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Octadentate chelators for zirconium- and other metal-based radiopharmaceuticals Buchwalder, Christian 2018

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 OCTADENTATE CHELATORS FOR ZIRCONIUM- AND OTHER  METAL-BASED RADIOPHARMACEUTICALS  by  Christian Buchwalder  B.Sc., University of Zurich, Switzerland, 2011 M.Sc., University of Zurich, Switzerland, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2018  © Christian Buchwalder, 2018  ii Committee Page The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  Octadentate Chelators for Zirconium- and Other Metal-Based Radiopharmaceuticals  submitted by Christian Buchwalder  in partial fulfillment of the requirements for the degree of Doctor of Philosophy  in Pharmaceutical Sciences   Examining Committee: Prof. Urs Häfeli, Pharmaceutical Sciences Supervisor  Prof. Paul Schaffer, TRIUMF Supervisory Committee Member  Prof. Chris Orvig, Chemistry Supervisory Committee Member Prof. W. Russell Algar, Chemistry University Examiner Prof. Adam Frankel, Pharmaceutical Sciences University Examiner   Additional Supervisory Committee Members: Prof. David Grierson, Pharmaceutical Sciences Supervisory Committee Member Prof. Daniel Renouf, BC Cancer Agency Supervisory Committee Member   iii Abstract Metal-based radiopharmaceuticals are critical in nuclear medicine for non-invasive diagnosis and treatment of serious diseases such as cancer. To be successfully deployed in such radioactive drugs, radiometal ions need to be stably sequestered by a suitable chelating agent. This work investigates a new class of octadentate chelating agents containing four 3-hydroxy-4-pyridinone (3,4-HOPO) entities with a focus on the development of new zirconium(IV) chelates. The diagnostic radionuclide zirconium-89 (89Zr, t1/2 78.41 h, Iβ+ 22.7%, Eβ+ mean 395.5 keV) is of particular interest for antibody-targeted positron emission tomography (immunoPET). The developed tetrakis(3,4-HOPO) chelator is capable of quantitatively sequestering 89Zr within 10 min at ambient temperature. The resultant ZrIV complex was found to be of exceptional thermodynamic stability (log β 50.3), showed a favourable pharmacokinetic profile in vivo, and exceeded the current standard Zr-chelator in direct in vitro stability tests. The tetrapodal chelator was derivatized in a multi-step synthesis for covalent attachment to targeting vectors or other carriers. The conjugation and 89Zr-radiolabeling of the bifunctional chelator (BFC) was subsequently optimized and investigated in detail with two monoclonal antibodies, a model protein, and with polymeric nanoparticles. The long-term in vivo stability of the 89Zr-radiochelate was assessed in a novel way over six days by utilizing long-circulating hyperbranched polyglycerol (HPG) nanoparticles. The radiochelate-nanocarrier conjugates were examined over six days by non-invasive positron emission tomography (PET) and in a biodistribution study. Although in vitro exams demonstrated extended plasma stability, these data revealed a physiologic susceptibility of the tetrakis(HOPO) complex attributed to kinetic lability. In addition to investigations with ZrIV, the new ligand system was briefly explored with iv FeIII, GaIII, YIII, SmIII, GdIII, TbIII, LuIII, and BiIIII and indicated to be a promising chelator for iron(III), gallium(III), yttrium(III), and lanthanide ion coordination.  v Lay Summary Radiolabeled drugs are routinely used to detect, monitor, and treat a number of serious diseases such as cancer. To develop more sensitive and specific radiopharmaceuticals, researchers are exploring different isotopes and attach them to new targeting molecules that bind to diseased cells. As some of the current attachment methods are plagued by limited stability, we synthesized and explored here for the first time a new class of binding molecules to attach a diagnostic isotope of zirconium to different targeting molecules. We investigated the chemistry of these radioactive constructs in detail and tested how they can be coupled to different carriers. The stability of those complexes was further examined over several days in the blood stream. The findings of this work are expected to advance nuclear medicine and help develop better diagnostic positron emission tomography imaging agents and new targeted radioactive therapies.   vi Preface The presented work was conducted at the Faculty of Pharmaceutical Sciences at the University of British Columbia with the exception of animal studies that were performed at the UBC Centre for Comparative Medicine (Chapter 3) or at the BC Cancer Research Centre (Chapter 4). All animal studies were performed in accordance with the University of British Columbia’s Animal Care Committee (ACC) under the approved protocols A12-0172 and A16-0104. I, C. Buchwalder, was the lead investigator for the projects described herein and conducted this work with the following contributions by others: Sections of Chapter 1 were written by myself for a published work and are reproduced in part with permission from The Royal Society of Chemistry (C. Buchwalder, C. Rodríguez-Rodríguez, P. Schaffer, S. K. Karagiozov, K. Saatchi and U. O. Häfeli, Dalton Trans., 2017, 46, 9654-9663). In Chapter 2, K. Saatchi conceived an initial tetrakis(hydroxypyrone) chelator design, which I adapted for the intended bifunctional derivative. K. Saatchi and S. K. Karagiozov produced a starting material, compound 2.3. The synthetic route pursued was devised by myself and R. Gealageas. I conceptualized, executed, analyzed, and interpreted all syntheses and experiments and discussed findings with U. O. Häfeli and K. Saatchi.  Chapter 3 is, in part, an adaptation of published work reproduced with permission from The Royal Society of Chemistry (C. Buchwalder, C. Rodríguez-Rodríguez, P. Schaffer, S. K. Karagiozov, K. Saatchi and U. O. Häfeli, Dalton Trans., 2017, 46, 9654-9663). I conceptualized and wrote most of the manuscript with editorial help from C. Rodríguez-Rodríguez, P. Schaffer, and U. O. Häfeli. K. Saatchi suggested the initial ligand design, was involved in the study design, and harvested organs for the biodistribution study. K. Saatchi and S. K. Karagiozov produced a starting material, compound 3.1. C. Rodríguez-Rodríguez performed the vii computational study, operated the PET scanner, and wrote the corresponding experimental section. P. Schaffer was involved in the design of radiochemical experiments and provided access to 89Zr. U. O. Häfeli was the supervisory investigator and provided support and guidance. I selected the metal ion ZrIV and designed, conducted, evaluated, and interpreted all syntheses and experiments. I discussed findings with U. O. Häfeli, C. Rodríguez-Rodríguez, P. Schaffer, and K. Saatchi.  The thermodynamic solution studies in Chapter 3 are unpublished and will be reported together with results from Chapter 4. M. G. Jaraquemada-Peláez and I conducted all experiments, analyzed and interpreted the results, and wrote the corresponding sections together (sub-chapters 3.2.3 and 3.4.4). In Chapter 4, I designed and synthesized the bifunctional chelator with some suggestions from K. Saatchi. Polymeric nanoparticles were produced by K. Saatchi. S. K. Karagiozov produced a starting material, compound 4.6. J. Rousseau and H. Merkens were involved with in vivo studies, together with staff at the BC Cancer Research Centre. J. Rousseau and I analyzed the in vivo results together. U. O. Häfeli was the supervisory investigator and provided support and guidance in the study design and execution. I designed and conducted all other experiments including syntheses, characterizations, radiolabelings, and analyses. I evaluated and interpreted all data and discussed results with U. O. Häfeli, P. Schaffer, K. Saatchi, J. Rousseau, C. Rodríguez-Rodríguez, and T. V. Esposito. A manuscript reporting these findings is in preparation. All experiments in Chapter 5 were designed, conducted, and interpreted by myself.  viii Table of Contents Abstract....................................................................................................................................................... iii Lay Summary ............................................................................................................................................. v Preface ......................................................................................................................................................... vi Table of Contents ................................................................................................................................... viii List of Tables ............................................................................................................................................. xv List of Figures .......................................................................................................................................... xvi List of Schemes ......................................................................................................................................... xx List of Abbreviations .............................................................................................................................. xxi Acknowledgements ............................................................................................................................ xxvii Dedication .............................................................................................................................................. xxix Chapter 1: Introduction ......................................................................................................................... 1 1.1 Radiopharmaceuticals for Diagnostic Imaging ............................................................... 1 1.2 Radiopharmaceuticals for Therapy ................................................................................... 6 1.3 Design of Metal-based Radiopharmaceuticals .............................................................. 11 1.4 Hydroxypyridinones and Hydroxypyrones as Metal Binding Units ......................... 17 1.5 Zirconium-89 for PET Imaging ........................................................................................ 18 1.6 Reported Zirconium-89 Chelates ..................................................................................... 20 1.7 Dissertation Overview and Research Objectives ........................................................... 24 Chapter 2: Efforts Towards an Octadentate Hydroxypyrone-Based Ligand System ............... 25 2.1 Introduction ........................................................................................................................ 25 2.2 Results and Discussion ...................................................................................................... 25 ix 2.2.1 Synthesis and Characterization ................................................................................... 25 2.2.2 Ester Bond Instability .................................................................................................... 27 2.3 Conclusions ......................................................................................................................... 31 2.4 Experimental ....................................................................................................................... 32 2.4.1 Materials and Methods ................................................................................................. 32 2.4.2 Syntheses ........................................................................................................................ 33 2.4.2.1 N-(9-Fluorenylmethoxycarbonyl)iminodiacetic acid (2.2) .................................. 33 2.4.2.2 Bis((3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-yl)methyl) 2,2' N-(9-fluorenyl methoxycarbonyl)iminodiacetate (2.4) ................................................. 34 2.4.2.3 Bis((3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-yl)methyl) 2,2'-iminodiacetate (2.5) ................................................................................................... 35 2.4.3 Decomposition Study .................................................................................................... 36 Chapter 3: The Octadentate Tetrakis(3-Hydroxy-4-Pyridinone) Chelator THPN for Zirconium(IV) Complexation ......................................................................................... 37 3.1 Introduction ........................................................................................................................ 37 3.2 Results and Discussion ...................................................................................................... 38 3.2.1 Synthesis and Characterization of THPN and Zr-THPN ........................................ 38 3.2.2 Computational Studies ................................................................................................. 40 3.2.3 Thermodynamic Solution Studies ............................................................................... 43 3.2.3.1 THPN Ligand Protonation Constants .................................................................... 43 3.2.3.2 Complex Formation Equilibria with Fe3+ and Zr4+ ................................................ 45 3.2.4 Radiolabeling of THPN with 89ZrIV ............................................................................. 51 3.2.5 Concentration Dependence of Radiolabeling ............................................................ 52 x 3.2.6 Distribution Coefficients............................................................................................... 54 3.2.7 In Vitro Stability Experiments ..................................................................................... 54 3.2.7.1 Transchelation Competition Study .................................................................... 55 3.2.7.2 EDTA Challenge ..................................................................................................... 57 3.2.7.3 Serum Stability Study ............................................................................................ 58 3.2.8 In Vivo Behaviour ........................................................................................................... 58 3.3 Conclusions ......................................................................................................................... 60 3.4 Experimental ....................................................................................................................... 61 3.4.1 Materials and Methods ................................................................................................. 61 3.4.2 Syntheses ....................................................................................................................... 63 3.4.2.1 2,2',2'',2'''-((Propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-(phenylmethoxy)-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (3.2) ..... 63 3.4.2.2 2,2',2'',2'''-((Propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (THPN) .............. 64 3.4.2.3 Zr-THPN Complex ................................................................................................. 65 3.4.3 Computational Studies .............................................................................................. 65 3.4.4 Thermodynamic Solution Studies ............................................................................... 66 3.4.4.1 Materials and Methods ............................................................................................. 66 3.4.4.2 THPN Ligand Protonation Constants .................................................................... 67 3.4.4.3 Metal Complex Formation Constants .................................................................... 68 3.4.5 Concentration Dependence of 89Zr-Radiolabeling ............................................... 69 3.4.6 Human Serum Stability ............................................................................................. 69 3.4.7 Distribution Coefficients............................................................................................... 69 3.4.8 Transchelation Competition Study ........................................................................ 70 xi 3.4.9 EDTA Competition Study ......................................................................................... 71 3.4.10 In Vivo Study of 89Zr Complexes .................................................................................. 71 3.4.11 PET/CT Imaging ............................................................................................................ 72 Chapter 4: Bifunctionalization of THPN and Study of 89Zr-THPN Conjugates ....................... 73 4.1 Introduction ........................................................................................................................ 73 4.2 Results and Discussion ...................................................................................................... 74 4.2.1 Synthesis of Bifunctional THPN .................................................................................. 74 4.2.2 89Zr-Trastuzumab Radioimmunoconjugates ............................................................. 80 4.2.2.1 89Zr-Labeling of Trastuzumab by Postlabeling ..................................................... 82 4.2.2.2 89Zr-Labeling of Trastuzumab by Prelabeling ....................................................... 86 4.2.2.3 In Vitro Plasma Stability Study ................................................................................ 88 4.2.3 89Zr-Anti-PD-L1 Radioimmunoconjugates ................................................................ 89 4.2.4 89Zr-Mouse Serum Albumin (MSA) Radioconjugates .............................................. 93 4.2.5 89Zr-Hyperbranched Polyglycerol (HPG) Radioconjugates .................................... 95 4.2.5.1 Chelator Conjugation and Radiolabeling .............................................................. 95 4.2.5.2 In Vitro Plasma Stability of 89Zr-HPG Conjugates ................................................ 97 4.2.5.3 In Vivo Stability of 89Zr-HPG Conjugates ............................................................... 98 4.3 Conclusions ....................................................................................................................... 103 4.4 Experimental ..................................................................................................................... 105 4.4.1 Materials and Methods ............................................................................................... 105 4.4.2 Syntheses ...................................................................................................................... 108 4.4.2.1 Diethyl-2-(4-nitrobenzyl)malonate (4.1)............................................................... 108 4.4.2.2 2-(4-Nitrobenzyl)propanediamide (4.2) ............................................................... 109 xii 4.4.2.3 1,3-Diamino-2-(4-nitrobenzyl)propane dihydrochloride (4.3) .......................... 110 4.4.2.4 Tetra-tert-butyl 2,2',2'',2'''-((2-(4-nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))-tetraacetate (4.4) .................................................................. 111 4.4.2.5 2,2',2'',2'''-(2-(4-Nitrobenzyl)propane-1,3-diyl-bis(azanetriyl))-tetraacetic acid (4.5) .................................................................................................................... 111 4.4.2.6 2,2',2'',2'''-((2-(4-Nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-(phenylmethoxy)-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (4.7) ..................................................................................... 112 4.4.2.7 2,2',2'',2'''-((2-(4-Nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (4.8). ........................................................................................................................... 113 4.4.2.8 2,2',2'',2'''-((2-(4-Aminobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (4.9) ............................................................................................................................ 114 4.4.2.9 2,2',2'',2'''-((2-(4-Isothiocyanatobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (p-SCN-Bn-THPN, 4.10) ................... 115 4.4.2.10 2,2',2'',2'''-((2-(4-(3-(2-(2-(2-(2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-ethoxy)ethoxy)ethoxy)ethyl)thioureido)benzyl)propane-1,3-diyl)bis(azanetriyl))-tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)-acetamide) (Tz-THPN, 4.12) .............................. 116 4.4.3 89Zr-Trastuzumab Radioimmunoconjugates ........................................................... 117 4.4.3.1 Postlabeling Strategy .............................................................................................. 117 4.4.3.2 Prelabeling Strategy ................................................................................................ 118 xiii 4.4.3.3 In Vitro Plasma Stability Study .............................................................................. 119 4.4.3.4 Trans-Cyclooctene (TCO) Modification of Trastuzumab .................................. 120 4.4.3.5 Determination of Number of Reactive TCO Groups per mAb ......................... 121 4.4.3.6 Radiolabeling by Tetrazine/Trans-Cyclooctene Click Chemistry ..................... 121 4.4.4 89Zr-Anti-PD-L1 Radioimmunoconjugates .............................................................. 123 4.4.4.1 THPN-Modification of anti-PD-L1 mAb ............................................................. 123 4.4.4.2 89Zr-Labeling of THPN-anti-PD-L1 immunoconjugate...................................... 123 4.4.5 89Zr-Mouse Serum Albumin (MSA) Radioconjugates ............................................ 124 4.4.5.1 THPN-Modification of MSA .................................................................................. 124 4.4.5.2 89Zr-Radiolabeling of THPN-MSA ........................................................................ 124 4.4.6 89Zr-HPG Radioconjugates ......................................................................................... 125 4.4.6.1 Chelator Conjugation to HPG-Nanoparticles ..................................................... 125 4.4.6.2 89Zr-Radiolabeling of HPG-Chelator Conjugates................................................ 126 4.4.6.3 In Vitro Plasma Stability of 89Zr-HPG Conjugates .............................................. 126 4.4.6.4 In Vivo Study of 89Zr-HPG Conjugates ................................................................. 127 Chapter 5: THPN with Other Medically Relevant Metal Ions................................................... 129 5.1 Introduction ...................................................................................................................... 129 5.2 Results and Discussion .................................................................................................... 129 5.3 Conclusions ....................................................................................................................... 132 5.4 Experimental ..................................................................................................................... 133 5.4.1 Materials and Methods ............................................................................................... 133 5.4.2 Syntheses ..................................................................................................................... 133 Chapter 6: Conclusions and Outlook .............................................................................................. 136 xiv References ............................................................................................................................................... 140 Appendices ............................................................................................................................................. 157 Appendix A: Data for Zr-THPN and 89Zr-Labeling................................................................. 157 Appendix B: PET/CT Images from In Vivo Study of 89Zr-Chelates ....................................... 160 Appendix C: Spectra Pertaining to Thermodynamic Solution Studies ................................ 161 Appendix D: Inverse Electron Demand Diels-Alder Conjugation Conjugations ............... 163  xv List of Tables Table 1.1. Properties of radionuclides used for nuclear imaging by SPECT or PET, respectively. ........................................................................................................................... 4 Table 1.2. Properties of selected radionuclides appropriate for radionuclide therapy. ................... 8 Table 3.1. Comparison of Zr−O bond lengths for different Zr-complexes.a..................................... 42 Table 3.2. O−Zr−O bond angles for DFT-optimized Zr-THPN structure calculated with different DFT basis sets. ..................................................................................................... 42 Table 3.3. Protonation constants of THPN.a ......................................................................................... 43 Table 3.4. Stability constants for the complexes formed by THPN with Fe3+ and Zr4+ ions, respectively at 25 °C, I = 0.16 M NaCl. ............................................................................. 49 Table 3.5. Comparison of pM values for ZrIV-complexes with THPN or literature chelators. ...... 51 Table 3.6. Distribution coefficients logD7.4 measured for 89Zr-complexes.a ...................................... 54 Table 3.7. EDTA challenge.a .................................................................................................................... 58 Table 3.8. Biodistribution of 89Zr-THPN and 89Zr-DFO after 24 h post injection.a .......................... 59 Table 4.1. Plasma stability of 89Zr-chelate-trastuzumab radioimmunoconjugates.a ....................... 89 Table 4.2. In vitro plasma stability of 89Zr-chelate-HPG radioconjugates.a ....................................... 97 Table 4.3. Acute biodistribution data for 89Zr-HPG conjugates with values expressed as %ID/g.a ................................................................................................................................ 102  xvi List of Figures Figure 1.1. Working principle of SPECT, which relies on spatial detection of γ photons that pass a collimator. .................................................................................................................. 2 Figure 1.2. Working principle of PET, which relies on coincidence detection of annihilation photons. ................................................................................................................................. 3 Figure 1.3. A selection of diagnostic imaging agents clinically used for PET or SPECT. ................ 5 Figure 1.4. Comparison of ionization effects to DNA and linear energy transfer between a) β– emission, b) Auger electron emission, and c) α emission. .............................................. 7 Figure 1.5. A selection of therapeutic radiopharmaceuticals. .............................................................. 9 Figure 1.6. A clinical example demonstrating the huge potential of radiopharmaceuticals for therapy and diagnostic treatment monitoring. .............................................................. 10 Figure 1.7. A large number of elements possess useful radioisotopes for applications in nuclear medicine. ................................................................................................................ 11 Figure 1.8. Schematic overview of the components of metal-based radiopharmaceuticals using a bifunctional chelate approach. ............................................................................ 12 Figure 1.9. Gold-standard chelators and their bifunctional isothiocyanate derivatives that are commonly used with a range of radiometal nuclides. .................................................. 14 Figure 1.10. A selection of conjugation strategies commonly used for the attachment of chelates (dark-green) to targeting vectors (red) ............................................................. 15 Figure 1.11. Chemical structures of selected bidentate binding units .............................................. 16 Figure 1.12. Chemical structures of the two hydroxypyrones maltol and ethylmaltol, which are approved food additives, and of the 3,4-HOPO deferiprone, which is an approved medication for iron chelation therapy. .......................................................... 17 xvii Figure 1.13. Three forms of hydroxypyridinones in their neutral and zwitterionic aromatic resonance forms. ................................................................................................................. 18 Figure 1.14. A simplified nuclear decay scheme for 89Zr. ................................................................... 19 Figure 1.15. A selection of reported ZrIV-chelators. ............................................................................. 22 Figure 2.1. Retrosynthetic analysis of the intended tetrakis(hydroxypyrone) chelator p-SCN-Bn-THPO reveals the building blocks from which the chelator may be assembled. ........................................................................................................................... 26 Figure 2.2. HPLC analysis over 4 days showed decomposition of compound 2.5 ......................... 29 Figure 3.1. DFT-optimized Zr-THPN complex structures in top-down (left) and side views (right). ................................................................................................................................... 41 Figure 3.2. a-c) Representative spectra of the UV-potentiometric titration of an 8.47  10−5 M solution of THPN at different pH (at 25 °C, 0.16 M NaCl, l = 0.2 cm); d) Speciation plot for the THPN ligand ............................................................................... 45 Figure 3.3. Representative spectra of the UV-Vis titrations of the FeIII-THPN system at increasing pH values .......................................................................................................... 47 Figure 3.4. Metal-metal competition spectra of the FeIII-THPN system with increasing equivalents of Zr4+. ............................................................................................................. 48 Figure 3.5. Speciation plots for a) FeIII-THPN complexes and b) ZrIV-THPN complexes calculated with stability constants from Table 3.4. ........................................................ 50 Figure 3.6. UV-HPLC trace of non-radioactive Zr-THPN (top) compared with radio-HPLC trace of 89Zr-THPN (bottom in blue) .............................................................. 52 Figure 3.7. Radiochemical yield (RCY) of 89Zr-radiolabeling over time with THPN (left) or DFO ligand (right) at different ligand concentrations ...................................... 53 xviii Figure 3.8. ITLC chromatograms of A) 89Zr-THPN complex alone; B) 89Zr-DFO complex alone; C) transchelation challenge of 89Zr-THPN incubated with different amounts of DFO over time; D) transchelation challenge of 89Zr-DFO incubated with different amounts of THPN over time ......................................... 56 Figure 4.1. Retrosynthetic analysis of the intended bifunctional THPN derivative p-SCN-Bn-THPN reveals the building blocks from which the bifunctional chelator may be assembled. ........................................................................................................................... 74 Figure 4.2. Possible side products and by-products that can form during the amide coupling reaction between compounds 4.5 and 4.6 in addition to the desired product 4.7. .... 78 Figure 4.3. Two strategies can be pursued for the conjugation and radiolabeling of a bifunctional chelator. ......................................................................................................... 81 Figure 4.4. (Radio-)SE-HPLC chromatograms of a) THPN-, b) DFO-, and c) DFO*-modified trastuzumab before (top) and after 89Zr-radiolabeling by postlabeling (centre and bottom). ................................................................................................................................ 83 Figure 4.5. Radio-SE-HPLC chromatograms of 89Zr-THPN-trastuzumab using improved postlabeling conditions showed less aggregates on a small scale (left), but when repeated with more 89Zr, aggregate formation was again more pronounced (right). ................................................................................................................................... 85 Figure 4.6. Analyses of 89Zr-THPN-trastuzumab produced by a prelabeling approach (A) by radio-SE-HPLC and (B) by gel electrophoresis. ............................................................. 87 Figure 4.7. (Radio-)SE-HPLC chromatograms of a) unmodified anti-PD-L1 mAb, b) THPN-modified anti-PD-L1 mAb, and c & d) 89Zr-THPN-anti-PD-L1. .................................. 90 Figure 4.8. (Radio-)SE-HPLC chromatograms of a) unmodified anti-PD-L1 mAb, b) THPN-modified anti-PD-L1 mAb, c & d) 89Zr-THPN-anti-PD-L1. .......................................... 91 xix Figure 4.9. Analyses of anti-PD-L1 antibodies by SDS-PAGE ........................................................... 92 Figure 4.10. (Radio-)SE-HPLC chromatograms of unmodified MSA, THPN-modified MSA, and radiolabeled 89Zr-THPN-MSA. ................................................................................. 94 Figure 4.11. Analysis by SDS-PAGE (12%) of a) unmodified MSA (66 kDa), b) THPN-modified MSA, c & d) radiolabeled 89Zr-THPN-MSA .................................................. 95 Figure 4.12. (Radio-)SE-HPLC chromatograms of a) unconjugated HPG-NH2, b) HPG-chelator conjugates, and c) radiolabeled 89Zr-chelate-HPGs show no difference upon conjugation and radiolabeling. ............................................................................... 96 Figure 4.13. Bone uptake of 89Zr for the three radioconjugates measured in biodistribution studies after 1, 3, or 6 days post-injection. ...................................................................... 99 Figure 4.14. Schematic representation comparing thermodynamic and kinetic effects governing the stability of a radiometal complex in vivo, with the dissociation rate constant koff being a critical factor. .................................................................................. 100 Figure 4.15. Maximum intensity projections of PET (left) and fused PET/CT images (right) over six days of three mice injected i.v. with either 89Zr-THPN-HPG (top), 89Zr-DFO-HPG (centre), or 89Zr-DFO*-HPG (bottom). ........................................................ 101 Figure 6.1. Suggested structures for a bi-macrocyclic cage (left) or “clam-shell” like derivative (right) of THPN. ................................................................................................................ 138 Figure 6.2. DFT-optimized structures of Zr-chelates with the proposed bi-macrocyclic cage- (left) or “clam-shell”-like chelator (right). ..................................................................... 138    xx List of Schemes Scheme 2.1. Synthetic route towards the intended tetrakis(hydroxypyrone) chelator p-SCN-Bn-THPO. ............................................................................................................................ 26 Scheme 2.2. The two degradation products 2.3 and 2.6 formed by transesterification of intermediate 2.5. ................................................................................................................. 28 Scheme 2.3. Proposed mechanism of ester degradation by transesterification with methanol yielding the observed fragments 2.3 and 2.6. ................................................................. 30 Scheme 3.1. Two-step synthesis of the tetrakis(3,4-HOPO) ligand THPN. ............................. 39 Scheme 4.1. Nine-step synthesis of the bifunctional ligand p-SCN-Bn-THPN, 4.10. ..................... 75 Scheme 4.2. Alkylation of diethyl malonate greatly depends on reaction conditions leading to addition of either one or two equivalents of nitrobenzyl. ............................................ 76 Scheme 4.3. Modification of trastuzumab with the three chelators p-SCN-Bn-THPN, p-SCN-Phe-DFO, and p-SCN-Phe-DFO*. ..................................................................................... 82   xxi List of Abbreviations ~ Approximate α Alpha Å Ångström, 10−10 m β Cumulative stability constant β+ Positron, beta plus β− Beta minus γ Gamma ray δ Chemical shift (NMR)  ελ Extinction coefficient at wavelength λ λ Wavelength μ Micro (10−6) νmax Wavenumber of maximal absorption peak (IR) Ω Ohm AcOH Acetic acid AE Auger electron ATR Attenuated total reflectance avg. Average BC British Columbia Bn Benzyl Bq Becquerel br Broadened c Centi (10−2) °C Degree Celsius xxii calcd. Calculated Ci Curie CT Computed Tomography d Day(s), doublet (NMR), diameter (DLS) Da Dalton DCC N,N'-Dicyclohexylcarbodiimide DCM Dichloromethane DFO Desferrioxamine B, deferoxamine DFO* DFO-star, N1-[5-(Acetylhydroxyamino)pentyl]-N26-(5-aminopentyl)-N26,5,16-trihydroxy-4,12,15,23-tetraoxo-5,11,16,22-tetraazahexacosanediamide DFT Density functional theory DLS Dynamic light scattering DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DTPA Diethylenetriaminepentaacetic acid Eβ+ max Maximal positron energy Eβ+ mean Mean positron energy EC Electron capture EDTA Ethylenediaminetetraacetic acid EDTMP Ethylenediaminetetramethylenephosphonate, lexidronam EMA European Medicines Agency eq. Equivalent ESI Electrospray ionization  xxiii EtOAc Ethyl acetate eV Electronvolt FA Formic acid FDA U.S. Food and Drug Administration Fmoc 9-Fluorenylmethyloxycarbonyl FT-IR Fourier-transform infrared spectroscopy h Hour(s) HBED-CC N,N'-Bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N'-diacetic acid HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HER2 Human epidermal growth factor receptor 2 HOBt 1-Hydroxybenzotriazole HOPO Hydroxypyridinone HPG Hyperbranched polyglycerol HPLC High-performance liquid chromatography HR High-resolution Hz Hertz I Ionic strength Iβ+ Positron abundance Iγ Gamma emission intensity ID Injected dose IEDDA Inverse electron demand Diels-Alder IgG Immunoglobulin G ITLC Instant thin-layer chromatography xxiv IR Infrared IT Isomeric transition IUPAC International Union of Pure and Applied Chemistry i.v. Intravenous J Coupling constant (NMR) k Kilo (103) K Kelvin L Ligand l Path length (UV-Vis) LC Liquid chromatography LET Linear energy transfer Ln Lanthanide m Metre(s), milli (10−3), or multiplet (NMR) M Molarity, metal, or mega (106) mAb Monoclonal antibody MALDI Matrix-assisted laser desortpion/ionization MeCN Acetonitrile MeOH Methanol MIP Maximum intensity projection MS Mass spectrometry MSA Mouse serum albumin MW Microwave m/z Mass-to-charge ratio (MS) n Nano (10−9) xxv N Normality NHS N-Hydroxysuccinimide NMR Nuclear magnetic resonance NMWL Nominal molecular weight limit NSG NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ o ortho p para or probability value p Pico (10−12) PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline Pd/C Palladium on carbon PDI Polydispersity index PDTA 1,3-Propanediamine-N,N,N',N'-tetraacetic acid PEG Polyethylene glycol PET Positron emission tomography pH − log [H3O+] Phe Phenylene p.i. Post-injection pM − log[Mn+]free at pH 7.4 with [Mn+] = 1 μM and [Lx−] = 10 μM ppm Parts per million PSMA Prostate-specific membrane antigen Rβ+ mean Mean positron range RCP Radiochemical purity RCY Radiochemical yield xxvi Rf Retention factor (TLC) RP Reversed phase RT Room temperature s Second(s), singlet (NMR) SD Standard deviation SDS Sodium dodecyl sulfate SE Size-exclusion SEC Size-exclusion chromatography SPECT Single-photon emission computed tomography t Triplet (NMR) t1/2 Half-life TCO trans-Cyclooctene tert Tertiary TFA Trifluoroacetic acid THF Tetrahydrofuran THPN 2,2',2'',2'''-((Propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) TLC Thin-layer chromatography TOF Time of flight tR Retention time Tz 1,2,4,5-Tetrazine UBC University of British Columbia UV-Vis Ultraviolet-visible  xxvii Acknowledgements I am extremely grateful to many people who were instrumental in bringing this work to fruition and who encouraged and supported me throughout this journey. To my supervisor Dr. Urs Häfeli, thank you for all your help and guidance, for giving me the freedom to pursue this project, and for always being available for discussions. My sincerest gratitude also to my committee members, Drs. Paul Schaffer, Chris Orvig, David Grierson, Daniel Renouf, and Brian Cairns (chair). Thank you for all your input, time, and guidance. Paul, I appreciated our many meetings that inspired and truly motivated me when I needed it most. Thank you for the thought-provoking discussions looking over raw data with me and for suggesting many plan Bs. Special thanks also to Dr. Orvig for facilitating the collaboration to investigate the solution stabilities.  To Dr. Cristina Rodríguez-Rodríguez, a big thank-you for all your help in this project, many cafecitos, your encouragement, and for believing in me. Muchas gracias also to Dr. María de Guadalupe “Lupe” Jaraquemada-Peláez for all your help and hard work with the solution studies—even after it turned out to be a tad more complicated than we had thought. To Dr. Kathy Saatchi, thank you for your help and input, and for suggesting this ligand system. Thank you Dr. Julie Rousseau, Helen Merkens, Dr. François Bénard, and the other members of the Bénard lab for your help with the smooth in vivo study. To Dr. Veronika Schmitt, Tullio V. Esposito, Marta Bergamo, Jenna Neufeld-Peters, Zeynab Nosrati, Lennart Bohrmann, and all other members of the Häfeli lab, thank you for helpful discussions and your help in the lab. I also acknowledge Nick Zacchia and TRIUMF’s TR13 team for producing 89Zr, Dr. Stoyan K. Karagiozov for starting materials, Dr. Ronan Gealageas for helpful discussions on retrosyntheses, Dr. Caterina Ramogida for help with my first 89Zr- and 225Ac-labeling experiments, and members of the Orvig lab for help with semi-prep. HPLC-purification.  xxviii I would also like to thank Drs. Roger Alberto, Felix H. Zelder, and Jochen Müller for everything you taught me about chemistry, for instilling your fascination and passion for science in me, and for encouraging me to pursue this journey. I acknowledge financial support from the NSERC IsoSiM program as well as from UBC and generous donors through a Four-Year Doctoral Fellowship, a Mitchell-Dwivedi graduate award, a John H. McNeill scholarship, and symposium and travel awards. Computational resources are acknowledged from WestGrid and Compute Canada.  On a personal note, I would like to thank all the great friends I was fortunate to make here in Vancouver, many of whom have meanwhile moved on to conquer the world: Veronika, Jacob, and Jonathon, all of whom I was delighted to share this Ph.D. journey with, along with Shambhavi, Irene, Natalie, Meagan, Cristina, Ronan, Alice, and Bharat. Thank you all so much for helping make rough times bearable and most times phenomenal. I am also truly grateful to my friends back home, Tobi, Chäbe, Mario, Thierry, Maurice, Laura, Päsge, Dieter, Simon, Felix, and Majorie. Many thanks for your visits, Frässpäcklis, and ultimately, for making me feel as though it had only been a few months apart. Finally, I must express my deepest gratitude to my dear Tiffany and to my parents for all their support and love. Tiffany, thank you for your encouragement and for always being there to cheer me up. I am tremendously grateful for our love, friendship, and adventures. And to my dear parents, thank you for encouraging me to pursue my dreams and for being extremely supportive, kind, and loving throughout my life and in particular this journey. This thesis is for you and for my dear grandparents. xxix Dedication           Meiner Familie  A ma famille  To my family   1 Chapter 1: Introduction The concept of using radioactivity to study biological processes or to treat disease emerged shortly after the discovery of radioactivity itself.1-3 Nuclear medicine has since become an integral part in the research and clinical management of many diseases, such as cancer or cardiovascular disease. Radiopharmaceuticals are radioactive compounds that are administered to patients for the diagnosis or treatment of disease.4 The radiation is emitted from inside the patient’s body and is used either diagnostically for molecular imaging, therapeutically for treatment, or in a combination of the two, then referred to as a theranostic application.  1.1 Radiopharmaceuticals for Diagnostic Imaging  Diagnostic radiopharmaceuticals are based on radionuclides that emit penetrating radiation in the form of gamma rays (γ) or annihilation photons from positron decay (β+). This radiation is then detected outside the patient by an appropriate detector. Detection of γ photons is achieved with gamma cameras either by planar radioscintigraphy or by the more sophisticated three-dimensional single-photon emission computed tomography (SPECT). SPECT uses a collimator and an array of detectors to register incident gamma photons of specific energies. From these data, a three-dimensional volume rendering of the radiation distribution is then reconstructed (Figure 1.1).  2  Figure 1.1. Working principle of SPECT, which relies on spatial detection of γ photons that pass a collimator. Reproduced with permission, © 2018 Dr. Cristina Rodríguez-Rodríguez. In contrast, emission from β+ decay is typically detected employing positron emission tomography (PET). PET relies on the detection of annihilation photons that are the result of a β+ decay. Radionuclides that undergo β+ decay emit a positron and a neutrino. The positron (a positively charged electron) travels a short distance losing almost all of its energy, before it combines with an atomic electron from the medium in an annihilation event. In this process, two γ photons of 511 keV energy each are generated and emitted in opposite directions.5 PET scanners use coincidence detection of these two annihilation photons by a circular array of gamma detectors. The two impinging photons allow the calculation of a line of response along which the annihilation event must have taken place. By intersecting many such lines and taking attenuation and scatter factors into account, a computer can then back calculate the origin of the decay event and thus, generate a three-dimensional volume rendering of the radiation distribution in the patient (Figure 1.2).  3  Figure 1.2. Working principle of PET, which relies on coincidence detection of annihilation photons. Adapted with permission, © 2018 Dr. Cristina Rodríguez-Rodríguez. The positron energy dictates how far the β+ travels before it annihilates and hence directly affects the PET image resolution.6 Low-energy positron emitters (e.g., 18F, Eβ+ mean 249.8 keV (96.7%), Rβ+ mean 0.6 mm in water)7, 8 have lower positron range (R β+) and therefore provide PET images of higher resolution than higher energetic positron emitters (e.g., 82Rb, Eβ+ 1 mean 1167.6 keV (13.1%), Rβ+ 1 mean 5.0 mm in water; Eβ+ 2 mean 1534.6 keV (81.8%), Rβ+ 2 mean 7.1 mm in water).7, 8 In contrast to SPECT, PET uses coincident detection and therefore does not require a collimator. This results in an increased sensitivity of PET over SPECT and has secured PET recent popularity. Still, SPECT remains a powerful technique and both imaging modalities are routinely used in clinical practice.  A considerable number of radionuclides can be used for SPECT or PET, respectively. While some are already in widespread clinical use, others have only recently emerged and are still being investigated for new applications. Table 1.1 gives an overview of the decay characteristics of some of the most popular clinical and preclinical imaging nuclides used in SPECT or PET, respectively.  4 Table 1.1. Properties of radionuclides used for nuclear imaging by SPECT or PET, respectively. From refs.7, 9-12 SPECT nuclide Half-life Eγ [keV] (% Iγ) Production 67Ga 78.3 h 93.3 (38.8) 184.6 (21.4) 300.2 (16.6) Cyclotron  natZn(p,x)67Ga 68Zn(p,2n)67Ga 99mTc 6.01 h 140.5 (89) 99Mo/99mTc Generator 111In 67.4 h 245.4 (94.1) 171.3 (90.6) Cyclotron 111Cd(p,n)111m,gIn 112Cd(p,2n)111m,gIn 123I 13.2 h 159.0 (83.3) Cyclotron 124Xe(p,2n) 123Cs/123Xe/123I 124Xe(p,pn)123Xe/123I 131I 8.03 d 364.5 (81.5) 80.2 (2.6) Reactor 131Te(n,γ)131I 133Xe 5.25 d 81.0 (36.9) Fission 235U(n,f)133Xe 201Tl 73.1 h 167.4 (10.0) 135.3 (2.6) Cyclotron 203Tl(p,3n)201Pb/201Tl     PET nuclide Half-life Eβ+ mean [keV] (% Iβ+) Production 11C 20.4 min 385.7 (99.8) Cyclotron  14N(p,α)11C 13N 9.96 min 491.8 (99.8) Cyclotron  16O(p,α)13N 15O 122 s 735.3 (99.9) Cyclotron  15N(p,n)15O 14N(d,n)15O 18F 110 min 249.8 (96.7) Cyclotron  18O(p,n)18F 20Ne(d,α)18F 68Ga 67.7 min 836.0 (87.7) 68Ge/68Ga Generator 82Rb 1.27 min 1167.6 (13.1) 1534.6 (81.8) 82Sr/82Rb Generator 89Zr 78.4 h 395.5 (22.7) Cyclotron  89Y(p,n)89Zr 5  Figure 1.3. A selection of diagnostic imaging agents clinically used for PET or SPECT.  Five examples of diagnostic imaging agents that are used clinically for PET or SPECT are shown in Figure 1.3. The cationic technetium-99m sestamibi complex and rubidium-82 chloride are both used for myocardial perfusion imaging by SPECT or PET, respectively.4 The fluorine-18 labeled glucose analog fludeoxyglucose (FDG) is a PET tracer to image glucose metabolism and is widely applied for many conditions including diagnosis and management of cancer.4 The murine anti-CD20 monoclonal antibody ibritumomab-tiuxetan (Zevalin®) can be radiolabeled with indium-111 for SPECT of non-Hodgkin’s B-cell lymphoma. The antibody is modified with the DTPA chelator analog tiuxetan, which, alternatively, can be radiolabeled with the β− emitter yttrium-90, which turns it into the approved radioimmunotherapy (RIT) agent for the same disease.13 Currently in clinical trials, the gallium-68 PET tracer 68Ga-PSMA-11 has attracted much recent attention for diagnosis and therapy monitoring of recurrent prostate 6 cancer. The urea-based peptidomimetic is an inhibitor of prostate-specific membrane antigen (PSMA) and employs the acyclic HBED-CC chelator to coordinate radiogallium(III).14-18  1.2 Radiopharmaceuticals for Therapy  In contrast to diagnostic radiopharmaceuticals, which rely on nuclides that emit penetrating radiation, therapeutic radiopharmaceuticals use particulate radiation in the form of α, β–, or Auger electron emission. These cytotoxic emissions greatly differ in their energies, range, and biological effects. Alpha particle emissions possess a large amount of energy, which they deposit over a very short distance of ~50–100 μm and are therefore designated to have a high linear energy transfer (LET). Due to the short range of α particles, only a few cell diameters are being passed, where the radiation induces highly cytotoxic DNA double-strand breaks (Figure 1.4). Since α particles directly affect DNA, their cytotoxic effect is independent of oxygenation or active cell proliferation. This gives them a high relative biologic effectiveness (RBE) and renders them particularly promising to treat disseminated disease and micrometastases.19, 20 Beta emitters deposit their energy over a relatively long range in tissue and therefore have a relatively low LET (Figure 1.4). The range of their β– emission is dependent on the β– energy and the cytotoxic effect is mostly achieved indirectly by generating reactive oxygen species (ROS) which damage DNA. To achieve its best effect, β– emitters should concentrate inside or near a solid tumour so that crossfire and bystander effects can play together to damage surrounding malignant cells. Auger emitters have medium energy but a very low range and have therefore an intermediate LET (Figure 1.4). In order to be effective, Auger emitters have to localize in close proximity to radiosensitive cell components, such as the nucleus, or to cell membranes, which are less radiosensitive. Due to these stringent targeting requirements, only limited studies have been carried out with Auger emitters and they have not 7 been translated to the clinical setting yet. Still, they hold considerable therapeutic potential for specialized applications, too.21-25  Figure 1.4. Comparison of ionization effects to DNA and linear energy transfer between a) β– emission, b) Auger electron emission, and c) α emission. Reproduced with permission from ref.26 © 2011 Springer Nature. Table 1.2 presents some radionuclides that can be used for targeted radionuclide therapy. To date, a number of radiopharmaceuticals based on β− emitters gained regulatory approval but only one α therapy, based on the α emitter radium-223 (Xofigo®), is approved. This is bound to change in the near future as many new radiotherapeutic compounds are currently investigated in clinical trials. With new, highly specific targeting vectors becoming available and with increasing accessibility of more exotic therapeutic radionuclides, in particular the potent, underemployed α particle emitters, the nuclear medicine community sees itself at the brink of an exciting new therapeutic era.20, 27-29   8 Table 1.2. Properties of selected radionuclides appropriate for radionuclide therapy. Compiled with refs.11, 22, 25, 29-31 α Emitters  Nuclide Half-life Avg. α energy (MeV) Avg. α range (μm) Production  211At 7.2 h 6.79 60 Cyclotron  213Bi 45.6 min 8.32 84 Generator  223Ra 11.4 days 6.79 45 Cyclotron  225Ac 9.9 days 6.83 61 Generator  227Th 18.7 days 5.9 ~60 Generator        β− Emitters   Nuclide Half-life Max. β− energy (keV) Max. β− range (mm) Production  67Cu 61.9 h 575 2.1 Cyclotron  89Sr 50.5 days 1,491 7.0 Reactor  90Y 64.1 h 2,284 11.3 Generator  131I 8.0 days 606 2.3 Reactor  153Sm 46.3 h 803 8.7 Cyclotron  166Ho 28.8 h 1,854 9.0 Reactor  177Lu 6.6 days 497 1.8 Reactor  186Re 3.7 days 1,077 4.8 Reactor  188Re 17.0 h 2,120 10.4 Generator       Auger electron (AE) emitters  Nuclide Half-life AE energy/decay (keV) AE range Production # of AE/decay 67Ga 3.3 days 6.3 0.1 nm−2.7 μm Cyclotron 4.7 111In 2.8 days 6.8 0.3 nm−14 μm Cyclotron 14.7 125I 59.4 days 12.2 1.5 nm−14 μm Reactor 24.9 201Tl 3.0 days 15.3 3 nm−40 μm Cyclotron 36.9   9   Figure 1.5. A selection of therapeutic radiopharmaceuticals. Figure 1.5 shows four examples of therapeutic radiopharmaceuticals. As mentioned above, the only α emitting radiopharmaceutical approved to date is based on radium-223 in form of its dichloride salt. 223RaCl2 (Xofigo®) is used to treat bone metastases in metastatic castration-resistant prostate cancer.20, 32 The other approved therapeutic radiopharmaceuticals are all β– emitters and are also employed for internal radiation therapy in oncology. Just as 223RaCl2, the samarium-153 lexidronam (EDTMP) radiocomplex has bone-seeking properties and is used in palliative care of bone metastases.33 Lutetium-177 labeled peptide octreotate (177Lu-DOTATATE, Lutathera®) acts as somatostatin analog and was recently approved (EMA: 2017, FDA: 2018) for the treatment of neuroendocrine tumours (NETs). The peptide is closely related to octreotide, which was previously investigated for radionuclide therapy with the 10 higher energetic β− emitter yttrium-90 (90Y-DOTATOC).34-36 The investigational drug 225Ac-PSMA-617 is an α-emitting compound that is currently in clinical trials and shows very impressive results for salvage therapy of metastatic prostate cancer (cf., Figure 1.6). PSMA-617 is a urea-based antagonist of prostate-specific membrane antigen (PSMA). The attached DOTA chelator was radiolabeled with the α emitter actinium-225 or with the β– emitter lutetium-177 and both radiotherapeutics are currently being investigated clinically.37-42   Figure 1.6. A clinical example demonstrating the huge potential of radiopharmaceuticals for therapy and diagnostic treatment monitoring. A) Baseline PET scan with the radiotracer 68Ga-PSMA-11 (vide supra) before targeted radionuclide therapy shows a very high disease burden with a lot of distant metastases; B) After two cycles of β− therapy with 177Lu-PSMA-617, restaging of the same patient showed disease progression; C and D) Switching to α therapy with the 225Ac analog 225Ac-PSMA-617 led to an impressive treatment response. After three cycles of α therapy, levels of the biomarker prostate-specific antigen (PSA) dropped below the measurable level, corroborating the therapeutic response. PET images are shown as maximum intensity projections (MIPs). Reproduced with permission from ref.41 © 2016 SNMMI.  11 1.3 Design of Metal-based Radiopharmaceuticals When contemplating the development of a new radiopharmaceutical, a suitable radionuclide must be selected to meet the demands of the given application. Important selection criteria are decay properties such as half-life, and type and energy of emissions. Availability, the radiochemistry of a nuclide, cost, and specific activity are additional central factors to be considered.1 The majority of the periodic table and of the chart of nuclides is made up of metals, thus it is not surprising that a large number of nuclides with feasible properties for nuclear medicine applications are radiometals (Figure 1.7).10, 43-46   H   Imaging    Therapy    Imaging & Therapy      He Li Be           B C N O F Ne Na Mg           Al Si P S Cl Ar K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe Cs Ba  Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn Fr Ra                                     La Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu    Ac Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr  Figure 1.7. A large number of elements possess useful radioisotopes for applications in nuclear medicine. Elements with radioisotopes suitable for diagnostic imaging are shaded ochre in this periodic table, elements with therapeutic radioisotopes are shaded red, and elements that possess diagnostic and therapeutic isotopes are shaded blue. Many of the highlighted elements are metallic elements that require chelation for their use in radiopharmaceuticals. Compiled with refs.26, 47, 48 Radiometals typically require stable sequestration by a chelating agent (a chelator), with only few exceptions where the metal can simply be used as salt or colloid (e.g, 223RaCl2 and 12 99mTc-colloids). Robust complexation of a radiometal ion by a chelator is critical in modifying its biodistribution profile and avoiding undesired off-target distribution of the radionuclide. By means of a bifunctional chelator (BFC), a radiometal complex can be covalently tethered to carrier entities that can act as targeting vectors. Targeting vectors are often biomolecules such as antibodies, antibody-fragments, peptides, or nucleic acid oligomers. They are selected for their high affinity and specificity for features of the target site, such as overexpressed receptors on target cell surfaces. Using a bifunctional chelate approach, target-specific metallo-radiopharmaceuticals should therefore include the following components (Figure 1.8): (1) Radiometal nuclide: possesses desired decay properties  (2) Bifunctional chelator: sequesters the radiometal ion by matching its coordination chemistry and provides functionality for covalent attachment to targeting vector (3) Linker: provides spacing between chelator and targeting vector and may tune overall polarity (4) Targeting vector: provides specificity and affinity for target tissue   Figure 1.8. Schematic overview of the components of metal-based radiopharmaceuticals using a bifunctional chelate approach. (Targeting vector structure generated with QuteMol49 from protein databank entry 1igy50). 13 The thermodynamic stability and kinetic inertness of the radiometal complex is critical for the physiologic stability of metal-based radiopharmaceuticals.1, 10, 46, 51, 52 The chelator must strongly coordinate the radiometal ion to prevent chemical and/or recoil dissociation from the radiopharmaceutical. Released radionuclides would distribute away from the targeting vector and accumulate in non-target tissues. This would result in an increase in undesired background signal for imaging, and in detrimental long-term exposure of healthy tissues to radiation. The chelator must therefore be carefully crafted to best match the coordination chemistry of the radiometal ion for maximal physiologic and metabolic stability.  In the design of new chelates, the metal’s chemical properties need to be taken into account. Consideration should be given to the metal’s preference in valence and electronics, coordination number, relative hardness, Lewis basicity, and coordination geometry.45, 46, 51, 53 The overall charge should also be taken into account as it can affect the biodistribution of metallo-radiopharmaceuticals.11 To attain high thermodynamic stability, a coordinating ligand should provide donor groups that offer the metal ion its preferred coordination environment. The metal ion’s coordination sphere should be fully saturated and the metal core shielded from solvent access. Increased denticity of the ligand can thermodynamically stabilize the coordination complex further by virtue of the chelate effect. Hence, it is desirable to use a polydentate ligand that ideally matches in denticity with the metal ion’s maximal coordination number.54 Macrocyclic chelators typically tend to provide higher kinetic inertness than acyclic chelators since they provide a higher degree of preorganization. A higher kinetic inertness is critical to prevent the coordination complex from transchelation and transmetallation under the competitive dilute conditions encountered in biological systems. Compared to acyclic chelators, this preorganization reduces the entropic penalty paid upon metal ion coordination. This so-called macrocycle effect explains for example why, even though they have comparable 14 thermodynamic stabilities, macrocyclic 90Y-DOTA is kinetically more inert and physiologically stable, than the acyclic 90Y-DTPA chelate.51, 52, 55 On the contrary, macrocycles often require elevated reaction temperatures or prolonged reaction times to achieve radiometal chelation.56 These can be important limitations in the radiolabeling of heat-sensitive targeting vectors (e.g., proteins), or when working with short-lived radionuclides that mandate short reaction times.  A large number of chelators have been developed and studied with a broad range of metal ions. Figure 1.9 shows some of the most widely applied chelators with bifunctional derivatives. For certain radiometal ions these chelators have established themselves as gold-standards and are routinely used in clinical practice. For other metals ions, however, less than optimal complex stabilities are achieved and new ligands that better match the metal’s coordination preference are sought. A number of excellent reviews thoroughly discuss benefits and drawbacks of pertinent chelators and provide rankings with regards to different metal ions.11, 45, 56   Figure 1.9. Gold-standard chelators and their bifunctional isothiocyanate derivatives that are commonly used with a range of radiometal nuclides.   15  Figure 1.10. A selection of conjugation strategies commonly used for the attachment of chelates (dark-green) to targeting vectors (red): a) Amide coupling between a carboxylic acid and an amine with coupling reagents; b) amide coupling between an active ester and an amine; c) thiourea formation between an isothiocyanate and an amine; d) thioether formation between a maleimide and a thiol; e) copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) between an alkyne and an azide; f) strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC) between a strained alkyne and an azide; g) inverse electron demand Diels-Alder [4+2] cycloaddition (IEDDA) between a 1,2,4,5-tetrazine (Tz) and a trans-cyclooctene (TCO).11, 56, 57 In order to allow covalent attachment of chelates to targeting vectors, chelators can be functionalized by introducing a reactive conjugation group.56, 57 Such a chelator is then termed a bifunctional chelator (BFC) since it serves two functions: first, to stably coordinate the metal ion, and secondly, to provide a point for covalent conjugation to a targeting entity. Several different 16 strategies can be employed to (bio)conjugate BFCs to targeting entities, each coming with their individual requirements for reaction conditions. Figure 1.10 gives an overview of some of the most commonly used conjugation strategies that can be readily used with sensitive biomolecules. Of particular importance in the design of new chelators is the choice of the binding units that provide the donor atoms to coordinate the metal ion. The relative hardness and basicity of the donor groups should match the electronic requirements of the metal ion. A range of binding units have been employed in the design of polydentate chelators. Some common motifs include hydroxamic acids, catechols, picolinic acids, 8-hydroxyquinolines, 2-hydroxyisophthalamides, 2,3-dihydroxyterephthalamides, hydroxypyrones, and hydroxy-pyridinones (Figure 1.11). With the exception of 2-hydroxyisophthalamides, these bidentate compounds all form five-membered chelate rings with metal ions. For larger metal ions, such five-membered chelate rings are generally favourable over six-membered rings.58 In the following we will focus on hydroxypyridinones and hydroxypyrones, since key compounds in this work are based on these structures.  Figure 1.11. Chemical structures of selected bidentate binding units: (a) hydroxamic acid, (b) catechol, (c) picolinic acid, (d) 8-hydroxyquinoline, (e) 2-hydroxyisophthalamide, (f) 2,3-dihydroxyterephthalamide, (g) hydroxypyrone, and (h) hydroxypyridinone (HOPO). Donor groups are highlighted in boldface.  17 1.4 Hydroxypyridinones and Hydroxypyrones as Metal Binding Units Hydroxypyridinones and hydroxypyrones are two classes of N- and O-heterocycles, respectively, that feature a keto- and a hydroxy-group in ortho-position relative to each other (Figure 1.11g, h).59 Hydroxypyrones have been studied extensively and have been found to be effective chelators for various metal ions such as AlIII, FeIII, VIII/IV, ZnII, GaIII, and InIII.59-63 This compound class is considered to possess a very favourable toxicity profile as is evidenced by the two hydroxypyrones maltol and ethylmaltol (Figure 1.12) being approved as food flavouring additives.64   Figure 1.12. Chemical structures of the two hydroxypyrones maltol and ethylmaltol, which are approved food additives, and of the 3,4-HOPO deferiprone, which is an approved medication for iron chelation therapy. Hydroxypyridinones (HOPOs) are closely related structures, which are typically synthesized from the corresponding hydroxypyrones. Depending on the position of the ring nitrogen, three forms of HOPOs can be distinguished, which are the 1-hydroxy-2-pyridinone (1,2-HOPO), the 3-hydroxy-2-pyridinone (3,2-HOPO), and the 3-hydroxy-4-pyridinone (3,4-HOPO) form (Figure 1.13). Like hydroxypyrones, HOPOs possess a partially aromatic character, which becomes evident from their zwitterionic resonance structures (Figure 1.13). 18  Figure 1.13. Three forms of hydroxypyridinones in their neutral and zwitterionic aromatic resonance forms. HOPOs typically form even stronger metal complexes than their hydroxypyrone congeners and have therefore gained considerable attention as metal chelators.59, 65, 66 The 3,4-HOPO deferiprone (Ferriprox®, Figure 1.12) is an approved treatment for iron overload from thalassemia. Mono- and poly-HOPO ligands have been studied extensively since the late 1980s and have been investigated as metal chelators for a variety of metal ions including FeIII, AlIII, GaIII, PuIV, ZrIV, GdIII, and EuIII.31, 36-44 The intended medical applications of these HOPO ligands include as scavengers for chelation therapy of iron- and aluminium-overload, as actinide decorporation agents, as magnetic resonance contrast agents, as antimicrobials (by depriving microbes of FeIII), and for radiopharmaceutical applications.59, 63, 67-74   1.5 Zirconium-89 for PET Imaging  Among the many radionuclides that can be used for molecular imaging, zirconium-89 (89Zr) is a particularly interesting positron-emitter for PET imaging of antibodies and other long-circulating targeting vectors. Antibodies are immunoglobulin proteins with exquisite specificity and affinity for their target antigens. Besides acting as formidable targeting vectors that can 19 differentiate otherwise untargeted disease, many monoclonal antibodies (mAbs) are used therapeutically to trigger an immune response or to deliver cytotoxic cargo in antibody-drug conjugates (ADCs). Since such immunologic treatments are not equally effective in all patients and can lead to serious adverse effects (besides being very costly), having a non-invasive diagnostic tool available to assess whether a given patient would benefit from such an intervention is very valuable to personalize care. Antibody-based PET (immunoPET) has therefore gained much recent attention and 89Zr is a promising radionuclide to achieve immunoPET.75-78 89Zr decays with a half-life of 78.4 h by electron capture (EC, 76.2%) and positron decay (22.7%, Eβ+ mean 395.5 keV) to metastable yttrium-89m.7 This short-lived nuclide (t1/2 15.7 s) subsequently undergoes quantitative isomeric transition (IT) to give stable 89Y, under emission of a 909.0 keV photon (Iγ 99.2%) (Figure 1.14).7 The emissions of this gamma ray, as well as conversion and Auger electrons that accompany the electron capture event do not interfere with PET imaging.54  Figure 1.14. A simplified nuclear decay scheme for 89Zr. Values taken from ref.7 The relatively long half-life of 89Zr of over three days matches the slow pharmacokinetics of targeting vectors such as mAbs that have a long biological half-life. In addition to the matched half-life, 89Zr’s emitted positron is of relatively low energy compared to other long-lived radionuclides (e.g., 124I, t1/2 4.18 days, Eβ+ mean 687.0 keV (11.7%), 974.7 keV 20 (10.7%)). This low positron energy translates into favourably high PET image resolution and renders 89Zr a suitable radionuclide for immunoPET applications.79  89Zr can be readily produced in a cyclotron in a 89Y(p,n)89Zr transmutation reaction or, less frequently, in a 89Y(d,2n)89Zr reaction from a solid or liquid 89Y target, which has a 100% natural abundance.79-82 Purification of the produced 89Zr is then most commonly achieved by weak cation exchange chromatography over a hydroxamate-functionalized resin and is eluted with 1 M oxalic acid as [89Zr(oxalate)4]4− in high specific activity and radionuclidic and radiochemical purity.79, 80 The +IV oxidation state dominates zirconium’s aqueous chemistry, with the reduction potential to ZrIII being too high to be biologically relevant (−1.4–1.5 V vs. standard hydrogen electrode).54 ZrIV is a hard Lewis acid that prefers hard donor atoms such as oxygen donors and has a strong preference for high coordination numbers of 8–9. Its ionic radius is 84 and 89 pm when 8- and 9-coordinated, respectively.54, 56, 83 Experiments in mice showed that ‘free’ 89ZrIV accumulates in bone where it deposits into the hydroxyapatite matrix.84 Surprisingly, in humans, bone uptake of 89ZrIV appears to be much less pronounced; when administered as citrate the radionuclide was found to be mostly associated with blood serum proteins with a plasma half-life of around 1–2 days.83, 85  1.6 Reported Zirconium-89 Chelates In order to prevent loss of the radionuclide from 89Zr-targeting vector conjugates, it is of great importance that the radionuclide be stably complexed and remains chelated under physiologic conditions. Among the different chelators that have been studied for 89Zr complexation, the bacterial siderophore desferrioxamine B (DFO, Figure 1.9) is by far the most widely employed chelator. Besides its widespread use in preclinical studies, it has also been 21 studied in the clinical setting. To date, no 89Zr-based radiopharmaceutical has gained regulatory approval, but around 22 clinical trials are currently either underway or have already been completed.78 Among these clinical studies the vast majority investigate 89Zr-immunoconjugates that are radiolabeled with bifunctional versions of DFO. In spite of its popularity, preclinical results in mice consistently show considerable bone uptake, which suggests that 89Zr-DFO is not sufficiently stable in vivo and releases osteophilic 89ZrIV.79, 86-92 This is generally attributed to DFO’s hexadentate chelation, which cannot fully saturate ZrIV’s octadentate coordination preference. Computational studies by Holland et al.86 suggest that in aqueous medium ZrIV’s coordination sphere is complemented by two aquo ligands. While this octadentate complex was calculated to be thermodynamically stable, the two aquo ligands are likely kinetically labile, which may render the metal ion exposed to solvent exchange. These stability concerns have recently spurred a quest for alternative chelators that may provide improved in vivo stability.  Over the last around six years, a range of new chelators have been developed and investigated with 89ZrIV and the state of the art is the subject of several recent reviews.54, 84, 93 Figure 1.15 presents a selection of zirconium(IV) chelators. A coloured-coded ranking serves as an approximate indicator of chelate stability. Drawing from DFO’s success, many new ligands were designed based on similar scaffolds relying on bidentate hydroxamate functionalities. Decristoforo and co-workers94, 95 reported on fusarinine C (FSC), a hexadentate macrocyclic siderophore based on three hydroxamate groups, while Boros et al.96 studied a small library of macrocyclic ligands with three to four pendant hydroxamate groups. Rousseau et al.97, 98 and Guérard et al.99 developed several acyclic and macrocyclic tetrahydroxamic acid chelators. As a rational extension of DFO, Gasser, Mindt, and co-workers100 elongated the siderophore ligand by an additional hydroxamate group to make the octadentate congener DFO*, which shows particularly22    Figure 1.15. A selection of reported ZrIV-chelators. Colour-coding indicates long-term chelate stability: green: favourable long-term stability, superior to DFO; orange: intermediate stability or unexplored long-term stability; red: poor stability. 23 promising stability over DFO.100, 101 The same team recently improved the limited water solubility of DFO* by introducing oxygen atoms to the backbone to produce the chelator oxoDFO*.102 Rudd et al.103 reported on the squaramide ethyl ester derivative DFOSqOEt as a modified DFO ligand, while Allott et al.104 extended DFO by a 1,2-HOPO group. Pandya et al.105 have recently examined three tetraazamacrocycles as 89Zr-chelators and found the widely used ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to form a particularly stable 89Zr-complex. Others have studied systems based on picolinic acid,106 hydroxyisophthalamide,107 2,3-dihydroxyterephtalamide (TAM),108 or, perhaps most relevant here, hydroxypyridinone (HOPO) groups as alternative 89Zr-chelators. The HOPO chelators that have been investigated with 89Zr are based on three to four HOPO units (of either the 1,2-, the 3,2-, or the 3,4-HOPO positional isomers). Guérard et al.109 compared Zr(HOPO)4 complexes of individual HOPO ligands of either the 1,2-, the 3,2-, or the 3,4-HOPO isomers in terms of geometry, stability, and inertness. Deri et al.110, 111 studied 89Zr-complexation with the previously reported linear tetrakis(1,2-HOPO) ligand 3,4,3-(LI-1,2-HOPO), and Tinianow et al.112 reported on the di-macrocyclic ligand BPDETLysH22-3,2-HOPO, which is based on four 3,2-HOPO units. Ma et al.113 investigated the previously reported tripodal GaIII-ligand CP256 (and its bifunctional derivative YM103) with 89Zr as an example of a poly(3,4-HOPO) ligand. In contrast to the octadentate 1,2-HOPO and 3,2-HOPO ligands, which provided promising 89Zr complexes stability, 89Zr-CP256 showed insufficient in vivo stability. This finding was attributed to the three 3,4-HOPO units, which only provide hexadentate coordination and do not satisfy the octadentate coordination preference by ZrIV.63, 113 As a result of the work presented in this dissertation, we reported on the first octadentate tetrakis(3,4-HOPO) chelator THPN, which we investigated with 89Zr.114  24 1.7 Dissertation Overview and Research Objectives A range of bifunctional chelators are available for radiopharmaceutical development, however the coordination chemistry of several radionuclides is still not ideally matched or the required radiolabeling conditions are problematic with certain targeting vectors. In this work we aimed to develop alternative bifunctional chelators to expand the repertoire of chelating agents for the complexation of medically relevant metal ions, with a particular focus on new zirconium-89 chelators.   The underlying research hypothesis was therefore formulated as follows:  Bifunctional tetrapodal, octadentate ligands are suitable chelators to provide fast and stable complexation of radiometal ions and to allow their conjugation to targeting vectors for applications in nuclear medicine.   The specific research objectives were: (1) Synthesize and characterize a tetrapodal, octadentate ligand system and study (radio)complex formation and stability with radiopharmaceutically relevant metal ions (2) Develop a bifunctional derivative and investigate conjugation of the chelate to delivery vehicles (3) Evaluate the stability of the such formed radioconjugates both in vitro and in vivo 25 Chapter 2: Efforts Towards an Octadentate Hydroxypyrone-Based Ligand System  2.1 Introduction In our design of an octadentate ligand to chelate large metal ions we set out to produce a ligand based on a branched tetrapodal backbone bearing binding units on each of its four pendant arms. As binding units, we opted for bidentate 3-hydroxy-4-pyrone (discussed here) or 3,4-HOPO units (discussed in subsequent chapters) based on their favourable binding properties discussed above. Since the pyrone starting material was available earlier, we commenced with this binding unit.   2.2 Results and Discussion 2.2.1 Synthesis and Characterization We set out to directly develop a bifunctional chelator that would bear a pendant para-isothiocyanatobenzyl linker group on the backbone to allow covalent attachment to amino groups of carrier molecules (e.g., from lysine residues). Figure 2.1 shows the envisioned bifunctional tetrakis(3-hydroxy-4-pyrone) chelator, abbreviated as p-SCN-Bn-THPO. From our retrosynthetic analysis we devised a synthetic plan to produce p-SCN-Bn-THPO from four starting materials in seven steps. Scheme 2.1 depicts the synthetic strategy we pursued.  26  Figure 2.1. Retrosynthetic analysis of the intended tetrakis(hydroxypyrone) chelator p-SCN-Bn-THPO reveals the building blocks from which the chelator may be assembled.  Scheme 2.1. Synthetic route towards the intended tetrakis(hydroxypyrone) chelator p-SCN-Bn-THPO. Starting from commercially available iminodiacetic acid (2.1), the secondary amine should first be Fmoc-protected before a Steglich esterification115 would couple two equivalents of the benzyl-protected pyrone-alcohol 2.3. After deprotecting the amino group, two of such obtained equivalents (2.5) should be amine alkylated by nucleophilic substitution to 1,3-27 dichloroacetone. In the next step, the conjugation handle should be installed by reductive amination with 4-nitrobenzylamine. The last steps would involve hydrogenolysis of the benzyl protecting groups to produce the 3-hydroxy-4-pyrone moieties, as well as transformation of the nitrobenzyl to an aniline group and subsequent conversion to an activated isothiocyanatobenzyl functionality. The first reaction step was reproduced from a reported procedure116 and produced the Fmoc-protected intermediate 2.2 in excellent yield of 92%. The following ester coupling was relatively efficient and although two chromatographic separations were required to remove all coupling reagent residues, intermediate 2.4 could still be obtained in a yield of 79% and was fully characterized. The subsequent step involved the cleavage of the Fmoc protecting group with piperidine, which proceeded fairly quickly, as observed by HPLC. While intermediate 2.5 could be characterized, we observed that the compound was degrading during and after purification. Acidic aqueous workup compromised product stability considerably and led to very poor isolated yields. When this workup procedure was avoided and the product was instead isolated by column chromatography, decomposition could be minimized and better yields were achieved. However, also in this case, the product was found to already having partially degraded during purification.   2.2.2 Ester Bond Instability In order to better understand this degradation behaviour, we incubated a small amount of purified intermediate 2.5 (which already contained some degradation products) in methanol and analyzed aliquots over time by HPLC (Figure 2.2). After four days at ambient temperature, the HPLC peak for compound 2.5 (tR 14.2 min) had disappeared almost completely 28 and two new species had emerged at earlier retention times (tR 12.5 min and 13.4 min). We separated and isolated the two degradation products by column chromatography and analyzed them by 1H NMR spectroscopy and mass spectrometry. The two isolated degradation products were identified as the pyrone alcohol 2.3 and a methyl ester 2.6, suggesting that intermediate 2.5 underwent transesterification with methanol (Scheme 2.2).  Scheme 2.2. The two degradation products 2.3 and 2.6 formed by transesterification of intermediate 2.5.  29  Figure 2.2. HPLC analysis over 4 days showed decomposition of compound 2.5 (tR 14.2 min, which already partially degraded at the beginning of the experiment) into two fragments (tR 12.5 and 13.4 min) that could be identified as compounds 2.6 and 2.3, respectively. UV-Absorbance UV-Absorbance InitialUV-Absorbance 4 h in MeOH3 days in MeOH0 5 10 15UV-Absorbance Retention time [min]4 days in MeOH14.2 min13.4 min13.4 min12.5 min13.4 min14.2 min12.5 min14.2 min13.4 min12.5 min30 Although ester bonds are known to be prone to transesterification and hydrolysis, these reactions usually require the presence of catalytic amounts of acid or base in order for the reaction to proceed at a noticeable rate. A possible explanation for the accelerated reaction rate could be the presence of the free amino group after deprotection. The secondary amine could act as a Lewis base and polarize a solvent molecule (methanol) to facilitate its nucleophilic attack at the nearby ester carbonyl centre. Due to the proximity of the basic amine to the ester groups it could likely act as an intramolecular catalyst in this transesterification reaction (Scheme 2.3).   Scheme 2.3. Proposed mechanism of ester degradation by transesterification with methanol yielding the observed fragments 2.3 and 2.6. Avoiding protic solvents during purification could alleviate this problem and we indeed found the compound to remain stable for several hours when dissolved in aprotic solvents such as acetonitrile, acetone, and ethyl acetate. From a synthetic perspective it should therefore be possible to overcome this limitation by conducting subsequent synthetic steps in aprotic solvents and using anhydrous techniques. However, considering the aqueous environment in the intended biological applications, the observed instability would arguably result in a major stability liability for the target chelator. We therefore deemed it sensible to adapt our synthetic strategy and revisit our ligand design.  31 One potential alternative to overcome the instability of ester bonds would be to construct the chelator using amides instead. Amide bonds are known to be much more resistant to cleavage than ester bonds. We therefore chose to base our subsequent ligand design on amide bonds to provide superior stability for the binding unit-backbone linkages.  In addition to this change, we also opted to switch the 3-hydroxy-4-pyrone binding units for 3-hydroxy-4-pyridinone groups instead. The pyridinone binding units generally provide stronger metal ion complexation than the pyrone analogs do.   2.3 Conclusions Our synthetic efforts towards a bifunctional tetrakis(3-hydroxy-4-pyrone) chelator based on four ester linkages (p-SCN-Bn-THPO) were abandoned after instability of a synthetic intermediate was observed. The ester bonds of intermediate 2.5 were found to be unstable in protic solvents. Analysis of the degradation products suggested that the compound was susceptible to transesterification reaction with methanol and hydrolysis in aqueous media. The intermediate was stable in aprotic solvents, thus the synthetic strategy could in theory still be pursued employing aprotic reaction and purification conditions. However, in view of the intended application of the target chelator in an aqueous, biologic environment, this ester bond instability was deemed a potential liability of the ligand system. This warranted the reconsideration of our ligand and synthesis design. Amide bonds were identified as potential solutions to this instability. An alternative ligand synthesis was therefore pursued which was to be designed based on amide linkages to connect the binding units to the ligand backbone. Besides this modification, 3-hydroxy-4-pyridinone groups were chosen as binding units instead of 3-hydroxy-4-pyrone groups since they generally form stronger metal complexes. The design, 32 synthesis, and evaluation of this modified chelator system are discussed in the following chapters.  2.4 Experimental 2.4.1 Materials and Methods All chemicals were used as received without further purification. Building block 2.2 (2-(hydroxymethyl)-6-methyl-3-(phenylmethoxy)-4(1H)-pyranone) was prepared by Drs. Stoyan Karagiozov and Katayoun Saatchi according to a published procedure117 with minor adjustments. Iminodiacetic acid was purchased from TCI. N,N'-Dicyclohexylcarbodiimide (DCC) was obtained from Acros. All other chemicals and solvents were purchased from Sigma-Aldrich. All water used was ultrapure (18.2 MΩ∙cm) and was purified with a Millipore Milli-Q Integral-10 water purification system. Parsability of chemical nomenclature was confirmed with OPSIN.118, 119 NMR spectra were recorded on a Bruker Ascend 400 spectrometer (400.13 MHz for 1H; 100.62 MHz for 13C) at 297.16 K. Chemical shifts (δ relative to residual solvent peak) are reported as parts per million (ppm) and coupling constants (J) in hertz (Hz). ESI-MS spectra were recorded on an AB Sciex QTrap 5500 mass spectrometer. High-resolution mass spectrometry (HR-MS) analysis was acquired on a Thermo Scientific Q Exactive mass spectrometer. IR spectra were recorded on a PerkinElmer Frontier FT-IR spectrometer equipped with an attenuated total reflectance (ATR) crystal. HPLC was performed on a Waters Alliance e2695 separations module coupled to a Waters 2489 UV/Vis-detector. The column was a reversed phase C18 Waters Atlantis T3, 100 Å, 5 μm particle size (4.6 × 150 mm), supported by a C18 guard cartridge and was operated in an oven (40 °C). The column was eluted with following gradient (method A): A = 0.1% trifluoroacetic acid (TFA) in water; B = methanol; flow 33 rate = 1 mL/min; 0–5 min 90% A; 5–15 min 10–100% B; 15–18 min 100% B. Flash chromatography was performed on a Biotage Isolera One system using Biotage SNAP KP-Sil or ZIP silica gel cartridges. Analytical thin layer chromatography (TLC) was performed using silica gel 60 F254 plates with aluminium backing obtained from Merck Millipore. Silica gel for gravity column chromatography was from SiliCycle.  2.4.2 Syntheses 2.4.2.1 N-(9-Fluorenylmethoxycarbonyl)iminodiacetic acid (2.2) This synthesis was adapted from a literature procedure.116 In a dry round-bottom flask TMS-Cl (trimethylchlorosilane, 406.1 μL, 3.2 mmol) was dropwise added to a suspension of iminodiacetic acid (2.1, 133.0 mg, 1 mmol) in anhydrous CH2Cl2 (4 mL) and it was heated at reflux for 3.5 h. The mixture was cooled in an ice batch and DIPEA (N,N-diisopropylethylamine, 522.5 μL, 3 mmol) was added dropwise, followed by addition of Fmoc-Cl (9-fluorenylmethyloxycarbonyl chloride, 310.4 mg, 1.2 mmol) as a powder. The reaction was stirred for one hour at 0 °C and then overnight at ambient temperature. The reaction mixture was evaporated under reduced pressure and the residue was washed with Et2O and extracted with 10% Na2CO3 (3 × 20 mL). The combined aqueous phases were washed one more time with Et2O (20 mL) and then precipitated by addition of concentrated HCl at 0 °C. The precipitate was collected on paper by filtration and was dried in a desiccator to give compound 2.2 as a white powder in 92% yield (328 mg, 0.92 mmol).  1H NMR (400 MHz, CD3OD): δ 4.11 (s, 4H, NCH2), 4.25 (t, J = 6.8 Hz, 1H, CHCH2), 4.36 (d, J = 6.8 Hz, 2H, OCH2), 7.31 (t, J = 7.4 Hz, 2H, arom.), 7.39 (t, J = 7.5 Hz, 2H, arom.), 7.61 (d, J = 7.4 Hz, 2H, arom.), 7.80 (d, J = 7.5 Hz, 2H, arom.); 13C NMR (100 MHz, CD3OD): δ 173.0 (COOH), 34 158.0 (COON), 145.1 (arom. C), 142.6 (arom. C), 128.9 (arom. CH), 128.2 (arom. CH), 126.1 (arom. CH), 121.0 (arom. CH), 69.6 (OCH2), 50.5 (NCH2), 50.2 (NCH2), 48.2 (CH2CH); FT-IR (neat, ATR): νmax/cm−1 1724 (s), 1696s, 1446m, 1417m, 1364s, 1266s, 1250s, 1135s, 972m; HR-ESI-MS: calcd. (m/z) for C19H16NO6− [M-H]−: 354.09831; found: 354.09851; ESI-MS (m/z) 353.7 [M-H]−; HPLC: tR = 15.3 min (method A).   2.4.2.2 Bis((3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-yl)methyl) 2,2' N-(9-fluorenyl methoxycarbonyl)iminodiacetate (2.4) N-(9-Fluorenylmethoxycarbonyl)iminodiacetic acid (2.2, 177.7 mg, 0.50 mmol) and 2-(hydroxymethyl)-6-methyl-3-(phenylmethoxy)-4(1H)pyranone (2.3, 258.6 mg, 1.05 mmol) were suspended in anhydrous CH2Cl2 (3 mL) and DMAP (4-dimethylaminopyridine, 12.2 mg, 0.1 mmol) was added. In an ice bath, DCC (N,N'-dicyclohexylcarbodiimide, (206.3 mg, 1.0 mmol) was added as a solid and the mixture was stirred overnight at ambient temperature. After HPLC analysis indicated complete consumption of the starting material, the reaction mixture was filtered, rinsed with DCM, and the filtrate was washed with 0.5 M HCl (2 × 20 mL) and saturated NaHCO3 (2 × 20 mL). The organic phase was dried over MgSO4, filtered, and evaporated. The resulting oil was purified by two consecutive flash chromatographic separations over silica (0–8% MeOH/DCM) and product fractions were pooled and evaporated to give the title compound 2.4 as a transparent oil in 79% yield (321 mg, 0.40 mmol).  1H NMR (400 MHz, CDCl3): δ 7.71 (d, J = 7.2 Hz, 2H, arom.), 7.47 (d, J = 7.6 Hz, 2H, arom.), 7.22-7.36 (m, 14H, arom.), 6.15 (d, J = 4.4 Hz, 2H, CHCO), 5.19 (s, 4H, OCH2Ph), 4.85 (d, J 35 = 6.8 Hz, 4H, OCH2CO), 4.37 (d, J = 6.8 Hz, 2H, Fmoc-CH2), 4.18 (t, J = 6.6 Hz, 1H, Fmoc-CH), 4.09 (s, 2H, NCH2), 4.02 (s, 2H, NCH2), 2.17 (s, 3H, CH3), 2.09 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 175.4 and 175.3 (CHCO), 168.4 and 168.2 (CCH2N), 164.7 and 164.6 (CCH3), 155.4 (NOCO), 152.7 and 152.6 (OCH2CO), 143.7 and 143.6 (COBn), 143.3 (Fmoc-C), 141.0 (Fmoc-C), 136.0 (CH2CCH), 128.9 and 128.8 (arom. CH), 128.3 and 128.3 (arom. CH), 127.6 (arom. CH), 126.9 (arom. CH), 124.6 (arom. CH), 119.8 (arom. CH), 114.9 and 114.9 (CHCO), 73.6 and 73.6 (OCH2Ph), 68.1 (Fmoc-CH2), 58.3 and 58.2 (OCH2CO), 48.7 and 48.4 (NCH2), 46.7 (Fmoc-CH), 19.3 and 19.3 (CH3). HR-ESI-MS: calcd. (m/z) for C47H42NO12+ [M+H]+: 812.27015; found: 812.26989, calcd. (m/z) for C47H41NNaO12+ [M+Na]+: 834.25210; found: 834.25084; ESI-MS (m/z) 812.5 [M+H]+, 834.5 [M+Na]+; HPLC: tR = 17.4 min (method A).  2.4.2.3 Bis((3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-yl)methyl) 2,2'-iminodiacetate (2.5) Compound 2.4 (~92 mg, 0.11 mmol) was dissolved in DCM (1 mL) and piperidine (33.6 μL, 0.34 mmol) was added at 0 °C. After stirring for 1.5 h the reaction mixture was evaporated, the residue was taken up in an EtOAc/hexane mixture and loaded on a short silica column. Impurities were washed off with EtOAc/hexane (1:1) before eluting the product with MeOH/DCM (10%). The eluate was evaporated and analyzed. Although degradation products were observed after purification, the identity and predominance of the intended product, title compound 2.5, could be confirmed. 1H NMR (400 MHz, CDCl3): δ 7.34 (m, 10H, arom.), 6.19 (s, 2H, CH), 5.19 (s, 4H, CH2Ph), 4.86 (s, 4H, OCH2-pyr), 3.45 (s, 2H, NCH2), 3.41 (s, 2H, NCH2), 2.22 (s, 6H, CH3). HR-ESI-MS: calcd. (m/z) for C32H32NO10+ [M+H]+: 590.20207; found: 590.20374, calcd. (m/z) for 36 C32H31NNaO10+ [M+Na]+: 612.18402; found: 612.18384; ESI-MS (m/z) 590.3 [M+H]+, 612.2 [M+Na]+; HPLC: tR = 14.2 min (method A).  2.4.3 Decomposition Study In an HPLC autosampler vial, a small amount of compound 2.5 was dissolved in MeOH (2 mL). The solution was kept at ambient temperature and the transesterification reaction was monitored over 4 days by regular HPLC injections, at which point HPLC indicated nearly all starting material had been consumed. The solution was evaporated and separated with EtOAc over a small silica gel column. Two fractions were isolated, pooled, evaporated, and analyzed.   Fraction 1 was identified as compound 2.3 (2-(hydroxymethyl)-6-methyl-3- (phenylmethoxy)-4(1H)pyranone): 1H NMR (400 MHz, CDCl3): δ 7.38 (s, 5H, arom.), 6.19 (s, 1H, CH), 5.22 (s, 2H, CH2Ph), 4.28 (d, J = 6.8 Hz, 2H, OCH2-pyr), 2.26 (s, 3H, CH3). ESI-MS (m/z) 268.6 [M+Na]+ (calcd. 269.1); HPLC: tR = 13.4 min (method A).  Fraction 2 was identified as compound 2.6 ((3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-yl)methyl 2-((2-methoxy-2-oxoethyl)amino)acetate): 1H NMR (400 MHz, CDCl3): δ 7.40-7.31 (m, 5H, arom.), 6.21 (s, 1H, CH), 5.22 (s, 2H, CH2Ph), 4.89 (s,2H, OCH2-pyr), 3.74 (s, 3H, OCH3), 3.51-3.42 (m, 2H, NCH2COOMe), 3.51-3.42 (m, 2H, NCH2COOR), 2.26 (s, 3H, CCH3); ESI-MS (m/z) 398.3 [M+Na]+ (calcd. 398.1); HPLC: tR = 12.5 min (method A). 37 Chapter 3: The Octadentate Tetrakis(3-Hydroxy-4-Pyridinone) Chelator THPN for Zirconium(IV) Complexation  This chapter is, in part, an adaptation of published work, reproduced with permission from The Royal Society of Chemistry (C. Buchwalder, C. Rodríguez-Rodríguez, P. Schaffer, S. K. Karagiozov, K. Saatchi and U. O. Häfeli; A new tetrapodal 3-hydroxy-4-pyridinone ligand for complexation of 89zirconium for positron emission tomography (PET) imaging, Dalton Transactions, 2017, 46, 9654-9663).  3.1 Introduction Zirconium-89 is an appealing radionuclide for PET with long-circulating targeting vectors (cf., Section 1.5). The most widely employed chelator to complex 89ZrIV is the linear hexadentate tris(hydroxamic acid) ligand desferrioxamine B (DFO). However, DFO is deemed not a perfect chelator for zirconium(IV) and alternative ligands that could provide improved in vivo complex stability are needed. We sought to develop a tetrapodal octadentate ligand for ZrIV complexation to potentially overcome DFO’s limitations. After the encountered stability issues in our initial synthetic efforts towards a tetrapodal 3-hydroxy-4-pyrone chelator (Chapter 2), the ligand design was revisited. In addition to replacing the ester bonds (which were prone to cleavage) by amide bonds, we took the opportunity to also exchange the four 3-hydroxy-4-pyrone groups for four 3,4-HOPO binding units. Hydroxypyridinones generally form stronger metal complexes than their hydroxypyrone analogs and we therefore selected 3,4-HOPO groups to coordinate the hard ZrIV metal ion. 38 Several hexadentate 3,4-HOPO ligands have been reported in the literature and have been mainly studied with iron(III) or gallium(III).63, 67, 120 Only one of those tris(3,4-HOPO) chelators, CP256 (and its bifunctional derivative YM103), has also been studied with ZrIV by Blower and co-workers.113 However, the hexadentate 89Zr-CP256 complex suffered from considerable in vivo instability and readily decomposed. We were hopeful that (as also speculated by Blower et al.) an octadentate 3,4-HOPO chelator could provide more robust complexation of 89Zr by satisfying ZrIV’s coordination preference for eight donor atoms. The only octadentate HOPO ligands reported prior to our work were based on either four 1,2- or 3,2-HOPO groups (cf., Chapter 1) and to the best of our knowledge, no octadentate 3,4-HOPO chelator had yet been reported. Before tackling the direct synthesis of a bifunctional chelator, we therefore set out to develop an octadentate 3,4-HOPO chelator. After successful synthesis and characterization of the tetrakis(3,4-HOPO) chelator we named THPN, we investigated the potential of this new chelator in depth with zirconium(IV). Contingent on strong metal ion complexation, a bifunctional derivative was synthesized (Chapter 4). In this chapter, the synthesis and characterization of the tetrapodal ligand THPN and its Zr-THPN complex is presented along with the measurements of thermodynamic stability, radiolabeling of THPN with 89Zr, and the in vitro stability and in vivo biodistribution of the 89Zr-THPN complex in mice.  3.2 Results and Discussion 3.2.1 Synthesis and Characterization of THPN and Zr-THPN The octadentate chelator THPN was designed to contain four pendant arms with 3,4-HOPO coordinating groups. The length and positioning of the linker arms ortho and meta to the 39 coordination groups were chosen to facilitate a monometallic coordination behaviour.121 THPN was synthesized in two steps from the previously reported117 building block 3.1 (2-(amino-methyl)-1,6-dimethyl-3-(phenylmethoxy)-4(1H)-pyridinone, Scheme 3.1). Four equivalents of this benzyl-protected 3,4-HOPO amine were conjugated to 1,3-propanediamine-N,N,N',N'-tetraacetic acid (PDTA) using DCC/HOBt. The coupling reaction was monitored by HPLC and reached completion after six days at ambient temperature. Inspired by a published report,122 we also performed the coupling in a microwave (MW) reactor at 55 °C, which decreased the reaction time considerably and led to completion after six hours of irradiation. Subsequently, hydrogenolysis of the benzyl groups gave the unprotected tetrakis(3,4-HOPO) ligand THPN, which was purified by reversed phase chromatography and recrystallization. Using microwave conditions for the first reaction also increased the cumulative yield from ~51% to ~64% over two steps.  Scheme 3.1. Two-step synthesis of the tetrakis(3,4-HOPO) ligand THPN. a) DCC, HOBt, DMF, RT or MW 55 °C; b) H2, Pd/C, MeOH, RT.  40 Next, we produced the non-radioactive Zr-THPN complex by adding an equimolar amount of a ZrCl4 solution to THPN. Formation of the monometallic complex was demonstrated by MALDI-TOF mass spectrometry, which confirmed a 1:1 metal-to-ligand ratio with characteristic Zr-isotope pattern (Appendix A, Figure A.1). Using HPLC, formation of the Zr-THPN complex was observed by a single peak, which eluted with a slight delay in retention time (34 s) compared to the ligand THPN. Notable changes were also observed in the IR spectrum, where frequencies of the HOPO groups shifted upon complexation (Appendix A, Figure A.2). 1H NMR spectroscopy of the Zr-THPN complex gave broad and complex signals that were difficult to analyze. However, upon complexation signal splitting was observed for the HOPO ring protons suggesting an asymmetric constitution. This is in agreement with the slightly distorted DFT-optimized structure (vide infra).  3.2.2 Computational Studies Since crystals suitable for X-ray diffraction analysis could unfortunately not be obtained, we conducted ab initio density functional theory (DFT) calculations of the Zr-THPN complex to simulate the coordination geometry around the ZrIV centre. Calculations were carried out using the Gaussian 09 suite123 with the B3LYP functional124-126 and two different basis sets. First, we used the LanL2DZ basis set127-130 with B3LYP as these are among the most common parameters used for Zr-chelates.96, 100, 131 Since Holland et al.131 reported that for their Zr-systems the computationally more demanding basis set DGDZVP132, 133 provided slightly more accurate results, we repeated our calculations using this DGDZVP/B3LYP methodology as well.  41   Figure 3.1. DFT-optimized Zr-THPN complex structures in top-down (left) and side views (right).  While the structures on top were calculated with LanL2DZ/B3LYP, the bottom structures were generated using the DGDZVP/B3LYP methodology. Images were rendered in CYLview134 and hydrogen atoms were omitted for clarity. The DFT-optimized Zr-THPN structure (Figure 3.1) shows a saturated coordination sphere around the Zr4+ ion by the octadentate THPN ligand. The complex looks fairly symmetric and displays a square antiprismatic geometry with a slight distortion of the HOPO groups. The structures obtained with the two different DFT basis sets differ only marginally from each other (Figure 3.1). The calculated Zr−O bond lengths range between 2.18–2.29 Å using the LanL2DZ and 2.20–2.30 Å using the DGDZVP basis set. These values are comparable 42 to Zr−O bond lengths reported for the crystal structure of Zr-(3,4,3-(LI-1,2-HOPO),110 or the DFT-optimized structure of Zr-DFO86 (Table 3.1). With both basis sets, the Zr−O bond lengths of the hydroxylic oxygen atoms (O1, O3, O5, O7) were calculated to be slightly shorter than those of the ketonic HOPO oxygen atoms. Table 3.2 compares the O−Zr−O bond angles around the ZrIV centre, which ranged between 69.8°–81.4° (LanL2DZ) and 70.2°–82.7° (DGDZVP) for Zr-THPN.  Table 3.1. Comparison of Zr−O bond lengths for different Zr-complexes.a Bond [Zr-THPN]b,c [Zr-THPN]b,d [Zr-DFO-cis(OH2)2]+b,e [Zr-(3,4,3-(LI-1,2-HOPO)]f,g Zr−O1 2.183 2.205 2.26 2.1929 Zr−O2 2.267 2.304 2.12 2.1957 Zr−O3 2.219 2.246 2.19 2.1821 Zr−O4 2.249 2.295 2.11 2.1838 Zr−O5 2.188 2.203 2.25 2.2432 Zr−O6 2.291 2.277 2.20 2.1721 Zr−O7 2.234 2.231 2.33h 2.1981 Zr−O8 2.286 2.268 2.47i 2.2036 a All bond lengths are given in Å; b by DFT calculations; c using basis set LanL2DZ; d using basis set DGDZVP; e from ref.86; f by X-ray crystallography; g from ref.110; h axial, and i equatorial water ligands coordinating to ZrIV. Table 3.2. O−Zr−O bond angles for DFT-optimized Zr-THPN structure calculated with different DFT basis sets. Bond angle [Zr-THPN]a [Zr-THPN]b  Bond angle [Zr-THPN]a [Zr-THPN]b O1−Zr−O2 72.9° 72.2°  O8−Zr−O2 74.9° 70.4° O2−Zr−O3 76.1° 80.6°  O8−Zr−O1 80.7° 82.7° O3−Zr−O4 71.9° 70.8°  O1−Zr−O3 73.5° 70.2° O4−Zr−O5 81.4° 82.1°  O3−Zr−O5 78.1° 74.1° O5−Zr−O6 72.6° 72.7°  O5−Zr−O7 69.8° 73.8° O6−Zr−O7 78.7° 77.9°  O7−Zr−O1 74.2° 77.8° O7−Zr−O8 70.8° 71.7°  O2−Zr−O4 79.1° 75.2° O6−Zr−O8 76.1° 77.9°  O4−Zr−O6 71.1° 74.5° a Calculated using LanL2DZ/B3LYP; b calculated using DGDZVP/B3LYP methodology 43 3.2.3 Thermodynamic Solution Studies In order to quantify the thermodynamic stability of the Zr-THPN complex, we investigated the THPN ligand and its Zr-complex formation in several thermodynamic solution studies. 3.2.3.1 THPN Ligand Protonation Constants Since the ZrIV-affinity of the THPN ligand, and in particular of the HOPO groups, depends on the acid-base properties, the protonation equilibria of the THPN ligand were first determined by combined spectrophotometric-potentiometric titrations in aqueous solution. The UV-potentiometric titrations of an acidic THPN solution (8.47  10−5 M) were carried out in the pH range 1.6−11.5 by addition of standardized NaOH and measured at 25 °C, I = 0.16 M NaCl. Analysis of the combined potentiometric-spectrophotometric data with the software HypSpec2014135 allowed us to determine all ten protonation constants of THPN (Table 3.3) and generate the corresponding speciation plot (Figure 3.2d).  Table 3.3. Protonation constants of THPN.a Equilibrium reaction log β log K L + H+ ⇆ HL 10.28(2) 10.28 HL + H+ ⇆ H2L 20.11(2) 9.83 H2L + H+ ⇆ H3L 29.41(2) 9.30 H3L + H+ ⇆ H4L 38.12(2) 8.71 H4L + H+ ⇆ H5L 42.43(3) 4.31 H5L + H+ ⇆ H6L 46.14(3) 3.71 H6L + H+ ⇆ H7L 49.35(2) 3.21 H7L + H+ ⇆ H8L 52.49(2) 3.14 H8L + H+ ⇆ H9L 54.97(1) 2.48 H9L + H+ ⇆ H10L 56.77(2) 1.80 a Determined by simultaneous UV-potentiometric titrations at 25 °C, I = 0.16 M NaCl. Charges are omitted for clarity.  44 The speciation plot shows that at a physiologic pH of 7.4, THPN predominantly exists in its neutral form H4THPN. From the analysis of the spectra, two different buffer regions could be distinguished between pH 1.6−6 and pH 6−11, which are characterized by spectral evolutions marked by the appearance of different isosbestic points. Six protonation constants could be calculated from spectra in the pH range 1.6−6 (Figure 3.2a-b). Comparison with similar spectroscopic features of reported HOPO analogues136-140 allowed to attribute these constants to the protonations of the 4-oxo groups in the HOPO rings and to the protonations of the tertiary amines of the backbone. The highest protonation value in this buffer region (log K = 4.31(3)), could be attributed to one of the tertiary amines, while the other tertiary amine was assigned the most acidic protonation constant, log K = 1.80(2). This difference in acidity between the two amines can be explained by electronic repulsion between the two protonated amines as well as the possibility for stabilization of the protonated amine by hydrogen bonding with a neighbouring amide carbonyl. The remaining four protonation constants in this region (log K = 2.48, 3.14, 3.21, 3.71) were assigned to the protonations of the 4-oxo groups of the four HOPO moieties.  In the first dissociation equilibrium, there are two bands present at λmax = 247 and 282 nm, and two isosbestic points at 221 and 240 nm (Figure 3.2a). The spectroscopic features for the five successive equilibria are characterized by a decrease of the band at 247 nm, the increase of the band at 282 nm, and the appearance of a new band at 225 nm, as well as three new isosbestic points at 219, 236, and 255 nm, respectively (Figure 3.2b). The second buffer region between pH 6–11 (Figure 3.2c), presents the spectroscopic evolutions for the four equilibria involving the hydroxy functionalities in the HOPO units. The spectrum at pH 5.60 presents two maxima at 221 and 282 nm (Figure 3.2c). Upon increasing the pH, these bands shift to higher wavelengths and new bands appear at 230 and 312 nm with the occurrence of three 45 new isosbestic points at 225, 250, and 295 nm, confirming the presence of different species in this pH interval.    Figure 3.2. a-c) Representative spectra of the UV-potentiometric titration of an 8.47  10−5 M solution of THPN at different pH (at 25 °C, 0.16 M NaCl, l = 0.2 cm); d) Speciation plot for the THPN ligand calculated with protonation constants in Table 3.3, [THPN] = 8.47  10−5 M; dashed line represents pH = 7.4.  3.2.3.2 Complex Formation Equilibria with Fe3+ and Zr4+ The strength of the metal complexes formed between THPN and Zr4+ made it necessary to employ a combination of different methods. Attempts to directly determine the ZrIV-THPN stability constants by proton competition experiments were not conclusive by 225 250 275 300 325 3500.000.250.500.751.001.25iso221 nmpH 2.19  1.67 1.80 1.89 2.0 2.19AbsorbanceWavelength (nm)pH 1.67iso240 nma)225 250 275 300 325 3500.000.250.500.751.001.25iso255 nmiso236 nm 2.30 2.65 2.86 3.05 3.33 3.76 5.60pH 5.60AbsorbanceWavelength (nm)pH 2.30iso219 nmb)225 250 275 300 325 3500.000.250.500.751.001.25iso295 nmiso250 nm 5.60 8.30 8.91 9.26 9.55 9.78 9.96 10.24 10.51pH 5.60AbsorbanceWavelength (nm)pH 10.51iso225 nmc)2 4 6 8 10020406080100L4-HL3-H2L2-H7L3+H6L2+H5L+H3L-H4LH8L4+H9L5+% Formation relative to ligandpHH10L6+d)46 themselves as ZrIV-complexation was completed immediately after addition of the Zr4+ solution and the complex formation could not be reversed even at the most acidic pH studied. As an alternative, we therefore first determined the stability constants of the FeIII-THPN system and then conducted an FeIII-ZrIV metal-metal competition experiment. From combining these results the ZrIV-THPN stability constants could then be determined indirectly. The FeIII-THPN formation constants were determined by direct UV-Vis batch proton competitions, as well as by direct simultaneous potentiometric-spectrophotometric titrations. Figure 3.3 shows representative spectra from the batch titration of the FeIII-THPN system as the pH was raised. From pH 0.16−1.47 a new band emerges centered at 510 nm (Figure 3.3b), which is characteristic of red coloured FeIII-HOPO complexes and indicates the formation of the [FeIII(H2THPN)]+ complex from the colourless [FeIII(OH2)6]3+ in solution. In the pH range 1.47−3.26, a new band appears at 458 nm and two isosbestic points are observed at 360 and 503 nm, which indicate two species in equilibrium (Figure 3.3a,c). This is attributed to the deprotonation of a hydroxy group from one HOPO unit (pKa = 2.01), which leads to the formation of the fully coordinated FeIII-complex [FeIII(HTHPN)]. In fact, the last hydroxy substituent deprotonates with pKa = 8.23 to form the negatively charged [Fe(THPN)]− species, for which the spectrum remains unchanged because the last HOPO unit is not involved in the coordination sphere (Appendix C, Figure C.1). The FeIII-complex formation by THPN is further confirmed by a red shift of the free protonated ligand from 282 nm to 290 nm as the pH is raised from 0.16 to 3.26 (Figure 3.3d). The spectroscopic features in the formation of the FeIII-THPN complexes evolve similarly to those of the clinically used iron chelator deferiprone and other reported deferiprone analogs in the formation of their 1:1, 1:2, and 1:3 metal-to-ligand complexes.138-140 Table 3.4 presents the stability constants determined for the FeIII-THPN 47 complexes by refinement of the spectroscopic data with the HypSpec2014 software.135 The corresponding speciation plot is shown in Figure 3.5a.     Figure 3.3. Representative spectra of the UV-Vis titrations of the FeIII-THPN system at increasing pH values : a) from 250 to 650 nm and pH range 0.16−3.03; b) from 350 to 650 nm and pH range 0.16−1.47; c) from 350 to 650 nm and pH range 1.47−3.26; d) from 250 to 350 nm and pH range 0.16−3.26. [THPN] = [Fe3+] = 7.16  10−5 M, 25 °C, I = 0.16 M NaCl, l = 1 cm. Next, an indirect metal-metal competition UV-Vis titration was performed to determine the stability constants of the ZrIV-THPN complexes, as the stability constants were too high to be determined by direct methods. This method exploited the fact that, other than the FeIII-THPN complexes, the ZrIV-THPN complexes are spectroscopically silent in the visible 250 300 350 400 450 500 550 600 6500.00.51.01.52.02.5pH 0.16pH 3.03 3.03 2.57 1.97 1.69 1.18 0.95 0.82 0.71 0.52 0.16AbsorbanceWavelength (nm)pH 3.04a)350 400 450 500 550 600 6500.000.050.100.150.200.250.300.350.40pH 0.16pH 1.47 1.47 1.30 1.0 0.95 0.82 0.71 0.52 0.16AbsorbanceWavelength (nm)b)350 400 450 500 550 600 6500.000.050.100.150.200.250.300.350.40360nmpH 1.47pH 3.26 3.26 2.57 2.14 1.97 1.86 1.69 1.47AbsorbanceWavelength (nm)503nmc)250 275 300 325 3500.00.51.01.52.02.5283nmpH 0.16pH 3.26 3.26 2.14 1.69 1.47 0.95 0.82 0.71 0.16AbsorbanceWavelength (nm)d)48 range. The competition between the FeIII- and the ZrIV-complexes can therefore be observed by the disappearance of the red ligand-to-metal charge transfer (LMCT) band of the [Fe(H2THPN)]+ and [Fe(HTHPN)] complexes upon addition of Zr4+ equivalents (Figure 3.4 and Appendix C, Figure C.2). In addition, we observed a slight red shift of the UV band centered at 290 to 295 nm upon ZrIV-transmetalation of the FeIII-THPN complexes (Figure 3.4 left inset, Appendix C, Figure C.2, and Figure C.3). The pH was held constant at pH 2 to avoid precipitation of iron hydroxides.   Figure 3.4. Metal-metal competition spectra of the FeIII-THPN system with increasing equivalents of Zr4+. [THPN] = [Fe3+] = 7.16  10−5 M; [Zr4+]/[ Fe3+] = 0–4.5; pH = 2, 25 °C, I = 0.16 M NaCl, l = 1 cm. The insets represent the change in absorbance with increasing equivalents of Zr4+ at λ = 279, 300, or 305 nm (left) and at λ = 427, 475, or 510 nm (right), respectively. The stability constant of the [Zr(H2THPN)]2+ species could then be calculated from comparison with the spectra of the FeIII-THPN complexes present at pH 2 and are presented in Table 3.4. In additional direct UV-potentiometric titrations we found that [Zr(H2THPN)]2+ deprotonates its two protons with dissociation constants of pKa = 3.32 and 3.85, respectively to yield the neutral [Zr(THPN)] complex. These deprotonations are accompanied by a shift of the  0 1 2 3 40.00.10.20.3AbsorbanceZr4+ equivalents 510 nm 427 nm 475 nm0 1 2 3 41.01.52.02.5AbsorbanceZr4+ equivalents 305 nm 300 nm 279 nm49 band at 295 nm to 305 nm and the occurrence of an isosbestic point at 298 nm (Appendix C, Figure C.3). At pH ~6.5 the neutral [Zr(THPN)] complex started to precipitate and the potentiometric data were therefore not included in the calculations. However, as shown by the UV spectra in Appendix C, Figure C.3, the [Zr(THPN)] complex does not exhibit further transformations and remains stable at higher pH. The distribution diagrams for the ZrIV-THPN complexes were calculated using the stability constants presented in Table 3.4 and are shown in Figure 3.5. Table 3.4. Stability constants for the complexes formed by THPN with Fe3+ and Zr4+ ions, respectively at 25 °C, I = 0.16 M NaCl.  FeIII-THPN ZrIV-THPN Model log β pK log β pK MH2L 54.95(1)a  57.51(5)c  MHL 52.94(1)a 2.01 54.2(1)b 3.3 ML 44.71(7)b 8.23 50.3(1)b 3.9 pM 38.0 42.8 a Determined by proton competition UV-Vis titrations; b determined by simultaneous UV-potentiometric titrations; c determined by metal-metal competition UV-Vis titrations. Charges are omitted for simplicity.  The stability constants of the [Zr(THPN)] and the [Fe(THPN)]− complexes are both very strong (log β110 = 50.3(2) and log β110 = 44.71(7), respectively), with the ZrIV complex stability exceeding that of the FeIII complex. This confirms that THPN is capable of forming a thermodynamically stable ZrIV-complex. To the best of our knowledge, the stability constant for the [Zr(THPN)] complex, is the highest reported stability constant for ZrIV complexes in aqueous solution thus far. The closest value reported is for the [Zr(3,4,3-LI(1,2-HOPO))] complex and is seven orders of magnitude lower (log β110 = 43.1(6)).141   50     Figure 3.5. Speciation plots for a) FeIII-THPN complexes and b) ZrIV-THPN complexes calculated with stability constants from Table 3.4. [THPN] = [Mn+] = 1.10  10−4 M; dashed line indicates pH = 7.4. While the stability constant is a good measure for the overall thermodynamic complex stability in solution, an even more effective way to compare different chelators for biological applications is by employing pM values. pM is linearly correlated to the stability constant of the metal complexes at physiologically relevant conditions, and is defined as pM = − log [Mn+]free at pH 7.4 with [Mn+] = 1 M and [Lx−] = 10 μM.142, 143 Besides the thermodynamic stability constant of the metal complexes, the pM value also takes ligand acid-base properties into account, allowing for the most suitable comparison of the ability of different ligands with diverse basicities and/or denticities and different metal-to-ligand stoichiometries.144, 145 Even though the [Zr(THPN)] complex has the higher overall stability constant, the higher ligand basicity of THPN results in a 1.2 unit lower pM value for [Zr(THPN)] when compared to [Zr(3,4,3-LI(1,2-HOPO))] (Table 3.5). Still, both HOPO complexes exert impressively high thermodynamic stability. Surprisingly, the stability constants and pM values for ZrIV-complexes with DFO or the recently introduced DFO* family of chelators have not been experimentally determined to date. Abergel and co-workers, however estimated the pM value for the ZrIV-0 2 4 6 8 100102030405060708090100[Fe(THPN)]-Fe(HTHPN)[Fe(H2THPN)]+% Formation relative to Fe3+pHFe3+a)2 4 6 8 10020406080100[Zr(H2THPN)]2+[Zr(HTHPN)]+Zr(THPN)% Formation relative to Zr4+pHb)51 complex of DFO to be between 29 and 31.141, 146 pM values based on computational approximations have also been reported for the Zr-complexes of DTPA and citrate141, 147 and the values are included in the comparison in Table 3.5. Hence, from a thermodynamic stability point of view, both THPN and 3,4,3-LI(1,2-HOPO) appear to be much more effective in sequestering Zr4+ and form much stronger ZrIV-complexes than DFO, DTPA, or citrate.147 Table 3.5. Comparison of pM values for ZrIV-complexes with THPN or literature chelators.  THPN 3,4,3-LI(1,2-HOPO) DTPA DFO Citrate pM 42.8 44.0a ~35.0a,b ~29–31c ~28.7a,b a From ref.141; b calculated, from ref.147; c estimated, from refs.141, 146  These results further indicate that THPN is also an excellent FeIII-chelator. The pM value for FeIII-THPN (pM 38.0) exceeds that of the iron(III) chelate with the siderophore enterobactin (pM 34.3),148, 149 one of the strongest iron chelators. THPN may therefore hold therapeutic potential for treatment of iron overload disease.  3.2.4 Radiolabeling of THPN with 89ZrIV Next, the radiolabeling of THPN was investigated with positron-emitting zirconium-89. To this end, a neutralized 89Zr-oxalate solution (1.1 MBq, 29 μCi, 72 μL, pH ~7.5) was added to a THPN solution (8 μL, 1 mM; final [THPN] = 100 μM in water). Within 10 min at ambient temperature this quantitatively produced the single radiochemical species 89Zr-THPN, as determined by radio-HPLC. The radiocomplex eluted at a nearly identical retention time as the non-radioactive complex, with a minor difference (15 s) between the UV- and the gamma-traces due to the sequential arrangement of the detectors (Figure 3.6). In contrast, 89Zr-oxalate alone eluted with the mobile phase front, confirming that THPN is in fact chelating 89Zr4+. On instant 52 thin-layer chromatography (ITLC) strips the chelated 89Zr-THPN complex remained at the origin, whereas 89Zr-oxalate moved along with the mobile phase front (Appendix A, Figure A.3), further supporting the radio-HPLC results.  Figure 3.6. UV-HPLC trace of non-radioactive Zr-THPN (top) compared with radio-HPLC trace of 89Zr-THPN (bottom in blue). 89Zr-oxalate alone elutes with the mobile phase front (bottom in black). Radiotraces were normalized.  3.2.5 Concentration Dependence of Radiolabeling The concentration dependence of the 89Zr-radiolabeling of THPN was studied and compared to DFO. Ligand solutions of four different concentrations (167 μM, 16.7 μM, 1.67 μM, and 167 nM final concentration, respectively) were incubated with a neutralized solution of 89Zr-oxalate (~0.37 MBq, 10 μCi, pH ~7.0) and left to react at ambient temperature. The radiochemical yield (RCY) of the labeling reactions was quantified by ITLC at 10 min, 30 min, 1 h, 2 h, and 24 h. The same was done with DFO chelator solutions of the same concentrations. 53 ITLC strips were developed and the peaks were integrated to quantify the RCY of chelated 89Zr. Figure 3.7 shows the RCY as a function of time with different concentrations of THPN or DFO, respectively.   Figure 3.7. Radiochemical yield (RCY) of 89Zr-radiolabeling over time with THPN (left) or DFO ligand (right) at different ligand concentrations: 167 μM ( ), 16.7 μM ( ), 1.67 μM ( ), and 167 nM  ( ). Error bars indicate standard deviations. At micromolar concentrations THPN provides faster radiolabeling kinetics than DFO. At a THPN concentration of 1.67 μM, >95% RCY was achieved within 30 min, whereas DFO solutions of the same concentration took 1 h to reach a similar labeling yield. At ten- or one hundred-fold higher concentrations (16.7 μM or 167 μM), both DFO and THPN achieve quantitative RCY within 10 min. At sub-micromolar concentrations (167 nM) both ligands lead to incomplete radiolabeling, with DFO ultimately achieving a higher (~61%) RCY by the end of the experiment. One data point from DFO’s data set was excluded after a Grubbs test identified it as a statistical outlier.  The fast, quantitative radiolabeling provided by THPN is of importance to provide high specific activity under mild conditions (temperature, pH, solvent), which is crucial for the radiolabeling of sensitive antibodies or other biological targeting vectors. 54  3.2.6 Distribution Coefficients As an estimate for lipophilicity, the distribution coefficients logD7.4 for 89Zr-THPN and 89Zr-DFO were measured between n-octanol and phosphate-buffered saline (PBS) at pH 7.4 (Table 3.6). Both radiocomplexes demonstrated hydrophilic character, which can be attributed to their polar nature. Our data suggest 89Zr-THPN to be a factor of three times more hydrophilic than 89Zr-DFO. The logD7.4 value we measured for 89Zr-DFO deviated by the same amount from previously reported data for the same complex (−3.1 ± 0.1),96 which leads us to conclude the two radiocomplexes are similar in terms of their lipophilicity. A hydrophilic distribution coefficient of the radiocomplex could be advantageous for immunoPET applications as it should be less likely to drastically alter the solubility and biodistribution of antibodies. Table 3.6. Distribution coefficients logD7.4 measured for 89Zr-complexes.a Complex  logD7.4 89Zr-THPN  −3.1 ± 0.2 (n = 3) 89Zr-DFO  −2.6 ± 0.2 (n = 4) a Distribution coefficients between n-octanol and PBS at pH 7.4. Values are reported as average ± standard deviation.  3.2.7 In Vitro Stability Experiments Next, the in vitro stability of the 89Zr-THPN complex was assessed in a series of in vitro challenge studies and the results were compared against 89Zr-DFO.  55 3.2.7.1 Transchelation Competition Study A direct transchelation competition study was performed between THPN and DFO. For this, increasing concentrations of the challenge ligand DFO were added to 89Zr-THPN in THPN solutions. Similarly, different concentrations of THPN as a challenge ligand were added to 89Zr-DFO in DFO solutions. The reactions were monitored by ITLC (Biodex (dark green), sodium citrate mobile phase, 100 mM, pH 5.5), where 89Zr-THPN remained at the origin as a single spot (Figure 3.8A), whereas any 89Zr-DFO present was evident by activity migration113 (Figure 3.8B). Since the 89Zr-DFO migration was diffuse, and a small amount potentially remained at the baseline, integration to quantify the ratio between the two radiocomplexes was not possible, however the different appearances allowed the qualitative distinction of the predominating radiocomplex. Other ITLC conditions were also tried with different mobile (DTPA solution, 50 mM, pH 7.0) and stationary phases (Biodex (black), silica plates, or Whatman paper strips), but under these conditions the two radiocomplexes could not be distinguished from one another. In the first three reactions, the 89Zr-THPN complex was first formed by adding 89Zr-oxalate (~0.37 MBq, 10 μCi, pH ~7) to a THPN solution (5 μL; 1 mM), which was confirmed by ITLC (Figure 3.8A). Aliquots of increasing concentration of DFO (5 μL; 100 mM, 10 mM, or 1 mM, respectively) were then added to these mixtures. Reactions were monitored by ITLC after 10 min, 30 min, 1 h, 2 h, and 73 h, which showed that 89Zr-THPN was stable to competition from free DFO ligand at all tested concentrations over the duration of the experiment (Figure 3.8C). Even when exposed to a one hundred-fold excess DFO compared to THPN, no transchelation was observed. 56  Figure 3.8. ITLC chromatograms of A) 89Zr-THPN complex alone; B) 89Zr-DFO complex alone; C) transchelation challenge of 89Zr-THPN incubated with different amounts of DFO over time; D) transchelation challenge of 89Zr-DFO incubated with different amounts of THPN over time. Whereas 89Zr-THPN resists transchelation, 89Zr-DFO loses 89ZrIV to the THPN competition at a 1:1 ligand ratio within 30 min. ITLC strips were developed with sodium citrate solution (100 mM, pH 5.5). Next, the reverse experiment was performed by challenging 89Zr-DFO with THPN. 89Zr-oxalate (~0.37 MBq, 10 μCi, pH ~7) was added to three vials containing DFO solutions (5 μL; 1 mM). Formation of the 89Zr-DFO complex was confirmed by ITLC (Figure 3.8B) and then aliquots of decreasing concentration of THPN (5 μL; 1 mM, 100 μM, or 10 μM, respectively) were added to the mixtures. This time we used lower concentrations of the challenge ligand such that the THPN to DFO ratio was 1:1, 1:10, or 1:100 in favor of DFO. Monitoring by ITLC showed that after 30 min 89ZrIV transchelation took place from DFO to THPN in the 1:1 competition (Figure 3.8D). Even when only a tenth of THPN was present, some transchelation was evident after 1–2 h. Under conditions in which THPN:DFO was 1:100, no 57 transchelation was observed. All experiments were repeated for triplicates (Appendix A, Figure A.4). These results show that when in direct competition, the new chelator THPN outperforms DFO in terms of its potential to bind 89ZrIV. Whereas 89Zr-THPN appears resistant to transchelation even when exposed to a one hundred-fold excess of DFO, the 89Zr-DFO complex does not withstand a 1:1 challenge with THPN.  3.2.7.2 EDTA Challenge In order to further examine the Zr-complex stability, we challenged aliquots of the 89Zr-THPN and 89Zr-DFO complexes with one hundred-fold excess of free EDTA. The mixtures were incubated at a range of pH values (pH 5.0–8.0) at 37 °C over a period of seven days and the quantity of intact radiocomplex was determined by ITLC (Table 3.7). Interestingly, we did not observe the extent of 89Zr-DFO instability reported by Deri et al.111 Over a period of 7 days both, 89Zr-THPN and 89Zr-DFO, remained >99% intact at pH 6.0–8.0 and resisted transchelation. Transchelation to EDTA was only observed at pH 5.0. At this pH, 89Zr-DFO disintegrated completely and after one day only 1.6 ± 0.5% remained intact. 89Zr-THPN was affected by transchelation at this pH, but exhibited much better stability than the DFO-complex. After seven days at pH 5.0, 41.6 ± 11.1% of 89Zr-THPN remained intact.  In the physiologically more relevant pH range 6.0–8.0 the 100-fold excess EDTA did thus not impair 89Zr-THPN or 89Zr-DFO. Under the more extreme conditions at pH 5.0, 89Zr-THPN exhibited superior complex stability compared to 89Zr-DFO.   58 Table 3.7. EDTA challenge.a Complex pH initial 1 h 1 d 3 d 5 d 7 d 89Zr-THPN 8.0 99.2 ± 0.6 >99.9 >99.9 >99.9 >99.9 >99.9 7.0 99.2 ± 0.4 >99.9 >99.9 >99.9 >99.9 >99.9 6.0 99.7 ± 0.3 99.9 ± 0.3 >99.9 >99.9 >99.9 >99.9 5.0 99.1 ± 0.2 68.3 ± 5.1 62.1 ± 3.4 52.8 ± 13.3 40.5 ± 6.1 41.6 ± 11.1 89Zr-DFO 8.0 99.6 ± 0.3 99.7 ± 0.6 99.9 ± 0.2 99.7 ± 0.3 99.7 ± 0.3 99.5 ± 0.2 7.0 99.5 ± 0.2 >99.9 99.9 ± 0.1 99.9 ± 0.1 99.9 ± 0.2 99.9 ± 0.2 6.0 99.9 ± 0.2 99.7 ± 0.3 99.7 ± 0.3 99.5 ± 0.4 99.5 ± 0.4 99.6 ± 0.7 5.0 99.5 ± 0.2 64.6 ± 3.1 1.6 ± 0.5 1.2 ± 0.6 1.1 ± 1.1 0.5 ± 0.1 a Values are reported as percentage of intact 89Zr-complex (average ± standard deviation) after incubation with a 100-fold excess of EDTA at 37 °C. The experiments were performed in triplicate.  3.2.7.3 Serum Stability Study Incubation of 89Zr-THPN in human blood serum for seven days at 37 °C did not impair the complex stability considerably. ITLC analysis after incubation showed that 93 ± 2% of 89Zr-THPN remained intact. This compares well to the 89Zr-DFO complex, which remained 94 ± 8% intact.   3.2.8 In Vivo Behaviour In order to investigate the in vivo biodistribution and clearance pathway of 89Zr-THPN, the radiocomplex (5.22 ± 0.59 MBq, 141 ± 16 μCi) was injected intravenously into four healthy mice and imaged via PET/CT for 30 min post-injection. Within this short amount of time, most of the activity (~75 ± 13%) had already reached the urinary bladder and no other organ accumulation was detected (Appendix B, Figure B.1). After 27 h post-injection, the mice were sacrificed, the activity in the whole carcass was measured, and an ex vivo biodistribution 59 study was performed. After this time, only 2.7 ± 1.3% of the injected dose (%ID) remained in the entire carcass as the rest was excreted during recovery from anesthesia. Biodistribution results are shown in Table 3.8. Table 3.8. Biodistribution of 89Zr-THPN and 89Zr-DFO after 24 h post injection.a Organ  89Zr-THPN (n = 4)b 89Zr-DFO (n = 1) Blood 0.01 ± 0.01 0.07 Heart 0.05 ± 0.01 0.03 Liver 1.08 ± 1.25 0.58 Kidneys 3.49 ± 0.28 4.81 Lungs 0.24 ± 0.31 0.04 Small intestine 0.08 ± 0.04 0.07 Bladder 0.13 ± 0.03 0.08 Muscle 0.02 ± 0.01 0.00 Spleen 0.75 ± 0.94 2.37 Bone 0.11 ± 0.01 0.12 Stomach 0.06 ± 0.02 0.03 Pancreas 0.02 ± 0.01 0.01 Feces 0.70 ± 0.47c 0.68 Urine 0.21 ± 0.05 0.32 a Values are reported as percentage of injected dose per gram of tissue (%ID/g). Healthy female C57BL/6 mice were injected with either 89Zr-THPN (n = 4), or 89Zr-DFO (n = 1) via tail vein injection. The animals were sacrificed 27 h, or 25 h p.i., respectively; organs were harvested, weighed, and their activity was measured in a γ counter. b Average value from four animals ± standard deviation. c One data point was excluded after a Grubbs test identified it as an outlier.  One mouse was injected with 89Zr-DFO (2.8 MBq, 77 μCi) as control. Image quantification and subsequent biodistribution confirmed rapid renal excretion as well. Within 60 the first 30 min, ~43% of the injected activity reached the urinary bladder (Appendix B, Figure B.2) and 25 h post-injection only 2.4%ID remained in the entire mouse. Necropsy showed that this remaining activity was mainly associated with kidneys and spleen (Table 3.8). As the in vivo behaviour of 89Zr-DFO in murine models has been reported in many previous reports80, 86, 96, 111, 150 we used a single animal in this study. The fast blood clearance of the two radiocomplexes via the kidneys was expected due to their low molecular weights (<1 kDa) and was consistent with previous reports for 89Zr-DFO and other 89Zr-chelates.80, 86, 96, 111, 150 The absence of significant residual organ uptake, in particular in the bones, indicates that the complexes were stable in vivo over the course of the experiment. The observed in vivo stability and the fast pharmacokinetic excretion of 89Zr-THPN were promising and encouraged us to further scrutinize its long-term in vivo stability. Due to the fast excretion of 89Zr-THPN, the radiochelate had to be conjugated to a long-circulating carrier molecule to allow a conclusive assessment of this long-term stability. For this, a bifunctional derivative of THPN had to be developed, which could then be conjugated to a carrier (Chapter 4).  3.3 Conclusions The new octadentate tetrakis(3,4-HOPO) chelator THPN was synthesized, characterized, and studied as a Zr-chelator. The thermodynamic stability of THPN complexes with ZrIV, as well as with FeIII, were determined experimentally and found to be exceptionally high (Zr-THPN: log β110 50.3; pM 42.8; Fe-THPN: log β110 44.7; pM 38.0). The chelator was further investigated in vitro and in vivo with radioactive 89ZrIV and quantitative radiolabeling was achieved with 16.7 μM THPN concentrations within 10 min at ambient temperature. The radiocomplex was stable over a week in human blood serum and outperformed DFO in an 61 EDTA challenge, as well as a direct transchelation competition study. While 89Zr-THPN resisted a one hundred-fold excess of DFO, an equimolar amount of THPN induced transchelation of 89ZrIV from DFO to THPN. The radiocomplex was stable in vivo and was excreted rapidly via the renal pathway. These results led us to conclude that the first octadentate 3,4-HOPO chelator THPN is a promising novel chelator for 89Zr-based PET tracers and we further examined this ligand system by developing a bifunctional derivative (Chapter 4).   3.4 Experimental 3.4.1 Materials and Methods All chemicals were used as received without further purification and all water used was ultrapure (18.2 MΩ∙cm). Building block 3.1 (2-(aminomethyl)-1,6-dimethyl-3-(phenylmethoxy)-4(1H)-pyridinone) was prepared by Dr. Stoyan Karagiozov according to a published procedure117 with minor adjustments or was purchased from Otava Ltd. 1,3-Propanediamine-N,N,N',N'-tetraacetic acid (PDTA) was purchased from TCI. 1-Hydroxybenzotriazole hydrate (HOBt∙H2O) was purchased from AC PharmaChem. N,N'-Dicyclohexylcarbodiimide (DCC) and palladium on carbon (Pd/C) were purchased from Aldrich. Desferrioxamine (DFO, as mesylate salt), anhydrous N,N-dimethylformamide (DMF), acetonitrile, and methanol were purchased from Sigma-Aldrich. Zirconium(IV) chloride was purchased from Strem Chemicals. Celite 545 was purchased from Fisher Scientific. Parsability of chemical nomenclature was confirmed with OPSIN.118, 119 NMR spectra were recorded on a Bruker Ascend 400 spectrometer (400.13 MHz for 1H; 100.62 MHz for 13C) at 297.16 K. Chemical shifts (δ relative to residual solvent peak) are reported as parts per million (ppm) and coupling constants (J) in hertz (Hz). ESI-MS spectra were recorded on an AB Sciex QTrap 5500 mass 62 spectrometer. High-resolution mass spectrometry (HR-MS) analysis was acquired on a Thermo Scientific Q Exactive mass spectrometer. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a Bruker autoflex by UBC Mass Spectrometry Centre. IR spectra were recorded on an Agilent Cary 660 FT-IR spectrometer equipped with an attenuated total reflectance (ATR) crystal. HPLC was performed on a Waters Alliance e2695 separations module coupled to a Waters 2489 UV/Vis-detector and, for radio-HPLC, a LabLogic Scan-RAM radio-detector. The column was a reversed phase C18 Waters Atlantis T3, 100 Å, 5 μm particle size (4.6 × 150 mm), supported by a C18 guard cartridge and was operated in an oven (40 °C). The column was eluted with following gradient: A = 0.1% trifluoroacetic acid (TFA) in water; B = methanol; flow rate = 1 mL/min; 0–5 min 90% A; 5–15 min 10–100% B; 15–18 min 100% B. Microwave reactions were conducted in a Biotage Initiator+ microwave synthesizer. Flash chromatography was performed on a Biotage Isolera One system using a Biotage SNAP Ultra C18 reversed phase cartridge. 89Zr was produced at TRIUMF on a TR13 cyclotron (Ebco Industries Ltd.) via the 89Y(p,n)89Zr reaction from a Y(NO3)3 solution target,82 or at the BC Cancer Agency on a TR19 cyclotron (Advanced Cyclotron Systems Inc.) from a solid yttrium target (American Elements). 89Zr was purified either following a procedure by Holland et al.80 or using the commercially available ZR Resin (TrisKem International) and eluting with 0.05 M oxalic acid. 89Zr-oxalate solutions were neutralized with Na2CO3 solutions. Activities were measured using a Capintec CRC-55tR or a Biodex Atomlab 500 dose calibrator. Instant thin-layer chromatography (ITLC) was carried out using Biodex Tec-Control chromatography strips (black: #150-005, or dark green: #150-771), which were developed with an aqueous mobile phase of either DTPA solution (50 mM, pH 7.0) or sodium citrate solution (100 mM, pH 5.5)113 and analyzed on a Packard Cyclone storage phosphor 63 screen imager. Biodistribution samples were counted on a calibrated Packard Cobra II gamma counter.  3.4.2 Syntheses 3.4.2.1 2,2',2'',2'''-((Propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-(phenylmethoxy)-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (3.2) (a) Using standard conditions: A mixture of PDTA (12.3 mg, 40.0 μmol, 1 eq.), HOBt∙H2O (29.4 mg, 192 μmol, 4.8 eq.), and DCC (39.6 mg, 192 μmol, 4.8 eq.) was suspended in anhydrous DMF (1 mL) and stirred at ambient temperature under N2. After 3 h, building block 3.1 (2-(aminomethyl)-1,6-dimethyl-3-(phenylmethoxy)-4(1H)-pyridinone, 49.6 mg, 192 μmol, 4.8 eq.) was added as a powder and the reaction mixture was stirred at ambient temperature under N2 for 6 days until monitoring by HPLC showed complete consumption of the starting material 3.1. At this point the white slurry was filtered over fritted glass, rinsed with acetonitrile, evaporated, and purified by reversed phase flash chromatography (eluted with a gradient of 100% water with 0.1% formic acid to 100% methanol) to yield the title compound 3.2 as a colorless oil, containing some N,N'-dicyclohexylurea impurity that was removed in the subsequent reaction step.  (b) Using microwave irradiation: In a conical microwave reaction vial, a mixture of PDTA (50.5 mg, 165 μmol, 1 eq.), HOBt∙H2O (121.3 mg, 792 μmol, 4.8 eq.), and DCC (163.4 mg, 792 μmol, 4.8 eq.) was suspended in anhydrous DMF (10 mL) and crimp capped under N2. The mixture was stirred for 5 min at ambient temperature and then irradiated in a microwave 64 synthesizer in five intervals (2 h, 4 × 1 h) at 55 °C for a total of 6 h. Reaction progress was monitored by HPLC between MW intervals and once complete consumption of starting material 3.1 was achieved, the white slurry was filtered over fritted glass, rinsed with acetonitrile, evaporated, and purified by reversed phase flash chromatography (eluted with a gradient of 100% water with 0.1% formic acid to 100% methanol) to yield the title compound 3.2 as a colorless oil, containing some N,N'-dicyclohexylurea impurity that was removed in the subsequent reaction step.   3.4.2.2 2,2',2'',2'''-((Propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (THPN) In a dry Schlenk flask under N2, Pd/C (10% w/w, 13.7 mg, 12.7 μmol Pd), was suspended in MeOH (3 mL). To this was added a solution of compound 3.2 (39.6 mg, 31.2 μmol) in MeOH (3 mL) and it was rinsed with MeOH (7 mL). The reaction vessel was placed under a hydrogen atmosphere and stirred vigorously at ambient temperature. After 2.5 h, HPLC indicated completion of the reaction and the mixture was filtered through a plug of Celite 545 and rinsed ad libitum with MeOH and water. The filtrate was concentrated in vacuo and purified by reversed phase chromatography (eluted with a gradient of 100% water to 100% methanol). The product fractions were pooled, evaporated, and recrystallized from MeOH/H2O (20:1) to give the title compound THPN as a white powder (~11.5 mg). Cumulative yield over two steps: 51% using standard method (a); 64% using microwave method (b). 65 1H NMR (400 MHz, CD3OD): δ 6.28 (s, 4H, CH), 4.60 (s, 8H, OCNCH2), 3.66 (s, 12H, NCH3), 3.21 (s, 8H, OCCH2), 2.51 (m, 4H, NCH2CC), 2.39 (s, 12H, CCH3), 1.51 (m, 2H, CCH2C); 13C NMR (100 MHz, CD3OD/D2O (4:1)): δ 174.0, 171.0, 149.0, 147.1, 132.5, 114.9, 59.6, 54.7, 37.3, 35.9, 25.9, 21.1; FT-IR (ATR): νmax/cm−1 1654.5s, 1624.3s, 1576.7s, 1503.0vs, 1241.4vs; HR ESI-MS: calcd. (m/z) for C43H59N10O12+ [M+H]+: 907.43084; found: 907.42932, (1.68 ppm); calcd. (m/z) for C43H57N10O12– [M–H]–: 905.41629; found: 905.41681, (0.57 ppm); ESI-MS (m/z) 907.6 [M+H]+, 929.3 [M+Na]+, 905.4 [M−H]−; elemental analysis (%) calcd. for C43H58N10O12∙2(CH3OH)∙H2O: C 54.65, H 6.93, N 14.16; found: C 54.85, H 6.81, N 14.31; HPLC: tR = 9.7 min.  3.4.2.3 Zr-THPN Complex An aqueous solution of ZrCl4 (669 μL, 10 mM, 6.69 μmol 1.05 eq.) was dropwise added to a stirring solution of THPN in 1:1 MeOH/water (637 μL, 10 mM, 6.37 μmol, 1.00 eq.), the pH was adjusted with 0.1 M Na2CO3 (170 μL) to pH ~6–7 and the solution was stirred overnight at ambient temperature. The cloudy mixture was centrifuged (5 min at 13,000 rpm) and the supernatant was removed. The precipitate was characterized as the Zr-THPN complex by MALDI-TOF MS, HPLC, and FT-IR spectroscopy. FT-IR (ATR): νmax/cm−1 1647.2s, 1560.7s, 1509.5vs, 1476.6s, 1300.0s, 1259.3s; MALDI-TOF MS: calcd. (m/z) for C43H55N10O12Zr+ [Zr-THPN+H]+: 993.3; found: 993.3; HPLC: tR = 10.3 min.   3.4.3 Computational Studies Density functional theory (DFT) calculations were conducted as implemented in the Gaussian 09 revision E.01 suite of ab initio quantum chemistry programs.123 Geometry optimizations were performed by using the B3LYP124-126 functional in combination with 66 LanL2DZ127-130 or DGDZVP132, 133 basis sets, which were used for description of the valence electrons as well as the effective core potentials of the ZrIV ion. Normal self-consistent field (SCF) and geometry convergence criteria were employed and Zr-THPN was optimized in the gas phase without the use of symmetry constraints.   3.4.4 Thermodynamic Solution Studies 3.4.4.1 Materials and Methods Protonation constants and metal stability constants were obtained by combined potentiometric-spectrophotometric titrations as described before.151 Measurements were conducted at 25 °C and an ionic strength of I = 0.16 M NaCl, using a Metrohm Titrando 809 equipped with a Ross combined electrode, a Metrohm Dosino 800, and a Varian Cary 60 UV-Vis spectrophotometer (200–800 nm spectral range) connected to a 0.2 cm path length (l) optic dip probe immersed in the titration cell. Additional batch experiments to study proton competition or metal-metal competition were carried out in a 1 cm cuvette at 25 °C and 0.16 M NaCl ionic strength to determine the formation constants of the FeIII-THPN and ZrIV-THPN systems. Metal ion solutions were prepared from atomic absorption (AA) standard metal ion solutions. The exact amount of acid present in the iron and zirconium standards was determined by Gran’s method,152 titrating equimolar solutions of either metal ion and Na2H2EDTA. All potentiometric measurements were processed using the Hyperquad2013 software,153 whereas spectrophotometric data were processed using the HypSpec2014 software.135 Speciation plots for the ligand and metal complexes were calculated with HySS154 using the constants in Table 3.3 and Table 3.4 and the metal hydrolysis equilibrium constants. The molar absorptivities of all protonated species of THPN were included in the metal stability calculations. Proton dissociation constants corresponding to hydrolysis of aqueous Fe3+ and Zr4+ ions included in the calculations were 67 taken from Baes and Mesmer.155 The species formed in the studied systems were characterized by the general equilibrium: mM + lL + hH ⇆ MmLlHh (charges omitted). For convention, a complex containing metal ion M, ligand L, and proton H has the general formula MmLlHh. The stoichiometric index m might also be 0 in the case of protonation equilibria. Negative values for the index h refer to proton removal or hydroxide ion addition during formation of a M-OH complex. The overall equilibrium constant for the formation of the complex MmLlHh from its components is designated as log β. Stepwise equilibrium constants log K correspond to the difference in log units between the overall constants of sequentially protonated (or hydroxide) species. pM is defined as − log [Mn+]free and is always calculated at [Mn+] = 1 μM, [Lx−] = 10 μM, pH 7.4, and 25 °C.143 To facilitate reading, ligand nomenclature is generally described using the neutral form THPN herein without reflecting its protonation state unless specific protonation of the ligand is being discussed.  3.4.4.2 THPN Ligand Protonation Constants Protonation equilibria of the ligand were studied by simultaneous potentiometric-spectrophotometric titrations of an acidic solution of THPN (8.47  10−5 M) at 25 °C, l = 0.2 cm and 0.16 M NaCl ionic strength. Electromotive force values and spectra were recorded after each addition of NaOH (0.1496 M) or HCl and both instruments were synchronized in order to have constant delays between each titrant addition and to allow enough time to reach equilibrium. The potentiometric and spectrophotometric data collected for at least three replicates were processed using the HypSpec2014 and Hyperquad2013 software suites.135, 153 The respective molar absorptivities of the differently protonated species were also determined.  68 3.4.4.3 Metal Complex Formation Constants The strength of the resulting metal complexes made it necessary to employ different methods to study the complexation of THPN with FeIII and ZrIV. UV-Vis experiments were conducted to determine the FeIII-THPN formation constants by direct proton competition and to study the FeIII/ZrIV competition. In addition to these experiments, direct simultaneous potentiometric-spectrophotometric titrations were carried out with both, the FeIII-THPN and the ZrIV-THPN systems, once their first formation constants were determined using the above methods. Direct proton competition experiments were carried out by UV-potentiometric batch experiments (spectral range 200–800 nm, 25 °C, l = 1 cm) on a set of 22 solutions containing [THPN] = [Fe3+] = 7.16  10−5 M with different amounts of standardized HCl to cover a pH range of 0.16–3.26. The ionic strength of each sample was adjusted to 0.16 M by addition of different amounts of NaCl.  To determine the formation constant of the [Zr(H2THPN)]2+ complex, metal-metal competition UV-Vis titrations were carried out. A set of 20 solutions containing [THPN] = [Fe3+] = 7.16  10−5 M with varying amounts of competing Zr4+ ions were prepared with [Zr4+]:[Fe3+] molar ratios ranging from 0 to 4.5. The pH was adjusted with standardized HCl to pH 2 and the ionic strength of each sample was adjusted to 0.16 M by addition of different amounts of NaCl as supporting electrolyte. The samples were left up to 24 h in order to reach equilibrium before measurement of pH and UV-Vis spectra. UV-Vis measurements were carried out in a 1 cm cuvette at 25 °C and 250–700 nm spectral range. Additional direct UV-potentiometric titrations were carried out for either the FeIII-THPN or the ZrIV-THPN system, by titrating equimolar solutions of THPN ligand and the metal ion with standardized NaOH ([THPN] = [Mn+] ≈ 2.11  10−4 M, 25 °C, 0.16 M NaCl, l = 0.2 cm).  69  3.4.5 Concentration Dependence of 89Zr-Radiolabeling Aliquots of a neutralized solution of 89Zr-oxalate (25 μL, ~0.37 MBq, 10 μCi, pH ~7) were added to aqueous THPN or DFO ligand solutions of different concentrations (5 μL; 1 mM, 100 μM, 10 μM, or 1 μM) and were left to react at ambient temperature. Each reaction mixture was analyzed by ITLC (black strips, DTPA mobile phase, 50 mM, pH 7.0) after 10 min, 30 min, 1 h, 2 h, and 24 h reaction time and the percentage of chelated 89Zr was determined by integration of the ITLC peaks. The experiments were performed in triplicate.  3.4.6 Human Serum Stability  A neutralized solution of 89Zr-oxalate (48 μL, ~0.85 MBq, 23 μCi, pH ~7) was added to aqueous ligand solutions of THPN or DFO, respectively (5.3 μL, 1 mM) and quantitative radiocomplex formation was confirmed by ITLC. Human serum (500 μL from a healthy male donor) was added to these mixtures and the vials were incubated for 7 days at 37 °C with agitation (500 rpm). Aliquots were analyzed by ITLC (black strips, DTPA mobile phase, 50 mM, pH 7.0) after 3, 5, and 7 days incubation and the percentage of chelated 89Zr was determined by integration. As a control, 89Zr-oxalate was incubated with human serum. The experiment was performed in duplicate.   3.4.7 Distribution Coefficients A neutralized solution of 89Zr-oxalate (102 μL, ~0.81 MBq, 22 μCi) was added to aqueous ligand solutions of THPN or DFO, respectively (20 μL, 1 mM) and quantitative radiocomplex formation was confirmed after 15 min at ambient temperature by ITLC (black 70 strips, DTPA mobile phase, 50 mM, pH 7.0). Aliquots of radiocomplex solutions in replicates (30 μL, ~6 μCi, THPN: n = 3; DFO: n = 4) were diluted with PBS (470 μL), 1-octanol (500 μL) was added, and the mixtures were thoroughly vortexed for 1 min at 3,000 rpm. After centrifugation for 5 min at 14,000 rpm, ~200 μL of both the organic and of the aqueous phases were removed and counted separately in a gamma counter. Results were corrected for decay and the distribution coefficients at pH 7.4 of [89Zr-THPN] and [89Zr-DFO] were then calculated as log D7.4 = log10 ([89Zr]octanol / [89Zr]aqueous).  3.4.8 Transchelation Competition Study  Aliquots of a neutralized solution of 89Zr-oxalate (30 μL, ~0.37 MBq, 10 μCi, pH ~7) were added to solutions of THPN (5 μL; 1 mM) and left to react for 15 min and formation of 89Zr-THPN was verified by ITLC. After this, aliquots of DFO (5 μL; 100 mM, 10 mM, or 1 mM) were added to the mixtures. Each reaction mixture was analyzed by ITLC (dark green strips, sodium citrate mobile phase, 100 mM, pH 5.5) after 10 min, 30 min, 1 h, 2 h, and 73 h reaction time at ambient temperature. The experiments were performed in triplicate. In a different set of experiments, aliquots of a neutralized solution of 89Zr-oxalate (30 μL, ~0.37 MBq, 10 μCi, pH ~7) were added to solutions of DFO (5 μL; 1 mM) and left to react for 15 min and formation of 89Zr-DFO was verified by ITLC. After this, aliquots of THPN (5 μL; 1 mM, 100 μM, or 10 μM) were added to the mixtures. Each reaction mixture was analyzed by ITLC (dark green strips, sodium citrate mobile phase, 100 mM, pH 5.5) after 10 min, 30 min, 1 h, 2 h, and 73 h reaction time at ambient temperature. The experiments were performed in triplicate.   71 3.4.9 EDTA Competition Study  Aliquots of a neutralized solution of 89Zr-oxalate (~0.67 MBq, 18 μCi, 35 μL, pH ~7.5) were added to solutions of either THPN or DFO (0.1 mM, 55 μL) and left to react for 15 min. Formation of 89Zr-THPN or 89Zr-DFO, respectively, was verified by ITLC. After this, EDTA solutions (5 mM, 110 μL) of different pH values (pH 5.0, 6.0, 7.0, or 8.0) were added as well as sodium acetate solutions (0.5 M, 50 μL) of the same pH value. The mixtures were incubated for 7 days at 37 °C with slight agitation (350 rpm) and samples were analyzed by ITLC (black strips, DTPA mobile phase, 50 mM, pH 7.0) after 1 h, 1 d, 3 d, 5 d, and 7 d. The percentage of intact radiocomplex was determined by integration. Whereas intact 89Zr-THPN and 89Zr-DFO remained at the ITLC baseline, transchelated 89Zr-EDTA moved with the eluent front. The experiments were performed in triplicate.  3.4.10 In Vivo Study of 89Zr Complexes  This animal study was performed in accordance with the animal care committee (ACC) of the University of British Columbia under the approved protocol A12-0172. Two-month-old healthy female C57BL/6 mice (Charles River Laboratories) were anesthetized using isoflurane inhalation and received a subcutaneous injection of lactated Ringer’s solution (0.5 mL) for hydration prior to the experiment. Sterile filtered (0.22 μm) solutions of 89Zr-THPN (5.22 ± 0.59 MBq, 141 ± 16 μCi, n = 4) or 89Zr-DFO (2.8 MBq, 77 μCi, n = 1) in PBS were intravenously administered via tail vein injection. Oxalate concentrations were minimized (<30 mM) to account for its toxicity. The animals were imaged on a PET/CT scanner during the first 30 min post-injection and recovered from anesthesia after the scan. After 27 h (89Zr-THPN) or 25 h post-injection (89Zr-DFO) the animals were sacrificed and the total body 89Zr activity was 72 measured in a dose calibrator. Then, organs were excised, weighed, and their activity was counted in a γ counter and decay corrected to conduct a biodistribution.  3.4.11 PET/CT Imaging  Animals were imaged on a dedicated small animal PET/SPECT/CT scanner (VECTor/CT, MILabs) equipped with a HEUHR high energy multipinhole collimator suitable for PET isotopes. Dynamic whole body scans were acquired in list-mode format over 30 min. Following each PET acquisition, a whole body CT scan was performed to obtain anatomical information and both images were registered. The photopeak energy window was centered at 511 keV with a 20% energy window width. Throughout the entire scanning procedure, mice were kept under isoflurane anesthesia and constant body temperature was maintained using a heating pad. For quantitative analysis, PET data were reconstructed with ordered subset expectation maximization logarithm (OS-EM) using ten iterations of 16 subsets and 0.4 mm3 voxel size. The images were decay corrected and after CT registration, attenuation correction was applied using MILabs proprietary software. Volumes of interest were manually defined using the software AMIDE (v.1.0.4) to determine the time activity pattern per target organ. Thus, the delineated regions were urinary bladder and background. In order to relate the scanner units (counts/pixel) to radioactivity concentration (mCi/mL), a calibration factor was determined by scanning a 12 mL syringe with a known concentration of 89Zr. 73 Chapter 4: Bifunctionalization of THPN and Study of 89Zr-THPN Conjugates  4.1 Introduction After the study of THPN with zirconium(IV) identified the tetrakis(3,4-HOPO) ligand as a very promising ZrIV-chelator (Chapter 3), we set out to investigate THPN in conjunction with delivery vehicles. For this, a bifunctional derivative of THPN was first developed to allow covalent attachment to carrier molecules. In a nine-step synthesis, we produced a bifunctional THPN derivative bearing an isothiocyanate group on the backbone. This functional group is reactive to amino groups and can thus be tethered to any amine-bearing delivery vehicle. Once a bifunctional THPN ligand was produced, we investigated its conjugation and 89Zr-radiolabeling with four different carrier molecules; the two monoclonal antibodies trastuzumab and anti-programmed death ligand-1 (anti-PD-L1), the protein mouse serum albumin, as well as polymeric nanoparticles based on hyperbranched polyglycerol (HPG). The radiolabeling of HPG radioconjugates was found to be particularly robust and thus, HPG was chosen as carrier molecule to examine the long-term radiocomplex stability in vivo. The behaviour of 89Zr-THPN-HPG radioconjugates was investigated in healthy mice in an acute biodistribution and PET/CT imaging study and was compared against two radiolabeled HPG conjugates prepared with literature chelators, 89Zr-DFO-HPG and 89Zr-DFO*-HPG.  74 4.2 Results and Discussion 4.2.1 Synthesis of Bifunctional THPN In order to allow covalent conjugation of THPN to delivery vehicles, a bifunctional derivative of THPN had to be produced. The central carbon of THPN’s propyl backbone was chosen as position to install a conjugation handle in the form of a para-isothiocyanatobenzyl group. This positioning should allow the conjugation group to point away from the metal coordination groups to minimize interference between conjugated targeting molecules and metal complexation. Furthermore, this is the only position that maintains the symmetry of the molecule and thereby avoids the creation of chiral centres. Figure 4.1 depicts the intended bifunctional THPN derivative p-SCN-Bn-THPN 4.10 and the building blocks it may be synthesized from.  Figure 4.1. Retrosynthetic analysis of the intended bifunctional THPN derivative p-SCN-Bn-THPN reveals the building blocks from which the bifunctional chelator may be assembled.  75  Scheme 4.1. Nine-step synthesis of the bifunctional ligand p-SCN-Bn-THPN, 4.10. i) NaH, THF, 35 °C; ii) NH3, MeOH, RT; iii) BH3·THF, THF, RF; iv) Na2CO3, DMF, RT; v) H3PO4, MeCN, 45 °C; vi) DCC, HOBt, DMF, MW 55 °C; vii) AcOH/HCl (conc.), 50 °C; viii) H2, Pd/C, MeOH, RT; ix) CSCl2, CHCl3/H2O, RT. Cumulative yield over nine steps: ~7%. The nine-step synthesis of the bifunctional chelator p-SCN-Bn-THPN 4.10 is presented in Scheme 4.1. The benzyl-protected HOPO-methylamine building block 4.6 is a relatively expensive starting material and was the limiting reagent in this synthesis. We therefore introduced the HOPO motif only at a late stage in the synthesis to reduce losses of this starting material. We chose to install the conjugation handle at the beginning of the synthesis, similarly 76 to a strategy pursued by Price et al.156 The conjugation handle should be introduced in the form of a protected para-nitrobenzyl group which could be converted in the last reaction steps into a reactive para-isothiocyanatobenzyl group. After installation of the para-nitrobenzyl group, the four arms of the backbone should be assembled to allow subsequent coupling with four HOPO binding units. The final steps involved deprotections and activation of the conjugation handle as isothiocyanate group, which is reactive towards conjugation to amino group bearing delivery vehicles. The first step of the reaction sequence involved the alkylation of the C3 building block diethyl malonate with 4-nitrobenzyl bromide (Scheme 4.2). Initially, we tried to reproduce a literature procedure156 that uses sodium ethoxide as a base in ethanol. In our hands, this procedure, however, only led to the doubly alkylated product (Scheme 4.2). Variation of stoichiometry or temperature did not change this, but a workaround was found by changing the base and solvent. Following a different literature procedure,157 we employed sodium hydride as base with the solvent tetrahydrofuran. This approach produced the intended mono-alkylated product 4.1 in 53% yield, similar to the literature protocol (52%).   Scheme 4.2. Alkylation of diethyl malonate greatly depends on reaction conditions leading to addition of either one or two equivalents of nitrobenzyl. 77 The next step involved the preparation of the diamide 4.2 by conversion of the two ethyl ester groups of 4.1 into amides. Following a reported procedure,156 compound 4.1 was treated with methanolic ammonia to produce the diamide 4.2 in 89% yield (literature: 83%). Next, the amide groups were reduced to primary amines. Similarly to reported procedures,156, 158 compound 4.2 was reacted with borane in THF to produce the diamine, which was isolated as dihydrochloride salt in 66% yield (literature: 60%). In order to introduce the four ligand arms, the two amino groups were then alkylated each with two equivalents of tert-butyl bromoacetate using sodium carbonate as a base. This produced the tetrakis(tert-butyl ester) 4.4 in ~46% yield. In the next step the tert-butyl ester protection groups were hydrolyzed to give the corresponding carboxylic acids. Attempts to saponify 4.4 using lithium hydroxide remained unsuccessful. This could, however, be overcome by employing aqueous ortho-phosphoric acid, which Li et al.159 described as a mild deprotection reagent for tert-butyl esters. In this way, the deprotected tetraacid 4.5 was obtained in near-quantitative yield (97%). Next, four equivalents of the benzyl-protected HOPO-methylamine 4.6 (2-(aminomethyl)-1,6-dimethyl-3-(phenylmethoxy)-4(1H)-pyridinone) were conjugated to the tetraacid 4.5, similar to the amide couplings in the THPN synthesis (Chapter 3). The reaction was conducted in a microwave reactor with gentle heating and the coupling reagents DCC/HOBt were employed as before. The carbodiimide DCC is a reliable and widely used amide coupling reagent. It can, however, also lead to side reactions and forms different by-products that can be difficult to remove (Figure 4.2).  78  Figure 4.2. Possible side products and by-products that can form during the amide coupling reaction between compounds 4.5 and 4.6 in addition to the desired product 4.7.  Several steps were taken to minimize the formation of side products. The coupling mechanism of carbodiimide-mediated amide formation proceeds via an O-acylurea intermediate. This intermediate can either react with the amine to form the intended amide, or it can convert into an unreactive N-acylurea in an irreversible side reaction. In order to minimize this side reaction, HOBt was used as a coupling additive. HOBt is believed to react with the O-acylurea to form a more stable active ester intermediate, which can then react with the amine to produce the desired amide product.160-163 Not only the acid, but also the amine 4.6 appears to react with DCC to irreversibly form a guanidine side product (Figure 4.2). To minimize this loss of valuable starting material, the tetraacid 4.5 was first reacted with DCC and HOBt alone for two hours to allow reaction time to form the active ester intermediate, before addition of the amine 4.6. The reaction was closely monitored by HPLC to follow the formation of product, as 79 well as side products. HPLC also proved very helpful in the optimization of the purification of the fairly complex reaction mixture. Filtration and purification by reversed phase flash chromatography was found to be an effective purification method. Elution with an acetonitrile gradient produced the tetraamide product 4.7 pure in ~83% yield. Using acetonitrile instead of methanol as organic component of the eluent was found to separate the product better from the coupling by-product N,N'-dicyclohexylurea (DCU), which is well soluble in MeOH but only sparingly soluble in MeCN. The next two reactions involved the debenzylation of the HOPO hydroxyls and the reduction of the p-nitrobenzyl group. Both of these conversions can be conducted in a single reaction step by acidic hydrogenation. Yet, we chose to perform the reactions in two separate steps, which allowed better control over the reaction and improved yield. First, the four debenzylations were performed by heating overnight in a mixture of glacial acetic acid and concentrated hydrochloric acid. This gave the tetrakis(HOPO) compound 4.8 in excellent yield (94%). Next, the nitro group of 4.8 was reduced to its aniline analog by hydrogenation over a heterogeneous palladium catalyst to quantitatively produce compound 4.9. It should be noted that the choice of the filter aid used during workup was found to be of importance. When the reaction mixture was passed through a filter plug of regular Celite® 545, a red-brown coloured filtrate was obtained and 1H NMR peaks showed peak broadening. This may be the result of complexation of metal ion impurities (e.g., Fe3+) that may be present in the filter material. This assumption is supported by the very similar colour of the Fe-THPN complex. When instead an acid-washed diatomaceous earth was used as filter aid, namely AW Standard Super-Cel®, the product was obtained colourlessly and NMR peaks were markedly sharper, suggesting no metal complexation. 80 Lastly, in order to activate the conjugation handle, the aniline group of 4.9 was converted into its isothiocyanate derivative by reaction with thiophosgene in a biphasic chloroform/water mixture. Purification in several batches by semi-preparative HPLC and reversed phase flash chromatography gave the activated bifunctional chelator p-SCN-Bn-THPN (4.10) in ~65% yield.  Cumulative yield over the entire nine-step synthesis was ~7%. It is worth mentioning that most of the lower yielding reactions occurred at the beginning of the multi-step synthesis where starting materials were inexpensive and readily available. The valuable HOPO-building block 4.6 was introduced at a later stage during the synthesis and the cumulative yield over the remaining four reaction steps of 50% was relatively high in comparison.  4.2.2 89Zr-Trastuzumab Radioimmunoconjugates Once the bifunctional derivative p-SCN-Bn-THPN was produced, its conjugation and 89Zr-radiolabeling was investigated with several carrier molecules. First, the antibody trastuzumab was explored as a targeting vector. Trastuzumab is a humanized monoclonal antibody (mAb) that targets the human epidermal growth factor receptor 2 (HER2).164 The transmembrane tyrosine kinase receptor HER2 is involved in the regulation of cell proliferation and survival. HER2 can be overexpressed in a variety of cancers and in particular in breast and stomach cancers, HER2 overexpression is associated with poor prognosis.165, 166 Thus, the immunoglobulin G (IgG) trastuzumab (Herceptin®) is an approved treatment for HER2-positive breast and metastatic stomach cancers. Conjugation and radiolabeling of trastuzumab with bifunctional THPN was explored pursuing either a postlabeling or a prelabeling strategy (Figure 4.3). In the postlabeling approach, the chelator is first tethered to the targeting vector and the resulting 81 immunoconjugate is radiolabeled in the second step. In contrast, in the prelabeling approach, the bifunctional chelator is first radiolabeled, following conjugation to the targeting vector.167 The postlabeling approach is more commonly used for 89Zr-radiolabeling as it allows for better control of the conjugation stoichiometry and for purification of the immunoconjugate from unreacted chelator. This often leads to higher radiochemical yields and specific activities (i.e., [labeled mAb]/[unlabeled mAb]). A prelabeling strategy, on the other hand, also holds advantages. It can be particularly useful when only small amounts of targeting vector are available (such as expensive investigational mAbs) or when high toxicity is of concern as this approach reduces the number of handling steps.101 Prelabeling is also an advantageous strategy if the radiolabeling requires harsh reaction conditions such as temperatures, solvents, or a pH that would compromise targeting vector integrity.167  Figure 4.3. Two strategies can be pursued for the conjugation and radiolabeling of a bifunctional chelator. In the postlabeling approach (top), the chelator is first tethered to the targeting vector and the immunoconjugate is radiolabeled in the second step. In contrast, in the prelabeling approach (bottom), the bifunctional chelator is first radiolabeled and conjugated to the targeting vector in the second step. (mAb structure generated with QuteMol49 from protein data bank entry 1igy50).  82 4.2.2.1 89Zr-Labeling of Trastuzumab by Postlabeling First, a postlabeling strategy was pursued since THPN exhibited fast radiolabeling kinetics with 89Zr at ambient temperature (Chapter 3). In analogy to a protocol by Vosjan et al.,168 we modified trastuzumab with a five-fold molar excess of p-SCN-Bn-THPN by conjugation of the chelator’s isothiocyanate group to amino groups of random mAb lysine residues (Scheme 4.3). The product was purified by size-exclusion chromatography (SEC) and/or ultrafiltration and the THPN-immunoconjugate was analyzed by SE-HPLC and by gel electrophoresis (SDS-PAGE) in which no changes were observed from unmodified trastuzumab. Analysis by MALDI-TOF mass spectrometry according to a published procedure169 also confirmed a slight   Scheme 4.3. Modification of trastuzumab with the three chelators p-SCN-Bn-THPN, p-SCN-Phe-DFO, and p-SCN-Phe-DFO*. 83 increase in average protein mass from 147.8 kDa to 148.5 kDa due to the conjugated chelator. Equivalent conjugations were also performed with the two literature chelators p-SCN-Phe-DFO and p-SCN-Phe-DFO* as comparisons (Scheme 4.3). All three immunoconjugates showed no noticeable changes in their chromatograms on SE-HPLC.  Figure 4.4. (Radio-)SE-HPLC chromatograms of a) THPN-, b) DFO-, and c) DFO*-modified trastuzumab before (top) and after 89Zr-radiolabeling by postlabeling (centre and bottom). The three immunoconjugates were then radiolabeled with 89Zr following the method by Vosjan et al.168 and the reactions were monitored by ITLC (complexed 89Zr-immunoconjugate: Rf ~ 0; 89Zr-oxalate: Rf ~ 1). Once ITLC indicated satisfactory radiolabeling (typically near-Absorbance (280 nm)Absorbance (280 nm)0 5 10 15 20 25Radioactivity Retention time [min]Absorbance (280 nm)0 5 10 15 20 25Radioactivity Retention time [min]Absorbance (280 nm)Absorbance (280 nm)0 5 10 15 20 25Radioactivity Retention time [min]Absorbance (280 nm) UV-trace-trace89Zr-THPN-trastuzumab 89Zr-DFO-trastuzumab 89Zr-DFO*-trastuzumabTHPN-trastuzumab DFO-trastuzumab DFO*-trastuzumaba) b) c)84 quantitative), the mixtures were purified by size-exclusion chromatography and/or ultrafiltration and were analyzed by radio-SE-HPLC (Figure 4.4).  The chromatograms of the DFO- and DFO* radioimmunoconjugates looked as expected and showed a single major peak in the UV- and γ-traces that matched the unmodified and chelator-conjugated mAbs before labeling. Radiolabeling of the THPN-conjugate, on the other hand, led to multiple peaks in the radiochromatogram. While one peak matched the retention time of the intended radioimmunoconjugate, another large peak eluted at an earlier retention time, indicating the presence of species of larger molecular size. A small peak at a later retention time matched in retention time with 89Zr-oxalate and indicated presence of free 89Zr4+ or small sized 89Zr species. Moreover, this peak grew considerably after storage over three days at ambient temperature, suggesting a release of 89Zr from the larger species. We speculate that the large species are 89Zr-labeled antibody aggregates, such as antibody dimers or trimers, which is in line with analyses by gel electrophoresis (SDS-PAGE). Various efforts were made to minimize the formation of aggregates and the radiolabeling was repeated with different batches of chelator and with modifications to a range of radiolabeling parameters. These modifications included: the THPN/mAb stoichiometry, the radiolabeling pH, the order of reagent addition, the concentration of immunoconjugate, the 89Zr activity concentration, the labeling buffer (PBS, HEPES, acetate), presence or absence of citrate as a transfer ligand, presence or absence of gentisic acid as protectant from radiolysis, the purification method (SEC vs. ultrafiltration), the form of 89Zr starting material (89Zr-oxalate vs. 89ZrCl4), and the 89Zr sourcing and production method (solid vs. liquid 89Y target). Under certain conditions, the formation of aggregates could be reduced for small radiolabeling reactions (albeit with some inseparable free 89Zr), yet when the reactions were scaled up to larger amounts of 89Zr, the aggregate formation increased again (Figure 4.5).  85        Figure 4.5. Radio-SE-HPLC chromatograms of 89Zr-THPN-trastuzumab using improved postlabeling conditions showed less aggregates on a small scale (left), but when repeated with more 89Zr, aggregate formation was again more pronounced (right). To explore a different conjugation chemistry, a tetrazine-bearing THPN derivative (Tz-THPN) was produced. The 1,2,4,5-tetrazine group (Tz) can undergo an inverse electron demand [4 + 2] Diels-Alder (IEDDA) cycloaddition reaction with trans-cyclooctene (TCO). This conjugation reaction has recently been introduced as a bioorthogonal click reaction for pretargeting and in vivo click coupling.170-175 To this end, we synthesized a PEG4-spaced THPN-tetrazine derivative 4.12 (Tz-THPN) by reacting p-SCN-Bn-THPN with a commercially available methyl-tetrazine amine (MeTz-PEG4-NH2) and purified it by reversed phase flash chromatography (Appendix D, Figure D.1). Meanwhile, trastuzumab was modified with TCO groups by reaction with a commercially available TCO active ester (TCO-NHS, Appendix D, Figure D.1). A colorimetric assay with a dye-tetrazine revealed an average presence of 1.8 reactive TCO groups per mAb when a 35 molar excess of TCO active ester was used.  Following this click conjugation strategy, 89Zr-radiolabeling was pursued both in a prelabeling and in a postlabeling approach (Appendix D, Figure D.1). Yet the results were not 0 5 10 15 20 25Radioactivity Retention time [min]UV-Absorbance 0 5 10 15 20 25Radioactivity Retention time [min]UV-Absorbance 86 satisfactory and the produced radioimmunoconjugates still showed aggregates as well as a tendency to stick to the HPLC tubing rendering such analyses difficult to interpret. The limitations encountered with the postlabeling strategy and the persistent challenges with the Tz/TCO click conjugation approach motivated us to investigate radiolabeling of trastuzumab by a prelabeling approach.  4.2.2.2 89Zr-Labeling of Trastuzumab by Prelabeling With the aim of reducing the aggregate formation, a prelabeling approach was explored for the radiolabeling of THPN and trastuzumab. The bifunctional chelator (~51 nmol) was first complexed with a neutralized 89Zr4+ solution (~11 MBq, 300 μCi) and analyzed after ten minutes by ITLC, which showed quantitative radiolabeling. At this point, unmodified trastuzumab (~1.5 mg) was added and the pH was raised to pH ~9 to allow thiourea bond formation between the radiocomplex and trastuzumab. After purification by size-exclusion chromatography, 89Zr-THPN-trastuzumab was isolated in 52% radiochemical yield and radio-SE-HPLC showed that the formation of aggregates could indeed be suppressed and the product was obtained in ~93% radiochemical purity (HPLC). The radioimmunoconjugate eluted as a single peak with only a very small shoulder (Figure 4.6A). The peak matched the retention time of unmodified trastuzumab and remained stable over three days when stored at 4 °C. Analysis by gel electrophoresis (SDS-PAGE, 7.5%) furthermore confirmed that trastuzumab was successfully radiolabeled and autoradiographic development clearly showed that the 89Zr activity was associated with the antibody band of around 146 kDa (Figure 4.6B). Only a very faint second band at around 250–300 kDa suggested minimal amount of presumably dimerized mAb, which is in line with the small shoulder observed in the radiochromatogram. 87         Figure 4.6. Analyses of 89Zr-THPN-trastuzumab produced by a prelabeling approach (A) by radio-SE-HPLC and (B) by gel electrophoresis. Coomassie blue staining of the SDS-PAGE (7.5%) gel shows a) unmodified trastuzumab (146 kDa) and b) 89Zr-THPN-trastuzumab of equivalent size. Autoradiography c) of lane b) shows clear association of 89Zr with the mAb band. Scaling the radiolabeling up with more 89Zr (~47 MBq, 1.3 mCi) and less mAb was successful but remained challenging. Relatively large amounts of chelator (~17 nmol) were required to obtain sufficient radiolabeling and avoid presence of free 89Zr. This had to be pursued since the removal of unchelated 89Zr was found to be challenging and inefficient. A larger amount of chelator in turn required more antibody (~0.5 mg) to maintain a feasible conjugation ratio between mAb and radiochelate and therefore limited the attainable specific activity. Additionally, considerable amounts of activity were lost adhering to the reaction vial (~40%) or stuck to the size-exclusion column (~20%) during purification. Thus, the radioimmunoconjugate was isolated in ~22% radiochemical yield with a radiochemical purity of around 80% (radio-SE-HPLC). The specific activity was estimated as ~10 MBq/mg, 260 μCi/mg. The losses of 89Zr during the synthesis might be caused by hydrolysis of the isothiocyanate group, which at alkaline pH directly competes with the conjugation reaction to the antibody. 0 5 10 15 20 25Radioactivity Retention time [min]89Zr-THPN-TrastuzumabγUV-Absorbance 89Zr-THPN-TreastuzumabUVA88 Additionally, the solubility of 89Zr-THPN-NCS appeared to be limited and could explain precipitation and adhesion of the radiocomplex to the reaction vial, rendering it unavailable for reaction with the mAb and, thus, lowering the yield. The relatively low specific activity limited the possibility to perform a quantitative in vivo study by PET/CT imaging. Instead, an in vitro plasma stability study was conducted.  4.2.2.3 In Vitro Plasma Stability Study The stability of the 89Zr-THPN-trastuzumab radioimmunoconjugate was assessed in an in vitro plasma stability study. As a comparison, radioimmunoconjugates were also produced with the two literature chelators DFO and DFO*. For consistency, these were also produced in a prelabeling strategy for this study. Vugts et al.101 reported that the prelabeling approach was only successful with DFO* and did not work with DFO. In our hands, however, both chelators could be prelabeled with 89Zr (5.4 MBq, 145 μCi) and be conjugated to trastuzumab (~1.5 mg). The DFO- and DFO*-radioimmunoconjugates were isolated in 58% and 55% radiochemical yield, respectively, and radiochemical purities of >99% (radio-SE-HPLC). To conduct the in vitro plasma stability study the radioimmunoconjugates with either THPN, DFO, or DFO* as chelator were diluted in mouse plasma and were incubated for seven days at 37 °C. The integrity of the radiocomplexes was assessed by analyzing aliquots by ITLC after 1, 3, 5, and 7 days of incubation. The experiment was conducted in duplicate and the results are presented in Table 4.1. As control, a freshly neutralized solution of 89Zr-oxalate was also incubated with mouse plasma and showed only very minor 89Zr-binding over seven days. The THPN and DFO radioimmunoconjugates remained largely intact and the DFO* conjugate showed no signs of demetallation. Thus, all three radioimmunoconjugates remained over 95% 89 intact over seven days indicating reliable complex stability under these simulated physiologic conditions. Table 4.1. Plasma stability of 89Zr-chelate-trastuzumab radioimmunoconjugates.a Radioimmunoconjugate Day 0 Day 1 Day 3 Day 5 Day 7 89Zr-THPN-trastuzumab >99% >99% 99% 99% 95% 89Zr-DFO-trastuzumab 99% >99% >99% 94% 99% 89Zr-DFO*-trastuzumab >99% >99% >99% >99% >99% 89Zr-oxalate (control) 0% 0% 4% 6% 6% a Stability over time is reported as percentage bound 89Zr after incubation in mouse plasma at 37 °C, 550 rpm. Radioimmunoconjugate experiments were performed in duplicate and are reported as mean,  while the control experiment with 89Zr-oxalate was performed once.  4.2.3 89Zr-Anti-PD-L1 Radioimmunoconjugates In order to explore whether the postlabeling approach for THPN could be more successful with another antibody instead of trastuzumab, a postlabeling strategy was investigated with a monoclonal anti-mouse antibody that targets programmed death ligand-1 (PD-L1). PD-L1 is a transmembrane protein that acts as a ligand for the immune checkpoint receptor programmed death-1 (PD-1). Inhibition of PD-1 or PD-L1 has recently gained attention as a promising target for cancer immunotherapy and new methods to monitor their expression levels are currently being sought.176-185 In analogy to the postlabeling method used with trastuzumab, anti-PD-L1 mAb was first modified according to the protocol by Vosjan et al.168 by reacting it with a ten-fold molar excess of p-SCN-Bn-THPN. The antibody size was assessed by SE-HPLC, where no change was detected upon chelator conjugation (Figure 4.7). The immunoconjugate was then radiolabeled in HEPES buffer (0.5 M, pH 7.0) with neutralized 89Zr-oxalate. After purification by SEC and/or ultrafiltration, only small amounts of aggregates were detected. In a first batch, when ~100 μg 90 immunoconjugate was radiolabeled with 7.5 MBq, 202 μCi 89Zr-oxalate, the purified product (recovered in 44%) showed three peaks on radio-SE-HPLC (Figure 4.7). The main peak (~64%) matched the retention time of the mAb and confirmed radiolabeling of the THPN-anti-PD-L1 immunoconjugate. A small shoulder peak (~10%) detected at an earlier retention time indicates presence of some larger molecular size compounds, presumably antibody aggregates. Another small peak (~10%) was detected at a later retention time and was attributed to small molecular size 89Zr species, such as free 89ZrIV or 89Zr-EDTA (from quenching) that could not be removed during purification. The product was recovered in 44% radiochemical yield with a radiochemical purity of around 64% as indicated by radio-SE-HPLC    Figure 4.7. (Radio-)SE-HPLC chromatograms of a) unmodified anti-PD-L1 mAb, b) THPN-modified anti-PD-L1 mAb, and c & d) 89Zr-THPN-anti-PD-L1. When in a different batch, larger amounts of both, immunoconjugate (~320 μg) and 89Zr activity (18 MBq, 476 μCi) were used, the formation of aggregates was further reduced. However, a large second peak indicated more uncomplexed or free 89Zr species that could not be removed by SEC or ultrafiltration (Figure 4.8). It should be mentioned that a different SE-UV-Absorbance 0 5 10 15 20 25Radioactivity Retention time [min]UV-Absorbance UV-Absorbance anti-PDL1 mAbUVTHPN-PDL1-mAbUV89Zr-THPN-PDL1-mAb89Zr-THPN-PDL1-mAbUVa)b) d)c)91 HPLC column was used compared to the previous batch in an attempt to better separate the species, explaining the difference in retention times. The HPLC chromatogram further showed some streaking and adhesion of activity to the radiation detector, rendering this method not very conclusive (Figure 4.8). Analysis by SDS-PAGE, however, confirmed that the immunoconjugate was radiolabeled and only a small band at larger molecular size indicated the presence of larger sized aggregates (Figure 4.9). Small molecular sized activity may have been missed with this analytical method missed as it could have run off the gel and thus were excluded from autoradiographic exposure.   Figure 4.8. (Radio-)SE-HPLC chromatograms of a) unmodified anti-PD-L1 mAb, b) THPN-modified anti-PD-L1 mAb, c & d) 89Zr-THPN-anti-PD-L1.  UV-Absorbance anti-PDL1 mAbUVUV-Absorbance 0 5 10 15 20 25Radioactivity Retention time [min]UV-Absorbance THPN-PDL1-mAbUV89Zr-THPN-PDL1-mAb89Zr-THPN-PDL1-mAbUVa)b) d)c)92  Figure 4.9. Analyses of anti-PD-L1 antibodies by SDS-PAGE (7.5%): a) unmodified anti-PD-L1 mAb, b) THPN-modified anti-PD-L1 mAb, c & d) 89Zr-THPN-anti-PD-L1; a-c) Coomassie blue stained, d) autoradiography of lane c). These findings show that the radiolabeling of THPN-anti-PD-L1 by postlabeling remains challenging, but in contrast to trastuzumab, leads to considerably less antibody aggregation. The substantial aggregate formation observed for postlabeling of trastuzumab may therefore be the result of a particular incapability of trastuzumab’s protein structure with the physicochemical properties of the 89Zr-THPN complex.  Proteins and antibodies in particular, are known to be susceptible to aggregation when modified with conjugates. This can be particularly often observed in the development of antibody drug conjugates (ADCs) with hydrophobic drugs, which induce a strong tendency to aggregate.186, 187 Antibody aggregation was also reported as a potential pitfall for the production of radioimmunoconjugates.103, 187, 188 The physicochemical properties of the attached chelates affect the native antibody structure and can lead to unfolding of subdomains or exposure of regions that can increase the tendency for aggregation.187 Different antibodies that possess a 93 different amino acid sequence and tertiary structure may be more or less susceptible to a given modification.  4.2.4 89Zr-Mouse Serum Albumin (MSA) Radioconjugates To explore 89Zr-labeling of a different protein than an antibody with THPN, mouse serum albumin (MSA) was radiolabeled employing a postlabeling approach. MSA was modified with a tenfold molar excess of p-SCN-Bn-THPN and purified by size-exclusion chromatography. Analysis by SE-HPLC and gel electrophoresis indicated no noticeable changes upon modification. The THPN-MSA conjugate (~264 μg) was then radiolabeled in HEPES buffer (0.5 M, pH 7.0) with a neutralized solution of 89Zr-oxalate (5.0 MBq, 134 μCi). After purification by size-exclusion chromatography, the product was obtained in an excellent radiochemical yield of 98% and in radiochemical purity of ~88% (HPLC) with a specific activity of ~15 kBq/μg; 0.42 μCi/μg. Analysis by radio-SE-HPLC showed a matching retention time of the radiolabeled conjugate with unmodified and THPN-modified MSA (Figure 4.10). The only difference observed was some peak tailing upon radiolabeling, which might be due to a change in overall polarity that led to difference in the interaction with the column material. Analysis by SDS-PAGE also showed a consistent molecular size between the unmodified (66 kDa), THPN-modified, and radiolabeled MSA (Figure 4.11). Autoradiography clearly associated the large majority of activity with the MSA protein. Only a faint band was noticed at a larger molecular size. However, this larger band was also observed for the unmodified MSA and might be due to impurity of the starting material. Storage of the 89Zr-THPN-MSA radioconjugate for six days at ambient temperature or at 4 °C did not cause changes in radio-SE-HPLC indicating a good stability of the radioconjugate. 94  Figure 4.10. (Radio-)SE-HPLC chromatograms of unmodified MSA, THPN-modified MSA, and radiolabeled 89Zr-THPN-MSA. 0 5 10 15 20 25Retention time [min]89Zr-THPN-MSAUVTHPN-MSAUVMSAUV89Zr-THPN-MSAγ95  Figure 4.11. Analysis by SDS-PAGE (12%) of a) unmodified MSA (66 kDa), b) THPN-modified MSA, c & d) radiolabeled 89Zr-THPN-MSA; a-c) Coomassie blue stained, d) autoradiography of lane c) showing clear association of 89Zr with the MSA protein.  4.2.5 89Zr-Hyperbranched Polyglycerol (HPG) Radioconjugates 4.2.5.1 Chelator Conjugation and Radiolabeling In order to evaluate the long-term stability of the 89Zr-THPN radiocomplex and compare it to the literature chelators DFO and DFO*, polymeric nanoparticles based on hyperbranched polyglycerol (HPG) were selected as carrier molecules. HPG particles are highly biocompatible189-193 and can be readily synthesized as globular macromolecules with narrow size distributions. They have been investigated as drug delivery vehicles,194 as blood plasma expanders,195 as cell surface protectants,196 or as carrier molecules for imaging agents.193, 197, 198 Owing to the long circulation half-life of large molecular weight HPG, these nanoparticles are suitable carriers to test the long-term stability of attached cargo molecules. 96 The three chelators p-SCN-Bn-THPN, p-SCN-Phe-DFO, and p-SCN-Phe-DFO* were conjugated to amine-functionalized HPG particles by thiourea bond formation. After incubation overnight, the modified HPG particles were purified by SEC and ultrafiltration. Analysis by dynamic light scattering (DLS) indicated that chelator modifications did not significantly affect the particle size and size distribution, which was corroborated by indistinguishable SE-HPLC chromatograms (Figure 4.12).  Figure 4.12. (Radio-)SE-HPLC chromatograms of a) unconjugated HPG-NH2, b) HPG-chelator conjugates, and c) radiolabeled 89Zr-chelate-HPGs show no difference upon conjugation and radiolabeling. The chelator-modified HPG nanoparticles were then radiolabeled at neutral pH with 89Zr-oxalate solutions (~32 MBq, ~870 μCi). Once quantitative radiolabeling was reached, the reaction mixtures were purified by SEC and the radioconjugates were recovered in 97% 97 radiochemical yield for all three conjugates. The products were analyzed by ITLC and radio-SE-HPLC, which indicated >99% radiochemical purity and showed retention times and peak shapes that matched those of amino-HPG and chelator modified HPG particles (Figure 4.12). DLS analysis showed that the hydrodynamic diameter and polydispersity of the particles remained largely constant upon radiolabeling.  4.2.5.2 In Vitro Plasma Stability of 89Zr-HPG Conjugates As a first indication of physiologic stability of the HPG radioconjugates, an in vitro plasma stability study was conducted. 89Zr-HPG particles with either THPN, DFO, or DFO* as chelator were diluted four-fold in human blood plasma and were incubated for five days at 37 °C. Aliquots were analyzed by ITLC after 1, 3, and 5 days. The fraction of intact radiocomplex was quantified by integration of the bound fraction of 89Zr on ITLC plates, where complexed 89Zr remained close to the baseline (Rf ~ 0). The obtained results for the three radioconjugates are shown in Table 4.2. The DFO* radioconjugate showed no signs of dissociation and also the other two radioconjugates remained largely intact and prevailed with >95% integrity after five days of exposure to plasma proteins. Table 4.2. In vitro plasma stability of 89Zr-chelate-HPG radioconjugates.a Radioconjugate initial 1 d 3 d 5 d 89Zr-THPN-HPG >99% 97% 96% 96% 89Zr-DFO-HPG >99% >99% >99% 97% 89Zr-DFO*-HPG >99% >99% >99% >99% a Stability over time is reported as percentage of bound 89Zr after incubation in human plasma at 37 °C, 550 rpm. Measurements were performed in triplicate and are reported as mean.   98 4.2.5.3 In Vivo Stability of 89Zr-HPG Conjugates In order to investigate the long-term in vivo stability of the 89Zr-THPN radiocomplex and compare it against the radiocomplexes with DFO and DFO*, healthy NSG mice were administered 89Zr-HPG nanoparticles with either of the chelates. For each radioconjugate, twelve mice were used for an acute biodistribution study and were sacrificed after either 1, 3, or 6 days post-injection (n = 4 per group and time point). One animal per group received a larger dose of radioconjugate and was imaged by in vivo PET/CT after 1 h, 1, 3, and 6 days post-injection, before being also included in the biodistribution study.  PET images at 1 h and 1 day post-injection showed the majority of activity for all three radioconjugates to be associated with the blood circulation as hearts and carotid arteries were clearly distinguishable (Figure 4.15). After 3 and 6 days post-injection, 89Zr uptake in the joints and bones became particularly prominent in the mouse that received 89Zr-THPN-HPG conjugate. The other two mice showed much less bone uptake at these time points. All three mice also showed some activity uptake by liver and spleen. The data from the acute biodistribution study confirmed these findings. For the mice that were administered the THPN-based radioconjugate, 89Zr sequestration to the bones increased over the six days and was significantly higher compared to the other two groups of mice (Figure 4.13). The full biodistribution data is presented in Table 4.3. Outliers were identified using a Grubbs test (p < 0.01) using the software R199 and were excluded from the reported results. Since released 89Zr tends to accumulate in bones, it is reasonable to regard bone uptake as a measure for the release of 89Zr from the complex. The in vivo data therefore suggest that the DFO- and DFO*-radioconjugates possess superior in vivo stability over the THPN-radioconjugate. 99  Figure 4.13. Bone uptake of 89Zr for the three radioconjugates measured in biodistribution studies after 1, 3, or 6 days post-injection. Error bars indicate standard deviations. We did not anticipate these findings since the thermodynamic stability of Zr-THPN was determined to be exceptionally high (log β 50.3, Chapter 3). It is, however, understood that the kinetic inertness can often have an even more profound effect on the in vivo stability of metallo-radiopharmaceuticals than the thermodynamic stability itself.1, 46, 48, 51, 56, 167, 200 Diagnostic radiopharmaceuticals are administered in extremely low quantities which are further diluted as  the compounds enter circulation. Under these highly dilute conditions, equilibrium conditions are no longer applicable and the rate of dissociation (koff) predominantly dictates in vivo stability (Figure 4.14).1, 48, 51, 167 The radiometal complex, moreover, faces fierce competition from orders of magnitude higher concentrations of metal ions and native chelators for potential transmetallation or transchelation.  100  Figure 4.14. Schematic representation comparing thermodynamic and kinetic effects governing the stability of a radiometal complex in vivo, with the dissociation rate constant koff being a critical factor. Reproduced with permission from ref.51 © 2015 John Wiley & Sons.     101                 Figure 4.15. Maximum intensity projections of PET (left) and fused PET/CT images (right) over six days of three mice injected i.v. with either 89Zr-THPN-HPG (top), 89Zr-DFO-HPG (centre), or 89Zr-DFO*-HPG (bottom).  015%ID/g015%ID/g015%ID/g1 h Day 6Day 389Zr-THPN-HPG89Zr-DFO-HPG89Zr-DFO*-HPGDay 1 1 h Day 1 Day 6Day 389Zr-THPN-HPG89Zr-DFO-HPG89Zr-DFO*-HPG015%ID/g015%ID/g015%ID/g102  Table 4.3. Acute biodistribution data for 89Zr-HPG conjugates with values expressed as %ID/g.a  Day 1  Day 3  Day 6  THPN DFO DFO*  THPN DFO DFO*  THPN DFO DFO* Blood 20.0 ± 0.8 19.7 ± 1.3 5.8 ± 1.3  10.7 ± 1.3 9.0 ± 1.3 11.2 ± 2.3  2.9 ± 1.1 4.0 ± 1.3 6.4 ± 1.2 Fat 0.6 ± 0.1 0.7 ± 0.3 0.1 ± 0.0  0.6 ± 0.1 0.8 ± 0.2 0.6 ± 0.2  0.7 ± 0.2 0.6 ± 0.3 0.8 ± 0.0 Uterus 11.4 ± 4.4 7.1 ± 0.8 2.0 ± 0.3  11.1 ± 5.5 13.9 ± 4.4 8.2 ± 2.3  9.9 ± 1.9 7.8 ± 0.8 9.1 ± 0.3 Ovaries 8.9 ± 2.4 8.0 ± 1.1 2.2 ± 0.7  8.5 ± 2.7 12.8 ± 1.8 7.4 ± 2.0  8.4 ± 1.3 8.7 ± 1.1 18.1 ± 17.0 Intestine 1.8 ± 0.1 1.6 ± 0.2 0.5 ± 0.1  1.4 ± 0.1 1.2 ± 0.0 1.2 ± 0.3  0.8 ± 0.1 0.8 ± 0.2 1.1 ± 0.1 Spleen 8.3 ± 1.4 12.0 ± 3.2 3.4 ± 0.9  25.8 ± 16.8 18.6 ± 2.8 13.0 ± 5.8  21.8 ± 2.8 19.6 ± 3.2 22.7 ± 7.7 Liver 8.0 ± 0.6 3.9 ± 4.2 2.0 ± 0.5  9.5 ± 0.6 6.9 ± 0.9 6.4 ± 1.4  8.4 ± 0.7 5.9 ± 4.2 5.7 ± 0.6 Pancreas 1.6 ± 0.2 1.7 ± 0.6 0.4 ± 0.1  1.6 ± 0.3 1.8 ± 0.2 1.9 ± 0.4  1.7 ± 0.3 1.5 ± 0.6 1.7 ± 0.3 Stomach 2.2 ± 0.4 1.3 ± 0.9 0.6 ± 0.1  2.3 ± 0.2 2.0 ± 0.2 1.8 ± 0.6  1.4 ± 0.1 1.4 ± 0.9 1.8 ± 0.3 Adrenal glands 8.7 ± 2.6 10.5 ± 3.8 4.6 ± 4.1  13.6 ± 4.4 16.5 ± 13.1 9.3 ± 3.4  13.6 ± 4.7 13.7 ± 3.8 20.3 ± 10.0 Kidney 5.3 ± 1.0 4.3 ± 0.2 1.9 ± 0.4  3.9 ± 0.3 3.6 ± 0.2 3.7 ± 1.1  2.6 ± 0.4 2.0 ± 0.2 2.4 ± 0.3 Lungs 6.1 ± 0.8 5.9 ± 0.6 1.9 ± 0.5  5.0 ± 0.7 4.5 ± 0.8 4.4 ± 1.3  2.8 ± 0.3 2.6 ± 0.6 4.0 ± 1.2 Heart 4.0 ± 0.3 3.9 ± 0.5 1.2 ± 0.1  3.7 ± 0.5 3.3 ± 0.7 3.6 ± 1.0  2.6 ± 0.3 2.5 ± 0.5 3.1 ± 0.6 Muscle 1.0 ± 0.2 0.7 ± 0.1 0.3 ± 0.1  0.8 ± 0.1 1.2 ± 0.2 0.8 ± 0.1  1.0 ± 0.2 0.8 ± 0.1 0.9 ± 0.2 Bone 3.9 ± 0.3 2.5 ± 0.4 0.6 ± 0.2  7.1 ± 0.8 3.8 ± 0.3 2.3 ± 0.7  8.4 ± 2.2 3.3 ± 0.4 3.1 ± 0.7 Brain 0.3 ± 0.0 0.4 ± 0.1 0.1 ± 0.0  0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.1  0.1 ± 0.0 0.1 ± 0.1 0.2 ± 0.0 Tail 3.1 ± 0.4 2.4 ± 0.2 0.7 ± 0.1  4.0 ± 0.3 2.5 ± 0.0 2.2 ± 0.3  4.2 ± 0.4 1.7 ± 0.2 2.0 ± 0.2 a Studies were performed in healthy female NSG mice administered with 89Zr-THPN-HPG, 89Zr-DFO-HPG, or 89Zr-DFO*-HPG nanoparticles via i.v. tail vein injection (n = 4 per group). Results are reported as mean ± SD.  103 4.3 Conclusions In order to study the Zr-THPN complex in combination with different carrier molecules, a bifunctional version of the chelator was produced. The novel isothiocyanate functionalized derivative p-SCN-Bn-THPN was synthesized in a nine step synthesis in ~7% cumulative yield and was, along with all synthetic intermediates, fully characterized. The isothiocyanate functionality enables covalent conjugation to amino group bearing carrier molecules by thiourea bond formation. The conjugation and 89Zr-radiolabeling of p-SCN-Bn-THPN was then evaluated with four different carriers.  First, the anti-HER2 antibody trastuzumab was radiolabeled with THPN as chelator using either a post- or a prelabeling approach. The postlabeling strategy led to considerable antibody aggregation, which could not be avoided even by extensive optimization efforts. Instead, a prelabeling approach was more successful and trastuzumab could be 89Zr-radiolabeled with THPN. Successful prelabeling with trastuzumab was achieved for the two literature chelators, DFO and DFO*, even though other authors reported problems with DFO. The specific activities obtained for THPN using the prelabeling approach were too low for a meaningful in vivo imaging study. Instead, the three radioimmunoconjugates were examined in an in vitro mouse plasma stability study. All three radioimmunoconjugates remained 95% or more complexed over seven days at 37 °C. The DFO* radioimmunoconjugate displayed the highest plasma stability as no transmetallation was observed.  Second, a monoclonal anti-mouse anti-PD-L1 antibody was 89Zr-radiolabeled with THPN by a postlabeling approach. For this antibody, the postlabeling approach produced a much smaller amount of aggregates. Some aggregates and/or small molecular weight activity, however, remained problematic although these experiments showed that the extreme aggregation observed for trastuzumab does not apply to all antibodies. 104 Third, mouse serum albumin (MSA) was successfully 89Zr-radiolabeled with THPN by a postlabeling approach. No major aggregation products were detected and MSA thus constitutes a model protein for which the postlabeling approach leads to high radiochemical yield and purity. Fourth, in order to evaluate the in vivo long-term stability of the 89Zr-THPN complex and compare it to the radiocomplexes with DFO and DFO*, the three chelators were conjugated to hyperbranched polyglycerol (HPG) nanoparticles. These conjugates were successfully radiolabeled with radiochemical yields of 97% and radiochemical purities of >99%. Chelator conjugation and radiolabeling did not affect the HPG particle size or size distribution. In an in vitro human plasma stability study, all three radiochelate-HPGs retained >95% integrity after five days incubation at 37 °C, yet DFO* appeared the most stable showing no signs of demetallation. The long-term in vivo stability of the three radiochelate-HPG conjugates was assessed over six days in healthy, immunocompromised mice by in vivo PET/CT imaging and an acute biodistribution study. PET images and the biodistribution results both revealed a significant 89Zr uptake by the bones for the 89Zr-THPN-HPG nanoparticles when compared to the DFO and DFO* counterparts. This in vivo instability was not anticipated since the thermodynamic stability for Zr-THPN was experimentally determined to be exceptionally high. Yet, the in vivo stability is known to often be dominated by the kinetic inertness of a radiocomplex over its thermodynamic stability. Thus, these findings suggest that the kinetic stabilities of DFO and DFO* exceed that of THPN and limit its biologic applicability. At the same time this reinforces the need to conduct such in vivo experiments to carefully evaluate physiologic stability of radiochelates over extended periods of time. For this purpose, we identified long-circulating HPG nanoparticles as a robust nanocarrier. Their high 105 biocompatibility, high solubility, and long circulation time render HPG nanoparticles a suitable investigative tool to assess the in vivo stability of radiochelates over several days.  4.4 Experimental 4.4.1 Materials and Methods All reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, TCI, AC PharmaChem, Acros, Strem Chemicals, Fisher Scientific) and were used as received without further purification. All water used was ultrapure (18.2 MΩ∙cm) and was purified with a Millipore Milli-Q Integral-10 water purification system. Building block 4.6 (2-(aminomethyl)-1,6-dimethyl-3-(phenylmethoxy)-4(1H)-pyridinone) was purchased from Otava Ltd or was prepared by Dr. Stoyan Karagiozov according to a published procedure117 with minor adjustments. Trastuzumab (Herceptin) was generously supplied by BC Cancer Agency and was purified by ultrafiltration (Amicon Ultra 100K). Anti-PD-L1 mAb was purchased from Bio X Cell as InVivoMAb anti-mouse PD-L1 (B7-H1) solution (5.5 g/L in PBS, pH 6.5). Mouse serum albumin (MSA) was purchased from Innovative Research, Inc. as lyophilized powder and was reconstituted with water. Hyperbranched polyglycerol (HPG) was synthesized and amino-functionalized by Dr. Katayoun Saatchi according to published procedures.193, 197, 201 In brief, HPG of approximately 800 kDa was synthesized by ring-opening multi-branching polymerization of glycidol. Hydroxyl groups were oxidized with NaIO4, followed by reductive amination with 1,10-diaza-4,7-dioxadecane and NaCNBH3. p-SCN-Phe-DFO (p-SCN-Bn-Deferoxamine) was purchased from Macrocyclics and p-SCN-Phe-DFO* (N1-(8,19-dihydroxy-1-((4-isothiocyanato-phenyl)amino)-9,12,20,23-tetraoxo-1-thioxo-2,8,13,19,24-pentaazanonacosan-29-yl)-N1-hydroxy-N4-(5-(N-hydroxyacetamido)pentyl)succinamide) was produced by Pharma 106 Inventor Inc. and was generously supplied by Dr. François Bénard’s lab (BC Cancer Agency). Methyltetrazine-PEG4-amine (4.11, 2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy] ethoxy]ethoxy]-ethanamine), TCO-NHS (succinimidyl (E)-cyclooct-4-en-1-yl carbonate), and sulfo-Cy3-methyltetrazine (2-[3-[1,3-dihydro-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-2H-indol-2-ylidene]-1-propen-1-yl] 3,3-dimethyl-1-[6-[[[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl] amino]-6-oxohexyl]-5-sulfo-3H-indolium) were purchased from Broadpharm. Parsability of chemical nomenclature was confirmed with OPSIN.118, 119 NMR spectra were recorded on a Bruker Ascend 400 spectrometer (400.13 MHz for 1H; 100.62 MHz for 13C) at 297.16 K. Chemical shifts (δ relative to residual solvent peak) are reported as parts per million (ppm) and coupling constants (J) in hertz (Hz). ESI-MS spectra were recorded on an AB Sciex QTrap 5500 mass spectrometer. High-resolution mass spectrometry (HR-MS) analyses were acquired by UBC Mass Spectrometry Centre on a Bruker HTCultra PTM Discovery mass spectrometry system. IR spectra were recorded on an Agilent Cary 660 FT-IR spectrometer equipped with an attenuated total reflectance (ATR) crystal. Microwave reactions were conducted in a Biotage Initiator+ microwave synthesizer. Hydrodynamic diameter measurements were carried out by dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS. Measurements were performed in triplicate in water at 25 °C using a 173° backscatter angle and the material refractive index of polystyrene latex (1.590). Particle size diameters are reported as Z-average (Z-avg. (d)). Data were analyzed using the software Malvern Zetasizer (v. 7.12). Protein concentrations were quantified either by measuring absorbance at 280 nm on a Thermo Scientific NanoDrop 2000 spectrophotometer, or by Bradford assay measured at 595 nm on a BioTek SynergyMx 96-well plate reader and comparing against a bovine serum albumin calibration curve. HPLC was performed on a Waters Alliance e2695 separations module coupled to a Waters 2489 UV/Vis-detector and, for 107 radio-HPLC, a LabLogic Scan-RAM radio-detector. For reversed phase HPLC, the column was a C18 Waters Atlantis T3, 100 Å, 5 μm particle size (4.6 × 150 mm), supported by a C18 guard cartridge and was operated in an oven (40 °C). Method A used the following gradient: A = 0.1% trifluoroacetic acid (TFA) in water; B = methanol; flow rate = 1 mL/min; 0–5 min 90% A; 5–15 min 10–100% B; 15–18 min 100% B. Method B used the following gradient: A = 0.1% trifluoroacetic acid (TFA) in water; B = acetonitrile; flow rate = 1 mL/min; 0–5 min 90% A; 5–15 min 10–100% B; 15–18 min 100% B. Method C used the following gradient: A = 10 mM (NH4)HCO3 in water; B = 10 mM (NH4)HCO3 in 2:1 methanol/water; flow rate = 1 mL/min; 0–13 min 14–100% B; 13–18 min 100% B. Size-exclusion HPLC (SE-HPLC) was performed at ambient temperature on the same radio-HPLC system but using either a Waters Ultrahydrogel Linear column (method D, 10 μm, 7.8 × 300 mm) or a Waters Ultrahydrogel 250 column (method E, 250 Å, 6 μm, 7.8 × 300 mm). The mobile phase for methods D and E was an isocratic gradient consisting of a filtered aqueous phosphate buffer (50 mM NaH2PO4, 50 mM Na2HPO4, 150 mM NaCl, 10 mM NaN3, pH 6.2–7.0) and flow rate was 0.5 mL/min. Semi-preparative HPLC was performed on a Phenomenex Synergi Hydro-RP column (80 Å, 21.1 × 250 mm) connected to a Waters 600 controller and a Waters 2487 dual wavelength absorbance detector using the following gradient: A = 0.1% TFA in water; B = methanol; flow rate = 10 mL/min; 5–100% B over 25 min. Flash chromatography was performed on a Biotage Isolera One system using Biotage SNAP KP-Sil or ZIP silica gel cartridges (normal phase) or Biotage SNAP Ultra C18 cartridges (reversed phase). Solid phase extraction cartridges (Chromafix C18-ec) were obtained from Machery-Nagel. Analytical thin layer chromatography (TLC) was performed using silica gel 60 F254 plates with aluminium backing obtained from Merck Millipore. AW Standard Super-Cel filter aid was purchased from Sigma-Aldrich. Semi-preparative size-exclusion chromatography was performed on PD-10 or PD MiniTrap G-25 desalting columns 108 (Sephadex G-25, GE Healthcare). Ultrafiltration was performed using Amicon Ultra centrifugal filters of 30 kDa, 50 kDa, or 100 kDa NMWL of 0.5 mL or 2 mL volume (EMD Millipore).  89Zr was obtained either from TRIUMF, BC Cancer Agency, Sherbrooke University, or PerkinElmer Health Sciences Canada, Inc. and was produced via the 89Y(p,n)89Zr reaction. 89Zr was either received already purified in 1 M oxalic acid or was purified either following a reported procedure80 or using commercially available ZR Resin (TrisKem International) and eluted with 0.05 M oxalic acid. 89Zr-oxalate solutions were neutralized with Na2CO3 solutions. Activities were measured using a Capintec CRC-55tR or a Capintec CRC-25R/W dose calibrator. Instant thin-layer chromatography (ITLC) was carried out using Biodex Tec-Control chromatography strips (#150-005, black). Unless otherwise mentioned, ITLC strips were developed using an aqueous DTPA solution as mobile phase (50 mM, pH 7.0). ITLC strips were analyzed on a Packard Cyclone storage phosphor screen imager with the OptiQuant software. Biodistribution samples were counted on a calibrated PerkinElmer 2480 Wizard2 automated gamma counter. NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were obtained through an in-house breeding program at the Animal Research Centre, BC Cancer Research Centre. PET/CT images were acquired on a Siemens Medical Solutions Inveon PET/CT small animal scanner.  4.4.2 Syntheses 4.4.2.1 Diethyl-2-(4-nitrobenzyl)malonate (4.1) This synthesis was adapted from a literature procedure.157 Diethyl malonate (3.20 g, 20.0 mmol) was dropwise added to a suspension of sodium hydride (60% in oil, 545 mg, 13.6 mmol) in anhydrous THF (7.5 mL) and was stirred 1 h at ambient temperature under argon. A solution of 4-nitrobenzyl bromide (2.55 g, 11.8 mmol) in THF (7.5 mL) was dropwise added while cooling in 109 an ice bath. After complete addition, the mixture was heated for 1.5 h at 35 °C. Once TLC indicated completion of the reaction, the mixture was evaporated, taken up in diethyl ether (20 mL) and washed with water (2 x 20 mL). The organic phase was dried with MgSO4, filtered, and evaporated. Recrystallization from 1:1 Et2O/hexane gave the title compound 4.1 as yellow needles in 53% yield (1.84 g, 6.24 mmol).  1H NMR (400 MHz, CDCl3): δ = 8.14 (d, J = 8.8 Hz, 2H, -Ph-H), 7.39 (d, J = 8.8 Hz, 2H, -Ph-H), 4.23−4.10 (m, 4H, -O-CH2-), 3.66 (t, J = 7.8 Hz, 1H, -CH-), 3.31 (d, J = 7.6 Hz, 2H, -CH2-Ph-), 1.21 (t, J = 7.2 Hz, 6H, -CH3). 13C NMR (100 MHz, CDCl3): δ = 168.2, 146.9, 145.6, 129.8, 123.7, 61.8, 53.1, 34.3, 14.0. HPLC (method A): tR = 15.9 min. ESI-MS: m/z 318.2 [M+Na]+; 294.2 [M−H]−.  4.4.2.2 2-(4-Nitrobenzyl)propanediamide (4.2)  This synthesis was adapted from a literature procedure.156 Compound 4.1 (1.64 g, 5.56 mmol) was suspended in methanol (10 mL) and was placed in an ice bath. An ammonia solution (30 mL, 7 N in methanol) was dropwise added to this. The mixture was allowed to warm to ambient temperature and was stirred overnight. HPLC indicated consumption of the starting material but the presence of an intermediary product, which converted to the product after stirring for another night at ambient temperature as observed by HPLC. At this point, the suspension was filtered over fritted glass and washed with methanol and boiling acetonitrile. Drying in vacuo gave the title compound 4.2 as a white powder in 89% yield (1.18 g, 4.97 mmol).  1H NMR (400 MHz, DMSO-d6): δ = 8.14 (d, J = 8.8 Hz, 2H, -Ph-H), 7.48 (d, J = 8.8 Hz, 2H, -Ph-H), 7.29 (s, 2H, -NH2), 7.09 (s, 2H, -NH2), 3.38 (t, J = 7.6 Hz, 1H, -CH-), 3.10 (d, J= 7.6 Hz, 2H, -CH2-). 13C NMR (100 MHz, DMSO-d6): δ = 170.2, 148.0, 146.1, 130.2, 123.3, 54.0, 34.5. HPLC (method A): tR = 11.46 min. ESI-MS: m/z 260.0 [M+Na]+. 110  4.4.2.3 1,3-Diamino-2-(4-nitrobenzyl)propane dihydrochloride (4.3) This synthesis was adapted from reported procedures.156, 158 A dry round-bottom flask was charged with compound 4.2 (129 mg, 542 μmol) and was placed under nitrogen. To this was slowly added a solution of borane stabilized in THF (BH3·THF, 1 M in THF, 1.2 mL, 1.2 mmol) and it was stirred 45 min at ambient temperature. Some more anhydrous THF (5 mL) was added and it was heated at reflux overnight. The reaction progress was monitored by HPLC and another aliquot of borane solution (0.2 mL) was added to the mixture. It was heated for another night at reflux after which the reaction was quenched by dropwise addition of concentrated HCl (2 mL). The mixture was heated for an hour at reflux and volatiles were evaporated in vacuo. The yellow milky residue was taken up in water (3 mL) and at 0 °C, NaOH (6 M, 2.7 mL) was added using a dropping funnel. The mixture was extracted with DCM (5×15 mL), the organic phases were combined, dried over MgSO4, and evaporated. The resulting yellow oil was suspended in ethanol (3 mL), a drop of concentrated HCl was added, and the mixture was left to precipitate over 3 days at ‒20 °C. The supernatant was removed and the precipitate was triturated with diethyl ether and then dried in vacuo to give the title compound 4.3 as a light yellow powder in 66% yield (101 mg, 357 μmol).  1H NMR (400 MHz, D2O): δ = 8.23 (d, J = 8.8 Hz, 2H, -Ph-H), 7.50 (d, J = 8.8 Hz, 2H, -Ph-H), 3.15 (dd, J = 13.6 Hz, 6.7 Hz, 2H, -N-CH2-), 3.02 (dd, J = 13.6 Hz, 6.5 Hz, 2H, -N-CH2-), 2.95 (d, J = 7.6 Hz, 2H, -CH2-), 2.57-2.46 (m, 1H, -CH-). 13C NMR (100 MHz, D2O): δ = 146.7, 145.2, 130.0, 124.0, 39.9, 36.6, 34.7. HPLC (method A): tR = 6.64 min. ESI-MS: m/z 210.0 [M+H]+.  111 4.4.2.4 Tetra-tert-butyl 2,2',2'',2'''-((2-(4-nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))-tetraacetate (4.4) Compound 4.3 (56 mg, 0.20 mmol) was dissolved in anhydrous DMF (4 mL) and placed under N2. To this was added tert-butyl bromoacetate (129 μL, 0.87 μmol) and sodium carbonate (~0.39 g) and it was stirred overnight at ambient temperature. HPLC indicated completion of the reaction and the mixture was filtered and rinsed ad libitum with acetone. The filtrate was evaporated and purified by flash chromatography over silica with 0–20% ethyl acetate/hexane to give the title compound 4.4 as a yellow oil in 46% yield (60 mg, 90 μmol).  1H NMR (400 MHz, CDCl3): δ = 8.10 (d, J = 8.8 Hz, 2H, -Ph-H), 7.41 (d, J = 7.6 Hz, 2H, -Ph-H), 3.35 (s, 8H, -N-CH2-CO-), 2.88 (d, J = 6.0 Hz, 2H, -CH2-Ph-), 2.74 (m, 2H, -N-CH2-CH-), 2.47 (m, 2H, -N-CH2-CH-), 1.96 (m, 1H, -CH-), 1.43 (s, 36H, -CH3). 13C NMR (100 MHz, CDCl3): δ = 170.6, 149.6, 146.1, 130.2, 123.3, 80.9, 56.6, 56.4, 38.3, 36.6, 28.1. HPLC (method A): tR = 16.57 min. Rf = 0.37 (20% EtOAc/hexane). ESI-MS: m/z 666.1 [M+H]+, 688.1 [M+Na]+.  4.4.2.5 2,2',2'',2'''-(2-(4-Nitrobenzyl)propane-1,3-diyl-bis(azanetriyl))-tetraacetic acid (4.5) Compound 4.4 (249 mg, 374 μmol) was dissolved in 4.5 mL acetonitrile and ortho-phosphoric acid (~1.5 mL, 85 wt%) was dropwise added. This mixture was stirred one night at ambient temperature and then heated another night at 45 °C. Once HPLC indicated complete conversion, 3 mL water were added and the mixture was concentrated in vacuo. The residual oil was taken up in (NH4)HCO3 (15 mL, 0.1 M), washed with ethyl acetate (10 mL) and extracted with another 3 × 10 mL 112 (NH4)HCO3 solution. The combined aqueous layer was concentrated in vacuo and purified by reversed phase chromatography (eluted with a gradient of 100% water with 0.1% formic acid to 100% methanol). Fractions were pooled and evaporated to give the title compound 4.5 as a fluffy white powder in 97% yield (173 mg, 364 μmol).  1H NMR (400 MHz, CD3OD): δ = 8.17 (d, J = 8.8 Hz, 2H, -Ph-H), 7.51 (d, J = 8.8 Hz, 2H, -Ph-H), 3.81 (d, J = 16.8 Hz, 4H, -N-CH2-CO-), 3.68 (d, J = 16.8 Hz, 4H, -N-CH2-CO-), 3.48 (dd, J = 13.2, 3.6 Hz, 2H, -N-CH2-CH-), 3.09-3.03 (m, 2H, -N-CH2-CH-), 2.75 (d, J = 7.2 Hz, 2H, -CH2-Ph-), 2.7-2.6 (m, 1H, -CH-). 13C NMR (100 MHz, CD3OD): δ = 172.8, 148.2, 148.0, 131.3, 124.7, 59.6, 56.8, 37.6, 33.4. HPLC: tR = 11.76 min. ESI-MS: m/z 442.1 [M+H]+, 464.2 [M+Na]+. Elemental analysis (%) calcd. for C18H23N3O10∙H2O∙½(CH3OH): C 46.77, H 5.72, N 8.84; found: C 46.97, H 5.77, N 9.01.  4.4.2.6 2,2',2'',2'''-((2-(4-Nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-(phenylmethoxy)-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (4.7) A microwave vial was charged with compound 4.5 (108 mg, 226 μmol), which was suspended in anhydrous DMF (4 mL). To this was added HOBt∙H2O (173 mg, 1.13 mmol, 5.0 eq.) and a solution of DCC (233 mg, 1.13 mmol, 5.0 eq.) in DMF (4.5 mL). The mixture was stirred for 2 h at ambient temperature under N2. At this point, 2-(aminomethyl)-1,6-dimethyl-3-(phenylmethoxy)-4(1H)-pyridinone (4.6, 292 mg, 1.13 mmol, 5.0 eq.) was added, it was rinsed with another 2 mL DMF and the vial was crimp capped under N2. The mixture was heated in a microwave reactor for 4 h at 55 °C and then stirred at ambient 113 temperature overnight. The mixture was heated another 2 h at 55 °C in the microwave reactor and then stirred a second night at ambient temperature. At this point, HPLC indicated complete conversion and the mixture was filtered over a cotton plug, rinsed with acetonitrile, evaporated, and purified by reversed phase flash chromatography (eluted with a gradient of 100% water with 0.1% formic acid to 40% acetonitrile). Product fractions were pooled and concentrated in vacuo to give the title compound 4.7 as a yellow oil in ~83% yield (262 mg, 187 μmol).  1H NMR (400 MHz, CD3OD): δ = 8.01 (d, J = 8.4 Hz, 2H, NO2-Ph-H), 7.41-7.39 (m, 8H, Bn-H), 7.29-7.26 (m, 12H, Bn-H), 7.16 (d, J = 8.8 Hz, 2H, NO2-Ph-H), 6.40 (s, 4H, -CO-CH-), 5.14 (s, 8H, -O-CH2-Ph), 4.42 (s, 8H, -CO-N-CH2-), 3.53 (s, 12H, -N-CH3), 3.02 (s, 8H, -N-CH2-CO-), 2.54 (d, J = 6.4 Hz, 2H, -CH2-Ph-NO2), 2.47-2.43 (m, 2H, -N-CH2-CH-), 2.33 (s, 12H, -C-CH3), 2.21-2.16 (m, 2H, -N-CH2-CH-), 1.64-1.57 (m, 1H, -CH-CH2-N-). 13C NMR (100 MHz, CD3OD): δ = 174.7, 173.4, 150.9, 149.9, 147.7, 147.3, 143.5, 138.4, 131.0, 130.0, 129.5, 129.4, 124.6, 119.2, 74.6, 60.5, 60.4, 39.5, 37.9, 37.5, 36.2, 20.9. HPLC (method B): tR = 11.52 min. ESI-MS: m/z 1402.8 [M+H]+, 1424.7 [M+Na]+; 1400.8 [M−H]−. HR ESI-MS: calcd.(m/z) for C78H88N11O14+ [M+H]+: 1402.6512; found: 1402.6532, (1.4 ppm).  4.4.2.7 2,2',2'',2'''-((2-(4-Nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (4.8). Compound 4.7 (262 mg, 187 μmol) was dissolved in glacial acetic acid (2.5 mL) and concentrated HCl (2.5 mL) and was stirred 24 h at 50 °C. The solution was removed from heat and a stream of nitrogen was blown over it 114 overnight before evaporating the residue in vacuo. The residue was taken up in a small amount of methanol (~3.5 mL), precipitated by dropwise addition to diethyl ether (~40 mL), and placed in a freezer at ‒20 °C for 20 min. The supernatant was removed and the precipitate was dried in vacuo to give the title compound 4.8 as white hydrochloride salt in ~94% yield (243 mg, ~176 μmol).  1H NMR (400 MHz, CD3OD): δ = 8.19 (d, J = 8.0 Hz, 2H, NO2-Ph-H), 7.65 (d, J = 7.6 Hz, 2H, NO2-Ph-H), 7.18 (s, 4H, -CO-CH-), 4.81 (s, 8H, -CO-N-CH2-), 4.11 (sb, 8H, -N-CH2-CO-), 4.05 (s, 12H, -N-CH3), 3.44-3.32 (sb, 4H, -N-CH2-CH-), 3.04-2.94 (m, 1H, -CH-CH2-N-), 2.90-2.81 (m, 2H, -CH2-Ph-NO2), 2.68 (s, 12H, -C-CH3). 13C NMR (100 MHz, CD3OD): δ = 168.6, 160.5, 150.7, 148.2, 147.2, 144.8, 140.3, 131.8, 124.8, 114.1, 61.1, 57.3, 40.5, 37.3, 36.1, 33.3, 21.4. HPLC (method A): tR = 11.07 min. ESI-MS: m/z 521.8 [M+2H]2+, 1042.4 [M+H]+, 1064.4 [M+Na]+. HR ESI-MS: calcd.(m/z) for C50H64N11O14+ [M+H]+: 1042.4634; found: 1042.4619, (1.4 ppm). Elemental analysis (%) calcd. for C50H63N11O14∙7.4(HCl)∙2.15(CH3OH): C 45.36, H 5.77, N 11.16; found: C 45.55, H 5.54, N 10.94.  4.4.2.8 2,2',2'',2'''-((2-(4-Aminobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (4.9) In a Schlenk flask under inert atmosphere, compound 4.8 (~240 mg, ~174 μmol) was dissolved in methanol (~20 mL) and Pd/C was added (10% w/w, 19.6 mg, 18.4 μmol). The reaction vessel was sealed, placed under a hydrogen atmosphere (balloon), and the mixture was stirred vigorously at ambient 115 temperature. After 2.5 h, the reaction mixture was vented to nitrogen, filtered through a plug of filter aid, and generously rinsed with methanol. The filtrate was purified by reversed phase flash chromatography (eluting with 0–100% methanol/water with 0.1% formic acid). Product fractions were pooled, evaporated, and dried in vacuo to give the title compound 4.9 in its formic acid salt as an off-white solid in ~99% yield (~188 mg, ~173 μmol). (Molecular weight estimated from NMR integrals). 1H NMR (400 MHz, CD3OD): δ = 6.75 (d, J = 8.4 Hz, 2H, -Ph-H), 6.64 (d, J = 8.0 Hz, 2H, -Ph-H), 6.35 (s, 4H, -CO-CH-), 4.61 (s, 8H, -CO-N-CH2-), 3.66 (s, 12H, -N-CH3), 3.19 (s, 8H, -N-CH2-CO-), 2.53-2.48 (m, 2H, -N-CH2-CH-), 2.36 (s, 12H, -C-CH3), 2.40-2.31 (m, 2H, -N-CH2-CH-), 2.40-2.31 (m, 2H, -CH2-Ph-), 1.64-1.59 (m, 1H, -CH-CH2-N-). 13C NMR (100 MHz, CD3OD): δ = 173.7, 170.6, 148.8, 147.3, 144.2, 132.7, 132.4, 130.7, 117.9, 114.8, 61.0, 60.5, 39.6, 37.4, 37.4, 35.8, 21.0. HPLC (method A): tR = 9.79 min. ESI-MS: m/z 1012.5 [M+H]+, 1034.5 [M+Na]+. HR ESI-MS: calcd. (m/z) for C50H66N11O12+ [M+H]+: 1012.4892; found: 1012.4908, (1.6 ppm).  4.4.2.9 2,2',2'',2'''-((2-(4-Isothiocyanatobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)acetamide) (p-SCN-Bn-THPN, 4.10) A solution of 4.9 (~116 mg, ~115 μmol) in water (~10 mL) was added to a solution of thiophosgene (131.8 μL, 1.719 mmol, 15 eq.) in CHCl3 (1.5 mL) [note: thiophosgene is highly toxic and care must be exercised in its handling]. The reaction mixture was stirred vigorously overnight at ambient temperature. The mixture was transferred with water (5 mL) to a conical 116 centrifuge tube and was washed with CHCl3 (4 × 1 mL) by biphasic stirring using a vortex and removing the organic layers with a pipette. The aqueous layer was diluted with H2O to ~25 mL and was purified in separate batches either by semi-preparative HPLC as described above or by reversed phase flash chromatography (eluted with a gradient of 5–70% methanol with 0.1% TFA/water with 0.1% TFA). Product fractions were pooled, concentrated by rotary evaporation, and dried overnight by lyophilization to yield the title compound 4.10 (p-SCN-THPN) as a white powder in ~65% yield (~79 mg, ~75 μmol).  1H NMR (400 MHz, CD3CN/D2O, 11:1 v/v): δ = 7.22 (d, J = 8.4 Hz, 2H, -Ph-H), 7.16 (d, J = 8.4 Hz, 2H, -Ph-H), 7.00 (s, 4H, -CO-CH-), 4.61-4.52 (m, 8H, -CO-N-CH2-), 3.79 (s, 12H, -N-CH3), 3.69-3.59 (m, 8H, -N-CH2-CO-), 3.01-2.97 (m, 2H, -N-CH2-CH-), 2.85-2.79 (m, 2H, -N-CH2-CH-), 2.48 (s, 12H, -C-CH3), 2.46-2.43 (m, 2H, -CH2-Ph-), 2.32-2.27 (m, 1H, -CH-CH2-Ph-). 13C NMR (100 MHz, CD3CN/D2O, 11:1 v/v): δ = 170.3, 161.9, 150.4, 144.4, 139.5, 138.9, 131.3, 130.0, 126.8, 116.1, 114.3, 60.8, 57.1, 39.6, 36.6, 36.0, 33.8, 21.4. HPLC (method C): tR = 13.15 min. ESI-MS: m/z 527.8 [M+2H]2+, 1054.4 [M+H]+, 1076.4 [M+Na]+. HR ESI-MS: calcd. (m/z) for C51H64N11O12S+ [M+H]+: 1054.4457; found: 1054.4493.  4.4.2.10 2,2',2'',2'''-((2-(4-(3-(2-(2-(2-(2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-ethoxy)ethoxy)ethoxy)ethyl)thioureido)benzyl)propane-1,3-diyl)bis(azanetriyl))-tetrakis(N-((1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydropyridin-2-yl)methyl)-acetamide) (Tz-THPN, 4.12) To a solution of p-SCN-Bn-THPN (~12.4 mg, ~11.8 μmol) in anhydrous DMSO (~0.4 mL) was added a solution of methyltetrazine-PEG4-amine·TFA (4.11, 2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-ylphenoxy]ethoxy] ethoxy] ethoxy]-ethanamine tetrafluoroacetate) in anhydrous DMSO (123.4 μL, 12.9 μL, 1.1 eq., 50 mg/mL). The pH was raised to pH ~9 by 117 addition of Et3N and the reaction mixture was incubated for 2 h at ambient temperature with agitation (650 rpm). Although HPLC indicated some remaining starting material, it was decided to proceed with work up since the tetrazine moiety is to sensitive towards alkaline conditions. The reaction mixture was diluted with water, loaded on a small C18-ec solid-phase extraction cartridge, and purified by reversed phase flash chromatography using a gradient of 0–100% MeOH/water. Product fractions were pooled, evaporated, and dried in vacuo to give the title compound 4.12 as a pink solid with minor impurities (~2.3 mg, ~1.6 μmol, ~14%). 1H NMR (400 MHz, CD3OD): δ = 8.44 (d, J = 8.8 Hz, 2H, arom.), 7.29 (d, J = 8.0 Hz, 2H, arom.), 7.13 (d, J = 8.8 Hz, 2H, arom.), 7.03 (d, J = 8.0 Hz, 2H, arom.), 6.78 (s, 4H, HOPO-C5-H), 4.68 (s, 8H, HOPO-CH2-), 4.24 (m, 2H, terminal PEG), 3.90-3.87 (m, 2H, PEG), 3.85 (s, 12H, -N-CH3), 3.76-3.70 (m, 4H, PEG), 3.70-3.63 (m, 8H, 4 PEG), 3.55-3.46 (m, 8H, -N-CH2-CON-), 3.00 (s, 3H, -tetrazine-CH3), 2.80-2.72 (m, 4H, -N-CH2-CH), 2.51 (s, 12H, HOPO C6-CH3), 2.47-2.43 (m, 2H, -CH2-Ph-), 2.04 (m, 1H, -CH). HPLC (method A): tR = 13.7 min. ESI-MS: m/z 709.4 [M+2H]2+, 720.5 [M+H+Na]2+.  4.4.3 89Zr-Trastuzumab Radioimmunoconjugates 4.4.3.1 Postlabeling Strategy Chelator-modifications of trastuzumab. In analogy to a reported method by Vosjan et al.,168 purified trastuzumab (~2 mg) was modified at pH ~9 and 37 °C with a five-fold molar 118 excess of either p-SCN-Bn-THPN, p-SCN-Phe-DFO, or p-SCN-Phe-DFO* as bifunctional chelator. The immunoconjugates were purified by ultrafiltration and/or size-exclusion chromatography and concentrations were measured by absorbance at 280 nm or by Bradford assay. The purified immunoconjugates were analyzed by SE-HPLC (method E): THPN-trastuzumab: tR = 13.1 min; DFO-trastuzumab: tR = 13.1 min; DFO*-trastuzumab: tR = 13.2 min; unmodified trastuzumab: tR = 13.0 min.  Radiolabeling of chelator-trastuzumab immunoconjugates. In three separate micro-centrifuge tubes, a solution of 89Zr-oxalate (~78 MBq, ~2.1 mCi) in 0.05 M oxalic acid was neutralized with 0.1 M Na2CO3 to pH ~7–7.5. To this was added either of the immunoconjugates, i.e., THPN-, DFO-, or DFO*-trastuzumab (~500 μg, ~3.4 nmol). The mixtures were diluted with PBS to a total volume of 1 mL and incubated for one hour at ambient temperature with gentle agitation (300 rpm). At this point, an aliquot was removed and analyzed by ITLC to measure the radiolabeling yield. If the yield was not high enough, more immunoconjugate was added and the reaction was given another incubation period. Once ITLC indicated satisfactory radiolabeling, the mixtures were purified by size-exclusion chromatography and/or ultrafiltration. The purified radioimmunoconjugates were analyzed by ITLC and radio-SE-HPLC (method E): 89Zr-THPN-trastuzumab: tR = 13.2 min, aggregates: tR ≈ 11.8 min, small/free 89Zr-species: tR ≈ 16.8 min; 89Zr-DFO-trastuzumab: tR = 13.4 min; 89Zr-DFO*-trastuzumab: tR = 13.4 min; 89Zr-oxalate (negative control): tR = 17.1 min.  4.4.3.2 Prelabeling Strategy A 89Zr-oxalate solution in 1 M oxalic acid was neutralized with 2 M Na2CO3 to pH ~6.5, diluted with 0.9% saline (376 μL), and mixed to dissolve precipitated sodium oxalate. A 119 solution of p-SCN-Bn-THPN (10.2 μL, ~51 nmol, 5 mM in anhydrous DMSO) was added to the 89Zr solution (11 MBq, 297 μCi) in portions with mixing in between additions. The mixture was incubated at ambient temperature with agitation (550 rpm). After 10 min, the reaction was analyzed by ITLC, which indicated quantitative radiolabeling. Trastuzumab (38.7 μL, 38.7 g/L, ~1.5 mg, ~10 nmol) was added with mixing and the pH was raised to pH ~9 with 0.1 M Na2CO3 (~18 μL). The mixture was incubated for 1 h at 37 °C with agitation (550 rpm) before purification by size-exclusion chromatography (PD MiniTrap G-25) eluted with sterile 0.9% saline (1 mL). The product fraction was recovered in 52% radiochemical yield and was analyzed by SDS-PAGE (7.5%), ITLC, and radio-SE-HPLC (method E, tR = 13.3 min). Radiochemical purity was determined as ~93% by HPLC (~99% by ITLC).  4.4.3.3 In Vitro Plasma Stability Study The stability of radioimmunoconjugates with either THPN, DFO, or DFO* as chelator was assessed in an in vitro plasma stability study. The three radioimmunoconjugates were prepared following a prelabeling approach. DFO- and DFO*-radioimmunoconjugates were prepared by prelabeling similarly to the prelabeling method reported for THPN. In short, a neutralized 89Zr solution (5.4 MBq, 145 μCi) was diluted with 0.9% saline (375 μL) and a solution of either p-SCN-Phe-DFO or p-SCN-Phe-DFO* (10.3 μL, ~52 nmol, 5 mM in DMSO) was added. The mixture was incubated at ambient temperature with agitation (550 rpm). After 15 min, ITLC indicated >97% radiolabeling and trastuzumab (38.7 μL, 38.7 g/L, ~1.5 mg, ~10 nmol) was added. The pH was raised to pH ~9 with Na2CO3 and the mixture was incubated for 1 h at 37 °C with agitation (550 rpm). The products were purified by size-exclusion chromatography (PD MiniTrap G-25) with sterile 0.9% saline (1 mL) to give the DFO and DFO* 120 radioimmuno-conjugates in 58% and 54% radiochemical yield, respectively, and radiochemical purities of >99% (radio-SE-HPLC). The three radioimmunoconjugates with THPN, DFO, or DFO* chelates were diluted (1:4 v/v) with mouse plasma. The mixtures were incubated for seven days at 37 °C and aliquots were analyzed by ITLC (mobile phase: EDTA, 50 mM, pH 5.0) at the start and after 1, 3, 5, and 7 days of incubation. The experiment was conducted in duplicate. As a control, 89Zr-oxalate was incubated in mouse plasma.  4.4.3.4 Trans-Cyclooctene (TCO) Modification of Trastuzumab Trastuzumab was modified with TCO groups similarly to published procedures.172, 202-204 A solution of freshly purified trastuzumab (210 μL, 2.80 mg, 19.2 nmol) was diluted with PBS (413 μL) that was adjusted to pH 8.8 with 0.1 M NaHCO3. To this was added a solution of TCO-NHS (succinimidyl (E)-cyclooct-4-en-1-yl carbonate) in anhydrous DMSO (7.2 μL, 25 g/L, 673 nmol) to give a 35:1 molar ratio of TCO:mAb, while keeping the total DMSO concentration below 2%. The mixture was briefly vortexed and then incubated in the dark for 1 h at 21 °C with agitation (500 rpm). The TCO-mAb conjugate was purified by size-exclusion chromatography (PD-10), which was eluted in fractions with PBS (pH 7.4) and antibody concentrations were determined spectrophotometrically. The most concentrated fraction was analyzed for its number of reactive TCO groups/mAb, which was determined as 1.8. It was further analyzed by SE-HPLC (method E): tR = 13.0 min; (unmodified mAb: tR = 12.8 min).  121 4.4.3.5 Determination of Number of Reactive TCO Groups per mAb This experiment was inspired by a procedure reported elsewhere.204 An aliquot of TCO-modified trastuzumab (28.7 μL, ~50 μg) was diluted in PBS (pH 7.4, 271 μL). To this was added a large excess of sulfo-Cy3-methyltetrazine (99 eq., 3.0 μL, 11.0 mM in anhydrous DMSO, sulfo-Cy3 dye: ελ = 555 nm = 150,000 L·mol−1·cm−1). The mixture was briefly vortexed and incubated in the dark for 20 min at 21 °C with agitation (500 rpm). The solution was submitted to size-exclusion chromatography (PD MiniTrap G-25) and it was eluted with 2 x 0.5 mL PBS, pH 7.4. The eluted fractions were further purified by ultrafiltration on an Amicon Ultra centrifugal filter (100 kDa NMWL) and washed with PBS (0.5 mL, pH 7.4). The recovered sulfo-Cy3-mAb conjugate was analyzed by SE-HPLC (method E): tR = 13.1 min. Using the Proteins and Labels function of a Thermo Scientific NanoDrop 2000 spectrophotometer, the antibody concentration was measured as 4.5 μM and sulfo-Cy3 dye concentration was measured as 8.3 μM. Thus, the relative number of reactive TCO groups per mAb was determined as 1.8.  4.4.3.6 Radiolabeling by Tetrazine/Trans-Cyclooctene Click Chemistry [A reaction scheme for orientation is provided in Appendix D, Figure D.1.] Postlabeling approach. To a solution of TCO-modified trastuzumab in PBS pH 7.4 (284 μL, ~500 μg, ~3.33 nmol, ~1.8 TCO/mAb) was added in five portions a solution of Tz-THPN in DMSO (4.12, 4.1 nmol, 10 μL, 10 mM) with immediate mixing between additions. The volume was adjusted with PBS, pH 7.4 to a total of 300 μL and the reaction was incubated in the dark at ambient temperature with agitation (500 rpm). After 30 min, the mixture was purified by size-exclusion chromatography (PD MiniTrap G-25) eluting with PBS, pH 7.4, followed by ultrafiltration (100 kDa NMWL) and the centrifugal filter was rinsed once with PBS, pH 7.4. The recovered immunoconjugate was analyzed by SE-HPLC and its concentration was determined 122 by a Bradford assay. A solution of 89Zr-oxalate in 1 M oxalic acid was then neutralized with 2 M Na2CO3 to pH 6.5–7.0, left to react for several minutes, and centrifuged. The supernatant (~60 μL, 13.5 MBq, 365 μCi) was transferred to a solution of the immunoconjugate in PBS, pH 7.4 (28.9 μL, ~50 μg, ~1.73 mg/mL), was immediately briefly vortexed, and was incubated at 21 °C with agitation (650 rpm). The radiolabeling yield was monitored by ITLC at several time intervals and two more aliquots of immunoconjugate were added for a total of ~100 μg (57.8 μL) immunoconjugate until ITLC indicated 91% of bound 89Zr. After a total incubation period of 2 h 15 min, the reaction was quenched by addition of ~5 μL 50 mM EDTA ,pH 7.0. The mixture was purified by size-exclusion chromatography (PD MiniTrap G-25) and the product was recovered in 74% radiochemical yield (10.0 MBq, 269 μCi). Analyses by radio-SE-HPLC indicated the presence of radiolabeled mAb with some aggregates but were hampered by adhesion of activity to the detector tubing.  Prelabeling approach. A solution of 89Zr-oxalate in 1 M oxalic acid was neutralized with 2 M Na2CO3 to pH ~7.5, was spun, and the supernatant (~56 μL, 16.3 MBq, 441 μCi) was transferred to a separate reaction vial. To this were added aliquots of Tz-THPN (4.12, total of 4.1 nmol, 8.22 μL as 0.25 mM solution in 1:4 DMSO/HEPES (0.5 M, pH 7.0) and 2.06 μL as 1 mM solution in DMSO) and the mixture was incubated in the dark at 21 °C with agitation (650 rpm). Small amounts of 1 M oxalic acid were added to adjust the reaction pH to 7.5–8. Radiolabeling yield was monitored by ITLC at several time intervals and aliquots of Tz-THPN were added until ITLC indicated 96% of bound 89Zr. Total reaction time was 2 h. This mixture was then added to a solution of TCO-modified trastuzumab in PBS, pH 7.4 (210 μL, ~371 μg, ~2.54 nmol, ~1.8 TCO/mAb) for an intended molar ratio of ~1.1 tetrazine groups per reactive TCO groups. However, ~58% of activity were lost to adhesion to the previous reaction vial, even after rinsing 123 with HEPES buffer (152 μL, 0.5 M, pH 7.0), which was added to the reaction mixture. The reaction was incubated in the dark for 1 h at 21 °C with agitation (650 rpm) and was then purified by size-exclusion chromatography (PD MiniTrap G-25). The product was eluted with 1 mL HEPES buffer and was recovered in overall 20% radiochemical yield (3.23 MBq, 87.3 μCi). Analyses by radio-SE-HPLC indicated presence of radiolabeled mAb with a limited amount of aggregation but were hampered by adhesion of activity to the detector tubing.  4.4.4 89Zr-Anti-PD-L1 Radioimmunoconjugates 4.4.4.1 THPN-Modification of anti-PD-L1 mAb A solution of anti-mouse PD-L1 mAb (45.4 μL, ~250 μg, 5.5 g/L in PBS pH 6.5) was diluted with NaHCO3/Na2CO3 buffer (50 μL, 0.1 M, pH ~9) and a freshly prepared p-SCN-Bn-THPN solution (8.3 μL, ~10 eq., 2 mM) in 10% DMSO was added. The solution was carefully mixed, the pH was adjusted to pH ~8.7–9.0 (0.1 M Na2CO3) and the reaction was incubated for 2 h at 37 °C with gentle agitation (350 rpm). The mixture was purified by size-exclusion chromatography over a PD-10 desalting column and/or by ultrafiltration over an Amicon Ultra 100K centrifugal filter, rinsed twice, and recovered with HEPES buffer (0.5 M, pH 7.0). The immunoconjugate product was analyzed by SE-HPLC (method E): tR = 12.8 min (unmodified anti-PD-L1 mAb: tR = 12.8 min).  4.4.4.2 89Zr-Labeling of THPN-anti-PD-L1 immunoconjugate A stock solution of 89Zr-oxalate in 1 M oxalic acid was neutralized with 2 M Na2CO3 to pH ~7 and the supernatant (~36 μL, 7.5 MBq, 202 μCi) was added to a solution of THPN-modified anti-PD-L1 mAb (~50 μg) in HEPES buffer (48.6 μL, 0.5 M, pH 7.0). It was briefly 124 mixed and then incubated at 22 °C with gentle agitation (350 rpm). After 15 min, the reaction mixture was analyzed by ITLC and another aliquot of THPN-anti-PD-L1 (~50 μg, 16.4 μL) was added. After another 15 min, ITLC indicated a radiolabeling yield of 70%, at which point the reaction was quenched by adding EDTA (5 μL, 50 mM, pH 7.0). The mixture was purified by ultrafiltration (Amicon Ultra 100K centrifugal filter) and analyzed by radio-SE-HPLC (method E): tR = 12.7 min. Isolated radiochemical yield was 47% and radiochemical purity was ~64% (HPLC).  4.4.5 89Zr-Mouse Serum Albumin (MSA) Radioconjugates 4.4.5.1 THPN-Modification of MSA A freshly prepared p-SCN-Bn-THPN solution in DMSO (11.4 μL, 20 mM, 10 eq.) was diluted with water (138.6 μL) and carbonate buffer (225 μL, pH ~8.7). To this, an aqueous solution of mouse serum albumin (MSA) was added (75 μL, 20 g/L, 1.5 mg). The pH of the mixture was adjusted to pH 8.3–8.5 (0.1 M Na2CO3) and the reaction was incubated overnight at 21 °C with gentle agitation (350 rpm). The mixture was purified by size-exclusion chromatography over a PD-10 desalting column, eluted with HEPES buffer (0.5 M, pH 7.0), and the product fraction was analyzed by SDS-PAGE (Figure 4.11) and SE-HPLC (method E): tR = 13.5 min (unmodified MSA: tR = 13.3 min).  4.4.5.2 89Zr-Radiolabeling of THPN-MSA To a freshly neutralized solution of 89Zr-oxalate (5.0 MBq, 134 μCi) was added a solution of THPN-modified MSA (142 μL, ~264 μg, ~1.8 g/L) in HEPES buffer (0.5 M, pH 7.0). The mixture was very briefly vortexed and incubated at 21 °C with gentle agitation (350 rpm). 125 After 20 min, an aliquot was analyzed by ITLC, which indicated 99% radiolabeling yield. The reaction mixture was purified by size-exclusion chromatography over a PD-10 desalting column with HEPES buffer (25 mM, pH 7.0) as eluent. The product was isolated in 98% radiochemical yield and was analyzed by SDS-PAGE (Figure 4.11) and radio-SE-HPLC (method E): tR = 13.6 min. Radiochemical purity (HPLC) was ~88% and specific activity was determined as ~15 kBq/μg; 0.42 μCi/μg by measuring a known volume in a dose calibrator and determining the protein concentration in a Bradford assay.  4.4.6 89Zr-HPG Radioconjugates 4.4.6.1 Chelator Conjugation to HPG-Nanoparticles THPN-, DFO-, and DFO*-HPG Conjugates. In three separate reaction vials, aqueous solutions (100 μL, 40 g/L, ~5 nmol) of amine-functionalized hyperbranched polyglycerol (HPG, ~800 kDa molecular weight) were diluted with sodium carbonate buffer (150 μL, 0.1 M, pH 9) and ligand solutions of either p-SCN-Bn-THPN, p-SCN-Phe-DFO, or p-SCN-Phe-DFO* were added (50 μL, 0.5 M, ~5 eq.). DFO and DFO* stock solutions were in anhydrous DMSO, while the THPN stock solution was in freshly diluted 5% DMSO (aq.). The pH of the reaction mixtures was adjusted to pH 8.5–9.0 with Na2CO3 and the mixture was incubated overnight at 37 °C with gentle agitation (350 rpm). After 21 h, the mixture was loaded on a PD MiniTrap G-25 size-exclusion column, eluted with HEPES buffer (0.5 M, pH 7.0), and further purified by ultrafiltration over an Amicon Ultra 100K centrifugal filter (rinsed with 3 × 0.4 mL HEPES buffer). The recovered HPG conjugates were analyzed by SE-HPLC and DLS.  THPN-HPG Conjugate: SE-HPLC (method D): tR = 15.3 min; DLS: Z-avg. (d) 15.4 ± 0.5 nm; PDI: 0.15 ± 0.04. DFO-HPG Conjugate: SE-HPLC (method D): tR = 15.4 min; DLS: Z-avg. (d) 19.2 ± 0.6 nm; PDI: 0.32 ± 0.05. DFO*-HPG Conjugate: SE-HPLC (method D ): tR = 15.4 min; 126 DLS: Z-avg. (d) 16.9 ± 0.4 nm; PDI: 0.23 ± 0.02. Unmodified HPG-NH2 (control): SE-HPLC (method D ): tR = 15.2 min; DLS: Z-avg. (d) 16.7 ± 0.2 nm; PDI: 0.23 ± 0.01.  4.4.6.2 89Zr-Radiolabeling of HPG-Chelator Conjugates In three separate reaction vials, HPG-chelator solution (THPN, DFO, or DFO*) was added to a neutralized 89Zr-oxalate solution (~170 μL, ~32 MBq, ~870 μCi, pH ~7), it was briefly vortexed and the mixtures were incubated at 21 °C with gentle agitation (350 rpm). Aliquots were analyzed by ITLC and more HPG-chelator solution was added in portions until quantitative radiolabeling was achieved. Added HPG-chelate volumes were 67.4, 75.6, and 66.6 μL for the THPN, DFO, and DFO* conjugates, respectively and total reaction volumes were ~250 μL. After 3 h reaction time, the reactions were purified by size exclusion chromatography (PD MiniTrap G-25), and eluted with water in seven fractions. Isolated radiochemical yields for all three reactions were 97% and radiochemical purities were >99% (HPLC). The most active product fractions were pooled and analyzed by radio-SE-HPLC, ITLC, and DLS. 89Zr-THPN-HPG Conjugate: radio-SE-HPLC (method D): tR = 15.3 min; DLS: Z-avg. (d) 16.2 ± 1.0 nm; PDI: 0.25 ± 0.02. 89Zr-DFO-HPG Conjugate: radio-SE-HPLC (method D): tR = 15.3 min; DLS: Z-avg. (d) 14.8 ± 0.2 nm; PDI: 0.22 ± 0.01. 89Zr-DFO*-HPG Conjugate: radio-SE-HPLC (method D): tR = 15.3 min; DLS: Z-avg. (d) 18.3 ± 0.7 nm; PDI: 0.29 ± 0.03.  4.4.6.3 In Vitro Plasma Stability of 89Zr-HPG Conjugates 89Zr-chelate-HPG conjugates with THPN, DFO, or DFO* prepared as described above were diluted 1:4 (v/v) with human blood plasma from a healthy donor. The mixtures were incubated for 5 days at 37 °C with gentle agitation (350 rpm). Aliquots were analyzed by ITLC 127 (black strips, DTPA mobile phase, 50 mM, pH 7.0) at the beginning of the experiment and after 1, 3, and 5 days incubation and the percentage of chelated 89Zr was determined by integration. Measurements were performed in triplicates.  4.4.6.4 In Vivo Study of 89Zr-HPG Conjugates This animal study was performed in accordance with the animal care committee (ACC) of the University of British Columbia under the approved protocol A16-0104. 89Zr-chelate-HPG conjugates with THPN, DFO, or DFO* chelators were prepared as described above on the day prior to the experiment and were stored overnight at 4 °C. On the day of administration, quality control of the radioconjugates was performed by radio-SE-HPLC and ITLC. Immediately before preparation of the injection doses, the aqueous radioconjugate stock solutions (~490 μL) were diluted with sterile-filtered (0.22 μm) concentrated PBS (10x, 54.4 μL). Eleven-week-old female, healthy, immunodeficient NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were anesthetized using isoflurane inhalation (2% in O2) and were administered 89Zr-THPN-HPG, 89Zr-DFO-HPG, or 89Zr-DFO*-HPG conjugate solutions via tail vein injection. For biodistribution studies, each mouse was injected ~1.5 MBq (~40 μCi, median, ~40 μL) 89Zr-chelate-HPG solution and mice were euthanized after 1, 3, or 6 days post-injection by CO2 inhalation, followed by blood withdrawal by cardiac puncture (n = 4 per group and time point). Tissues and organs were harvested, washed in Dulbecco’s PBS, dried on paper, and weighed. 89Zr activities were measured using a calibrated γ counter. Data were decay corrected and compiled in Microsoft Excel and evaluated in GraphPad Prism and Excel. Statistical analysis of bone uptake was performed by multiple t-tests assuming equal variance with a significance level of α = 0.05. Activity distributions are expressed as percentage of the injected dose per gram of tissue (%ID/g).  128 For PET/CT imaging, each mouse was injected ~5 MBq (~135 μCi, ~120 μL) of 89Zr-chelate-HPG solution (n = 1 per group). Prior to imaging, mice received a subcutaneous injection of 0.9% NaCl (0.2 mL) for hydration. Mice were imaged under isoflurane anesthesia with a CT scan followed by a 20 min PET acquisition at 1 h, 1 d, 3 d, and 6 d post-injection. After the last imaging time point, mice were euthanized by CO2 inhalation followed by blood withdrawal by cardiac puncture and were included in the respective biodistribution studies described above. Images were reconstructed using a three-dimensional ordered subsets expectation maximization (OSEM3D, 2 iterations), followed by a fast maximum a priori algorithm (FastMAP, 18 iterations) and attenuation correction was performed based on the CT image. Images were rendered using the Inveon Research Workplace software.    129 Chapter 5: THPN with Other Medically Relevant Metal Ions  5.1 Introduction To explore the potential of THPN as a chelator for metals other than Zr, THPN was screened with several medically interesting metal ions and the formation of monometallic complexes was investigated. The chelation potential of THPN for a few metal ions that have not been tested experimentally is also briefly discussed.  5.2 Results and Discussion The two preceding chapters focused on the complexation of Zr4+ by THPN. As a preliminary assessment of the potential of THPN as a chelator for other metals and to provide a starting point for future development of this chelator, we explored the THPN complexation of eight other metal ions (Fe3+, Ga3+, Y3+, Sm3+, Gd3+, Tb3+, Lu3+, and Bi3+) that are also of interest for applications in (nuclear) medicine. Formation of the iron(III) complex was studied in the context of the thermodynamic stability study of Zr-THPN (Section 3.2.3.2). In that study, the formation constants of the Fe-THPN complex were determined and presented in Table 3.4 along with the speciation plot shown in Figure 3.5a. The thermodynamic complex stability of Fe-THPN was very high (log β110 44.71, pM 38.0) and THPN merits further exploration of its therapeutic potential to treat for example iron overload. THPN also formed a monometallic complex with gallium(III), which shares a similar coordination chemistry with FeIII. After incubation overnight at ambient temperature, electrospray ionization (ESI) mass spectrometry (MS) showed the formation of the 130 monometallic Ga-THPN complex. Signals at m/z 973.3 and 995.3 Da featured Ga-distinctive isotope patterns and were assigned to the double-proton- and proton-sodium-adducts, respectively. Gallium complexes could be useful for PET with the β+-emitting 68Ga, or for SPECT and Auger electron radionuclide therapy with 67Ga. The group 3 transition metal ion yttrium(III) was complexed with THPN at ambient temperature and analyzed by mass spectrometry. In ESI-MS, the monometallic [Y(H2THPN)]+ complex† was detected in positive mode at m/z 993.5 Da. Other signals also indicated the presence of a trinuclear [Y3(THPN)2]+ species and two peaks at m/z 1036.5 and 1047.5 Da were assigned to the doubly-charged proton and sodium adducts, respectively. Only the doubly-charged adducts could be detected since the singly-charged trinuclear complex exceeded the detection limit of the mass spectrometer of m/z 1250 Da. Yttrium complexes with the high-energy β− emitter 90Y are of interest to improve 90Y-microspheres for selective internal radiation therapy (SIRT) of solid tumours such as the radioembolization of hepatocellular carcinoma. The β+-emitting 86Y, in turn, is a PET nuclide and can be employed as imaging surrogate for 90Y therapy.205 Complexation of the lanthanide ions samarium(III), gadolinium(III), terbium(III), and lutetium(III) with THPN also produced the monometallic [Ln(H2THPN)]+ complexes as indicated by ESI-MS. Yet all of these lanthanides also showed some—albeit sometimes small—peaks attributed to trinuclear M3L2 species detected as [Ln3(THPN)2H]2+. The MS peaks of the Gd and Tb complexes also featured the characteristic isotopic patterns distinctive for these two lanthanides. Gadolinium complexes have desirable properties for contrast enhancement in magnetic resonance imaging (MRI).70 Metal complexes with the β−-emitters 153Sm and 177Lu are                                                      † In this discussion of THPN metal complexes, THPN in complexes refers to the deprotonated ligand THPN4−. 131 feasible for targeted radionuclide therapy and each have one approved therapeutic radiocomplex (153Sm-lexidronam and 177Lu-DOTATATE). Terbium complexes are compelling from a radiopharmaceutical perspective in that four Tb radionuclides cover all decay modes for nuclear medicine. 155Tb and 152Tb are suited for SPECT and PET, respectively, while the β−- and Auger emitter 161Tb and the α-emitter 149Tb have promising therapeutic emissions.206-208 In contrast to the metal ions discussed above, complexation of bismuth(III) appeared to form a binuclear M2L species, which was detected as a major peak at m/z 659.8 Da assigned to the doubly-charged bimetallic ion [Bi2(THPN)]2+. Complexes with the potent bismuth nuclides 213Bi or 212Bi could be useful for targeted alpha therapy.20, 27, 209 With the exception of BiIII, all other metal ions discussed above formed monometallic complexes with THPN at ambient temperature. This renders THPN a promising ligand to be further explored with these metals. With the exception of GaIII complexation, no free THPN ligand was detected by ESI-MS, supporting THPN’s expected preference for complexation of the metal ions. The small ligand peak observed for GaIII complexation may be owed to an overestimation of the metal stock concentration since the number of crystallization water molecules in the metal salt was unknown. The observation of trinuclear species needs to be further investigated and complexation conditions may have to be optimized to avoid their formation. A possible explanation for their formation could be that even though an equimolar reaction stoichiometry was intended, inaccuracies in the preparation of THPN stock solutions due to the small scale of these experiments could have resulted in an excess of metal ions over ligand molecules.  In addition to iron, gallium, yttrium, and lanthanides, THPN may be an interesting ligand for the complexation of other metal ions such as actinides. The complexation of actinides has been explored with a number of other octadentate HOPO ligands by the groups of 132 Raymond, Abergel, and others.68, 141, 146, 210-215 The recent development of a tetrapodal octadentate 3,2-HOPO ligand for chelation of the α particle emitter 227ThIV also looks promising and the resulting 227Th complex is believed to be investigated for radioimmunotherapy in a first-in-human clinical trial by the pharmaceutical company Bayer.214-216 All octadentate HOPO chelators investigated with actinides are based on 1,2- or 3,2-HOPO groups, while THPN is thus far the only reported octadentate 3,4-HOPO chelator. Based on the promising results with those chelators, THPN is envisioned to also hold potential as an actinide chelator. Preliminary radiolabeling attempts with the α particle emitter 225AcIII were, unfortunately, not conclusive. The absence of a stable Ac isotope renders the exploration of its coordination chemistry particularly challenging and chelation was not further explored. But due to structural similarities to those literature chelators, and given THPN’s octadentate O8 donor set, THPN may also be a feasible ligand to be explored with ThIV, as well as other actinides. Such complexes would hold potential for targeted alpha particle therapy or for actinide decorporation.  5.3 Conclusions To assess the potential of THPN as a chelator for medically relevant metals other than Zr, we studied the complex formation with eight additional metal ions (Fe3+, Ga3+, Y3+, Sm3+, Gd3+, Tb3+, Lu3+, and Bi3+). The very high thermodynamic stability of the iron(III) complex suggests the exploration of THPN’s therapeutic potential to treat iron overload or to deprive microorganisms of Fe3+ for antimicrobial applications. Monometallic complexes were also obtained with gallium(III), yttrium(III), and the lanthanides SmIII, GdIII, TbIII, and LuIII but complexation conditions need to be optimized to avoid formation of trinuclear species. From a radiopharmaceutical perspective, Ga and Tb complexes could be particularly interesting for 133 imaging and Y, Tb, and Lu complexes for targeted radionuclide therapy. Likewise, the potential for actinide chelation also merits exploration to further harness the therapeutic potency of actinide α particle emitters.  5.4 Experimental 5.4.1 Materials and Methods All chemicals were used as received without further purification. Metal salts were purchased from Strem Chemicals, Alfa Aesar, or Aldrich as YCl3∙6H2O, Ga(NO3)3∙xH2O, SmCl3∙6H2O, GdCl3∙6H2O, TbCl3∙6H2O, LuCl3∙6H2O, and anhydrous BiCl3. All water used was ultrapure (18.2 MΩ∙cm). ESI-MS spectra were recorded on an AB Sciex QTrap 5500 mass spectrometer in positive mode.  5.4.2 Syntheses GaIII complexation. A solution of Ga(NO3)3 (10.5 μL, 0.5 M, ~5.3 μmol) in 50% methanol/water was added to a solution of THPN in 5:1 methanol/water (600 μL, 8 mM, ~4.8 μmol). The mixture was briefly vortexed and left stirring overnight at ambient temperature. The solution was then precipitated by dropwise addition to THF (~4 mL), centrifuged, and the supernatant was removed. The precipitate was air-dried, followed by freeze-drying, and the resulting white powder was analyzed by mass spectrometry. ESI-MS (m/z) 973.3 [ML+2H]+ (calcd. 973.3); 995.3 [ML+H+Na]+ (calcd. 995.3). YIII complexation. An aqueous solution of YCl3 (220 μL, 5 mM, ~1.1 μmol) was added to a solution of THPN in water (400 μL, 2.8 mM, ~1.1 μmol). The mixture was briefly vortexed and then incubated for 1.5 h at ambient temperature with agitation (700 rpm). At this point, an 134 aliquot was removed, diluted with methanol, and analyzed by mass spectrometry. ESI-MS (m/z) 993.5 [ML+2H]+ (calcd. 993.3); 1036.5 [M3L2+H]2+ (calcd. 1036.2); 1047.5 [M3L2+Na]2+ (calcd. 1047.2). SmIII complexation. A solution of SmCl3 in D2O (220 μL, 5 mM, ~1.1 μmol) was added to a solution of THPN in a D2O/H2O (4:1) mixture (250 μL, 4.4 mM, ~1.1 μmol). The mixture was incubated for 1 h at ambient temperature with agitation (700 rpm). An aliquot was removed, diluted with methanol, and analyzed by mass spectrometry. ESI-MS (m/z) 1056.4 [ML+2H]+ (calcd. 1056.3); 1129.9 [M3L2+H]2+ (calcd. 1129.3). GdIII complexation. A solution of GdCl3 in water (276 μL, 4 mM, ~1.1 μmol) was added to a solution of THPN in D2O (100 μL, 11 mM, ~1.1 μmol). The mixture was incubated overnight at ambient temperature with agitation (700 rpm). An aliquot was removed, diluted with methanol, and analyzed by mass spectrometry. ESI-MS (m/z) 1062.5 [ML+2H]+ (calcd. 1062.3); 1140.5 [M3L2+H]2+ (calcd. 1139.8). TbIII complexation. A solution of TbCl3 in D2O (220 μL, 5 mM, ~1.1 μmol) was dropwise added to a solution of THPN in D2O (250 μL, 4.4 mM, ~1.1 μmol). The mixture was incubated for 25 min at ambient temperature with agitation (700 rpm). A portion of the reaction mixture (200 μL) was removed, basified by addition of NaOH to pH ~8 (~20 μL, 0.1 M), and the mixture was incubated for another 25 min at ambient temperature with agitation. An aliquot was removed, diluted with methanol, and analyzed by mass spectrometry. ESI-MS (m/z) 1063.5 [ML+2H]+ (calcd. 1063.3); 1085.3 [ML+H+Na]+ (calcd. 1085.3); 1141.4 [M3L2+H]2+ (calcd. 1141.3); 1152.5 [M3L2+Na]2+ (calcd. 1152.3). LuIII complexation. THPN (5.3 mg, 5.4 μmol) was dissolved with heating in a mixture of methanol, water, and 0.1 M Na2CO3 (20:5:1) and was allowed to cool to ambient temperature. To this ligand solution was dropwise added a solution of LuCl3 in methanol (410 μL, 13.1 mM, 135 ~5.4 μmol) and Na2CO3 (6.8 μL, 0.1 M) was added. The mixture was briefly vortexed and was then incubated overnight at ambient temperature with agitation (700 rpm). A solution of Na2CO3 (80.4 μL, 0.1 M) was added which caused turbidity and the mixture was mixed another night at ambient temperature. The slurry was then transferred to a microcentrifuge tube, centrifuged, the supernatant was removed, and the white precipitate was analyzed by mass spectrometry. ESI-MS (m/z) 1079.4 [ML+2H]+ (calcd. 1079.3); 1101.4 [ML+H+Na]+ (calcd. 1101.3); 1176.9 [M3L2+Na]2+ (calcd. 1176.3). BiIII complexation. A freshly prepared solution of anhydrous BiCl3 in methanol (275 μL, 4 mM, ~1.1 μmol) was dropwise added to a solution of THPN in methanol (300 μL, 3.7 mM, ~1.1 μmol). The mixture was incubated for 1.5 h at ambient temperature with agitation (700 rpm). An aliquot was removed, diluted with methanol, and analyzed by mass spectrometry. ESI-MS (m/z) 659.8 [M2L]2+ (calcd. 660.2).   136 Chapter 6: Conclusions and Outlook Metallic radiopharmaceuticals play a critical role in nuclear medicine and are desired for applications in imaging and therapy. To be utilized in most radiopharmaceutical compounds, radiometals need to be stably sequestered by chelating agents to avoid off-target distribution. The long-lived radionuclide zirconium-89 possesses compelling decay characteristics for antibody-targeted PET but is in need for alternative chelating agents. Through the work presented herein, a new class of octadentate chelators was introduced to expand the repertoire of available chelating agents and these chelators were investigated in vitro and in vivo with zirconium(IV).  Chapter 3 discussed the synthesis and characterization of the novel tetrapodal 3,4-HOPO ligand THPN and the examination of its complex with ZrIV. Using micromolar concentrations, the chelator rapidly forms a monometallic Zr-complex that is of exceptional thermodynamic stability. In challenge experiments, the new chelator outperformed the current literature standard and short-term in vivo stability showed promising rapid excretion of the 89Zr-radiocomplex without signs of residual organ uptake. In Chapter 4, this chelator was further functionalized in a multistep synthesis to introduce a reactive isothiocyanate handle to the backbone. This bifunctionalization then enabled (bio)conjugation to targeting entities. The bifunctional chelator was successfully conjugated to antibodies, a model protein, and polymeric nanoparticles and radiolabeling of these conjugates with 89Zr was optimized. Subsequently, the stability of the radioconjugates was investigated in vitro and, if feasible, in vivo and was compared against two literature chelators. Polymeric hyperbranched polyglycerol nanoparticles were identified as a suitable platform to assess the physiologic stability of radiochelates. Their prolonged circulation time, robust chemistry, and innocuous toxicity profile render them suitable nanocarriers to evaluate the 137 long-term stability of conjugated radiocomplexes (or other cargo) in the blood stream over several half-lives. A six-day mouse study revealed inferior physiologic stability of 89Zr-THPN-nanoparticle conjugates compared to corresponding radiochelate-particles with two literature chelators. Since the thermodynamic stability of the radiocomplex was previously determined to be exceptionally high, the observed instability was attributed to suboptimal kinetic inertness, which is of particular importance for the in vivo stability of radiopharmaceuticals.  In order to improve this kinetic aspect, a more preorganized structure of the chelator may be beneficial. This could reduce the entropic penalty accrued upon metal ion complexation and should convey a higher kinetic inertness, as often seen for macrocycles.51 Thus, a bi-macrocyclic cage or “clam-shell” like version of the ligand could be a rational extension of THPN. An imaginable strategy could be to connect the 3,4-HOPO groups not only at the C2 ring position, but also at the C5 position. Two potential ligand structures are suggested in Figure 6.1 and their Zr-complex geometries were simulated by DFT (Figure 6.2). Connecting the same linker as used for the THPN backbone at the C5 positions would give a bi-macrocyclic cage, while using two different linkers could give access to a “clam-shell” like bi-macrocycle, somewhat resembling a ligand reported by Tinianow et al.112 Analogous to THPN, a conjugation handle could be introduced at the centre of one linking group. It should be taken into account, however, that the improved kinetic inertness of macrocycles commonly comes at the cost of more stringent reaction conditions for metal ion coordination. This aspect, together with synthetic feasibility, hydrophilicity, solubility, and geometric factors, therefore needs to be carefully considered when designing the next generation of this ligand system. 138  Figure 6.1. Suggested structures for a bi-macrocyclic cage (left) or “clam-shell” like derivative (right) of THPN.   Figure 6.2. DFT-optimized structures of Zr-chelates with the proposed bi-macrocyclic cage- (left) or “clam-shell”-like chelator (right). Calculations were performed with Gaussian 16123 using B3LYP/LANL2DZ theory. Hydrogen atoms are omitted for clarity.  Apart from such ligand modifications, we believe that THPN itself holds the potential to form strong and inert complexes with a range of other metal ions. 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ClinicalTrials.gov, First-in-human Study of BAY2287411 Injection, a Thorium-227 Labeled Antibody-chelator Conjugate, in Patients With Tumors Known to Express Mesothelin, https://clinicaltrials.gov/ct2/show/NCT03507452, (accessed Sep. 17, 2018).   157 Appendices Appendix A: Data for Zr-THPN and 89Zr-Labeling   Figure A.1. MALDI-MS of Zr-THPN. The monometallic complex shows the expected [M+H]+ peak and isotope pattern at m/z 993.3 that matches the predicted pattern (inset). 158  Figure A.2. FT-IR comparison of THPN (top) and Zr-THPN (bottom).  Figure A.3. Comparison of ITLC chromatograms of 89Zr-oxalate, 89Zr-THPN, and 89Zr-DFO. Biodex Tec-Control (black #150-005) ITLC strips developed with aqueous DTPA solution (50 mM, pH 7.0). 159       Figure A.4. ITLCs of transchelation competition (repetitions of Figure 3.8 for n = 3). Biodex Tec-Control (dark green, #150-771) ITLC strips developed with aqueous sodium citrate solution (100 mM, pH 5.5).   160 Appendix B: PET/CT Images from In Vivo Study of 89Zr-Chelates  Figure B.1. Fused PET/CT images (MIP) of all four mice injected with 89Zr-THPN (0–30 min post injection).  Figure B.2. Fused PET/CT image (MIP) of the mouse injected with 89Zr-DFO (0–30 min post injection).  161 Appendix C: Spectra Pertaining to Thermodynamic Solution Studies  Figure C.1. Representative spectra of the direct UV-Vis potentiometric titration of the FeIII-THPN system ([THPN] = [Fe3+] = 2.08  10−4 M) as the pH is raised from 1.93 to 10.3, 25 °C, I = 0.16 M (NaCl), l = 0.5 cm.   Figure C.2. Representative spectra of the metal-metal competition of the FeIII-THPN system by increasing the equivalents of Zr4+. [THPN] = [Fe3+] = 7.16  10−5 M, [Zr4+]/[ Fe3+] = 0–4.5, pH = 2, 25 °C, I = 0.16 M (NaCl), l = 1 cm.  350 400 450 500 550 600 650 7000.00.20.40.60.8pH 10.3iso = 363 nmAbsorbanceWavelength (nm) 1.93 1.99 2.05 2.12 2.20 2.30 2.44 2.64 3.09 3.82 4.75 10.30iso = 503 nmpH 1.93250 275 300 325 3500.00.51.01.52.02.5AbsorbanceWavelength (nm) 4 3 2.22 1.75 1.25 1 0.5 0.25 0Zr4+ eq.350 400 450 500 550 600 6500.000.050.100.150.200.250.300.350.40AbsorbanceWavelength (nm) 0 0.25 0.5 1 1.25 1.75 2.22 3 4Zr4+ eq.162  Figure C.3. Representative spectra of the combined UV-potentiometric titration of the ZrIV-THPN system in the pH range 2.07–6.64. [THPN] = [Zr4+] = 2.11  10−4 M, at 25 °C, I = 0.16 M (NaCl), l = 0.2 cm.    250 275 300 325 3500.000.250.500.751.001.251.501.75 Zr(THPN)AbsorbanceWavelength (nm) 6.64 5.56 4.50 3.90 3.69 3.55 3.44 3.35 3.27 3.14 2.85 2.24 2.07[Zr(H2THPN)]2+163 Appendix D: Inverse Electron Demand Diels-Alder Conjugation Conjugations  Figure D.1. Reaction sequences pursued for tetrazine/trans-cyclooctene conjugation strategies to radiolabel trastuzumab. a) Synthesis of tetrazine-THPN derivative 4.12; b) TCO-modification of trastuzumab; c) postlabeling strategy by IEDDA cycloaddition between 4.12 and TCO-trastuzumab, followed by 89Zr-labeling; d) prelabeling strategy by IEDDA cycloaddition between TCO-modified trastuzumab and radiolabeled 89Zr-THPN-tetrazine (shown schematically). 

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