3+ Fe binding efficiency is achieved 3,4-HP motifs in a tripodal topology. binding efficiency is achieved usingusing 3,4-HP motifs in a tripodal THPTHP topology.

3+ ions. Figure 7. 7. Dendritic Dendritic 3,4-HPs 3,4-HPs such such as as (36) (36) can can coordinate coordinate multiple multiple Fe Fe3+ Figure ions.

4.3. Derivatising 4.3. Derivatising THP THP Ligands Ligands THP compounds compoundsofof same topology and substitution as THP-Ac (26) are synthetically THP thethe same topology and substitution as THP-Ac (26) are synthetically accessible accessible from the β-alanine derivative, THP-NH 2 (37, Figure 8) using similar reaction routes to that from the β-alanine derivative, THP-NH2 (37, Figure 8) using similar reaction routes to that described described Scheme [51]. THP derivatives such as 33 oramenable 34 are lesstoamenable to derivatisation in in Scheme in 2 [51]. THP2 derivatives such as 33 or 34 are less derivatisation in this fashion. this fashion. The presence of an apical primary amine in THP-NH 2 allows attachment to dendritic The presence of an apical primary amine in THP-NH2 allows attachment to dendritic scaffolds [51], scaffolds [51],[13–16], biomolecules [13–16], polymer units [57], and [54]. This ability to biomolecules polymer units [57], and fluorophores [54].fluorophores This ability to functionalise THP functionalise THP compounds makes them attractive for technological applications where trivalent compounds makes them attractive for technological applications where trivalent metal ion complexes metal complexes of high affinity and kinetic are required. We haveforexplored THP of highion affinity and kinetic stability are required. We stability have explored THP derivatives PET imaging 3+ derivatives with Ga3+ . for PET imaging with Ga .

Int. J. Mol. Sci. 2017, 18, 116

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Int. J. Mol. Sci. 2017, 18, 116

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Figure 8. THP derivatives.

Figure 8. THP derivatives. 5. Tris(hydroxypyridinone) Ligands for Radiolabelling with 68Ga3+

5. Tris(hydroxypyridinone) Ligands for Radiolabelling with 68 Ga3+ 5.1. Radiolabelling Peptides with 68Ga for PET Imaging: The Case for Tris(hydroxypyridinone) Derivatives

5.1. Radiolabelling Peptides with 68 Ga for PET Imaging: The Case for Tris(hydroxypyridinone) Peptide-based molecular imaging agents can rapidly accumulate at target tissue and clearDerivatives from circulation within 1–2 h. The 68 min half-life and positron emission properties of

Ga (β+ 90%,

68

Peptide-based molecular imaging agents can rapidly accumulate at target tissue and clear from Emax = 1880 KeV) match these requirements for PET imaging of peptide receptor expression. 68 Ga (β+ 90%, circulation within68Ga 1–2ish.conveniently The 68 minavailable half-lifebyand positron of produce 68Ge/68Ga properties Moreover, elution of theemission generator to Emax = 1880 KeV) match these requirements for PET imaging no-carrier-added solutions of 68Ga3+ in hydrochloric acid [58]. of peptide receptor expression. Moreover, 68 Ga is conveniently 68 Ge/ 3+ Hydrated Ga species such as [Ga(Hof 2O) 6]3+ exist in 68 aqueous solution below pH 4. As the pH is available by elution the Ga generator to produce no-carrier-added 68 3+ raised poorly soluble hydroxide solutions of above Ga 4,inthe hydrochloric acid [58]. species Ga(OH)3 predominates in solution, until pH > 6.3, where tetradentate [Ga(OH)4]− predominates [59,60]. For efficient 68Ga3+ radiolabelling of Hydrated Ga3+ species such as [Ga(H2 O)6 ]3+ exist in aqueous solution below pH 4. As the pH chelate-peptide conjugates at neutral or near neutral pH, chelate complex formation must effectively is raised above 4, the poorly soluble hydroxide species Ga(OH)3 predominates in solution, until compete with unreactive 68Ga-colloid formation. Preferably, the rate of chelation will be − predominates [59,60]. For efficient 68 Ga3+ radiolabelling pH > 6.3, where tetradentate [Ga(OH) ] diffusion-controlled, so that complex4formation outcompetes 68Ga3+ colloid formation. Additionally, 68Ga eluted from of chelate-peptide at neutral or nearare neutral pH, ofchelate formation must the amounts ofconjugates clinical generators in the range 200–2000complex MBq, approximately equivalent to 2–20 of 68Ga3+ 68 inGa-colloid 1–5 mL of solution. Highly efficient chelators to will be effectively compete withpmol unreactive formation. Preferably, the rateare ofrequired chelation 68 3+ quantitatively complex such low concentrations of metal ion without using excessively high chelator diffusion-controlled, so that complex formation outcompetes Ga colloid formation. Additionally, concentration. In clinical formulations of chelator bioconjugates, low concentrations of the amounts of 68 Ga eluted from clinical generators are in the range of 200–2000 MBq, approximately chelator-peptide conjugate are important. Clinical formulations do not usually separate unlabelled equivalent to 2–20 pmol of 68 Ga3+ in 1–5 mL of solution. Highly efficient chelators are required to bioconjugate from labelled conjugate, and large amounts of unlabelled conjugate can lead to saturation quantitatively such of target complex receptors in vivo.low concentrations of metal ion without using excessively high chelator 3+ has similar coordination concentration. clinical formulations of chelator bioconjugates, low Ga concentrations of chelator-peptide As In THP ligands have extraordinarily high pFe3+ values, and preferences to Fe3+, it was reasoned that such couldseparate be very efficient at quantitatively conjugate are important. Clinical formulations dochelators not usually unlabelled bioconjugate from 68Ga3+ at low chelator concentrations [13]. The acyclic nature of THP, and hence coordinating labelled conjugate, and large amounts of unlabelled conjugate can lead to saturation of target receptors flexibility compared to macrocyclic ligands, results in low activation barriers to complexation, in vivo. resulting in rapid rates of reaction at room temperature. As THP have high pFe3+ values, andstable Ga3+ has coordination It isligands also critical thatextraordinarily the 68Ga3+ complex is sufficiently kinetically over thesimilar period of time 3+ preferences to for Fe imaging , it was that transchelation such chelators could beendogenous very efficient at quantitatively required (1–2reasoned h) to withstand by competing proteins, such as 68 Gaand 3+ atother transferrin, that compete for Ga3+ in vivo The iron transport protein coordinating lowligands chelator concentrations [13]. The[4]. acyclic nature of THP, andtransferrin hence flexibility 3+ (log β1 = 22.8, log β2 = is abundant in serum and its two metal binding sites have high affinity for Fe compared to macrocyclic ligands, results in low activation barriers to complexation, resulting in rapid and Ga3+ (log β1 = 20.3, log β2 = 39.6) [61]. Transchelation of 68Ga3+ to endogenous ligands results rates of 44.3) reaction at room temperature. in increased non-target tissue uptake and lower tumour/diseased tissue uptake, decreasing PET It isimage alsoquality. critical that the 68 Ga3+ complex is sufficiently kinetically stable over the period of time required forAs imaging (1–2 h) to withstand by competing endogenous 3,4-HP and THP derivatives can transchelation bind Fe3+ under physiological conditions, with theproteins, resulting such as 3+ in vivo transferrin, and other ligands that compete for [4]. Thecould iron transport protein complexes excreted, it was hypothesised thatGa a THP Ga3+ complex be sufficiently stabletransferrin in 3+ 67Ga3+ (half-life = 78 h), comparing vivo. Mouse biodistribution studies with the γ-emitting isotope, is abundant in serum and its two metal binding sites have high affinity for Fe (log β1 = 22.8, 3+ to endogenous biodistribution of [67βGa(deferiprone) 3] with [67Ga(citrate)3] show that mice 68 administered the log β2 =the 44.3) and Ga3+ (log 1 = 20.3, log β2 = 39.6) [61]. Transchelation of Ga deferiprone complex intravenously have lower 67Ga blood activity one day post-administration than ligands results in increased non-target tissue uptake and lower tumour/diseased tissue uptake, animals administered the citrate complex [37]. Subsequent studies demonstrate that 67Ga: (i) clears decreasing imagefrom quality. morePET rapidly animals administered the deferiprone complex compared to animals 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, 67 Ga3+ (half-life = 78 h), comparing the biodistribution of [67 Ga(deferiprone)3 ] with [67 Ga(citrate)3 ] show that mice administered the deferiprone complex intravenously have lower 67 Ga blood activity one day post-administration than animals administered the citrate complex [37]. Subsequent studies demonstrate that 67 Ga: (i) clears

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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 67 Ga3+ in vivo, with biodistribution of radioactivity modified by N1 substituents [62]. Such results suggest that [67 Ga(deferiprone)3 ] has appreciable stability in vivo, and that 3,4-HPs can effectively compete with endogenous protein ligands for Ga3+ [37,62]. Int. J. Mol. Sci. 2017, 18, 116

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5.2. Tris(hydroxypyridinone) Bioconjugates administered the citrate complex; and

(ii) clears predominantly via a renal pathway in animals administered the the deferiprone complexand [37,62]. N1-functionalised derivatives of pathway 3,4-HP are also administered citrate complex; (ii) clears predominantly via a renal in animals We first reported the utility67Ga of3+ THP-Ac (26,biodistribution Figure 8) ofasradioactivity a basis for highly efficient 68 Ga efficacious at sequestering in vivo, with administered the deferiprone complex [37,62]. N1-functionalised derivatives modified of 3,4-HPbyareN1also labelling under very[62]. mild conditions undertaking side-by-side comparisons 67Ga3+ after substituents results suggest that [67Ga(deferiprone) 3] has appreciable stability in vivo, and efficacious at Such sequestering in vivo, with biodistribution of radioactivity modified by of N1 THP-Ac 68 Ga3+ thatsubstituents 3,4-HPs can[62]. effectively compete with protein ligands for Ga3+ [37,62]. Such results suggest that [67Ga(deferiprone) 3:] has appreciable stability in vivo, and with chelators already commonly used toendogenous complex macrocyclic derivatives DOTA (41) that 3,4-HPs can effectively compete with endogenous protein ligands for (1,4,7-triazacyclononane-1,4,7Ga [37,62]. (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and NOTA (42) 5.2. Tris(hydroxypyridinone) Bioconjugates triacetic acid), and the acyclic chelator HBED (43) (bis(2-hydroxybenzyl)ethylenediaminediacetic acid) 5.2. Bioconjugates WeTris(hydroxypyridinone) first reported the utility of THP-Ac (26, Figure 8) as a basis for highly efficient 68Ga 68 Ga3+ at progressively lower (Figure 9) [13]. Each chelator was reacted with generator-produced labellingWe under mild conditions undertaking side-by-side of THP-Ac with68Ga firstvery reported the utility after of THP-Ac (26, Figure 8) as comparisons a basis for highly efficient chelators already commonly used toeach complex Ga3+corresponding : macrocyclic derivatives DOTA (41) (1,4,7,10chelator concentrations (100 nM–1 10068undertaking µL tocomparisons amounts ofTHP-Ac 10 pmol–100 nmol) labelling under very mildmM, conditions after side-by-side of with 68Ga3+ acid) and NOTA (42) (1,4,7-triazacyclononane-1,4,7chelators already commonly used to complex : macrocyclic derivativeschelators DOTA (41) would (1,4,7,10-maintain (Figure 10), tetraazacyclododecane-1,4,7,10-tetraacetic with the reasoning that the most efficient, rapidly complexing triacetic acid), and the acyclic chelator HBEDacid) (43) (bis(2-hydroxybenzyl)ethylenediaminediacetic tetraazacyclododecane-1,4,7,10-tetraacetic and NOTA (42) (1,4,7-triazacyclononane-1,4,7high labelling efficiency the lowest ligand Optimised reaction 68Ga3+ at conditions acid) (Figureacid), 9)at[13]. chelator wasconcentrations. reacted progressively (as reported triacetic andEach the acyclic chelator HBEDwith (43)generator-produced (bis(2-hydroxybenzyl)ethylenediaminediacetic 68 3+ at progressively in the radiochemical literature) each were employed. At a concentration ofof1010µM, THP-Ac lower chelator concentrations (100chelator nM–1 each 100generator-produced µL corresponding to acid) (Figure 9) [13].for Each chelator wasmM, reacted with Gaamounts 3+ innmol) (Figure 10), with the reasoning the most efficient, rapidly chelators chelator concentrations (100 nM–1 that mM, each 100 µL at corresponding to amounts of 10 complexes 68pmol–100 Galower 5 min in >98% radiochemical yield at pH 6.5 room complexing temperature. Radiochemical would maintain high labelling at the lowest ligand Optimised reaction pmol–100 nmol) (Figure 10), efficiency with the reasoning that the mostconcentrations. efficient, rapidly complexing chelators yields for DOTA are(as >95% at thethe same chelator concentration, but this requires heating at 100 ◦ C conditions reported radiochemical each concentrations. chelator were employed. a would maintain highinlabelling efficiency atliterature) the lowestforligand Optimised At reaction 68 3+ 68Ga3+ in 5 min at pH 4.4. NOTA has ofbeen reported toradiochemical coordinate Gain >98% efficiently at room but at concentration 10reported µM, THP-Ac complexes radiochemical yieldemployed. attemperature, pH 6.5 atAt a conditions (as in the literature) for each chelator were 68Gaare 3+ in>95% room temperature. yields for DOTA the same chelator concentration, but concentration ofRadiochemical 10 temperature, µM, THP-Ac complexes 5 minatin >98% radiochemical yield at pH 6.5 atAt 10 µM, 10 µM concentration at room pH 4.4, radiochemical yields only average 80%. 68Ga3+ efficiently at thisroom requires heating atRadiochemical 100 °C at pH 4.4. NOTA has been coordinate temperature. yields for DOTA arereported >95% at to chelator concentration, but 68 Ga 3+ in HBED is able to complex >96% radiochemical yield atthe pHsame 4.6. Whilst HBED radiochemical 68Ga3+ efficiently room temperature, but atat10 µM at room temperature, 4.4, radiochemical yields at this requires heating 100 °C concentration at pH 4.4. NOTA has been reported pH to coordinate 68 3+ yields underonly acidic conditions are comparable to those of THP-Ac at near neutral pH, HBED forms average 80%. At 10but µM, able to complex Ga temperature, in >96% radiochemical yield at pH 4.6. room temperature, at HBED 10 µMisconcentration at room pH 4.4, radiochemical yields 3+ 68 3+ Whilst HBED radiochemical yields under acidic conditions are comparable to those of THP-Ac at only average At 10 µM, HBED is able[4]. to complex in >96% radiochemical yield atthe pH presence 4.6. geometric isomers when80%. complexed to Ga From aGaregulatory perspective, of nearWhilst neutral pH, HBED forms geometric isomers when complexed to Ga3+ [4]. to From a regulatory HBED radiochemical yields under conditions are the comparable those of THP-Ac different geometric isomers is undesirable, as itacidic is possible that different isomers haveat different perspective, the pH, presence different geometric isomers is complexed undesirable,toasGait3+ is[4]. possible the near neutral HBEDofforms geometric isomers when From athat regulatory pharmacological profiles. different isomersthe have differentofpharmacological profiles. perspective, presence different geometric isomers is undesirable, as it is possible that the 3+

different isomers have different pharmacological profiles.

Figure 9. Chelators for 68Ga3+.

Figure 9. Chelators for 6868Ga3+ . 3+ Figure 9. Chelators for Ga .

Figure 10. Radiolabelling yield versus ligand concentration for 68Ga-DOTA (pH 4.4, 30 min, 100 °C), 68Ga-NOTA (pH 3.6, 10 min, room temperature), 68Ga-HBED (pH 4.6, 68Ga-DOTA 10 min, room Figure 10. Radiolabelling yield versus ligand concentration for 68 (pHtemperature) 4.4, 30 min, and 100 °C), 68Ga-THP, Figure 10. Radiolabelling yield versus ligand concentration for Ga-DOTA (pH 4.4, batch 30 min, 100 ◦ C), 68Ga-NOTA 68Ga-HBED (pH 6.5, min,10 room All experiments were same (pH5 3.6, min, temperature). room temperature), (pH conducted 4.6, 10 min,with roomthe temperature) and 68 Ga-NOTAof(pH 68 6868 Ga eluate. All radiolabelling buffers were 0.2 acetic acid/sodium acetate. Reprinted with 3.6, 10(pH min, temperature), Ga-HBED (pHconducted 4.6, 10 min, room temperature) Ga-THP, 6.5, 5room min, room temperature). AllM experiments were with the same batch permission Berry, D.J.; et al. Efficient bifunctional gallium-68 chelators for positron emissionwith 68 eluate. All radiolabelling buffers were 0.2 M acetic acid/sodium acetate. Reprinted of 68Ga from

and Ga-THP, (pH 6.5, 5 min, room temperature). All experiments were conducted with the same permission from Berry, D.J.; et al. Efficient bifunctional gallium-68 chelators for positron emission batch of 68 Ga eluate. All radiolabelling buffers were 0.2 M acetic acid/sodium acetate. Reprinted with permission from Berry, D.J.; et al. Efficient bifunctional gallium-68 chelators for positron emission tomography: tris(hydroxypyridinone) ligands. Chem. Commun. 2011, 47, 7068–7070. Copyright Royal Society of Chemistry.

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tomography: ligands. Chem. Commun. 2011, 47, 7068–7070. Copyright Royal 13 of 23 Int. J. Mol. Sci. 2017, 18,tris(hydroxypyridinone) 116 Society of Chemistry.

The β-alanine-derived (37)[51], [51],can canbebefunctionalised functionalised bioconjugation The β-alanine-derivedcompound, compound, THP-NH THP-NH22(37) forfor bioconjugation a maleimide [13,16] and isothiocyanates [14,15] (Figure The bifunctional chelators THP-mal viavia a maleimide [13,16] and isothiocyanates [14,15] (Figure 8).8). The bifunctional chelators THP-mal (38), (38), THP-Ph-NCS (39) and THP-NCS (40) have all been conjugated to biomolecules. Amide THP-Ph-NCS (39) and THP-NCS (40) have all been conjugated to biomolecules. Amide conjugation of conjugation (37) to activated carboxylates of peptides and proteins is also a viable THP-NH to THP-NH activated2 carboxylates of peptides and proteins is also a viable conjugation strategy. 2 (37)of conjugation strategy. The protein C2A, containing an engineered cysteine residue, has been conjugated to THP-mal The protein anof engineered has been conjugated to THP-mal (38), resulting in a C2A, singlecontaining equivalent THP-mal cysteine attachedresidue, per protein molecule [13]. The conjugate (38), resulting in a single equivalent of THP-mal attached per protein molecule [13]. The conjugate 68 3+ can be radiolabelled with Ga at pH 5.5 (6 nmol of protein in 100 µL, at a concentration of 60 µM), 3+ at pH 5.5 (6 nmol of protein in 100 µL, at a concentration of 60 µM), can bequantitative radiolabelledlabelling with 68Gaafter giving 5 min. In vivo PET imaging in healthy mice demonstrates that giving quantitative labelling after 5 min. In vivo PET imaging in healthy mice demonstrates that 68 Ga-THP-mal-C2A clears to the kidneys, and does not release any 68 Ga3+ over a 90 min period. 68Ga-THP-mal-C2A clears to the kidneys, and does not release any 68Ga3+ over a 90 min period. In 68 3+ In contrast, in mice administered solutions of unchelated68 Ga , radioactivity is distributed throughout contrast, in mice administered solutions of unchelated Ga3+, radioactivity is distributed throughout the body 90 min post-injection. In competition studies where6868 Ga-THP-Ac is incubated with the body 90 min post-injection. In competition studies where Ga-THP-Ac is incubated with transferrin, [6868Ga(THP-Ac)] remains intact. On the other hand, when6868 Ga-transferrin is incubated transferrin, [ Ga(THP-Ac)] remains intact. On the other hand, when Ga-transferrin is incubated with THP-Ac, 6868Ga is quickly transchelated to form a complex with THP-Ac [13]. with THP-Ac, Ga is quickly transchelated to form a complex with THP-Ac [13]. THP-Ph-NCS been attached attachedtotothe thecyclic cyclicpentapeptide, pentapeptide, c(RGDfK) THP-Ph-NCSand andTHP-NCS THP-NCS have have both both been c(RGDfK) viavia lysine sidechains [14]. The “RGD” peptide motif targets α β integrin receptors that are expressed lysine sidechains [14]. The “RGD” peptide motif targets αvvβ3 3integrin receptors that are expressed on on thethe surface as well wellas asinflamed inflamedtissue tissueand and blood vessels undergoing surfaceofofmany manymetastatic metastatictumour tumour cells, cells, as blood vessels undergoing angiogenesis. Both THP-Ph-NCS-RGD (41, Figure 11) and THP-NCS-RGD (42, Figure angiogenesis. Both THP-Ph-NCS-RGD (41, Figure 11) and THP-NCS-RGD (42, Figure 11)11) cancan be be 68 3+ eluate at conjugate concentrations of 4–5 µM, or total 68 3+ radiolabelled with generator-produced Ga radiolabelled with generator-produced Ga eluate at conjugate concentrations of 4–5 µM, or total amounts of of 10–12 nmol, giving radiochemical yields of 95%–99%. TheseThese reactions proceed in aqueous amounts 10–12 nmol, giving radiochemical yields of 95%–99%. reactions proceed in solution, 5.5–6.5 pH in less thanin 5 min ambient to give a single product corresponding aqueouspH solution, 5.5–6.5 less at than 5 min temperature at ambient temperature to give a single product to either [68Ga(THP-Ph-NCS-RGD)] or [68Ga(THP-NCS-RGD)]. to corresponding either [68 Ga(THP-Ph-NCS-RGD)] or [68 Ga(THP-NCS-RGD)].

Figure11. 11. Representative maximum intensity projection of Balb/c bearing U87MG Figure RepresentativePET PET maximum intensity projection of nu/nu Balb/cmice nu/nu mice bearing Ga(THP-Ph-NCS-RGD)]; and (b)and tumourstumours on right at 1 at h post-injection of: (a) U87MG on flank right flank 1 h post-injection of: [68(a) [68 Ga(THP-Ph-NCS-RGD)]; 68 Ga(THP-NCS-RGD)]. BlackBlack arrow,arrow, tumour; grey arrow, arrow, kidneys and kidneys liver. (b)[68[Ga(THP-NCS-RGD)]. tumour; greybladder; arrow, dashed bladder; dashed arrow, Reprinted with permission a Creative Commons Attribution (CC-BY) License from Ma, M.T.; and liver. Reprinted with under permission under a Creative Commons Attribution (CC-BY) License et al. New tris(hydroxypyridinone) bifunctional chelators containing isothiocyanate groups provide from Ma, M.T.; et al. New tris(hydroxypyridinone) bifunctional chelators containing isothiocyanate 68Ga3+. Bioconjugate Chem. a versatile platform for rapid one-step and PET labelling imaging with groups provide a versatile platform forlabelling rapid one-step and PET imaging with 68 Ga3+ . 2016, 27, 309–318. Copyright American Chemical Society. Bioconjugate Chem. 2016, 27, 309–318. Copyright American Chemical Society.

Both [68 Ga(THP-Ph-NCS-RGD)] and [68 Ga(THP-NCS-RGD)] retain affinity for αv β3 integrin receptors in vitro and in vivo [14]. PET imaging and biodistribution studies of mice bearing U87MG

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Both [68Ga(THP-Ph-NCS-RGD)] and [68Ga(THP-NCS-RGD)] retain affinity for αvβ3 integrin receptors in vitro and in vivo [14]. PET imaging and biodistribution studies of mice bearing U87MG tumours demonstratethatthat [68Ga(THP-Ph-NCS-RGD)] and [68Ga(THP-NCS-RGD)]: (i) tumours demonstrate bothboth [68 Ga(THP-Ph-NCS-RGD)] and [68 Ga(THP-NCS-RGD)]: (i) selectively selectively αvβreceptors; 3 integrinand receptors; (ii)the clear from the 1–2 body within 1–2 h predominantly post-injection, target αv β3target integrin (ii) clearand from body within h post-injection, predominantly a renal route (Figure 11). via a renal routevia (Figure 11). The The molecular molecular imaging imaging agent, agent, [[6868Ga(DOTA-TATE)] Ga(DOTA-TATE)] isis routinely routinely used used for for PET PET imaging imaging of of neuroendocrine neuroendocrine tumours. tumours. DOTA-TATE DOTA-TATE (43, (43, Figure Figure 12) 12) isis aa conjugate conjugate of of the the macrocyclic macrocyclic chelator chelator DOTA DOTA (27, Figure Figure 9) and and Tyr Tyr33-octreotate, -octreotate, an an eight eight amino amino acid acid cyclic cyclic peptide peptide that that targets targets somatostatin somatostatin 22 receptors of neuroendocrine neuroendocrine tumours. tumours. Using receptors (SSTR) (SSTR) overexpressed overexpressed on the surface of Using THP-NCS, THP-NCS, THP-TATE to to RGD conjugates, THP-TATE cancan be THP-TATE (44, (44, Figure Figure 12) 12)has hasbeen beensynthesised synthesised[15]. [15].Similar Similar RGD conjugates, THP-TATE radiolabelled at room temperature at concentrations of 5 µM nmol conjugate) at near neutral be radiolabelled at room temperature at concentrations of (10 5 µM (10ofnmol of conjugate) at near pH in less 2 min. In contrast, DOTA-TATE requires temperatures of 80–90 °C atofpH 3–5, ◦with neutral pHthan in less than 2 min. In contrast, DOTA-TATE requires temperatures 80–90 C at reaction of 5–10 times min, and in clinical radiolabelling protocols, post-synthetic purifications pH 3–5, times with reaction of 5–10 min, and in clinical radiolabelling protocols, post-synthetic procedures invariablyare employed [15]. PET imaging andimaging biodistribution experimentsexperiments show that purificationsare procedures invariably employed [15]. PET and biodistribution 68Ga(THP-TATE)] [show has similar in SSTR-positive tumours to to thetheclinical that [68 Ga(THP-TATE)] has uptake similar uptake in SSTR-positive tumours clinical standard standard 68 68 68 68 [[ Ga(DOTA-TATE)]. Ga(DOTA-TATE)].[[ Ga(THP-TATE)] Ga(THP-TATE)]clears clearsvia viaaarenal renalpathway, pathway, but but significantly significantly higher higher kidney kidney 68Ga(THP-TATE)] and and liver liver retention retention of [68 Ga(THP-TATE)] is is observed observed (Figure (Figure 12). In In PET PET images images of of mice mice bearing bearing 68Ga(THP-TATE)], SSTR2-positive SSTR2-positive tumours tumours administered administered [68 Ga(THP-TATE)], tumours tumours can be clearly delineated.

Figure 12. 12. Representative Representative PET PET maximum nu/numice mice bearing bearing AR47J AR47J Figure maximum intensity intensity projections projections of of Balb/c Balb/c nu/nu 68 Ga(DOTA-TATE)]; and (b) [68 Ga(THP-TATE)]. Black arrow, tumours on the right flank 1 h PI of: (a) [ 68 68 tumours on the right flank 1 h PI of: (a) [ Ga(DOTA-TATE)]; and (b) [ Ga(THP-TATE)]. Black tumour; grey arrow, dashed dashed arrow, kidneys. Reprinted with permission under a Creative arrow, tumour; grey bladder; arrow, bladder; arrow, kidneys. Reprinted with permission under a 68 Ga-labelling and PET Commons Attribution (CC-BY) License from Ma, M.T.; et al. Rapid kit-based Creative Commons Attribution (CC-BY) License from Ma, M.T.; et al. Rapid kit-based 68Ga-labelling 3 -octreotate: a preliminary comparison with DOTA-Tyr3 -octreotate. EJNMMI imaging THP-Tyr 3-octreotate: a preliminary comparison with DOTA-Tyr3-octreotate. and PET with imaging with THP-Tyr Res. 2015, 5, 52. Copyright. EJNMMI Res. 2015, 5, 52. Copyright.

In all all of of these radiolabelled radiolabelled derivatives, derivatives, the the radiotracer radiotracer demonstrates demonstrates high high serum serum stability stability and and In 68 3+ 68 3+ in vivo vivo stability, stability, with no evidence of dissociation of Ga Ga from fromthe theTHP THPchelator. chelator. in 5.3. Preparation, Preparation, Radiolabelling Radiolabelling and and In In Vitro Vitro Uptake Uptake of of aa Trastuzumab Trastuzumab Immunoconjugate 5.3. Immunoconjugate New protein receptors with highhigh affinity and specificity have similar utility New protein constructs constructsthat thattarget target receptors with affinity and specificity have similar to peptides in molecular imaging. imaging. For proteins short circulation and rapid accumulation utility to peptides in molecular Forwith proteins with short times circulation times and rapid 68 Ga PET imaging 68 at target tissue, will be clinically viable, provided that appropriate radiolabelling accumulation at target tissue, Ga PET imaging will be clinically viable, provided that appropriate

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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 Int. J. Mol. Sci. 2017, 18, 116 15 of 23 of Ga3+ are unstable [65]. Trastuzumab is aprotocols therapeutic monoclonal antibody used for treatment of to breast radiolabelling are available. The sensitivity of proteins’ tertiary structures acidiccancer. pH and It targets extremes of temperature mild radiolabelling conditions, problems whenasusing the human epidermal growth requires factor receptor 2 (HER2). In vivo,raising antibodies such trastuzumab 3+ chelators. HBED, NOTA and DFO are capable of radiolabelling Ga3+ isotopes at conventional Ga require extended periods of time (6 to 48 h) to clear circulation and accumulate at HER2-positive target room temperature, but HBED [63] and NOTA [64] require low pH for reactions to proceed tissue, and given the short half-life of 68 Ga, it is likely impractical to use 68 Ga-labelled trastuzumab quantitatively, and DFO complexes of Ga3+ are unstable [65]. to image HER2 expression. Nonetheless, it is instructive to radiolabel Trastuzumab is a therapeutic monoclonal antibody used for treatmentTHP-PhNCS-trastuzumab of breast cancer. It the ahuman epidermalconjugate growth factor 2 (HER2). In vivo, such to assess targets whether THP protein thatreceptor is sensitive to acidic pHantibodies (less than pHas 5) can be trastuzumab periods of time to 48 to clear acirculation and accumulate at injection radiolabelled rapidlyrequire underextended mild conditions (pH (66–7) to h) provide formulation suitable for HER2-positive target tissue, and given the short half-life of 68Ga, it is likely impractical to use without further purification. 68Ga-labelled trastuzumab to image HER2 expression. Nonetheless, it is instructive to radiolabel The bifunctional chelator THP-PhNCS hasa THP beenprotein conjugated to that the ismonoclonal antibody THP-PhNCS-trastuzumab to assess whether conjugate sensitive to acidic pH (mAb), (less than pH 5) can bea radiolabelled rapidly under mildcontaining conditions (pH 6–7)reagents to provideunder a trastuzumab by incubating HEPES buffered solution both mild formulation suitable for injection without further purification. conditions [66,67]. The immunoconjugate, THP-PhNCS-trastuzumab has been isolated using solid The bifunctional chelator THP-PhNCS has been conjugated to the monoclonal antibody (mAb), phase size exclusion chromatography [66,67]. Addition of generator-produced 68 Ga3+ (~10 MBq, trastuzumab by incubating a HEPES buffered solution containing both reagents under mild 50 µL, aqueous 0.1[66,67]. M HCl) solutions of THP-PhNCS-trastuzumab (50 isolated µL, 0.65 mgsolid ·mL−1 , 0.2 M conditions The to immunoconjugate, THP-PhNCS-trastuzumab has been using ammonium acetate, pH 6–7)chromatography results in formation of [68 Ga(THP-PhNCS-trastuzumab)], with specific phase size exclusion [66,67]. Addition of generator-produced 68Ga3+ (~10 MBq, −1, 0.2 M − 1 0.1 M HCl) to solutions of THP-PhNCS-trastuzumab (50as µL, 0.65 mg·mLby activities 50 of µL, up aqueous to 50 MBq ·nmol and radiochemical yields of 99% measured size exclusion ammonium acetate, pH 6–7) results in formation of [68Ga(THP-PhNCS-trastuzumab)], with specific HPLC (Figure 13a, red trace). The major signal at 7.98 min in the radiochromatogram matches the activities of up to 50 MBq·nmol−1 and radiochemical yields of 99% as measured by size exclusion retention HPLC time (Figure of native andsignal the minor signal atradiochromatogram 6.83 min is typical of formation of 13a, trastuzumab, red trace). The major at 7.98 min in the matches the 68 Ga3+ solutions to samples containing radiolabelled immunoconjugate aggregates [66]. Addition of retention time of native trastuzumab, and the minor signal at 6.83 min is typical of formation of radiolabelled immunoconjugate aggregates [66]. Addition of 68antibody Ga3+ solutions to samples native, unconjugated trastuzumab do not provide labelled (Figure 13a,containing black trace)—the 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 mobile phase in these experiments contains ethylenediaminetetraacetate (EDTA),68and the single− in the radiochromatogram with a retention time of 12.08 min corresponds to [ Ga(EDTA)] . signal in the radiochromatogram with a retention time of 12.08 min corresponds to [68Ga(EDTA)]−.

Figure 13. (a) Size exclusion radio-HPLC traces of [68Ga(THP-PhNCS-trastuzumab)] (red) and

Figure 13. (a) Size exclusion radio-HPLC traces of [68 Ga(THP-PhNCS-trastuzumab)] (red) [68Ga(EDTA)]− (black); and (b) In vitro uptake of [68Ga(THP-PhNCS-trastuzumab)] and 68 − (black); and (b) In vitro uptake of [68 Ga(THP-PhNCS-trastuzumab)] and and [ Ga(EDTA)] [68Ga(THP-PhNCS-trastuzumab)] in the presence of an inhibitory concentration of trastuzumab, and 68 68Ga3+ in HER2-positive HCC1954 cells, and [68Ga(THP-PhNCS-trastuzumab)] in HER2-negative [ Ga(THP-PhNCS-trastuzumab)] in the presence of an inhibitory concentration of trastuzumab, and 68 Ga3+ in HER2-positive HCC1954 cells, and [68 Ga(THP-PhNCS-trastuzumab)] in HER2-negative A375 cells, after 30 min incubation. Uptake is expressed as a percentage of added radioactivity (AR)/1 × 106 cells (n = 3, error bars correspond to standard error of the mean).

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A375 cells, after 30 min incubation. Uptake is expressed as a percentage of added radioactivity (AR)/1 × 106 cells (n = 3, error bars correspond to standard error of the mean). Int. J. Mol. Sci. 2017, 18, 116

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To establish that the labelled immunoconjugate, [ Ga(THP-PhNCS-trastuzumab)], retains affinity for HER2 receptors, [68Ga(THP-PhNCS-trastuzumab)] has been incubated with To establish that the labelled immunoconjugate, [68 Ga(THP-PhNCS-trastuzumab)], retains HER2-positive HCC1954 cells and HER2-negative A375 cells. Additionally, a blockade experiment affinity for HER2 receptors, [68 Ga(THP-PhNCS-trastuzumab)] has been incubated with HER2-positive where [68Ga(THP-PhNCS-trastuzumab)] is incubated with HCC1954 cells in the presence of a large HCC1954 cells and HER2-negative A375 cells. Additionally, a blockade experiment where excess of native trastuzumab has been undertaken. Uptake of [68Ga(THP-PhNCS-trastuzumab)] in [68 Ga(THP-PhNCS-trastuzumab)] is incubated with HCC1954 cells in the presence of a large excess of HCC1954 cells measures 35.12 ± 0.53 percentage68 of added radioactivity per one million cells native trastuzumab has been undertaken. Uptake of [ Ga(THP-PhNCS-trastuzumab)] in HCC1954 cells (%AR/million cells), whereas uptake in A375 cells measures 1.98% ± 0.02 %AR/million cells and measures 35.12 ± 0.53 percentage of added radioactivity per one million cells (%AR/million cells), whereas uptake in the trastuzumab blockade measures 0.30% ± 0.03 %AR/million, indicating that uptake of uptake in A375 cells measures 1.98% ± 0.02 %AR/million cells and uptake in the trastuzumab blockade [68Ga(THP-PhNCS-trastuzumab)] is receptor-mediated, and that in vitro [68Ga(THP-PhNCS68 measures 0.30% ± 0.03 %AR/million, indicating that uptake of [ Ga(THP-PhNCS-trastuzumab)] is trastuzumab)] retains affinity for68HER2-expressing cells (Figure 13b). Addition of a solution receptor-mediated, and that in vitro [ Ga(THP-PhNCS-trastuzumab)] retains affinity for HER2-expressing containing 68Ga3+ to HCC1954 cells does not result in68significant uptake of activity (1.09% ± 0.05 cells (Figure 13b). Addition of a solution containing Ga3+ to HCC1954 cells does not result in %AR/million cells). significant uptake of activity (1.09% ± 0.05 %AR/million cells). 3+ radiolabelling of proteins under mild, aqueous Thus, THP enables efficient and rapid 6868Ga3+ Thus, THP enables efficient and rapid Ga radiolabelling of proteins under mild, aqueous conditions. This radiolabelling strategy will have utility for 68 Ga PET imaging of proteins such as conditions. This radiolabelling strategy will have utility for 68 Ga PET imaging of proteins such as fusion proteins and antibody fragments that have shorter clearance times than full length antibodies. fusion proteins and antibody fragments that have shorter clearance times than full length antibodies. 68

5.4. 5.4. Other Other THP THP Derivatives Derivatives Alternative 3 (45) have been reported for complexation of the Alternative THP THP chelators chelatorssuch suchasasNTP(PrHP) NTP(PrHP) 3 (45) have been reported for complexation of 67Ga 67 [68,69]. In these derivatives (Figure 14), the 3,4-HP groupsgroups are attached to the SPECT isotope, the SPECT isotope, Ga [68,69]. In these derivatives (Figure 14), the 3,4-HP are attached 1 ring atoms, tripodal scaffold via the N and the chelating O atoms are meta and para to the linker. 1 to the tripodal scaffold via the N ring atoms, and the chelating O atoms are meta and para to the Such topology can lead to lead formation of eitherofdinuclear structures or structures where one 3,4-HP linker.a Such a topology can to formation either dinuclear structures or structures where one unit has dissociated [55]. Both species are generally more kinetically labile than species of a 1:1 3,4-HP unit has dissociated [55]. Both species are generally more kinetically labile than species of stoichiometry and are ideal for for in vivo applications. The presence a 1:1 stoichiometry and not are not ideal in vivo applications. The presenceofofmore morethan thanone one complex complex structure also undesirable, undesirable, as as these these species species can can have have different different biological biological behaviours. behaviours. NTP(PrHP) NTP(PrHP)3,, structure is is also 3 with its tripodal topology centred on a tertiary amine rather than carbon, is not readily adapted to with its tripodal topology centred on a tertiary amine rather than carbon, is not readily adapted to use 67Ga3+ indicate that use a bifunctional chelator. SPECT imaging and biodistribution studies as a as bifunctional chelator. SPECT imaging and biodistribution studies with 67with Ga3+ indicate that most 67 most [ Ga-NTP(PrHP) 3] rapidly clears circulation within 1 h, and remaining [67Ga-NTP(PrHP)3] 67 [67 Ga-NTP(PrHP) 3 ] rapidly clears circulation within 1 h, and remaining [ Ga-NTP(PrHP)3 ] does not 67Ga3+ over 24 h [69]. does not67release release Ga3+ over 24 h [69].

Figure 14. 14. Structure of NTP(PrHP) NTP(PrHP)3.. Figure Structure of 3 89 4+ 6. 6. Hydroxypyridinones Hydroxypyridinones for for Radiolabelling Radiolabelling 89Zr Zr4+ We havealso alsoinvestigated investigatedTHP-Ac THP-Ac and a THP-mal-trastuzumab conjugate for radiolabelling We have and a THP-mal-trastuzumab conjugate for radiolabelling with 89Zr4+) (half-life = 78 h) [16]. The Zr4+ ion is oxophilic, with the long-lived PET isotope, zirconium-89 ( 89 4+ 4+ the long-lived PET isotope, zirconium-89 ( Zr ) (half-life = 78 h) [16]. The Zr ion is oxophilic, and and bifunctional hexadentate derivatives are most commonly utilised for incorporating Zr4+ 89 Zr4+89into bifunctional hexadentate DFODFO derivatives are most commonly utilised for incorporating

antibodies. There is evidence that [89 Zr(DFO)]+ derivatives are unstable in vivo, with observations of

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into antibodies. There is evidence that [89Zr(DFO)]+ derivatives are unstable in vivo, with 89 Zr accumulation in accumulation the skeleton over the course of over a week Here, is assumed to observations of 89Zr in the skeleton the[16]. course ofskeletal a weekuptake [16]. Here, skeletal 89 Zr4+ from a chelator-conjugate. 89 4+ be indicative of dissociation of uptake is assumed to be indicative of dissociation of Zr from a chelator-conjugate. 89 4+ at room Both THP-Ac and THP-mal-trastuzumab can be be radiolabelled radiolabelled with with 89Zr 4+ at room temperature and near neutral pH in quantitative yield. For THP-Ac, this was achieved at 1 mM concentration and near neutral pH in quantitative yield. For THP-Ac, this was achieved at 1 mM concentration and and 10 µL volume, corresponding to 10 nmol of ligand. Competition experiments indicated 10 µL volume, corresponding to 10 nmol of ligand. Competition experiments indicated a 4+ coordination to THP-Ac over DFO. This is consistent with a thermodynamic preference thermodynamic preference for for Zr4+Zr coordination to THP-Ac over DFO. This is consistent with higher higher ion metal ion affinities of 3,4-HPs compared to hydroxamates. However, experiments in metal affinities of 3,4-HPs compared to hydroxamates. However, experiments in which 89 89 4+ 89 89 which Zr-THP-mal-trastuzumab was administered to healthy animals indicate thatinin vivo, Zr Zr-THP-mal-trastuzumab was administered to healthy animals indicate that Zr4+ dissociates from from THP-mal-trastuzumab THP-mal-trastuzumab within within 48 48 h, h, with with activity activity accumulating accumulating in in the skeleton dissociates 89 + complex is inferior to that of the [89 + 89 + (Figure 15). In In vivo stability of the [ Zr(THP)] of the [ complex is of the [89Zr(DFO)]+ complex, possibly a result of the different chelator topologies.

Figure 15. PET scans of C57Bl/6j mice administered: (a) [89Zr(oxalate) 4]4−; (b) Figure 15. PET scans of C57Bl/6j mice administered: (a) [89 Zr(oxalate)4 ]4− ; 89Zr-THP-mal-trastuzumab; and (c) 89Zr-DFO-mal-trastuzumab. For the animal administered 89 89 (b) Zr-THP-mal-trastuzumab; and (c) Zr-DFO-mal-trastuzumab. For the animal administered 4− 98% of the injected dose remains 4 h post-injection, and is associated predominantly with [[89 89Zr(ox) Zr(ox)44] ]4− 98% of the injected dose remains 4 h post-injection, and is associated predominantly with 89Zr-THP-mal-trastuzumab, the skeleton is clearly visible the skeleton. For animal administered administered 89 the skeleton. For the the animal Zr-THP-mal-trastuzumab, the skeleton is clearly visible 4+ from THP-mal-trastuzumab over Zr4+ from days post-injection, post-injection, indicative indicative of of dissociation dissociation of of8989Zr from three three days from THP-mal-trastuzumab over 89Zr-DFO-trastuzumab, where the blood time. administered 89 time. This This is is in in marked marked contrast contrast to to the the animal animal administered Zr-DFO-trastuzumab, where the blood pool to seven seven days days post-injection. post-injection. Reprinted pool remains remains visible visible out out to Reprinted with with permission permission under under aa Creative Creative Commons Attribution(CC-BY) (CC-BY)License License from al. Tripodal tris(hydroxypyridinone) Commons Attribution from Ma,Ma, M.T.;M.T.; et al. et Tripodal tris(hydroxypyridinone) ligands 89Zr4+: comparison with desferrioxamine-B. Dalton 89 4+ ligands for immunoconjugate PET imaging with for immunoconjugate PET imaging with Zr : comparison with desferrioxamine-B. Dalton Trans. Trans. 2015, 44, 4884–4900. Copyright Royal Society of Chemistry. 2015, 44, 4884–4900. Copyright Royal Society of Chemistry.


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4+ to eight donordonor atoms atoms in its coordination sphere, andsphere, these coordination Zr4+ can can accommodate accommodateupup to eight in its coordination and these 89 Zr(THP)]+ in 89 + vivo. requirements are likely to be a factor in the instability of [ The presence of coordination requirements are likely to be a factor in the instability of [ Zr(THP)] in vivo. The two coordination sites unoccupied by THP chelator allows coordination of endogenous ligands, and presence of two coordination sites unoccupied by THP chelator allows coordination of endogenous providesand pathways to transmetallation/ligand exchange. exchange. ligands, provides pathways to transmetallation/ligand It is possible possible that that aa tetrakis(3,4-hydroxypyridinone) tetrakis(3,4-hydroxypyridinone) ligand ligand could could impart impart greater greater inin vivo butthere thereareare reports of ligands such ligands date.researchers Other researchers have studied stability, but no no reports of such to date.toOther have studied octadentate 89 Zr4+ incorporating 1,2-HPs and 3,2-HPs. 89 4+ octadentate tetrakis(hydroxypyridinone) derivatives for tetrakis(hydroxypyridinone) derivatives for Zr incorporating 1,2-HPs and 3,2-HPs. The The bifunctional open chain (1,2-HP) Figure 16)can: can:(i)(i)coordinate coordinateZr Zr4+4+ininan an octadentate octadentate bifunctional open chain (1,2-HP) 4 chelator (46,(46, Figure 16) 4 chelator environment; and (ii) be -trastuzumab can environment; be conjugated conjugated to to trastuzumab. trastuzumab. Immunoconjugate Immunoconjugate (1,2-HP) (1,2-HP)44-trastuzumab 89Zr be radiolabelled with 89 Zr4+4+ atat room room temperature, temperature, and and PET PET imaging imaging and biodistribution studies demonstrate high in vivo stability of the complex, with concomitant high tumour uptake [70].

Figure 16. Structures of octadentate (HP)4 derivatives for 89Zr4+ immunoconjugate Figure 16. Structures of octadentate (HP)4 derivatives evaluatedevaluated for 89 Zr4+ immunoconjugate radiolabelling. radiolabelling.

Bifunctional macrobicyclic octadentate (3,2-HP) (47, Figure 16) has also been derivatised for Bifunctional macrobicyclic octadentate (3,2-HP)44 (47, Figure 16) has also been derivatised for conjugation to trastuzumab [71]. The coordination environment of the Zr4+ complex is not well defined, conjugation to trastuzumab [71]. The coordination environment of the Zr4+ complex is not well and chromatographic analysis indicates that multiple Zr-bound (3,2-HP)4 species form under the mild defined, and chromatographic analysis indicates that multiple Zr-bound (3,2-HP)4 species form reaction conditions described (room temperature, 15 min incubation). PET imaging and biodistribution under the mild reaction conditions described (room temperature, 15 min incubation). PET imaging studies in mice using 89 Zr-labelled (3,2-HP) 4 -antibody conjugates suggest that at least one form of 89Zr-labelled and biodistribution studies in mice using (3,2-HP) 4-antibody conjugates suggest that at 89 Zr4+ -(3,2-HP) is unstable in vivo, as significantly higher bone uptake is observed for (3,2-HP) 4 89 4+ least one form of Zr -(3,2-HP)4 is unstable in vivo, as significantly higher bone uptake is observed4 conjugates compared to DFO conjugates. This highlights the difficulty in interpreting in vivo results for (3,2-HP)4 conjugates compared to DFO conjugates. This highlights the difficulty in interpreting in based on radiolabelled compounds that contain metal complexes in more than one conformation. 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 7. Concluding Remarks including the proton and Fe3+ affinities, and in vivo distribution and reactivity towards endogenous Derivatisation of 3,4-HPs via substitution of ring protons allows for tailoring of chelator enzymes. 3,4-HPs can be further functionalised with fluorescent tags and biologically active motifs, properties including the proton and Fe3+ affinities, and in vivo distribution and reactivity towards and can be incorporated into chelators of higher denticity. Hexadentate THP chelators based on endogenous enzymes. 3,4-HPs can be further functionalised with fluorescent tags and biologically 3,4-HPs possess extraordinarily high pFe3+ values, and are very potent Fe3+ scavengers. As Fe3+ and active motifs, and can be incorporated into chelators of higher denticity. Hexadentate THP chelators Ga3+ have similar coordination preferences, THP chelators are ideal candidates for development of 3+ based on 3,4-HPs possess extraordinarily high pFe values, and are very potent Fe3+ scavengers. As PET radiopharmaceuticals based on 68 Ga3+ , and direct translation of this chemistry has enabled rapid 3+ 3+ Fe and Ga have similar coordination preferences, THP chelators are ideal candidates for development of bioconjugates of THP chelators for 68 Ga PET imaging. development of PET radiopharmaceuticals based on 68Ga3+, and direct translation of this chemistry Simplicity of radiolabelling with minimal need for complex equipment and radiochemical has enabled rapid development of bioconjugates of THP68chelators for 68Ga PET imaging. expertise is likely to be a key to the wider availability of Ga PET, and this is afforded by appropriate Simplicity of radiolabelling with minimal need for complex equipment and radiochemical design of a 68 Ga chelator. THP derivatives fulfil these requirements whereas other established chelator 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

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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 68 Ga3+ 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 68 Ga-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. Acknowledgments: Ruslan Cusnir acknowledges the Swiss National Science Foundation for an Early Postdoc Mobility Fellowship (P2LAP3-168441). Cinzia Imberti acknowledges the National Institute for Health Research Biomedical Research Centre for a PhD studentship. This research was supported by the Centre of Excellence in Medical Engineering Centre funded by the Wellcome Trust and the Engineering and Physical Sciences Research Council (WT088641/Z/09/Z), the King’s College London and University College London Comprehensive Cancer Imaging Centre funded by Cancer Research UK and the Engineering and Physical Sciences Research Council in association with the Medical Research Council and Department of Health (England), and by the National Institute for Health Research Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the National Health Service, the National Institute for Health Research or the Department of Health. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations Bn DFO EDTA GIT HER2 HP HPLC PET RGD TATE THP SSTR2

Benzyl Deferrioxamine B Ethylenediaminetetraacetate Gastrointestinal tract Human epidermal growth factor receptor 2 Hydroxypyridinone High performance liquid chromatography Positron Emission Tomography Cyclic RGDfK peptide Octreotate Tris(hydroxypyridinone) Somatostatin 2 receptor

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