Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Ingham, Aidan
Doctor of Philosophy - PhD
University of British Columbia
Metal-based radiopharmaceuticals provide a vital option to treat patients suffering from metastatic cancers. This class of drugs can both diagnose and destroy widely disseminated tumour cells in a targeted manner, including tumours that demonstrate a resistance to other forms of treatments such as chemotherapy. To selectively seek out tumour cells, most metal-based radiopharmaceuticals are designed to involve four components: a radiometal, a bifunctional chelator, a linker, and a targeting vector. The bifunctional chelator coordinates the metal and keeps it attached to the drug whilst in the body. There are many different types of diagnostic and therapeutic radiometals. Thus, to save time and resources, a non-bifunctional version of the chelator is tested with the selected radiometal to ensure they are a strong match before a bifunctional version is constructed. This thesis focuses on the design, synthesis, and evaluation of (non-bifunctional) open-chain chelators, which have been paired with large (radio)metal ions. Amide-based chelators EGTAm and ampam were matched with [ⁿᵃᵗᐟ²⁰³Pb]Pb²⁺ ions, and their respective complexes were studied via NMR spectroscopy, X-ray diffraction, potentiometric titrations, and radiolabeling studies. Neither chelator was found to be a suitable match, likely due to their large scaffolds and amide donor groups being mismatched with one another for Pb²⁺ ion chelation. A diprotic analogue of ampam, known as H₂ampa, was then tested with [ⁿᵃᵗᐟ²⁰³Pb]Pb²⁺, [²²⁵Ac]Ac³⁺ and [ⁿᵃᵗᐟ²¹³Bi]Bi³⁺ ions. To evaluate these complexes, NMR spectroscopy, X-ray diffraction, thermodynamic solution studies, and radiolabeling studies were carried out. From these preliminary studies, H₂ampa was found to be a versatile chelator demonstrating a high affinity for all three radiometals. Finally, H₅decapa and H₄noonpa were tested with [²²⁵Ac]Ac³⁺ ions via radiolabeling and serum stability studies. Both chelators demonstrated a high affinity for [²²⁵Ac]Ac³⁺ ions and formed kinetically inert complexes; however, H₄noonpa was deemed more favourable due to the simpler and more reliable radiochemistry methodologies that could be applied to study its monoanionic complex compared to the dianionic complex H₅decapa formed.
LONE PAIR ACTIVITY, VERSATILITY, AND CHARGE: LESSONS FROM CHELATORS DESIGNED FOR RADIOPHARMACEUTICALS by Aidan Ingham MSci, University of Bristol, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2022 © Aidan Ingham, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Lone Pair Activity, Versatility, and Charge: Lessons from Chelators Designed for Radiopharmaceuticals submitted by Aidan Ingham in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Dr. Chris Orvig, Chemistry, UBC Supervisor Dr. Glenn Sammis, Chemistry, UBC Supervisory Committee Member Dr. Parisa Mehrkhodavandi, Chemistry, UBC University Examiner Dr. Marcel Bally, Experimental Therapeutics, BC Cancer University Examiner Additional Supervisory Committee Members: Dr. Jay Kizhakkedathu, Pathology, UBC Supervisory Committee Member Dr. Zachary Hudson, Chemistry, UBC Supervisory Committee Member iii Abstract Metal-based radiopharmaceuticals provide a vital option to treat patients suffering from metastatic cancers. This class of drugs can both diagnose and destroy widely disseminated tumour cells in a targeted manner, including tumours that demonstrate a resistance to other forms of treatments such as chemotherapy. To selectively seek out tumour cells, most metal-based radiopharmaceuticals are designed to involve four components: a radiometal, a bifunctional chelator, a linker, and a targeting vector. The bifunctional chelator coordinates the metal and keeps it attached to the drug whilst in the body. There are many different types of diagnostic and therapeutic radiometals. Thus, to save time and resources, a non-bifunctional version of the chelator is tested with the selected radiometal to ensure they are a strong match before a bifunctional version is constructed. This thesis focuses on the design, synthesis, and evaluation of (non-bifunctional) open-chain chelators, which have been paired with large (radio)metal ions. Amide-based chelators EGTAm and ampam were matched with [nat/203Pb]Pb2+ ions, and their respective complexes were studied via NMR spectroscopy, X-ray diffraction, potentiometric titrations, and radiolabeling studies. Neither chelator was found to be a suitable match, likely due to their large scaffolds and amide donor groups being mismatched with one another for Pb2+ ion chelation. A diprotic analogue of ampam, known as H2ampa, was then tested with [nat/203Pb]Pb2+, [225Ac]Ac3+ and [nat/213Bi]Bi3+ ions. To evaluate these complexes, NMR spectroscopy, X-ray diffraction, thermodynamic solution studies, and radiolabeling studies were carried out. From these preliminary studies, H2ampa was found to be a versatile chelator demonstrating a high affinity for all three radiometals. Finally, H5decapa and H4noonpa were tested with [225Ac]Ac3+ ions via radiolabeling and serum stability studies. Both chelators demonstrated a high affinity for [225Ac]Ac3+ ions and formed kinetically inert complexes; however, H4noonpa was deemed more iv favourable due to the simpler and more reliable radiochemistry methodologies that could be applied to study its monoanionic complex compared to the dianionic complex H5decapa formed. v Lay Summary The development of drugs based on radioactive metal atoms, or “radiometals”, are of interest because they can diagnose and treat patients suffering from widely spread cancers. Most radiometal-based drugs contain a “chelator” component and a targeting vector. The chelator wraps around the radiometal and keeps it attached to the drug, while the targeting vector selectively seeks out and binds to tumour cells. Before these drugs are developed, the chelator must demonstrate it is a suitable match with the selected radiometal. Within this thesis, several chelators have been paired with radiometal atoms to determine their compatibility with one another. Two were designed for lead atoms, two for actinium atoms, and one for lead, actinium, and bismuth atoms. All the chelators were found to wrap around the (radio)metal atoms under mild temperature conditions and in short reaction times, although further studies are necessary for radioactive drug development. vi Preface Chapter 2 is an adaption of published work and is reproduced in part from A. Ingham, T. I. Kostelnik, B. L. McNeil, B. O. Patrick, N. Choudhary, M. de G. Jaraquemada-Peláez and C. Orvig; Getting a lead on Pb2+-amide chelators for 203/212Pb radiopharmaceuticals, Dalton Trans., 2021, 50, 11579-11595, Copyright 2021 The Royal Society of Chemistry. I am co-first author as I designed and synthesized EGTAm and ampam, obtained their respective Pb2+ complex NMR spectra and crystal structures, performed their [203Pb]Pb2+ radiolabeling studies at TRIUMF, and co-wrote the manuscript. Dr. T. Kostelnik is the other co-first author as he designed and synthesized DTPAm, obtained its Pb2+ complex NMR spectra and crystal structure, and co-wrote the manuscript. Dr. B. O. Patrick and Dr. N. Choudhary solved the crystal structures. In addition to conducting [203Pb]Pb2+ radiolabeling studies with DTPAm, W. Fu, Dr. A. Robertson and B. McNeil produced and purified the [203Pb]Pb2+ for all the radiolabeling studies at TRIUMF. Dr. M. de G. Jaraquemade-Peláez and I executed the EGTAM and ampam solution studies together, including the NMR spectroscopy titration studies. In addition to conducting the DTPAm solution studies, Dr. M. de G. Jaraquemade-Peláez independently fitted the corresponding data for all three chelators. Dr. C. Orvig gave input throughout the process of editing the manuscript leading to publication. Chapter 3 is a manuscript in preparation and is based on worked conducted in Dr. C. Orvig’s laboratory and the TRIUMF radiochemistry laboratories. I am co-first author as I designed H2ampa, established its synthetic route, assisted with its synthesis and metal complexation studies, performed its [203Pb]Pb2+ radiolabeling studies at TRIUMF, and wrote the majority of the manuscript. An undergraduate student who worked in the group for one year, T. El Sayed, is a co-first author as he synthesized H2ampa, helped obtain metal complex NMR spectra, provided an X-vii ray quality metal complex crystal structure, and wrote part of the manuscript while co-supervised by me, L. Wharton, and Dr. C. Orvig. The third co-first author, L. Wharton, assisted T. El Sayed with the metal complexation studies, set up an 225Ac/213Bi generator at TRIUMF, performed the [213Bi]Bi3+ radiolabeling studies, and wrote part of the manuscript. L. Southcott conducted the [225Ac]Ac3+ radiolabeling studies at TRIUMF, Dr. B. O. Patrick solved the crystal structure, and B. McNeil produced and purified the [203Pb]Pb2+ at TRIUMF. T. El Sayed, Dr. M. de G. Jaraquemade-Peláez and I executed potentiometric and spectrophotometric studies involving the ligand and its Pb2+ complex as a group, while T. El Sayed and Dr. M. de G. Jaraquemade-Peláez conducted the La3+ complex study and fitted all the data together. Dr. C. Orvig gave input throughout the process of editing the manuscript. Chapter 4 is based on unpublished work conducted in Dr. C. Orvig’s laboratory and the TRIUMF radiochemistry laboratories. A former graduate student from the group, Dr. E. Price, originally designed and synthesized H5decapa. I designed an alternative synthetic scheme and re-synthesized H5decapa. Another former graduate student from the group, Dr. X. Wang, designed H4noonpa while I independently designed and conducted the synthetic experiments, including full characterization of the products. I carried out all the non-radioactive metal complexation studies with La3+ ions, performed the [225Ac]Ac3+ radiochemistry studies involving H5decapa at TRIUMF, and wrote the chapter. L. Southcott performed the [225Ac]Ac3+ radiochemistry studies with H4noonpa and Dr. C. Orvig supervised the project and helped edit the chapter. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Schemes .......................................................................................................................... xvii List of Charts ............................................................................................................................ xviii List of Symbols and Abbreviations .......................................................................................... xix Glossary ..................................................................................................................................... xxii Acknowledgements .................................................................................................................. xxiv Dedication ................................................................................................................................. xxvi Chapter 1: Introduction ................................................................................................................1 1.1 Nuclear Medicine and Radiopharmaceuticals ................................................................ 1 1.1.1 Imaging ................................................................................................................... 2 1.1.2 Radiation Therapies ................................................................................................ 5 1.2 Non-Metallic Radionuclides Versus Radiometals .......................................................... 8 1.3 Metalloradiopharmaceuticals ........................................................................................ 11 1.4 Chelator Design and Radiometal Complexation........................................................... 14 1.5 Metal Complex Evaluation ........................................................................................... 17 1.5.1 Thermodynamic Stability...................................................................................... 17 ix 1.5.2 Radiolabeling Ability ............................................................................................ 19 1.5.3 Kinetic Inertness and In Vivo Performance .......................................................... 20 1.6 Thesis Overview ........................................................................................................... 21 Chapter 2: An Evaluation of Open-Chain Amide Chelators Designed for [203/212Pb]Pb2+ Ions ................................................................................................................................................23 2.1 Introduction ................................................................................................................... 23 2.1.1 Lead (Pb) in Medicine .......................................................................................... 23 2.1.2 Lone Pair Activity ................................................................................................. 24 2.1.3 Amide Chelators ................................................................................................... 25 2.2 Results and Discussion ................................................................................................. 27 2.2.1 Ligand Synthesis ................................................................................................... 27 2.2.2 Metal Complexation.............................................................................................. 31 2.2.3 X-ray Crystal Structures ....................................................................................... 33 2.2.4 Radiolabeling with [203Pb]Pb2+ ............................................................................. 36 2.2.5 Solution Thermodynamics .................................................................................... 40 2.3 Conclusions ................................................................................................................... 46 2.4 Experimental ................................................................................................................. 47 2.4.1 Synthetic Materials and Methods.......................................................................... 47 2.4.2 Synthesis and Characterization of EGTAm and ampam ...................................... 48 2.4.3 Metal Complexation and NMR Titration Solutions.............................................. 53 2.4.4 X-ray Crystallography .......................................................................................... 54 2.4.5 Radiolabeling Studies ........................................................................................... 55 2.4.6 Potentiometric Titration Studies ........................................................................... 55 x Chapter 3: H2ampa: a Versatile Chelator Capable of Binding [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+ Ions ............................................................................................................................58 3.1 Introduction ................................................................................................................... 58 3.1.1 Selection of Targeted Alpha Therapy Candidates ................................................ 58 3.1.2 Design of H2ampa ................................................................................................. 60 3.2 Results and Discussion ................................................................................................. 62 3.2.1 Ligand Synthesis ................................................................................................... 62 3.2.2 Metal Complexation Studies ................................................................................. 64 3.2.3 X-ray Crystallography Study ................................................................................ 70 3.2.4 Radiolabeling Studies with [203Pb]2+, [213Bi]Bi3+, and [225Ac]Ac3+ ...................... 72 3.2.5 Thermodynamic Solution Studies ......................................................................... 78 3.3 Conclusion .................................................................................................................... 81 3.4 Experimental ................................................................................................................. 82 3.4.1 Synthetic Materials and Methods.......................................................................... 82 3.4.2 Synthesis and Characterization of H2ampa ........................................................... 82 3.4.3 H2ampa Metal Complexation Studies ................................................................... 86 3.4.4 X-ray Crystallography Study of [Bi(ampa)](NO3) ............................................... 87 3.4.5 Radiolabeling Experiments with [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+ ......... 87 3.4.6 Thermodynamic Solution Studies ......................................................................... 89 Chapter 4: Impact of Charge on [225Ac]Ac3+ Radiolabeling Studies ......................................91 4.1 Introduction ................................................................................................................... 91 4.2 Results and Discussion ................................................................................................. 94 4.2.1 Synthesis ............................................................................................................... 94 xi 4.2.2 Metal Complexation.............................................................................................. 97 4.2.3 [225Ac]Ac3+ Radiolabeling and Serum Stability Studies ..................................... 102 4.3 Conclusion .................................................................................................................. 105 4.4 Experimental ............................................................................................................... 106 4.4.1 Synthetic Materials and Methods........................................................................ 106 4.4.2 Synthesis and Characterization of H5decapa and H4noonpa ............................... 106 4.4.3 Metal Complexation............................................................................................ 112 4.4.4 Radiolabeling and Human Serum Stability Experiments ................................... 112 Chapter 5: Conclusion and Future Work ................................................................................116 5.1 Lone Pair Activity ....................................................................................................... 116 5.2 Versatility .................................................................................................................... 117 5.3 Importance of Charge ................................................................................................. 119 5.4 Bifunctional Chelator Development ........................................................................... 121 Bibliography ...............................................................................................................................124 Appendices ..................................................................................................................................137 Appendix A. Supplementary Data for Chapter 2 .................................................................... 137 Appendix B. Supplementary Data for Chapter 3 .................................................................... 145 Appendix C. Supplementary Data for Chapter 4 .................................................................... 157 xii List of Tables Table 1.1 Frequently considered non-metallic radionuclides.18,42–45 ........................................... 10 Table 1.2 Popular radiometals with medically valuable decay properties.16,46–52 ........................ 10 Table 2.1 Bond lengths of each Pb2+ complex formed from Pb(NO3)2. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. .................. 35 Table 2.2 Protonationa and formationb constants of ligands and their corresponding Pb2+ complexes. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry..................................................................................................................... 42 Table 3.1 Key bond distances found in [Bi(ampa]+ as determined by X-ray diffraction, including a comparison to those bonding distances found for [Bi(macropa]+. ............................................. 72 Table 3.2 H2ampa achieved near-quantitative radiolabeling (RCC > 95%) of [203Pb]Pb2+ ions at 10-5 M, making it comparable to DOTA and p-SCN-Bn-TCMC. ................................................ 74 Table 3.3 Protonation constants (log K)a of H2ampa reveal four protonation sites. .................... 78 Table 3.4 Formation constantsa of various H2ampa/Pb2+ and H2ampa/La3+ species. .................. 79 Table 4.1 Serum stability of H4noonpa and H5decapa with [225Ac]Ac3+. .................................. 105 xiii List of Figures Figure 1.1 SPECT (above) involves a collimator on its detector to manage a range of  energies, while PET (below) uses coincidence detectors to better measure antiparallel  photons that are produced because of the +-e- annihilation reaction. ...................................................................... 4 Figure 1.2 Simplified illustration of the three different types of particle-based radiotherapies highlighting how far the different types of particles travel in biological tissue, as well as their linear energy transfer ionising interactions with DNA. ............................................................................ 6 Figure 1.3 Generalized depiction of a four-component metalloradiopharmaceutical. ................ 13 Figure 1.4 Chelators must first demonstrate they are compatible in binary with a radiometal (Phase 1) before four-component radiopharmaceuticals are developed (Phase 2). .................................. 14 Figure 2.1 A) 1H NMR spectra of EGTAm (top) and [Pb(EGTAm)]2+ (bottom) (300 MHz, 298 K, D2O, pD 6-7); B) 1H NMR spectra of ampam (top) and [Pb(ampam)]2+ (bottom) (300 MHz, 298 K, D2O, pD 6-7). Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. ......................................................................................................... 32 Figure 2.2 ORTEP-style diagrams of A) [Pb(EGTAm)(NO3)](NO3), B) [Pb(ampam)](NO3)2, C) [Pb(DTPAm)](NO3)2; for clarity, ellipsoids are drawn at 50% probability, the Pb2+ ion at 80%, and hydrogen atoms are omitted except on amide groups. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. ................................................... 34 Figure 2.3 HPLC traces: A = free [203Pb]Pb2+, B = 10-3 mol dm-3 [203Pb][Pb(EGTAm)]2+ and C = 10-3 mol dm-3 [203Pb][Pb(ampam)]2+. Analytical column used; flow rate = 1 mL/min; gradient: A = H2O (0.1% TFA), B = MeCN (0.1% TFA), A = 95%, 0-2 min, A = 95-80%, 2-18 min. [203Pb]Pb2+ elutes at tR ~ 3.2 min, [203Pb][Pb(EGTAm)]2+ elutes at tR ~ 4.0 min, and [203Pb][Pb(ampam)]2+ xiv elutes at tR ~ 4.9 min with a peak that streaks. Reproduced from Ref. 80 (ESI section) with permission from the Royal Society of Chemistry. ........................................................................ 38 Figure 2.4 Speciation plots (A) EGTAm and (B) ampam, calculated with protonation constants in Table 2.2, [L] = 0.001 M; dashed line indicates pH = 7.4. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. .......................................... 41 Figure 2.5 Speciation plots of (A) Pb2+-EGTAm and (B) Pb2+-ampam, calculated with stability constants in Table 2.2, [Pb2+] = [L] = 0.001 M; dashed line indicates pH = 7.4. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. ........ 43 Figure 2.6 Portion of the 1H NMR spectra for EGTAm and [Pb(EGTAm)]2+ vs pH (300 MHz, 298 K, D2O). Reproduced from Ref. 80 with permission from the Royal Society of Chemistry. ...... 45 Figure 3.1 Stacked 1H NMR of H2ampa and corresponding complexes with metals Pb2+, Bi3+, and La3+ (400 MHz, 298 K, D2O). The pD of H2ampa, [Pb(ampa)], [Bi(ampa)]+ and [La(ampa)]+ solutions were 6, 7, 5, and 7, respectively. Only the amide and picolinate methylene peaks have been highlighted for clarity. .......................................................................................................... 65 Figure 3.2 Variable temperature 1H NMR of [Pb(ampa)] (400 MHz, D2O, pD 7). Aside from the large D2O solvent peak at 4.79 ppm, the proton signal peaks of the complex have a general trend of becoming sharper and shifting downfield with increasing temperature. .................................. 67 Figure 3.3 HSQC spectrum of [Bi(ampa)]+ with focus on the aliphatic section (400 MHz, 298 K, D2O, pD 7). ................................................................................................................................... 68 Figure 3.4 ORTEP-style diagrams showing two different views of ten-coordinate [Bi(ampa)]NO3. Thermal ellipsoids are shown at 50% probability, except for the Bi3+ ion. Methylenic hydrogens (-CH2-) and co-crystallized solvent molecules have been omitted for clarity. ................................ 70 xv Figure 3.5 Radio-HPLC traces: A = free [203Pb]Pb2+, while B and C = [203Pb][Pb(ampa)] at 10-4 and 10-5 mol dm-3, respectively. Mobile phase used: A) H2O 0.1% TFA, B) MeCN 0.1% TFA; gradient: 82% A 0-3 minutes, 82% to 72% A, 3-23 minutes, 3 mL/min flow rate passing through a semi-prep column. Under these conditions, [203Pb]Pb2+ has a retention time (tR) ~ 4.6 min and [203Pb][Pb(ampa)] has a tR = 6.0 min. ........................................................................................... 73 Figure 3.6 Using ~20 kBq of [213Bi]Bi3+ and NH4OAc buffer (0.4 M, pH 7) in each concentration-dependent study, H2ampa and DOTA both achieved high radiochemical conversions (RCC > 90%) except at very dilute ligand concentrations (~10-7 M). H2ampa solutions were mixed with [213Bi]Bi3+ for 7 minutes at room temperature (25 ℃), while DOTA solutions were mixed for 10 minutes at high temperatures (90 ℃). .......................................................................................... 76 Figure 3.7 Using ~40 kBq of [225Ac]Ac3+, NH4OAc buffer (0.2 M, pH 7) and 30 minute mixing times in each concentration-dependent study, H2ampa achieved higher radiochemical conversions (RCC) compared to DOTA at low ligand concentrations (10-5 - 10-7 M). Room temperature (25 ℃) radiolabeling conditions were used for H2ampa, while DOTA required high temperatures (85 ℃). ................................................................................................................................................ 77 Figure 3.8 Speciation plots showing the deprotonation events of [H4ampa]2+ to ampa2- (top), as well as metal complexation species for the Pb2+/ampa2- and La3+/ampa2- systems (bottom left and bottom right, respectively). Calculated with protonation constants and formation constants from Tables 3.3 and 3.4, and [Ligand] = [Metal] = 0.001 M. Dotted line in graphs indicates physiological pH (7.4). ........................................................................................................................................ 81 Figure 4.1 1H NMR spectra of H5decapa (400 MHz, 298 K, D2O, pD 1, top) and [La(decapa)]2- (400 MHz, 298 K, D2O, pD 6, bottom). ....................................................................................... 99 xvi Figure 4.2 HSQC spectrum of [La(decapa)]2- focused on the aliphatic region (400 MHz, 298 K, D2O, pD 6). ................................................................................................................................... 99 Figure 4.3 1H NMR spectra of H4noonpa (top) (400 MHz, 298 K, D2O, pD 1) and [La(noonpa)]- (bottom) (400 MHz, 298K, D2O, pD 6). ..................................................................................... 101 Figure 4.4 13C{1H} NMR spectra of H4noonpa (top) (400 MHz, 298 K, D2O, pD 1) and [La(noonpa)]- (bottom) (400 MHz, 298K, D2O, pD 6) focused on the aliphatic region. ........... 101 Figure 4.5 Using ~40-60 kBq of [225Ac]Ac3+, NH4OAc buffer (0.2 M, pH 7), 15 minute mixing times and RT conditions in each concentration-dependent study, H4noonpa and H5decapa achieved similarly high radiochemical conversions (RCCs) at low ligand concentrations (10-5-10-7 M), but the TLC conditions used for H4noonpa produced less error than the Chelex-resin method used for H5decapa. .................................................................................................................................... 102 xvii List of Schemes Scheme 2.1 Synthetic route to afford EGTAm (2) with an overall yield of 79%. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry ......... 28 Scheme 2.2 Synthetic route to afford ampam (9) with an overall yield of 12%. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry ......... 29 Scheme 3.1 Synthesis of H2ampa (10) obtained in a cumulative yield of 21% ........................... 63 Scheme 4.1 The five-step synthetic route used to produce H5decapa (15) in an overall yield of 11% ............................................................................................................................................... 95 Scheme 4.2 The five-step synthetic route used to produce H4noonpa (19) in an overall yield of 22% ............................................................................................................................................... 97 Scheme 5.1 Proposed route to form a modified picolinic acid moiety (23) that can be incorporated into bifunctional H4noonpa, inspired by a similar synthetic route established by Li et al.159 .... 122 Scheme 5.2 Part of the established route used to synthesize H4noonpa (involving compounds 5, 16 and 17) ................................................................................................................................... 122 Scheme 5.3 Proposed route to synthesize bifunctional H4noonpa (29) from compound 17, inspired by a similar synthetic route established by Li et al.159 .................................................. 123 xviii List of Charts Chart 1.1 Chemical structures of metal-essential radiopharmaceuticals: 99mTc-sestamibi and Xofigo. The former is a -emitting cationic complex that is naturally taken up by the heart with use in myocardial perfusion procedures. The latter is an -emitting radium chloride salt able to treat patients suffering from prostate cancer that has spread to the bone.55–57 ............................. 12 Chart 1.2 DOTA is one of the most frequently used macrocyclic chelators in nuclear medicine but suffers from slow metal complexation kinetics, while DTPA is one of the oldest and most pervasive open-chain chelators used in nuclear medicine but has been made redundant by new chelators that form complexes with an improved in vivo stability.35,41,53,65–67 ............................. 17 Chart 2.1 Carboxylic acid (top) and amide derivatives (bottom) of frequently discussed ligands. Reproduced from Ref. 80 with permission from the Royal Society of Chemistry. ...................... 27 Chart 3.1 Chemical structures of the chelators frequently discussed in this work. Collectively, there is a range in denticity, functional groups, charge, and non-macrocyclic versus macrocyclic backbones allowing for comparative studies to aid in the chelator design discussions ................ 62 Chart 4.1 Several of the chelators used in initial 225Ac chelation studies ................................... 91 Chart 4.2 Contemporary chelators used for 225Ac chelation studies ........................................... 94 Chart 5.1 Chemical structures of py-macrodipa (left) and its bifunctional analogue (right), a suggested chelator system for future [203/212Pb]Pb2+ chelation studies ....................................... 119 xix List of Symbols and Abbreviations ° degrees ℃ degrees Celsius ~ approximate Å angstrom (10-10 metre)  alpha particle + positron - beta particle  gamma δ chemical shift in parts per million µ micro (10-6) 2D 2-dimensional AA atomic absorption BFC bifunctional chelator Bq Becquerel calcd calculated CN coordination number COSY correlation spectroscopy d day(s) or doublet (NMR) e- electron equiv equivalent(s) eV electron volts xx FDA United States Food and Drug Administration g gram(s) h hour(s) HPLC high performance liquid chromatography HR-ESI-MS high resolution electrospray ionization mass spectroscopy HSAB hard-soft acid-base HSQC heteronuclear single quantum correlation Hz hertz I ionic strength iTLC instant thin layer chromatography J coupling constant K Kelvin k kilo (103) KML formation constant (metal-ligand) L ligand or litre LET linear energy transfer LR-ESI-MS low resolution electrospray ionization mass spectroscopy log K log stability constant m/z mass to charge ratio MΩ cm–1 water resistivity (purity measure) M molarity (mol/L) or mega (106) m milli (10-3), metre(s) or multiplet (NMR) min minute(s) xxi mol moles n nano (10-9) NMR nuclear magnetic resonance ORTEP Oak Ridge Thermal-Ellipsoid Plot Program p pico (10-12) PET positron emission tomography ppm parts per million PSMA prostate specific membrane antigen q quartet (NMR) RCC radiochemical conversion RP reverse phase RPT radiopharmaceutical therapy RT room temperature s singlet (NMR) SPECT single photon emission computed tomography T temperature t triplet (NMR) t1/2 half-life TAT targeted alpha therapy TLC thin layer chromatography tR retention time (HPLC) UV-Vis ultraviolet-visible XRD X-ray diffraction xxii Glossary ampam 3,12-bis((6-carbamoylpyridin-2-yl)methyl)-6,9-dioxa-3,12-diazatetradecanediamide Bn benzyl crown 1,10-dioxa-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetraacetic acid DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DTPA 1,1,4,7,7-diethylenetriaminepentaacetic acid DTPAm diethylenetriamine-N,N’,N’,N’’,N’’-pentaacetamide EDTA ethylenediaminetetraacetic acid EGTA 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid EGTAm 3,12-bis(2-amino-2-oxoethyl)-6,9-dioxa-3,12-diazatetradecanediamide EtOAc ethyl acetate EtOH ethanol H2ampa 6,6'-(2,11-bis(2-amino-2-oxoethyl)-5,8-dioxa-2,11-diazadodecane-1,12-diyl)dipicolinic acid H2macropa 6,6'-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid H4noonpa 3,12-bis((6-carboxypyridin-2-yl)methyl)-6,9-dioxa-3,12-diazatetradecanedioic acid H4octapa 6,6’-((ethane-1,2-diylbis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid H4py4pa 6,6',6'',6'''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis(methylene))tetrapicolinic acid xxiii H5decapa 6,6’-(((((carboxymethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid HEHA 1,4,7,10,13,16-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid MeCN acetonitrile MeOH methanol Nosyl 2-nitrobenzenesulfonamide pH -log[H3O]+ pKa -logKa; protonation constant pM -log[metal] py-macrodipa 6,6'-(6,9,12-trioxa-3,15-diaza-1(2,6)-pyridinacyclohexadecaphane-3,15-diylbis(methylene))dipicolinic acid TATE Tyr3-octreotate TCMC 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane TEA triethylamine THF tetrahydrofuran TFA trifluoroacetic acid xxiv Acknowledgements I would like to thank… Dr. Chris Orvig and the group members (past and present) for their efforts and contributions towards my PhD. More specifically, I thank: Dr. Orvig for accepting me into his highly sought-after research group, for hosting incredible group activities, and for giving me the support I needed to reach the finish line; my inspiring mentor, Dr. Xiaozhu Wang, who was the ‘column’ that supported me, keeping the ‘fire’ of belief alight while I was learning new areas of chemistry; Dr. Thomas Kostelnik and Dr. Neha Choudhary for acting as my family away from home – Tom has been my main man in Vancouver, from backflips on trampolines to the high-spirited night outs and Neha provided much needed emotional support over the years and made for one heck of a housemate; Jeff Jang for his involvements in running a smoother operating lab; Alex Dimmling for his warm heart that greatly improved the sense of lab camaraderie; Tarek El Sayed for his hard work and fearlessness to learn new techniques; Dr. Lupe Jaraquemada, who caringly supported us all over the years; Luke Wharton for his scientific advice and collaborative efforts; Desiree Fiaccabrino for being one of my lunch and Loafe buddies; and finally, Lily Southcott for her group leadership qualities, research support, and for taking me on an unforgettable hike. Dr. Glenn Sammis, Dr. Jay Kizhakkedathu and Dr. Zachary Hudson who provided crucial encouragement and advice in my comprehensive exam and fourth year meeting. xxv Dr. Maria Ezhova for being an anchor of support for me at UBC. Every year was tough, but her willingness to help and unfailing professionalism supported me and inspired me while I grew as a scientist. Dr. David Bauer, Mo Talha Chaudhry, Dr. Duane Hean and Brooke McNeil David elated those around him, both inside and outside of the lab; Mo and Duane offered me help and advice purely out of kindness; and Brooke was a massive support producing lead for my projects and being a great friend down at TRIUMF. They are all exemplary researchers whose involvement in science make it a better discipline. my partner Katrina Reyes who enhances my sense of vitality. She has been there beside me from start to finish of this journey, with her creativity and natural tendency for fun bringing so much joy into my life. I am fortunate to be with someone so special. my mum (Dr. Susan Ingham) for creating a strong platform to help me over the hurdles of moving abroad and doing a PhD in North America. As someone who balanced an impressive work career with raising a family, goes into tough times head on, and makes time to care for others, my mum has been an excellent role model to me. She has inspired me to be hard working, to have grit when faced with challenges, but to also to live a life that feels fulfilling. I cannot really thank her enough. xxvi Dedication To my parents, for all their love and support. 1 Chapter 1: Introduction Cancer is a vastly varying disease that requires personalized medicine to remove or kill cancer cells while leaving the patient in as safe a condition as possible. Non-targeted treatments, such as chemotherapy, are routinely applied to kill cancer cells but they often subject patients to harsh side effects since they also target healthy cells.1–3 Alternatively, targeted treatments can be used but even these face a major challenge since tumours exhibit high levels of genetic and molecular heterogeneity.2,4 There are also selected cancer types that have poor survival rates. For example, lung cancer has a 5-year survival rate of 21% due to the large proportion of patients diagnosed with metastatic disease.5 Challenges such as these have led to a continuous need for research to either improve or establish new cancer therapies. In the first part of this chapter, a medical specialty known as nuclear medicine and its contributions to cancer treatments will be covered. After, the growing motivations for radiometal-based drug developments are discussed. The designs of such metalloradiopharmaceuticals are then examined with an emphasis on the role both coordination chemistry and medicinal inorganic chemistry play in their construction, including the metal complex evaluation tests necessary for metalloradiopharmaceutical development. Finally, a thesis overview is provided. 1.1 Nuclear Medicine and Radiopharmaceuticals Nuclear medicine involves the use of radioactive drugs, or radiopharmaceuticals, for diagnostic and treatment purposes. Radionuclides constitute the basis of these drugs with radiation being emitted from their nuclei during their decay. Those that decay to emit or indirectly produce penetrating photons may be applied in nuclear imaging modalities, while those that decay to release therapeutic particle emissions can be used as a form of (internal) radiation therapy. 2 1.1.1 Imaging Radiopharmaceutical imaging agents have the capability to visualize and measure the biochemical function of cells.6 These drugs are therefore hugely beneficial to cancer treatment strategies since molecular/cellular changes occur at an early stage of disease development. An early diagnosis allows for an appropriate treatment to be selected, guided, and optimized for improved patient survival rates.7–9 In contrast, most other cancer imaging techniques are more structurally informative than radiopharmaceutical imaging procedures but they are limited to diagnosing cancer when tumour growth is anatomically observable at a later stage of development, which often correlates to a more severe prognosis.6,9,10 The ability of radiopharmaceuticals to diagnose diseases lies in their specificity to localize only at the desired target, such as an organ or type of cell receptor. When the radiopharmaceutical decays, it produces photons that minimally interact with biological tissue and travel out of the body to reach external detectors. These detectors then translate absorbed radiation events into spatial representations of the drug biodistribution, highlighting the diseased state of the body. Consequently, it is important these drugs remain attached or incorporated into their target so that most decay events take place in that location, leading to a signal to noise ratio that will enable a successful diagnosis.11–13 The two main imaging modalities that apply this practice are single photon emission computed tomography (SPECT) and positron emission tomography (PET). The detectors used in SPECT and PET scans are highly sensitive to radiopharmaceutical emissions within the body, meaning extremely low concentrations can be administered (between the micromolar to picomolar 3 range).14 At such low concentrations, the toxic chemical effects of radiopharmaceuticals are minimal and they rarely show any measurable off-target pharmacological effects.13 Radiopharmaceuticals used as SPECT imaging agents contain a radionuclide that directly produces gamma () photon emission(s) during its electron capture (EC) or isomeric transition (IT) decay processes. Low energy -photons are necessary in SPECT imaging, which ideally fall within the range of 100-250 keV.15 When the energy is lower than 100 keV, the risk of photon absorption by the body or scattering in the crystal detector increases; while if it is higher than 250 keV, the probability of photons penetrating the collimator without being attenuated to a suitable energy for detection also increases (Figure 1.1).16 PET imaging involves radionuclides that decay to emit positron (+) particles. These particles are the antimatter counterpart to electrons and have an electric charge of +1. Upon emission, + particles travel a short distance until they undergo an annihilation reaction with an electron (e-) in the surrounding media to produce two  photons. These photons are produced with an energy of 511 keV due to the conservation of matter in the annihilation reaction and are emitted anti-parallel to one another. The photon signals are then measured “coincident” in time, 180 apart by a circular PET detector (Figure 1.1).17,18 4 Figure 1.1 SPECT (above) involves a collimator on its detector to manage a range of  energies, while PET (below) uses coincidence detectors to better measure antiparallel  photons that are produced because of the +-e- annihilation reaction. PET imaging exhibits a higher sensitivity than SPECT imaging. The use of a collimator in SPECT decreases the detector’s -photon measurement efficiency, while PET imaging does not involve a collimator as detection is based on measuring two photons in coincidence.13 According to various literature reports, PET also produces images with better spatial resolution compared to SPECT;14,17,18 however, it should be noted that the type of radionuclide used in PET affects the resolution. Radionuclides that decay to release high energy + particles result in lower imaging resolution, while radionuclides that release low energy + particles result in higher imaging resolution. This is because decay events in PET are recorded when an annihilation reaction has occurred a short distance away (usually a few millimetres) from the radionuclide, and the energy at which positrons are emitted affect the distance they travel until an annihilation reaction occurs.18 5 1.1.2 Radiation Therapies Once a cancer has been diagnosed, it needs to be treated. There are various forms of therapies available, such as surgery, chemotherapy, external beam radiotherapy, and radiopharmaceutical therapy (RPT). The best option to treat cancer will depend on the location, grade of tumour, stage of the disease, and the condition of the patient.19 When it comes to metastatic cancers, especially those that demonstrate resistance to chemotherapeutic drugs, RPT has tremendous potential to treat the widely disseminated disease in a targeted manner.3,20–24 The strategy of administering therapeutic radiopharmaceuticals is similar to their nuclear imaging analogues. For RPT to be effective, the radiopharmaceutical must locate at cancerous sites with a maximized residence time while having minimal interactions with healthy cells; however, unlike their diagnostic counterparts, the emissions of therapeutic nuclides entail particles that transfer a localised dose of radiation over short distances to induce cell death.3 It is important to note therapeutic nuclides undergoing a particle decay process may or may not produce -photon emissions, as it is common for a radionuclide to involve more than one decay process to form a stable nuclide (referred to as its branching ratio or “pathways”).20,25 These -photon emissions can be an advantage or a disadvantage depending on the emission energies. Those energies that fit into the acceptable ranges for detectors may enable the location of the drugs to be detected, while those that are outside this range augment irradiation to non-target tissue. There are three different types of particle emissions associated with RPT: alpha () particles, beta (-) particles, and Auger electrons (Figure 1.2). Alpha particles are helium nuclei (4He) that are predominantly emitted from the nuclei of heavy radionuclides (Z > 82);26 - particles are electrons that are emitted from the nuclei of “neutron rich” radionuclides; and Auger electrons 6 are low energy electrons ejected from the electron shells of daughter nuclides as they continue to decay to their most stable configuration.13 Each type of particle has a distinctive effect on biological tissue that is largely summarized by its characteristic kinetic energy and linear energy transfer (LET) values.27–29 The kinetic energy of a particle is the energy at which it is emitted from the radionuclide and the LET reflects the energy transferred from the particle to its surroundings per unit length. These two properties influence how far a particle can travel within the body, as well as its ability to damage cancer cells. Generally, when a particle of high LET radiation traverses through the nucleus of a cancer cell it irreparably breaks the double-stranded DNA within the nucleus, whereas a particle of low LET radiation mainly produces single-strand breaks and exhibits approximately 500 times lower cytotoxic potency.25,30 Figure 1.2 Simplified illustration of the three different types of particle-based radiotherapies highlighting how far the different types of particles travel in biological tissue, as well as their linear energy transfer ionising interactions with DNA. 7 Matching the LET and kinetic energy of the particle to the type of treatment or size of tumour is important. Alpha particles are emitted at higher energies than - particles (5-9 MeV versus 0.3-2.3 MeV) but their mass is much greater, meaning they have a LET value that is around 100-1000 times greater than - particles (60-230 keV/µm versus 0.1-1 keV/µm). As a result,  particles transverse a much shorter range in biological tissue than - particles (2-10 cell diameters versus 10-1000 cell diameters) (Figure 1.2).31–34 This short range enables greater selectivity since  therapeutics attached or internalized within a cluster of tumours will release a radiotoxic payload to the tumour cells while minimally irradiating off-target healthy cells. Alpha therapeutics are therefore of interest to treat micrometastatic diseases.35 In addition, studies have shown  particles demonstrate potential to destroy tumours presenting areas of hypoxia, a feature often associated with chemotherapy- and radiotherapy-resistant tumours.36 The disadvantage of  therapeutics is they may require internalization by cancer cells. Alpha particles are emitted with a large recoil energy that causes the radionuclide to dissociate away from the drug, which is concerning for those that decay to form radioactive daughter nuclides.37,38 In contrast, the lengthy range - particles travel have worse selectivity at treating small tumours but they have a much smaller recoil energy, which combined with their track length allows them to damage large, poorly vascularized tumours with less need for cellular internalization.34 Auger electron therapy is the least established form of RPT, largely due to the need for Auger electron emitters to be internalized by the nucleus of the cancer cell to exert a toxic effect. This internalization requirement comes from the low kinetic energies (~1 keV) at which Auger electrons are emitted from radionuclei, as well as their high LET values (~4-25 keV/µm).39 With this combination, Auger electrons travel very short distances (1-1000 nm) from the radionuclide, meaning they need to be in the cancer cell nucleus where they are in close proximity to the DNA.3 8 Although these short ranges have limited the development of Auger electron emitters, if able to be utilized effectively, Auger radiation is an appealing form of cancer treatment in which non-targeted cells experience minimal radiation.19,40 1.2 Non-Metallic Radionuclides Versus Radiometals In addition to radionuclide emission types (EC, IT, + decay, - decay, and  decay), the half-life and chemical properties of a radionuclide must be considered. Radionuclides require a half-life long enough for their incorporation into a drug and for target localization but not much longer than the time necessary to complete the procedure.16 Understanding the chemical properties of each radionuclide is also important since these influence the pharmacological properties and the construction of the radiopharmaceutical. Although there are several exceptions, radionuclides generally cannot be freely administered into the body due to undesired uptake by endogenous ligands, accumulation in off-target tissues/bone, or rapid excretion.3,41 These radionuclides therefore require attachment to the organic framework of a drug if they are to be safely administered for medical applications. Based on their chemical properties, non-metallic radionuclides and radiometals are split into two different groups. Non-metallic radionuclides compose a much smaller section of the periodic table and chart of nuclides compared to radiometals, and accordingly exhibit a narrower range of nuclear properties. The range of clinically relevant non-metallic radionuclides predominantly offers imaging agents (largely involving EC, IT, and + decay processes), and a sizeable portion have short half-lives (< 30 min) (Table 1.1).18,42–45 On the other hand, the radiometals comprise an array of diagnostic and therapeutic radiometals (collectively involving EC, IT, + decay, - decay, and  decay processes) with a greater range of half-lives (Table 9 1.2).16,46–52 The chemical behaviour of non-metallic nuclides presents an additional issue since their incorporation into a radiopharmaceutical often requires complex and lengthy synthetic procedures to form a new covalent bond, which is exacerbated by those with short half-lives.11,53 To overcome these drawbacks, much effort is being made towards the development of new metalloradiopharmaceuticals. 10 Table 1.1 Frequently considered non-metallic radionuclides.18,42–45 Radionuclide Main decay mode(s) Half-life Application 11C + 20.4 min PET 13N + 10.0 min PET 15O + 2.0 min PET 18F + 1.8 h PET 123I EC 13.2 h SPECT 131I -, IT 8.0 d - therapy 133Xe -, IT 5.2 d SPECT Table 1.2 Popular radiometals with medically valuable decay properties.16,46–52 Radionuclide Main decay mode(s) Half-life Application 44Sc + 4.0 h PET 47Sc - 3.4 d - therapy 68Ga + 67.8 min PET 89Zr + 3.3 d PET 90Y - 2.7 d - therapy 99mTc IT 6.0 h SPECT 111In EC 2.8 d SPECT 177Lu - 6.6 d - therapy 203Pb EC 2.2 d SPECT 213Bi , - 45.6 min  therapy 223Ra  11.4 d  therapy 225Ac  10.0 d  therapy 11 1.3 Metalloradiopharmaceuticals There are two categories of metalloradiopharmaceuticals: metal essential and metal tagged.54 The former category is distinguished by the radiometal ion having an essential role in directing the drug to its biological target. The latter category requires the use of a radioactively tagged designer molecule to transport the metal ion specifically to its intended target within the body. Illustrative examples of metal essential drugs include the imaging agent 99mTc-sestamibi and the therapeutic drug Xofigo (Chart 1.1). 99mTc-sestamibi is used for myocardial perfusion imaging due to its -emitting [99mTc]Tc+ centre, which is coordinated by six lipophilic methoxyisobutylisonitrile (MIBI) ligands for effective heart uptake.55 Even more simple in design, Xofigo is a [223Ra]Ra2+ -emitting chloride salt able to treat metastatic prostate cancer that has spread to the bone.56,57 While their simplicity is a strength, metal essential drugs are limited to target physiological processes associated with certain organs or bone and leave many types of cancers untreated. 12 Chart 1.1 Chemical structures of metal-essential radiopharmaceuticals: 99mTc-sestamibi and Xofigo. The former is a -emitting cationic complex that is naturally taken up by the heart with use in myocardial perfusion procedures. The latter is an -emitting radium chloride salt able to treat patients suffering from prostate cancer that has spread to the bone.55–57 To increase the targeting capabilities of radiopharmaceuticals, most metal tagged radiopharmaceuticals comprise four-components: a radiometal, a bifunctional chelator (BFC), a linker, and a targeting vector (Figure 1.3). The choice of radiometal determines the purpose of the radiopharmaceutical. The BFC sequesters the radiometal, keeping it hidden from competing bioligands and strapped to the drug when in the body. In addition, the BFC incorporates a point of derivatization to allow for attachment of a targeting vector via a linker. Ideally the linker bridges the metal complex to the targeting vector without impacting the coordination properties of the chelator and has pharmacokinetic properties that improve the drug. The targeting vector is the designer molecule that specifically transports the radiometal to the tumour. Accordingly, the targeting vector must exhibit a high affinity for only the cancer-associated target, such as over-expressed tumour cell surface receptors. 13 Figure 1.3 Generalized depiction of a four-component metalloradiopharmaceutical. To employ a radiometal as part of a successful four component radiopharmaceutical, the other components must be carefully matched to mutually complement one another. For example, the biological half-life of the targeting vector must be well matched with the physical half-life of the radiometal. Generally, antibodies take days to circulate the blood and accumulate at tumour sites so are better matched with radiometals of longer half-life, whereas peptides and small molecules more rapidly circulate around the body and accumulate at the site of disease within a time frame more comparable to those radiometals of a shorter decay half-life.15,19,25,53,58 The development of four-component radiopharmaceuticals is a highly interdisciplinary process. Radiochemists and nuclear physicists enhance their development by offering new methods to produce, purify and store radiometals; immunologists contribute by advancing targeting vector capabilities and by furthering our understanding on the mechanisms of cancer cells at the molecular level; and medicinal inorganic chemists play a crucial role in designing chelators that strap the radiometals to the organic framework of the drug for safe administration. This latter area in which the fields of nuclear medicine and medicinal inorganic chemistry overlap is the focus of this thesis. Bifunctional ChelatorTargeting VectorLinkageRadiometalThe four-component radiopharmaceutical approach6 14 1.4 Chelator Design and Radiometal Complexation The design of four-component radiopharmaceuticals is vast and there is immense potential to tune each of the components (except for the radiometal) for an improved in vivo performance. In addition, most of the drug can be constructed prior to being tagged with the radiometal, saving many half-lives of radioactivity. Prior to radiopharmaceutical formation, a chelator must first prove it coordinates the radiometal to form a thermodynamically stable and kinetically inert complex that prevents the metal dissociating from the drug when in the body (Figure 1.4). Figure 1.4 Chelators must first demonstrate they are compatible in binary with a radiometal (Phase 1) before four-component radiopharmaceuticals are developed (Phase 2). Each type of radiometal has different physical and chemical properties that should be addressed prior to designing a chelator to target the metal ion in question. Metal ions can often form various complexes, exhibiting a range of coordination numbers, geometries, and ionic radii. In addition, some types of metal ions prefer more ionic interactions, while other types of metal Developing a four-component radiopharmaceuticalinvolves two phasesPhase 1:Phase 2:7 15 ions prefer more covalent bonding interactions. In 1968, Pearson established a hard-soft acid-base (HSAB) theory,59,60 which is a convenient way to approximate which donor atoms and metal ions should be matched together. According to this theory, hard oxygen and nitrogen groups favour less polarizable, harder metal ions, whereas softer sulfur groups are better suited with more polarizable, softer metal ions. In most instances, chelator components for radiopharmaceuticals are designed to be polydentate structures that form a one-to-one complex with the radiometal. Polydentate chelators can be thought of as preorganized binding pockets or claws for the radiometal. The motivation to form a single, polydentate ligand is summarized by “the chelate effect”, which in a simplified sense demonstrates that there is a favourable entropic contribution when an n-dentate chelator coordinates to a metal to replace n unidentate ligands.61 The size and denticity of the chelator should match the metal ion size and coordination number (CN) ranges. Preferably, the metal complex will have no vacant coordination sites that encourage bioligands to bond to the metal, which may cause the complex to unravel. Also, the chelator should not have too many of its donor atoms uncoordinated to the metal, given these may lead to transchelation with biometals found within the body.62 Understanding the role of ligand scaffold architecture was advanced by Hancock and Martell in 1989, who highlighted the importance of steric strain in complex formation.63 When two donor atoms of a chelating ligand (ligands that are not unidentate) bond to a metal ion, the skeletal structure between the donor atoms is also part of the newly formed chelate ring. Through molecular mechanic calculations and empirical observations, larger metal ions have been demonstrated to most prefer five-membered chelate rings, while smaller metal ions generally have less of a preference between five- and six-membered chelate rings. At first, it seems more intuitive 16 to propose a larger chelator ring will be more suitable for a large metal ion; however, larger metal ions tend to form complexes with higher coordination numbers than smaller metal ions and as the coordination number increases, the donor-metal-donor bond angle within the complex becomes smaller. For example, an octahedral structure (CN 6) has bond angles of 90°, while a square-prismatic structure (CN 8) has bond angles of ~70°. Five-membered chelate rings form bond angles much more compatible with those bond angles favoured by large metal ions. The bond distances involved in five-membered chelate rings are also longer than six-membered chelate rings. Thus, five-membered chelate rings allow for more donor atoms to surround large metal ions with less strain and form more favourable complexes.61,63,64 Another decision regarding scaffold architecture is whether to design a macrocyclic chelator or an open-chain chelator. Traditionally, macrocyclic chelators with pendent donor groups were considered to offer a higher amount of preorganization compared to open-chain chelators and produce more thermodynamically stable and kinetically inert complexes.63 However, open-chain chelators were commonly found to have faster complexation kinetics over their macrocyclic counterparts.35 Slow complexation rates are particularly an issue for radiopharmaceuticals designed to incorporate heat-sensitive targeting vectors, such as antibodies, since high temperatures cannot be used to increase the rate at which the radiometal is taken up by the chelator without damaging the targeting vector. In addition, radiometals with short half-lives would be incompatible due to the slow uptake. These respective drawbacks are best illustrated by the archetypal chelators of nuclear medicine: macrocyclic DOTA and open-chain DTPA (Chart 1.2).35,41,53,65–67 Fortunately, chelator design has advanced to the stage where there is very little to distinguish new macrocyclic and open-chain chelators from one another in terms of complexation performance, which will be discussed and demonstrated in Chapter 4. 17 Chart 1.2 DOTA is one of the most frequently used macrocyclic chelators in nuclear medicine but suffers from slow metal complexation kinetics, while DTPA is one of the oldest and most pervasive open-chain chelators used in nuclear medicine but has been made redundant by new chelators that form complexes with an improved in vivo stability.35,41,53,65–67 1.5 Metal Complex Evaluation Once a chelator has been designed for a certain radiometal, it must be synthesized, and the metal complex evaluated. The fewer the number of synthetic steps, the better. Chelators are also usually designed to be symmetric for simpler characterization, and the product should be soluble in aqueous solution given it is to be administered into the body. After a chelator is synthesized, various screening experiments are carried out to determine its thermodynamic stability, radiolabeling ability, kinetic inertness, and in vivo performance. 1.5.1 Thermodynamic Stability Potentiometric and spectrophotometric titrations are commonly employed to obtain thermodynamic stability constant values of metal complexes. The overall equilibrium formation constants (log ) reveal the extent of the equilibrium between metal ions, protons, and chelating 18 ligands and are obtained by Equation 1.1, where M is the metal ion, H is the proton, L is the fully deprotonated ligand, and p, q and r are the stoichiometric numbers: 𝑝𝑀 + 𝑞𝐻 + 𝑟𝐿 ⇌ 𝑀𝑝𝐻𝑞𝐿𝑟  (𝑀𝑝𝐻𝑞𝐿𝑟) =[𝑀𝑝𝐻𝑞𝐿𝑟][𝑀]𝑝[𝐻]𝑞[𝐿]𝑟⁄ (1.1) In Equation 1.1, p can be 0 in the case of protonation equilibria (HqL), q can be negative when hydroxide species form (MpHq and MpHqLr), and q can be 0 in the case of deprotonated metal complex states (MpLr). In addition, stepwise equilibrium constants (log K) are calculated from the difference in log units between the accumulative constants (log ) of sequentially protonated (or hydroxide) species. The donor groups of a ligand can either coordinate to a metal ion or bond to a proton in solution, while hydroxides and ligands can coordinate to a metal ion. This means metal-ligand complexation is pH dependent since these species are in competition with one another (as shown in Equation 1.1). Accordingly, high log K values of metal-ligand systems correspond to a ligand that has a high drive for complex formation (low concentrations of M and L; high concentration of ML). Within the context of radiopharmaceuticals, an additional thermodynamic parameter is often determined for metal complexes: the pM value (pM = -log[M]).68 These values are linked to the stability constant but account for variables and conditions such as metal ion hydrolysis, pH, and stoichiometry. The pM therefore more accurately quantifies complex formation under physiologically relevant conditions ([L] = 10 µM, [M] = 1 µM, pH 7.4).68–70 Similarly, higher pM 19 values are indicative of more favourable metal complexation by a chelating ligand as these values are obtained when uncoordinated metal concentrations are low. It should be noted, while thermodynamic parameters provide meaningful insight into metal complex stability, they are lower on the hierarchy of evaluation tests when compared to kinetic inertness and in vivo performance. In other words, if one chelator formed a thermodynamically stable metal complex but was kinetically labile, while a different chelator formed a less stable complex with the same metal ion but was kinetically inert, the latter chelator would have a higher chance of being put forward for the second phase of radiopharmaceutical development. 1.5.2 Radiolabeling Ability Radiolabeling experiments are important to determine the binding affinity of a chelator. An optimal chelator will bind a radiometal at low concentrations (~10-5-10-7 M) in quantitative yields, under mild temperature conditions (25-37 ℃) and in short mixing times (~5-15 min). These conditions are less essential for radiometals with relatively long half-lives and targeting vectors exhibiting a robustness to heat; however, they are the most convenient and a chelator demonstrating these radiolabeling abilities has more opportunities to be employed as the chelator component of metalloradiopharmaceuticals. Caution is advised when referring to radiolabeling abilities of chelators since in many instances, the yield or conversion of the radiolabeling procedure is referenced at the lowest chelator concentration; however, the amount of radioactivity and the total reaction volume must be taken into consideration. A 10-5 M chelator solution one millilitre in volume will contain more moles of chelator than a 10-5 M chelator solution one microlitre in volume. A solution of low radioactivity will also be easier to quantitatively radiolabel than a solution of high radioactivity. Thus, to refer 20 to the affinity of a chelator for a radiometal more accurately, a molar activity (radioactivity per mole) may be calculated. Unfortunately, even molar activities must be compared with caution if the radiolabeling experiments are conducted across different production procedures or different production runs. Each production procedure may have variation in the radiochemical purity, and radiolabeling experiments carried out at different times will have different amounts of contamination. Molar activities are therefore used more as a guide to describe the affinity of a chelator for a radiometal, and their validity is increased if a chelator achieves a consistent radiolabeling performance/molar activity each time the radiometal is produced. 1.5.3 Kinetic Inertness and In Vivo Performance Once the affinity of a chelator for a radiometal has been determined, various in vitro competition experiments are usually applied to probe the degree and rate at which extraneous factors induce metal complex dissociation. For example, acid dissociation experiments specifically measure the relative kinetic inertness of a metal complex under acidic conditions (pH < 2) and measure whether the donor groups of the chelator prefer to coordinate with the metal or with the high concentrations of protons.65,71 These experiments are not commonly performed because low pH environments are rarely encountered within the blood and most organs; however, acid catalyzed dissociation has been noted to be an issue once a radiopharmaceutical has been internalized by target cells.72 Given the radiopharmaceutical has to be transported around the body via the blood, one of the most pertinent in vitro studies involves incubating the metal complex in blood serum to test whether radiometal transmetallation occurs by native serum proteins.73–77 Depending on the radiometal complex, the kinetic inertness can be tested with more specific mixtures, such as apo-transferrin and hydroxyapatite (bone).74,78 21 While all these former evaluation tests are useful, in vivo studies are at the top of the hierarchy and will ultimately determine whether a chelator is suitable for radiopharmaceutical applications. A radiometal complex (with no targeting vector) can be injected into animals, most commonly mice, for pre-clinical biodistribution studies.73,76,78 The clearance and uptake profiles of tissue, organs and bone are assessed to evaluate whether the radiometal dissociates from the chelator. Metal complexes are cleared largely based on their polarity and charge, and highly polar complexes often have rapid clearance making it difficult to assess whether it would remain a complex upon prolonged exposure.79 In addition, highly lipophilic radiometal complexes may accumulate in tissue and organs such as the liver, which would make it hard to differentiate between dissociated radiometal and radiometal complex.76 Thus, the most reliable in vivo studies involve injecting the four-component radiopharmaceutical itself. As long as the targeting vector has been established to have a high affinity for its target, the radiometal complex will be maintained in the body for longer, and any off-target radiometal accumulation in the body can more reliably be linked to inadequate in vivo behaviour of the metal complex. 1.6 Thesis Overview The research presented in this thesis focuses on the ability of several multidentate (octadentate and decadentate) chelators to coordinate large radiometals ([nat/203Pb]Pb2+, [nat/213Bi]Bi3+, [225Ac]Ac3+/natLa3+), and examines whether the chelators are suitable to move towards bifunctional development so that the metal complexes can be tested in vivo. Many chelator design aspects are covered, such as denticity, scaffold size, and so forth; however, each research chapter (Chapters 2-4) is based on a particular chelator design lesson. The thesis ends with a conclusion and further works section (Chapter 5), as well as references and appendices. 22 Chapter 2, An Evaluation of Open-Chain Amide Chelators Designed for [203/212Pb]Pb2+ Ions, supports the idea that Pb2+ ion chelation is complicated by its lone pair of electrons. The chapter involves two new amide chelators (EGTAm and ampam), which were designed and synthesized for Pb2+ ion chelation. The structure and stability of the respective metal complexes were evaluated by 1H NMR spectroscopy, X-ray diffraction, and potentiometric titrations. The affinity of EGTAm and ampam for [203Pb]Pb2+ ions were also explored in respective radiolabeling studies. Chapter 3, H2ampa: a Versatile Chelator Capable of Binding [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+ Ion, is based on a diprotic decadentate chelator (H2ampa), which was designed to target three chemically different radiometal ions. The versatile ability of H2ampa to coordinate these metal ions were demonstrated via NMR spectroscopy, X-ray diffraction, concentration-dependent radiolabeling studies, and thermodynamic solution studies. Chapter 4, Impact of Charge on [225Ac]Ac3+ Radiolabeling Studies, details how metal complex charge affects radiochemistry methodologies. Within this chapter, a tetraprotic chelator (H4noonpa) forms a monoanionic complex with [225Ac]Ac3+ ions ([225Ac][Ac(noonpa)]-), while a pentaprotic chelator (H5decapa) forms a dianionic complex ([225Ac][Ac(decapa)]2-). The monoanionic complex was able to be studied via TLC techniques, whereas the dianionic complex required Chelex-resin columns and a gamma spectrometer. This unconsidered impact of complex charge became apparent during concentration-dependent radiolabeling and human serum stability studies. Chapter 5, Conclusion and Future Work, summarizes the lessons offered from each research chapter and suggests further work based on the findings, including a proposed bifunctional route for one of the chelators. 23 Chapter 2: An Evaluation of Open-Chain Amide Chelators Designed for [203/212Pb]Pb2+ Ions Chapter 2 is an adaptation on the paper originally titled “Getting a lead on Pb2+ - Amide Chelators for 203/212Pb Radiopharmaceuticals” by A. Ingham, T. Kostelnik, B. L. McNeil, B. O. Patrick, N. Choudhary, M. de G. Jaraquemada-Peláez, and C. Orvig.80 It has been adapted to focus on my contributions to the paper with the other co-first author contributions (by T. Kostelnik) having either been removed or referenced. 2.1 Introduction 2.1.1 Lead (Pb) in Medicine Developing drugs based on Pb2+ ions may at first seem counterintuitive. As an element, lead is infamously poisonous with blood concentration levels of 10 µg/dL (or above) being a concern for most adults.81 However, radiopharmaceuticals typically achieve their function within the body at micromolar to picomolar concentrations and the chemical toxicity threshold of lead may be avoided toward the lower end of this concentration range.6,14 As a result, radioactive Pb2+ ions are still frequently considered for the basis of new radiopharmaceuticals, with their development being particularly boosted by the pair of radioisotopes 212Pb (t1/2 = 10.6 h) and 203Pb (t1/2 = 51.9 h).51,82 Lead-212 and 203Pb are referred to as a ‘theranostic’ pair of radioisotopes. Lead-212 is a neutron rich radionuclide that involves multiple therapeutic particles (two - particles and one  particle) being emitted until stable 208Pb is formed at the end of its decay chain; 203Pb is a neutron deficient radionuclide that decays via electron capture to form excited 203Tl, which emits diagnostic γ-rays (279 keV; 81%) suitable for SPECT imaging as it decays to its ground state.83,84 A chemically identical theranostic pair of radioisotopes comprising a source of  particles and an 24 imaging agent with moderate half-lives is highly desired for cancer treatments but is rare.85 For example, 225Ac (t1/2 = 9.9 d) is a promising  therapeutic but no actinium isotope exists that is suitable for diagnostic purposes.86,87 Other illustrative examples are 211At (t1/2 = 7.2 h), 223/224Ra (t1/2 = 11.4 d and 3.6 d, respectively), and 227Th (t1/2 = 18.7 d); all are promising  emitters but none have a chemically identical diagnostic radioisotope.46,47,88,89 At the clinical level, to accurately monitor the progress of cancer treatments while keeping radiation dose exposure at a minimum, several  therapeutics require switching to a chemically different diagnostic radiometal.27,85 This is less preferable because changing the radiometal changes the chemical properties of the radiopharmaceutical. Not only is a versatile chelator component required to coordinate two chemically different radiometals, but the pharmacokinetics of the drug are likely to change. Radiopharmaceuticals based on [203/212Pb]Pb2+ ions avoid these issues since the metal complexes formed will have the same chemical properties. Use of [203/212Pb]Pb2+ radiopharmaceuticals have therefore been explored in various cancers treatments, such as prostate cancer and melanoma.90–92 2.1.2 Lone Pair Activity Numerous design aspects must be considered when targeting Pb2+ ions. As a group 14, post-transition metal, Pb2+ ions form a wide range of complexes with coordination numbers ranging from 2-10, and they have a 6s2 lone pair of electrons that are either stereochemically active or inactive.93,94 The lone pair orbital is said to be active when it has a degree of 6p orbital character and the complex formed has directionality.95 Such complexes are aptly referred to as being “hemidirected” since the chelators predominantly bind to one hemisphere of the Pb2+ ion with the other hemisphere being occupied by the active lone pair. When the lone pair orbital has little to no 25 6p character, it is said to be inactive and more spherical geometries form.96 These complexes are described as “holodirected” because the chelator encompasses both hemispheres of the metal ion. The degree of lone pair orbital hybridization can be approximated using computational techniques. Such calculations have shown p character contributions ranged from 2.6-15.7% when investigating different four-coordinate hemidirected complexes with anionic ligands.94 Accordingly, lone pair activity is more evident in certain Pb2+ complex geometries than others, and viewing lone pair activity as a scale may become more common when analyzing Pb2+ complex directionality.95 Shimoni-Livy et al. have summarized a list of factors that favour hemidirected or holodirected arrangements of chelators around a Pb2+ ion, including ligand denticity and the HSAB properties of the ligand donor groups.59,60,94,95 Spatially, chelators with low denticities (less than six) usually form hemidirected complexes, while chelators with high denticities (greater than eight) tend to form holodirected complexes. Chelators that are hexadentate to octadentate are in the intermediate range, and lone pair activation is more likely to be determined by other ligand properties within this range.96 Alternatively, Pb2+ complexes formed in this intermediate range may compromise to form a geometry on the frontier.95 2.1.3 Amide Chelators Currently, the gold standard chelator used for [203/212Pb]Pb2+ radiopharmaceutical applications is octadentate TCMC (Chart 2.1).41 TCMC contains the macrocyclic cyclen backbone and four pendant amide groups. It is therefore an amide derivative of carboxylate based DOTA and is alternatively referred to as DOTAm. Compared to DOTA, the softer amide donor groups of TCMC have shown a great improvement in their Pb2+ ion chelation abilities with the chelator 26 radiolabeling [203/212Pb]Pb2+ ions more effectively and forming a more kinetically inert complex that can withstand acidic pH environments.72,97 Whether [Pb(TCMC)]2+ is hemidirected or holodirected has caused much debate in the literature since the macrocyclic backbone makes it difficult to determine whether the void is due to an active lone pair or a geometric constraint.98,99 More recently, Moncomble et al. extensively investigated the geometry of [Pb(TCMC)]2+ using computational techniques and a frontier geometry was suggested with neither a fully activated or deactivated lone pair confirmed.95 A non-macrocyclic chelator known as DTPAm, an amide derivative of DTPA, has also been investigated for its [nat/203Pb]Pb2+ coordination properites.80 This octadentate chelator was first published by Burdinski et al. for Yb3+-based MRI contrast agents investigations;100 however, Kostelnik and co-authors more recently resynthesized DTPAm for novel [nat/203Pb]Pb2+ studies.80 In its [nat/203Pb]Pb2+ studies, DTPAm was found to form a hemidirected Pb2+ complex and demonstrated promising radiolabeling abilities, although still less effective than TCMC.52,80 Inspired by TCMC and DTPAm, two new amide chelators were designed. One of the chelators is an amide derivative of EGTA and is known as EGTAm (Chart 2.1), while the other chelator is a hybrid between an amido-version of H4octapa and EGTAm and is referred to as ampam (Chart 2.1). These two chelators are based on the same large acyclic backbone since Pb2+ ions have particularly large ionic radii in their eight- and ten-coordinate complexes (~1.3 Å and ~1.4 Å, respectively).101,102 27 Chart 2.1 Carboxylic acid (top) and amide derivatives (bottom) of frequently discussed ligands. Reproduced from Ref. 80 with permission from the Royal Society of Chemistry. The research presented in this chapter focuses on the suitability of EGTAm and ampam as chelates for [203/212Pb][Pb2+ radiopharmaceuticals. Reported herein are the syntheses of EGTAm and ampam; their metal complexation studies with Pb2+ ions involving both 1H nuclear magnetic resonance (NMR) spectroscopy and crystal structure analysis; radiolabeling studies between the chelators and [203Pb]Pb2+ ions; and finally, potentiometric titrations of the chelators and their Pb2+ complexes. 2.2 Results and Discussion 2.2.1 Ligand Synthesis EGTAm (2) was synthesized in an overall yield of 79% using a simple two step derivatization strategy involving its commercially available, carboxylic acid analogue EGTA (Scheme 2.1). First, the carboxylic acids of EGTA were converted to ethyl ester groups via Fischer esterification, forming compound 1. The esters of 1 were then converted into primary amides using NH3 (7 N) in methanol to form EGTAm (2). The amidation reaction required stirring times as long 28 as 14 days for complete product formation; however, the leaving groups and solvent were simply removed under vacuum conditions and pure ligand (2) was obtained in a quantitative yield. Scheme 2.1 Synthetic route to afford EGTAm (2) with an overall yield of 79%. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. The synthesis of ampam (Scheme 2.2) was more demanding due to its unique scaffold, which could not be directly derivatized from any commercially available chelators. Overall, ampam (9) was synthesized in five steps to afford a cumulative yield of 12%. The approach involved adding functional groups onto the commercially available 2,2’-(ethane-1,2-diylbis(oxy))bis(ethan-1-amine) backbone, also referred to as the “ether-amino” or “NOON” backbone within this document. 29 Scheme 2.2 Synthetic route to afford ampam (9) with an overall yield of 12%. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. Before adding functional groups onto the NOON backbone, the methyl(6-bromomethyl) picolinate arm (4) was synthesized in two steps following published procedures but with minor adaptations.62 2,6-Dimethylpyridinecarboxylate is asymmetrically reduced with NaBH4 to form compound 3; the hydroxyl group of 3 is then brominated with PBr3 to produce the desired picolinate arm (4). After, the five-step synthesis of ampam proceeded as follows. First, 2-nitrobenzenesulphonyl (nosyl) groups were used to protect each of the primary amines of the NOON backbone, generating compound 5. The secondary amines of 5 were then alkylated with 4 30 via SN2 reactions, thereby forming compound 6. After, both nosyl protecting groups were removed using thiophenol to reform the secondary amines and produce compound 7. In the penultimate step, the amines of 7 were alkylated with 2-bromoacetamide via SN2 reactions to form compound 8. Finally, the methyl ester groups on the picolinic arms were converted into primary amides using NH3 (7 N) in methanol and the final product (ampam, 9) was formed. Overall, the five-step synthesis of ampam was convenient in that every product formed (5 to 9) had an aromatic ‘arm’ (nosyl and/or pyridine functional groups). These aromatic arms meant all the compounds were UV-active, making their product formation easy to identify via thin layer chromatography (TLC). Additionally, the products formed did not require storage under an inert atmosphere to prevent decomposition. Individually, 5 and 6 were generated in high yields (> 80%), while 7, 8, and 9 had more moderate yields (~50-60%). The lower yield of 7 is due to the liquid-liquid extractions applied to remove the K2CO3 base. Its secondary amines had a high affinity for the aqueous layer resulting a sizeable portion of 7 not being extracted despite multiple organic washes. Filtration was also not a suitable alternative since 7 would stick to the solid inorganic salts, resulting in even lower yields. Compound 8 was formed in a moderate yield because the two secondary amines of 7 did not react quantitatively with 2-bromoacetamide, even when in excess (> 2 equivalents). A mixture of 8 and a singly added acetamide arm product formed, making purification via silica or alumina column chromatography difficult since these two products had a similar affinity for the solid phase(s). Finally, 9 was formed in quantitative yield during its amidation reaction but a large amount of product (~39%) was lost during its HPLC purification. This purification process involved injecting 15-25 mg/mL solutions per injection due to the loading capacity of the semi-prep C-18 column; there was also a degree of human error with product peak collection being done manually. 31 2.2.2 Metal Complexation Complexation of Pb2+ ions by EGTAm and ampam was studied by 1H NMR spectroscopy to gain structural insights into their Pb2+ ion coordination abilities. Given radiopharmaceuticals will be administered at physiological pH (7.4), the free ligands and their respective metal complexes were measured close to this value at pD 6-7 (with pD = pHmeasured + 0.4). The spectra obtained for [Pb(EGTAm)]2+ and [Pb(ampam)]2+ showed few indicators of complex formation (Figure 2.1). No diastereotopic splitting patterns were observed, and broad signals were produced in the 1H NMR spectrum of [Pb(ampam)]2+; however, complexation can be confirmed in both instances by downfield shifts of the signals relative to the free ligand. The most notable downfield shifts are from the methylene groups adjacent to the coordinating amides [N-(CH2)-CONH2], which shift to overlap with the methylene groups between the ethers [O-(CH2)-(CH2)-O]. Broad peaks and a lack of signal splitting are indicative of fluxional coordination on the NMR timescale. The methylenic hydrogens adjacent to each donor group also appear to remain in equivalent environments upon complexation. Due to the simplicity of the metal complex spectra, it is not possible to determine whether the donor groups are dissociating and re-coordinating rapidly for these signals to average out or whether symmetric complexes have formed with the chelator demonstrating fluxional behaviour while bound to the metal centre. In the former case, the ligands would not be suitable for radiopharmaceutical purposes given the complex must be kinetically inert; however, more comprehensive studies are required to determine the suitability of these chelators since broad 1H NMR peaks are a common feature of Pb2+ complexes.103,104 32 Figure 2.1 A) 1H NMR spectra of EGTAm (top) and [Pb(EGTAm)]2+ (bottom) (300 MHz, 298 K, D2O, pD 6-7); B) 1H NMR spectra of ampam (top) and [Pb(ampam)]2+ (bottom) (300 MHz, 298 K, D2O, pD 6-7). Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. 33 2.2.3 X-ray Crystal Structures X-ray quality crystals of both metal complexes were obtained. Single colourless needle crystals of [Pb(EGTAm)(NO3)]NO3 were obtained through a two-vial vapour diffusion technique, in which acetone slowly diffused into an aqueous solution containing the Pb2+ complex, while irregular crystals of [Pb(ampam)](NO3)2 were isolated by slow evaporation of an aqueous solution at ambient temperature. EGTAm coordinates Pb2+ with all eight donor atoms but the structure also contains a bidentate NO3- group bound to the metal centre resulting in a ten-coordinate complex with a distorted bicapped square antiprismatic geometry (Figure 2.2a). The two backbone amines (N2 and N5) form the distorted bicapped axis (N2-Pb-N5 = 172) and the remaining eight O atoms are split into two planes (O4-O6-O5-O7 and O1-O3-O2-O8) with the chelator encompassing both hemispheres of the Pb2+ to form a holodirected geometry. In addition, the asymmetric structure formed misaligns with the symmetry observed in the 1H NMR spectrum obtained for this complex. This disconnect is not uncommon and can be explained by the crystal structure of [Pb(EGTAm)(NO3)]+ providing a fixed view of ligand coordination, while the 1H NMR spectrum observes the dynamic behaviour of the metal-ligand complex in solution. Decadentate ampam coordinates the Pb2+ ion to form a symmetric binary ten-coordinate complex with a distorted bicapped square antiprism geometry (Figure 2.2b). Again, the ether groups strap to the Pb2+ ion extending the two backbone amines (N2 and N2’) to coordinate the metal ion from opposite directions along a distorted bicapped axis (N2-Pb-N5 = 168), with the remaining atoms (N2O6) split into two planes (N3-O1-O2’-O3 and N3’-O1’-O2-O3’). The structure formed is holodirected, which was expected given the high denticity of ampam. There is also a two-fold 34 rotation (C2) axis right in the middle of the ether atoms (O3 and O3’, Figure 2.2b), which is consistent with the symmetry observed in the solution state 1H NMR spectrum. A structure of [Pb(DTPAm)](NO3)2, (obtained and solved by T. Kostelnik and N. Choudhary, respectively),80 has been included to provide an illustration of a hemidirected geometry (Figure 2.2c). The diethylene triamine backbone of DTPAm coordinates to one hemisphere of the Pb2+ ion and the amide arms form an eight-coordinate complex with a distorted square antiprism geometry. The complex is hemidirected as evinced by a void in the coordination sphere that is characteristic of lone pair stereochemical activity. By comparing EGTAm, ampam and DTPAm, the size of the backbone scaffold seems to have a large impact on deciding whether these chelators predominantly coordinate from one hemisphere or encompass both hemispheres. Figure 2.2 ORTEP-style diagrams of A) [Pb(EGTAm)(NO3)](NO3), B) [Pb(ampam)](NO3)2, C) [Pb(DTPAm)](NO3)2; for clarity, ellipsoids are drawn at 50% probability, the Pb2+ ion at 80%, and hydrogen atoms are omitted except on amide groups. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. A BC 35 Table 2.1 Bond lengths of each Pb2+ complex formed from Pb(NO3)2. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. [Pb(EGTAm)(NO3)]NO3 Bond length to Pb2+ (Å) [Pb(ampam)](NO3)2 Bond length to Pb2+(Å) [Pb(DTPAm)](NO3)2 Bond length to Pb2+ (Å) O1Amide 2.559(3) O1/O1’Amide 2.734(2) O1Amide 2.727(2) O2Amide 2.772(3) O2/O2’Amide 2.700(2) O2Amide 2.613(2) O5Amide 2.611(3) O3/O3’Ether 2.673(2) O3Amide 2.578(2) O6Amide 2.697(3) N2/N2’Amine 2.803(3) O4Amide 2.568(2) O3Ether 2.726(3) N3/N3’Pyr 2.763(3) O5Amide 2.921(2) O4Ether 2.803(3) N2Amine 2.696(2) N2Amine 2.754(4) N4Amine 2.664(2) N5Amine 2.792(4) N6Amine 2.836(2) O7Nitrate 2.809(3) O8 Nitrate 2.818(3) OAve 2.72 OAve 2.70 OAve 2.68 NAve 2.77 NAve 2.78 NAve 2.73 Despite the three open-chain amido chelators forming diverse geometries with Pb2+ ions, they present an overarching trend; average Pb-O bond lengths are shorter than Pb-N bond lengths (Table 2.1). Interestingly, the literature shows a number of cyclen-based macrocycle chelators that coordinate to Pb2+ ions with their Pb-Oave bond lengths being significantly longer than their Pb-Nave bond lengths.104–107 This includes TCMC and DOTA. [Pb(TCMC)]2+ has Pb-Oave and Pb-Nave bond lengths of 2.75 Å and 2.63 Å, respectively; while [Pb(DOTA)]2- has Pb-Oave and Pb-Nave bond lengths of 2.77 Å and 2.66 Å, respectively.105,106 Even with their difference in chemical hardness, TCMC and DOTA both form a saturated eight-coordinate sphere around the Pb2+ ion, with similar geometries and their Pb-O and Pb-N bond lengths following the same trend. This 36 further demonstrates the importance of the ligand architecture since one of the main factors determining these bonding distances is the geometric constraint of the macrocycle. Overall, the crystallography results show EGTAm may be unsuitable for 203/212Pb radiopharmaceuticals while ampam holds more promise. The denticity of EGTAm is inadequate to completely encapsulate the Pb2+ ion as evidenced by NO3- coordination. An identifiable vacant coordination site on the metal centre is of concern for radiopharmaceutical applications due to the high competition of biological ligands that may also coordinate to the Pb2+ ion. As a decadentate chelator, ampam fully encapsulates the Pb2+ ion demonstrating chelators involving the NOON backbone may benefit from an increased denticity compared to octadentate EGTAm. 2.2.4 Radiolabeling with [203Pb]Pb2+ Both EGTAm and ampam were found to radiolabel [203Pb]Pb2+ (45-50 kBq) and achieved quantitative radiochemical conversions108 (RCC > 99%) at 10-3 M ligand concentration (Figure 2.3). The radiolabeling reactions were carried out under mild temperature conditions (37 ℃) and relatively short time periods (30 min) in MES buffer (0.5 M, pH 5.5-6). The molar activities achieved were low at ~0.25 MBq/µmol. EGTAM and ampam therefore demonstrated an affinity for [203Pb]Pb2+ ions inferior to DTPAm, with the latter achieving a molar activity up to 3.5 MBq/µmol.80 These molar activities are even lower when compared to p-SCN-Bn-TCMC, which obtained values around 50 MBq/µmol.52 The molar activities referenced for DTPAm and p-SCN-Bn-TCMC involved [203Pb]Pb2+ produced and purified from irradiated thallium targets at TRIUMF using procedures established by McNeil et al.52,80 As discussed in Chapter 1.5.2, comparing molar activities should be done with caution since contamination levels can vary from one production to another. To make molar 37 activity comparisons truly valid, radiolabeling studies must be done with the same production run. Although radiolabeling studies with DTPAm, p-SCN-Bn-TCMC, and my two chelators were not carried out in the same production run, EGTAm and ampam radiolabeling studies were conducted using the same published [203Pb]Pb2+ production and purification procedure.52 In addition, the radiolabeling experiments with EGTAm and ampam were carried out in parallel over several [203Pb]Pb2+ production trials. With the total reaction volumes (100-200 µL) being comparable to those used for p-SCN-Bn-TCMC (100 µL), and the [203Pb]Pb2+ activity being around 20-100 kBq per sample, EGTAm and ampam consistently radiolabeled [203Pb]Pb2+ at 10-3 M but not at more dilute concentrations. HPLC conditions were required to separate solvated [203Pb]Pb2+ ions from the [203Pb][Pb(EGTAm)]2+ and [203Pb][Pb(ampam)]2+ complexes formed in their respective radiolabeling studies (Figure 2.3). An analytical column was used with a slow flow rate and a polar gradient. Under these conditions, [203Pb][Pb(EGTAm)2+ had a retention time (tR) ~ 4.0 min and [203Pb]Pb2+ had a tR ~ 3.2 min. While these times are close, identification was further helped by the complex product having a sharper peak compared to the solvated radiometal peak. At ligand concentrations lower than 10-3 M, the retention time of the peak changed to match that of the control and exhibited slight broadening indicative of no complexation. As a result, the ability of EGTAm to uptake [203Pb]Pb2+ ions for radiopharmaceutical application was deemed ineffective. During the ampam radiolabeling studies, [203Pb][Pb(ampam)]2+ eluted with a very broad peak showing the complex species streaks on the column. It is possible this peak is a one to one chelator-metal complex since broad HPLC peaks have been observed in other [203Pb]Pb2+ radiolabeling studies, such as those conducted by McNeil, et al.52 However, it is suspected a dimeric or polymeric metal-ligand species forms with incomplete coordination of donor groups 38 affecting the C18 column interactions. Regardless of the product species formed, at lower ligand concentrations (10-4 and 10-5 M) the broad peak is not observed and the retention time matches that of solvated [203Pb]Pb2+ ions, meaning ampam was also unsuitable for 203/212Pb radiopharmaceuticals. Figure 2.3 HPLC traces: A = free [203Pb]Pb2+, B = 10-3 mol dm-3 [203Pb][Pb(EGTAm)]2+ and C = 10-3 mol dm-3 [203Pb][Pb(ampam)]2+. Analytical column used; flow rate = 1 mL/min; gradient: A = H2O (0.1% TFA), B = MeCN (0.1% TFA), A = 95%, 0-2 min, A = 95-80%, 2-18 min. [203Pb]Pb2+ elutes at tR ~ 3.2 min, [203Pb][Pb(EGTAm)]2+ elutes at tR ~ 4.0 min, and [203Pb][Pb(ampam)]2+ elutes at tR ~ 4.9 min with a peak that streaks. Reproduced from Ref. 80 (ESI section) with permission from the Royal Society of Chemistry. CBA [203Pb]Pb2+(control)[203Pb][Pb(EGTAm)]2+(10-3 M)[203Pb][Pb(ampam)]2+(10-3 M) 39 Comparing the [203Pb]Pb2+ radiolabeling results amongst p-SCN-Bn-TCMC, DTPAm, EGTAm, and ampam provides a clear preliminary assessment of their suitability for [203/212Pb]Pb2+ radiopharmaceuticals, especially given they range over two orders of magnitude. The high molar activities produced with p-SCN-Bn-TCMC can largely be explained by the preorganized macrocyclic backbone of TCMC coordinating Pb2+ ions in a cage-like fashion;95,99 however, understanding the higher molar activity achieved by DTPAm compared to EGTAm and ampam, requires a more careful examination. They are all open-chain chelators with relatively minor variations in terms of their denticity, basicity, and hardness of their functional groups, meaning a straightforward comparison of their donor sets is insufficient to explain the radiolabeling results. Since the crystal structures of [Pb(EGTAm)(NO3)]+ and [Pb(ampam)]2+ revealed holodirected complexes, while [Pb(DTPAm)]2+ formed a hemidirected complex, a comparison of their radiolabeling results indicates the nitrogen and oxygen rich donor sets of these chelators were more compatible when the complex formed was hemidirected over holodirected. This outcome aligns with the findings of Shimoni-Livy et al., who propose hard donor (nitrogen and oxygen) atoms favour hemidirected complexation while soft donor (halogen) atoms encourage holodirected complexation.94 Accordingly, the differences in affinities for [203Pb]Pb2+ ions exhibited by these chelators is explained by how well the scaffolds sterically accommodate the bonding preferences of their hard donor groups upon complexation. The large NOON backbone of EGTAm (N2O6) and ampam (N4O6) sterically dominates to force holodirected complexation even though the hard donor atoms energetically favour hemidirected complexation, and this mismatch likely resulted in a low affinity for [203Pb]Pb2+ ions.96,109 In contrast, the smaller diethylene triamine scaffold of DTPAm (N3O5) coordinates the Pb2+ ion to form a hemidirected complex that energetically aligns with its hard donor groups; hence its higher affinity for [203Pb]Pb2+ ions. 40 2.2.5 Solution Thermodynamics Potentiometric titrations were first conducted with EGTAm and ampam to determine their protonation constants. After, titrations were carried out to obtain stability constants of their respective Pb2+ complexes. The stability constants quantify the thermodynamic stability of the metal complexes, and all the titrations were studied over a broad pH range (1.8–11) to provide much insight into their speciation formations. Two pKa values were calculated for H2EGTAm2+, pKa = 4.32(1), 3.53(1), which were attributed to the amine groups, and three deprotonation events were noted in the titration of H3ampam3+, pKa = 5.61(1), 5.05(1), 2.44(2), corresponding to the backbone amines and the pyridine ring of a picolinamide group (Table 2.2). The backbone amines are therefore more basic when two of the acetamide groups have been replaced by picolinamide groups, although the overall pKa values for both chelators indicate they have relatively low basicity. In general, the overall basicity of polydentate amide chelators tends to be lower than their carboxylate analogues, both due to a decrease in amine basicity as well as a lack of carboxylate groups susceptible to protonation.110–113 The speciation plots detailing the deprotonation events of these chelators are shown in Figure 2.4. The plots were produced using the pKa values calculated from the potentiometric titrations (found in Table 2.2) and provide a visual representation of how much each de/protonated species is formed at a given pH. The dotted lines in the speciation plots also highlight physiological pH (7.4). 41 Figure 2.4 Speciation plots (A) EGTAm and (B) ampam, calculated with protonation constants in Table 2.2, [L] = 0.001 M; dashed line indicates pH = 7.4. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. [Pb(EGTAm)]2+ and [Pb(ampam)]2+ were then calculated to have stability constants or log K values of 6.24(3) and 9.21(4) from their respective titration studies (Table 2.2). The pPb values, which measure metal sequestering ability under standard conditions ([L] = 10 µM, [M] = 1 µM, pH = 7.4), were found to mimic this outcome with ampam obtaining a pPb value 3 units higher than EGTAm. In addition, the stability constant of [Pb(ampam)]2+ is similar to that obtained for [Pb(DTPAm)]2+ (log KML = 8.79(2));80 however, the thermodynamic stability of [Pb(TCMC)]2+ (log KML > 19)112 far exceeds these open-chain chelators and provides further support that TCMC forms the most effective complex.61,99 A B 42 Table 2.2 Protonationa and formationb constants of ligands and their corresponding Pb2+ complexes. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. log KHL log KH2L log KH3L log KML log KMHL log KM(OH)L pMc (pH 7.4) EGTAm 4.32 (1) 3.53 (1) - 6.24 (3) - - 7.2 ampam 5.61 (1) 5.05 (1) 2.44(2) 9.21 (4) 3.51 (5) - 10.2 a Values were obtained by pH-potentiometry (25 C, I = 0.16 M NaCl). KHqL defined as [HqL]/([H][Hq-1L])). b KML defined as [ML]/([M][L]); KMHL defined as [MHL]/([ML][H]); KM(OH)L defined as [M(OH)L]/([ML][OH]) (charges omitted for clarity). c pM defined as –log[Pbfree] when [L] = 10 µM; [M] = 1 µM at pH 7.4. 43 In the speciation trends obtained for the metal complex solutions (Figure 2.5), EGTAm directly coordinates Pb2+ ions to form [Pb(EGTAm)]2+ and becomes the dominant species in solution by pH 3. This dicationic species is nearly quantitatively formed between pH 4-8.5 but by pH 10 the complex has dissociated to a significant degree. For ampam, the chelator initially coordinates Pb2+ ions to form the monoprotonated species [PbH(ampam)]3+ at low pH (~0-4.5). This species has a pKa = 3.51(5) and deprotonates to form [Pb(ampam)]2+ as the dominant species in solution from pH ~3.5-10.5. Beyond pH 11, hydrolysis of Pb2+ occurs to a significant extent. These speciation plots show that the molar activities obtained in the radiolabeling studies are unlikely to be significantly increased by changing the pH from the original values used. EGTAm and ampam radiolabeled [203Pb]Pb2+ ions at pH 5.5-6 in MES buffer,114 and at these pH values the dicationic metal complex species are nearly quantitatively formed relative to the amount of free Pb2+. Figure 2.5 Speciation plots of (A) Pb2+-EGTAm and (B) Pb2+-ampam, calculated with stability constants in Table 2.2, [Pb2+] = [L] = 0.001 M; dashed line indicates pH = 7.4. Reproduced (with slight modifications) from Ref. 80 with permission from the Royal Society of Chemistry. A B 44 NMR titrations are convenient to validate findings from potentiometric studies, achieved by comparison of 1H NMR spectra (of increasing pD) with speciation diagrams of ligands and metal complexes to discern if significant thermodynamic events (i.e., deprotonations, complex formation) coincide with drastic spectral changes in the expected pD region(s). Using the 1H NMR spectra of EGTAm at various pD values (Figure 2.6) as an example, there is negligible spectral change when the pD is increased from 1.9 to 2.4. This correlates with the diprotonated species, H2EGTAm2+, being the dominant species in this pD range. As the pD increases from 2.4 to 4.4 there is a drastic shift in the proton environments. The upfield shift of the peaks in the spectra is indicative of deprotonation events occurring. Furthermore, with a slight increase of pD from 4.4 to 4.6 there is still a significant shift in the proton environments. This supports one of the donor atoms has a pKa value close to 4.4. Finally, there are spectral differences from pD 4.6 to 6.4 as the deprotonated neutral species, EGTAm, increasingly becomes the only species to exist in solution. The 1H NMR spectra also indicate that the amines are the sites of deprotonation because there is a more dramatic upfield shift of the methylene groups adjacent to amine, corresponding to an increase in electron density upon their deprotonation. This NMR titration analysis aligns with the speciation plot of EGTAm (Figure 2.4a) as well as its potentiometric titration results, in which its amine pKa values were calculated to be 4.32(1) and 3.53(1). 45 Figure 2.6 Portion of the 1H NMR spectra for EGTAm and [Pb(EGTAm)]2+ vs pH (300 MHz, 298 K, D2O). Reproduced from Ref. 80 with permission from the Royal Society of Chemistry. 1H NMR analysis of the Pb2+/EGTAm system is in similar agreement with speciation diagrams. Slight spectral changes are noted for a solution containing EGTAm and Pb2+ (1:1.1 ratio) between pD 1.8 and 2.2, which can be attributed to the onset of complex formation. A drastic change occurs between pD 2.2 and 4.4, attributed to complete complex formation. This is further supported by comparison of the ligand spectrum at pD 4.4, which is clearly indicative of a different species, as evidenced by the two singlet peaks overlapping in the complex spectrum compared to 46 the ligand spectrum where all four distinct proton environments are observed. The speciation diagram of [Pb(EGTAm)]2+ predicts a dominant 1:1 species across a broad pH range, which is similarly noted in the 1H NMR spectra as no spectral change occurs between pD 4.4 and 6.3. Parallel analysis is possible with ampam and [Pb(ampam)]2+; relevant 1H NMR spectra appear in Figure A.7. 2.3 Conclusions Various [nat/203Pb]Pb2+ complexation studies were carried out with EGTAm and ampam to assess their compatibility for radiopharmaceutical applications. The 1H NMR spectra of [Pb(EGTAm)]2+ and [Pb(ampam)]2+ revealed spectral features indicative of fluxional behaviour. An X-ray diffraction study of [Pb(EGTAm)(NO3)](NO3) then showed the amines of the amino-ether (NOON) backbone coordinated to the Pb2+ ion from opposite hemispheres to form a holodirected complex. Unfortunately, this resultant complex had a vacant coordination site that was filled by a NO3- ion. Within the context of radiopharmaceutical applications, a vacant coordination site is considered problematic since many biomolecules may be able to coordinate the Pb2+ ion and change the properties of the complex. Use of the same backbone as part of decadentate ampam was more sterically compatible since the crystal structure of [Pb(ampam)](NO3)2 showed a holodirected ten-coordinate complex, in which the chelator fully encapsulated the Pb2+ ion. In the potentiometric titration studies, [Pb(EGTAm)]2+ and [Pb(ampam)]2+ obtained low to modest pM values of 7.2 and 10.2, respectively. Despite the higher stability constant achieved with ampam, both chelators demonstrated a low affinity for [203Pb]Pb2+ ions (~0.25 MBq/µmol) in their respective radiolabeling studies. Overall, these results aligned to 47 show both EGTAm and ampam are unsuitable matches with Pb2+ ions, and will not be suitable chelator components for [203/212Pb]Pb2+-based radiopharmaceuticals. 2.4 Experimental 2.4.1 Synthetic Materials and Methods All solvents and reagents were purchased from commercial suppliers (Sigma-Aldrich, Fisher Scientific, TCI America, Alfa Aesar, AK Scientific, Fluka) and were used as received. Synthetic reactions were monitored by thin-layer chromatography (TLC) (MERCK Silicagel 60 F254, aluminum sheet). Flash chromatography was performed using Redisep Rf high performance (HP) silica and alumina columns and a Teledyne Isco (Lincoln, NE) Combiflash Rf automated system. Water was ultrapure (18.2 MΩ cm–1 at 25 ℃, Milli-Q, Millipore, Billerica, MA). 1H and 13C{1H} NMR spectra were recorded at ambient temperature on Bruker AV300 and AV400 instruments; unless otherwise specified, the NMR spectra are expressed on the δ scale and referenced to residual solvent peaks. LR-ESI-MS was performed using a Waters ZG spectrometer with an electrospray chemical ionization (ESCI) source, and HR-ESI-MS was performed on a Micromass LCT time-of-flight instrument. Microanalyses for C, H, and N were performed on a Carlo Erba elemental analyzer EA 1108. The HPLC system used for analysis and purification of ampam (9) consisted of a Waters 600 controller, A Waters 2487 dual wavelength absorbance detector and a Waters delta 600 pump with a Phenomenex Synergi 4 μm hydro-RP 80 Å column (250 mm × 21.2 mm semipreparative). 48 2.4.2 Synthesis and Characterization of EGTAm and ampam Diethyl 3,12-bis(2-ethoxy-2-oxoethyl)-6,9-dioxa-3,12-diazatetradecanedioate (1). [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (0.572 g, 1.50 mmol) was mixed with EtOH (99 mL). Concentrated H2SO4 (1 mL) was then added, and the mixture was heated to 60 ℃. The mixture turned from a cloudy to clear solution within 30 minutes. The reaction mixture was left to stir for seven days being monitored by mass spectroscopy. The EtOH was removed in vacuo and the remaining crude product was mixed with EtOAc (50 mL) and sat. Na2CO3 (50 mL). The aqueous layer was then extracted with EtOAc (3 x 50 mL). The organic layers were combined, dried with Na2SO4, filtered, and concentrated in vacuo to give a clear oil product (0.589 g, 1.19 mmol, 80%). 1H NMR (400 MHz, 298 K, CDCl3):  4.14 (q, 3J = 7.1 Hz, 8H), 3.59 (t, 3J = 5.8 Hz, 4H), 3.59 (s, 8H), 3.53 (s, 4H), 2.94 (t, 3J = 5.7, 4H), 1.25 (t, 3J = 7.2, 12H). 13C{1H} NMR (100 MHz, 298 K, CDCl3):  171.5, 70.5, 70.3, 60.5, 56.0, 53.7, 14.4. HR-ESI-MS: calcd for [C22H40N2O10 + H]+: 493.2756; found [M + H]+: 493.2757. EGTAm (2). NH3 (7 N) in MeOH (10 mL) was added to compound 1 (0.554 g, 1.12 mmol). The reaction mixture was left to stir at room temperature for fourteen days with white precipitate observed during the final two days of stirring. The MeOH was removed in vacuo and the product was dried on a high vacuum line. The product was then redissolved in H2O and lyophilized to give a white solid product (0.423 g, 1.12 mmol, 99%). 1H NMR (400 MHz, 298 K, D2O, pD 7):  3.66 (s, 4H), 3.64 (t, 3J = 5.3 Hz, 4H), 3.36 (s, 8H), 2.84 (t, 3J = 5.2, 4H). 13C{1H} NMR (100 MHz, 298 K, D2O, pD 7):  176.9, 69.5, 68.6, 58.2, 54.7. HR-ESI-MS: calcd for [C14H28N6O10]: 376.2070; found [M]: 376.2074. Elemental analysis: calcd % for EGTAm•0.25H2O•0.25MeOH: C 44.01, H 7.64, N 21.61; found: C 44.33, H 7.30, N 21.29. 49 Methyl 6-(hydroxymethyl)picolinate (3). Compound 3 was prepared with slight modifications to the literature with characteristic spectra confirmation.62 Pyridine-2,6-dicarboxylic acid (9.58 g, 49.1 mmol, 1.0 equiv) was dissolved in a 3:2 mixture of CH2Cl2:MeOH (240 mL:160 mL) and the solution was cooled to 0 ℃ in an ice bath. NaBH4 (2.17 g, 57.4 mmol, 1.2 equiv) was added in portions over 1 hour. The ice bath was then allowed to melt while the reaction progress was monitored by TLC. After one more hour, the water bath was removed and CH2Cl2 (100 mL) was added to the reaction mixture. Immediately afterwards, sat. Na2CO3 (100 mL) was added over 5-10 minutes quenching the reaction mixture. The organic layer was collected, and liquid-liquid extractions were carried out on the aqueous phase using CH2Cl2 (4 x 100 mL). The organic layers were all combined, dried over MgSO4, filtered, and concentrated in vacuo to obtain a crude product. Purification was carried out using silica chromatography (CombiFlash Rf automated column system; 2 x 80 g HP silica columns; A: CH2Cl2, B: MeOH; 0% B to 5% B). The product was collected and concentrated in vacuo to yield a white solid product (4.62 g, 27.6 mmol, 56%). 1H NMR (400 MHz, 298 K, CDCl3):  8.04 (d, 3J = 7.7 Hz, 1H), 7.86 (t, 3J = 7.8 Hz, 1H), 7.53 (d, 3J = 7.8 Hz, 1H), 4.87 (s, 2H), 4.01 (s, 3H). 13C{1H} NMR (100 MHz, 298 K, CDCl3):  165.6, 160.6, 146.9, 137.9, 124.2, 123.9, 64.7, 53.0. HR-ESI-MS: calcd for [C8H9NO3 + H]+: 168.0654; found [M + H]+: 168.0654. Methyl 6-(bromomethyl)picolinate (4). Compound 4 was prepared with slight modifications to the literature with characteristic spectra confirmation.62 Compound 5 (4.257 g, 25.47 mmol, 1.0 equiv) was dissolved in MeCN (200 mL) and cooled to 0 ℃. PBr3 (2.6 mL, 28 mmol, 1.1 equiv) was added dropwise over 15 minutes and the solution became an increasingly intense yellow colour throughout the addition. The reaction mixture was left to warm to room temperature. After five hours, the mixture was slowly quenched with Na2CO3 (100 mL) over 40 50 minutes due to effervescence occurring upon its addition. EtOAc (100 mL) was added, and the organic layer was collected. The aqueous layer was then extracted with more EtOAc (2 x 100 mL). The organic layers were combined, dried with MgSO4, filtered, and concentrated in vacuo to give a white solid crude product. Purification was carried out using silica chromatography (CombiFlash Rf automated column system; 2 x 80 g HP silica columns; A: hexanes, B: EtOAc; 100% A to 50% A). The white solid final product was then collected (5.56 g, 24.2 mmol, 95%). 1H NMR (400 MHz, 298 K, CDCl3):  8.06 (d, 3J = 8.7 Hz, 1H), 7.87 (t, 3J = 7.8 Hz, 1H), 7.69 (d, 3J = 7.8 Hz, 1H), 4.65 (s, 2H), 4.02 (s, 3H). 13C{1H} NMR (400 MHz, 298 K, CDCl3):  165.4, 157.5, 147.8, 138.3, 127.3, 124.5, 53.2, 33.3. HR-ESI-MS: calcd for [C8H8BrNO2 + Na]+: 251.9630; found [M + Na]+: 251.9629. N,N'-((Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-nitrobenzenesulfonamide) (5). 2,2’-(Ethylenedioxy)bis(ethylamine) (3.00 mL, 20.5 mmol, 1.0 equiv) was mixed with CH2Cl2 (75 mL). Na2CO3 (5.47 g, 51.6 mmol, 2.5 equiv) was added and the solution was cooled to 0 ℃. 2-Nitrobenzene sulfonyl chloride (10.15 g, 45.80 mmol, 2.2 equiv) dissolved in CH2Cl2 (100 mL) was added dropwise. The mixture was left to warm to room temperature and was stirred for 24 hours, being monitored by TLC. The mixture was then filtered and concentrated in vacuo. The yellow oil was diluted in CH2Cl2 (~20 mL) and refluxed to give a clear yellow solution. The solvent was left to evaporate until small crystals were observed, then the mixture was placed into a freezer to be left overnight. The white crystals were then collected by filtration and washed with cold CH2Cl2 (~15 mL) (8.42 g, 16.2 mmol, 81%). 1H NMR (400 MHz, 298 K, CDCl3):  8.13 (m, 2H), 7.88 (m, 2H), 7.73 (m, 4H), 5.89 (t, 3J = 5.6 Hz, 2H), 3.57 (t, 3J = 5.1 Hz, 4H), 3.51 (s, 4H), 3.28 (q, 3J = 5.3 Hz, 4H). 13C{1H} NMR (75 MHz, 298 K, CDCl3):  148.1, 133.8, 133.7, 133.0, 131.2, 51 125.7, 70.5, 69.3, 43.7. HR-ESI-MS: calcd for [C18H22N4O10S2 + H]+: 519.0850; found [M + H]+: 519.0853. Dimethyl 6,6'-(2,11-bis((2-nitrophenyl)sulfonyl)-5,8-dioxa-2,11-diazadodecane-1,12-diyl)dipicolinate (6). Compound 4 (1.788 g, 7.773 mmol, 2.0 equiv) and K2CO3 (3.23 g, 23.4 mmol, 6 equiv) were mixed in dry MeCN (25 mL). The mixture was heated to 50 ℃ and compound 5 (2.00 g, 3.85 mmol, 1 equiv) was added. The mixture was left to stir overnight before the MeCN was removed in vacuo. The remaining mixture was dissolved in CH2Cl2 (80 mL) and H2O (60 mL). The aqueous layer was then extracted using CH2Cl2 (3 x 80 mL). The organic layers were combined, dried with MgSO4, filtered, and concentrated in vacuo. The crude product was then purified using silica chromatography (CombiFlash Rf automated column system; 2 x 48 g HP liquid injection column; A: CH2Cl2, B: MeOH; 100% A to 5% B) to give a white sticky solid product (2.58 g, 3.16 mmol, 82%). 1H NMR (400 MHz, 298 K, CDCl3):  8.12 (m, 2H), 7.99 (d, 3J = 7.7 Hz, 2H), 7.81 (t, 3J = 7.8 Hz, 2H), 7.66 (m, 8H), 4.83 (s, 4H), 3.97 (s, 6H), 3.55 (t, 3J = 5.4 Hz, 4H), 3.47 (t, 3J = 5.3 Hz, 4H), 3.23 (s, 4H). 13C{1H} NMR (75 MHz, 298 K, CDCl3):  165.5, 157.4, 148.2, 147.5, 138.1, 133.7, 133.5, 132.0, 131.1, 125.5, 124.4, 124.2, 70.1, 69.0, 53.9, 53.0, 48.2. HR-ESI-MS: calcd for [C34H36N6O14S2 + Na]+: 839.1629; found [M + Na]+: 839.1625. Dimethyl 6,6'-(5,8-dioxa-2,11-diazadodecane-1,12-diyl)dipicolinate (7). Compound 6 (2.07 g, 2.54 mmol, 1 equiv) and K2CO3 (1.11 g, 8.03 mmol, 3.2 equiv) were mixed in dry THF (16 mL). Thiophenol (0.60 mL, 5.8 mmol, 2.3 equiv) was added, turning the mixture yellow within seconds. The mixture was then heated to 50 ℃ and left to stir for two days. After, the solvent was removed in vacuo. The leftover yellow mixture was dissolved in H2O (20 mL) and CH2Cl2 (40 mL). The aqueous phase was then extracted with CH2Cl2 (4 x 40 mL) and EtOAc (100 mL). Saturated Na2CO3 (10 mL) was then added to the aqueous layer which was then further extracted 52 with CH2Cl2 (3 x 40 mL). The organic phases were collected, dried with Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by alumina chromatography (CombiFlash Rf automated column system; 24 g HP alumina column; A: CH2Cl2, B: MeOH; 100% A to 5% B). The product eluted off at ~1.5% MeOH and was concentrated in vacuo, giving a pale-yellow oil product (0.62 g, 1.39 mmol, 55%). 1H NMR (300 MHz, 298 K, CDCl3):  7.99 (d, 3J = 7.6 Hz, 2H), 7.79 (t, 3J = 7.7 Hz, 2H), 7.63 (d, 3J = 7.6 Hz, 2H), 4.04 (s, 4H), 3.98 (s, 6H), 3.63 (t, 3J = 5.3 Hz, 4H), 3.62 (s, 4H), 2.86 (t, 3J = 5.3 Hz, 4H), 2.53 (s, 2H). 13C{1H} NMR (75 MHz, 298 K, CDCl3):  166.0, 160.6, 147.6, 137.6, 125.8, 123.7, 70.6, 70.4, 55.1, 53.0, 48.9. HR-ESI-MS: calcd for [C22H30N4O6 + H]+: 447.2244; found [M + H]+: 447.2247. Dimethyl 6,6'-(2,11-bis(2-amino-2-oxoethyl)-5,8-dioxa-2,11-diazadodecane-1,12-diyl)dipicolinate (8). Compound 7 (0.595 g, 1.3 mmol, 1 equiv) was mixed with K2CO3 (0.82 g, 6.0 mmol, 4.5 equiv) in dry MeCN (5 mL). As the mixture was heating up to 60 ℃, 2-bromoacetamide (0.51 g, 3.7 mmol, 2.8 equiv) was added. The reaction mixture was then left to stir overnight. The next day, the reaction mixture was centrifuged and decanted to collect the MeCN solution containing the product. The remaining salt was sonicated and rinsed with more MeCN (2 x 5 mL) to repeat the centrifugation process. The solvent was then removed in vacuo and the remaining product was purified using silica chromatography (CombiFlash Rf automated column system; 24 g HP silica column liquid injection; A: CH2Cl2, B: MeOH; 100% A to 10% B). The final product was a very pale-yellow oil (0.39 g, 0.69 mmol, 53%). 1H NMR (400 MHz, 298 K, CD2Cl2):  8.12 (s, 2H), 7.97 (d, 3J = 7.7 Hz, 2H), 7.81 (t, 3J = 7.8 Hz, 2H), 7.57 (d, 3J = 7.7 Hz, 2H), 5.80 (s, 2H), 3.94 (s, 4H), 3.94 (s, 6H), 3.54 (t, 3J = 5.2 Hz, 4H), 3.53 (s, 4H), 3.24 (s, 4H), 2.82 (t, 3J = 5.2 Hz, 4H). 13C{1H} NMR (100 MHz, 298 K, CD2Cl2):  175.0, 166.1, 160.1, 53 148.1, 138.1, 126.6, 124.1, 70.9, 69.6, 61.5, 59.3, 55.5, 53.1. HR-ESI-MS: calcd for [C26H36N6O8 + H]+: 561.2667; found [M + H]+: 561.2671. ampam (9). NH3 (7N) in MeOH (6 mL) was added to compound 8 (0.28 g, 0.49 mmol, 1 equiv). The reaction mixture was left to stir at room temperature for six days, being monitored by mass spectroscopy. The solvent was removed, and the product was dried on a high vacuum line. The product was then dissolved in H2O, acidified using a few drops of HCl (1 M) and purified via HPLC (A: H2O, B: MeCN; 95% A to 0% A over 30 min, tR ~ 16.5 min). The fractions containing the product were collected and the solvent was removed. The product was then redissolved in H2O before being lyophilized. The final product was a fluffy white solid (0.16 g, 0.30 mmol, 61%). 1H NMR (300 MHz, 298 K, D2O, pD 7):  7.88 (m, 4H), 7.62 (m, 2H), 3.91 (s, 4H), 3.64 (t, 3J = 5.2 Hz, 4H), 3.60 (s, 4H), 3.28 (s, 4H), 2.84 (t, 3J = 5.3 Hz, 4H). 13C{1H} NMR (100 MHz, 298 K, DMSO):  172.9, 166.0, 158.3, 149.5, 137.9, 125.4, 120.1, 69.6, 68.6, 60.0, 58.0, 53.9. HR-ESI-MS: calcd for [C24H34N8O6]: 530.2601; found [M]: 530.2601. Elemental analysis: calcd % for ampam•H2O: C 52.55, H 6.61, N 20.43; found: C 52.68, H 6.46, N 20.32. 2.4.3 Metal Complexation and NMR Titration Solutions The NMR spectra of EGTAm and [Pb(EGTAm)]2+ measured over a series of pD values involved a number of solutions: D2O (0.9 mL) was added to ten separate vials containing portions of EGTAm•0.25H2O•0.25MeOH (3.0-3.4 mg, 7.7-8.7 μmol, 8.5-9.6 mM). The ligand solution from five of these vials were added to five new vials containing a slight excess of Pb(NO3)2 (L:M molar ratio ~ 1:1.1). The pD values of the five remaining ligand solutions and the five new metal complex solutions were then adjusted with DCl (0.1 M and 0.2 M; diluted from 40 wt.% DCl) to give a range of pD values = 1.9-6.4 and 1.8-6.5, respectively. 54 The same process was applied to obtain NMR spectra for ampam and [Pb(Hqampam)]2+q (q = 1, 0) with minor changes in the quantities used: D2O (0.7 mL) was added to ten vials containing portions of ampam•H2O (2.0-3.2 mg, 3.6-5.8 μmol, 5.1-8.3 mM). Five ligand solutions were then added to separate new vials containing Pb(NO3)2 in excess (L:M molar ratio ~ 1:1.3). For ampam and [Pb(Hqampam)]2+q (q = 1, 0) solutions, the pD values were adjusted with DCl (6 M or 1 M) and NaOD (0.2 M; diluted from 40 wt. % NaOD) to give pD ranges of 1.4-7.0 and 1.4-6.5, respectively. All pD values were obtained using a Ross combined electrode and the equation pD = pHmeasured + 0.4. Furthermore, all solutions were vortexed and allowed to stand for at least 15 minutes at room temperature before collecting their NMR spectra. 2.4.4 X-ray Crystallography Single colourless needle crystals of [Pb(EGTAm)(NO3)](NO3) were recrystallized from slow evaporation of acetone into an aqueous solution of the metal complex (1 mL, 38 mM, pD ~6-7). Single colourless irregular crystals of [Pb(ampam)](NO3)2 were recrystallized by slow evaporation of an aqueous solution (1 mL, 6.6 mM, pD ~6-7). The [Pb(EGTAm)(NO3)](NO3) crystal analyzed was 0.29 × 0.15 × 0.08 mm3 in size and the [Pb(ampam)](NO3)2 crystal was 0.19 × 0.15 × 0.07 mm3. Crystals were mounted on a Mylar loop in oil on a Bruker APEX II area detector diffractometer. The [Pb(EGTAm)(NO3)](NO3) and [Pb(ampam)](NO3)2 crystals were kept at steady temperatures of T = 110(2) K and T = 115(2) K, respectively, during data collection. The structures were solved with the XT 2018/2 solution program using Intrinsic Phasing methods and by using Olex2 as the graphical interface.115,116 Finally, the models were refined with XL using full matrix least squares minimization on F2.115 55 2.4.5 Radiolabeling Studies Lead-203 was produced via irradiation of a thallium target (nat 203Tl) with 12.8 MeV protons using a cyclotron [203Tl(p,n)203Pb] reaction and was purified using techniques described elsewhere.52 Before radiolabeling studies were conducted, [203Pb]Pb2+ radioactivity was quantified using a high-purity germanium (HPGe) detector (Mirion Technologies (Canberra) Inc.) with Genie 2000 software via measurement of 203Pb decay events (t1/2 = 52 h, 279 keV, 81% abundance). Radiolabeling studies with EGTAm and ampam were conducted by making 10-2 M stock ligand solutions in ultrapure deionized H2O. In each study, a 20 µL portion of ligand solution was diluted in 170 µL MES buffer (0.5 M, pH 5.5-6) before being mixed with 10 µL of 203Pb solution (45-50 kBq). Control solutions contained 20 µL of ultrapure deionized water, 170 µL MES buffer (0.5 M, pH 5.5-6), and 10 µL of 203Pb solution (45-50 kBq). All solutions were incubated for 30 minutes at 37 ℃ and RCCs were determined by reverse-phase HPLC (A: H2O (0.1% TFA), B: MeCN (0.1% TFA); 95% A, 0-2 min, 95% A to 80% A, 2-18 min), with a flow rate of 1 mL/min passing through a 250 mm Synergi hydro analytical column. Under these conditions, [203Pb]Pb2+ elutes at tR ~ 3.2 min, [203Pb][Pb(EGTAm)]2+ elutes at tR ~ 4.0 min, and [203Pb][Pb(ampam)]2+ elutes at tR ~ 4.9 min with a peak that streaks. 2.4.6 Potentiometric Titration Studies Protonation constants and metal complex stability constants were determined from potentiometric data obtained using a Metrohm Titrando 809 with a Ross combined electrode. A 20 mL 25 ℃ thermostated glass cell with an inlet—outlet tube for nitrogen gas (purified through a 10% NaOH solution to exclude CO2 prior to and during the titration) was used as a titration cell. 56 The electrode was calibrated daily in hydrogen ion concentration by direct titration of HCl with freshly prepared NaOH solution, and the results were analyzed with the Gran procedure117 in order to obtain the standard potential (Eo) and the ionic product of water pKw, T = 25 ℃, with 0.16 M NaCl used as a supporting electrolyte. Solutions were titrated with carbonate-free NaOH (∼0.16 M) that was standardized against freshly recrystallized potassium hydrogen phthalate. Protonation constants were obtained from individual solutions containing EGTAm or ampam at 9.13-9.99  10-4 M (25 ℃ and 0.16 M NaCl ionic strength). Each titration consisted of 100-150 equilibrium points in the pH range 1.8-11. Up to three titrations were performed for each ligand and electromotive force values were recorded after 2 min of each NaOH addition. Metal complexation studies with Pb2+ were carried out in 1:1 metal to ligand molar ratio aqueous solutions ([ampam] = 6.78  10-4 M and [EGTAm] = 6.63  10-4 M) at 25 ℃ and I = 0.16 M NaCl as supporting electrolyte. Metal solutions were prepared by adding the atomic absorption (AA) standard metal ion solution to a ligand solution of known concentration in the 1:1 metal to ligand molar ratio. The exact amount of acid present in the lead standard was determined by Gran’s method117 titrating equimolar solution of the metal ion and Na2H2EDTA. All the potentiometric measurements were performed in duplicate with the electromotive force values recorded up to 5 min after each NaOH addition, and were processed using the Hyperquad2013 software.118 Speciation plots in Figure 2.4 and Figure 2.5 were generated with Hyss119 using the protonation constants and stability constants in Table 2.2. Dissociation constants corresponding to hydrolysis of Pb2+ ions included in the calculations were taken from Baes and Mesmer.120 The species formed in the studied systems are characterized by the general equilibrium pM + qH + rL = MpHqLr (charges omitted). For convention, a complex containing a metal ion, M, proton, H and ligand, L, has the general formula MpHqLr. The stoichiometric index p might also be 0 in the case 57 of protonation equilibria, and negative values of q refers to proton removal from coordinated water, equivalent to hydroxide ion addition during formation of the complex. The overall equilibrium constant for the formation of the complexes MpHqLr 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 calculated at [Mn+] = 1 µM, [Lx-] = 10 µM, pH 7.4 and 25 ℃.68 58 Chapter 3: H2ampa: a Versatile Chelator Capable of Binding [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+ Ions 3.1 Introduction 3.1.1 Selection of Targeted Alpha Therapy Candidates As discussed in Chapter 1.1.2,  therapy is a form of radiation therapy that has great potential to treat a broad range of late-stage cancers.31,32 Currently, there is just one FDA approved  therapeutic, known as “Xofigo” ([223Ra]RaCl2).57 This drug has proven targeted  therapy (TAT) is applicable to the clinic and is used to treat patients suffering from metastatic castration-resistant prostate cancer that had spread to the bone.56 Due to the medical and commercial success of [223Ra]RaCl2, the nuclear medicine community has strengthened its commitment to develop alternative  therapeutics that can target other types of cancers.57,121–123 The list of  emitters that can act as the basis of new  therapeutics is not extensive. Those  emitters that are frequently considered belong to the heavy element section of the periodic table, such as the post-transition metal and actinide sections.48 These include 212Pb (t1/2 = 10.6 h), 212Bi (t1/2 = 60.6 min), 213Bi (t1/2 = 45.6 min), and 225Ac (t1/2 = 9.9 days).46,47 These radiometals are often selected as the basis of four-component radiopharmaceutical that can utilize their radioactive properties in more of a targeted manner.11 Access to 212Pb is best known to come from 224Ra/212Pb generators, which have relatively short half-lives (224Ra (t1/2 = 3.4 day)) and provide on-site production of 212Pb or 212Bi, depending on the eluent conditions chosen.84 Technically, 212Pb is not an  emitter but decays via - particle emission to form the  emitting daughter radionuclide 212Bi. Lead-212 is often desired to be employed as an in-situ generator of 212Bi since it has a longer half-life that gives the 59 radiopharmaceutical more time to be constructed and delivered to its intended target site; hence its frequent association with TAT.124 The recoil energy of - particle emissions is not as great as  particle emissions (as mentioned in Chapter 1.1.2); however, there are still concerns about whether 212Pb can decay to 212Bi and stay coordinated by the chelator of a radiopharmaceutical to remain at the cancer site for  particle treatment. Lead-212 radiopharmaceuticals may therefore have to rely on being internalized by the cancer cells so that the 212Bi remains inside the cell even if it released from the chelator upon decay. As discussed in Chapter 2, Pb2+-based radiopharmaceuticals are still highly desirable because of the chemically identical theranostic pair of radioisotopes: 203/212Pb. Accordingly, [203/212Pb]Pb2+ radiopharmaceuticals have been developed and tested in various cancers treatments, such as prostate cancer and melanoma, but a [203/212Pb]Pb2+ drug is yet to be clinically approved.90–92 Another useful  emitting bismuth isotope is 213Bi. Access to 213Bi comes from 225Ac/213Bi generators.125 The availability of 212Bi and 213Bi complement one another to promote the development of 212/213Bi radiopharmaceuticals. Between the two, 213Bi is usually the favourable choice for TAT because its biodistribution can more easily be monitored. While 213Bi decays to stable 209Bi to release therapeutic particles, it also releases -rays (440 keV) that can be detected by SPECT gamma cameras.126,127 Bismuth-213 has therefore been involved in various cancer studies. These studies range from radiolabeled antibodies ([213Bi]anti-CD45), which induce cell apoptosis in chemoresistant and radioresistant leukemia cells, to the formation of [213Bi][Bi(DOTATATE)], which improves the overall survival of mice expressing neuroendocrine tumours (when pretreated with L-lysine).128,129 In addition to its use as a generator, 225Ac is a valuable radionuclide for its own direct in vivo administration. There are four  particles released during its decay to stable 209Bi, meaning 60 225Ac can be administered at lower quantities compared to most other  therapeutics.130 On the other hand, the production of  emitting daughter radionuclides raises concern since the release of an  particle from 225Ac has a high recoil energy that will break apart the metal complex, allowing undesirable redistribution of the daughter radionuclides. Currently, clinical trials of a drug known as “225Ac-PSMA” showed a couple of patients suffering from metastatic prostate cancer responded well to TAT using this radionuclide,131 although moving forward, cellular internalization mechanisms are likely to be sought-after to make 225Ac-based therapeutics more reliably safe for patients. Perhaps the main limiting factor affecting 225Ac radiopharmaceutical development is the limited access of 225Ac itself. The primary source of 225Ac is derived from limited 233U stockpiles, which do not produce enough 225Ac for a radiopharmaceutical to reach the commercial scale. Motivated by impressive TAT milestones such as those achieved by [223Ra]RaCl2 and 225Ac-PSMA, alternative 225Ac production methods are now being developed to counteract its limited supply and to accelerate the development of 225Ac radiopharmaceuticals.123 3.1.2 Design of H2ampa To our knowledge, very few chelators have been designed to target [nat/203/212Pb]Pb2+, [nat/212/213Bi]Bi3+ and [225Ac]Ac3+ ions, although Pb2+/Bi3+ and Bi3+/Ac3+ have been targeted as pairs.103,104,132 In terms of their HSAB properties, Pb2+ and Bi3+ ions are considered as borderline acids, while Ac3+ ions are somewhere between being intermediately hard to hard.11,127,130 In additional to this range in hardness, both Pb2+ and Bi3+ ions have a 6s2 lone pair of electrons that should be considered during chelator design (as demonstrated for Pb2+ ions in Chapter 2) and no stable isotopes of Ac3+ are available hindering structural studies of its complexes. 61 Despite these challenges, we wanted to explore the versatility of H2ampa with these contemporary radiometal ions for potential radiopharmaceutical development(s). H2ampa is a newly synthesized, decadentate chelator based on a large open-chain scaffold with two acetamide groups and two picolinic acid moieties (Chart 3.1). For Pb2+ ion chelation, neutrally charged TCMC demonstrated an improved kinetic inertness compared to anionically charged DOTA (technically H4DOTA or DOTA4-); however, as discussed in Chapter 2, neutrally charge ampam (Chart 3.1) coordinated Pb2+ ions but demonstrated a low affinity in its radiolabeling studies.72,80,96,97 To investigate the impact of charge on Pb2+ chelation H2ampa (or ampa2-) was designed to be the same as ampam, except the picolinamide groups of ampam were replaced with protic picolinic acid groups. In addition, H2ampa is essentially a non-macrocyclic analogue of H2macropa (Chart 3.1); thus the remarkable effective complexation of H2macropa with [213Bi]Bi3+ and [225Ac]Ac3+ inspired us to test H2ampa with these same radiometals.133,134 Reported herein, is the synthesis of H2ampa; its extensive NMR spectroscopy metal complexation studies with Pb2+, Bi3+, and La3+ ions; a crystal structure analysis of [Bi(ampa)](NO3); its radiolabeling studies involving [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+; and finally, the thermodynamic solution studies of H2ampa and its metal complex formations with Pb2+ and La3+ ions. 62 Chart 3.1 Chemical structures of the chelators frequently discussed in this work. Collectively, there is a range in denticity, functional groups, charge, and non-macrocyclic versus macrocyclic backbones allowing for comparative studies to aid in the chelator design discussions. 3.2 Results and Discussion 3.2.1 Ligand Synthesis H2ampa (10) was synthesized in a cumulative yield of 21% (Scheme 3.1). The strategy used to synthesize H2ampa largely follows synthetic schemes established for methyl(6-bromomethyl) picolinate62 and ampam80 (covered in Chapter 2), which involve two and five steps, respectively. Compared to ampam, the synthesis of H2ampa entails some minor variations in the first, third and fourth steps, such as the type of base used, and purification techniques applied etc.; the final step is also completely changed from an amidation reaction to an ester hydrolysis reaction. 63 Scheme 3.1 Synthesis of H2ampa (10) obtained in a cumulative yield of 21%. Briefly, the five steps entail: N-nosyl protection of the NOON backbone to form compound 5; N-alkylation with methyl(6-bromomethyl) picolinate (4) to produce compound 6; nosyl deprotection using three equivalents of thiophenol per nosyl group to form 7; N-alkylation with 2-bromoacetamide to synthesize 8; and finally, deprotection of the picolinate arm methyl ester groups using LiOH to afford H2ampa (10). With the variations applied to synthesize 10, four of the five steps are high yielding (> 70%). One subtle but significant improvement involved the number of thiophenol equivalents used in the nosyl deprotection step when forming 7. Using one equivalent of thiophenol per nosyl group led to reaction times that were 2-4 days long but when three equivalents of thiophenol were used per nosyl group, the reaction times were less than 24 hours. Removal of the inorganic salt from 7 64 was also changed from liquid-liquid extractions to a centrifugation method. This compound has a high affinity for the aqueous layer in a liquid-liquid extraction, so the yields were greatly improved (55% to 74%) by centrifuging the salt to the bottom of the solution and collecting the supernatant (see experimental section for more detail). The overall route used to synthesize 10 avoided amide hydrolysis. As expected, the amides were stable enough to not be hydrolyzed during the final step involving LiOH deprotection (in which compound 8 was converted into compound 10). Fortunately, the amide groups of 10 were also resistant to hydrolysis in the acidic conditions required for reverse-phase HPLC purification (0.1% TFA was used to acidify the semi-prep column), even when heated for solvent removal in vacuo. 3.2.2 Metal Complexation Studies The complexes H2ampa forms with Pb2+, Bi3+, and La3+ ions in the solution phase were studied via NMR spectroscopy. Conveniently, non-radioactive Pb2+ and Bi3+ ions are readily accessible; however, La3+ ions had to be used instead of Ac3+ ions since only radioactive isotopes of Ac3+ exist. The [Pb(ampa)] complex produced a unique 1H NMR spectrum with broad signals (Figure 3.1), so variable temperature (VT) NMR experiments were also carried out to gain more structural information (Figure 3.2). The metal complexes involving the trivalent metals were studied using a combination of 1H, 13C{1H} and 2D (COSY, HSQC) NMR techniques to assign the number of isomers as well as the symmetry of the complexes. The methylenic protons adjacent to the picolinate and acetamide donor groups of H2ampa have been highlighted for both the free ligand as well as the metal complexes to rationalize key aspects of the ligand’s structural behaviour in the complexes (Figure 3.1). 65 Figure 3.1 Stacked 1H NMR of H2ampa and corresponding complexes with metals Pb2+, Bi3+, and La3+ (400 MHz, 298 K, D2O). The pD of H2ampa, [Pb(ampa)], [Bi(ampa)]+ and [La(ampa)]+ solutions were 6, 7, 5, and 7, respectively. Only the amide and picolinate methylene peaks have been highlighted for clarity. Compared to H2ampa, the 1H NMR spectrum of [Pb(ampa)] exhibits a broadening of the signals and a large downfield shift of the methylenic protons adjacent to the picolinate (Hd and Hm) and acetamide (He and Hl) donor groups at 298 K, pD 6-7 (Figure 3.1). The methylene groups adjacent to the amides shift to overlap with the methylene groups between the ether groups so only 66 four aliphatic signals are observed. While the coordination number of [Pb(ampa)] cannot be determined from these broad signals, a similar shifting pattern was observed for [Pb(ampam)]2+, which formed a decadentate complex with a C2 axis of symmetry in its crystal structure.80 To further probe the structure [Pb(ampa)] a variable temperature (VT) NMR experiment was performed (Figure 3.2). The VT 1H NMR spectra indicates [Pb(ampa)] is a fluxional symmetric complex. As temperature increases to 333 K, the signals sharpen. This is interpreted as fluxional signals averaging out on the NMR timescale. Also, as the temperature is lowered to 283 K, the aliphatic peaks further broaden. At an even lower temperature it is suspected the fluxionality would further reduce and some of the peaks would eventually separate to show the diastereotopic splitting of the methylenic protons, such as those adjacent to the picolinates (Hd/d’ and Hm/m’). However, the complex was recorded in D2O so temperatures could not be decreased much below 283 K. As mentioned in Chapter 2, a fluxional complex is usually concerning for radiopharmaceutical applications but in the case of Pb2+ complexes, NMR spectroscopy is usually insufficient to determine coordination behaviour as evinced by the broad 1H NMR signals obtained for a number of different chelators.52,80,103,104 67 Figure 3.2 Variable temperature 1H NMR of [Pb(ampa)] (400 MHz, D2O, pD 7). Aside from the large D2O solvent peak at 4.79 ppm, the proton signal peaks of the complex have a general trend of becoming sharper and shifting downfield with increasing temperature. At pD 5, the 1H NMR spectrum of [Bi(ampa)]+ exhibits sharply resolved signals evident of no fluxional behaviour on the NMR timescale (at room temperature) (Figure 3.1). The methylenic protons adjacent to the picolinate (Hd and Hm) and acetamide (He and Hl) donor groups show diastereotopic splitting, whereby each methylene group appears as a pair of chemically distinct, mutually coupled doublets. One set of methylenic protons associated with a picolinate donor (Hd/Hd’) appears in a more deshielded environment and is obscured by the residual solvent; however, these are clearly resolved in the 2D HSQC spectrum (Figure 3.3). In addition, the HSQC spectrum distinguishes each of the methylenic protons associated with the donor arms according to their expected 13C and 1H signals. In this spectrum, the acetamide donors He/e’ and Hl/Hl’ are 68 correlated to the 13C signals at 57.8 and 56.8 ppm, while picolinate donors Hd/Hd’ and Hm/Hm’ are correlated to more deshielded 13C signals at 62.8 and 62.1 ppm. With these assignments, it was found that the acetamide donor group with the most shielded 13C signal of 56.8 ppm also has 1H signals in a more upfield position (< 4 ppm) relative to the other three donor groups (> 4 ppm), implying less electron donation to the Bi3+ centre and comparatively weaker coordination. Figure 3.3 HSQC spectrum of [Bi(ampa)]+ with focus on the aliphatic section (400 MHz, 298 K, D2O, pD 7). The remaining signals corresponding to the backbone ethylene and ethereal protons appear as clearly resolved sharp signals in the 1H NMR spectrum and exhibit typical 2J and 3J splitting patterns due to the asymmetric environment generated on coordination to the Bi3+ ion. These 69 signals can also be assigned in the HSQC spectra: the four 13C signals observed between 66.2-67.8 ppm correlate to the four methylene groups adjacent to the ether groups, with the final two methylene groups adjacent to the less electronegative amines being observed at 53.3 and 57.2 ppm. Each of these six 13C signals has a set of methylenic protons showing diastereotopic splitting. Thus, with all the NMR spectra taken into consideration, H2ampa appears to be able to fully saturate the inner coordination sphere as of Bi3+ ions, with [Bi(ampa)]+ forming a single asymmetric complex that has a coordination number as high as ten. Compared to [Bi(ampa)]+, H2ampa appears to be less well matched with La3+ ions according to its NMR studies. At pD 7, the 1H NMR spectrum of [La(ampa)]+ shows two metal complex isomers have formed (Figure 3.1). These isomers were determined to be one symmetric and one asymmetric isomer formed in an approximate ratio of 1:7. For the predominantly formed asymmetric complex, the picolinate donor groups coordinate the La3+ ion to adopt similar environments as captured by the comparable chemical shifts of the aromatic picolinate signals (Ha/Hp, Hb/Ho, Hc/Hn). The 1H NMR spectrum of this complex shows further evidence of the similar picolinate arm arrangements in the metal complex since the methylenic protons neighbouring these donor arms (Hd/d’ and Hm/m’) both have pairs of doublets with similar 1H NMR chemical shifts (d/d’ ~0.6 ppm and m/m’ ~0.5 ppm). Conversely, the two acetamide donor groups show distinctly different behaviour to one another, whereby one acetamide remains unbound to the metal centre, as indicated by the appearance of a singlet at 3.49 ppm (Hl), while the other acetamide coordinates to the metal ion with its doublet pair (He/e’) observed at distinctly different chemical shifts (e/e’ ~0.6 ppm). 70 3.2.3 X-ray Crystallography Study The solid-state structure of [Bi(ampa)]+ was investigated using X-ray crystallography studies to provide a more defined assessment of the coordination environment around the central Bi3+ metal ion. Single colourless plate-shaped crystals were obtained by slow evaporation from an aqueous solution containing an equimolar amount of Bi(NO3)3 and H2ampa, adjusted to pH 5 with NaOH (0.1 M). Analysis of a suitable crystal revealed [Bi(ampa)]+ as an asymmetric, ten-coordinate complex, belonging to the centrosymmetric P21/n space group (Figure 3.4). Figure 3.4 ORTEP-style diagrams showing two different views of ten-coordinate [Bi(ampa)]NO3. Thermal ellipsoids are shown at 50% probability, except for the Bi3+ ion. Methylenic hydrogens (-CH2-) and co-crystallized solvent molecules have been omitted for clarity. The X-ray crystal structure aligns with the 1H NMR spectral assignment of the [Bi(ampa)]+ complex, which also provided evidence of an asymmetric, ten-coordinate metal complex. The solid-state structure shows four of the ten solid-state bond lengths are elongated (Table 3.1); with Bi-O4Ether, Bi-O5Ether, Bi-O8Amide, and Bi-N5Amine bond distances between 2.772 Å to 2.891 Å. 71 Accordingly, it is most accurate to describe the structure of [Bi(ampa)]+ as ten-coordinate but with some of the coordinate bonds better described as weak bond interactions. As a group 15, post-transition metal, Bi3+ has a lone pair of 6s2 electrons that can be stereochemically active or inactive in its complexes.135 For [Bi(ampa)]+, the crystallography results indicate the presence of a partially stereochemically active lone pair on the Bi3+ ion since there is an asymmetry in the metal-ligand bonding distances. The previously mentioned long bond distances are all situated on the same face of the metal ion and have an average bond distance of 2.828 Å. On the opposite face, the bond lengths are much shorter with an average bond length of 2.470 Å. Thus, this asymmetry can be explained by partial lone pair activation, with increased electron density in the region of space occupied by the neutral oxygen donors on one face of the metal, and decreased electron density on the opposite face that is occupied by the other donor groups including two anionic picolinates. To further illustrate the presence of the Bi3+ lone pair of electrons, the X-ray crystal structure of [Bi(ampa)]+ is compared to the X-ray crystal structure of [Bi(macropa)]+ reported by Fiszbein, et al. (Table 3.1).133 [Bi(macropa)]+ has a greater range in bond lengths than [Bi(ampa)]+ (0.816 Å versus 0.585 Å, respectively),) due to an emphasized effect of the lone pair activation on the Bi3+ ion. Interestingly, H2ampa and H2macropa also have different coordination preferences with the Bi3+ ion despite both being diprotic and decadentate. For [Bi(ampa)]+, it is more energetically favourable for Bi3+ to have a partially activated lone pair in a ten-coordinate complex; while for [Bi(macropa)]+, it is more energetically favourable for Bi3+ to have a fully stereochemically active 6s2 lone pair in a six-coordinate complex.133 72 Table 3.1 Key bond distances found in [Bi(ampa]+ as determined by X-ray diffraction, including a comparison to those bonding distances found for [Bi(macropa]+. [Bi(ampa)]NO3 Bond length to Bi3+ (Å) [Bi(macropa)]NO3133 Bond length to Bi3+ (Å) O1Amide 2.502(5) O1Ether 2.987(2) O2COOH 2.404(5) O2Ether 2.895(2) O4Ether 2.809(5) O3Ether 2.754(2) O5Ether 2.891(5) O4Ether 3.048(2) O6COOH 2.306(5) O5COOH 2.258(2) O8Amide 2.840(5) O6COOH 2.232(2) N2Amine 2.681(6) N1Amine 2.930(3) N3Pyr 2.427(6) N2Pyr 2.442(2) N4Pyr 2.500(6) N3Amine 2.688(2) N5Amine 2.772(6) N4Pyr 2.413(2) 3.2.4 Radiolabeling Studies with [203Pb]2+, [213Bi]Bi3+, and [225Ac]Ac3+ Preliminary radiolabeling experiments with [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+ were performed to assess the affinity of H2ampa for each radiometal. While [203Pb]Pb2+ is not a therapeutic radionuclide, it gives a valuable reference for the ability of H2ampa to chelate [212Pb]Pb2+ ions; however, it is important to note that the different production methods and specific activities obtained for the [203/212Pb]Pb2+ radioisotopes are likely to cause variations in radiolabeling experiments. Concentration-dependent radiolabeling studies of H2ampa with [203Pb]Pb2+ (~20 kBq) demonstrated highly effective metal ion chelation (Figure 3.5). The results have been compared to DOTA and p-SCN-Bn-TCMC as they are established Pb2+ ion chelators (Table 3.2). H2ampa was able to radiolabel [203Pb]Pb2+ ions within 30 minutes at 37 ℃ to generate a near-quantitative RCC (> 96%) at 10-5 M. It was therefore found to produce molar activities up to ~20 MBq/µmol. H2ampa 73 therefore has an affinity for [203Pb]Pb2+ ions that is within the same order of magnitude as DOTA and p-SCN-Bn-TCMC, which were reported to achieve high RCCs at 10-5 M, corresponding to molar activities of ~40 and ~50 MBq/µmol, respectively.52 Figure 3.5 Radio-HPLC traces: A = free [203Pb]Pb2+, while B and C = [203Pb][Pb(ampa)] at 10-4 and 10-5 mol dm-3, respectively. Mobile phase used: A) H2O 0.1% TFA, B) MeCN 0.1% TFA; gradient: 82% A 0-3 minutes, 82% to 72% A, 3-23 minutes, 3 mL/min flow rate passing through a semi-prep column. Under these conditions, [203Pb]Pb2+ has a retention time (tR) ~ 4.6 min and [203Pb][Pb(ampa)] has a tR = 6.0 min. The radiolabeling experiments carried out for H2ampa involved the same [203Pb]Pb2+ production and purification procedures as those carried out for DOTA and p-SCN-Bn-TCMC.52 ‘Free’ [203Pb]Pb2+(Negative control)[203Pb][Pb(ampa)]([L] = 10-4 M)[203Pb][Pb(ampa)]([L] = 10-5 M)ABC 74 The proportions of ligand, buffer and [203Pb]Pb2+ solution were also the same; although the activity of [203Pb]Pb2+ was lower in the H2ampa experiments (20 kBq compared to 50 kBq) and MES buffer (0.5 M, pH 5.5) was used, whereas NH4OAc buffer (1 M, pH 7) was used for DOTA and p-SCN-Bn-TCMC. For H2ampa, the RCCs had to be determined by HPLC, while the RCCs of both DOTA and p-SCN-Bn-TCMC were determined by TLC. The radiolabeling conditions used for each chelator are summarized in Table 3.2. While different production runs will show variations in the molar activities achieved, H2ampa was found to consistently radiolabel [203Pb]Pb2+ ions at 10-4 M to 10-5 M, for activities between 20-100 kBq, and using a total reaction volume of 100 µL. Table 3.2 H2ampa achieved near-quantitative radiolabeling (RCC > 95%) of [203Pb]Pb2+ ions at 10-5 M, making it comparable to DOTA and p-SCN-Bn-TCMC. Ligand concentration (M) Radiochemical conversion (RCC) (%)a H2ampab DOTAc p-SCN-Bn-TCMC c 10-4 > 99 96 ± 1 97 ± 1 10-5 97 ± 1 76 ± 9 97 ± 1 a Each sample consisted of: 10 µL [203Pb]Pb2+, 10 µL [L] = 10-3-10-4 M, 80 µL buffer. b Reaction conditions: ~20 kBq [203Pb]Pb2+ per reaction, MES buffer (0.5 M, pH 6), 30 min and 37 ℃. c Reaction conditions: ~50 kBq [203Pb]Pb2+ per reaction, NH4OAc buffer (1 M, pH 7), 60 min and 25 ℃.52 Repeatedly, the molar activities obtained for [Pb(ampa)] (up to 20 MBq/µmol) were higher than those obtained for [Pb(ampam)]2+ (up to 0.25 MBq/µmol, as shown in Chapter 2.2.4). We postulate the improved radiolabeling affinity of ampa2- over ampam is due to the anionic charge influencing the geometry of [Pb(ampa)] to be different to [Pb(ampam)]2+. In Chapter 2, the crystal structure of [Pb(ampam)]2+ revealed a holodirected geometry, in which the 6s lone pair of electrons 75 on Pb2+ is stereochemically inactive. The low molar activity achieved with ampam was therefore explained by the thermodynamic preferences of its hard nitrogen and oxygen donor groups (N4O6) being mismatched with the holodirected geometry formed with Pb2+ ions, which tend to favour soft donor atoms such as halogens.80 As a result, it is proposed ampa2- coordinates Pb2+ to form a complex that has a degree of lone pair orbital activity, since its anionic charge would further exacerbate the donor mismatch with a holodirected complex. Unfortunately, a crystal of [Pb(ampa)] was not obtained for confirmation but additional support for this suggestion comes from Esteban-Gómez et al. who also investigated the impact of chelator charge on Pb2+ ions.96 They compared the crystal structures of two different Pb2+ complexes coordinated by octadentate chelators. The neutrally charged chelator formed a holodirected dicationic complex, but the chelator with an anionic charge favoured a hemidirected complex. Hence, the high affinity achieved for [Pb(ampa)] is supportive of a hemidirected geometry being formed. To accommodate a hemidirected geometry and a degree of lone pair activation, it is likely certain backbone donor atoms are weakly coordinated to the Pb2+ ion, which would explain the small chemical shifts of the backbone methylene groups and the general fluxionality observed in the NMR spectroscopy studies. Concentration-dependent radiolabeling studies of H2ampa with [213Bi]Bi3+ (15 - 20 kBq) and [225Ac]Ac3+ ions (~40 kBq) also demonstrated highly effective metal ion chelation, with mixing times of 7 minutes and 30 minutes, respectively. For both radiometals, near-quantitative RCCs (> 96%) were achieved across a broad concentration range (10-3-10-6 M), with lower RCC values being achieved at 10-7 M (78% and 33%, respectively) (Figure 3.6 and Figure 3.7). By contrast, parallel studies with macrocyclic DOTA showed a steep drop in overall RCC for [213Bi]Bi3+ at 10-7 M (12%) and was not even able to quantitatively radiolabel [225Ac]Ac3+ at 10-5 76 M concentration (79%). The RCCs produced with DOTA for each of these radiometals also required much higher temperature conditions (> 80 ℃). Figure 3.6 Using ~20 kBq of [213Bi]Bi3+ and NH4OAc buffer (0.4 M, pH 7) in each concentration-dependent study, H2ampa and DOTA both achieved high radiochemical conversions (RCC > 90%) except at very dilute ligand concentrations (~10-7 M). H2ampa solutions were mixed with [213Bi]Bi3+ for 7 minutes at room temperature (25 ℃), while DOTA solutions were mixed for 10 minutes at high temperatures (90 ℃). 0204060801004 5 6 7 8Ligand Concentration (10-x M)RCC(%)[213Bi][Bi(DOTA)]-[213Bi][Bi(ampa)]+ 77 Figure 3.7 Using ~40 kBq of [225Ac]Ac3+, NH4OAc buffer (0.2 M, pH 7) and 30 minute mixing times in each concentration-dependent study, H2ampa achieved higher radiochemical conversions (RCC) compared to DOTA at low ligand concentrations (10-5 - 10-7 M). Room temperature (25 ℃) radiolabeling conditions were used for H2ampa, while DOTA required high temperatures (85 ℃). Molar activities of ~370 MBq/µmol and ~130 MBq/µmol were achieved for [213Bi][Bi(ampa)]+ and [225Ac][Ac(ampa)]+, respectively. The molar activity obtained for [213Bi][Bi(ampa)]+ is low by comparison to other established chelating ligands. This value is likely a limitation of the available activity used in the generator rather than a constraint dictated by the ligand meaning no design aspects influencing lone pair activity can be drawn from these results alone. On the other hand, the molar activity produced for [225Ac][Ac(ampa)]+ at 10-6 M is within the same order of magnitude as the highly effective 225Ac chelator, H2macropa, which was documented to produce molar activities ranging from ~100-230 MBq/µmol in its preliminary studies.134 Again, comparing molar activities cannot say which chelator performs better unless they are tested in parallel for the same production run; however, the similar molar activities of 0204060801004 5 6 7 8[225Ac][Ac(ampa)]+[225Ac][Ac(DOTA)]-Ligand Concentration (10-x M)RCC(%) 78 H2macropa and H2ampa are still encouraging as it appears the affinity of H2ampa is not negatively impacted by lacking a preorganized macrocyclic backbone while coordinating [225Ac]Ac3+ ions. 3.2.5 Thermodynamic Solution Studies Combined spectrophotometric UV-Vis and potentiometric titrations were conducted with H2ampa to determine its pKa values. After its protonation constants were calculated, further solution studies probing its complex formation equilibria with Pb2+ and La3+ ions were studied over a broad pH range (1.8–11). While titrations were repeatedly attempted for Bi3+ ions, no accurate formation constants and speciation plots were obtained due to precipitation issues when its hydroxide species formed in the higher pH range. For H2ampa, there are four possible sites of protonation which consist of the picolinic acid and tertiary amine protons (Table 3.3). The picolinic acid groups have pKa values of 2.65 and 3.34, which are in an expected range according to the literature.69,136–138 The pKa values of the tertiary amines were then found to be 5.15 and 5.97, which closely match the pKa values calculated for the tertiary amines of the picolinic amide analogue, ampam: 5.05 and 5.61.80 With these pKa values, the ligand was found to exist in its fully deprotonated state as ampa2- at physiological pH (7.4) (Figure 3.8). Table 3.3 Protonation constants (log K)a of H2ampa reveal four protonation sites. Equilibrium reaction log K Assignment H4L2+ ⇆ H3L+ + H+ 2.65(2) Pyr-CO2 H3L+ ⇆ H2L + H+ 3.34(2) Pyr-CO2 H2L ⇆ HL- + H+ 5.15(1) NAmine HL- ⇆ L2- + H+ 5.97(1) NAmine a Values obtained by pH-potentiometric titrations (25 C and I = 0.16 M NaCl). KHqL defined as [HqL]/([H][Hq-1L])). 79 With the protonation constants calculated, the formation constants (log KML values) and pM values of the metal complexes H2ampa forms could be calculated. The pM value quantifies the metal scavenging ability of H2ampa under standard conditions ([L] = 10 µM; [M] = 1 µM; pH = 7.4) (Table 3.4) and represent a more accurate measure of the stability of the metal complexes under in vivo conditions. In general, the pM values mimic the trend noted for formation constants; however, comparing a ligand’s suitability between metals based on these thermodynamic parameters should be done with caution. More acidic metal ions generally form complexes with a higher formation constant and pM value than less acidic metal ions. Similarly, the basicity of ligands also influences these thermodynamic parameters. Table 3.4 Formation constantsa of various H2ampa/Pb2+ and H2ampa/La3+ species. Pb2+ La3+ log KML 13.84(3) 14.12(3) log KMHL 3.43(1)b 3.09(1)b log KM(OH)L 10.70(4) 9.87(4) pMc 14.8 15.1 a Values obtained from UV-potentiometric titrations (25 C, I = 0.16 M NaCl). KML defined as [ML]/([M][L]); KMHL defined as [MHL]/([ML][H]); KM(OH)L defined as [M(OH)L]/([ML][OH]) (charges omitted for clarity). b Values obtained from UV-acidic batch titrations (25 C, I = 0.16 M NaCl). c pM defined as –log[Pb2+free] when [L] = 10 µM; [M] = 1 µM at pH 7.4. To evaluate the stability of the metal complexes formed with H2ampa, a comparison to other ligands can be a useful guide. H2ampa pPb value calculated for macrocyclic ligands DOTA and TCMC both have pPb ≥ 18.4, while open-chain DTPA and EDTA have pPb values 15.0 and 15.9, respectively.112,139 Thus, the pPb value of H2ampa (14.8) is comparable (< 1.1 units 80 difference) to the highly protic open-chain chelators (technically H5DTPA and H4EDTA). In my attempts at designing a chelator with the optimal hardness to match the borderline soft nature of Pb2+ ions, the simple substitution of switching two picolinamide groups for two picolinic acids (from ampam to H2ampa) was also found to increase the pPb by 4.6 units. H2ampa complexed La3+ ions to form a stability constant and pM value which were modest to high when compared to macrocyclic chelators, such as DOTA and H2macropa. DOTA coordinates La3+ ions to form a complex with a higher stability constant (log KLaL ~24.3),140 while [La(ampa)]+ (log KLaL = 14.12(3), pLa = 15.1) has a stability constant comparable to [La(macropa)]+ (log KLaL = 14.99, pLa = 15.6).141,142 As an open-chain chelator, H2ampa forming a similarly stable complex compared to H2macropa is encouraging since crown ether-based macrocycles possess a high level of preorganization that often improves the stability of their complexes.136 Comparing H2ampa to H2macropa is also a better evaluation of their geometric preferences upon metal ion coordination opposed to comparing H2ampa and DOTA, since the former two chelators are diprotic and decadentate, unlike DOTA, which is tetraprotic and octadentate. Complexation of Pb2+ and La3+ by H2ampa showed similarities in their speciation plots, which can be used to visualize the species formed at physiological pH (7.4) under specified conditions (Figure 3.8). For both metal ions, log K values were calculated for three different complex species ([Pb(Hqampa)]q+ and [La(Hqampa)]q+1 where q = 1, 0, -1). They initially form a monoprotonated state at low pH (~0–4), then the deprotonated complex is the dominant species in solution across a broad pH range (~4-10), until the hydroxo species predominates in solution at high pH (~10-12). 81 Figure 3.8 Speciation plots showing the deprotonation events of [H4ampa]2+ to ampa2- (top), as well as metal complexation species for the Pb2+/ampa2- and La3+/ampa2- systems (bottom left and bottom right, respectively). Calculated with protonation constants and formation constants from Tables 3.3 and 3.4, and [Ligand] = [Metal] = 0.001 M. Dotted line in graphs indicates physiological pH (7.4). 3.3 Conclusion A new decadentate chelator, H2ampa, was synthesized incorporating both amide and picolinate functional groups on a long, ether-bridged backbone, in a cumulative yield of 21%. Its ability to coordinate Pb2+, Bi3+ and La3+ ions, respectively, was studied via NMR spectroscopy. The NMR spectroscopic studies revealed [Pb(ampa)] as a fluxional, symmetric complex; [La(ampa)]+ as a mixture of isomers showing rigid complexation; and [Bi(ampa)]+ as a rigid 82 asymmetric, ten-coordinate complex. Confirmation of the fully saturated Bi3+ ion coordination sphere was confirmed by its crystal structure, which was studied by X-ray diffraction. Solution studies were then carried out to gain insight into the thermodynamic drive of metal complexation between H2ampa/Pb2+ and H2ampa/La3+ systems. The metal formation constants and pM values obtained for H2ampa from the solution studies were encouraging since pM values of 14.8 and 15.1 were obtained for Pb2+ and La3+ ions, respectively. In addition, only one species predominated at physiological pH (7.4) for these metal complexes. Most informatively, H2ampa radiolabeled [203Pb]Pb2+, [213Bi]Bi3+ and [ 225Ac]Ac3+ ions under mild conditions to generate molar activities of ~20 MBq/µmol, ~370 MBq/µmol, and ~130 MBq/µmol, respectively. These preliminary studies therefore show H2ampa is a versatile chelator, and further studies with all three of the radiometals are warranted to determine the suitability of H2ampa as a chelator component for radiopharmaceuticals more comprehensively. 3.4 Experimental 3.4.1 Synthetic Materials and Methods For details regarding synthetic materials or instrumentation, see Chapter 2.4.1. 3.4.2 Synthesis and Characterization of H2ampa N,N'-((Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-nitrobenzenesulfonamide) (5). Compound 5 was prepared with slight modifications to the literature with characteristic spectra confirmation.80 2,2’-(Ethylenedioxy)bis(ethylamine) (5.00 g, 33.8 mmol, 1.0 equiv) was mixed with CH2Cl2 (250 mL). Triethylamine (12.0 mL, 118 mmol, 3.5 equiv) was added dropwise and the solution was cooled to 0 C. 2-Nitrobenzene sulfonyl chloride (14.9 g, 67.5 mmol, 2.0 equiv) 83 dissolved in CH2Cl2 (120 mL) was added dropwise over 45 minutes. The mixture was then left to warm to room temperature and was stirred overnight. HCl (175 mL, 1 M) was added, quenching the reaction mixture, and turning the clear solution cloudy. The organic and aqueous layers were separated before the aqueous layer was further extracted with CH2Cl2 (3 x 150 mL). The organic layers were all combined, then washed with brine (200 mL), dried over MgSO4, and evaporated in vacuo to afford a white solid. This crude solid product was refluxed in EtOAc (100 mL) and recrystallized to yield white crystals of 5 (13.4 g, 77%). 1H NMR (400 MHz, 298 K, CDCl3):  8.13 (m, 2H), 7.88 (m, 2H), 7.73 (m, 4H), 5.89 (t, 3J = 5.6 Hz, 2H), 3.57 (t, 3J = 5.1 Hz, 4H), 3.51 (s, 4H), 3.28 (q, 3J = 5.3 Hz, 4H). 13C{1H} NMR (75 MHz, 298 K, CDCl3):  148.1, 133.8, 133.7, 133.0, 131.2, 125.7, 70.5, 69.3, 43.7. HR-ESI-MS: calcd for [C18H22N4O10S2 + H]+: 519.0850; found [M + H]+: 519.0853. Dimethyl 6,6'-(2,11-bis((2-nitrophenyl)sulfonyl)-5,8-dioxa-2,11-diazadodecane-1,12-diyl)dipicolinate (6). Compound 6 was prepared with slight modifications to the literature with characteristic spectra confirmation.80 Methyl 6-(bromomethyl)picolinate (5.0 g, 22 mmol, 2.0 equiv), K2CO3 (6.49 g, 47.0 mmol, 4.3 equiv) and compound 5 (5.66 g, 10.9 mmol, 1 equiv) were mixed together in dry MeCN (55 mL) under an inert atmosphere. The mixture was then heated to 50 C and left to stir overnight. The K2CO3 was then removed via filtration and the volatiles were evaporated in vacuo until a yellow oil remained. The oil was dissolved in CH2Cl2 (80 mL) and washed with deionized H2O (80 mL). After collecting the organic layer, the aqueous layer was further extracted with CH2Cl2 (2 x 80 mL). The combined organic layers were subsequently washed with brine (130 mL), dried over MgSO4, filtered, and concentrated in vacuo to yield a white fluffy solid product (7.48 g, 84%). 1H NMR (400 MHz, 298 K, CDCl3):  8.12 (m, 2H), 7.99 (d, 3J = 7.7 Hz, 2H), 7.81 (t, 3J = 7.8 Hz, 2H), 7.66 (m, 8H), 4.83 (s, 4H), 3.97 (s, 6H), 3.55 (t, 3J 84 = 5.4 Hz, 4H), 3.47 (t, 3J = 5.3 Hz, 4H), 3.23 (s, 4H). 13C{1H} NMR (75 MHz, 298 K, CDCl3):  165.5, 157.4, 148.2, 147.5, 138.1, 133.7, 133.5, 132.0, 131.1, 125.5, 124.4, 124.2, 70.1, 69.0, 53.9, 53.0, 48.2. HR-ESI-MS: calcd for [C34H36N6O14S2 + Na]+: 839.1629; found [M + Na]+: 839.1625. Dimethyl 6,6'-(5,8-dioxa-2,11-diazadodecane-1,12-diyl)dipicolinate (7). Compound 7 was prepared with slight modifications to the literature with characteristic spectra confirmation.80 Compound 6 (1.06 g, 1.37 mmol, 1 equiv) and K2CO3 (0.79 g, 5.72 mmol, 4.4 equiv) were mixed in dry THF (38 mL). Thiophenol (0.67 mL, 6.5 mmol, 5 equiv) was added, turning the reaction mixture an intense yellow colour. The mixture was heated to 50 C and left to stir overnight. After, the solvent was removed in vacuo. The leftover yellow mixture was dissolved in MeCN (20 mL) and centrifuged. The supernatant was collected, separating the crude product from the inorganic salts. The salts were then mixed with further portions of MeCN (~15 mL), sonicated and vortexed, before being centrifuged again. This process was repeated two more times, with the supernatant layers being collected each time. The combined organic layers were then concentrated in vacuo to dryness and the subsequent crude product was purified by alumina chromatography (CombiFlash Rf automated column system; 24 g HP neutral alumina column; A: CH2Cl2, B: MeOH; 100% A to 10% B). The product eluted off at ~2% MeOH and was concentrated in vacuo to yield a pale-yellow oil product (0.44 g, 74%). 1H NMR (300 MHz, 298 K, CDCl3):  7.99 (d, 3J = 7.6 Hz, 2H), 7.79 (t, 3J = 7.7 Hz, 2H), 7.63 (d, 3J = 7.6 Hz, 2H), 4.04 (s, 4H), 3.98 (s, 6H), 3.63 (t, 3J = 5.3 Hz, 4H), 3.62 (s, 4H), 2.86 (t, 3J = 5.3 Hz, 4H), 2.53 (s, 2H). 13C{1H} NMR (75 MHz, 298 K, CDCl3):  166.0, 160.6, 147.6, 137.6, 125.8, 123.7, 70.6, 70.4, 55.1, 53.0, 48.9. HR-ESI-MS: calcd for [C22H30N4O6 + H]+: 447.2244; found [M + H]+: 447.2247. Dimethyl 6,6'-(2,11-bis(2-amino-2-oxoethyl)-5,8-dioxa-2,11-diazadodecane-1,12-diyl)dipicolinate (8). Compound 8 was prepared with slight modifications to the literature with 85 characteristic spectra confirmation.80 Compound 7 (1.15 g, 2.50 mmol, 1 equiv), 2-bromoacetamide (0.80 g, 5.8 mmol, 2.3 equiv), and N,N-diisopropylethylamine (1.83 mL, 10.5 mmol, 4.2 equiv) were all mixed in dry MeCN (20 mL). The reaction mixture was heated at 50 C over two days. Upon completion, the volatiles were evaporated in vacuo to yield a yellow oil. This oil was dissolved in CH2Cl2 (30 mL) and washed with deionized H2O (30 mL). The organic layer was separated, and the aqueous layer was further extracted with CH2Cl2 (2 x 30 mL). The organic layers were collected and washed with brine (100 mL). After, the organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The yellow oil crude product was purified using alumina chromatography (CombiFlash Rf automated column system; 24 g HP neutral alumina column; A: CH2Cl2, B: MeOH; 100% A to 10% B). The product eluted at ~3% MeOH and was concentrated in vacuo to produce compound 8 as a pale-yellow oil (0.80 g, 57%). 1H NMR (400 MHz, 298 K, CD2Cl2):  8.12 (s, 2H), 7.97 (d, 3J = 7.7 Hz, 2H), 7.81 (t, 3J = 7.8 Hz, 2H), 7.57 (d, 3J = 7.7 Hz, 2H), 5.80 (s, 2H), 3.94 (s, 4H), 3.94 (s, 6H), 3.54 (t, 3J = 5.2 Hz, 4H), 3.53 (s, 4H), 3.24 (s, 4H), 2.82 (t, 3J = 5.2 Hz, 4H). 13C{1H} NMR (100 MHz, 298 K, CD2Cl2):  175.0, 166.1, 160.1, 148.1, 138.1, 126.6, 124.1, 70.9, 69.6, 61.5, 59.3, 55.5, 53.1. HR-ESI-MS: calcd for [C26H36N6O8 + H]+: 561.2667; found [M + H]+: 561.2671. H2ampa (10). LiOH (0.12 g, 5.00 mmol, 4 equiv) was added to compound 8 (0.70 g, 1.3 mmol) in a mixture of H2O and THF in a 3:1 ratio (11.5 mL). The reaction mixture was stirred at room temperature for 3 h and the crude mixture was monitored by mass spectrometry. The solvent was removed, and the product was dried on a high vacuum line. The product was then dissolved in H2O and purified via HPLC (A: H2O (0.1% TFA), B: MeCN; 5% B to 40% B, over 35 min, tR ~ 16.5 min). The fractions containing the product were collected and the solvent was removed. The product was converted into an HCl salt using a few drops of HCl (1 M), following evaporation 86 in vacuo. This process was repeated two more times, before being redissolved in H2O and lyophilized. The final product was a sticky off-white solid (0.73 g, 76%). 1H NMR (400 MHz, 298 K, D2O, pD 6):  7.92 (t, 3J = 7.7 Hz, 2H), 7.84 (d, 3J = 7.6 Hz, 2H), 7.61 (d, 3J = 7.6 Hz, 2H), 4.02 (s, 4H), 3.66 (t, 3J = 5.2, Hz 4H), 3.58 (s, 4H), 3.42 (s, 4H), 2.94 (t, 3J = 5.0, 4H). 13C{1H} NMR (100 MHz, 298 K, D2O, pD 0-1):  167.7, 167.3, 149.4, 147.0, 140.2, 128.1, 125.6, 69.6, 64.5, 58.6, 55.3, 54.9. HR-ESI-MS: calcd for [C24H32N6O8]: 532.2282; found [M]: 532.2286. Elemental analysis: calcd % for H2ampa•4HCl•1.3H2O: C 41.07, H 5.54, N 11.97; found: C 41.19, H 5.57, N 11.80. 3.4.3 H2ampa Metal Complexation Studies NMR spectra for [Pb(ampa)] were obtained by dissolving H2ampa•4HCl•1.3H2O (13.1 mg, 18.8 µmol) in D2O (800 µL) and adding a portion (400 µL) to a vial containing Pb(NO3)2 (3.4 mg, 10.3 µmol, 1.1 equiv) with the pD subsequently adjusted to 7. The remaining portion (400 µL) was then added to a separate vial containing Bi(NO3)3•5H2O (5.1 mg, 10.5 µmol, 1.1 equiv). After the metal salt had dissolved, the pD value was adjusted to 5. Finally, NMR spectra for [La(ampa)](NO3) were obtained by dissolving another portion of H2ampa•4HCl•1.3H2O (8.0 mg, 11.5 µmol) in D2O (400 µmol) and adding the solution to La(NO3)3•6H2O (6.51 mg, 15.2 µmol, 1.3 equiv) with the pD increased to 7. The pD values of these respective metal complex solutions were adjusted using NaOD solutions (0.2 M and 0.1 M; diluted from 40 wt. % NaOD) and all metal complex solutions were vortexed prior to observing their NMR spectra. 87 3.4.4 X-ray Crystallography Study of [Bi(ampa)](NO3) Single colourless plate-shaped crystals of [Bi(ampa)](NO3) were recrystallized by slow evaporation of water. A suitable crystal with dimensions 0.18 × 0.13 × 0.02 mm3 was selected and mounted on a Mylar loop in oil on a Bruker APEX II area detector diffractometer. The crystal was kept at a stedy T = 90(2) K during data collection. The structure was solved with the XT 2018/2 solution program using Intrinsic Phasing methods and by using Olex2 as the graphical interface.115,116 The model was refined with XL using full matrix least squares minimization on F2.115 3.4.5 Radiolabeling Experiments with [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+ Lead-203 was cyclotron-produced at TRIUMF via irradiation of 203Tl targets using 12.8 MeV protons, while 225Ac was produced at the TRIUMF 500 MeV Isotope Production Facility (IPF) via irradiation of 232Th foils; full details of their productions have been described elsewhere.52,143,144 A small-scale 225Ac/213Bi generator was also prepared using well established methodologies based on AGMP-50 cation exchange chromatography. Actinium-225 (t1/2 = 9.92 days) decays via the two short-lived daughter radionuclides, 221Fr (t1/2 = 4.8 min) and 217At (t1/2 = 32.3 ms), to give the longer-lived daughter nuclide 213Bi (t1/2 = 45.6 min). The suitable half-lives of 225Ac and 213Bi, and short-lived nature of 221Fr and 217At, allow for a generator to be prepared which will attain radioactive secular equilibrium after 5 hours. The 213Bi radioactivity is eluted in the form of [BiI4]- and [BiI5]2- by washing the resin with freshly prepared NaI/HCl (0.1 M) solution, while 225Ac is retained on the cation exchange column. An optimal equilibration time of 3 hours is typically used, as 90% of the total 213Bi radioactivity is achieved in this time, allowing for 88 multiple elutes to be performed on the same day. This shortened time also yields the highest specific activity 213Bi eluate, while extended time delays between generator milking allow for the ingrowth of the pure --emitting daughter nuclide 209Pb, and ultimately stable 209Bi, which are co-eluted from the generator. [203Pb][Pb(ampa)] radiolabeling. H2ampa was dissolved in ultrapure deionized H2O (1.01262 Supelco) to form a 10-2 M ligand stock solution. A portion of this stock solution was used to give a serial dilution series ranging from 10-3 to 10-5 M. In the concentration-dependent radiolabeling studies, a 10 µL portion of ligand solution (10-3 to 10-5 M) was diluted in 80 µL MES buffer (0.5 M, pH 5.5-6) before being mixed with 10 µL of 203Pb solution (20 kBq). Control solutions involved 10 µL H2O, 80 µL MES buffer (0.5 M, pH 5.5-6) and 10 µL of 203Pb solution (20 kBq). All solutions were incubated for 30 minutes at 37 ℃. After, RCCs were determined by reverse-phase HPLC (A: H2O (0.1% TFA), B: MeCN (0.1% TFA); 82% A, 0–3 min, 82% A to 72% A, 3-23 min) with a flow rate of 3 mL/min passing through a 250 x 10.0 mm Luna C18(2) semi-prep column. Under these conditions, ‘free’ [203Pb]Pb2+ eluted with a retention time of ~4.6 min, while [203Pb][Pb(ampa)] eluted with a retention time of ~6.0 min. [213Bi][Bi(ampa)]+ and [225Ac][Ac(ampa)]+ radiolabeling. In their respective concentration dependent reactions, H2ampa stock solutions of 10-2 M were made in ultra-pure deionized H2O, with a portion of the stock solution used to make a serial dilution series ranging from 10-3 to 10-7 M. In the 213Bi radiolabeling study, reactions involved a total volume of 60 µL, with 10 µL of ligand solution (10-3 to 10-8 M), 30 µL of MES buffer (0.5 M, pH 5.5), and 20 µL of 213BiI4-/213BiI52- solution (~20 kBq). The control solutions used 10 µL of ultra-pure deionized H2O, 30 µL of MES buffer (0.5 M, pH 5.5), and 20 µL of 213BiI4-/213BiI52- solution (~20 kBq). 213BiI4-/213BiI52- solution was added within 5 minutes post-elution of the 225Ac/213Bi generator and 89 the mixtures were left for 5-7 minutes at room temperature. In the 225Ac radiolabeling study, reactions involved a total volume of 300 µL, with 30 µL ligand solution (10-3 to 10-7 M), 269 µL of NH4OAc buffer (pH 7, 0.2 M) and 1 µL of 225Ac stock (~40 kBq). Again, control solutions were used in which the only difference was ultra-pure deionized H2O replaced ligand solution. All solutions were left for 30 minutes at room temperature. The RCCs in these respective concentration dependent studies were monitored by instant thin-layer chromatography (iTLC) using silicic acid (SA)-impregnated paper TLC plates and EDTA mobile phase (50 mM) as the eluant. In the 213Bi studies, EDTA mobile phase was pH 5.5, while in the 225Ac studies it was pH 7. The TLC plates were scanned using an AR-2000 imaging scanner equipped with PD-10 gas, and analysis of their RCCs were carried out using WinScan V3 14 software. In the chromatograms produced, [213Bi][Bi(ampa)]+ and [225Ac][Ac(ampa)]+ remained at the baseline (Rf = 0), while other 213Bi and 225Ac species were found to migrate with the solvent front (Rf = 1). 3.4.6 Thermodynamic Solution Studies See Chapter 2.4.5 for details regarding set up and calibration procedure. Protonation equilibria of H2ampa solutions were studied by titrations of solutions containing ([L] = 1.002 x 10-3 M) at 25 ℃ and I = 0.16 M NaCl using a joint potentiometric-spectrophotometric procedure. Each titration consisted of 100-150 equilibrium points in the pH range 1.8-11 and electromotive force values were recorded after 2 min of each NaOH addition. Spectra were recorded in the 200-450 nm wavelength range with a 0.2 cm path length optic dip probe connected to a Varian Cary 60 UV-Vis spectrophotometer. Subsequent spectrophotometric and potentiometric data were 90 analyzed with HypSpec2014145 and HyperQuad2013118 to obtain the protonation constants (Table 3.3). Complex formation equilibria of H2ampa with Pb2+ and La3+ ions were studied using two different methods. First, UV-Vis spectrophotometric measurements (l = 1 cm) were carried out on a set of aqueous solutions containing 1:1 metal to ligand molar ratios ([H2ampa = [Mn+] = 1.44 x 10-4 M). These solutions involved different amounts of standardized HCl and NaCl to set the ionic strength constant at 0.16 M when possible. The molar absorptivities of all the protonated species were calculated with HypSpec2014 using the protonation constant from Table 3.3. The second method involved combined UV-potentiometric titrations (25 ℃, l = 0.2 cm, and I = 0.16 M NaCl) being carried out for the complex formation studies. Metal concentrations were: [La3+] = 7.46 x 10-4 M, [Y3+] = 8.58 x 10-4 M, and [Pb2+] = 6.01 x 10-4 M. Each metal solution was prepared by adding the atomic absorption (AA) standard metal ion solution to a ligand solution of known concentration in a 1:1 metal to ligand molar ratio. The exact amount of acid present in the metal standards were determined by Gran’s method117 titrating equimolar solution of the metal ion and Na2H2EDTA. Each titration consisted of 100-150 equilibrium points in the pH range 1.8-11.0, with equilibration time set up to 5 minutes. All the potentiometric measurements were processed using the Hyperquad2013 software.118 Speciation plots in Figure 3.8 were generated with Hyss119 using the protonation constants and stability constants in Table 3.3 and Table 3.4. For further details regarding equation conventions, see Chapter 2.4.5. 91 Chapter 4: Impact of Charge on [225Ac]Ac3+ Radiolabeling Studies 4.1 Introduction As discussed in Chapter 3.1.1, notable milestones have been achieved to establish targeted  therapy (TAT) as a form of radiation therapy within the clinic; however, the approval of an  emitting radionuclide that is part of a four-component radiopharmaceutical has yet to be achieved.31,56,57 Also covered in Chapter 3 were the promising decay properties of 225Ac, its successful clinical studies and the challenges 225Ac radiopharmaceuticals face due to limited access of 225Ac from 233U stockpiles.38,123,131,146 In this chapter, the additional challenge 225Ac radiopharmaceuticals face to reach the clinic will be addressed, which is the need for chelating agents that possess the ability to both rapidly radiolabel [225Ac]Ac3+ ions under mild conditions as well as form a kinetically inert complex. The majority of initial 225Ac chelation studies involved commercially available polydentate macrocyclic and open-chain chelators, such as DOTA, HEHA, EDTA, and DTPA (Chart 4.1).79,147–149 These studies showed DOTA was the most promising match. As a result, DOTA was conjugated to various targeting vectors and was the chelator used in the initial 225Ac-PSMA clinical trials that saw two patients with complete remission of their metastatic prostate cancer.131,150 Chart 4.1 Several of the chelators used in initial 225Ac chelation studies. 92 Actinium-225 chelation by DOTA still presents an issue for clinical applications. DOTA tends to require high temperatures and high ligand concentrations to effectively radiolabel [225Ac]Ac3+ ions.151–153 This becomes problematic when DOTA is conjugated to antibody targeting vectors, which are well-matched with 225Ac (t1/2 = 9.9 d) due to their slow blood circulation times and relatively long biological half-lives.36,154 There are alternates to avoid this issue. For example Maguire et al. have found conditions under which DOTA can radiolabel [225Ac]Ac3+ at room temperature, but the mixing times are still long at 2 hours.150 Another option is to initially radiolabel [225Ac]Ac3+ with DOTA at high temperatures, then undergo a bioconjugation reaction to link the radiometal-complex to the antibody; however, this procedure requires an additional purification step, which is more time consuming and significantly reduces the radiochemical yield of the radioactive drug. To surpass the radiolabeling abilities of DOTA, 18-membered macrocyclic scaffolds have been constructed, which also incorporate pendant functional groups that form singly charged or neutral complexes with Ac3+ ions. Notable examples of these macrocyclic chelators include H2macropa and crown (Chart 4.2).134,152 Despite having macrocyclic scaffolds, H2macropa and crown impressively radiolabel 225Ac at low concentrations (5.9 x 10-7 M and 10-6 M, respectively), within short times (15 min) and under room temperature conditions. In addition, both chelators form complexes that have demonstrated promising in vivo behaviour, although further studies are still required. As discussed in Chapter 1.5, the typical drawback of open-chain chelators is that they form less kinetically inert complexes than macrocyclic chelators. To compensate for this, new open-chain chelators are often designed to have an increased level of preorganization as binding pockets 93 (compared to more traditional chelators such as EDTA and DTPA). For example, open-chain chelators can be based on a rigid backbone that involves fewer bond rotations, such as a pyridine-based backbone. Alternatively, open-chain chelators can involve rigid arms, such as the picolinic acid moiety. Both of these choices have been applied in the design of H4py4pa (Chart 4.2), which for an open-chain chelator has few bonds able to rotate meaning less distortion is required for metal complexation and all 10 of the five-chelate membered rings formed upon complexation have a degree of rigidity.151 Owed to its donor groups and its relatively rigid structure, H4py4pa has demonstrated the ability to form a kinetically inert [225Ac]Ac3+ complex whilst maintaining its ability to radiolabel [225Ac]Ac3+ ions at low concentrations (10-6 M) and room temperature conditions. Thus, there is little to separate contemporary open-chain and macrocyclic chelators (Chart 4.2) in terms of their performance with [225Ac]Ac3+ ions. To offer more variation in [225Ac]Ac3+ complexation properties, such as polarity and charge, two open-chain chelators are reported herein. The first is H5decapa, which was originally published by Price et al. for 111In chelation.62 In that study, the biodistribution of [111In][In(decapa)]2- (amongst other results) showed H5decapa was incompatible with In3+ ions, most likely due this decadentate chelator being too large for In3+ ions. The large diethylene triamine backbone and high denticity of H5decapa was thought to be a better match with Ac3+ ions (106-122 pm for CN 6-9, respectively) since they have larger ionic radii than In3+ ions (62-92 pm for CN 4-8, respectively) and Ac3+ ions have been documented to form effective complexes with decadentate chelators, such as crown and H2macropa.102,130,134,151,152,155 The second chelator is H4noonpa, which was also considered an attractive candidate for 225Ac chelation due to its large scaffold and high denticity. This decadentate chelator was designed as a simpler synthetic alternative to H5decapa since the backbone only involves two primary amines. 94 Chart 4.2 Contemporary chelators used for 225Ac chelation studies. 4.2 Results and Discussion 4.2.1 Synthesis H5decapa (15) was produced in an overall yield of 11% (Scheme 4.1). The synthetic scheme relied on adding functional groups onto a diethylene triamine backbone over five-steps. These five steps involved: N-nosyl protection of the two primary amines belonging to the backbone, generating compound 11; N-alkylation of the three secondary amines with tert-butyl bromoacetate groups to form compound 12; thiophenol deprotection of the two nosyl groups to generate 13; a second set of N-alkylation reactions involving the regenerated secondary amines and compound 4, forming the ester protected ligand (14); and finally, ester hydrolysis reactions using heated HCl (6 M) solution to form H5decapa (15). 95 Scheme 4.1 The five-step synthetic route used to produce H5decapa (15) in an overall yield of 11%. This synthetic approach was inspired by the original scheme established by Price.62 In the original scheme, H5decapa was also developed over five-steps and involved the same alkyl halide SN2 reactions, in which tert-butyl acetate and picolinate moieties were added onto a diethylene triamine backbone; however, different amine protection group chemistry was applied. The original scheme relied on N-benzyl protection and hydrogenation deprotection steps, whereas Scheme 4.1 is based on N-nosyl protection (to form 11) and thiophenol deprotection (to form 13). This change of protection group led to H5decapa being synthesized in an improved yield, increasing from ~2.5% to 11%.62 There are two important points centered around the thiophenol deprotection step that led to this improved yield. First, thiophenol deprotection of nosyl groups is much more selective than hydrogenation deprotection of benzyl groups, which is evident from the yields at which compound 13 was formed. In Scheme 4.1, 13 was formed in a yield of 70%, whereas in the original route, 13 96 was obtained in a yield of 30% due to significant cleavage of the backbone.62 Second, there are two sets of N-alkylation reactions, one on either side of the deprotection step. It was found adding the tert-butyl acetate “arms” onto the backbone (to generate 12) before the picolinate arms were added onto the backbone (to form 14) was much more effective than adding these arms in the reverse order. Compound 13 has two secondary amines and three large hydrophobic tert-butyl ester groups to increase its partition coefficient for an organic phase during a liquid-liquid extraction. This enabled the K2CO3 base to more easily be separated from 13, which was necessary for subsequent column chromatography purification. The alternate order requires adding three nosyl groups onto the backbone (one on each primary amine and one on the central secondary amine), followed by N-alkylation with 4, and a subsequent deprotection step in which three secondary amines are regenerated. This route was considered since the deprotected intermediate is UV-active (due to the picolinate arms), whereas 13 is minimally UV-active (due to a lack of aromatic groups). However, with three secondary amines and just two methyl ester picolinate groups, the purification of this deprotected intermediate was considerably more challenging. The hydrophilic product formed stuck to the K2CO3 base during filtration and centrifugation removal techniques. The product also had little to no affinity for organic phases in liquid-liquid extractions. Upon developing an improved scheme for H5decapa (15) (Scheme 4.1), which was the best of three routes, the same scheme was applied to synthesize H4noonpa (19) (Scheme 4.2). H4noonpa was synthesized in a higher cumulative yield (22%) than H5decapa (11%). The main step that was easier to carry out for H4noonpa compared to H5decapa, was the N-nosyl protection step to form 5 versus 11, respectively. The ether-amino or NOON backbone of H4noonpa only has two terminal amines, whereas the diethylene triamine backbone of H5decapa has two terminal amines and a central secondary amine. This central secondary amine could react with the nosyl group during the 97 N-nosyl protection step, making synthesis and purification of 11 more challenging. As a result, 5 was formed in a yield of 81% (Scheme 4.2), while 11 was synthesized in a yield of 64% (Scheme 4.1). Scheme 4.2 The five-step synthetic route used to produce H4noonpa (19) in an overall yield of 22%. 4.2.2 Metal Complexation The respective complexes H5decapa and H4noonpa form with La3+ ions in the solution phase were studied via NMR spectroscopy. Non-radioactive La3+ ions were used as Ac3+ ion substitutes since only radioactive Ac3+ ions exist. The metal complexes were intensively studied using a combination of 1H, 13C{1H} and 2D (COSY, HSQC) NMR techniques to assign the number of isomers as well as the symmetry of the complexes. Radiolabeling studies for [225Ac]Ac3+ are usually performed at pH values near-physiological conditions since [225Ac]Ac3+ ions have a high 98 resistance to hydrolysis;130 thus, solutions of the metal complexes were adjusted to pD 6-7 using NaOD before collecting each spectrum, forming Na2[La(decapa)] and Na[La(noonpa)]. The 1H NMR spectrum of Na2[La(decapa)] displays several sharply resolved doublet signals and a broad complicated splitting pattern in the aliphatic region that reveal a single asymmetric metal complex (Figure 4.1). The doublets correspond to diastereotopic splitting (2J coupling) of the methylene groups associated with the picolinate and carboxylate arms of the ligand, while the broad signal between 2.93-3.27 ppm is largely due to the similar chemical shifts of the ethylene bridge methylenic protons (N-CH2-CH2-N) that experience 2J and 3J coupling. On the y-axis of the HSQC spectrum obtained, seven aliphatic 13C{1H} NMR signals are observed (Figure 4.2). For an asymmetric complex, nine aliphatic signals are expected; however, upon closer inspection the 13C{1H} NMR signal at 54.4 ppm corresponds to two backbone methylene groups, and the two 13C{1H} signals at 59.1 and 59.2 ppm correspond to three methylene groups. According to the COSY NMR spectrum, these three methylene groups at 59.1 and 59.2 ppm comprise one picolinate, one carboxylate and one backbone methylene group. Also, at least eight of the carbon signals in the aliphatic region of the HSQC spectrum have their related pair of protons split into two signals with different chemical shifts, confirming diastereotopic splitting. As a result, nine aliphatic signals can be accounted for, corresponding to a single asymmetric complex with rigid donor-metal interactions. 99 Figure 4.1 1H NMR spectra of H5decapa (400 MHz, 298 K, D2O, pD 1, top) and [La(decapa)]2- (400 MHz, 298 K, D2O, pD 6, bottom). Figure 4.2 HSQC spectrum of [La(decapa)]2- focused on the aliphatic region (400 MHz, 298 K, D2O, pD 6). 100 Compared to H4noonpa, the 1H NMR spectrum of Na[La(noonpa)] greatly increases in complexity due to the formation of one symmetric and one asymmetric isomer formed in an approximate ratio of 1:3 (Figure 4.3). The formation of one symmetric and one asymmetric isomer were also found in the 1H NMR spectrum of [La(ampa)](NO3), although in a ratio that strongly favoured asymmetric formation (1:7, respectively; discussed in Chapter 3.2.2). Consequently, switching the acetamides of H2ampa into the acetate groups of H4noonpa changes the HSAB preferences for La3+ ions so that the NOON scaffold more evenly forms two different isomers. Unlike H2ampa, which had a distinct singlet due to an unbound acetamide arm, there are no identifiable singlets to suggest any of the acetate arms of H4noonpa are unbound. Due to the relatively even formation of two different isomers, the 1H NMR spectrum of Na[La(noonpa)] and even its 2D (COSY, HSQC) NMR techniques have several indistinguishable signals. Fortunately, 13C{1H} NMR spectra provides more clear evidence to confirm one symmetric and one asymmetric complex have formed. Upon asymmetric coordination, the H4noonpa scaffold forms ten different 13C{1H} NMR signals in the aliphatic region, while upon symmetric coordination the aliphatic 13C{1H} environments remain as five signals (Figure 4.4). 101 Figure 4.3 1H NMR spectra of H4noonpa (top) (400 MHz, 298 K, D2O, pD 1) and [La(noonpa)]- (bottom) (400 MHz, 298K, D2O, pD 6). Figure 4.4 13C{1H} NMR spectra of H4noonpa (top) (400 MHz, 298 K, D2O, pD 1) and [La(noonpa)]- (bottom) (400 MHz, 298K, D2O, pD 6) focused on the aliphatic region. 102 4.2.3 [225Ac]Ac3+ Radiolabeling and Serum Stability Studies Both H5decapa and H4noonpa demonstrated high capabilities to radiolabel [225Ac]Ac3+ ions in their respective concentration-dependent radiolabeling studies. With an interest in 225Ac-radioimmunotherapy, the radiolabeling experiments were conducted under conditions amenable with antibodies: ambient temperatures (25-37 ℃) and aqueous buffer solution (NH4OAc, 0.2 M, pH 7). H5decapa radiolabeled [225Ac]Ac3+ ions within 15 minutes at 37 ℃ to generate high RCCs (> 85%) up to concentrations as dilute as 10-6 M, with RCCs dropping off at 10-7 M ligand concentration (~80%; Figure 4.5). H4noonpa radiolabeled [225Ac]Ac3+ ions within 15 minutes at room temperature, producing even higher RCCs with near quantitative radiolabeling (> 97%) being achieved up until extremely dilute concentrations (10-4-10-7 M). Figure 4.5 Using ~40-60 kBq of [225Ac]Ac3+, NH4OAc buffer (0.2 M, pH 7), 15 minute mixing times and RT conditions in each concentration-dependent study, H4noonpa and H5decapa achieved similarly high radiochemical conversions (RCCs) at low ligand concentrations (10-5-10-7 M), but the TLC conditions used for H4noonpa produced less error than the Chelex-resin method used for H5decapa. Ligand Concentration (10-x M)RCC (%)[225Ac][Ac(noonpa)]- TLC[225Ac][Ac(decapa)]2- Chelex resin0204060801004 5 6 7 8 9 103 The radiolabeling experiments carried out for H5decapa and H4noonpa involved the same [225Ac]Ac3+ production and purification procedures; however those conducted with H4noonpa were monitored by TLC while those involving H5decapa required a combination of Chelex-resin columns and gamma spectroscopy. Consequently, precise RCCs with very little variation between their duplicate trials were calculated in the H4noonpa radiolabeling studies, while imprecise RCCs were measured in the duplicate trials involving H5decapa. In the latter case, these varying RCCs are primarily due to the gamma spectrometry measurements. The RCCs were quantified based on 221Fr 218 keV gamma emissions involved in the 225Ac decay chain (221Fr, t1/2 = 4.8 min, 218 keV, 11.4% abundance) after secular equilibrium had been reached.143,144,156 Although error in the detection events can be reduced by increasing the amount of activity in each sample, or by measuring the samples for longer times, the logistics of conducting preliminary concentration-dependent studies in duplicates meant each vial (20 vials in total; ~30 kBq each) was only measured for 5-10 minutes to give errors between 5-10%; hence the large error bars observed for [225Ac][Ac(decapa)]2- in Figure 4.5. TLC conditions were explored for H5decapa, but separating [225Ac][Ac(decapa)]2- from solvated [225Ac]Ac3+ ions was found to be challenging. Although oppositely charged, both species are very hydrophilic and migrated in solvent fronts with streaking that prevents accurate RCCs to be determined. In addition, radiolabeling with [225Ac]Ac3+ requires halting the reactions after the desired mixing time (for example 15 min), but the RCCs cannot be measured until at least five hours afterwards to ensure secular equilibrium between 225Ac and its decay chain. As a result, HPLC was also not deemed a suitable method to monitor the reactions with H5decapa. 104 Given the compatibility of 225Ac-radioimmunotherapy comes from matching the long half-life of [225Ac]Ac3+ (t1/2 = 9.9) with the slow blood circulation times of antibodies, measuring the kinetic inertness of [225Ac][Ac(noonpa)]- and [225Ac][Ac(decapa)]2- in human blood serum was deemed necessary.35,150,157 For these in vitro competition experiments, the respective metal complex solutions were incubated in excess human blood serum proteins at 37 ℃. The amount of [225Ac]Ac3+ transchelated by native serum proteins was measured to be less than 2% for [225Ac][Ac(noonpa)]-, even after an incubation period as long as 10 days.41 On the other hand, ~7-13% of [225Ac]Ac3+ was released from [225Ac][Ac(decapa)]2- over its measured time points (Table 4.1). At first appearance, H4noonpa seems to form a more inert metal complex than H5decapa. Unfortunately, it was difficult to obtain precise values for the kinetically inertness of [225Ac][Ac(decapa)]2-, whereas precise values for [225Ac][Ac(noonpa)]- were easier to obtain. This was due to the methodologies applied when measuring [225Ac]Ac3+ transchelation. For [225Ac][Ac(noonpa)]-, iTLC was used to determine the % intact, while for [225Ac][Ac(decapa)]2- a combination of MeCN precipitation and gamma spectroscopy measurements were applied (see Chapter 4.4.3). The MeCN precipitation approach has several drawbacks. For example, the 7-13% of [225Ac]Ac3+ measured as being coordinated by proteins instead of H5decapa may have been [225Ac][Ac(decapa)]2- stuck to the precipitated serum proteins or the reaction vessel. Conversely, in the control studies, ~5-6% of [225Ac]Ac3+ was released by the proteins upon precipitation. Thus, using this method shows [225Ac][Ac(decapa)]2- forms a kinetically inert complex but quantifying the amount of [225Ac]Ac3+ that remains intact is limited to a range opposed to a precise value. 105 Table 4.1 Serum stability of H4noonpa and H5decapa with [225Ac]Ac3+. Time (h) Human serum stability of [225Ac][Ac(noonpa)]- (%) Human serum stability of [225Ac][Ac(decapa)]2- (%) 1 100 ± 0 87 ± 10 24 98.8 ± 1.2 90 ± 9 72 99.6 ± 0.1 - 120 - 86 ± 3 168 99.5 ± 0.1 - 192 - 93 ± 1 240 98.5 ± 0.8 - 4.3 Conclusion A new synthetic scheme was established to obtain H5decapa in an improved yield compared to its original scheme (~11% versus ~2.5%, respectively). Using the same sequence of reactions, H4noonpa was obtained in a higher overall yield (22%). Both chelators then demonstrated their capabilities to coordinate large La3+ ions in NMR spectroscopy studies. In these studies, [La(decapa)]2- formed a single asymmetric complex, while [La(noonpa)]- formed one symmetric complex and one asymmetric complex, approximately in a ratio of 1:3. In its [225Ac]Ac3+ concentration-dependent radiolabeling studies, H4noonpa obtained near quantitative RCCs within 15 minutes at room temperature, even at extremely low concentrations (10-7 M). H5decapa also demonstrated a high affinity for [225Ac]Ac3+ ions; however, the dianionic metal complex formed required more intricate methodologies to determine its RCCs. In addition, [225Ac][Ac(noonpa)]- was found to be a kinetically inert complex, with less than 2% of [225Ac]Ac3+ being transchelated by human proteins in the in vitro serum competition studies over one whole half-life (10 d). Again, [225Ac][Ac(decapa)]2- also demonstrated a high degree of kinetic inertness but the studies were hindered by the combination of MeCN precipitation and gamma spectroscopy 106 required to conduct measurements opposed to simple TLC measurements. Out of the two chelators, H4noonpa would therefore be more suitable to advance to the next stage of development, in which a bifunctional analogue is to be synthesized and tested for in vivo studies. 4.4 Experimental 4.4.1 Synthetic Materials and Methods For details regarding synthetic materials or instrumentation, see Chapter 2.4.1. 4.4.2 Synthesis and Characterization of H5decapa and H4noonpa N,N'-(Azanediylbis(ethane-2,1-diyl))bis(2-nitrobenzenesulfonamide) (11). Diethylene triamine (1.0 mL, 9.3 mmol, 1.0 equiv) was suspended in THF (40 mL), followed by the addition of Na2CO3 (2.04 g, 19.2 mmol, 2.1 equiv). 2-Nitrobenzene sulfonyl chloride (4.06 g, 18.3 mmol, 2.0 equiv), dissolved in THF (70 mL), was added dropwise over two hours at room temperature and the reaction mixture was left to stir overnight. The mixture was then filtered and the THF solvent was removed in vacuo, leaving an orange oil. CH2Cl2 (~20 mL) was added to the oil and the solution was heated at reflux for 20 minutes. The hot solution was filtered, left to cool, and put into a freezer overnight. Off-white crystals of 11 were obtained by filtration and dried on a high vacuum line (2.8 g, 5.9 mmol, 64%). 1H NMR (400 MHz, 298 K, (CD3)2CO):  8.12 (m, 2H), 7.97-7.87 (m, 6H), 3.10 (t, 3J = 6.1 Hz, 4H), 2.67 (t, 3J = 6.1 Hz, 4H). 13C{1H} NMR (100 MHz, 298 K, (CD3)2CO):  149.1, 134.8, 134.0, 133.5, 131.4, 125.8, 48.4, 43.9. HR-ESI-MS: calcd [C16H19N5O8S2 + H]+: 474.0748; found [M + H]+ 474.0749. Di-tert-butyl 2,2'-((((2-(tert-butoxy)-2-oxoethyl)azanediyl)bis(ethane-2,1-diyl))bis(((2-nitrophenyl)sulfonyl)azanediyl))diacetate (12). Compound 11 (2.64 g, 5.58 mmol, 1.0 equiv) was 107 dissolved in MeCN (30 mL), followed by the addition of K2CO3 (~3.0 g). Tert-butyl bromoacetate (3 mL, 20 mmol, 3.6 equiv) was added and the reaction mixture left to stir overnight, heated at 50 ℃. The solvent was removed in vacuo, and both CH2Cl2 (60 mL) and H2O (35 mL) were added to the leftover mixture. The organic layer was collected before the aqueous layer was further extracted with CH2Cl2 (60 mL) and EtOAc (60 mL). The organic layers were combined, dried with Na2SO4, filtered, and concentrated in vacuo to form an orange oil. The crude product was purified by silica chromatography (CombiFlash Rf automated column system; 2 x 40 g HP silica column; A: hexanes, B: EtOAc; 0% B to 60 % B) and the product fractions were collected and concentrated to give 12 as a pale-yellow oil product (3.72 g, 4.55 mmol, 82%). 1H NMR (400 MHz, 298 K, CDCl3):  8.09 (m, 2H), 7.68 (m, 4H), 7.58 (m, 2H), 4.16 (s, 4H), 3.46 (t, 3J = 6.8 Hz, 4H), 3.25 (s, 2H), 2.87 (t, 3J = 6.9 Hz, 4H), 1.45 (s, 9H), 1.35 (s, 18H). 13C{1H} NMR (100 MHz, 298 K, (CDCl3):  170.3, 167.9, 148.1, 133.5, 133.4, 131.9, 131.0, 82.3, 81.5, 56.3, 53.2, 49.6, 46.8, 28.2, 27.9. HR-ESI-MS: calc [C34H49N5O14S2 + H]+ 816.2790, found [M + H]+ 816.2788. Di-tert-butyl 2,2'-((((2-(tert-butoxy)-2-oxoethyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))diacetate (13). K2CO3 (1.95 g, 14.1 mmol, 3.7 equiv) and compound 12 (3.10 g, 3.80 mmol, 1 equiv) were mixed in dry THF (15 mL). As the mixture was heated to 50 ℃, thiophenol (2.0 mL, 19 mmol, 5 equiv) was added, turning the reaction mixture a strong yellow colour within seconds. The reaction mixture was left to stir overnight. The next day, the solvent was removed in vacuo. The mixture was then dissolved in CH2Cl2 (50 mL) and H2O (15 mL). The organic layer was collected before the aqueous layer was further washed with CH2Cl2 (4 x 50 mL) and EtOAc (2 x 50 mL). The organic layers were combined, dried with Na2SO4, filtered, and concentrated in vacuo. The mixture was purified by alumina chromatography (CombiFlash Rf automated column system; 2 x 48 g HP neutral alumina column; A: CH2Cl2, B: MeOH; 0% B to 108 5% B). Ninhydrin was used to help identify the fractions containing product, which were collected and concentrated to give a pale-yellow oil product (1.18 g, 2.65 mmol, 70%). 1H NMR (400 MHz, 298 K, CDCl3):  3.34 (s, 2H), 3.31 (s, 4H), 2.81 (t, 3J = 5.9 Hz, 4H), 2.66 (t, 3J = 5.9 Hz, 4H), 1.84 (s, 2H), 1.46 (s, 18H), 1.45 (s, 9H). 13C{1H} NMR (100 MHz, 298 K, CDCl3):  171.8, 171.0, 81.0, 80.9, 55.8, 54.3, 51.8, 47.5, 28.3, 28.2. HR-ESI-MS: calc [C22H43N3O6 + H]+ 446.3224, found [M + H]+ 446.3226. N,N’’-((6-Methoxycarbonyl)pyridine-2-yl)methylamino)-N,N’,N’’-((tert-butoxycarbonyl)methyl))diethylenetriamine (14). Compound 13 (0.751 g, 1.69 mmol, 1 equiv) was dissolved in dry MeCN (16 mL). K2CO3 (0.86 g, 6.2 mmol, 3.7 equiv) was added, followed by methyl 6-(bromomethyl)picolinate (0.78 g, 3.4 mmol, 2.0 equiv). The reaction mixture was heated to 40 ℃ and left to stir overnight. The following day, MeCN was removed in vacuo and the mixture was dissolved in CH2Cl2 (40 mL) and H2O (8 mL). The organic layer was collected, and the aqueous layer was further extracted with CH2Cl2 (3 x 40 mL) and EtOAc (50 mL). All the organic layers were collected, dried with Na2SO4, filtered, and concentrated in vacuo to an orange oil. The crude product was purified by silica chromatography (CombiFlash Rf automated column system; 40 g HP silica column; A: CH2Cl2, B: MeOH; 0% B to 10% B). The fractions containing pure product were collected and concentrated to afford a yellow oil product (0.53 g, 0.71 mmol, 42%). 1H NMR (400 MHz, 298 K, CDCl3):  7.98 (d, 3J = 8.6 Hz, 2H), 7.84 (d, 3J = 6.8 Hz, 2H) 7.78 (t, 3J = 7.7 Hz, 2H), 3.99 (s, 4H), 3.98 (s, 6H), 3.30 (s, 4H), 3.25 (s, 2H), 2.74 (s, 8H), 1.43 (s, 18H), 1.39 (s, 9H). 13C{1H} NMR (100 MHz, 298 K, CDCl3):  170.9, 170.7, 166.0, 161.2, 147.3, 137.5, 126.2, 123.7, 81.0, 80.8, 60.7, 56.6, 56.0, 53.0, 52.9, 52.8, 28.3, 28.3. HR-ESI-MS: calc [C38H57N5O10 + H]+ 744.4178, found [M + H]+ 744.4179. 109 H5decapa (15). 6 N HCl (8 mL) was added to compound 14 (0.488 g, 0.656 mmol, 1 equiv). The reaction mixture was stirred overnight at 50 ℃, monitored by mass spectroscopy. The mixture was then concentrated in vacuo, leaving an off-white product, which was purified by reverse phase HPLC (A: H2O (0.1% TFA), B: MeCN; 5 B to 50% B over 30 min with tR ~ 14.2 min). Pure fractions were combined and concentrated in vacuo to produce a white solid product. The TFA salt was converted into an HCl salt by adding 3 N HCl (5 mL) to the product, which was then reconcentrated in vacuo. This was repeated two more times. The solid product was dissolved one last time in H2O to be lyophilized. White crystalline solid H5decapa•4.4HCl was formed (0.326 g, 0.465 mmol, 71%). 1H NMR (400 MHz, 298 K, D2O, pD 0-1):  8.23-8.16 (m, 4H), 7.82 (d, 3J = 7.6 Hz, 2H), 4.61 (s, 4H), 3.95 (s, 4H), 3.73 (s, 2H), 3.50 (t, 3J = 6.3 Hz, 4H), 3.36 (t, 3J = 6.3 Hz, 4H). 13C{1H} NMR (75 MHz, 298 K, D2O, pD 0-1):  172.8, 170.1, 166.3, 150.7, 146.2, 141.8, 128.4, 126.0, 57.7, 54.9, 54.0, 51.9, 50.5. HR-ESI-MS: calc [C24H29N5O10 + H]+ 548.1993, found [M + H]+ 548.1995. Elemental analysis: calcd % for H5decapa•4.4HCl: C 40.72, H 4.76, N 9.89; found: C 40.84, H 4.80, N 9.82. N,N'-((Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-nitrobenzenesulfonamide) (5). See Chapter 2.4.2 to see experimental preparation of compound 5. Di-tert-butyl 3,12-bis((2-nitrophenyl)sulfonyl)-6,9-dioxa-3,12-diazatetradecanedioate (16). Compound 5 (1.008 g, 1.944 mmol, 1.0 equiv) was dissolved in dry MeCN (14 mL) and heated to 35 ℃. K2CO3 (1.17 g, 8.47 mmol, 4.4 equiv) was added, followed by tert-butyl bromoacetate (0.7 mL, 5 mmol, 2.5 equiv). The mixture was left to stir overnight. The next day, MeCN was removed in vacuo and the sticky mixture was dissolved using portions of acetone (4 x 25 mL) that were passed through a filter. The filtrate was concentrated in vacuo and dried before being purified using silica chromatography (CombiFlash Rf automated column system; 24 g HP 110 silica column; A: hexanes, B: EtOAc; 0% B to 100% B) to give a white sticky solid product (1.328 g, 1.78 mmol, 91%). 1H NMR (400 MHz, 298 K, CDCl3):  8.07 (m, 2H), 7.68 (m, 4 H), 7.71 (m, 2H), 4.21 (s, 4H), 3.65 (t, 3J = 4.9 Hz, 4H), 3.59 (t, 3J = 5.0 Hz, 4H), 3.50 (s, 4H), 1.36 (s, 18H). 13C{1H} NMR (100 MHz, 298 K, CDCl3):  168.0, 148.1, 133.7, 133.6, 131.8, 131.0, 124.1, 82.3, 70.5, 50.3, 48.2, 28.0. HR-ESI-MS: calcd [C30H42N4O14S2 + Na]+: 769.2032; found [M + H]+: 769.2030 Di-tert-butyl 6,9-dioxa-3,12-diazatetradecanedioate (17). Compound 16 (1.271 g, 1.702 mmol, 1 equiv) was mixed with dry THF (10 mL) and K2CO3 (0.71 g, 5.1 mmol, 3 equiv). As the mixture was heated to 45 ℃, thiophenol (0.79 mL, 7.7 mmol, 4.5 equiv) was added and the reaction mixture was left to stir overnight. TLC indicated the reaction progress had gone to completion and the solvent was removed in vacuo. A liquid-liquid extraction was carried out using H2O (10 mL), CH2Cl2 (5 x 60 mL), and a final organic wash with EtOAc (50 mL). The organic layers were collected, dried with Na2SO4, filtered, and concentrated in vacuo. The product was then purified using alumina chromatography (CombiFlash Rf automated column system; 40 g HP neutral alumina column; A: CH2Cl2, B: MeOH; 0% B to 10% B) to give a yellow oil product (0.495 g, 1.32 mmol, 77%). 1H NMR (400 MHz, 298 K, CDCl3):  3.66 (t, 3J = 5.1 Hz, 4H), 3.64 (s, 4H), 3.39 (s, 4H), 2.86 (t, 3J = 5.2 Hz, 4H), 1.47 (s, 18H). 13C {1H} NMR (100 MHz, 298 K, CDCl3):  171.6, 81.1, 70.8, 70.4, 51.8, 48.8, 28.2. HR-ESI-MS: calcd [C18H36N2O6 + H]+: 377.2645; found [M + H]+: 377.2648. Di-tert-butyl 3,12-bis((6-(methoxycarbonyl)pyridin-2-yl)methyl)-6,9-dioxa-3,12-diazatetradecanedioate (18). Compound 17 (0.459 g, 1.22 mmol, 1 equiv) was mixed with dry MeCN (5 mL) and K2CO3 (0.80 g, 5.7 mmol, 4.7 equiv). Methyl 6-(bromomethyl)picolinate (0.62 g, 2.7 mmol, 2.2 equiv) was added, and the reaction mixture was left to stir overnight, heated at 111 50 ℃. After, the solvent was removed in vacuo and a liquid-liquid extraction was carried out using H2O (10 mL) and CH2Cl2 (50 mL). The aqueous layer was further washed with CH2Cl2 (4 x 50 mL) and EtOAc (50 mL). The organic layers were collected, dried with Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica chromatography (CombiFlash Rf automated column system; 40 g HP silica column; A: CH2Cl2, B: MeOH; 0% B to 10% B). The product came off at ~2% MeOH, and the appropriate fractions were collected and concentrated to produce an oil product (0.434 g, 0.643 mmol, 53%). 1H NMR (300 MHz, 298 K, CDCl3):  7.98 (d, 3J = 7.5 Hz, 2H), 7.89 (d, 3J = 7.3 Hz, 2H), 7.79 (t, 3J = 7.7 Hz, 2H), 4.06 (s, 4H), 3.98 (s, 6H), 3.55 (t, 3J = 5.8 Hz, 4H), 3.50 (s, 4H), 3.38 (s, 4H), 2.90 (t, 3J = 5.8 Hz, 4H), 1.44 (s, 18H). 13C {1H} NMR (75 MHz, 298 K, CDCl3):  170.9, 166.1, 161.4, 147.3, 137.5, 126.4, 123.7, 81.1, 70.4, 70.1, 61.0, 57.0, 53.8, 53.0, 28.3. HR-ESI-MS: calcd [C34H50N4O10 + H]+: 675.3600; found [M + H]+: 675.3604. H4noonpa (19). 5 N HCl (3 mL) was added to compound 18 (0.208 g, 0.308 mmol, 1 equiv). The reaction mixture was then left to stir overnight, heated at 50 ℃. The solvent was removed in vacuo to form an off-white solid. The crude product was dissolved in H2O (15 mL) and purified by HPLC (A: H2O (0.1% TFA), B: MeCN; 5% B to 50% B over 30 min with tR ~ 15.6 min). The fractions containing pure product were collected and concentrated in vacuo. The product was then redissolved in H2O and lyophilized to yield H4noonpa•2.9TFA•H2O (0.193 g, 0.225 mmol, 73%). 1H NMR (400 MHz, 298 K, D2O, pD 0-1):  8.15 (d, 3J = 6.9 Hz, 2H), 8.08 (t, 3J = 7.8 Hz, 2H), 7.73 (d, 3J = 7.7 Hz, 2H), 4.77 (s, 4H), 4.16 (s, 4H), 3.88 (t, 3J = 4.7 Hz, 4H), 3.62 (t, 3J = 4.7 Hz, 4H), 3.60 (s, 4H). 13C {1H} NMR (75 MHz, 298 K, D2O, pD 0-1):  168.9, 167.4, 149.7, 146.9, 140.3, 128.3, 125.8, 69.8, 64.7, 58.7, 55.2, 55.1. HR-ESI-MS: calcd [C24H30N4O10]: 534.1962; 112 found [M]: 534.1964. Elemental analysis: calcd % for H4noonpa•2.9TFA•H2O: C 40.53, H 3.98, N 6.34; found: C 40.39, H 4.17, N 6.47. 4.4.3 Metal Complexation The NMR spectrum of Na2[La(decapa)] was obtained by dissolving H5decapa•4.4HCl (13.18 mg, 18.8 µmol) in D2O (0.7 mL) and adding the solution to La(NO3)3•6H2O (9.99 mg, 23.1 µmol, 1.2 equiv) with the pD increased to 6 using NaOD solutions (1 M and 0.1 M; diluted from 40 wt. % NaOD). The NMR spectrum of Na[La(noonpa)] was obtained by dissolving H4noonpa•2.9TFA•H2O (21.2 mg, 26.8 µmol) in D2O (1.0 mL) and adding the solution to La(NO3)3•6H2O (13.2 mg, 30.5 µmol, 1.1 equiv) with the pD increased to 7 using NaOD solutions (1 M and 0.1 M). Both metal complex solutions were sonicated at 20-25 ℃ for 1 minute prior to obtaining their NMR spectra. 4.4.4 Radiolabeling and Human Serum Stability Experiments 225Ac was produced at the TRIUMF 500 MeV Isotope Production Facility (IPF) via irradiation of 232Th foils with full details described elsewhere.143,144 Before radiolabeling studies were conducted, [225Ac]Ac3+ radioactivity was quantified using a high-purity germanium (HPGe) detector (Mirion Technologies (Canberra) Inc.) with Genie 2000 software after the attainment of secular equilibrium with daughters 221Fr (t1/2 = 4.8 min, 218 keV, 11.4% abundance) and 213Bi (t1/2 = 45.6 min, 440 keV, 25.9% abundance). [225Ac][Ac(noonpa)]- Radiochemistry. A stock solution of H4noonpa was prepared to 10-3 M in ultrapure deionized H2O (1.01262 Supelco). A portion of this stock solution was diluted to give a serial dilution series ranging from 10-4 to 10-7 M. Radiolabeling reactions in the 113 concentration dependent study were then prepared to a total volume of 300 µL, using 30 µL ligand solution (10-3 to 10-7 M), 269 µL of NH4OAc buffer (0.2 M, pH 7) and 1 µL of 225Ac stock (~40 kBq). Reactions were left for 15 minutes at room temperature. After, the RCCs were determined by TLC (SA paper plates, EDTA mobile phase, 50 mM, pH 7). The TLC plates were scanned with an AR-2000 imaging scanner equipped with PD-10 gas, and analysis of RCCs were carried out using WinScan V3 14 software. Under these conditions, [225Ac][Ac(noonpa)]- remained at the baseline (Rf = 0), while other 225Ac species migrated with the solvent front (Rf = 1). For the serum stability challenge experiment involving H4noonpa, a radiolabeling reaction was prepared with 800 µL NH4OAc buffer (0.2 M, pH 7), 90 µL of 10-4 M ligand stock solution, and 12 µL of 225Ac stock (~400 kBq). The radiolabeling reaction was left for 1 h, and quantitative yield was confirmed by TLC (SA paper plates; EDTA mobile phase, 50 mM, pH). Since the RCC had to be determined after 6 h, prior to its calculation, the reaction was split into three vials (300 µL each), and an equivalent amount of human serum (300 µL) was added. The samples were incubated at 37 ℃ and monitored for stability against serum proteins by TLC over a 10-day period. [225Ac][Ac(decapa)]2- Radiochemistry. Each Chelex-100 column was prepared by measuring out 100-150 mg of Chelex-100 resin and packing it into a reservoir column (1 mL) on top of a frit. A second frit was pushed into the column to form a tightly packed resin layer, which was then pre-equilibrated with NH4OAc buffer (2 mL, 0.2 M, pH 7). The columns were prepared the day of radiolabeling experiments but were still sealed while stored. A stock solution of H5decapa was prepared to 10-2 M in ultrapure deionized water. Using a portion of this stock solution, a dilution series from 10-3 to 10-7 M was prepared. Each radiolabeling reaction involved 20 µL of ligand solution (10-3 to 10-7 M), 170 µL of NH4OAc buffer (0.2 M, pH 7), and 10 µL of 225Ac solution (~60 kBq). With staggered timing, the reactions 114 were left for 15 minutes at 37 ℃ before a 90 µL portion of the reaction mixture was loaded onto a Chelex-100 packed column. Using a syringe and adapter to push air through the column, NH4OAc buffer (2 mL, 0.2 M, pH 7) was used to wash [225Ac][Ac(decapa)]2- off the resin and into a scintillation vial. Each elution vial was then diluted to 20 mL with ultrapure deionized H2O. An equal portion of reaction mixture (90 µL) was directly added to a different scintillation vial containing 20 mL of ultrapure deionized H2O. This procedure was applied to each reaction and all the vials were left overnight before their activities were quantified via gamma spectroscopy. In the gamma spectrometer, the geometry and height of the vials were calibrated prior to measurement. The samples were counted until the uncertainty was ~5-10% of the signal, with a dead time ~1-5%. The RCCs were then determined using Equation 4.1: 𝑅𝐶𝐶 = (𝑒𝑙𝑢𝑡𝑒𝑑 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦⁄ ) × 100 (4.1) In the control samples, the elute vials found ~97% of solvated [225Ac]Ac3+ ions were effectively retained by the resin meaning this approach was relatively effective; however, the RCCs were determined in large errors given each vial was counted until the uncertainty was ~5-10% of the signal (Equation 4.2). 𝑅𝐶𝐶 𝑒𝑟𝑟𝑜𝑟 = √(𝑒𝑙𝑢𝑡𝑒𝑑 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑒𝑟𝑟𝑜𝑟)2 + (𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 𝑎𝑐𝑡𝑣𝑖𝑡𝑦 𝑒𝑟𝑟𝑜𝑟)2 × 100 (4.2) For the serum stability challenge experiment involving H5decapa, a radiolabeling reaction was prepared with 1320 µL NH4OAc buffer (0.2 M, pH 7), 150 µL of 10-3 M ligand stock solution, and 30 µL of 225Ac stock (~1.5 MBq). The radiolabeling reaction was left for 3 h, until a 100 µL 115 portion was transferred to a Chelex-resin column and another 100 µL portion was directly added into a total activity vial. These vials were used to confirm [225Ac]Ac3+ had been quantitatively radiolabeled. Since the RCC had to be determined after 6 h, prior to its measurement, the remaining reaction solution was split into three vials (400 µL each), and an equivalent amount of human serum (400 µL) was added. The samples were incubated at 37 ℃ and monitored for stability against serum proteins by TLC over an 8-day period. For each time point measurement involving [225Ac][Ac(decapa)]2-, a 90 µL portion of the solution was transferred to a new Eppendorf vial and 90 µL of MeCN was added to precipitate the serum proteins out of solution. This allowed for the separation of supernatant (containing [225Ac][Ac(decapa)]2-) from the precipitate ([225Ac]Ac3+ chelated by proteins) via centrifugation (14000 rpm, 20 min). The supernatant layer was added to a scintillation vial containing ultrapure deionized H2O (20 mL). At the same time point, another 90 µL portion from the competition mixture was directly transferred to a different scintillation vial containing ultrapure deionized H2O (20 mL). The activities in each vial were then quantified using the high-purity germanium (HPGe) gamma detector and the % remaining intact was determined by Equation 4.3: % 𝑖𝑛𝑡𝑎𝑐𝑡 (𝑝𝑒𝑟 𝑡𝑖𝑚𝑒 𝑝𝑜𝑖𝑛𝑡) = (𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦⁄ ) × 100 (4.3) Control trials were conducted in parallel using with 1320 µL NH4OAc buffer (0.2 M, pH 7), 150 µL ultrapure deionized water, and 30 µL of 225Ac stock (~1.5 MBq). The same approach applied to [225Ac][Ac(decapa)]2- solutions were carried out for the controls, including the measurements being carried out in triplicate. 116 Chapter 5: Conclusion and Future Work 5.1 Lone Pair Activity Chapter 2 focused on the chelating ability of EGTAm and ampam to coordinate [nat/203Pb]Pb2+ ions. Both chelators were found to coordinate non-radioactive Pb2+ ions in various studies. The most structurally informative studies were the X-ray diffraction studies involving crystal structures of their respective Pb2+ complexes. In these studies, EGTAm and ampam were revealed to fully coordinate to the metal ion to form geometries in which the Pb2+ lone pair of electrons was stereochemically inactive. Although able to encapsulate Pb2+ ions, both chelators demonstrated a low affinity for [203Pb]Pb2+ ions (~0.25 MBq/µmol) in their respective radiolabeling studies and neither chelator was a compatible match with radioactive Pb2+ ions. Despite their poor radiolabeling abilities, the results obtained for EGTAm and ampam were still informative and provided an important point of reference to suggest how amides may be better utilized when paired with borderline acidic Pb2+ ions. The literature was an essential aid to Chapter 2, as there were previous [nat/203Pb]Pb2+ ion chelator coordination studies and a developed theory on how the lone pair is influenced by various chelator properties.80,94 Guided by the literature, the low affinities of EGTAm (N2O6) and ampam (N4O6) for [203Pb]Pb2+ ions were explained by their large NOON backbones sterically dominating to force holodirected complexation, despite their donor atoms energetically favouring hemidirected complexation. Although EGTAm and ampam were designed to be softer than carboxylate analogues, according to this analysis, amides may be considered as relatively hard and are better utilized on chelator scaffolds that encourage a degree of lone pair activation on the Pb2+ ion.59 117 Since both EGTAm and ampam were deemed unsuitable with [203/212Pb]Pb2+ ions, further studies with these chelators are not necessary, although modifications to these chelators may still be worth exploring as demonstrated by H2ampa in Chapter 3. 5.2 Versatility H2ampa was designed to be a modified version of ampam, in which the picolinamide groups of ampam were replaced by picolinic acid groups. The initial motivation for this adaptation was to assess how its anionic charge (as ampa2-) would affect its affinity for Pb2+ ions. It was found that H2ampa achieved a molar activity with [203Pb]Pb2+ ions over two orders of magnitude higher than ampam (~20 MBq/µmol versus ~0.25 MBq/µmol, respectively). This result contrasts with the improved molar activities achieved with neutrally charged TCMC compared to anionic DOTA.52 Furthermore, the high denticity of H2ampa encourages holodirected complexation; however, it has hard donor groups and is anionically charged (as ampa2-), which favour hemidirected complexation.94 Given the poor affinity of ampam, which is known to form a Pb2+ complex with an inactive lone pair, it was suggested H2ampa forms a Pb2+ complex in which there is at least partial lone pair activity. These studies therefore indicate that not only is the optimal hardness of donor groups dependent on the ligand scaffold, but chelator charge may take more priority on determining the lone pair activity opposed to other design features.96 Given the promising results obtained with picolinate-based chelators with trivalent metals, H2ampa was also applied to [213Bi]Bi3+ and [225Ac]Ac3+ chelation.133,134 From its preliminary radiolabeling studies, H2ampa proved to be a versatile chelator able to effectively coordinate at least three different radiometals that varied in size, charge, and hardness. Each of the  therapeutics have their own unique advantages, making the versatility of H2ampa highly appealing medically. 118 Targeting Pb2+ ions is an ongoing investigation for at least three reasons. First, many different donor groups can coordinate these metal ions, ranging from hard anionic oxygen donors to soft neutral sulfur donor groups.158 Second, there are several chelator properties that impact the stereochemical activity of the lone pair.94 Third, it appears there is a hierarchy amongst the chelator properties that influence lone pair activity.96 With these reasons in mind, obtaining a crystal structure of [Pb(ampa)] is a vital part of future work since the structure would provide more definitive insight into the directionality of this complex and may explain why H2ampa has a much greater affinity for Pb2+ ions than does ampam. In addition, TLC separation of solvated [203/212Pb]Pb2+ ions from the metal complex species would be highly beneficial. An effective TLC system would enable radiolabeling conditions to be optimized more easily as well as provide a convenient method for in vitro stability studies. Other further work should include in vitro stability studies involving [225Ac]Ac3+ ions. [213Bi]Bi3+ in vitro stability studies are optional given this radionuclide has a short half-life. If any of the in vitro stability studies involving these radiometals were found to be promising, a bifunctional analogue of H2ampa that could be conjugated to biomolecules would be the next step to further establish its potential as a radiopharmaceutical component. From the results obtained with H2ampa, further work involving a different chelator is suggested. A chelator known as py-macrodipa (Chart 5.1) coordinates metal ions of disparate sizes.142 Although macrocyclic, it has a long flexible tri-ether-bridged backbone that provides the option to bind to larger metals, such as [132/135La]La3+, or for the ether groups to remain unbound when the main binding cavity is coordinated to smaller metals, such as [44Sc]Sc3+. To my knowledge, this chelator has not been tested with Pb2+ ions. This chelator would be an interesting study since it offers Pb2+ ions a choice. The backbone is likely to be flexible enough to allow 119 certain ether groups to remain unbound to the Pb2+ ion, enabling the lone pair to be stereochemically active, or it can bind as a decadentate chelator and suppress lone pair activity. In addition, the picolinic acids may bind from the same plane or one may bind above and one below. This is a rare case in which the chelator can choose the optimal geometry, which may be particularly useful for Pb2+ ions given the plethora of chelator properties. Chart 5.1 Chemical structures of py-macrodipa (left) and its bifunctional analogue (right), a suggested chelator system for future [203/212Pb]Pb2+ chelation studies. Py-macrodipa is also well designed regarding the synthesis of a bifunctional analogue. Developing the same chelator but with a backbone pyridine-4-ol (Chart 5.1) would allow facile attachment of a linker from this hydroxyl group in the para-position, which also maintains the denticity of chelator and is unlikely to interfere with any donor group interactions upon metal ion coordination.142,151,159 5.3 Importance of Charge Chapter 4 focused on [225Ac]Ac3+ chelation and involved two decadentate chelators: H5decapa and H4noonpa. H5decapa was matched with [225Ac]Ac3+ ions due to its relatively large scaffold, high denticity and highly protic functional groups. This chelator (as decapa5-) forms a dianionic complex when coordinated to [225Ac]Ac3+ ions. The synthesis of H5decapa presented 120 many challenges and inspired the synthesis of H4noonpa, a simpler alternative. Unexpectedly, the monoanionic complex formed when noonpa4- coordinates [225Ac]Ac3+ ions turned out to be much more favourable for radiochemistry investigations compared to those carried out with decapa5-. When designing chelators to coordinate metal ions, charge is usually considered in relation to the HSAB properties of the chelator and its target metal ion, as well as how the charge will affect the pharmacokinetics of the drug. From the challenges presented by [225Ac][Ac(decapa)]2- compared to [225Ac][Ac(noonpa)]-, anticipating how charge will impact the ease of radiolabeling studies at a practical level is also important. Not only was determining the suitability of H5decapa for [225Ac]Ac3+ ions affected by its lack of TLC measurements, but the methods involving gamma spectroscopy measurements were more time consuming and generated large amounts of radioactive waste given each measurement requires two 20 mL scintillation vials. TLC measurements therefore have better transferability when expanding from research experiments to more commercial scales. Given the challenges [225Ac]Ac3+ already presents for HPLC measurements, designing the overall [225Ac]Ac3+ complex charge to be singly charged or neutral is advised since this increases the chance of obtaining successful TLC conditions.156 As shown with [225Ac]Ac3+, H5decapa may also have difficulties establishing TLC conditions with other trivalent radiometals such as [213Bi]Bi3+ ions. Instead, [226/227Th]Th4+ may be a stronger match. Few chelators are pentaprotic like H5decapa, thus [226/227Th][Th(decapa)]- offers a relatively rare monoanionic thorium complex, and the large scaffold, high denticity and highly protic functional groups of H5decapa are also likely to be well-suited with a highly charged Th4+ ion, which has been documented to form several ten-coordinate metal complexes.160 There are various further studies that could be conducted with H4noonpa. Firstly, H2ampa showed a high affinity for [213Bi]Bi3+ ions. It is therefore likely H4noonpa will similarly be well- 121 matched with [213Bi]Bi3+ ions. Investigating its affinity with [203/212Pb]Pb2+ ions is a possibility but it would form a dianionic complex that may also present challenges for the radiochemistry studies. 5.4 Bifunctional Chelator Development Of all the chelators studied within this document, H4noonpa is likely to be best chelator to put forward for bifunctional development. This suggestion is based on H4noonpa forming an [225Ac]Ac3+ complex that has excellent stability in human serum, and its superior radiolabeling ability: H2ampa, crown and H4py4pa radiolabel 225Ac at 10-6 M, while H4noonpa consistently radiolabels 225Ac at 10-7 M under similar conditions.151,152 To synthesize a bifunctional version of H4noonpa, a set of schemes has been recommended (Schemes 5.1-5.3). Many of the reactions proposed are inspired by the work of Li et al., who published a synthetic scheme for bifunctional H4pypa.159 Li et al. synthesized compounds 20 and 21 in yields of 99% and 64%, respectively (Scheme 5.1). Compounds similar to 22 and 23 were then synthesized in yields of 82% and 70%, respectively. The synthesis of compound 17 has already been established by the route used to form H4noonpa (19), discussed in Chapter 4.2.1 (Scheme 5.2, or part of Scheme 4.2). According to the synthetic route used to make bifunctional H4pypa,159 synthesizing bifunctional H4noonpa (29) should be feasible; however, Scheme 5.3 does involve adaptations so there is a large uncertainty in the overall yield 29 can be synthesized in. An additional reason Schemes 5.1-5.3 have been recommended is because of how 29 would be derivatized. In this design, one of the chelator arms has a linker attached at the para-position of the pyridine ring. This maintains the denticity of the chelator and sterically places the linker away from the metal ion coordination pocket to give the best chance of preserving the coordinating properties of the H4noonpa. After 29 is synthesized, the next steps would involve a bioconjugation 122 reaction with a targeting vector, then a radiolabeling study with [225Ac]Ac3+, followed by tests that evaluate the radiopharmaceutical’s performance in vivo. Scheme 5.1 Proposed route to form a modified picolinic acid moiety (23) that can be incorporated into bifunctional H4noonpa, inspired by a similar synthetic route established by Li et al.159 Scheme 5.2 Part of the established route used to synthesize H4noonpa (involving compounds 5, 16 and 17). 123 Scheme 5.3 Proposed route to synthesize bifunctional H4noonpa (29) from compound 17, inspired by a similar synthetic route established by Li et al.159 124 Bibliography 1) M. R. Gill, N. Falzone, Y. Du and K. A. Vallis, Lancet Oncol., 2017, 18, e414–e423. 2) W. 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Supplementary Data for Chapter 2 Table A.1 Crystallographic information for [Pb(EGTAm)(NO3)](NO3).H2O and [Pb(ampam)](NO3)2.xH2O Compound [Pb(EGTAm)(NO3)](NO3).H2O [Pb(ampam)](NO3)2.xH2O Formula C14H30N8O13Pb C24H38.17N10O14.67Pb Dcalc/g cm-3 2.008 1.833 /mm-1 7.108 5.208 Formula Weight 725.65 908.68 Colour colourless colourless Shape needle irregular Size/mm3 0.29x0.15x0.08 0.19x0.15x0.07 T/K 110(2) 115(2) Crystal System monoclinic monoclinic Space Group P21/c C2/c a/Å 11.2179(2) 14.0872(7) b/Å 27.5158(5) 22.5964(10) c/Å 7.7860(2) 12.3531(9) /° 90 90 /° 92.6860(10) 123.133(2) /° 90 90 V/Å3 2400.66(9) 3292.9(3) Z 4 4 Z' 1 0.5 Wavelength/Å 0.71073 0.71073 Radiation type MoK MoK min/° 1.962 1.802 max/° 30.553 30.705 Measured Refl. 34585 19580 Independent Refl. 7328 5069 Reflections with I > 2(I) 5751 4486 Rint 0.0689 0.0615 Parameters 344 333 Restraints 0 139 Largest Peak 2.514 1.568 Deepest Hole -2.005 -2.072 GooF 1.075 1.022 wR2 (all data) 0.0785 0.0742 wR2 0.0735 0.0710 R1 (all data) 0.0600 0.0388 R1 0.0400 0.0319 138 Table A.2 Selected crystallographic bond angle information for [Pb(EGTAm)(NO3)](NO3).H2O Atom Atom Atom Angle/° Atom Atom Atom Angle/° O1 Pb1 O2 90.19(11) O5 Pb1 O4 123.17(10) O1 Pb1 O3 92.53(10) O5 Pb1 O6 90.30(11) O1 Pb1 O4 135.14(10) O5 Pb1 O7 88.98(10) O1 Pb1 O5 72.28(10) O5 Pb1 O8 128.52(10) O1 Pb1 O6 71.91(11) O5 Pb1 N2 112.19(11) O1 Pb1 O7 154.91(11) O5 Pb1 N5 60.37(10) O1 Pb1 O8 138.45(10) O6 Pb1 O2 159.46(10) O1 Pb1 N2 62.55(11) O6 Pb1 O3 76.60(10) O1 Pb1 N5 111.51(10) O6 Pb1 O4 66.66(10) O2 Pb1 O4 133.24(10) O6 Pb1 O7 126.05(10) O2 Pb1 O7 68.29(10) O6 Pb1 O8 132.68(10) O2 Pb1 O8 67.65(10) O6 Pb1 N2 117.56(10) O2 Pb1 N5 117.91(10) O6 Pb1 N5 62.55(10) O3 Pb1 O2 115.19(9) O7 Pb1 O8 45.51(9) O3 Pb1 O4 62.15(9) N2 Pb1 O2 58.96(10) O3 Pb1 O7 107.95(9) N2 Pb1 O4 124.61(10) O3 Pb1 O8 68.61(9) N2 Pb1 O7 112.38(11) O3 Pb1 N2 65.72(11) N2 Pb1 O8 75.91(10) O3 Pb1 N5 120.67(10) N2 Pb1 N5 172.31(11) O4 Pb1 O7 69.14(10) N5 Pb1 O4 62.93(10) O4 Pb1 O8 69.07(10) N5 Pb1 O7 70.71(10) O5 Pb1 O2 74.30(11) N5 Pb1 O8 109.92(10) O5 Pb1 O3 162.59(11) 139 Table A.3 Selected crystallographic bond angle information for [Pb(ampam)](NO3)2.xH2O Atom Atom Atom Angle/° Atom Atom Atom Angle/° O1 Pb1 O11 84.37(10) O31 Pb1 O1 143.73(7) O11 Pb1 N21 59.12(7) O31 Pb1 O11 116.90(7) O1 Pb1 N21 111.33(7) O3 Pb1 O11 143.73(7) O11 Pb1 N2 111.33(7) O31 Pb1 O2 67.83(7) O1 Pb1 N2 59.12(7) O3 Pb1 O21 67.83(7) O11 Pb1 N3 70.95(7) O3 Pb1 O2 77.57(7) O11 Pb1 N31 94.98(8) O31 Pb1 O21 77.57(7) O1 Pb1 N3 94.98(8) O31 Pb1 O3 63.73(10) O1 Pb1 N31 70.95(7) O31 Pb1 N21 64.55(7) O21 Pb1 O11 147.99(7) O3 Pb1 N21 126.95(7) O21 Pb1 O1 70.68(7) O31 Pb1 N2 126.95(7) O2 Pb1 O11 70.68(7) O3 Pb1 N2 64.55(7) O2 Pb1 O1 147.99(7) O3 Pb1 N31 119.02(8) O2 Pb1 O21 139.16(10) O31 Pb1 N31 77.84(8) O21 Pb1 N2 73.05(8) O3 Pb1 N3 77.84(8) O21 Pb1 N21 111.22(8) O31 Pb1 N3 119.02(8) O2 Pb1 N2 111.22(8) N2 Pb1 N21 168.42(11) O2 Pb1 N21 73.05(8) N3 Pb1 N2 58.72(8) O21 Pb1 N3 129.36(8) N31 Pb1 N2 119.12(8) O21 Pb1 N31 58.57(8) N3 Pb1 N21 119.12(8) O2 Pb1 N31 129.36(8) N31 Pb1 N21 58.72(8) O2 Pb1 N3 58.57(8) N3 Pb1 N31 161.40(12) O3 Pb1 O1 116.90(7) Table A.4 HR-ESI-MS data for EGTAm and ampam, as well as their respective Pb2+ complexes. Species Formula Calcd (m/z) Found (m/z) EGTAm C14H28N6O6 376.2074 376.2070 EGTAm-Pb C14H27N6O6Pb 579.1722 579.1724 ampam C24H34N8O6 530.2601 530.2606 ampam-Pb C24H33N8O6Pb 733.2253 733.2256 140 Figure A.1 1H NMR spectrum of EGTAm/compound 2 (300 MHz, 298 K, D2O, pD 6-7). Figure A.2 13C{1H} NMR spectrum of EGTAm/compound 2 (100 MHz, 298 K, D2O, pD 6-7). 141 Figure A.3 1H NMR spectrum of ampam/compound 9 (300 MHz, 298 K, D2O, pD 6-7). Figure A.4 13C{1H} NMR spectrum of ampam/compound 9 (100 MHz, 298 K, DMSO). 142 Figure A.5 A) Titration plot of ~11 mL of [EGTAm] = 9.99 × 10-4 M with [NaOH] = 0.16 M at 25 ℃ and I = 0.16 M (NaCl); B) Titration plot of ~15 mL of [Pb2+] = [EGTAm] = 6.63 × 10-4 M with [NaOH] = 0.16 M at 25 ℃ and I = 0.16 M (NaCl). EGTAM_2B0 0.2 0.4 0.6titre volume357911pH0 0.2 0.4 0.6titre volume-0.200.2Weighted residuals0102030405060708090100% formation relative to EGTAMPb_EGTAM_1B0 1.0 2.0titre volume246810pH0 1.0 2.0titre volume-101Weighted residuals0102030405060708090100% formation relative to PbA B 143 Figure A.6 A) Titration plot of ~11 mL of [ampam] = 9.50 × 10-4 M with [NaOH] = 0.16 M at 25 ℃ and I = 0.16 M (NaCl); B) Titration plot of ~15 mL of [Pb2+] = [ampam] = 6.78 × 10-4 M with [NaOH] = 0.16 M at 25 ℃ and I = 0.16 M (NaCl). ampam_1B0 0.2 0.4 0.6 0.8titre volume357911pH0 0.2 0.4 0.6 0.8titre volume-0.200.2Weighted residuals0102030405060708090100% formation relative to ampamPb_ampam_1B0.6 1.0 1.4 1.8titre volume246810pH0.6 1.0 1.4 1.8titre volume-0.500.5Weighted residuals0102030405060708090100% formation relative to PbA B 144 Figure A.7 1H NMR spectra of ampam versus [Pb(ampam)]2+ at various pD values (300 MHz, 298 K, D2O). O ON NH2NONH2ON NH2NONH2OO ON NH2NONH2ON NH2NONH2OPb2+ampam Pb2+ / ampampD2.61.91.46.52.82.01.81.47.05.0pD 145 Appendix B. Supplementary Data for Chapter 3 Table B.1 Crystallographic information for [Bi(ampa)](NO3).5H2O Formula C24H40BiN7O16 Dcalc./ g cm-3 1.851 /mm-1 5.595 Formula Weight 891.61 Colour colourless Shape plate-shaped Size/mm3 0.18×0.13×0.02 T/K 90(2) Crystal System monoclinic Space Group P21/n a/Å 9.2754(5) b/Å 14.2171(8) c/Å 24.6287(13) /° 90 /° 99.863(2) /° 90 V/Å3 3199.8(3) Z 4 Z' 1 Wavelength/Å 0.71073 Radiation type MoK min/° 1.660 max/° 30.531 Measured Refl's. 30772 Indep't Refl's 30772 Refl's I≥2 (I) 25578 Rint 0.064 Parameters 478 Restraints 728 Largest Peak 3.881 Deepest Hole -3.702 GooF 1.055 wR2 (all data) 0.1137 wR2 0.1078 R1 (all data) 0.0631 R1 0.0478 146 Table B.2 Selected crystallographic bond angle information for [Bi(ampa)](NO3).5H2O Atom Atom Atom Angle/° Atom Atom Atom Angle/° O1 Bi1 O4 104.07(16) O6 Bi1 N5 129.81(17) O1 Bi1 O8 65.26(16) O8 Bi1 O5 72.34(14) O1 Bi1 N2 66.46(17) N2 Bi1 O4 62.11(16) O1 Bi1 N5 88.15(17) N2 Bi1 O5 118.01(16) O2 Bi1 O1 148.40(17) N2 Bi1 O8 87.35(16) O2 Bi1 O4 107.30(15) N2 Bi1 N5 144.03(18) O2 Bi1 O5 62.38(15) N3 Bi1 O1 130.23(18) O2 Bi1 O8 129.06(15) N3 Bi1 O4 68.86(18) O2 Bi1 N2 133.55(17) N3 Bi1 O5 81.89(17) O2 Bi1 N3 66.84(18) N3 Bi1 O8 129.50(17) O2 Bi1 N4 73.28(17) N3 Bi1 N2 67.42(18) O2 Bi1 N5 81.10(17) N3 Bi1 N4 122.2(2) O4 Bi1 O5 56.78(14) N3 Bi1 N5 141.54(18) O4 Bi1 O8 60.65(15) N4 Bi1 O1 75.34(18) O6 Bi1 O1 75.93(18) N4 Bi1 O4 166.38(18) O6 Bi1 O2 88.33(17) N4 Bi1 O5 114.27(16) O6 Bi1 O4 126.60(16) N4 Bi1 O8 107.98(18) O6 Bi1 O5 146.85(16) N4 Bi1 N2 127.71(18) O6 Bi1 O8 140.52(16) N4 Bi1 N5 63.17(19) O6 Bi1 N2 70.21(17) N5 Bi1 O4 103.29(17) O6 Bi1 N3 71.86(18) N5 Bi1 O5 64.13(16) O6 Bi1 N4 66.78(18) N5 Bi1 O8 58.11(16) Table B.3 HR-ESI-MS data for H2ampa and its respective metal complexes. Species Formula Calcd (m/z) Found (m/z) H2ampa C24H32N6O8 532.2282 532.2286 ampa-Pb C24H30N6O8Pb 734.1855 734.1856 ampa-Bi C24H30N6O8Bi 739.1929 739.1929 ampa-La C24H30N6O8La 668.1196 668.1203 147 Figure B.1 1H NMR spectrum of H2ampa/compound 10 (400 MHz, 298 K, D2O, pD 6). Figure B.2 13C{1H} NMR spectrum of H2ampa/compound 10 (100 MHz, 298 K, D2O, pD 1). 148 Figure B.3 1H NMR spectrum of [Bi(ampa)]+ (400 MHz, 298 K, D2O, pD 5). Figure B.4 1H-1H COSY NMR spectrum of [Bi(ampa)]+ (400 MHz, 298 K, D2O, pD 5). 149 Figure B.5 HSQC NMR spectrum of [Bi(ampa)]+ (400 MHz, 298 K, D2O, pD 5). Figure B.6 1H NMR spectrum of [La(ampa)]+ (400 MHz, 298 K, D2O, pD 7). 150 Figure B.7 13C{1H} NMR spectrum of [La(ampa)]+ (400 MHz, 298 K, D2O, pD 7). Figure B.8 1H-1H COSY NMR spectrum of [La(ampa)]+ (400 MHz, 298 K, D2O, pD 7). 151 Figure B.9 HSQC NMR spectrum of the aliphatic region of [La(ampa)]+ (400 MHz, 298 K, D2O, pD 7). Figure B.10 HSQC NMR spectrum of [La(ampa)]+ (400 MHz, 298 K, D2O, pD 7). 152 Figure B.11 iTLC traces of [213Bi][Bi(ampa)]+ radiolabeling after 5 min at RT (A) 5.27 × 10-4 M, (B) 5.27 × 10-5 M, (C) 5.27 × 10-6 M, (D) 5.27 × 10-7 M, (E) 5.27 × 10-8 M, and (F) free [213Bi]Bi3+ control. 153 Figure B.12 iTLC traces of [225Ac][Ac(ampa)]+ radiolabeling after 30 min at (A) 1 × 10-3 M, (B) 1 × 10-4 M, (C) 1 × 10-5 M, (D) 1 × 10-6 M, (E) 1 × 10-7 M, and (F) free [225Ac]Ac3+ control. 154 Figure B.13 Potentiometric titration plot of a solution containing H2ampa, [H2ampa] = 8.93 × 10-4 M, at 25 ℃ and I = 0.16 M (NaCl). Figure B.14 Representative spectra of H2ampa UV-potentiometric titration, [H2ampa] = 8.93 × 10-4 M, at 25 ℃, l = 0.2 cm and I = 0.16 M (NaCl). H2ampa_1B20 60 100 140point number357911pH20 60 100 140point number-0.500.5Weighted residuals0102030405060708090100% formation relative to ampa225 250 275 300 3250.000.250.500.751.001.251.501.752.00pH 4.47 2.43 2.80 3.08 3.30 4.47AbsorbanceWavelength (nm)pH 2.43A225 250 275 300 3250.000.250.500.751.001.251.501.752.00pH 4.47 4.47 5.15 5.54 6.06 10.72AbsorbanceWavelength (nm)pH 10.72B 155 Figure B.15 Potentiometric titration plot of a solution containing [La3+] = [H2ampa] = 7.37 × 10-4 M, at 25 ℃ and I = 0.16 M (NaCl). Figure B.16 Spectra of La3+/H2ampa UV-acidic batch titration [La3+] = [H2ampa] = 1.44 × 10-4 M, at 25 ℃, l = 1 cm and I = 0.16 M (NaCl). La_ampa20 60 100 140 180point number246810pH20 60 100 140 180point number-1.0-0.500.51.0Weighted residuals0102030405060708090100% formation relative to La225 250 275 300 3250.000.250.500.751.001.25pH 0.35 0.35 0.51 1.16 1.25 1.33 1.48 1.58 1.75 2.06 2.28AbsorbanceWavelength (nm)pH 2.28A 156 Figure B.17 Potentiometric titration plot of a solution containing [Pb2+] = [H2ampa] = 6.43 × 10-4 M, at 25 ℃ and I = 0.16 M (NaCl). Figure B.18 Spectra of Pb2+/H2ampa UV-acidic batch titration [Pb2+] = [H2ampa] = 1 × 10-4 M, at 25 ℃, l = 1 cm and I = 0.16 M (NaCl). Pb_ampa_1B20 60 100 140 180point number246810pH20 60 100 140 180point number-101Weighted residuals0102030405060708090100% formation relative to Pb225 250 275 300 3250.000.250.500.751.001.25pH 0.69 0.69 0.97 1.33 1.76 2.00 2.08AbsorbanceWavelength (nm)pH 2.08A 157 Appendix C. Supplementary Data for Chapter 4 Table C.1 HR-ESI-MS data for H5decapa and H4noonpa and their respective La3+ complexes Species Formula Calcd (m/z) Found (m/z) H5decapa C24H30N5O10 548.1993 548.1995 decapa-La C24H24N5O10Na3La 750.0280 750.0278 H4noonpa C24H30N4O10 534.1962 534.1964 noonpa-La C24H27N4O10La 669.0798 669.0800 Figure C.1 1H NMR spectrum of H5decapa/compound 15 (400 MHz, 298 K, D2O, pD 1). 158 Figure C.2 13C{1H} NMR spectrum of H5decapa/compound 15 (100 MHz, 298 K, D2O, pD 1). Figure C.3 1H NMR spectrum of H4noonpa/compound 19 (400 MHz, 298 K, D2O, pD 1). 159 Figure C.4 13C{1H} NMR spectrum of H4noonpa/compound 19 (100 MHz, 298 K, D2O, pD 1). Figure C.5 1H NMR spectrum of [La(decapa)]2- (400 MHz, 298 K, D2O, pD 6). 160 Figure C.6 13C{1H} NMR spectrum of [La(decapa)]2- (100 MHz, 298 K, D2O, pD 6). Figure C.7 1H-1H COSY NMR spectrum of [La(decapa)]2- (400 MHz, 298 K, D2O, pD 6). 161 Figure C.8 HSQC NMR spectrum of [La(decapa)]2- (400 MHz, 298 K, D2O, pD 6). Figure C.9 1H NMR spectrum of [La(noonpa)]- (400 MHz, 298 K, D2O, pD 6). 162 Figure C.10 13C{1H} NMR spectrum of [La(noonpa)]- (100 MHz, 298 K, D2O, pD 6). Figure C.11 HSQC NMR spectrum of [La(noonpa)]- (400 MHz, 298 K, D2O, pD 6). 163 Figure C.12 1H-1H COSY NMR spectrum of [La(noonpa)]- (400 MHz, 298 K, D2O, pD 6). 164 Figure C.13 Concentration-dependent radiolabeling of H4noonpa and 225Ac (~40 kBq), in NH4OAc buffer (0.2 M, pH 7), RT, 15 min. RCC obtained using iTLC SA-impregnated paper plates and EDTA mobile phase (50 mM, pH 7). 165 Figure C.14 Representative iTLC traces of [225Ac][Ac(noonpa)]- incubated in serum: A) control [225Ac]Ac3+ at 24 h, B) [L] = 1 × 10-5 M at 24 h, C) control [225Ac]Ac3+ at 7 d, D) [L] = 1 × 10-5 M at 7 d. iTLC system involved SA paper plates and EDTA mobile phase (50 mM, pH 7). 0 50 100 150 200050010001500Position (mm)CountsLS_225Ac - SSCTRL_D1.R001A0 50 100 150 2000500100015002000Position (mm)CountsLS_225Ac - DECSS1_D1.R001A0 50 100 150 20005001000150020002500Position (mm)CountsLS_225Ac - SSCTRL_D7_R.R001A0 50 100 150 20005001000150020002500Position (mm)CountsLS_225Ac - DECSS1_D7.R001AA BC D
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Orvig, Chris
Lone pair activity, versatility, and charge : lessons from chelators designed for radiopharmaceuticals
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