Inorganic Chemistry of the Tripodal Picolinate Ligand Tpaa with Gallium(III) and Radiolabeling with Gallium-68

We report here the improved synthesis of the tripodal picolinate chelator Tpaa, with an overall yield of 41% over five steps, in comparison to the previously reported 6% yield. Tpaa was investigated for its coordination chemistry with Ga(III) and radiolabeling properties with gallium-68 (68Ga). The obtained crystal structure for [Ga(Tpaa)] shows that the three picolinate arms coordinate to the Ga(III) ion, fully occupying the octahedral coordination geometry. This is supported by 1H NMR which shows that the three arms are symmetrical when coordinated to Ga(III). Assessment of the thermodynamic stability through potentiometry gives log KGa-Tpaa = 21.32, with a single species being produced across the range of pH 3.5–7.5. Tpaa achieved >99% radiochemical conversion with 68Ga under mild conditions ([Tpaa] = 6.6 μM, pH 7.4, 37 °C) with a molar activity of 3.1 GBq μmol–1. The resulting complex, [68Ga][Ga(Tpaa)], showed improved stability over the previously reported [68Ga][Ga(Dpaa)(H2O)] in a serum challenge, with 32% of [68Ga][Ga(Tpaa)] remaining intact after 30 min of incubation with fetal bovine serum.


■ INTRODUCTION
Generator-produced gallium-68 ( 68 Ga) is an exciting isotope for positron emission tomography (PET), 1,2 a highly sensitive imaging modality.The benchtop production of 68 Ga using a generator has the potential for application in the kit-type production of radiotracers for medical application, as has been seen for the widely popular technetium-99m. 3This would enable the realization of PET in hospitals and other institutes without direct access to the extensive infrastructure required for other radioisotopes, such as fluorine-18.
The most widely used chelate for 68 Ga is DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid, Figure 1). 4,5onjugates of DOTA have been applied to 68 Ga PET of a variety of targets, 6,7 with particular success seen for somatostatin targeting probes such as DOTATATE. 8However, radiolabeling conditions for DOTA with 68 Ga are harsh, requiring acidic conditions (pH 4.0) and high temperatures (80 °C) for efficient radiolabeling, due to the poor size match of the cavity of the DOTA ligand for Ga(III) coordination. 4,9,10−15 The choice of chelator has been reported to have an impact upon the localization and clearance of the radiotracer; 16−21 therefore, having a library of efficient chelators will aid in the rapid development of novel radiotracers with optimized uptake in target tissues.
To contribute to a library of chelators that can be labeled with 68 Ga under mild conditions, we have recently reported the complexation of 68 Ga by the tripodal picolinate chelate Dpaa (6,6′-{[(carboxymethyl)azanediyl]bis(methylene)}dipicolinic acid, Figure 1) and bifunctional derivatives. 22,23This system was also reported by the Orvig group. 24While Dpaa could be radiolabeled at neutral pH, the serum stability of the resulting complex was unsatisfactory for in vivo imaging, with [ 67/68 Ga]-[Ga(Dpaa)(H 2 O)] being reported as being only 58% intact after 2 h in 50% human serum 24 and being completely decomplexed within 30 min in 90% fetal bovine serum. 22−27 The linear chelator dedpa (6,6′-{[ethane-1,2-diylbis(azanediyl)]bis(methylene)}dipicolinic acid, Figure 1 and Table 1) was shown to label at very low ligand concentrations under acidic conditions, 25 with the resulting complex remaining 77.8% intact after 2 h in 50% human serum. 26A more rigid version of this chelator, CHXdedpa (6,6′-{[((1R,2R)-cyclohexane-1,2-diyl)bis-(azanediyl)]bis(methylene)}dipicolinic acid, Figure 1), re-quired slightly higher ligand concentrations for high radiochemical conversion (RCC) but yielded a complex that was more resistant to decomplexation, with 90.5% [ 67 Ga][Ga-(CHXdedpa)] remaining intact after 2 h in 50% human serum. 26Increasing the number of coordinating atoms to 8, by adding two acetate arms to form CHXoctapa (6,6′-{[((1R,2R)-cyclohexane-1,2-diyl)bis({carboxymethyl}azanediyl)]bis(methylene)}dipicolinic acid, Figure 1), reduced the stability of the resulting 67 Ga complex to 74.7% after 2 h in 50% human serum. 26hese results encouraged us to develop chelators based upon the Dpaa system to improve the stability of the 68 Ga complex upon exposure to serum.In the crystal structure of [Ga(Dpaa)(H 2 O)], the central amine of the Dpaa ligand was shown not to coordinate to the Ga(III) center, 22,24 resulting in one of the coordination sites of the metal being occupied by water, and further evidence of this was seen through potentiometry. 22,24While examples of stable 68 Ga systems featuring coordinated water or hydroxide molecules have been reported, 29,30 in the case of the Ga-Dpaa system this is a potential cause of the low stability of the complex in biological media.With a view to developing the tripodal picolinate family of ligands further, we have investigated a picolinate chelate with an increased number of coordinating atoms, Tpaa (6,6′,6″-(nitrilotris(methylene))tripicolinic acid, Figure 1).These additional coordinating atoms should compensate for the noncoordinating central amine while retaining the favorable radiolabeling properties of the picolinate functionalities.
Complexes of Tpaa have been previously reported with a variety of other metals; this ligand has been applied to the chelation of Pb 2+ , 31 Mn 2+ , 32,33 Ca 2+ , 31−35 and a series of Ln 3+ ions (Ln = La, Pr, Nd, Eu, Gd, Tb, Ho, Tm, Yb, Lu), 34,35 resulting in complexes with coordination numbers from 6 to 10, with the metal ion being coordinated by 5−7 ligand atoms.−35 We herein report and characterize the novel Ga(III) complex with Tpaa.The Tpaa ligand should form a [Ga(Tpaa)] complex where the central Ga(III) ion is bound solely through the picolinate pendant arms and the amine  group serves as a scaffold arranging all the donor atoms to form a coordination polyhedron close to the ideal octahedral geometry.The incorporation of an additional picolinate arm should increase the stability of the complex in comparison with the previously reported [Ga(Dpaa)(H 2 O)] and improve its potential to be used as a 68 Ga ligand.

■ RESULTS AND DISCUSSION
Ligand and Complex Synthesis.Tpaa was synthesized in two steps from a previously described precursor, ethyl 6-(chloromethyl)picolinate (3, Scheme 1; for a modified synthesis, see the Supporting Information); 36 reaction with ammonia in acetonitrile followed by deprotection under acidic conditions yielded Tpaa in a 71% yield over two steps.This results in an overall yield of 41% from commercially available 2,6-pyridinedicarboxylic acid in five steps, 36,37 in contrast to an overall yield of 6% over nine steps starting from 2,6-lutidine reported previously. 34omplexation of Ga(III) by this chelate was achieved at pH 4.5 overnight, with the formation of [Ga(Tpaa)] confirmed by high-resolution mass spectrometry (m/z = 489.0321,[M + H] + , 489.0320 expected for 69 GaC 21 H 16 N 4 O 6 ; Figure S16).The 1 H NMR spectrum of [Ga(Tpaa)] displayed characteristic geminal coupling of the methylene group between the central amine and the picolinate arms ( 2 J HH = 17.4 Hz; Figure S12).
The picolinate arms were shown to be symmetrical, suggesting complete coordination of the Ga(III) center by the picolinates.
Crystal Structure.A crystal of suitable quality for X-ray diffraction analysis was obtained by the slow evaporation of an acidic solution of the complex.a The structure crystallizes in the noncentric space group P2 1 .The asymmetric unit contained two symmetry-unique [Ga(Tpaa)] complexes and 7 water molecules (Figure 2).In both unique complexes, the Ga(III) center is coordinated by three chelating picolinate arms of the ligand; the overall coordination geometry is that of a distorted octahedron.The central amine of the ligand is not involved in complexation in this structure (Ga1 2).This is in agreement with the previously reported structures for [Ga(Dpaa)(H 2 O)] in which the central amine is not involved in coordination of the Ga(III) ion (Ga−N distance 2.4880(11) Å). 22,24 The Ga−N bond length of the coordinating picolinate arms is decreased (mean Ga−N bond length 2.08 Å) when compared to the bond length reported for [Ga(Dpaa)(H 2 O)] (mean Ga−N bond length 2.22 Å), 22 suggesting an improved coordination; the Ga−O distance does not significantly change (mean Ga−O 2.01 vs 2.03 Å).This suggests that overall there is an improved interaction between the picolinate arms and the Ga(III) center.Furthermore, the mean picolinate bite angle is  increased (78.66°compared to 74.46°) 22 �this is closer to the ideal 90°for an octahedral geometry, showing reduced strain in the [Ga(Tpaa)] complex than in [Ga(Dpaa)(H 2 O)].There is no evidence for a coordinated water molecule, in contrast to the reported crystal structures for [Ga(Dpaa)(H 2 O)].Further details of the crystal structure are contained in the Supporting Information.
Thermodynamic Stability.Protonation constants of ligand Tpaa (Table 2, Table S2, and Figure S20A) were determined by potentiometry.Four protonation constants were found in the studied pH range.The first protonation is likely localized on the amine nitrogen atom; the remaining three protonations occur on pyridine or carboxylate groups and they cover the whole acidic region.The determined protonation constants are in a good agreement with the previously published data. 35Overall, the ligand basicity is rather low, which might be beneficial for complexation of metal radioisotopes in acidic solutions.
The coordination properties of the Tpaa ligand toward Ga(III), Cu(II), and Zn(II) ions were studied in solution.Determination of the Zn(II) complex stability constant was disabled by extensive precipitation of the complex.Stability constants of the Ga(III)-Tpaa complexes (Table 2 and Tables S3 and S4) were determined by potentiometry.The out-of-cell method was used due to the long time required to reach equilibrium (competition between [Ga(L)] and tetrahydroxidogallate).Despite the low ligand basicity, the stability constant of [Ga(L)] is rather high and it is the dominant species along the whole acidic pH region (Figure 3).In addition, the protonated [Ga(HL)] + species was identified in the strongly acidic region.As no free Ga(III) ion is present at the start of the titration, the stability constants were calculated from the equilibrium in the weakly alkaline region where the complex [Ga(L)] undergoes dissociation forming tetrahydroxidogallate at pH > 8.The absence of the mixed hydroxido complexes, [Ga(L)(OH) n ] n− , in the chemical model is ascribed to their low abundance in solution.Some evidence of their formation can be seen through variable pH* 1 H NMR (Figure S24).This result is in contrast to that previously reported for [Ga(Dpaa)(H 2 O)], where the mixed hydroxido species [Ga(L)(OH)] − was the major species from pH 5−9. 22his corroborates the absence of a coordinated water molecule in the structure of [Ga(Tpaa)], as addition of a hydroxide to the Ga(III) coordination sphere would necessitate the removal of an N-or O-donor atom of a picolinate arm, ultimately leading to dissociation of the complex. 1 H and 71 Ga studies of the Ga-Tpaa system across a pH* range (Figures S24 and S25) reveals that some decomplexation can be seen in samples at pH* = 7.62 and higher, as evidenced by the formation of [Ga(OH) 4 ] − (δ 71 Ga = 224, Figure S25) with increasing decomplexation under more basic conditions.The formation of free ligand can be seen in samples at pH* values of 8.97 and 10.07 (Figure S24).No evidence of the formation of Ga(OH) 3 can be seen across the pH range studied.Note that the 71 Ga signal corresponding to [Ga-(Tpaa)] could not be observed due to the quadrupolar nature of 71 Ga, making this peak very broad.
Variable temperature NMR of [Ga(Tpaa)] (D 2 O, pH* = 6.81, Figure S26) shows that with increasing temperature, no significant differences are observed in the 1 H NMR. A minor change (0.05 ppm) is seen in the chemical shift of the CH 2 protons when the sample is heated to 65 °C, but no change is observed for the protons corresponding to the picolinic acid arms.This indicates a high rigidity and kinetic inertness of the complex.
Comparison with NOTA, which is the gold standard for Ga(III) complexation, shows that the stability constants of Tpaa are several orders of magnitude lower (Table 2).However, one must consider that the ligand overall basicity (expressed as ∑(log K 1−4 )) is much lower than that of NOTA (∼16.5 vs ∼24, respectively).This indicates that the conditional stability constants might be comparable for the title ligand; indeed, pGa (calculated as −log[Ga(OH) 4 ] − at a ligand concentration of 1 mM, a Ga(III) concentration of 0.1 mM, pH 7.4, and 25 °C) is 9.19 for [Ga(Tpaa)] and 11.82 for [Ga(NOTA)] (Table 2).
Stability constants of the Cu(II)-Tpaa complexes (Tables S3  and S4) were determined through a combination of potentiometry and UV−vis spectroscopy.The high complex stability combined with the low ligand basicity result in the complex being fully formed even in strongly acidic solutions, and only ∼20% of free Cu(II) ions are present even at pH 0 (Figure S18).Therefore, the stability constant of the [Cu(L)] complex was determined by a UV−vis competition titration with H 4 edta (Figures S19 and S20) following equilibration overnight.The wavelength corresponding to the absorption band maximum of [Cu(edta)] 2− at 740 nm was chosen for the calculation.Potentiometry was performed under both equimolar conditions and with the metal ion in excess, and thus, dinuclear species were also found in the chemical model; however, the second metal ion is coordinated only weakly   (Tables S3 and S4).Despite the low ligand basicity, stability constants of the mononuclear complexes are rather high.The presence of the dinuclear species indicates that several ligand donor groups remain noncoordinated in the mononuclear species; this is expected due to the high ligand denticity.This is also the reason protonated complexes are formed in the very acidic region (Table S4).However, the rigidity of the pendant arms probably does not allow complete saturation of the Cu(II) coordination sphere, as could be concluded from the presence of the mixed hydroxido species in the alkaline region.
Radiolabeling Studies with 68 Ga.Radiolabeling of Tpaa with 68 Ga was performed at pH 4.0 and 7.4.[ 68 Ga][Ga(Tpaa)] was produced with a radiochemical conversion of >99% using 100 μM Tpaa and 68 Ga in 15 min at both pH 4.0 (25 °C) and 7.4 (37 °C) (Figure 4).These results compare favorably to those of the standard chelators DOTA and NOTA and to other picolinate ligands for 68 Ga, which are typically labeled at acidic pH, albeit at lower ligand concentrations (Table 1).Isolation of the radiolabeled species gave the product with a molar activity of 3.1 GBq/μmol.This is similar to the molar activity reported for [ 68 Ga][Ga(Dpaa)(H 2 O)]. 22he effect of ligand concentration on the radiolabeling efficiency of Tpaa was assessed at both pH 4.0 and 7.4 (Figure 5).At pH 4.0, Tpaa could be effectively radiolabeled at concentrations as low as 3.3 μM, comparable to the radiolabeling reported for CHXdedpa or NOTA. 26,28At pH 7.4, a concentration of 6.6 μM Tpaa was required for radiolabeling >99%, a significant improvement upon that reported for Dpaa and approaching the concentrations reported for NOTA under acidic conditions. 22,28hen assessed for stability to 90% FBS, 32% of [ 68 Ga][Ga-(Tpaa)] was found to be intact after 30 min; however, after 2 h less than 5% of the radiolabeled complex remained intact (Figure S23).This is a significant improvement on the similar Dpaa picolinate system with 68 Ga, which shows no stability to FBS after 30 min. 22However, this stability is ultimately insufficient for further development of this system as a 68 Garadiotracer; other chelates show significantly higher stability to serum (e.g., [ 67 Ga][Ga(DOTA)] retains 80% of 67 Ga after 2 h and [ 67 Ga][Ga(NOTA)] 98% after 2 h following incubation in 50% human serum), 24 and this is a key requirement for the development of effective imaging agents.
■ CONCLUSIONS Tpaa, a tripodal picolinate-based chelator, has been synthesized in a shorter, five-step, route with an improved overall yield of 41%.Tpaa produces an octahedral complex upon coordination of Ga(III) despite having seven potential coordinating atoms.The [Ga(Tpaa)] system has an increased thermodynamic stability, with log K GaL = 21.32,compared to that of [Ga(Dpaa)(H 2 O)], a similar system featuring a tripodal picolinate ligand with six potential coordinating atoms.Furthermore, there is no evidence of water coordination in the [Ga(Tpaa)] system when it is assessed by potentiometry or in the crystal structure, supporting the conclusion that the additional picolinate arm eliminates the coordinated water molecule seen in the [Ga(Dpaa)(H 2 O)] system.
Tpaa can be efficiently radiolabeled with 68 Ga under mild conditions, with a >99% radiochemical conversion being achieved at pH 7.4 in PBS at a ligand concentration of 6.6 μM.The resulting radiolabeled complex, [ 68 Ga][Ga(Tpaa)], has a molar activity of 3.1 GBq μmol −1 .Serum stability was assessed using fetal bovine serum, with 32% of [ 68 Ga][Ga(Tpaa)] remaining intact after incubation for 30 min, which is an improvement upon the previously reported [ 68 Ga][Ga(Dpaa)-(H 2 O)] that showed no stability under these conditions.
We have shown that for tripodal picolinate chelators, increasing the number of coordinating picolinate arms can improve the thermodynamic stability of the resulting Ga(III) complex, the radiolabeling properties with 68 Ga, and the serum stability of the resulting 68 Ga complex.
The versatility demonstrated by this chelator for the coordination of metals will likely lead to diverse applications in the future.These applications may depend upon the conjugation of Tpaa to small biomolecules, and such a bifunctional analogue should be developed.We envisage that the modification of a number of the picolinic acid arms into picolinic amides may be a successful route to develop mono-or multimeric targeted agents in the future.
Experimental details, synthetic methodology, potentiometric data, and crystal structure data (PDF)    ; data collected using a Stoe IPDS2 diffractometer and processed using Stoe X-AREA and Sortav; 41 structure solved using dualspace methods implemented within SHELXT 42 and refined with SHELXL-2014. 43

Figure 1 .
Figure 1.Structures of chelators discussed in this manuscript.

Figure 5 .
Figure 5.Effect of the ligand concentration on radiochemical conversion.Black open squares: Tpaa labeled with 68 Ga at pH 4 (I = 0.1 M sodium acetate buffer, T = 25 °C, t = 15 min).Red filled circles: Tpaa labeled with 68 Ga at pH 7.4 (I = PBS, T = 37 °C, t = 15 min).Dashed lines are for a guide to the eye.

Table 2 .
Comparison of Stepwise Protonation and Stability Constants of the Discussed Ligands CCDC 1878045 contains the supplementary crystallographic data for this paper.These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, King's College London, London SE1 7EH, United Kingdom; orcid.org/0000-0002-0076-2246;Email: graeme.stasiuk@kcl.ac.ukT026367/1) for funding this work.The authors thank Dr. Kevin Welham and Dean Moore of the University of Hull Mass Spectrometry service for conducting the HRMS measurements; the authors also thank Professor John Greenman for useful discussions.The authors thank Dr. Tom Eykyn of King's College London for his assistance setting up 71 Ga NMR experiments.[Ga(Tpaa)] 2 •7H 2 O crystal structure data (CCDC Reference 1878045): refined formula C 42 H 44 Ga 2 N 8 O 19 ; M r = 1104.29;crystal dimensions 0.430 × 0.150 × 0.120 mm 3 ; monoclinic; a