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Metal chelating agents for medicinal radionuclides Li, Lily 2020

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Metal Chelating Agents for Medicinal Radionuclides by LILY LI B.S., Hong Kong Baptist University, 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)  July 2020 © LILY LI, 2020   ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a dissertation entitled:  Metal Chelating Agents for Medicinal Radionuclides  submitted by LILY LI in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemistry Examining Committee:  Chris Orvig, Professor, Department of Chemistry, UBC Supervisor  Michael O Wolf, Professor, Department of Chemistry, UBC Supervisory Committee Member  Judy Wong, Professor, Department of Pharmaceutical Science, UBC University Examiner  Michael D Fryzuk, Professor, Department of Chemistry, UBC University Examiner  Additional Supervisory Committee Members:   Prof. Jennifer Love, Professor, Department of Chemistry, University of Calgary Supervisory Committee Member  Prof. Raymond J Andersen, Professor, Department of  Earth Ocean & Atmospheric Sciences, UBC Supervisory Committee Member    iii  Abstract      Medical radionuclides can be classified into non-metallic and metallic. Due to the much broader decay spectrum, radiometal ions are generally of greater interest in radiopharmaceutical designs. Since free radionuclides are rapidly taken up by the non-targeting organs/tissues such as bone, liver and kidney, causing unnecessary radiation burden to the body, they need to be stably coordinated by a suitable bifunctional chelator conjugated to a biological targeting vector, which directs the radiation doses specifically to the tumor site. This thesis describes several picolinate-based ligand scaffolds (H4octapa, H4pypa and H4py4pa) with denticity from eight to eleven to accommodate radionuclides as small as scandium-44 and as large as actinium-225. Apart from rapid and stable chelation, versatile bifunctionalization is a crucial but often under-valued property of a ligand. The chemical properties of the linker can significantly alter the biodistribution of the chelate-bioconjugate, while the position of the linker in the ligand can dictate the coordination geometry. The second effect was observed in two newly developed 177Lu-labeled H4octapa peptide conjugates discussed in this thesis. This observation prompted the development of two functionally versatile chelators, H4pypa and H4py4pa, with increased rigidity imposed by the central pyridyl moiety that connects the pendent chelating arms. The central pyridyl moiety also serve as a convenient and robust bioconjugation spot. Studies of H4pypa with scandium-44, lutetium-177, indium-111, yttrium-86, zirconium-89, and H4py4pa with actinium-225 were conducted, while their biological properties were evaluated with either the peptide- or the antibody-conjugates, showing favorable radiolabeling results and stability which even surpassed the current “gold-standard” chelator, DOTA, rendering them highly promising for the applications in nuclear medicine. iv  Lay Summary      Nuclear medicine utilizes radioactivity to diagnose and treat cancer. A metal-binding agent is a vital component in metal-based radiopharmaceuticals. In this thesis, different newly developed metal-binding agents with different sizes of binding cavity will be explored and studies with a number of useful medicinal radiometal ions will be discussed, including the affinity of the binding agents for the metal ions of interest, the stability and geometry of the resulting metal complexes. Finally, selected metal complexes were attached to a tumor-targeting molecule and its biological behavior was evaluated in mice.   v  Preface Chapter 2 contains an adaptation of a published manuscript and is reproduced in part from:  Li, L.; Kuo, H.-T.; Wang, X.; Merkens, H.; Colpo, N.; Radchenko, V.; Schaffer, P.; Lin, K.-S.; Bénard, F.; Orvig, C. tBu4octapa-alkyl-NHS for Metalloradiopeptide Preparation. Dalton Trans. 2020, 49, 7605-7619. Dr. Caterina Ramogida designed the ligand and Lily Li performed the synthesis and characterizations. Dr. Xiaozhu Wang performed the DFT calculations and Lily Li analyzed the calculation results. Lily Li performed the radiolabeling, human serum stability studies and lanthanum challenge studies. Bioconjugations were performed by Lily Li and Dr. Hsiou-Ting Kuo, while the biodistribution studies and the imaging studies were contracted to the teams of Dr. François Bénard and Dr. Kuo-Shyan Lin (Dr. Hsiou-Ting Kuo , Helen Merkens, Nadine Colpo) in BC Cancer. Dr. Valery Radchenko and Dr. Paul Schaffer approved the radiochemical studies in TRIUMF.  Chapter 3 contains an adaption of published work and is reproduced in part from:  Li, L.; Jaraquemada-Peláeza, M. G.; Kuo, H.-T., Merken, H.;, Choudhary, N.; Gitschtaler, K.; Jermilova, U.; Colpo, N.; Uribe-Munoz, C.; Radchenko, V.; Schaffer, P.; Lin, K.-S.;  Bénard, F.; Orvig, C. Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-principle Prostate Specific Membrane Antigen Targeting. Bioconjugate Chem. 2019, 30, 1539-1553 (ACS Editors’ Choice). The ligands were designed, synthesized and characterized by Lily Li. The metal complexation, characterizations, as well as the synthesis of the crystal were performed by Lily Li, while the crystal structure was solved by Neha Choudhary. The major contribution on the solution vi  thermodynamics was from Dr. María de Guadalupe Jaraquemada-Peláez, with some help from Lily Li. The radiolabeling and serum stability studies were performed by Lily Li and Una Jermilova, while the bioconjugations were performed by Dr. Hsiou-Ting Kuo and Lily Li. The binding affinity assay was performed by Helen Merkens, while the biodistribution studies and imaging studies were contracted to the teams of Dr. François Bénard and Dr. Kuo-Shyan Lin (Dr. Hsiou-Ting Kuo, Helen Merkens, Dr. Nadine Colpo, Katrin Gitschtaler, Dr. Carlos Uribe-Munoz) in BC Cancer. Dr. Valery Radchenko and Dr. Paul Schaffer approved the radiochemical studies in TRIUMF.  Chapter 4 contains an adaption of a published manuscript and is reproduced in part from: Li, L.; de G. Jaraquemada-Peláez, M.; Aluicio-Sarduy, E.; Wang, X.; Jiang, D.; Sakhie, M.; Kuo, H.-T.; Barnhardt, T. E.; Cai, W.; Radchenko, V.; Schaffer, P.;  Lin, K.-S.; Engle, J. W.; Bénard, F.; Orvig, C. [nat/44Sc(pypa)]- : Thermodynamic Stability, Radiolabeling and Biodistribution of a Prostate-Specific-Membrane-Antigen-Targeted Conjugate.  Inorg. Chem. 2020, 59, 1985-1995. The metal complexation and characterizations were performed by Lily Li with the help from Dr. Maria Ezhova. DFT calculations were performed by Dr. Xiaozhu Wang, while the results were analyzed by Lily Li. Solution thermodynamics were performed by Dr. María de Guadalupe Jaraquemada-Peláez with some help from Lily Li. The bioconjugations were performed by Lily Li and Dr. Hsiou-Ting Kuo from the teams of Dr. François Bénard and Dr. Kuo-Shyan Lin, and the sample was sent to the team of Dr. Jonathan W. Engle in the University of Wisconsin for further biological studies. The isotope was produced by Meelad Sakheie, Dr. Eduardo Aluicio-Sarduy and Todd Barnhardt, while the radiolabeling and serum stability studies were performed by Lily Li. Dr. vii  Valery Radchenko, Dr. Paul Schaffer and Dr. Jonathan W. Engle approved the radiochemical studies. The biodistribution and imaging studies were performed by Dr. Eduardo Aluicio-Sarduy. The binding assay was performed by Dr. Dawei Jiang from the team of Dr. Weibo Cao.   Chapter 5 contains works performed during an internship in the Helmholtz-Zentrum Dresden Rossendorf (HZDR) under the supervision of Dr. Holger Stephan: [89Zr][Zr(pypa)]-: Non-Macrocyclic  Picolinate-Based Chelator for 89Zr-PET Imaging. The metal complexation and characterizations were performed by Lily Li. DFT calculations were performed by Dr. Xiaozhu Wang, while the results were analyzed by Lily Li. The radiolabeling and all the challenge studies were performed by Lily Li.  Chapter 6 contains an adaptation of a published manuscript and is reproduced in part from: Li, L.; Jaraquemada-Peláeza, M. G.; Aluicio-Sarduy, E.; Wang, X.; Barnhart, T. E.; Cai, W.; Radchenko, V.; Schaffer, V.; Engle, J. W.; Orvig, C. Coordination Chemistry of [Y(pypa)]- and Comparison Immuno-PET Imaging of 44Sc- and 86Y-pypa-phenyl-TRC105. Dalton Trans. 2020, 49, 5547-5562. The ligand was designed, synthesized and characterized by Lily Li. The metal complexation and characterizations were performed by Lily Li. DFT calculations were performed by Dr. Xiaozhu Wang, while the results were analyzed by Lily Li. Solution thermodynamics were performed by Dr. María de Guadalupe Jaraquemada-Peláez with some help from Lily Li. The isotopes were produced by Dr. Eduardo Aluicio-Sarduy and Todd Barnhardt, while the radiolabeling and serum stability studies were performed by Lily Li. Dr. Valery Radchenko, Dr. Paul Schaffer and Dr. viii  Jonathan W. Engle approved the radiochemical studies. The bioconjugation, biodistribution and imaging studies were performed by Dr. Eduardo Aluicio-Sarduy. Dr, Weibo Cai provided cell samples.  Chapter 7 contains an adaptation of an accepted manuscript and is reproduced in part from:  Li, L.; Rousseau, J.; de G. Jaraquemada-Peláez, M.; Wang, X.; Robertson, A.; Radchenko, V.; Schaffer, P.; Lin, K.-S.; Bénard, F.; Orvig, C. 225Ac-H4py4pa for Targeted-Alpha-Therapy. Bioconjugate Chem. The ligands were designed, synthesized and characterized by Lily Li. The metal complexation and characterizations were performed by Lily Li. The DFT calculations were performed by Dr. Xiaozhu Wang, while the results were analyzed by Lily Li. The solution thermodynamics was performed by Dr. María de Guadalupe Jaraquemada-Peláez. The isotope for radiolabeling the non-bifunctional chelator was produced by Andrew Robertson. The radiolabeling and serum stability studies were performed by Lily Li and Dr. Julie Rousseau, while the bioconjugations and the radioimmunoreactivity assays were performed by Dr. Julie Rousseau. The biodistribution studies were contracted to the teams of Dr. Kuo-Shyan Lin and Dr. François Bénard in BC Cancer. Dr. Valery Radchenko and Dr. Paul Schaffer approved the radiochemical studies.  Chapter 8 contains some other work performed during the PhD program. Part I: Improved synthesis of H2dedpa-benzyl-NCS and H4octapa-benzyl-NCS. The synthesis and characterizations of the ligands were performed by Lily Li. Part II contains works performed during an internship in HZDR under the supervision of Dr. Holger Stephan: Comparison of 177Lu-ix  labeled H4pypa-phenyl-Panitumumab and H4pypa-PEG4-benzyl-Panitumumab. H4pypa-PEG4-benzyl-NCS synthesis and 177Lu labeling. The ligand was designed, synthesized and characterized by Lily Li. The antibody conjugation, radiolabeling, human serum challenge studies, electrophoresis were performed by Lily Li, while the binding affinity assay was performed by Dr. Kristof Zarschler. All animal studies performed in Chapters 2, 3 and 7 used a protocol approved by the Animal Ethics Committee of the University of British Columbia (protocol # A10-0171) and was performed by the BC Cancer following the Animal Care Guidelines established by the Canadian Council. All animal studies in Chapters 4 and 6 were performed by the University of Wisconsin and approved by the Animal Ethics Committee of the University of British Columbia (protocol # A10-0171) and the Institutional Animal Care and Use Committee the University of Wisconsin.    x  Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface ............................................................................................................................................ v Table of Contents .......................................................................................................................... x List of Tables .............................................................................................................................. xvi List of Figures ........................................................................................................................... xviii List of Schemes .......................................................................................................................... xxv List of Symbols and Abbreviations ....................................................................................... xxvii Acknowledgements ................................................................................................................ xxxiii Dedication ................................................................................................................................ xxxv Chapter 1. Introduction ............................................................................................................... 1 1.1.   Nuclear Imaging and Therapy ............................................................................................ 1 1.2.   Metallo-Radiopharmaceutical Design ................................................................................ 4 1.2.1.   Diagnostic and Therapeutic Radionuclides of Emerging Interest ............................... 5 1.2.2.   Metal Chelating Agents ............................................................................................... 9 1.2.3.   Biological Targeting Vectors..................................................................................... 12 1.2.4.   Bioconjugations ......................................................................................................... 14 1.3.   Thesis Overview ............................................................................................................... 17 Chapter 2. tBu4octapa-alkyl-NHS for Metalloradiopeptides Preparation. ........................... 19 2.1.   Introduction ...................................................................................................................... 19 2.2.   Results and Discussion ..................................................................................................... 24 2.2.1.   Synthesis and Characterization .................................................................................. 24 2.2.2.   DFT Calculations ....................................................................................................... 26 2.2.3.   Radiolabeling, Human Serum Stability and Lanthanum Challenge Experiments ..... 31 xi  2.2.4.   SPECT/CT MIP Imaging and Ex vivo Biodistribution Studies ................................. 35 2.3.   Conclusions ...................................................................................................................... 39 2.4.   Experimental Section ....................................................................................................... 40 2.4.1.   Materials and Methods .............................................................................................. 40 2.4.2.   Synthesis and Characterization .................................................................................. 41 2.4.3.   DFT Calculations ....................................................................................................... 49 2.4.4.   Solid-Phase Peptide Coupling ................................................................................... 50 2.4.5.   Radiolabeling Studies ................................................................................................ 51 2.4.6.   In vitro Human Serum Challenge .............................................................................. 51 2.4.7.   In vitro Lanthanum(III) Challenge ............................................................................ 52 2.4.8.   Radiolabeling of Conjugates for In vivo Study ......................................................... 52 2.4.9.   SPECT/CT Imaging Studies ...................................................................................... 52 2.4.10.   Biodistribution Studies ............................................................................................ 54 Chapter 3. Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-principle Prostate Specific Membrane Antigen Targeting ..................................................... 55 3.1.   Introduction ...................................................................................................................... 55 3.2.   Results and Discussion ..................................................................................................... 59 3.2.1.   Synthesis and Characterization .................................................................................. 59 3.2.2.   Metal Complexation and Characterization ................................................................ 63 3.2.3.   X-ray Crystallography of H[Lu(pypa)] ..................................................................... 65 3.2.4.   Solution Thermodynamics ......................................................................................... 68 3.2.5.   Radiolabeling and Human Serum Challenge Experiments ....................................... 76 3.2.6.   SPECT/CT Imaging, Biodistribution Studies and Binding Affinity ......................... 80 3.3.   Conclusions ...................................................................................................................... 83 3.4.   Experimental Section ....................................................................................................... 84 3.4.1.   Materials and Methods .............................................................................................. 84 3.4.2.   Synthesis and Characterization .................................................................................. 85 3.4.3.   Solid-Phase Peptide Coupling ................................................................................... 97 3.4.4.   X-ray Crystallography ............................................................................................... 98 xii  3.4.5.   Solution Thermodynamics ......................................................................................... 99 3.4.6.   Radiolabeling Studies .............................................................................................. 101 3.4.7.   In Vitro Human Serum Challenge ........................................................................... 103 3.4.8.   In Vitro Competition Binding Assays ..................................................................... 103 3.4.9.   Radiotracer Preparation for Biodistribution Studies ............................................... 104 3.4.10.   Biodistribution Studies .......................................................................................... 104 3.4.11.   SPECT/CT Imaging Studies .................................................................................. 105 Chapter 4. [nat/44Sc(pypa)]- : Characterization and Evaluation of Thermodynamic Stability, Radiolabeling and Biodistribution of a Prostate-Specific-Membrane-Antigen-Targeting Conjugate ................................................................................................................................... 107 4.1.   Introduction .................................................................................................................... 107 4.2.   Results and Discussion ................................................................................................... 111 4.2.1.   Metal Complexation and Characterization .............................................................. 111 4.2.2.   DFT Calculations ..................................................................................................... 114 4.2.3.   Solution Thermodynamics ....................................................................................... 117 4.2.4.   Radiolabeling and Mouse Serum Challenge Experiment ........................................ 120 4.2.5.   PET/CT Imaging, Biodistribution Studies and Binding Affinity ............................ 123 4.3.   Conclusions .................................................................................................................... 128 4.4.   Experimental Section ..................................................................................................... 129 4.4.1.   Materials and Methods ............................................................................................ 129 4.4.2.   Na[natSc(pypa)] Complexation Reaction ................................................................. 130 4.4.3.   DFT Calculations ..................................................................................................... 130 4.4.4.   Solution Thermodynamics ....................................................................................... 131 4.4.5.   Production and Radiochemical Isolation of Scandium-44 ...................................... 133 4.4.6.   Radiolabeling Studies .............................................................................................. 134 4.4.7.   In vitro Mouse Serum Challenge ............................................................................. 134 4.4.8.   In vitro Competition Binding Assays ...................................................................... 135 4.4.9.   Radiolabeling of Conjugates for In vivo Study ....................................................... 135 4.4.10.   PET/CT Imaging Studies ....................................................................................... 136 4.4.11.   Ex Vivo Biodistribution Studies ............................................................................. 136 xiii  Chapter 5. [89Zr][Zr(pypa)]- : Nonmacrocyclic  Picolinate-Based Chelator for 89Zr-PET Imaging. ..................................................................................................................................... 138 5.1.   Introduction .................................................................................................................... 138 5.2.   Results and Discussion ................................................................................................... 141 5.2.1.   Metal Complexation and Characterization .............................................................. 141 5.2.2.   DFT Calculations ..................................................................................................... 145 5.2.3.   Radiolabeling and Challenge Experiments .............................................................. 146 5.3.   Conclusions .................................................................................................................... 150 5.4.   Experimental Section ..................................................................................................... 150 5.4.1.   Materials and Methods ............................................................................................ 150 5.4.2.   [natZr(pypa)] Complexation and Characterization ................................................... 151 5.4.3.   DFT Calculations ..................................................................................................... 152 5.4.4.   Radiolabeling Experiments ...................................................................................... 152 5.4.5.   EDTA (100-fold) Challenge Experiments ............................................................... 153 5.4.6.   DFO (100- and 1000-fold) Challenge Experiments ................................................ 153 5.4.7.   In vitro Human Serum Challenge ............................................................................ 154 Chapter 6. Coordination Chemistry of [Y(pypa)]- and Comparison Immuno-PET Imaging of 44Sc- and 86Y-pypa-phenyl-TRC105 .................................................................................... 155 6.1.   Introduction .................................................................................................................... 155 6.2.   Results and Discussion ................................................................................................... 159 6.2.1.   Synthesis and Characterization ................................................................................ 159 6.2.2.   Complexation and Characterization ........................................................................ 162 6.2.3.   DFT Calculations ..................................................................................................... 164 6.2.4.   Solution Thermodynamics ....................................................................................... 167 6.2.5.   Radiolabeling of [86Y][Y(pypa)]- and Mouse Serum Challenge Experiments ........ 171 6.2.6.   PET/CT Imaging and Biodistribution Studies ......................................................... 174 6.3.   Conclusions .................................................................................................................... 178 6.4.   Experimental Section ..................................................................................................... 179 6.4.1.   Materials and Methods ............................................................................................ 179 xiv  6.4.2.   Synthesis and Characterization ................................................................................ 180 6.4.3.   DFT Calculations ..................................................................................................... 187 6.4.4.   Solution Thermodynamics ....................................................................................... 188 6.4.5.   Production and Radiochemical Separation of Yttrium-86 ...................................... 190 6.4.6.   Production and Radiochemical Separation of Scandium-44 ................................... 190 6.4.7.   Radiolabeling Studies .............................................................................................. 191 6.4.8.   In Vitro Mouse Serum Challenge ............................................................................ 191 6.4.9.   Bioconjugation of H4pypa-phenyl-TRC105 and Radiolabeling with Yttrium-86 and Scandium-44 ........................................................................................................................ 192 6.4.10.   PET/CT Imaging and Biodistribution Studies ....................................................... 193 Chapter 7. 225Ac-H4py4pa for Targeted-Alpha-Therapy ...................................................... 194 7.1.   Introduction .................................................................................................................... 194 7.2.   Results and Discussion ................................................................................................... 198 7.2.1.   Synthesis and Characterization ................................................................................ 198 7.2.2.   Metal Complexation and Characterizations ............................................................ 203 7.2.3.   DFT Calculations ..................................................................................................... 206 7.2.4.   Solution Thermodynamics ....................................................................................... 208 7.2.5.   Radiolabeling H4py4pa and In vitro Serum Stability of [225Ac][Ac(py4pa)]- ......... 213 7.2.6.   In vitro Characterization of [225Ac][Ac(py4pa-phenyl-Trastuzumab)] ................... 215 7.2.7.   In vivo Biodistribution Studies ................................................................................ 219 7.3.   Conclusions .................................................................................................................... 222 7.4.   Experimental Section ..................................................................................................... 223 7.4.1.   Materials and Methods ............................................................................................ 223 7.4.2.   Synthesis and Characterization ................................................................................ 225 7.4.3.   DFT Calculations ..................................................................................................... 238 7.4.4.   Solution Thermodynamics ....................................................................................... 239 7.4.5.   225Ac Quantification ................................................................................................ 241 7.4.6.   Radiolabeling of H4py4pa and DOTA ..................................................................... 241 7.4.7.   Antibody Conjugation with the Bifunctional Chelators .......................................... 242 7.4.8.   Immunoconjugate Radiolabeling with 225Ac ........................................................... 242 xv  7.4.9.   Antibody Immunoreactivity .................................................................................... 243 7.4.10.   Mouse Serum Challenge Experiments .................................................................. 243 7.4.11.   Assessment of Radiopharmaceutical Biodistributions .......................................... 244 7.4.12.   Data Analysis ......................................................................................................... 245 Chapter 8: Other Work ............................................................................................................ 246 8.1.   Improved Synthesis of H2dedpa-benzyl-NCS and H4octapa-benzyl-NCS .................... 246 8.1.1.   Results and Discussion ............................................................................................ 246 8.1.2.   Experimental Procedures ......................................................................................... 250 8.2.   Comparison of 177Lu-labeled H4pypa-phenyl-Panitumumab and H4pypa-PEG4-benzyl-Panitumumab ........................................................................................................................... 257 8.2.1.   Results and Discussion ............................................................................................ 257 8.2.2.    Experimental Procedures ........................................................................................ 263 Chapter 9. Conclusions and Future Studies ........................................................................... 272 Bibliography .............................................................................................................................. 275 Appendices ................................................................................................................................. 290 Appendix A: Supplementary Data for Chapter 3 .................................................................... 290 Appendix B: Supplementary Data for Chapter 4 .................................................................... 294 Appendix C: Supplementary Data for Chapter 6 .................................................................... 300    xvi  List of Tables  Table 1.1 A list of diagnostic radionuclides for PET and SPECT imaging.5,6,15 ........................... 6 Table 1.2 A list of therapeutic radionuclides.5,6,15 .......................................................................... 7 Table 2.1 Selected DFT-calculated bond lengths and bond angles. ……………………………...28 Table 2.2 Tumor-to-background ratios at different p.i. timepoints (n = 5). ………..…………....38 Table 3.1 Selected bond lengths and bond angles in [Lu(pypa)]-. ………………………………..67 Table 3.2 Protonation constants of H4pypa at 25.0 °C, I = 0.16 M NaCl. ................................... 69 Table 3.3 Stepwise stability constants (log K) of H4pypa complexes with In3+, Lu3+ and La3+. . 75 Table 4.1 Comparison of DFT calculated Sc-O and Sc-N bond lengths in structures A and B. ...116 Table 4.2 Stepwise stability constants (log K) of H4pypa with Sc3+ ion. .................................. 120 Table 5.1 DFT-calculated metal-donor bond lengths for the [Zr(pypa)]. ………………………146 Table 5.1 DFT-calculated X-Zr-Y bond angles for the [Zr(pypa)]. ........................................... 146 Table 6.1 DFT-calculated metal-donor bond lengths for the [M(pypa)]- (M = Sc, Lu, Y) anions. ………………………………………………………………………………………….166 Table 6.2 DFT-calculated X-M-Y bond angles for the [M(pypa)]- (M = Sc, Lu, Y) anions. .... 167 Table 6.3 Stepwise stability constants (log K) of H4pypa with Sc3+, Lu3+, Y3+ and La3+ ions. . 171 Table 6.4 Radiochemical yield% of [86Y][Y(pypa)]- at pH=7. .................................................. 174 Table 7.1 Bond lengths in [La(py4pa)]- calculated by DFT. …………………………………... 207 Table 7.2 Protonation constants of H4py4pa at 25°C, I = 0.16 M (NaCl). The numbers in parentheses indicated the standard deviation of the last digit. .................................................... 210 Table 7.3 Stepwise stability constants (log K) of H4py4pa complexes with La3+. The numbers in parentheses indicated the standard deviation of the last digit. .................................................... 212 Table 7.4 Mouse serum stability results of [225Ac][Ac(L)]- (L = DOTA and py4pa) at 37°C over 9 d (n = 3). ................................................................................................................................... 215 xvii  Table 7.5 Biodistribution data of the 225Ac-labeled DOTA and H4py4pa conjugated Trastuzumab in SKOV-3 tumor bearing mice at 1, 3, 6 and 10 days post-injection. Data are presented as mean±SD %ID/g (n=3-4). Mice were injected with similar amount of antibody (50.3±3.6 μg) and similar injected activities (10.1±0.7 kBq). Significant difference between [225Ac]Ac-py4pa-Trastuzumab and the DOTA construct are highlighted: * p<0.01 and ** p<0.001. NA: not available as counts were in the background which did not allow for proper quantification. ..... 222 Table A.1 Crystallographic data for H[Lu(pypa)]. …………………………………………..293      xviii  List of Figures  Figure 1.1 Depiction of PET and SPECT imaging modalities. ..................................................... 2 Figure 1.2 Depiction of the relative path lengths of -- and -particles, and Auger electrons. ..... 4 Figure 1.3 Schematic design of a metallo-radiopharmaceutical. ................................................... 5 Figure 1.4 Chemical structures of selected chelators. .................................................................. 12 Figure 1.5 Possible bioconjugation techniques for metallo-radiopharmaceuticals. ..................... 16 Figure 2.1 Chemical structures of selected ligands. ……………………………………………23 Figure 2.2 DFT-calculated structures for S-DBTA and S-DSA geometries of [Lu(octapa-alkyl-benzyl-ester)]- (Same geometry adopted for the corresponding R-configuration). ...................... 29 Figure 2.3 DFT-calculated structures for [Lu(S-benzyl-octapa)]- (top) and [Lu(octapa)]- (bottom)........................................................................................................................................................ 30 Figure 2.4 (A) Concentration-dependent radiolabeling of H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 with lutetium-177 in 15 min at room temperature and pH = 7 (0.2 M NH4OAc). (B) Intact percentage of the radiocomplexes upon human serum challenge over 7 days at 37oC. Lanthanum challenges on 177Lu-labeled (C) H4octapa, H4octapa-alkyl-PSMA-ureido, H4octapa-alkyl-PSMA617, and (D) H4pypa, H4pypa-C7-PSMA-ureido, H4pypa-C7-PSMA617, over 7 days. ................................................................................................................................... 34 Figure 2.5 SPECT/CT MIP Images of (A) [177Lu][Lu(octapa-alkyl-PSMA-ureido)] (qualitative imaging) (B) [177Lu][Lu(octapa-alkyl-PSMA-617)] (quantitative imaging) at different post-injection time points. ..................................................................................................................... 37 Figure 2.6 Ex vivo biodistribution data of (A) [177Lu][Lu(octapa-alkyl-PSMA-ureido)] (B) [177Lu][Lu(octapa-alkyl-PSMA-617)] at different post-injection time points. ............................. 37 Figure 2.7 Bone uptake (%ID/g) comparison between the 177Lu-labeled octapa-alkyl-PSMA-ureido and octapa-alkyl-PSMA617 over time. ............................................................................. 38 Figure 3.1 Chemical structures of selected chelators. …………………………………………..59 Figure 3.2 (A) Partial 1H NMR spectra of [La(pypa)]-, [In(pypa)] -, [Lu(pypa)] -, H4pypa (top-bottom) (D2O, 400 MHz, 298 K). ................................................................................................. 64 xix  Figure 3.3 A) Partial 1H NMR spectra of [Lu(pypa)]- at pH=11.5, 2.1 and 1.5 (top-bottom)  (D2O, 400 MHz, 298 K). B) Potential structure of [Lu(Hpypa)] in aqueous solution at pH < 2. ........... 65 Figure 3.4 ORTEP diagrams of the anion in C50H79ClLu2N10O33 ............................................... 66 Figure 3.5 ORTEP diagrams of the anion in H[Lu(pypa)].. ........................................................ 67 Figure 3.6 A) and B) Representative spectra of the in batch acidic titration of H4pypa at [L] = 1.07  10-4 M as the pH is raised at 25 °C and l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. C) and D) Representative spectra of the combined UV-potentiometric titration of H4pypa at [L] = 6.34  10-4 M at 25 °C, l = 0.2 cm and I = 0.16 M NaCl. ..................................................................................................... 70 Figure 3.7 A) Titration curve of an acidic solution of H4pypa, [H4pypa] = 6.34  10-4 M, at 25 °C and I = 0.16 M NaCl. B) Speciation plots of H4pypa calculated with protonation constants on Table 3.2. [H4pypa] = 1  10-3 M. Dashed line indicates pH 7.4. ........................................................... 70 Figure 3.8 Representative spectra of the in-batch UV-titration of A) the In3+-pypa system as the pH is raised. [L] = [In3+] = 1.33  10-4 M; B) and C) the Lu3+-pypa system as the pH is raised. [L] = [Lu3+] = 1.33  10-4 M; D) and E) the La3+-pypa system as the pH is raised. [L] = [La3+] = 1.27  10-4 M, at 25 °C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. Distribution diagram of the In3+-pypa system. Dashed line indicates physiological pH (7.4). ........................................................................................... 73 Figure 3.9 Distribution diagram of A) the In3+-pypa system; B) the Lu3+-pypa system; C) the La3+-pypa system. Dashed line indicates physiological pH (7.4). ........................................................ 74 Figure 3.10 (A) pM values vs ionic radius119 for M3+- ligand complexes (CN = 8); (B) Lu3+ scavenging ability of different ligands as the pH is raised from 0-12, [Lu3+] = 1  10-6 M and [ligand] = 1  10-5 M. Solid line in B at indicates physiological pH (7.4).................................... 75 Figure 3.11 (A) Concentration dependent radiolabeling of H4pypa and H4pypa-C7-PSMA617 (10 min, RT) in NH4OAc solution (0.15 M, pH = 7) with 177Lu/111In. (B) Human serum challenge of the radiolabeled complexes over 5-7 d (37 oC). ............................................................................ 78  Figure 3.12 RP-rHPLC (A: ACN/0.1% TFA B: H2O/0.1% TFA) of (A) free 177Lu (0-100% A over 30 min, 1 mL/min, tR =  6.44 min); (B) [177Lu][Lu(pypa)] (10-6 M, 0-100% A over 30 min, 1 mL/min, tR =  12.9 min, RCY = 98%); (C) [177Lu][Lu(pypa-C7-PSMA617)] (10-6 M, 0-80% A over 30 min, 1 mL/min, tR =  15.0 min, RCY = >99%); (D) free 111In (0-100% A over 20 min, 1 mL/min, tR =  7.67 min); (E) [111In][In(pypa)] (10-6 M, 0-100% A over 20 min, 1 mL/min, tR =  10.4 min, RCY = 98%); (F) [111In][In(pypa-C7-PSMA617)] (10-6 M, 0-100% A over 20 min, 1 mL/min, tR =  14.1 min, RCY = >99%). ....................................................................................... 79 xx  Figure 3.13 Representative SPECT/CT images (MIP, coronal) of [AE][E(pypa-C7-PSMA617)] [AE=111In (left, 24.9 MBq), 177Lu (right, 44.1 MBq)] in LNCaP-tumor-bearing mice at different p.i. time points............................................................................................................................... 81 Figure 3.14 Ex vivo biodistribution data [AE][E(pypa-C7-PSMA617)] [AE=177Lu (A), 111In (B)] in LNCaP-tumor-bearing mice at selected p.i. time points (n=5 per time point). ........................ 82 Figure 4.1 Chemical structures of the discussed chelators. …………………………………..110 Figure 4.2 [Sc(pypa)]- 1H and 13C NMR assignments (See Figures 2 and S2(A-C) for labels). 113 Figure 4.3 [Sc(pypa)]- variable temperature 1H NMR (A) upfield and (B) downfield spectra (400 MHz, D2O, pH 7) (25, 35, 45, 65, 85˚C from bottom to top). .................................................... 114 Figure 4.4 Two isomeric species of the [Sc(pypa)]- anion simulated using DFT calculations. . 116 Figure 4.5 A) Representative spectra of the in-batch UV-titration of the Sc3+-H4pypa system as the pH is raised. [L] = [Sc3+] = 1.33  10-4 M at 25˚C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. B) pM vs. ionic radius119 for M3+ ions of interest and different ligands in discussion. ........................................ 119 Figure 4.6 Distribution diagram of the Sc3+-pypa system calculated with stability constants in Table 4.2, [L] = [Sc3+] = 8.53  10-4 M Dashed line indicates physiological pH (7.4). ............. 119 Figure 4.7 Radio-HPLC chromatographs of (A) [44Sc][Sc(pypa)]- (tR = 13.41 min) (B) [44Sc][Sc(pypa-C7-PSMA617)] (tR = 23.67 min)  (A: H2O/0.1%TFA B:ACN/0.1%TFA. 5-65% B over 32 min, 1 mL/min. .............................................................................................................. 121 Figure 4.8 (A) Concentration-dependent radiolabeling studies of H4pypa and H4pypa-C7-PSMA617 with 44Sc at room temperature in NH4OAc buffer (0.5 M, pH=5.5) over 5 min. (B) Concentration-dependent radiolabeling studies of H4pypa with 44Sc at room temperature in NH4OAc solution (0.5 M, pH = 2, 4, 5.5, 7) over 15 min. (C) Mouse serum stability of the corresponding complexes over 24 h at 37 oC.............................................................................. 122 Figure 4.9 Representative PET/CT images (MIP, coronal) of [44Sc][Sc(pypa-C7-PSMA617)] (7.4 (top) and 74 (bottom) GBq/mol)] in PC3-PIP (left shoulder) and PC3-Flu (right shoulder) tumor-bearing mice at different p.i. time points. ................................................................................... 126 Figure 4.10 Biodistribution data of [44Sc][Sc(pypa-C7-PSMA617)] (7.4 (left) and 74 (right) GBq/mol)] in PC3-PIP-tumor-bearing mice at selected p.i. time points (n = 3 per time point)...................................................................................................................................................... 126 xxi  Figure 4.11 Ex vivo biodistribution data of [44Sc][Sc(pypa-C7-PSMA617)] (7.4 and 74 GBq/mol)] in PC3-PIP-tumor-bearing mice at selected p.i. time points (n = 3 per time point)...................................................................................................................................................... 127 Figure 4.12 Tumor (PSMA+)-to-kidney ratio over time. .......................................................... 127 Figure 5.1 Chemical structures of selected chelators. ………………………………………....141 Figure 5.2 A) [Zr(pypa)] 1H and 13C NMR assignment (See Figures 5.2B and 5.3 for labels). B) 1H NMR spectra of [Zr(pypa)] (top) and H4pypa (bottom). ....................................................... 143 Figure 5.3 [Zr(pypa)] 13C{1H} NMR spectrum (100 MHz, 298 K, D2O). ................................. 143 Figure 5.4 [Zr(pypa)] COSY spectrum (400 MHz, 298 K, D2O). ............................................. 144 Figure 5.5 [Zr(pypa)] 1H-13C HMBC (red)/ HSQC (green-blue) spectra overlap (400/100 MHz, 298 K, D2O) ................................................................................................................................ 144 Figure 5.6 DFT-calculated geometry for the [Zr(pypa)]. ........................................................... 145 Figure 5.7 Radio-HPLC chromatograph of free zirconium-89 (A: H2O/0.1% TFA B: ACN/0.1% TFA, 0-70% B over 20 min, 1 mL/min) ..................................................................................... 148 Figure 5.8 HPLC chromatograph of [89Zr][Zr(pypa)] (Top : 10-5 M, bottom : 10-6 M) (A: H2O/0.1% TFA B: ACN/0.1% TFA, 0-70% B over 20 min, 1 mL/ min). ................................. 148 Figure 5.9 Concentration-dependent radiolabeling of zirconium-89 with H4pypa at A) 40˚C, pH = 5.5, B) RT, pH = 7, C) 40˚C, pH = 7; D) zirconium-89 with DFO at 40˚C, pH = 7. Challenge studies of E) [89Zr][Zr(DFO)]+ with EDTA (100×) and F) [89Zr][Zr(pypa)] with EDTA (100×) and DFO (100× and 1000×). .............................................................................................................. 149 Figure 6.1 Chemical structures of subject ligands. …………………………………………….159 Figure 6.2 1H NMR spectra (400 MHz, D2O) of H4pypa (RT), [Y(pypa)]- (RT), [Lu(pypa)]- (RT) and [Sc(pypa)]- (RT & 85˚C). ..................................................................................................... 164 Figure 6.3 (A) DFT calculated geometry for the [Y(pypa)]- anion. (Same geometries calculated for the [Sc(pypa)]- and [Lu(pypa)]- anions). (B) [Y(pypa)]- coordination environment showing only Y3+ and the donor atoms (A perspective looking through the central pyridine). ................ 166 Figure 6.4 Distribution diagram of the Y3+-H4pypa system calculated with stability constants in Table 6.3, [L] = [Y3+] = 1  10-3 M. Dashed line indicates physiological pH (7.4). .................. 169 xxii  Figure 6.5 (A) and (B) Representative spectra of the in-batch UV-titration of the Y3+-pypa system as the pH is raised. [L] = [Y3+] = 1.33  10-4 M at 25 °C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. ....................... 170 Figure 6.6 pM vs. ionic radius119 for M3+ and ligands of interest. ............................................. 170 Figure 6.7 (A) Concentration-dependent radiolabeling studies of H4pypa with 44Sc in NH4OAc buffer (0.5 M, pH=5.5) and 86Y in HEPES buffer (0.5 M, pH=7) at room temperature over 15 min. (B) Mouse serum stability of [86Y][Y(pypa)]-over 48 h at 37˚C ................................................ 173 Figure 6.8 PET/CT MIP Images of (A) [44Sc][Sc(pypa-phenyl-TRC105)] (B) [86Y][Y(pypa-phenyl-TRC105)] at different post-injection time points. .......................................................... 176 Figure 6.9 Quantitative ROI analysis of the in vivo PET imaging data of (A) [44Sc][Sc(pypa-phenyl-TRC105)] (B) [86Y][Y(pypa-phenyl-TRC105)] at different post-injection time points. 177 Figure 6.10 Ex vivo biodistribution data of [44Sc][Sc(pypa-phenyl-TRC105)] (18 h p.i.) and [86Y][Y(pypa-phenyl-TRC105)] (48 h p.i.). ............................................................................... 177 Figure 7.1 Chemical structures of selected chelators. …………………………………………198 Figure 7.2 1H NMR spectra of H4py4pa (bottom) and [La(py4pa)]- (top) (400 MHz, 298 K, D2O)...................................................................................................................................................... 204 Figure 7.3 Na[La(py4pa)] 13C NMR spectrum (100 MHz, 298 K, D2O). ................................. 204 Figure 7.4 Na[La(py4pa)] COSY NMR spectrum (400 MHz, 298 K, D2O). ............................ 205 Figure 7.5 Na[La(py4pa)] 1H-13C HSQC NMR spectrum (400/100 MHz, 298 K, D2O). ......... 205 Figure 7.6  DFT calculated structure of [La(py4pa)]- anion. ..................................................... 207 Figure 7.7 (A, B and C) Representative spectra of the combined UV-potentiometric titration of H4py4pa at [L] = 4.05  10-4 M at 25°C, l = 0.2 cm and I = 0.16 M NaCl. (D) Speciation plot of H4py4pa calculated with protonation constants on Table 7.2. [H4pypa] = 1  10-3 M. Dashed line indicates pH 7.4. ......................................................................................................................... 209 Figure 7.8 Normalized curve of the basic titration curve of H4py4pa · 4TFA · H2O. [H4py4pa] = 4.05  10-4 M at 25°C and I = 0.16 M NaCl. .............................................................................. 210 Figure 7.9 (A) and (B) Representative spectra of the in-batch UV-titration of the La3+-py4pa system as the pH is raised. [L] = [La3+] = 7.86  10-5 M at 25°C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. (C) Distribution diagram of the La3+-py4pa system calculated with stability constants in Tables 7.2 and 7.3. [L] = [La3+] = 1  10-3 M. Dashed line indicates physiological pH (7.4). .................... 212 xxiii  Figure 7.10 Concentration-dependent radiolabeling of H4py4pa and DOTA at room temperature, pH = 7 (NH4OAc, 1 M) in 30 min. ............................................................................................. 214 Figure 7.11 Size-exclusion HPLC profiles of (A) [225Ac][Ac(DOTA-benzyl-Trastuzumab)] and (B) [225Ac][Ac(py4pa-phenyl-Trastuzumab)]. For each panel, the top trace shows the 225Ac radioactive peak and the bottom one the protein UV280nm peak. For both products, an expected peak at 8.3-8.4 min post-injection that corresponds to an intact IgG is observed. For the radiotrace, the additional small peak around 12 min corresponds to the 225Ac daughters released from each chelators after 225Ac decay. The time difference between the UV and the radioactive peaks for each compound is due to the time needed for the antibody to travel from the absorbance detector to the scintillation detector. ................................................................................................................... 218 Figure 7.12 Mouse serum challenge of 225Ac labeled DOTA- and H4py4pa-phenyl-Trastuzumab at 37 oC over 11 days (n=3 per time points). .............................................................................. 218 Figure 7.13 Biodistribution of [225Ac][Ac(DOTA-benzyl-Trastuzumab)] (black) and [[225Ac][Ac(py4pa-phenyl-Trastuzumab)]  (grey) at (A) day 1, (B) day 3, (C) day 6 and (D) day 10 post-injection in an ovarian cancer xenografts model (SKOV-3). Data are presented as percentage of the injected dose per gram of tissue (% ID/g) for the tumor and the main organs (mean ± SD, n=3-4) and statistical differences between the two tracers are highlighted with * p<0.01 and ** p<0.001. NA: not available as the counts were in the background, i.e very low radioactivity concentration that cannot be quantified. ................................................................ 221 Figure 8.1 1H NMR spectrum of H4pypa-PEG4-benzyl-NCS (400 MHz, CD3CN, 298K). …..260 Figure 8.2 Radio-TLC plate showing the radiochemical yield percentage of S5, S20, L5, L20, as well as the control samples of 177Lu-Panitumumab (Panitumumab) and free 177Lu in buffer (Lu)...................................................................................................................................................... 261 Figure 8.3 A) Electrophoresis Phosphor scan (radioactive) and B) SDS-PAGE Scan. ............. 262 Figure 8.4 Binding Affinity curves for H4pypa-phenyl-Panitumumab and H4pypa-PEG4-benzyl-Panitumumab at 5:1 and 20:1 chelator:antibody ratios. .............................................................. 263 Figure A.1 [Lu(pypa)]- 13C NMR spectrum (100 MHz, 298 K, D2O). ……………………….290 Figure A.2  [Lu(pypa)]- 1H-13C HSQC NMR spectra (400/100 MHz, 298 K, D2O). ............... 290 Figure A.3 [La(pypa)]- 13C NMR spectrum (100 MHz, 298 K, D2O). ..................................... 291 Figure A.4 [In(pypa)]- 13C NMR spectrum (100 MHz, 298 K, D2O). ...................................... 291 Figure A.5 [In(pypa)]- COSY NMR spectra (400 MHz, 298 K, D2O). .................................... 292 Figure A.6 [In(pypa)]- 1H-13C HSQC NMR spectra (400/100 MHz, 298 K, D2O). ................. 292 xxiv   Figure B.1 [Sc(pypa)]- 13C{1H} NMR spectrum (100 MHz, 343 K, D2O). …………………..294 Figure B.2 [Sc(pypa)]- 13C{1H} NMR spectrum (partial) (100 MHz, 343 K, D2O). ................ 294 Figure B.3 [Sc(pypa)]- 13C{1H} NMR spectrum (partial) (100 MHz, 343 K, D2O). ................ 295 Figure B.4 [Sc(pypa)]- 1H-13C{1H} HSQC NMR spectrum (400/100 MHz, 343 K, D2O). ..... 295 Figure B.5 [Sc(pypa)]- 1H-13C{1H} HMBC NMR spectrum (400/100 MHz, 343 K, D2O). .... 296 Figure B.6 [Sc(pypa)]- COSY NMR spectrum (400 MHz, 343 K, D2O). ................................ 296 Figure B.7 [Sc(pypa)]- NOESY NMR spectrum (400 MHz, 343 K, D2O). .............................. 297 Figure B.8 [Sc(pypa)]- 13C{1H} NMR spectrum (400 MHz, 298 K, D2O). .............................. 297 Figure B.9 [Sc(pypa)]- 1H-13C{1H} HSQC NMR spectrum (100 MHz, 298 K, D2O). ............. 298 Figure B.10 [Sc(pypa)]- COSY NMR spectrum (400 MHz, 298 K, D2O). .............................. 298 Figure B.11 [Sc(pypa)]- NOESY NMR spectrum (400 MHz, 298 K, D2O). ............................ 299 Figure C.1 [Y(pypa)]- 13C NMR spectrum (100 MHz, 298 K, D2O). …………………………300 Figure C.2 [Y(pypa)]- 1H-13C HSQC spectrum (400/100 MHz, 298 K, D2O). ......................... 300 Figure C.3 [Y(pypa)]- 1H-1H COSY spectrum (100/100 MHz, 298 K, D2O). ........................... 301    xxv  List of Schemes  Scheme 2.1 Reagents and conditions. i) THF, nosyl chloride, ambient temp, 18 h, 89%; ii) tert-butyl bromoacetate, TEA, dry ACN, ambient temp, 18 h, 63%; iii) DCC, tert-butyl alcohol, DCM, ambient temp, overnight, 50%; iv) NaBH4, dry MeOH, ambient temp, 3-4 h, 72%; v) PBr3, dry ACN, 0 oC-ambient temp, 54%; vi) K2CO3, dry ACN, 60oC, 24 h, 70%; vii) PhSH, K2CO3, THF, 18 h, ambient temp, 63%; viii) NaBr, 2 N HBr, NaNO2, conc H2SO4, 18 h, ambient temp, 45%; ix) TBTA/cyclohexane, DMA, BF3-etherate, CHCl3, ambient temp, 3d ; x) K2CO3, dry ACN, 60 oC, 48 h, 30%; xi) Pd/C, H2, MeOH, ambient temp, 18 h, 87%; xii) NHS, EDC ˑ HCl, dry ACN, 69%........................................................................................................................................................ 24 Scheme 3.1 Reagents and conditions: i) DCC, tert-butyl alcohol, DCM, RT, overnight, 50%; ii) NaBH4, dry MeOH, RT, 3-4 h, 72%; iii) SeO2, 1,4-dioxane, 100oC, overnight, 56%; iv) 1. Dry MeOH, RT, 1 h; 2. NaBH3CN, dry MeOH, 3 h, 70%; v) NaBH4, dry MeOH, RT, 12 h, 92%; vi) PBr3, dry CHCl3/ACN, 60 oC, 18 h, 70%; vii) K2CO3, dry ACN, 60 oC, 24 h, 70%; viii) TFA/DCM, RT, overnight, 70%........................................................................................................................60 Scheme 3.2 Reagents and conditions. i) SOCl2, MeOH, RT-60 oC, 26 h, >99%; ii) BnBr, ACN, K2CO3, 60 oC, overnight, 64%; iii) NaBH4, dry MeOH, RT, overnight, 82%; iv) PBr3, dry CHCl3/dry ACN, 60 oC, overnight, 70%; v) K2CO3, dry ACN, 30 oC, 24 h, 73%; vi) Pd/C (10% w/w), H2 (g), MeOH, RT, overnight; vii) K2CO3, dry THF, RT-35 oC, 24 h, 90%; viii) Pd/C, MeOH, RT, overnight, 88%; ix) NHS, EDCI, dry ACN, RT, overnight, 86%. ........................................ 62 Scheme 6.1 Reagents and conditions. i) SOCl2, MeOH, RT-60 oC, 26 h, >99%; ii) BnBr, ACN, K2CO3, 60 oC, overnight, 64%; iii) NaBH4, dry MeOH, RT, overnight, 82%; iv) PBr3, dry CHCl3/dry ACN, 60 oC, overnight, 70%; v) K2CO3, dry ACN, RT, 24 h, 73%; vi) Pd/C (10% w/w), H2 (g), MeOH, RT, overnight; vii) K2CO3, dry THF, RT, 24-48 h, 90%; viii) p-TsCl, THF, 6 M NaOH, 71%; ix) TFA/DCM, overnight, 50%; x) CSCl2, 1 M HCl/ glacial AcOH/CHCl3, RT, 24 h, 30%..............................................................................................................................................161 Scheme 7.1 Reagents and conditions: i) NaBH4, dry DCM/dry MeOH, 0 oC-ambient temp, 4 h, 60%; ii) PBr3, dry ACN, 0 oC- ambient temp, 6 h, 80%; iii) DIPEA, dry ACN, ambient temp, 24 h, 91%; iv) Pd/C, H2(g), glacial AcOH, ambient temp, 3-4 h, 83%; v) NaBH4, dry MeOH, 0 oC-ambient temp, 12 h, 92%; vi) PBr3, dry CHCl3/ACN, 0-60 oC, 18 h, 87%; vii) DIPEA, KI, dry ACN, ambient temp, 24 h, 70%; viii) LiOH, H2O/THF, ambient temp, 24 h, 50%....................200 Scheme 7.2 Unsuccessful dipicolinate arm (7.4) synthetic routes. ............................................ 201 Scheme 7.3 Reagents and conditions: i) SOCl2, MeOH, ambient temp-65 oC, 26 h, >99%; ii) BzBr, K2CO3, dry ACN, 60 oC, 24 h, 64%; iii) NaBH4, dry MeOH, ambient temp, 24 h, 82%; iv) PBr3, dry ACN/CHCl3, 0-60 oC, 70%; v) DIPEA, KI, dry ACN, ambient temp, 24 h, 71%; vi) Pd/C, xxvi  H2(g), dry MeOH, ambient temp, 24 h, 78%; vii) TsCl, 6 M NaOH, THF, 0 oC-ambient temp, 24 h, 71%; viii) K2CO3, dry ACN, ambient temp, 48 h; ix) 1. LiOH, THF/D.I. H2O, ambient temp, 24 h 2. TFA/DCM, ambient temp, 24 h, 50 %; x) CSCl2, 1 M HCl/glacial AcOH/CHCl3, ambient temp, 24 h, 30% .......................................................................................................................... 202 Scheme 8.1 Reagents and conditions: i) 7N NH3 in MeOH, 85 %; ii) BH3-THF, Dry THF, N2(g), 77%; iii) 1. MeOH 2. NaBH3CN, MeOH, sat. NaHCO3, 64%; iv) Pd/C, H2, MeOH; v) TFA/DCM, o/n; vi) CSCl2, CHCl3, glacial AcOH, 3M HCl, RT, o/n 30%; vii) tert-butyl bromoacetate, K2CO3, Dry ACN, 48 h, 40%; viii) Pd/C, H2, MeOH; ix) CSCl2, CHCl3, EtOAc, RT, 4h; x) TFA/DCM, o/n, RT, 70%; xi) 1. 4-(2-aminoethyl)-benzenesulfonamide, DIEA, dry ACN, RT, o/n, 32%; 2. TFA/DCM, RT, o/n; xii) 1. tBu-(Lys-CO-Glu), DIEA, dry ACN, RT, o/n; 2. TFA/DCM, RT, o/n, 65%..............................................................................................................................................249 Scheme 8.2 Reagents and conditions: i) 2 M NaOH, p-TsCl, THF, 0˚C, 3h, 51%; ii) potassium phthalimide, dry ACN, 60˚C, 18 h, 72%; iii) TEA, DMAP, p-TsCl, 0˚C-RT, 48% .................. 257 Scheme 8.3 Reagents and conditions: iv) K2CO3, dry THF, 25˚C, 48 h; v) hydrazine, EtOH, 60˚C, overnight; vi) NHS, EDC, dry ACN, N2(g), RT, overnight, 86%; vii) DIPEA, DCM,  N2(g), RT, overnight; viii) TFA/DCM, RT, overnight, 65%; ix) CSCl2,1 M HCl/ glacial acetic acid/CHCl3, 24% ............................................................................................................................................. 258    xxvii  List of Symbols and Abbreviations  %ID/g % Injected-dose-per-gram  -particles  Alpha Particles +-particle Positron particles -rays Gamma rays δ Chemical shift A Ampere Å Angstrom a.k.a. As known as AAS Atomic absorption spectroscopy AAZTA 6-[Bis(hydroxycarbonyl-methyl)amino]-1,4-bis(hydroxycarbonylmethyl)-6-methylperhydro-1,4-diazepine ACN Acetonitrile AcOH Acetic acid Ambient temp Ambient temperature   Bmax  Maximum specific binding  BFC Bifunctional chelator BnBr Benzyl bromide boc Tert-butyloxycarbonyl  Bq Becquerel  BSA Bovine serum albumin BWXT BWX Technologies, Inc. c(RGDyK)  An αvβ3 Integrin Binding RGD Peptide calcd Calculated CHX-A”-DTPA 2,2'-((2-(((1S,2S)-2-(Bis(carboxymethyl)amino)cyclohexyl)(carboxymethyl)amino)ethyl)azanediyl)diacetic acid Ci Curie CN  Coordination number COSY Correlated Spectroscopy CT Computed tomography CuAAC Copper-catalyzed azide-alkyne cycloaddition  d Day or doublet D.I. water  Deionized water DBTA Distorted bicapped trigonal antiprism  DCC N,N’-dicyclohexylcarbodiimide  DCE 1,2-Dichloroethane  DCM Dichloromethane xxviii  DFO Desferrioxamine B  DFT Density-functional theory  DGA-branched N,N,N’,N’-tetrakis-2-ethylhexyldiglycolamide functionalized resin DIPEA N,N-diisopropylethylamine  DMAP N,N'-dicyclohexylcarbodiimide  DMF Dimethylformamide DMSO Dimethyl sulfoxide DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DSA Distorted square antiprism  DTPA Diethylenetriaminepentaacetic acid E- Beta-particle energy E+ Prositron energy EC Electron capture EDC HCl N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride EDCl 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide EDTA Ethylenediamine EGFR Epidermal growth factor receptor  EoB End-of-bombardment  EPR effect Enhanced permeability and retention effect equiv Equivalent ESCI Electrospray/chemical-ionization  EtOAc  Ethyl acetate EtOH Ethanol eV Electron volt Fab Fragment antigen binding  FBS Fetal Bovine Serum Fc Fragment crystallizable  FDA Food and Drug Administration  FDG Fluorodeoxygluose 18F-DCFPyL  2-(3-{1-carboxy-5-[(6-[(18)F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid Fmoc Fluorenylmethoxycarbonyl protecting group Fmoc-2-Nal-OH  Fmoc-3-(2-naphthyl)-L-alanine Fmoc-Lys(ivDde)-Wang resin  N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine resin g Gram h Hour H0 Acidity scale  xxix  H2bp18c6 and H2macropa N,N′-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6 HAMA Human anti-mouse antibody HATU O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluroniumhexa-fluorophosphate HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HEHA 1,4,7,10,13,16-Hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HER2 Human epidermal growth factor receptor 2  Hex Hexanes HMBC Heteronuclear Multiple Bond Correlation HOBt Hydroxybenzotriazole HPGe High purity germanium  HPLC High-performance liquid chromatography  HR-ESI-MS High-resolution electrospray-ionization mass spectrometry  HSQC Heteronuclear single quantum coherence spectroscopy Hz Hertz I Ionic strength Ib+  Prositron fraction IEDDA Inverse electron demand Diels-Alder reaction  IgG Immunogloblin  Immuno-PET Immuno-positron emission tomography IRDye800CW  A near infrared fluorescence dye IT Isomeric Transition ITG Isotope Technologies Garching  iTLC Instant thin-layer chromatography  iTLC-SA Instant thin-layer chromatography plates impregnated with silicic acid  iTLC-SG Instant thin-layer chromatography plate, impregnated with silica gel  J Coupling constant k Kilo- K Kelvin KD Equilibrium dissociation constant  Ki Inhibitory constant L Liter LET Linear energy transfer LD50 Lethal dose  LNCaP Lymph Node Carcinoma of the Prostate log K Stepwise formation constant log β  Overall equilibrium (formation) constants  xxx  LR-ESI-MS Low-resolution electrospray-ionization mass spectrometry  m Milli- m Micro- M Molar or mega- or metal ion m Multiplet m/z Mass per unit charge mAb Monoclonal antibody mCRPC  Metastatic castration-resistant prostate cancer MeOH  Methanol min Minute MIP Maximum intensity projection mol Mole MP-AES Microwave plasma atomic emission spectroscopy  MQ water MilliQ water MS Mass spectrometry n Nano- or number of unit n.c.a No-carrier-added NCS N-isothiocyanate  NHL  Non-Hodgkin lymphoma NHS N-hydroxysuccinimide  NMR Nuclear Magnetic resonance NODSCID A brand of immunodeficient laboratory mice NOESY Nuclear overhauser effect spectroscopy  NsCl 2-Nitrobenzenesulfonyl chloride  ORTEP Oak Ridge Thermal Ellipsoid Plot Program p Para- p p-value p-Bn-NCS p-benzyl-N-isothiocyanate PBr3 Phosphorus tribromide PBS Phosphate-buffered saline PC3-Flu A human prostate cancer cell line (PSMA-) PC3-PIP  A human prostate cancer cell line (PSMA+) Pca Prostate cancer Pd/C  Palladium/carbon PD10 column  Desalting Column PEG Poly(ethyleneglycol)  PET Positron emission tomography  pH Negative log [H]+ PhSH Thiophenol p.i.  Post-injection xxxi  pKa Protonation constant pM -log[free metal], or picomolar (10-12 M) PMOD PMOD Technologies, Switzerland ppm Parts per million PRIT Pretargeting radioimmunotherapy PRMI 1640 Roswell Park Memorial Institute 1640 Medium  p-SCN-Bn-CHX-A″-DTPA (R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid PSMA Prostate Specific membrane antigen RCY Radiochemical yield RIT Radioimmunotherapy ROI Region of interest RP-rHPLC  Reverse-phase radioactive high-performance liquid chromatography RS- Thiolate RT Room temperature s Singlet or second SCF Self-consistent field  scFv Single chain fragment variable SCID Severe combined immunodeficiency disease SDS-PAGE  Sodium dodecylsulfate-polyacrylamide gel electrophoresis SKOV-3  Human ovarian cancer cell line  SPE Solid phase extraction  SPECT  Single-photon emission computed tomography SPPS Solid phase peptide synthesis t Triplet t1/2  Decay half-life TAT Targeted alpha therapy  TBTA Tert-butyl-2,2,2-trichloroacetimidate  TCO Trans-cycloocten TEA Triethylamine  TFA Trifluoroacetic acid TFP Tetrafluorophenyl THF Tetrahydrofuran TIS Triisopropylsilane TLC Thin-layer chromatography  TMAA Tris(hydroxymethyl)aminomethane  tR Retention time TRC105 An anti-CD105 monoclonal antibody  TRIS Tris(hydroxymethyl)aminomethane  TRT Targeted radionuclide therapy xxxii  TTHA Triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic Acid Tz 1,2,4,5-Tetrazine  UV-Vis  Ultraviolet-Visble VT-NMR  Variable-temperature nuclear magnetic resonance     xxxiii  Acknowledgements       First and foremost, I would like to offer my enduring gratitude to my supervisor, Prof. Chris Orvig for his guidance and support over the course of my PhD studies. Thank you for all the advice, freedom in research, encouragement and last but not least, the fun skiing memories together. Not only has the experience of being your mentee enriched my knowledge in nuclear medicine, but also raised my overall confidence and made me a more independent individual.       I would also like to thank every member in my research group for all the support and valuable discussions at different points of my PhD. In particular, I would to thank Neha Choudhary for the crystal work and all the positivity; Lily Southcott and Aidan Ingham for proof-reading my thesis; Tom Kostelnik for all the joy and support; Jefferey Jang for solving the mechanical problems; Dr. Sarah Spreckelmeyer for helping me to adapt in my first year; Dr. Xiaozhu Wang for accepting my endless requests for DFT calculations during his busy time and having interesting discussions not only about chemistry, but from cultural knowledge to daily news; Dr. María de Guadalupe Jaraquemada-Peláez for the titration studies, all the support, useful advice and being good company; Una Jermilova for helping me start radiochemical studies in TRIUMF.       Thank you to all my collaborators without whom my thesis would not have been completed. Particularly, I would like to thank Dr. Valery Radchenko for the supervision in TRIUMF, all the “6AM rides” to TRIUMF for the biological sample preparations and all the joy working/skiing together; Dr. Jonathan W. Engle and Dr. Eduardo Aluicio-Sarduy for all the help, guidance and support when I was working in their lab in Wisconsin; Dr. Hsiou-Ting Kuo, Dr. Chengcheng Zhang, Dr. Julie Rousseau and Dr. Kuo-Shyan Lin from the BC Cancer for all the helps when I xxxiv  was doing my internship there; Dr. Holger Stephan and Dr. Kristof Zarschler for their supervision when I was working in Helmholtz-Zentrum Dresden Rossendorf (HZDR).        I must also thank the staffs in the Chemistry Department for their assistance, including Dr. Yun Ling and especially Marshall Lapawa in the MS lab, Dr. Maria Ezhova in the NMR lab, Dr. Elena Polishchuk and Dr. Jessie Chen in the Bioservices lab, the staff in the chemical stores especially George Kamel and Pat for all the help purchasing and shipping, and the friendly conversations; the staff in the mechanical shop especially Ken Love for the efficient maintenance of the vacuum pumps and rotary evaporators, as well as the smiles and friendly conversations.        Thank you to my committee members Prof. Michael Wolf, Prof. Raymond Anderson, Prof. Judy Wong, Prof. Michael Fryzuk and my external examiner, Prof. Paul Donnelly, for the time to read my thesis and attend my committee meeting.       Thank you to my partner (Divyanshu Sharma) and his parents (Dr. Manju Sharma and Dr. Nitya Nand) for the support, understanding and positivity whenever I am down and stressed. My partner, in particular, has been very accepting to my work hours and “moodiness” due to stress.        Most importantly, I must thank my parents (Mandy Cheng and Jimmy Li), my brother (Jeff Li) and my sister (Wai Ni Li). Their support of me traveling abroad to pursue my study and career have brought me to where I am. My parents, in particular, have always provided me a worry-free environment so that I could focus on study, while my brother never stops bringing joy to me. I am eternally grateful for everything they have taught me throughout my life.   xxxv  Dedication            To my family, partner, friends, collaborators, and mentors, for endless support and encouragement. 1  Chapter 1. Introduction 1.1.   Nuclear Imaging and Therapy      Radionuclides possess radioactivity that can be used for cancer diagnosis or therapy, depending on the decay products.       Nuclear imaging is a non-invasive and highly sensitive method used to assess the disease stage and the therapeutic effects of radiotracers. Typically, diagnostic radiopharmaceuticals are administered at a very low concentration (i.e. nanomolar to picomolar) to avoid any pharmacological effects. There are two main clinical radio-imaging modalities, single-photon emission computed tomography (SPECT) and positron emission tomography (PET) (Figure 1.1). SPECT imaging is the oldest of the two, relying on a -ray emitter that emits low-energy -rays (100-250 keV) as a result of electron capture (EC) or isomeric transition (IT).1 The -rays are collected by a gamma-camera/detector and are reconstructed to a three-dimensional image showing the origination of the -rays in the body.2 To date, the majority of clinical SPECT scans (~80%) are based on technetium-99m (t1/2 = 6 h).1,3 Owing to the success of 99mTc-labeled radiotracers, such as the myocardial perfusion agent 99mTc-sestamibi;4 the applicability of other SPECT-radionuclides that have different decay properties (such as the half-life) is continuously explored. As opposed to single emission events recorded in SPECT, PET detectors collect simultaneous events. As implied in the name, PET imaging requires a positron emitter that decays to give a +-particle. When a +-particle encounters an electron, an annihilation event occurs. The two resulting anti-parallel -rays (511 keV) are detected concomitantly at two locations 180° away in the circular PET detectors.1,5,6 The glucose analogue, [18F]FDG (18F-fluorodeoxygluose) is the 2  most clinically used PET-radiotracer and its localization within the body is based on the enhanced metabolic rate in the cancer cells.7       Figure 1.1 Depiction of PET and SPECT imaging modalities.        Targeted radionuclide therapy (TRT) is an important field in nuclear medicine that has been recognized for many decades. The earliest interest of using radioactivity to cure cancer was using radium-223 (t1/2 = 11.4 d) in the early 1900s.8 However, it was not until the 1940s that the first clinical application took place using iodine-131 (t1/2 = 8.0 d) to treat patients with thyroid diseases.9 Afterwards, the field called radioimmunotherapy (RIT) was recognized. It is a distinguished subsection of TRT that employs radiolabeled monoclonal antibodies for targeted therapy. 90Y-ibritumomab tiuxetan (Zevalin) was first approved by the Food and Drug Administration (FDA), followed by 131I-tositumomab (Bexxar), to treat the non-Hodgkin lymphoma (NHL).9 Today, interest in TRT and RIT continues to grow, and therapeutic radionuclides with different decay characteristics are actively explored. 3       There are three types of therapeutic radiations applied in the field of TRT, including alpha-particles (), beta-particles (−) and Auger electrons (Figure 1.2). They possess different linear energy transfers (LETs), a parameter that quantifies the energy deposition into biological tissues per unit length of travel, typically expressed in keV/m. Beta-particles have a relatively low LET (0.2 keV m-1) that allows the particles to travel approximately 50 cell diameters, rendering them ideal in treating larger and poorly vascularized tumors.1,10 However, one potential disadvantage of the long tissue penetration range is the bystander effects on the neighboring healthy cells when targeting the small tumors.10 Alpha-particles have much higher LET (~100 keV m-1) and shorter effective ranges of <100 m (<10 cell diameters).1,10 As a result, alpha-therapy is ideal for treating small clusters of tumor cells and micro-metastases, even under hypoxic conditions.10,11,12,13 The -particles are also more likely to inflict irreparable DNA double-strand breaks compared to the --particles.14  Auger-electron emitters have very low energies and high LET (4-26 keV m-1).1,13  Therefore, the path-lengths of Auger electrons are mostly <1 m, meaning that they are only effective once internalized into the cells and in close proximity to the DNA strands themselves.13  4     Figure 1.2 Depiction of the relative path lengths of -- and -particles, and Auger electrons.  1.2.   Metallo-Radiopharmaceutical Design       The fundamental structure of a metal-based radiopharmaceutical includes a radionuclide and a bifunctional chelator (BFC), which is composed of a chelating agent that binds the radiometal ion, and a linker connecting the chelator to a biological targeting vector.5 The modular nature of this design permits independent modifications on each component to adjust the targeting properties.    5  Figure 1.3 Schematic design of a metallo-radiopharmaceutical.  1.2.1.   Diagnostic and Therapeutic Radionuclides of Emerging Interest      When selecting a medical radionuclide, the intrinsic decay characteristics are important considerations - the decay mode, decay half-life, decay energy, as well as the production methods and costs. Collectively these factors determine the therapeutic efficacy, the image quality and the radionuclide availability. Tables 1.1 and 1.2 outline some popular diagnostic and therapeutic radionuclides, respectively, along with their decay properties and production method.5,6 Although only a handful of them have been FDA-approved, many of them have demonstrated promising preclinical results.     6  Table 1.1 A list of diagnostic radionuclides for PET and SPECT imaging.5,6,15 Radionuclide Half-life (h) Application Branching and maximun energy of decay particle Production method 99mTc 6.00 SPECT EC (99%) 99Mo/99mTc generators 67Ga  78.2  SPECT EC (100%) 67Zn(p,n)67Ga 111In 67.2 SPECT EC (100%) natCd(p,xn)111In 155Tb 128 SPECT EC (100%) Spallation of Ta-foil 155Gd(p,n)155Tb 159Tb(p,5n)155Dy→155Tb 152Tb 17.5 SPECT PET EC (80%) + (20%, 1.33 MeV) Spallation of Ta-foil 152Gd(p,n)152Tb 44Sc 4.04 PET + (94%, 632 keV) EC (6%) 44Ca(p,n)44Sc 44Ti/44Sc generator 66Ga 9.49 PET + (57%, 1.90 MeV) EC (43%) 66Zn(p,n)66Ga 68Ga 1.13 PET + (89%, 836 keV) EC (11%) 68Ge/68Ga generator 61Cu 3.34 PET + (60%, 1.22 MeV) EC (40%) 61Ni(p,n)61Cu 60Ni(d,n)61Cu 64Cu 12.7 PET + (19%, 656 keV) EC (38%) 64Ni(p,n)64Cu 51Mn 0.770 PET + (97%, 2.21 MeV) 50Cr(d,n)51Mn 54Fe(p,α)51Mn 52Mn 134 PET + (30%, 242 keV) natCr(p,n)52/52mMn 7  Radionuclide Half-life (h) Application Branching and maximun energy of decay particle Production method 55Co 17.5 PET + (77%, 1.5 MeV) EC (23%) 54Fe(d,n)55Co 58Ni(p,α)55Co 86Y 14.7 PET + (32%, 1.44 MeV) EC (68%) 86Sr(p,n)86Y 89Zr 78.4 PET + (23%, 396 keV) EC (77%) 89Y(p,n)89Zr 90Nb 14.6 PET + (53%, 350 keV) 90Zr(p,n)90Nb 18F 1.83 PET + (97%, 250 keV) 18O(p,n)18F   Table 1.2 A list of therapeutic radionuclides.5,6,15 Radionuclide Half-life (h) Application Branching and maximum energy of decay particle Production method 47Sc 80.4 −−therapy − (100%, 204 keV) 47Ti(n,p)47Sc 46Ca(n,)47Ca→47Sc 90Y 64.0 −−therapy − (100%, 934 keV) 90Sr/90Y generator 111In 67.2 Auger electron therapy 14.7 e-/decay e- = 6.8 keV natCd(p,xn)111In 67Ga 78.2 Auger electron therapy 4.7 e-/decay e- = 6.3 keV 67Zn(p,n)67Ga 8  Radionuclide Half-life (h) Applications Branching and maximum energy of decay particle Production method 149Tb 4.12 −therapy  (100%, 3.97 MeV) Spallation of Ta-foil 152Gd(p,4n)149Tb 142Nd(12C,5n)149Dy→149Tb 161Tb 165 −−therapy Auger electron therapy − (100%, 157 keV) 12.4 e-/decay e- = 46.5 keV 160Gd(n,)161Gd→161Tb 177Lu 159 −−therapy  − (100%, 149 keV)  176Lu(n,γ)177Lu 176Yb(n,)177Yb→177Lu 213Bi 0.760 −therapy −−therapy  (2%, 5.88 MeV) − (98%, 492 keV) 225Ac/213Bi generator 225Ac 238 −therapy  (100%, 5.83 MeV)  229Th/225Ac generator 226Ra(p,2n)225Ac 232Th(p,2p6n)225Ac 227Th 449 −therapy  (100%, 6.04 MeV) 227Ac/227Th generator 105Rh 35.4 −−therapy − (100%,566 keV) 104Ru(n,)105Ru→105Rh 109Pd 13.7 −−therapy − (100%, 360 keV) 108Pd(n, γ)109Pd 186Re 89.2 −−therapy − (93%, 359 keV) 186W(p,n)186Re 188Re 17.0 −−therapy − (100%, 795 keV) 188W/188Re generator 9   1.2.2.   Metal Chelating Agents      Dissociation of metal ions from administered complexes in vivo is undesirable since the free radioactive metal ions are rapidly taken up by non-target organs or tissues such as liver and bone, resulting in unnecessary radiation burden to the patient and false information in the imaging results. Therefore, the affinity of the chelator for the radiometal ion must be strong enough to avoid decomplexation or transchelation in vivo.       When assessing the metal-chelate complex, the thermodynamic stability and the kinetic inertness are two important considerations. The thermodynamic stability of a metal complex can be measured by potentiometric and UV-Vis spectrophotometric titrations to determine the complex formation constant (log KML, KML = [ML]/[M][L]) and the pM value (-log[M]free).1,16,17,18 The pM value is a measure of free metal ion concentration in the solution under specific conditions accounting for important variables such as the ligand basicity and pH.16 Therefore, it is a more accurate parameter than the formation constant in predicting in vivo thermodynamic stability. The higher is the pM value, the lower is the free metal ion concentration and the higher is the affinity of the chelator for the metal ion. However, the thermodynamic stability of a complex alone does not represent the biological stability since the radiotracer is usually prepared in extremely low concentration (i.e. M or lower) and is subjected to further dilution upon injected into the biological system due to circulation, metabolism and excretion.19,20,21 In addition, there are orders-of-magnitude higher concentrations of endogenous proteins competing with the chelator for the radiometal ion such as transferrin and superoxide dismutase.1,22 Under these conditions and competitions, the complex stability is no longer solely dictated by the thermodynamic stability but 10  also by the kinetic inertness (i.e. the rate of dissociation of the radionuclides from the chelator) of the metal complex.22                            Common methods to assess the kinetic inertness of the complex include the acid-dissociation experiments to measure the rate of metal dissociation at a specific pH,23,24 and competitive radiolabeling experiments in which radiolabeling is performed in the presence of excess non-radioactive, non-targeting metal ions.25 The in vitro transchelation experiments with the blood serum proteins16,17,26,27 and the in vivo biodistribution experiments17,27,28,29 are also useful in assessing the complex inertness under physiological conditions. Apart from the complex stability, appropriate complexation kinetics is also a determining factor when evaluating a chelator. Fast radiolabeling kinetics under mild conditions (~RT, pH~7) are advantageous, and crucial when using immuno-constructs and short-lived radionuclides. Immuno-constructs such as conjugates with antibodies and antibody-fragments are highly temperature- and pH-sensitive; thus, radiolabeling must be conducted under mild conditions to avoid protein degradation.  When short-lived radionuclides are used, such as gallium-68 (t1/2 = 67.7 min), radiolabeling should ideally complete within 15 min.27 Although rapid and mild radiolabeling might not be necessary with the long-lived radionuclides such as lutetium-177 (t1/2 = 6.65 d) and actinium-225 (t1/2 = 9.92 d), or with small peptide conjugates, it is still an asset in facilitating the radiotracer preparation in a clinical setting. Other factors such as reliable and versatile linker-attachment strategies, stable chelation with both therapeutic and diagnostic radionuclides are also advantageous properties of a medically useful chelating agent.      Chelators can be either macrocyclic or non-macrocyclic. Macrocyclic chelators have a more pre-organized binding cavity than the acyclic analogues; therefore, the entropy penalty upon 11  complexation is less, which usually leads to more thermodynamically favorable complexation.1,16 However, the complexation kinetics at room temperature, especially with low chelator concentrations, tend to be very slow and prolonged heating at elevated temperature is necessary for quantitative radiolabeling.1,16,30,31,32,33,34,35,36 Research efforts have thus been dedicated to strike a balance between the stability and the complexation kinetics through developing non-macrocyclic chelators with higher rigidity and a more pre-organized binding cavity. To name a few, the hexadentate H2dedpa (N4O2, Figure 1.4) is a promising example that demonstrated high affinity for gallium-68 and a favorable in vivo biodistribution using the small cyclic peptide c(RGDyK) as a targeting vector (i.e. [68Ga][Ga(RGD-1)], Figure 1.4).37 The same authors also reported another 68Ga-labeled c(RGDyK)-conjugate of H2dedpa (i.e. H2RGD-2, Figure 1.4) with inferior stability (73% intact after 2 h) against transferrin compared to [68Ga][Ga(RGD-1)] (92% intact after 2 h),37 which raised the awareness of potential alterations of the complex coordination environment during bifunctionalization. Another hexadentate oxine-based chelator H2hox (Figure 1.4) also demonstrated stable chelation with gallium-68.38 [68Ga][Ga(hox)]+ is not only promising for PET imaging, but also potentially useful for fluorescence imaging due to the intrinsic fluorescence properties of the complex.38 An  octadentate picolinate-based chelator, H4octapa (N4O4, Figure 1.4), is a successful development showing stable chelation with indium-111, lutetium-177 and yttrium-90.26,39,40  3,4,3-(LI-1,2-HOPO) (O8, Figure 1.4) is another octadentate ligand that exploits the hard-oxygen donor atoms from the hydroxypyridinone moieties to stably coordinate the hard and oxophilic zirconium-89.41,42 12    Figure 1.4 Chemical structures of selected chelators.  1.2.3.   Biological Targeting Vectors      Cancerous cells express many biomarkers that are either mutated or upregulated. These distinctive and/or abundant biomarkers become useful targets for radiopharmaceuticals to specifically detect the malignant cells. Various types of biological targeting vectors have been developed for tumor targeting, such as antibodies, antibody fragments, peptides and nanoparticles. Monoclonal antibodies (mAb) are large proteins (~150 kDa) with exquisite sensitivity and selectivity for targeted cell surface receptors.43,44 To match the long circulation half-life (days to 13  weeks) of mAbs,43 the radionuclides should be comparatively long-lived in order to allow treatments and imaging at late timepoints when most of the radioactivity is cleared from the background tissues/organs.45 111In-capromab pendetide (ProstaScint), 99mTc-fanolesomab (NeutroSpec), 90Y-ibritumomab tiuxetan (Zevalin) and 131I-tositumomab (Bexxar) are four radiolabeled antibodies approved by FDA.46,47,48,49         Antibody fragments are small parts of the full antibodies that retain the antigen-specificity.50 Examples of antibody fragments are the fragment antigen binding (Fab), the single chain fragment variable (scFv), the single domain antibody and the fragment crystallizable (Fc).51 Compared to the full mAbs, the smaller sizes of the antibody fragments result in higher tumor penetration, reduced immunogenicity and shorter biological half-life which can be used in combination with shorter-lived radionuclides.50,51       Peptides are amino acid chains that possess specificity to desired antigens.  In contrast to larger protein ligands, peptides are more easily synthesized and modified, with higher tumor diffusibility and biocompatibility.1,52 Due to the fast tumor localization and background clearance of peptides, they are compatible with, but not limited to, short-lived radionuclides such as gallium-68.53,54 One of the major concerns of peptides is in vivo degradation, but it can potentially be prevented by structural modifications such as incorporation of the D-amino acids or cyclization.52,55  Examples of FDA-approved radiopeptides are 111In-pentetreotide (Octreoscan), 68Ga-dotatate (Netspot) and 177Lu-dotatate (Lutathera).56,57,58      Instead of targeting specific antigens, nanoparticles accumulate in the tumor cells through the enhanced vascular permeability of tumor blood vessels and poor lymphatic drainage systems (a.k.a. the enhanced permeability and retention effect, EPR effect) due to their large sizes.59 The growing 14  attention on radiolabeled nanoparticles is partially due to the long blood circulation and biological stability.59,60    1.2.4.   Bioconjugations      Bioconjugation in metallo-radiopharmaceuticals connects the chelator and the biological targeting vector through a covalent bond. There are several important considerations when choosing a bioconjugation strategy. First and foremost, the conjugation should not impede either the chelation or the targeting properties of the biomolecules. Second, the coupling reaction should be reasonably fast under mild conditions to avoid potential degradations. Third, the spacer and the bioconjugate should be stable in vivo.       The list of possible bioconjugation techniques is outlined in Figure 1.5.      The formation of a peptide bond is commonly used in peptide-chelator conjugates. This can be done by coupling the free primary amine in the peptide to the carboxylic acid group in the chelator using HATU (O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluroniumhexa-fluorophosphate), HOBt (hydroxybenzotriazole) or EDCl (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) as the coupling agents.61 Alternatively, the carboxylic acid group in the chelator can be first activated as esters of N-hydroxysuccinimide (NHS) or tetrafluorophenyl (TFP), which is subsequently coupled to the primary amine in the peptides.61,62 These coupling methods are compatible with solid-phase peptide synthesis (SPPS), provided that the chelator is soluble under the SPPS conditions and can be deprotected with trifluoroacetic acid (TFA).61 Thiourea bonds are another common linkage formed between the primary amine in a protein or a peptide and the N-isothiocyanate group (NCS) 15  in a chelator.26,42 Apart from the primary amine, the thiol group in the protein can also be harnessed for bioconjugation via Michael addition of a thiolate (RS-) to the double bond of the maleimide resulting in a succinimide thioether bond.63,64 Click chemistry of the copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition is also a useful tool for efficient and selective bioconjugation via triazole linker.65 Cu(I) is necessary for fast and regioselective azide-alkyne cycloaddition (CuAAC),65 and can potentially be removed with Na2S as cupric sulfide precipitates;1 however, it can be problematic when used in combination with chelators that exhibit high affinity for copper. There are also concerns on the potential contamination of trace copper in the labeled radiotracers. Therefore, the field has progressed to adopt copper-free click chemistry using the strained alkyne group instead of the terminal alkyne group.66,67 In fact, the concept of click chemistry has become a very popular bioconjugation approach in nuclear medicine, particularly in pre-targeting radioimmunotherapy (PRIT). The literature shows different in vivo pre-targeting examples using trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) based on the biorthogonal inverse electron demand Diels-Alder reaction (IEDDA).68,69,70,71 By using PRIT, the TCO-bearing radioimmunoconjugate is first administered, followed by a multiday interval period to allow accumulation in the targeted tissues. After that, the radiolabeled tetrazine is administered. Click ligation happens between the antibody and the radiocomplex, while the unreacted tetrazine is rapidly cleared from the biological system. This approach can significantly reduce the radiation doses delivered by the long-lived radioimmunoconjugates to the healthy tissues.   16   Figure 1.5 Possible bioconjugation techniques for metallo-radiopharmaceuticals.1,16,72   17  1.3.   Thesis Overview      This thesis presents the synthesis of non-macrocyclic bifunctional chelators for medicinally useful radiometal ions such as 44Sc, 111In, 177Lu, 225Ac, etc, along with characterization, radiochemical studies and biological studies; these developments follow our group’s previous work on the picolinate-based chelators.      Chapter 2 focuses on the synthesis of a new bifunctional H4octapa, tBu4octapa-alkyl-NHS, for PSMA (prostate-specific membrane antigen)-targeting peptide conjugation; DFT calculations, radiochemical studies with lutetium-177 and animal studies are included.        Chapter 3 focuses on the synthesis and characterization of a nonadentate chelator H4pypa and the bifunctional tBu4-pypa-C7-NHS for PSMA-targeting peptide conjugation; metal complexation, crystallography, solution thermodynamics, radiochemical studies with lutetium-177 and indium-111 and biodistributions are included.      Chapter 4 focuses on the synthesis and characterization of [Sc(pypa)]-; DFT calculations, solution thermodynamics, radiochemical studies and animal studies with [44Sc][Sc(pypa-C7-PSMA617)] are included.      Chapter 5 focuses on the synthesis and characterization of [Zr(pypa)], DFT calculations and radiochemical studies with 89Zr.            Chapter 6 focuses on the [Y(pypa)]- complexation and characterization, DFT calculations, solution thermodynamics, radiochemical studies of [86Y][Y(pypa)]- and comparative immuno-PET imaging using 44Sc- and 86Y-pypa-phenyl-TRC105. 18       Chapter 7 focuses on the synthesis and characterization of a 11-coordinating chelator H4py4pa and the bifunctional H4py4pa-phenyl-NCS; DFT calculations, metal complexation and characterizations with La3+ ion, solution thermodynamics, radiochemical studies with actinium-225 and animal studies of the 225Ac-labeled Trastuzumab are included.      Chapter 8 summarizes some other work performed during the PhD program. Part I: Improved synthesis of H2dedpa-benzyl-NCS and H4octapa-benzyl-NCS. The synthesis and characterization of the ligands are included.  Part II contains works performed during an internship in HZDR under the supervision of Dr. Holger Stephan: Comparison of 177Lu-labeled H4pypa-phenyl-Panitumumab and H4pypa-PEG4-benzyl-Panitumumab. Synthesis of H4pypa-PEG4-benzyl-NCS, radiochemical studies with 177Lu, electrophoresis and binding affinity studies are included.      Chapter 9 contains the overview of the chelators discussed in this thesis and suggests some future studies.   19  Chapter 2. tBu4octapa-alkyl-NHS for Metalloradiopeptides Preparation. This chapter contains an adaptation of a submitted manuscript and is reproduced in part from:  Li, L.; Kuo, H.-T.; Wang, X.; Merkens, H.; Colpo, N.; Radchenko, V.; Schaffer, P.; Lin, K.-S.; Bénard, F.; Orvig, C. tBu4octapa-alkyl-NHS for Metalloradiopeptide Preparation. Dalton Trans. 2020, 49, 7605-7619.  2.1.   Introduction      Radiometalated bioconjugates are useful constructs in nuclear imaging and therapy. The primary scaffold of a metallo-radiopharmaceutical consists of four synthetically exchangeable modules: the radiometal ion, the emission properties of which can be harnessed for diagnosis and/or therapy, the chelator which stably sequesters the radiometal ion, and the linker which tethers the chelator to the final module, the biological targeting vector for disease targeting.1,16 Peptides, in particular, have recently garnered considerable attention in targeted drug delivery,55,73,74 due to their high affinity and specificity, higher diffusibility, lower immunogenicity, and easier synthesis/ modifications.52,75 Unlike monoclonal antibodies (mAb), which necessitate the use of long-lived radionuclides due to the long blood circulation, peptide-conjugates are compatible with a wider range of isotope half-lives, from ~1 h (e.g. gallium-68),76 to >6 d (e.g. lutetium-177 and actinium-225).77,78 Besides a favorably long half-life, lutetium-177 is a highly potent theranostic radionuclide due to the low energy -particles (maximum 498 keV),78 imageable -rays (208 and 20  113 keV),79 and efficient production routes (i.e. [176Lu(n,γ)177Lu] or [176Yb(n,)177Yb→177Lu]) to meet a potentially large clinical demand.5,80,81        The essential component to join the otherwise incompatible radionuclide and targeting vector is a bifunctional chelator that secures the radiometal ion in vivo. To date, the macrocycle DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) dominates radiometal chelation (including with lutetium-177) in both preclinical and clinical studies, because its constrained binding cavity often yields metal complexes with higher stability.58,82,83,84  However, prolonged heating is often required in order to achieve quantitative radiochemical yield (RCY).16 Although peptide-conjugates generally have higher tolerance to the elevated temperature, fast RT-radiolabeling can facilitate radiotracer preparation in the clinical settings following “shake-and-shoot” protocols, and avoid potential degradation of the products. In this regard, non-macrocyclic chelators are preferable since, with enough rigidity, they can combine fast coordination kinetics with high complex stability.17,41,85       Multiple studies have proved that the chemical properties (e.g. hydrophilicity, charge) of the radiometal complex and the spacer can impact the drug biodistribution drastically, particularly when coupled to small peptides.77,86,87,88,89,90,91,92 As a result, expanding the chelator-library beyond the commercial chelating agents (e.g. DOTA or DTPA (diethylenetriamine-pentaacetic acid), Figure 2.1) is worthwhile. Often, the synthesis of a bifunctional chelator (i.e. chelator + linker) is more complicated than of the non-bifunctional analog (i.e. without the linker); therefore, the latter is usually employed in the preliminary chemical and radiolabeling studies to investigate the affinity of the binding cavity for the metal ion of interest.39,41, If the results are promising, a reactive coupling group will be incorporated for bioconjugation and subsequent in vivo evaluation.26,42 21  Since the placement of the linker can tremendously alter the metal-complex coordination sphere, and therefore the complex stability, while it is difficult to predict the kinetic inertness of the metal complex in the complicated biological environment using simple chemical settings,20,93 radiolabeling and in vitro stability experiments are usually repeated with the radiolabeled bioconjugates to assist interpretation of the in vivo biodistribution results.       A family of picolinate-based non-macrocyclic chelators that target various radionuclides with fast coordination kinetics at room temperature has been established.17,27,32,33 An octadentate member, H4octapa (Figure 2.1), has demonstrated excellent affinity for a range of radiometal ions, including that of lutetium-177 and the complex was thermodynamically stable (log KLu(octapa) = 20.1 and pLu = 19.8).26 In addition, the biological stability of [177Lu][Lu(octapa)]- was demonstrated with the conjugate of a monoclonal antibody, Trastuzumab, using N-isothiocyanate functionalized H4octapa-benzyl-NCS (Figure 2.1), in which the p-benzyl-N-isothiocyanate (p-Bn-NCS) linker was covalently attached to the ethylenediamine backbone; the bioconjugate was radiolabeled in high radiochemical yield (95%) with excellent in vitro serum and in vivo stability.26 Although the antibody-conjugation was reported to be efficient, difficulties were encountered when conjugating H4octapa-benzyl-NCS to a small glutamate-urea-lysine-based PSMA (prostate-specific membrane antigen)-targeting peptidomimetic, perhaps due to the interferences from multiple free carboxylic acid groups on both the chelator and the targeting pharmacophore. This prompted us to develop a new bifunctional octapa analogue in which carboxylic acid groups are protected as tert-butyl esters and the linker is attached through one of the acetate arms to maintain the overall denticity (i.e. tBu4octapa-alkyl-NHS, Scheme 2.1). Additionally advantageous in the design is its compatibility with solid-phase peptide synthesis (SPPS), allowing the metal-binding 22  unit to conveniently be incorporated site specifically into the amino acid sequence on a solid support. Incorporating the chelator prior to resin cleavage eliminates one exhaustive step of high-performance liquid chromatography (HPLC) purification of the individual peptide. In fact, most peptides are generated by SPPS;61 therefore, tBu4octapa-alkyl-NHS is anticipated to have widespread application in metallo-radiopeptides. Herein, the synthesis and characterization of tBu4octapa-alkyl-NHS are detailed. The bifunctional chelator was successfully coupled to two well-studied PSMA-targeting pharmacophores with different hydrophobicity (named as PSMA-ureido and PSMA617 herein, Figure 2.1) on solid supports.94,95 The more hydrophobic conjugate (PSMA617) has been shown to have improved pharmacokinetics, including longer biological half-life, higher tumor uptake and faster kidney clearance.95 Both PSMA targeting motifs can easily be synthesized via SPPS, and most importantly, extensive research has proven its targeting-specificity to PSMA which is up-regulated in prostate cancer cells. As a result, they are suitable candidates for proof-of-principle animal studies with a new chelator scaffold.96 Radiolabeling, in vitro serum challenge and lanthanum(III) challenge studies were performed with both peptide-conjugates to investigate their metal-scavenging abilities under extremely dilute conditions, and the kinetic inertness of the formed complexes. Both radiolabeled tracers were also individually injected into LNCaP-xenograft mice (n = 5) for biodistribution studies to evaluate in vivo stabilities. As well, in order to study the effects from the bifunctionalization on the metal coordination environment, density-functional theory calculations (DFT) were performed to compare the geometry of different 177Lu-labeled H4octapa complexes. 23   Figure 2.1 Chemical structures of discussed ligands. 24  2.2.   Results and Discussion 2.2.1.   Synthesis and Characterization  Scheme 2.1 Reagents and conditions. i) THF, nosyl chloride, ambient temp, 18 h, 89%; ii) tert-butyl bromoacetate, TEA, dry ACN, ambient temp, 18 h, 63%; iii) DCC, tert-butyl alcohol, DCM, ambient temp, overnight, 50%; iv) NaBH4, dry MeOH, ambient temp, 3-4 h, 72%; v) PBr3, dry ACN, 0 oC-ambient temp, 54%; vi) K2CO3, dry ACN, 60oC, 24 h, 70%; vii) PhSH, K2CO3, THF, 18 h, ambient temp, 63%; viii) NaBr, 2 N HBr, NaNO2, conc H2SO4, 18 h, ambient temp, 45%; ix) TBTA/cyclohexane, DMA, BF3-etherate, CHCl3, ambient temp, 3d ; x) K2CO3, dry ACN, 60 oC, 48 h, 30%; xi) Pd/C, H2, MeOH, ambient temp, 18 h, 87%; xii) NHS, EDC ˑ HCl, dry ACN, 69%. 25       The synthesis (Scheme 2.1) started with the asymmetric nosyl protection of the ethylenediamine backbone. About 0.3 equivalents of 2-nitrobenzenesulfonyl chloride (NsCl) dissolved in tetrahydrofuran (THF) was slowly added to the stirred ethylenediamine solution at 0°C to give compound 2.1 in high yield (89%). After selective protection, mono-alkylation of the primary amine in compound 2.1 was achieved by dropwise addition of tert-butyl bromoacetate in THF at ambient temperature in order to minimize over-alkylation (compound 2.2, 63%). After that, the secondary amines in compound 2.2 were alkylated with the bromo-picolinate derivative (compound 2.5) to give the nosyl-protected ligand scaffold (compound 2.6, 70%). To synthesize the bromo-picolinate 2,6-substitute (compound 2.5), the carboxylic acid groups in the commercially available 2,6-picolinate dicarboxylic acid were converted to tert-butyl esters using N,N’-dicyclohexylcarbodiimide (DCC) (compound 2.3, 50%). The resulting ester-derivative was selectively reduced to tert-butyl 6-(hydroxymethyl)picolinate (compound 2.4, 72%) using sodium borohydride (NaBH4), followed by bromination of the alcohol group with phosphorus tribromide (PBr3) in dry acetonitrile (ACN) (54%). To free the secondary amine in compound 2.6, the nosyl protecting group was removed with thiophenol at ambient temperature overnight (compound 2.7, 63%). Due to the strong affinity for silica, compound 2.7 was purified with the neutral alumina column eluted with dichloromethane (DCM) and methanol (MeOH). After that, (2S/2R)-5-benzyl 1-(tert-butyl) 2-bromopentanedioate (compound 2.9) derived from (L)-glutamic acid γ-benzyl ester was incorporated into compound 2.7 by refluxing the mixture with potassium carbonate (K2CO3) in dry ACN to give compound 2.10 in 30% yield. The unsatisfactory yield could be due to the steric hindrance between the crowded secondary amine and the secondary alkyl bromide. The benzyl ester in compound 2.10 was selectively removed through palladium/carbon (Pd/C, 10% w/w)-catalyzed hydrogenation to give compound 2.11 (87%), while the resulting free carboxylic 26  acid group was activated with N-hydroxylsuccinimide and N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (NHS/EDCl) to give tBu4octapa-alkyl-NHS (compound 2.12, 69%). For peptide-conjugation, tBu4octapa-alkyl-NHS was coupled to the PSMA-targeting-peptide-bound resin (the peptide was either PSMA-ureido or PSMA617, Figure 2.1) by using N,N-diisopropylethylamine (DIPEA) in dry dimethylformamide (DMF) overnight. After that, the peptide-bioconjugate was deprotected and simultaneously cleaved from the resin using 95/5 trifluoroacetic acid (TFA)/triisopropylsilane (TIS) at ambient temperature to give either H4octapa-alkyl-PSMA-ureido or H4octapa-alkyl-PSMA617 (Figure 2.1). It is noted that the compound 2.9 used for the reaction consisted of both R- and S-enantiomers because the intermediate compound 2.8 was synthesized via the diazotization reaction, in which a carbocation derivative was generated upon the release of N2(g) by the highly unstable diazonium cation. As a result, the final bifunctional chelator is a mixture of enantiomers; however, due to the difficulties in separating the mixture and for the purpose of preliminary investigations of this new chelator scaffold, the isomers were not isolated, but the effects of the stereochemistry were studied with DFT calculations.   2.2.2.   DFT Calculations      To study the effects of the bifunctionalization in tBu4octapa-alkyl-NHS on the metal coordination environment, DFT calculations were performed to predict the structures of different Lu-octapa configurations. [Lu(octapa-alkyl-benzyl-ester)] was used as the simulation model for the arm functionalized H4octapa (Figures 2.1 and 2.2). Calculations for the non-bifunctional [Lu(octapa)]- and [Lu(S-benzyl-octapa)]- (Figures 2.1 and 2.3) employed as the model for the complex with the ethylenediamine functionalized H4octapa-benzyl-NCS, were performed for 27  comparison. The selected bond lengths and bond angles are tabulated in Table 2.1. The functionalization on the acetate arm obviates the 2-fold rotational symmetry about the ethylenediamine bridge in the non-bifunctional H4octapa. Two acetate arms in H4octapa-alkyl-benzyl-ester (and tBu4octapa-alkyl-benzyl-NHS) become inequivalent, and therefore, their relative positions can alter the overall complex geometry, leading to two possible structural isomers as shown in Figure 2.2. Since the methylene-C on the functionalized acetate arm contains both S- and R-configurations, while for each S-/R-configuration, the functional arm can bind to the metal ion (i.e. Lu3+) from the front or the back with respect to the five-membered chelate ring of Lu-N1-C-C-N2. The front and the back positions are defined based on the presented figures (Figure 2.2). When the functional acetate arm chelates from the back of the Lu-N1-C-C-N2 plane (O1 into the page), the 8-coordinated complex adopts the geometry of distorted square antiprism (DSA). When the arm is positioned at the front of the plane (O1 out of the page), the structure is a distorted bicapped trigonal antiprism (DBTA) where N2, N3, N4, O1, O2 and O3 formed the distorted trigonal antiprism capped by N1 and O4. DBTA was also the calculated geometry adopted by both [Lu(octapa)]- and [Lu(S-benzyl-octapa)]- complexes with similar bond lengths (Table 2.1), which means that the linker in H4octapa-benzyl-NCS very possibly poses minimal disturbances in Lu3+ coordination. As for [Lu(octapa-alkyl-benzyl-ester)]-, the adopted molecular shape avoids the S-/R-stereochemistry. Indeed, upon comparing the Lu-X bond lengths between the S- and the R-geometries, except for the Lu-N2 bond which was 0.1628 Å shorter in R-DBTA than S-DBTA, the S-/R-configurations on the arm have minimal effects on the coordination bond distances (i.e. difference <0.1 Å). Within the same S-/R-configuration, most bond lengths are comparable between the two complex geometries (DBTA vs. DSA), except for those with the tertiary amine nitrogen atoms (N1 and N2), particularly between the S-DBTA and the S-DSA isomers, where the 28  Lu-N2 bond distance in the latter was shorter by almost 0.3 Å. Furthermore, the Lu-N2 bonds in both DSA geometries were also shorter than those in the [Lu(octapa)]- and [Lu(S-benzyl-octapa)]- complexes.  The shorter Lu-N1 and Lu-N2 bond distances in the S-/R-DSA geometries accompanied larger N1-Lu-N2 bond angles (~68˚), which could be slightly more favorable considering the ideal M-N bond length and N-M-N bond angle in a five-membered chelate ring with ethylenediamine being ~2.5 Å and 69˚, respectively.97,98 Nonetheless, all four acetate-functionalized Lu-octapa geometries (i.e. S-/R- DSA/DBTA) were calculated to be energetically similar, with the energy difference < 7 kcal/mol. Therefore, it is possible that they co-exist in solution. Importantly, the results revealed a significant difference between placing the linker on the ethylenediamine and functionalizing via the acetate moiety, where the latter has altered the bond geometry in the [Lu(octapa)]- coordination sphere, and the changes were regardless of the S-/R-stereochemistry. Table 2.1 Selected DFT-calculated bond lengths and bond angles.     Bond length Å / bond angle˚    Arm functionalization [Lu(S-benzyl-octapa)]- [Lu(octapa)]-  R-DBTA R-DSA S-DBTA S- DSA N1 Backbone 3o N* Lu 2.8942 2.7442 2.8541 2.7731 2.8245 2.8202 N2 Backbone 3o N Lu 2.7859 2.6712 2.9487 2.6604 2.9616 2.8219 N3 Picolinate-N* Lu 2.4903 2.4849 2.5775 2.4659 2.4836 2.4678 N4 Picolinate-N Lu 2.4582 2.4895 2.5443 2.4958 2.5626 2.4662 O1 Acetate-COO* Lu 2.2732 2.2211 2.2430 2.2227 2.2610 2.2737 O2 Acetate-COO Lu 2.2703 2.2388 2.2307 2.2372 2.2445 2.2721 O3 Picolinate-COO* Lu 2.2592 2.3320 2.2599 2.3367 2.2769 2.2704 O4 Picolinate-COO Lu 2.2675 2.3364 2.2777 2.3380 2.2492 2.2704  N1-Lu-N2 angle  63.5 67.9 61.9 68.1 62.3 63.4  O1-Lu-O2 angle  170 135 170 136 170 171 *The side of the chelator where the linker located (no difference for H4octapa as it is symmetric).  29     S-DBTA      S-DBTA    S-DSA     S-DSA  Figure 2.2 DFT-calculated structures for S-DBTA and S-DSA geometries of [Lu(octapa-alkyl-benzyl-ester)]- (Same geometry adopted for the corresponding R-configuration).   30     DBTA      DBTA     DBTA     DBTA  Figure 2.3 DFT-calculated structures for [Lu(S-benzyl-octapa)]- (top) and [Lu(octapa)]- (bottom).      31  2.2.3.   Radiolabeling, Human Serum Stability and Lanthanum Challenge Experiments       Rapid and quantitative radiolabeling under mild conditions (~RT, pH ~ 7) with low chelator concentration not only provides flexibility in tuning the apparent molar activity of the radiotracer which can considerably influence the biological profile, but also obviates complicated post-labeling purification using high-performance liquid chromatography (HPLC). It was shown previously that high apparent molar activity of [177Lu][Lu(PSMA-617)] correlated to higher tumor-to-background ratio, and therefore enhanced tumoricidal effects.99 However, this trend is not generalizable because the opposite was observed with [44Sc][Sc(pypa-C7-PSMA617)], with which lower apparent molar activity significantly reduced the background organ uptakes, especially the kidney.100 For that reason, it is important to be able to adjust the apparent molar activity while not compromising the radiochemical yield (RCY). Concentration-dependent radiolabeling experiments, in which a constant amount of radioactivity is added to a series of diluted chelator solutions, determines the lowest concentration of chelator required for quantitative radiolabeling. After the radiolabeling studies, it is important to evaluate the stability of the labeled complex against the serum proteins over time. In this work, both experiments were performed with 177Lu-labeled H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 (Figures 2.4A and 2.4B). The results were analyzed with instant thin-layer chromatography plates impregnated with silicic acid (iTLC-SA), developed in ethylenediaminetetraacetic acid (EDTA) buffer (50 mM, pH = 5.2). Attempts to use HPLC to identify the radiolabeled complexes failed, perhaps due to the decomplexation in the acidic HPLC column (mobile phase: water/acetonitrile/0.1%TFA). To validate the human serum challenge experiment, a control sample with only lutetium-177 in ammonium acetate (NH4OAc) buffer (0.2 M, pH = 7) and serum was prepared, and analyzed with 32  the same TLC technique, which confirmed that the EDTA buffer was able to carry both free and transchelated lutetium-177 to the solvent front (i.e. only one radioactivity spot at the solvent front on the developed TLC plate).      Previously, H4octapa has demonstrated efficient radiolabeling with lutetium-177 resulting in a reasonably stable complex upon challenged with serum proteins; while the biological stability of the formed complex was confirmed with H4octapa-benzyl-Trastuzumab, a mAb-conjugate using the ethylenediamine-functionalized H4octapa-benzyl-NCS.26,101 In the case of H4octapa-benzyl-Trastuzumab, the conjugation did not hamper the radiolabeling efficiency (95%, RT, 15 min, 74-111 MBq)26, nor did it significantly reduce the kinetic inertness (~89% intact in human serum on day 5, 37°C).101 However, with the linker placed on one of the acetate arms instead, both 177Lu-labeled H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 demonstrated transchelation to the human serum proteins within one day, with the PSMA617 conjugate more labile (18±10% intact on day 1, 37˚C) (Figure 2.4B). The complex lability was disappointing since the radiolabeling performance at room temperature was impervious to the bioconjugation with quantitative radiochemical yields achieved using 10-6 M chelator concentration, in 15 min with 2 MBq lutetium-177 for both conjugates (~80 GBq/mol). (Figure 2.4A). To understand such unexpected lability, the kinetic inertness of the 177Lu-labeled H4octapa, H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 (Figure 2.4C) were challenged with excess La3+ ions. To validate the challenge experiment and relate it to the in vitro, and even in vivo kinetic inertness of the complex, comparative experiments should be conducted on similar constructs that have shown promising stability results with lutetium-177. In this work, H4pypa, H4pypa-C7-PSMA-ureido and H4pypa-C7-PSMA617 were adopted for direct comparisons (Figures 2.1 and 2.4D) since the 33  corresponding 177Lu-labeled pypa-complexes/conjugates have demonstrated excellent serum and biological stability (data with  H4pypa-C7-PSMA-ureido are unpublished).17 The challenge studies were conducted by adding diluted natLa3+ AAS (atomic absorption spectroscopy) standard solution to the radiolabeled samples with 3:1 La3+:ligand (mol/mol) ratio and then incubating at 37˚C. Samples without natLa3+ ion were prepared as the control. As seen in Figure 2.4C, a substantial amount of lutetium-177 leached out of all the H4octapa complexes steadily in the presence of excess La3+ ions (intact % on day 7: octapa = 51±14%, PSMA-ureido = 17±6%, PSMA617 = 39±11%), implying a high rate of dissociation (off-rate) of the complexes. Surprisingly, even in the absence of La3+, both labeled octapa-PSMA conjugates still decomplexed over time in the NH4OAc buffer, albeit much slower (intact % on day 1 and 7: PSMA-ureido = 80% and 75%, PSMA617 = 88% and 57%, respectively) (Figure 2.4C). As for [177Lu][Lu(octapa)]-, the dissociation was completely stimulated by the La3+ ions. In contrast, the 9-coordinated 177Lu-pypa complexes/conjugates were highly inert not only in the buffer solution, but also under the La3+-competition (Figure 2.4D). The disturbances from the PSMA-targeting vectors on the coordination spheres, albeit present, was much less severe compared to the H4octapa-counterparts (intact % on day 7: PSMA-ureido = 92±2%, PSMA617 = 86±3%) (Figure 2.4D). One explanation for these observations, particularly when comparing [177Lu][Lu(L)]- (L = octapa or pypa), could be that a nine- rather than eight- coordinating environment might be more favorable for the Lu3+ ion. The pyridyl backbone in H4pypa might also contribute to a more rigid, and subsequently more inert, complex compared to the ethylenediamine backbone in H4octapa. Another possible reason is the destabilization resulting from the conjugation through the arm, which can also explain the discrepancy in the kinetic inertness between the 177Lu-labeled octapa-PSMA conjugates reported here and [177Lu][Lu(octapa-benzyl-Trastuzumab)] reported previously.26 In fact, DFT calculations 34  also predicted different complex geometries were adopted by the ethylenediamine-functionalized and acetate-functionalized octapa ligands (Figures 2.2 and 2.3). To validate the results from the competition and in vitro experiments, in vivo studies must be conducted.    Figure 2.4 (A) Concentration-dependent radiolabeling of H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 with lutetium-177 in 15 min at room temperature and pH = 7 (0.2 M NH4OAc). (B) Intact percentage of the radiocomplexes upon human serum challenge over 7 days at 37oC. Lanthanum challenges on 177Lu-labeled (C) H4octapa, H4octapa-alkyl-PSMA-ureido, H4octapa-alkyl-PSMA617, and (D) H4pypa, H4pypa-C7-PSMA-ureido, H4pypa-C7-PSMA617, over 7 days.  4 5 6 7020406080100RCY %-log[M] [177Lu][Lu(octapa-alkyl-PSMA617)] [177Lu][Lu(octapa-alkyl-PSMA-ureido)]A0 1 2 3 4 5 6 7020406080100% IntactDay [177Lu][Lu(octapa-alkyl-PSMA-ureido)] [177Lu][Lu(octapa-alkyl-PSMA617)]B0 1 2 3 4 5 6 7020406080100% IntactDay [177Lu][Lu(octapa)]- 0:1 [177Lu][Lu(octapa)]- 3:1 [177Lu][Lu(octapa-alkyl-PSMA-ureido)] 0:1 [177Lu][Lu(octapa-alkyl-PSMA-ureido)] 3:1 [177Lu][Lu(octapa-alkyl-PSMA617)] 0:1 [177Lu][Lu(octapa-alkyl-PSMA617)] 3:1 La:Ligand : 0:1 and 3:1C0 1 2 3 4 5 6 7020406080100% IntactDay [177Lu][Lu(pypa)]- 0:1 [177Lu][Lu(pypa)]- 3:1 [177Lu][Lu(pypa-C7-PSMA-ureido)] 0:1 [177Lu][Lu(pypa-C7-PSMA-ureido)] 3:1 [177Lu][Lu(pypa-C7-PSMA617)] 0:1 [177Lu][Lu(pypa-C7-PSMA617)] 3:1 La:Ligand : 0:1 and 3:1D35  2.2.4.   SPECT/CT MIP Imaging and Ex vivo Biodistribution Studies      Both 177Lu-labeled H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 were prepared at an apparent molar activity of ~124 GBq/mol and then injected into LNCaP-xenograft mice (n = 5) at a dose of approximately 1.5-2 MBq per mouse, except for the imaging mice (~38-48 MBq per mouse). Due to the fast clearance of [177Lu][Lu(octapa-alkyl-PSMA-ureido)], the mice were monitored only up to 24 h post-injection (p.i.), but 72 h for those injected with the PSMA617 analog which has a longer biological half-life. SPECT/CT maximum intensity projection (MIP) imaging (Figure 2.5) and ex vivo biodistribution studies (Figure 2.6) showed that both radiotracers were cleared renally and collected through the bladder. Additionally, they shared similar biodistribution patterns where the majority of radioactivity accumulations were observed in PSMA-expressing organs, including spleen, adrenal glands, kidney, tumor and salivary glands.102,103 At 1 h p.i., for the PSMA-ureido conjugate, the uptakes of those organs were 9.60±5.15, 5.83±2.60, 75.0±9.3, 4.92±0.88 and 6.68±1.13% injected-dose-per-gram (% ID/g), respectively, while 25.0±13.0, 8.03±3.38, 143±17, 13.5±1.1 and 8.90±2.90 % ID/g, respectively, for the PSMA617 analog. The accumulations, except for the tumor and the kidney, were eliminated quickly over time (≤1.53% ID/g at the last time point). Incorporated with the additional hydrophobic linker, [177Lu][Lu(octapa-alkyl-PSMA617)] yielded significantly higher tumor uptake than [177Lu][Lu(octapa-alkyl-PSMA-ureido)], peaked at 4 h p.i. (17.0±2.0 vs. 3.45±0.95% ID/g), and retained at 12.3±4.6% ID/g on day 3, resulting in considerably higher tumor-to-muscle and tumor-to-blood ratios (Table 2.2). The day-3 tumor uptake was comparable to [177Lu][Lu(pypa-C7-PSMA617)] (12.7±4.2% ID/g), although the uptake at 4 h p.i. was higher for the pypa-analog (24.0±7.6% ID/g).17  Regarding the kidney clearance, it was much faster for 36  [177Lu][Lu(octapa-alkyl-PSMA617)] than [177Lu][Lu(octapa-alkyl-PSMA-ureido)], from 143±17% ID/g at 1 h p.i. to 26.0±10.9% ID/g at 24 h p.i., and 7.22±3.43% ID/g at 72 h p.i., leading to a sharp increase in tumor-to-kidney ratio by 19-fold over the course of study, in contrast to only ~25% drop over 24 h for the PSMA-ureido counterpart (49.5±4.4% ID/g, 24 h p.i.). Unfortunately, although the uptakes of both octapa radiotracers were mostly PSMA-driven and the tumor accumulation for [177Lu][Lu(octapa-alkyl-PSMA617)] was higher than the DOTA-based [177Lu][Lu(PSMA-617)] (12.3±4.6% vs. 7.80±3.69% ID/g on day 3),104 the bone radioactivity of the labeled octapa-alkyl-PSMA617 rose over time (Figure 2.7), from 0.64±0.29% ID/g (1 h p.i.) to 3.62±2.06% ID/g (24 h p.i.)), resulting in a dramatic drop in the tumor-to-bone ratio (20.9 to 2.96). Of course, the tumor clearance also contributed to the reduced contrast ratio. On the other hand, the growth in the bone uptake was significantly less appreciable with the more hydrophilic [177Lu][Lu(octapa-alkyl-PSMA-ureido)], perhaps due to the rapid excretion of the tracer. These in vivo biodistribution were due to the metal ion leaching out of the chelator, which was consistent with the in vitro serum instability, implying that the bifunctionalization on the acetate arm in H4octapa was suboptimal for 177Lu-chelation – this is significant for future chelator design. The lanthanum challenge experiment also predicted the lability of the 177Lu-octapa PSMA-targeting conjugates seen in the biodistribution. As for the 177Lu-pypa-C7-PSMA analogs, the high inertness in the presence of excess La3+ ions was consistent with the in vivo stability (<1% ID/g bone uptake on day 3 for [177Lu][Lu(pypa-C7-PSMA617)]).17 Nonetheless, considering the observed differences in the in vivo stability between [177Lu][Lu(octapa-alkyl-PSMA-ureido)] and [177Lu][Lu(octapa-alkyl-PSMA617)], it should be noted that the biological instability of the complex might not be revealed if the circulation half-life of the radiotracer is very short, and 37  therefore, a reasonably long-lived targeting vector should be adopted for proof-of-principle biodistribution studies of a new chelator – another significant fact for future chelator design.                            Figure 2.5 SPECT/CT MIP Images of (A) [177Lu][Lu(octapa-alkyl-PSMA-ureido)] (qualitative imaging) (B) [177Lu][Lu(octapa-alkyl-PSMA-617)] (quantitative imaging) at different post-injection time points.         Figure 2.6 Ex vivo biodistribution data of (A) [177Lu][Lu(octapa-alkyl-PSMA-ureido)] (B) [177Lu][Lu(octapa-alkyl-PSMA-617)] at different post-injection time points.  BloodFatSeminalTestesIntestineStomachSpleenLiverPancreasAdrenalKidneyLungHeartTumourMuscleBoneBrainTailSalivaryLacrimal0105060708090% ID/g 1 h 4 h 24 hABloodFatSeminalTestesIntestineStomachSpleenLiverPancreasAdrenalKidneyLungHeartTumourMuscleBoneBrainTailSalivaryLacrimal010203040140150160170180230240% ID/g 1 h 4 h 24 h 72 hB1 h 4 h 24 h 4 h 24 h 72 h  % ID/g 50 0 38   Figure 2.7 Bone uptake (%ID/g) comparison between the 177Lu-labeled octapa-alkyl-PSMA-ureido and octapa-alkyl-PSMA617 over time.  Table 2.2 Tumor-to-background ratios at different p.i. timepoints (n = 5).          [177Lu][Lu(octapa-alkyl-PSMA-ureido)]  1 h 4 h 24 h - Tumor/bone 8.76 5.17 6.10 - Tumor/muscle 16.10 27.67 41.35 - Tumor/blood 5.36 14.55 68.20 - Tumor/kidney 0.07 0.06 0.08 - [177Lu][Lu(octapa-alkyl-PSMA617)]  1 h 4 h 24 h 72 h Tumor/bone 20.94 12.78 2.96 5.76 Tumor/muscle 28.53 54.64 71.47 107.69 Tumor/blood 8.75 17.91 135.66 907.86 Tumor/kidney 0.09 0.09 0.41 1.70 0 20 40 600123456Bone uptake (% ID/g)Time (h) [177Lu][Lu(octapa-alkyl-PSMA617)]  [177Lu][Lu(octapa-alkyl-PSMA-ureido)]39  2.3.   Conclusions      H4octapa has previously shown versatility in chelating a range of radiometal ions with high stability,26,39,40 including lutetium-177. Herein, a new bifunctional analogue, tBu4octapa-alkyl-NHS, was synthesized and completely characterized. The carboxylic acid moieties in the chelator were protected as tert-butyl esters, facilitating peptide conjugation via solid-phase peptide synthesis, which is valuable in metallo-radiopeptide preparation. In tBu4octapa-alkyl-NHS, the linker is covalently attached to the methylene carbon on one of the acetate arms to maintain the coordination integrity. This functionalization generates a chiral carbon center, which, based on the DFT calculations, did not cause significant changes to the complex geometry. Because the two acetate arms become inequivalent, depending on their relative position upon complexation, there could be four isomers (i.e. S-/R-DBTA and S-/R-DSA). For proof-of-principle biological evaluations of the new bifunctional 177Lu-octapa chelate, two easily accessible and well-studied Glu-urea-Lys-based PSMA targeting motifs (i.e. PSMA-ureido and PSMA617) were conjugated to tBu4octapa-alkyl-NHS. Both ligands coordinate lutetium-177 efficiently at room temperature in 15 min at micromolar chelator concentration, despite the decomplexation in the presence of serum proteins and excess La3+ ions. To confirm the in vitro results, both radiotracers were injected into a cohort of five LNCaP-xenografted mice for biodistribution studies. The pharmacokinetics patterns of both tracers resembled with expected uptakes in the PSMA-expressing organs. Similar to [177Lu][Lu(PSMA-617)],104 higher tumor uptake and faster kidney clearance were seen with [177Lu][Lu(octapa-alkyl-PSMA617)], while the increasing bone radioactivity concentration was also exclusively observed with the PSMA617-conjugate, perhaps due to the enhanced blood circulation. Since the in vivo decomplexation was not seen in the ethylenediamine-functionalized 40  [177Lu][Lu(octapa-benzyl-Trastuzumab)],26 it is concluded that the alkyl linker on the acetate arm perturbed the 177Lu-octapa coordination sphere. Considering these results, it is important to understand the potential alterations exerted by the linker on the metal coordination which is often overlooked; however, the results reported in this work are not conclusive to the analogous complexes with other radionuclides that H4octapa has demonstrated high affinity for, such as 111In and 86/90Y.  2.4.   Experimental Section 2.4.1.   Materials and Methods      All solvents and reagents were purchased from commercial suppliers (TCI America, Alfa Aesar, AK Scientific, Sigma-Aldrich, Fisher Scientific, Fluka) and were used as received. Deionized water was filtered through the PURELAB Ultra Mk2 system. NMR spectra were recorded at ambient temperature on Bruker AV400 instruments, as specified; NMR spectra are expressed on the δ scale and referenced to residual solvent peaks. Low-resolution (LR) mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray-ionization mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the Department of Chemistry, University of British Columbia. The HPLC system used for purification of non-radioactive compounds consisted of a Waters 600 controller, Waters 2487 dual wavelength absorbance detector, and a Waters delta 600 pump. Phenomenex Synergi 4  hydro-RP 80 Å column (250 mm × 21.2 mm semipreparative) was used for purification of H4octapa-alkyl-PSMA-ureido. Phenomenex Luna 5 m C18 100 Å LC column (250 mm × 10 mm) was used for purification of H4octapa-alkyl-PSMA617. Automated 41  column chromatography was performed using a Teledyne Isco (Lincoln, NE) Combiflash Rf automated system with solid load cartridges packed with Celite and RediSep Rf gold reusable normal-phase silica/neutral alumina columns (Teledyne Isco, Lincoln, NE). Analyses of radiolabeled compounds were performed with both thin layer chromatography (TLC) (i.e. iTLC-impregnated with silicic acid (iTLC-SA) strips) purchased from Agilent Technologies). The TLC scanner model was BIOSCAN (system 200 imaging scanner). 177LuCl3 was purchased from Isotope Technologies Garching (ITG) as no-carrier added (n.c.a.).    2.4.2.   Synthesis and Characterization N-(2-Aminoethyl)-2-nitrobenzenesulfonamide (2.1)       A solution of 2-nitro-phenylsulfonyl chloride (5.00 g, 22.6 mmol, 0.3 equiv) in tetrahydrofuran (THF) (25 mL) was added dropwise via a dropping funnel over 2 h to a solution of ethylenediamine (5.00 mL, 67.9 mmol, 1 equiv) in THF (12.5 mL) in a round-bottom flask at 0°C. Upon complete addition, the resulting mixture was warmed up to room temperature and stirred for an additional 30 min. The crude mixture was then concentrated, and the residue obtained was taken up in dichloromethane (DCM) (25 mL) and washed with H2O (25 mL). The aqueous layer was further extracted with DCM (25 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate (Na2SO4), and then clarified by filtration. The filtrate was concentrated in vacuo. The oily residue obtained was then treated with concentrated hydrochloric acid (7.50 mL). The precipitate instantaneously formed was separated by filtration to yield the by-product (N-[2-(2-nitrophenylsulfonyl)amino-ethyl]-2-nitro-phenyl-sulfonamide) as a whitish solid. The resulting orange filtrate was rotary-evaporated to give a bright yellow solid as the product (5.69 g, 89%). 42  1H NMR (400 MHz, 298 K, D2O): δ 8.10-8.05 (m, 1H), 8.01-7.96 (m, 1H), 7.94 – 7.81 (m, 2H), 3.39 – 3.31 (m, 2H), 3.18-3.14 (m, 2H). 13C NMR (100 MHz, 298 K, D2O) : δ 135.1, 133.8, 131.3, 130.7, 125.9, 40.4, 39.5. LR-ESI-MS: calcd for [C8H11N3O4S+H]+ 246.0; found 246.2. Tert-butyl-(2-((2-nitrophenyl)sulfonamido)ethyl)glycinate (2.2).       A solution of tert-butyl bromoacetate (2.98 mL, 20.2 mmol, 1 equiv) in THF (85 mL) was added dropwise via a dropping funnel to a suspension of compound 2.1 (5.00 g, 17.7 mmol, 0.9 equiv) and triethylamine (TEA) (8.45 mL, 60.6 mmol, 3 equiv) in THF (85 mL) in a round-bottom flask over 1 h at room temperature. Another portion of TEA (1.41 mL, 10.1 mmol, 0.5 equiv) was then added, followed by the dropwise addition of tert-butyl bromoacetate (1.49 mL, 10.1 mmol, 0.5 equiv) in THF (35 mL) over 30 min. The additional portions were necessary to achieve the complete conversion of compound 2.1. Stirred overnight at room temperature, the crude mixture was then concentrated in vacuo and the residue was taken up in DCM (100 mL) and washed with water (50 mL). The aqueous layer was further extracted with DCM (100 × 2 mL). The combined organic layers were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was concentrated in vacuo. The oily yellow residue obtained was purified with a silica column (A: hexanes B: ethyl acetate, 80-100% B). The product fractions were rotary-evaporated to give a yellow oil (4.46 g, 63%). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.18 – 8.11 (m, 1H), 7.89 – 7.82 (m, 1H), 7.75-7.71 (m, 2H), 3.19 (s, 2H), 3.17 – 3.10 (m, 2H), 2.80 – 2.73 (m, 2H), 1.44 (s, 9H). 13C NMR (100 MHz, 298 K, D2O) : δ 171.5, 148.2, 133.7, 133.6, 132.8, 131.2, 125.4, 81.8, 51.0, 48.0, 43.2, 28.2. LR-ESI-MS: calcd for [C14H21N3O6S+H]+ 360.1; found [M+H]+ 360.1.  43  Di-tert-butyl pyridine-2,6-dicarboxylate (2.3)       To a stirred suspension of 2,6-pyridinedicarboxylic acid (10.0 g, 59.8 mmol, 1 equiv) in DCM (30 mL) was added tert-butyl alcohol (22.6 mL) and 4-dimethylaminopyridine (DMAP) (3.65 g, 29.9 mmol, 0.5 equiv) at room temperature. Then, N,N'-dicyclohexylcarbodiimide (DCC) (27.2 g, 0.132 mol, 2.2 equiv) in DCM (30 mL) was added dropwise using a dropping funnel over 1 h. The mixture was left stirring at room temperature overnight, and then the precipitate was filtered off by vacuum filtration. The filtrate was concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 80 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to give an off-white solid (8.36 g, 50%).  1H NMR (400 MHz, 298 K, CDCl3): δ 8.18 (d, J = 7.8 Hz, 2H), 7.95 – 7.90 (m, 1H), 1.64 (s, 18H). 13C NMR (100 MHz, 298 K, CDCl3): δ 168.3, 150.0, 138.1, 127.3, 83.2, 27.9.  LR-ESI-MS: calcd for [C15H21NO4 + H]+ 280.1;  found [M + H]+ 280.2. Tert-butyl 6-(hydroxymethyl)picolinate (2.4)       Compound 2.3 (1.40 g, 5.00 mmol, 1 equiv) was dissolved in dry MeOH (150 mL) in a round-bottom flask. Sodium borohydride (NaBH4) (0.189 g, 5.00 mmol, 1 equiv) was added at 0°C. The mixture was stirred at room temperature for 1 h and then another equivalent of NaBH4 was added. The reduction continued until the mono-reduced picolinate dominated, as monitored by silica TLC (5% MeOH in DCM). The average reaction time was 3-4 h. The reaction mixture was then diluted with DCM (100 mL) and quenched with saturated sodium bicarbonate (NaHCO3) in water (100 mL). The organic phase was separated and the MeOH in the aqueous phase was removed in vacuo to give an aqueous layer which was then extracted with DCM (100 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration.  The filtrate was 44  concentrated and then purified through a silica column (CombiFlash Rf automated column system, 40 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary evaporated to give an off-white powder (2.25 g, 72%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.88 (d, J = 7.6 Hz, 2H), 7.77 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 4.82 (s, 2H), 1.59 (s, 9H). 13C NMR (100 MHz, 298 K, CDCl3): δ 164.1, 160.3, 148.3, 137.6, 123.6, 123.4, 82.4, 64.5, 28.2. LR-ESI-MS: calcd for [C11H15NO3 + Na]+ 232.1; found [M + Na]+ 232.2. Tert-butyl 6-(bromomethyl)picolinate (2.5)       Compound 2.4 (1.83 g, 8.73 mmol, 1 equiv) with a stir bar in a three-neck round-bottom flask was purged with N2(g). Dry acetonitrile (ACN) (35 mL) was added via a syringe to dissolve the starting material. Phosphorus tribromide (PBr3) (1.24 mL, 13.1 mmol, 1.5 equiv) was added in three portions via a syringe over 15 min to the stirred solution of compound 2.5 at 0°C. The mixture was stirred at room temperature for 4-5 h, followed by dilution with DCM (50 mL) and saturated sodium carbonate (Na2CO3) in water (70 mL). The organic layer was separated while the ACN in the aqueous layer was removed in vacuo. The residual aqueous layer was further extracted with DCM (50 mL × 3). The combined organic layers were dried over anhydrous magnesium sulfate (MgSO4), filtered and then concentrated in vacuo. The crude product was further purified by a silica column (CombiFlash Rf automated column system; 40 g HP silica; A: hexanes, B: ethyl acetate, 30-50% B). The product fractions were rotary-evaporated to yield a waxy faint-yellow solid (1.36 g, 54%). 1H NMR (400 MHz, 298 K, CDCl3):  δ 7.93 (d, J = 8.4 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 4.64 (s, 2H), 1.63 (s, 9H). 13C NMR (100 MHz, 298 K, CDCl3) δ 163.5, 157.4, 148.9, 138.0, 126.6, 123.9, 82.5, 33.3, 28.1. LR-ESI-MS: calcd for [C11H1481BrNO2+Na]+ 296.0; found [M(81Br)+Na]+ 296.1. 45  Tert-butyl-6-(((N-(2-((2-(tert-butoxy)-2-oxoethyl)((6-(tertbutoxycarbonyl)-pyridin-2-yl)-methyl)amino)ethyl)-2- nitrophenyl)sulfonamido)methyl)picolinate (2.6)       Compound 2.2 (1.08 g, 3.01 mmol, 1 equiv) was dissolved in anhydrous ACN in a round-bottom flask, followed by addition of compound 2.5 (1.80 g, 6.63 mmol, 2.2 equiv) and potassium carbonate (K2CO3) (1.25 g, 9.04 mmol, 3 equiv). The reaction mixture was stirred at 60°C for 48 h. Then, K2CO3 was separated by centrifugation and washed with DCM (5 mL). The combined supernatants were concentrated in vacuo and purified by a silica column (CombiFlash Rf automated column system; 24 g HP silica; A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to yield a yellow oil (1.56 g, 70%). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.11-8.08 (m, 1H), 7.85 (d, J = 7.5 Hz, 2H), 7.76-7.70 (m, 2H), 7.67-7.66 (m, 1H), 7.64 – 7.59 (m, 3H), 7.53 (d, J = 7.7 Hz, 1H), 4.77 (s, 2H), 3.91 (s, 2H), 3.56-3.51 (m, 2H), 3.24 (s, 2H), 2.83-2.79 (m, 2H), 1.60-1.59 (m, 18H), 1.39 (s, 9H). 13C NMR (100 MHz, 298 K, CDCl3): δ 170.3, 164.1, 163.6, 160.0, 156.9, 148.9, 148.6, 148.1, 137.7, 137.3, 133.5, 133.3, 131.9, 130.9, 125.7, 125.1, 124.1, 123.6, 123.2, 82.1, 81.9, 81.1, 60.1, 55.8, 53.2, 52.6, 46.4, 28.1, 21.0, 14.2. LR-ESI-MS: calcd for [C36H47N5O10S+H]+ 741.3; found [M+H]+ 742.3. Tert-butyl-6-(((2-((2-(tert-butoxy)-2-oxoethyl)((6-(tert-butoxycarbonyl)-pyridin-2-yl)-methyl)amino)ethyl)amino)methyl)picolinate (2.7)       To a stirred solution of compound 2.6 (233 mg, 0.313 mmol, 1 equiv) in dry THF in a round-bottom flask was added thiophenol (80 L, 0.780 mmol, 2.5 equiv) and K2CO3 (0.130 g, 0.942 mmol, 3 equiv). The mixture was stirred at room temperature overnight, followed by separation of K2CO3 through centrifugation. The K2CO3 salt was washed with DCM (5 mL) and the combined supernatants were concentrated in vacuo. The residue was purified through a neutral alumina 46  column. (CombiFlash Rf automated column system; 12 g neutral alumina; A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to yield a yellow oil (0.11 g, 63%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.88 (d, J = 7.8 Hz, 2H), 7.85 – 7.71 (m, 3H), 7.54 (d, J = 7.6 Hz, 1H), 4.05 (s, 2H), 3.96 (s, 2H), 3.35 (s, 2H), 2.90 (t, J = 5.7 Hz, 2H), 2.72 (t, J = 5.8 Hz, 2H), 1.63 (s, 18H), 1.46 (s, 9H). 13C NMR (100 MHz, 298 K, CDCl3): δ 170.8, 164.0, 160.6, 148.8, 148.5, 137.4, 137.1, 129.8, 128.5, 125.6, 125.0, 123.1, 123.0, 81.9, 81.0, 60.6, 56.5, 54.9, 54.3, 53.5, 47.1, 28.2, 28.1. LR-ESI-MS: calcd for [C30H44N4O6 +H]+ 557.3; found [M+H]+ 557.2. 5-benzyl 1-(tert-butyl) glutamate (2.8)       L-Glutamic acid γ-benzyl ester (4.66 g, 19.7 mmol, 1 equiv) was mixed with sodium bromide (6.01 g, 58.9 mmol, 3 equiv) in 1 N aqueous hydrobromic acid (59 mL, 60 mmol, 3 equiv) at 0°C in a round-bottom flask. Sodium nitrite (2.71 g, 39.3 mmol, 2 equiv) was slowly added over 30 min in three portions and the reaction was continuously stirred for 10 h at ambient temperature. The reaction mixture was treated with concentrated sulfuric acid (2.5 mL) and the acidified aqueous solution was extracted with diethyl ether (60 mL × 3). The combined organic phases were reduced in volume in vacuo before a wash with brine (20 mL × 2). The ether layer was dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was concentrated in vacuo and then purified by a silica column (CombiFlash Rf automated column system; 40 g HP silica, A: hexanes B: ethyl acetate, 0-35% B). The product fractions were combined, and rotary evaporated to yield a white solid (2.64 g, 45%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.39-7.33 (m, 5H), 5.14 (s, 2H), 4.43-4.39 (m, 1H), 2.64-2.59 (m, 2H), 2.48-2.39 (m, 1H), 2.36-2.37 (m, 1H). LR-ESI-MS: calcd for [C12H13Br81O4+Na]+ 325.0; found [M(81Br)+Na]+ 324.9.  47  5-Benzyl 1-(tert-butyl) 2-bromopentanedioate (2.9)       Compound 2.8 (2.64 g, 8.76 mmol, 1 equiv) in chloroform (CHCl3) (11 mL) was added dropwise over 20 min to tert-butyl-2,2,2-trichloroacetimidate (TBTA) (4.79 g, 21.9 mmol, 2.5 equiv) in cyclohexane (13 mL) in a round-bottom flask using a dropping funnel. Dimethylacetamide (2 mL) and boron trifluoride diethyl etherate (175.2 µL) were then added to the reaction mixture sequentially. The mixture was stirred for 3 d at room temperature, and then clarified by filtration. The filtrate was concentrated in vacuo and then re-dissolved in hexanes (15 mL), followed by washing with water (15 mL × 3) in a separating funnel. Following the evaporation of hexanes in vacuo, purification was performed with a silica column (CombiFlash Rf automated column system; 40 g HP silica, A: hexanes B: ethyl acetate, 0-30% B). The product fractions were rotary-evaporated to yield a colorless liquid (1.60 g, 51%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.36 (s, 5H), 5.13 (s, 2H), 4.25 (m, 1H), 2.59 – 2.52 (m, 2H), 2.43 – 2.18 (m, 2H), 1.47 (s, 9H). 13C NMR (100 MHz, 298 K, CDCl3) : δ 172.1, 168.4, 135.8, 128.8, 128.5, 128.4, 82.8, 66.7, 46.8, 31.8, 29.9, 27.9. LR-ESI-MS: calcd for [C16H21Br81O4+Na]+ 381.1; found [M(81Br)+Na]+ 381.0. 5-Benzyl 1-(tert-butyl) N-(2-((2-(tert-butoxy)-2-oxoethyl)((6-(tert-butoxy-carbonyl)-pyridin-2-yl)methyl)amino)ethyl)-N-((6-(tert-butoxycarbonyl)pyridin-2-yl)methyl)glutamate (2.10)            To a stirred solution of compound 2.7 (126 mg, 0.230 mmol, 1 equiv) in dry ACN(5 mL) in a round-bottom flask was added compound 2.9 (161 mg, 0.423 mmol, 1.8 equiv) and K2CO3 (95 mg, 0.690 mmol, 3 equiv). The reaction mixture was stirred at 60°C for 48 h. K2CO3 was separated through centrifugation and then washed with DCM (10 mL). The combined supernatants were concentrated in vacuo and then purified through a silica column. (CombiFlash Rf automated 48  column system; neutral alumina, A: diethyl ether B: ethyl acetate, 0-50% B). The product fractions were rotary-evaporated to a yellow oil (75.8 mg, 30%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.75 (d, J = 5.5 Hz, 2H), 7.61 (d, J = 4.4 Hz, 2H), 7.56-7.50 (m, 2H), 7.26 (s, 5H), 5.00 (s, 2H), 3.97 – 3.84 (m, 4H), 3.29 – 3.22 (m, 1H), 3.16 (s, 2H), 2.76-2.64 (m, 4H), 2.41-2.35 (m, 2H), 2.03-1.83 (m, 2H), 1.54 (s, 18H), 1.39-1.34 (2s, 18H). 13C NMR (100 MHz, 298 K, CDCl3): δ 173.0, 171.7, 170.5, 164.2, 161.4, 160.8, 148.6, 137.2, 136.0, 128.6, 128.3, 125.5, 125.1, 123.1, 123.0, 82.0, 81.4, 81.0, 66.3, 63.5, 60.6, 57.8, 56.3, 53.5, 50.2, 31.0, 28.4, 28.2, 28.2, 25.0. LR-ESI-MS: calcd for [C46H64N4O10+H]+ 833.5 found [M+H]+ 833.6. 5-(Tert-butoxy)-4-((2-((2-(tert-butoxy)-2oxoethyl)((6(tertbutoxycarbonyl)-pyridin-2-yl)-methyl)amino)ethyl)((6-(tertbutoxycarbonyl)pyridin-2-yl)-methyl)amino)-5-oxopentanoic acid (2.11)       Compound 2.10 (75.8 mg, 0.911 mol, 1 equiv) was dissolved in dry MeOH (7 mL) in a three-neck round-bottom flask under N2(g). Pd/C (10 % w/w, 10.9 mg, 0.1 equiv) was added under a stream of N2(g), and then the flask was filled with H2(g) with a balloon. The mixture was stirred vigorously for 6 h under H2(g) atmosphere, and then Pd/C was filtered off through a pre-wet (MeOH) Celite bed, washed with MeOH (10 mL). The filtrate was concentrated to a yellow oil (58.0 mg, 87%) and used without purification. LR-ESI-MS: calcd for [C39H58N4O10+H]+  743.4; found [M+H]+ 743.5. tBu4octapa-alkyl-NHS (2.12)       To a two-neck round-bottom flask with a stirred solution of compound 2.11 (58.0 mg, 78.0 mol) in dry ACN (2 mL) was added NHS (13.5 mg, 0.117 mmol, 1.5 equiv) and EDCl (22.5 mg, 49  0.118 mmol, 1.5 equiv). The mixture was stirred at room temperature under N2(g) overnight. After the reaction was complete, the solvent was removed in vacuo and the residue was redissolved in DCM (20 mL), and then washed with water (10 mL × 3) and brine (10 mL × 2) using a separatory funnel. The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to yield a yellow oil (45.3 mg, 69 %) and used directly without further purification. 1H NMR (400 MHz, 298 K, CDCl3): δ 7.70 (m, 6H), 4.03 – 3.90 (m, 4H), 3.44 – 3.40 (m, 1H), 3.20 (s, 2H), 2.80-2.69 (m, 10H), 2.15 – 1.96 (m, 2H), 1.58 (s, 18H), 1.44-1.38 (2s, 18H). 13C NMR (100 MHz, 298 K, CDCl3): δ 171.3, 170.6, 169.1, 168.6, 164.2, 161.1, 160.8, 148.7, 148.6, 137.4, 137.3, 125.6, 125.2, 123.1, 82.0, 82.0, 81.7, 81.0, 62.9, 60.6, 57.7, 56.4, 53.5, 50.3, 28.4, 28.2, 28.2, 27.8, 25.7, 24.7. HR-ESI-MS: calcd for [C43H61N5O12+H]+ 840.4395; found [M+H]+ 840.4398.  2.4.3.   DFT Calculations      All DFT simulations were performed as implemented in the Gaussian 09 revision D.01 suite of ab initio quantum chemistry programs (Gaussian Inc., Wallingford, CT). B3LYP functional105,106 and the Stuttgart/Dresden and associated effective core potentials’ basis set for Lu 107,108 were applied to optimize the structural geometry in the presence of water solvent (IEF PCM as implemented in G09) without the use of symmetry constraints. Normal self-consistent field (SCF) and geometry convergence criteria were conducted for all the calculations. The calculated structures were visualized using Mercury 4.1. 50  2.4.4.   Solid-Phase Peptide Coupling      Solid-phase syntheses of H4octapa-alkyl-PSMA-ureido and H4octapa-alkyl-PSMA617 were modified from literature procedures.86 Fmoc-Lys(ivDde)-Wang resin (0.046 mmol, 0.61 mmol/g loading) was suspended in dimethylformamide (DMF) for 30 minutes. Fmoc was then removed by treating the resin with 20% piperidine in DMF (3 × 8 min). The isocyanate derivative of di-tert-butyl ester of glutamate (0.138 mmol, 3 equiv) was prepared according to literature procedures86 and added to the lysine-immobilized resin to react for 16 h. After washing the resin with DMF, the ivDde-protecting group was removed with 2% hydrazine in DMF (5 × 5 min). To synthesize the H4octapa-alkyl-PSMA617, that step was followed by coupling of Fmoc-2-Nal-OH and Fmoc-tranexamic acid to the side chain of Lys using Fmoc-protected amino acid (0.138 mmol, 3 equiv), N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (0.138 mmol, 3 equiv), hydroxybenzotriazole (HOBt) (0.138 mmol, 3 equiv) and N,N-diisopropylethylamine (DIPEA) (0.368 mmol, 8 equiv.). Afterwards, the chelator tBu4octapa-alkyl-NHS (0.140 g, 0.167 mmol) was coupled to the peptide-bound resin by using DIPEA (0.460 mmol, 10 equiv) in DMF overnight. The peptide was then deprotected and simultaneously cleaved from the resin by treating with 95/5 TFA/triisopropylsilane (TIS) for 2 h at room temperature. After filtration, the peptide was precipitated by adding cold diethyl ether to the TFA solution. The crude conjugate was purified by semi-preparative HPLC (H4octapa-alkyl-PSMA-ureido: 5-95% ACN in water containing 0.1% TFA over 40 min at a flow rate of 10 mL/min, tR = 14.3 min; H4octapa-alkyl-PSMA617: 29% ACN in water containing 0.1% TFA at a flow rate of 4.5 mL/min, tR = 10.5 min). The eluates containing the desired peptide were collected, pooled, and lyophilized (H4octapa-alkyl-PSMA-ureido: 12.0 mg, 14.6 mol, H4octapa-alkyl-PSMA617: 10.1 mg, 8.73 51  mol) HR-ESI-MS: calcd for H4octapa-alkyl-PSMA-ureido [C35H45N7O16 + H+]+ 820.3001; found [M+H+]+ 820.2994; calcd for H4octapa-alkyl-PSMA617 [C56H69N9O18 + H+]+ 1156.4839; found [M+H+]+ 1156.4800  2.4.5.   Radiolabeling Studies      An aliquot of ligand solution (H4octapa-alkyl-PSMA-ureido or H4octapa-alkyl-PSMA617, 10-3 - 10-6 M, 25 L) was mixed with ammonium acetate (NH4OAc) solution (0.2 M, pH = 7, 223 L), followed by 177LuCl3 in HCl (aq, 0.04 M) (2 MBq, 2 L). The reaction was incubated at ambient temperature over 15 min. The mixture (3 L) was spotted on an iTLC-SA plate and then developed in EDTA buffer (50 mM, pH = 5.2). The TLC plate was read by a TLC reader, showing the free metal ion migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate RCY%.  2.4.6.   In vitro Human Serum Challenge      To the radiolabeled sample of H4octapa-alkyl-PSMA-ureido or H4octapa-alkyl-PSMA617 solution (100 L) was added an equal volume (100 L) of human serum. The mixture was incubated at 37°C, and then an aliquot (5 L) was collected at desired timepoints. The aliquot was spotted onto an iTLC-SA plate next to the control (lutetium-177 in buffer with serum) and then developed in an EDTA solution (50 mM, pH = 5.2). The TLC plate was read by a TLC reader. The free metal migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate % intact. 52  2.4.7.   In vitro Lanthanum(III) Challenge      To the radiolabeled sample of H4octapa-alkyl-PSMA-ureido or H4octapa-alkyl-PSMA617 solution (100 L, 0.25 nmol of conjugate) was added La3+ ions (standard AAS solution) in 0.04 M HCl (aq) (7.5 L of 10-4 M, 0.75 nmol of La3+). The mixture was incubated at 37°C, and then an aliquot (5 L) was collected at desired timepoints. The aliquot was spotted onto an iTLC-SA plate next to the control (without La3+ ions), and then developed in an EDTA solution (50 mM, pH = 5.2). The TLC plate was read by a TLC reader. The free metal migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate % intact.  2.4.8.   Radiolabeling of Conjugates for In vivo Study      An aliquot of H4octapa-alkyl-PSMA-ureido or H4octapa-alkyl-PSMA617 solution in MQ water (<5% DMSO) (4.83 × 10-5 M, 33.4 L, 1.62 nmol) was added to an NH4OAc solution (0.15 M, pH = 7, 170 L), followed by lutetium-177 solution in ~0.04 M HCl (200 MBq, 10 L). The mixture was incubated at room temperature for 10 min before measuring the RCY % (>98%) with iTLC-SA plate as described above.  2.4.9.   SPECT/CT Imaging Studies        Imaging and biodistribution experiments were performed using NODSCID IL2RγKO male mice. The mice were maintained, and the experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee 53  of the University of British Columbia. Mice were anesthetized by inhalation with 2% isoflurane in oxygen and implanted subcutaneously with LNCaP cells (1×107) behind left shoulder. Mice were imaged or used in biodistribution studies when the tumor grew up to reach 5-8 mm in diameter during 5-6 weeks. SPECT/CT imaging experiments were conducted using the MILabs (Utrecht, The Netherlands) U-SPECT+/CT scanner. Each tumor-bearing mouse was injected with 177Lu-labeled H4octapa-alkyl-PSMA-ureido or H4octapa-alkyl-PSMA617 (~38-48 MBq) through the tail vein under anesthesia of 2% isoflurane in oxygen. The mice were allowed to recover and roam freely in their cage and imaged at 1, 4, 24 and 72 h after injection. At each time point, the mice were sedated again with 2% isoflurane in oxygen and positioned in the micro-scanner. Body temperature was maintained via a heating pad and vital signs were monitored throughout the scan.  A baseline CT scan was obtained for localization and attenuation correction with voltage setting at 60 kV and current at 615 µA followed by a 60 min static emission scan acquired in list mode using an extra ultra-high sensitivity big mouse (2mm multi-pinhole) collimator.  Data was reconstructed using MILabs reconstruction software centered on the 208 keV (177Lu) photopeaks.  Reconstruction parameters used similarity regulated ordered subset expectation maximization (128 subsets, 3 iterations) and a post-processing filter (Gaussian blurring) of 0.6 mm.   Scatter correction was performed using the automatic triple energy window setting and a calibration factor was applied generating images in MBq/mL.  Images were decay-corrected to injection time and divided by the injected activity in PMOD (PMOD Technologies, Switzerland) to obtain quantitative images expressed as the percentage of the injected dose per gram of tissue (% ID/g).   Data were then converted to DICOM for visualization using Inveon Research Workplace (Siemens Medical Solutions USA, Inc.), a Guassian filter of 0.4 mm was applied and maximum intensity projection images were generated. 54  2.4.10.   Biodistribution Studies       The LNCaP tumor-bearing mice were injected with the radiotracer (1.5-2 MBq) through the tail vein under anesthesia (2% isoflurane in oxygen). The mice were allowed to recover and roam freely in their cages. At predetermined time points (1, 4, 24 and 72 hours p.i.), the mice were euthanized by CO2 inhalation. Blood was withdrawn immediately from the heart, and the organs/tissues of interest were collected. The collected organs/tissues were weighed and counted using a Perkin Elmer (Waltham, MA) Wizard2 2480 automatic gamma counter.       55  Chapter 3. Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-principle Prostate Specific Membrane Antigen Targeting  This chapter contains an adaptation of published work, and is reproduced in part from Li, L.; Jaraquemada-Peláez, M.G.; Kuo, H.T.; Merkens, H.; Choudhary, N.; Gitschtaler, K.; Jermilova, U.; Colpo, N.; Uribe-Munoz, C.; Radchenko, V.; Schaffer, P.; Lin, K.S.; Bénard, F.; Orvig, C. Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-Principle Prostate-Specific Membrane Antigen Targeting. Bioconjugate Chem. 2019, 30, 1539-1553. (ACS Editors’ Choice)  3.1.   Introduction      The potential of radionuclides in cancer diagnosis and therapy has been recognized for decades since the discovery of radioactivity in 1901.8 The specificity and minimal invasiveness of targeted radionuclide therapy compared to chemotherapy has poised the field for further growth, stimulated by the technological advancements in production of both radionuclides and biological targeting vectors (e.g. peptides and monoclonal antibodies). The biological safety of the radiometal is ensured by a stably bound chelator coupled to a bio-targeting vector via a covalent linkage, which also modulates the pharmacokinetics of the whole.1,16,26 56       An ideal chelator should possess rapid complexation kinetics and strong affinity for the radiometal ion at mild conditions (RT, <15 minutes complexation), as well as high versatility of linker incorporation (i.e. bifunctionalization) without sacrificing the coordination integrity. Although a small peptidomimetic conjugate usually has higher tolerance to the harsh radiolabeling conditions (e.g. 60-90°C for 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, Figure 3.1), fast and quantitative radiolabeling at room temperature facilitate handling and avoid potential degradation of the product. One of the benefits is that instead of relying on central manufacturing, clinicians can conveniently prepare the radioactive tracer in a local hospital, which mimics the existing practice for 99mTc kits.109 In terms of bifunctionalization, most of the commercial chelators including DOTA and diethylenetriamine-pentaacetic acid (DTPA), lack a convenient spot for adding a linker. The cumbersome functionalization on the polyamine backbone or the sacrifice of a pendant arm can complicate the synthesis while restricting the linker variety, or even reduce the complex stability.110,111,112 Additionally, a practically and clinically useful chelator should accommodate (a pair of) theranostic isotopes (e.g. 177Lu, 111In, 86/90Y, 44/47Sc) for an accurate study of dosimetry to achieve optimal therapeutic effects. In this regard, 177Lu (t1/2 ~ 6.64 days) is well-recognized for treating small and metastatic tumors with its low-energy - particles (maximum 498 keV),78 along with two useful  emissions (208 keV and 113 keV) for SPECT imaging, rendering it an excellent theranostic isotope with a favorable half-life.79 The high thermal neutron capture cross section of 176Lu also allows direct production of 177Lu [176Lu(n,γ)177Lu] with high specific activity at multi-Curie activity levels to meet the impending demand for targeted cancer therapy.5,80 Of course, no-carrier-added (n.c.a.) 177Lu can also be produced through an indirect route (176Yb(n,)177Yb→177Lu).5,81 Another potential theranostic radioisotope is the -ray and Auger-electron-emitting 111In  (t1/2 ~ 2.8 days), which has proved 57  induction of cytocidal double-strand break (DSBs) upon internalization and translocation to the nucleus of the tumor cells, besides its widespread applications in SPECT imaging.26,113,114,115       To overcome the practical concerns on the radiolabeling conditions and the complex stability encountered with the current commercial chelators, we report herein the synthesis and characterization of a new potentially nonadentate pyridinecarboxylate-based ligand, H4pypa and a bifunctional H4pypa conjugated to glutamate-urea-lysine-based PSMA (prostate-specific membrane antigen)-targeting pharmacophore for prostate cancer (PCa) targeting. PCa is the most common cancer in men in the United States accounting for around 26,730 deaths in 2017.116 PSMA is primarily expressed in normal human prostate epithelium, but is up-regulated in prostate cancer cells, even more in de-differentiated, metastatic and hormone-refractory carcinomas, rendering it an attractive target in PCa.96 The current FDA-approved 111In-capromab pendetide scan (ProstaScint® scan) has several drawbacks such as inducing the formation of human anti-mouse antibody (HAMA) and targeting the intracellular epitope of PSMA, which somewhat limit its applications.117,118 The Glu-urea-Lys PSMA inhibitor adopted in this study was initially developed by Kozikowski et al., as an inhibitor for Glutamate Carboxypeptidase II,119 while the use of the same targeting moiety in diagnostic nuclear medicine was done by Maresca et al.,94 and the lipophilic spacer was  later suggested to improve the pharmacokinetics by Benešová et al. who conjugated the whole moiety to DOTA (a.k.a. PSMA617) for 68Ga and 177Lu radiolabels.95 In this paper, we show that both H4pypa and its PSMA-targeting counterpart radiolabel 111In and 177Lu in excellent yield and stability with the LNCaP-tumor targeting ability preserved. Moreover, the thermodynamic stabilities of M-pypa systems (M=In3+, Lu3+, La3+) measured as pM are considerably higher than those with DOTA, DTPA, H4octapa, H4octox and H4neunpa. In addition 58  to chelation, a significant advantage of the pyridyl bridge is the facile and versatile linker attachment via the central hydroxyl group. Undeniably, linkages play a pivotal role in the biological behavior of the radiochelate, particularly the small peptide conjugates.86,87,95,120,121 Therefore, this versatility provides flexibility in not only the chemical properties of the whole (e.g. hydrophilic, hydrophobic), but also the targeting vectors associated (e.g. peptide, antibody, antibody fragment, nanoparticles).16 The combination renders H4pypa a promising theranostic chelating agent for many different cancer treatments, despite the focus here on prostate cancer.  59   Figure 3.1 Chemical structures of selected chelators. 3.2.   Results and Discussion 3.2.1.   Synthesis and Characterization      Our group has established a significant library of non-macrocyclic chelators, including picolinate and hydroxyquinoline derivatives, to target different radionuclides.38,39,122 H4pypa is a new member of the picolinate-arm-based ligand family, designed to accommodate medium-to-60  large radioisotopes (e.g. 177Lu, 111In, 44Sc, 86/89Y) in its potentially nine-coordinating cavity, securing the metal ion with the rigid pyridyl cap on which a variety of linkers can be attached efficiently for bioconjugation. As depicted in Scheme 3.1, the arm and pyridyl backbone of the non-bifunctional H4pypa were strategically synthesized individually and then assembled in one   Scheme 3.1 Reagents and conditions: i) DCC, tert-butyl alcohol, DCM, RT, overnight, 50%; ii) NaBH4, dry MeOH, RT, 3-4 h, 72%; iii) SeO2, 1,4-dioxane, 100°C, overnight, 56%; iv) 1. Dry MeOH, RT, 1 h; 2. NaBH3CN, dry MeOH, 3 h, 70%; v) NaBH4, dry MeOH, RT, 12 h, 92%; vi) PBr3, dry CHCl3/ACN, 60 oC, 18 h, 70%; vii) K2CO3, dry ACN, 60 oC, 24 h, 70%; viii) TFA/DCM, RT, overnight, 70% convergence step for higher synthetic efficiency. The backbone was synthesized by first reducing the starting material dimethyl 2,6-pyridinedicarboxylate to pyridine-2,6-diyl-dimethanol (3.5, 92%), followed by bromination with phosphorus tribromide, to give the dibromo-derivative (3.6, 70%) which was coupled with 2 equivalents of the picolinate-acetate arm moiety (3.4). The tert-butyl esters on arm 3.4 were selected for the compatibility with solid-phase peptide coupling and it was synthesized through reductive amination using the aldehyde (3.3) and tert-butyl glycinate 61  (70%). Compound 3.3 was prepared by converting 2,6-pyridinedicarboxylic acid to the corresponding tert-butyl-ester analogue (3.1), followed by monoreduction with sodium borohydride (NaBH4) to give compound 3.2 and subsequent oxidation to aldehyde (3.3) by selenium(IV) oxide (SeO2) (cumulative yield ~20%). Finally, both pendant arm (3.4) and pyridyl bridge (3.6) were connected through SN2 nucleophilic substitution (3.7, 70%) and then deprotection was followed to give H4pypa (3.8, 70%). Purification with high-performance liquid chromatography (HPLC) yielded the final product as a trifluoroacetic acid (TFA) salt (H2pypa·2TFA·1.7H2O as determined by elemental analysis). Regarding the bifunctional H4pypa (Scheme 3.2), commercially available chelidamic acid monohydrate was selected as the backbone starting material. After converting the carboxylic acids to methyl esters (3.9, >99%), the p-OH group on the pyridyl moiety was protected with benzyl bromide (3.10, 64%). Similar to the H4pypa preparation, the methyl esters in compound 3.10 were reduced to alcohols (3.11, 82%), and then brominated to give compound 3.12 (70%), which was coupled to arm 3.4 through the aforementioned protocol to yield the protected pypa (3.13, 73%). Following debenzylation with Pd/C-catalyzed hydrogenation, the alkyl linker was added through SN2 nucleophilic substitution to give compound 3.16 (90%). Deprotection and activation with 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride/ N-hydroxysuccinimide (EDCI/NHS) generated the activated ester (3.18, 76% over 2 steps), which was coupled to the PSMA-targeting-peptide-bound resin by using N,N-diisopropylethylamine (DIPEA) in dry DMF overnight. After the coupling finished, the peptide-bioconjugate was deprotected and simultaneously cleaved from the resin with 95/5 trifluoroacetic acid (TFA)/triisopropylsilane (TIS) for 2 h at room temperature to give H4pypa-C7-PSMA617 (Figure 3.1). 62                Scheme 3.2 Reagents and conditions. i) SOCl2, MeOH, RT-60 oC, 26 h, >99%; ii) BnBr, ACN, K2CO3, 60 oC, overnight, 64%; iii) NaBH4, dry MeOH, RT, overnight, 82%; iv) PBr3, dry CHCl3/dry ACN, 60 oC, overnight, 70%; v) K2CO3, dry ACN, 30 oC, 24 h, 73%; vi) Pd/C (10% w/w), H2 (g), MeOH, RT, overnight; vii) K2CO3, dry THF, RT-35 oC, 24 h, 90%; viii) Pd/C, MeOH, RT, overnight, 88%; ix) NHS, EDCI, dry ACN, RT, overnight, 86%.   63  3.2.2.   Metal Complexation and Characterization      The complexation with non-radioactive In3+, Lu3+ and La3+ ions was studied, and the complexes were characterized with NMR spectroscopy (1H, 13C and HSQC) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). Being the largest lanthanide, La3+ is also of interest in its complexation with H4pypa in comparison with that of Lu3+ which is the smallest in the series.123 All three complexes appeared to be rigid coordination complexes with no observable fluxionality within NMR timescale, as confirmed by the sharply resolved 1H NMR peaks and the absence of extra hydrogen and carbon signals in 1H and 13C NMR spectra (Figures 3.2, A1, A3, A4). In the case of [In(pypa)]-, all the carbons in the complex were chemically distinct from one another, while there were three pairs of visible diastereotopic methylene protons (2.94 and 3.22 ppm, 2J =17.6 Hz; 3.78 and 3.91 ppm, 2J =17.1 Hz; 4.10 and 4.44, 2J=17.4) (Figures 3.2), and each doublet accounted for only one hydrogen atom; the rest were overlapping with the water signal (revealed by the interaction with the adjacent carbon atom shown in 1H-13C HSQC, Figure A6), indicating the asymmetry in the complex in solution state. The asymmetry was also observed in [La(pypa)]- which appeared in solution as a single isomer (Figure 3.2). On the other hand, [Lu(pypa)]- presented in solution as a symmetric complex, marked by the unchanged number of carbon signals (Figure A1), as well as the consistent 1H aromatic pattern (two triplets - 8.21 and 7.85 ppm, and three doublets - 8.05, 7.80 and 7.44 ppm) compared with the uncomplexed chelator (Figure 3.2). Furthermore, the complex only exhibited two pairs of diastereotopic protons (3.49 and 3.98 ppm, 2J=17.0 Hz; 4.07 and 4.39 ppm, 2J=14.7 Hz), while the last pair of methylene-H appeared as a sharp singlet at pH = 1.5 but a clear doublet at pH = 11.5 (Figure 3.3A). However, the much smaller splitting of the peak compared to the other diastereotopic pairs implied a much 64  weaker, or perhaps farther interaction with the Lu3+ ion. Additionally, this pair of methylene protons belonged to a carbon that has a very similar chemical shift with another methylene carbon which suggested that both of them might be adjacent to the pyridyl group instead of the carboxylic acid on the acetate arms (Figure A2).  Interestingly, as determined by potentiometric titration, the [Lu(Hpypa)] species deprotonates at pH = 3.35-3.60 which can be reasonably assigned to the protonated central pyridine (Figure 3.3B). In this case, it could be inferred that the interaction between the pyridyl bridge and the Lu3+ ion is weaker when the central pyridine-N atom is protonated at low pH, and is increased when is deprotonated at higher pH, which however, was not as strong as that with the acetate and picolinate arms. This phenomenon could also be explained by the relatively small atomic size and the hard nature of Lu3+.123 Figure 3.2 (A) Partial 1H NMR spectra of [La(pypa)]-, [In(pypa)] -, [Lu(pypa)] -, H4pypa (top-bottom) (D2O, 400 MHz, 298 K).  65                          Figures 3.3 A) Partial 1H NMR spectra of [Lu(pypa)]- at pH=11.5, 2.1 and 1.5 (top-bottom)  (D2O, 400 MHz, 298 K). B) Potential structure of [Lu(Hpypa)] in aqueous solution at pH < 2.  3.2.3.   X-ray Crystallography of H[Lu(pypa)]      X-ray quality single crystals of H[Lu(pypa)] were obtained by the slow evaporation of 1:1 LuCl3 and H4pypa solutions in water after adjustment of pH to 2. The ORTEP diagram of [Lu(pypa)]- is shown in Figures 3.4 and 3.5 and its crystallographic data can be found in Appendix A. From Figure 3.4 and 3.5, it can be seen that at pH = 2, two of the carboxylic groups of the ligand are protonated. The H atom attached to O2 (picolinic -COOH) is half occupied in one of the asymmetric units, as is the case with Cl atom which is equally shared by two asymmetric units,  while the H atom attached to O6 (backbone -COOH) is fully occupied; hence the overall charge is neutral. It is noteworthy that the protonation site of the [Lu(Hpypa)] complex in the solid state is different from that predicted based on the pKa and 1H NMR spectrum (Figure 3.3B). Nonetheless, the crystal structure of the [Lu(pypa)]- anion clearly showed that the Lu(III) ion is nine-coordinated A B 66  by the N5O4 donor atoms of the ligand.  Selected bond distances and bond angles are provided in Table 3.1. The geometry is distorted but nevertheless, the structure provides the vital visual insight to the coordination environment of the Lu(III) ion. It can be clearly seen from the structure that Lu(III) sits in the cavity of the H4pypa ligand, capped by the pyridyl group. Also, the preference of Lu(III) for O-donor atoms can be seen from the shorter Lu-O bond distances compared to longer Lu-N bonds.    Figure 3.4 ORTEP diagrams of the anion in C50H79ClLu2N10O33.   67   Figure 3.5 ORTEP diagrams of the anion in H[Lu(pypa)].  Table 3.1 Selected bond lengths and bond angles in [Lu(pypa)]-.            Bond length/Å Bond Angle/°    Atom     Atom    Length/Å      Atom      Atom     Atom Angle/° Lu O1    2.3266(17) O3 Lu N2 70.14(6) Lu O3    2.3333(17) O5 Lu N5 129.28(6) Lu O5    2.3559(17) O8 Lu N5 68.64(7) Lu O8    2.2954(17) N1 Lu N2 67.31(6) Lu N1    2.4181(19) N3 Lu N2 67.44(7) Lu N2    2.5370(2) N3 Lu N4 66.17(6) Lu N3    2.4510(2)     Lu N4    2.5530(2)     Lu N5    2.3845(19)     68  3.2.4.   Solution Thermodynamics      When evaluating ligands for metal complexation, knowledge of the basicity of the different ionizable and non-ionizable protons is essential because the metal ion will compete for replacing them at the basic sites. H4pypa possesses nine protonation sites, and in this work, we determined the acidity constants for all of them. Combined potentiometric-spectrophotometric titrations were carried out by following spectral changes in the absorption band of the picolinate chromophore for the first seven protonation equilibria and UV in batch spectrophotometric titrations for the last two equilibria, as they deprotonate at a pH below the electrode threshold. The different absorption features related to the above-mentioned equilibria as the pH is raised are presented in Figure 3.6. Table 3.2 presents the protonation constants calculated from the experimental data using the HypSpec2014124 and Hyperquad2013125  programs. Figure 3.7A presents one of the titration curves of an acidified solution of H4pypa·2TFA·1.7H2O and it shows that nine equivalents of base (NaOH) were consumed in the titration. The speciation plots of different species of H4pypa in Figure 3.7B were calculated from the protonation constants in Table 3.2 with the Hyss software.126        The structure of H4pypa resembles that of H4octapa,39,127 with the difference in the backbone: in H4pypa, a pyridyl ring bridges the two tertiary nitrogen atoms. Not surprisingly, also in this case, the two most acidic protons (species H9L5+ and H8L4+, pK1 = -0.58 (2) and pK2 = -0.37 (1)) can be attributed to the deprotonation of the pyridine nitrogen atoms in the picolinate moieties based on the bigger spectral changes in the band of this chromophore. Following that, the species H7L3+ and H6L2+ deprotonate with pK3 = 1.70 (2) and pK4 = 2.06 (6), respectively, and they can be attributed to the deprotonation of the two acetate carboxylic acids. Species H5L+ and H4L deprotonate with pK5 = 2.23 (2) and pK6 = 3.02 (1), respectively, and are attributed to the picolinate-COOH. The 69  last three dissociation steps are assigned to the tertiary nitrogen atoms and the central pyridine nitrogen (species H3L-, H2L2- and HL3-). Comparing the pKa values for the tertiary nitrogen atoms of H4pypa (pK8 = 6.78 (1) and pK9 = 7.78 (1)) to those of H4octapa (pK7 = 5.43 (2) and pK8 = 8.58 (1)),127 the ΔpKa for those equilibria is of 1 unit in H4pypa while it is 3.1 units in H4octapa. This can be explained for the larger charge repulsion in H4octapa compared to H4pypa when the two nitrogen atoms are protonated. The pK7 = 3.69 (1) can be allocated therefore to the central pyridine nitrogen atom.  Table 3.2 Protonation constants of H4pypa at 25.0°C, I = 0.16 M NaCl. Equilibrium Reaction log β log K L4- + H+⇆ HL3- 7.78 (1) 7.78 (1) HL3- + H+ ⇆ H2L2- 14.56 (1) 6.78 (1) H2L2- + H+ ⇆ H3L- 18.25 (1) 3.69 (1) H3L- + H+ ⇆ H4L 21.27 (1) 3.02 (1) H4L + H+ ⇆ H5L+ 23.50 (2) 2.23 (2) H5L+ + H+ ⇆ H6L2+ 25.56 (6) 2.06 (6) H6L2+ + H+ ⇆ H7L3+ 27.26 (2) 1.70 (2) H7L3+ + H+ ⇆ H8L4+ 26.89 (1) (a) -0.37 (1) (a) H8L4+ + H+ ⇆ H9L5+ 26.31 (2) (a) -0.58 (2) (a) (a) from in batch UV spectrophotometric titrations, not evaluated at constant ionic strength (0.16 M NaCl).  70   Figure 3.6 A) and B) Representative spectra of the in batch acidic titration of H4pypa at [L] = 1.07  10-4 M as the pH is raised at 25 °C and l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. C) and D) Representative spectra of the combined UV-potentiometric titration of H4pypa at [L] = 6.34  10-4 M at 25 °C, l = 0.2 cm and I = 0.16 M NaCl.  Figure 3.7 A) Titration curve of an acidic solution of H4pypa, [H4pypa] = 6.34  10-4 M, at 25 °C and I = 0.16 M NaCl. B) Speciation plots of H4pypa calculated with protonation constants on Table 3.2. [H4pypa] = 1  10-3 M. Dashed line indicates pH 7.4. 225 250 275 300 3250.000.250.500.751.001.251.501.75pH 0.88 -0.99 -0.66 -0.41 -0.26 -0.07 0.20 0.41 0.58 0.88AbsorbanceWavelength (nm)H -0.99A225 250 275 300 3250.000.250.500.751.001.251.501.75pH 2.14 1.18 1.77 1.86 2.14AbsorbanceWavelength (nm)pH 1.18B225 250 275 300 3250.000.250.500.751.001.251.501.75pH 5.51 2.33 2.45 2.56 2.61 2.83 3.05 3.29 3.8 5.51AbsorbanceWavelength (nm)pH 2.33C225 250 275 300 3250.000.250.500.751.001.251.501.75pH 5.51 10.37 8.30 7.98 7.76 7.57 7.39 7.23 6.97 6.81 6.64 6.42 6.11 5.51AbsorbanceWavelength (nm)pH 10.37D0 1 2 3 4 5 6 7 8 9 10 1123456789101112pHmol NaOH/mol LigandA0 2 4 6 8 10020406080100L4-HL3-H2L2-H3L-H4LH5L+H6L2+H7L3+H8L4+H9L5+% Formation pHB71       Complex formation equilibria studies of H4pypa with metal ions of relevant interest in radiopharmaceutical chemistry (i.e. In3+, Lu3+ and La3+ ions) were carried out by different methods. The extent to which the metal complexation occurred even at pH ~2 was too high to allow a direct determination of the stability constants for the [ML]- species by simple potentiometric titrations of H4pypa with the respective metal ions. Protonated species of the metal complexes, MHL, were found with H4pypa and In3+, Lu3+ and La3+ ions by competition methods; ligand-ligand competition methods with the competing ligand TTHA were used for In3+ and Lu3+ ions, while for La3+, EDTA was used as a competitor. Additionally, acidic in batch UV spectrophotometric titrations were carried out for all M3+-H4pypa systems (M3+ = In3+, Lu3+ and La3+) (Figures 3.8-3.9). Once the stability constants for the MHL species (log KMHL) were known, the direct potentiometric method was used to determine the stability constants of the [ML]- and [M(OH)L]2- species for In3+ and Lu3+, and [ML]- and [M2L2(OH)]3- species for La3+. Potentiometric and spectrophotometric experimental data were refined using the HypSpec2014124 and Hyperquad2013125 programs and the stability constants are presented in Table 3.3. The metal complex stability of the [ML]- species formed (log KML) with H4pypa follows the order In3+ > Lu3+ > La3+. The size and the acid character of the metal ion definitely play a role on its metal complex stability, and from these results, the cavity of H4pypa seems best fitted for the smaller In3+.  In order to compare the excellent stability of M3+-H4pypa complexes with the stability of other metal complexes involving different chelators, it is necessary to use a parameter that takes into account not just the stability of the metal complexes, but also the basicity and the denticity of the different chelators to be compared. The parameter pM is widely used in medicinal inorganic chemistry for this purpose, and is a measurement of the metal sequestering ability of a determined chelator towards an specific metal ion; it is defined as –log [Mn+]free at [ligand] = 10 M and [Mn+]= 1 M 72  at pH = 7.4.128 In Figure 3.10A, the pM values for the most relevant chelators currently used in the radiopharmaceutical field have been plotted for the metals of our interest and the H4pypa performance exceeds that of all the other chelators. Additionally, Figure 3.10B shows the importance of the basicity of different chelators on the metal sequestration ability or pM values; in particular, it shows the metal sequestering ability in terms of pLu3+ as the pH is raised.  For ligands with lower overall basicities, the protons will compete less with the metal ion and will show complexation from lower pHs and in a broader pH range. This effect is more dramatic when comparing the metal affinity of H4pypa and H4octox towards Lu3+. Although the overall stability constant of [Lu(octox)]- (log K = 24.66(1))85 is higher than that of [Lu(pypa)]- (log K = 22.02(6)), the higher basicity of H4octox prevents Lu3+ scavenging at lower pHs and more importantly, at physiological pH = 7.4, the effectiveness of H4octox is 4.4 units lower than that of H4pypa. These promising solution thermodynamics findings reveal H4pypa as an excellent chelator for further studies in vitro and in vivo, particularly for the theranostic 177Lu isotope.    73     Figure 3.8 Representative spectra of the in-batch UV-titration of A) the In3+-pypa system as the pH is raised. [L] = [In3+] = 1.33  10-4 M; B) and C) the Lu3+-pypa system as the pH is raised. [L] = [Lu3+] = 1.33  10-4 M; D) and E) the La3+-pypa system as the pH is raised. [L] = [La3+] = 1.27  10-4 M, at 25 °C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. Distribution diagram of the In3+-pypa system. Dashed line indicates physiological pH (7.4).  250 275 300 3250.000.250.500.751.001.251.501.75pH 0.48  0.18 0.33 0.40 0.48AbsorbanceWavelength (nm)pH 0.18A250 275 300 3250.000.250.500.751.001.251.501.75pH 2.08 0.69 0.86 0.97 1.13 1.39 2.09AbsorbanceWavelength (nm)pH 0.69B250 275 300 3250.000.250.500.751.001.251.501.75pH 1.18 -0.24 -0.09 0 0.22 0.36 0.44 0.59 0.81 1.18AbsorbanceWavelength (nm)Ho -0.24A250 275 300 3250.000.250.500.751.001.251.501.75pH 1.18pH 2.23 1.18 1.34 1.44 1.59 1.72 1.84 1.9 2.1 2.23AbsorbanceWavelength (nm)BE D C B 250 275 300 3250.000.250.500.751.001.251.501.75H -0.27  -0.27 -0.23 -0.18 -0.07 0.05 0.28 2.22AbsorbanceWavelength (nm)pH 2.22AA 74      Figure 3. 9 Distribution diagram of A) the In3+-pypa system; B) the Lu3+-pypa system; C) the La3+-pypa system. Dashed line indicates physiological pH (7.4).           0 2 4 6 8 10 12020406080100InCl3InCl2+[In(OH)(L)]2-[In(L)]-In(HL)% Formation relative to In3+pHInCl2+B0 2 4 6 8 10 120255075100[Lu(OH)(L)]2-[Lu(L)]-Lu(HL)% Formation relative to Lu3+pHLu3+C0 2 4 6 8 10 120255075100[La2(OH)(L)2]3-[La(L)]-La(HL)% Formation relative to La3+pHLa3+CAc B  75  Table 3.3 Stepwise stability constants (log K) of H4pypa complexes with In3+, Lu3+ and La3+. Equilibrium reaction In3+ Lu3+  La3+  M3+ + L ⇆ ML 29.99(4)a; 30.13(3)b 22.02(6)a; 22.20(2)b 19.74(3)d; 19.54(2)b ML + H+ ⇆ MHL 4.06(5)a; 3.80(1)c 3.35(8)a; 3.60(6)c 2.99(4)d; 3.24(5)c M(OH)L + H+ ⇆ ML 10.59(7)a; 10.44(4)b 10.77(8)a; 10.86(3)b - M2L2(OH) + H+ ⇆ M2L2 - - 33.88(7)d; 34.40(6)b pMe 30.5 22.6 19.9 a) ligand-ligand potentiometric competition with H6ttha at I = 0.16 M (NaCl) and 25 °C; b) potentiometric titrations at I = 0.16 M (NaCl) and 25 °C; c) in-batch acidic spectrophotometric competition at 25 °C, not evaluated at constant I = 0.16 M (NaCl); d) ligand-ligand potentiometric competition with H4edta at I = 0.16 M (NaCl) and 25 °C; e) pM is defined as -log [M]free at [L] = 10 M, [M] = 1 M and pH = 7.4. Charges are omitted for clarity.    Figure 3.10 (A) pM values vs ionic radius123 for M3+- ligand complexes (CN = 8); (B) Lu3+ scavenging ability of different ligands as the pH is raised from 0-12, [Lu3+] = 1  10-6 M and [ligand] = 1  10-5 M. Solid line in B at indicates physiological pH (7.4).   0.90 0.95 1.00 1.05 1.10 1.15 1.201416182022242628303234 H4pypa H4octapa DTPA H4octox H4neunpa DOTALa3+Lu3+pM values Ionic radius (CN = 8) In3+A0 2 4 6 8 10 126810121416182022242628 H4pypa H4octapa DTPA H4octox DOTApLu3+ pHBpH = 7.476  3.2.5.   Radiolabeling and Human Serum Challenge Experiments       In most receptor targeting radiotracer formulations, the unlabeled receptor targeting vector is preferably kept minimum to ensure that the receptor is not saturated by unlabeled targeting motif, which in other words is to maximize the apparent molar activity.129 Quantitative radiolabeling at mild conditions (RT, 10 min) with low ligand concentration and low ligand/radiometal (mol/mol) (L/M)  ratio obviates the need of post-labeling HPLC purification to attain high apparent molar activity, which has significant impact on the biological profile. Fendler et al. reported that high apparent molar activity of 177Lu-PSMA-617 was associated with higher tumor uptake, more prominent DNA damage and more effective tumor growth inhibition.99 In our study, concentration-dependent radiolabeling was applied to determine the lowest ligand (H4pypa and H4pypa-C7-PSMA617) concentration required for quantitative radiometalation with both 177Lu and 111In, while the corresponding L/M ratio was adjusted afterwards using a mixture of no-carrier-added (n.c.a.) radioactive and non-radioactive isotopes from diluted AAS standards (i.e. 177/natLu or 111/natIn). All radiolabeling studies were performed in triplicate. Firstly, both H4pypa and H4pypa-C7-PSMA617 radiolabeled 177Lu and 111In quantitatively (>98% radiochemical yield, RCY) in 10 min with 10-6 M ligand concentration at RT and pH=7 (Figure 3.11A), indicated by a single sharp signal at the baseline of the iTLC-SA plate developed with EDTA solution (50 mM, pH = 5.5), which is consistent with the well-separated radiopeaks of the free metal and the complex on the HPLC radiotraces (tR = 12.9 and 10.4 minutes for 177Lu- and 111In-pypa complexes; 15.0 and 14.1 min for 177Lu- and 111In-pypa-C7-PSMA617 complexes) (Figure 3.12). The corresponding complexes were highly kinetically inert with <1% transmetalation to the serum proteins over at least 7 days for both 177Lu complexes and 5 days for [111In][In(pypa)]- (Figure 77  3.11B), while that of [111In][In(pypa-C7-PSMA617)] was not determined due to unsuccessful separation of the transchelated 111In and the radioactive complex with either PD10 column or iTLC-SA plate (EDTA and DTPA solution as mobile phase). The results are in significant contrast with the industrial “gold standard” macrocyclic chelator –DOTA – which required microwave heating at 80-100°C over 20-30 min for quantitative radiolabeling with both 111In and 177Lu.30,39 DTPA and the cyclohexyl analogue (CHX-A”-DTPA) are two widely adopted non-macrocyclic ligands in 111In radiopharmaceutical development in an effort to achieve high RCY at RT, but their inferior in vitro stabilities unfortunately limit their clinical potential (88.3% and 89.9% for [111In][In(DTPA)]2- and [111In][In(p-NH2−Bn−CHX-A″−DTPA)]2−, respectively, after 24 h).39,122 Encouraging radiolabeling results with 111In and 177Lu were reported with H4octapa, an octadentate ligand previously reported by our group with reported in vitro stability 92.3% and 87.7%, respectively, after one day.26,39 The nonadentate p-NO2−Bn−neunpa was developed for 111In with more favorable stability in human serum (97.8% over 5 d), but exhibited very low affinity to 177Lu and therefore, further study was precluded.122 As mentioned above, the apparent molar activity is a crucial parameter in preparing radiotracers. For [AE][E(pypa-C7-PSMA617)] (AE=177Lu, 111In), at the optimal radiolabeling concentration (i.e. 10-6 M), radiolabeling yields with different L/M ratios were tested using a mixture of no-carrier-added 177Lu or 111In, as well as non-radioactive natLu or natIn in an attempt to achieve the lowest L/M ratio. For both radiotracers, L/M ratio 2 has proved sufficient for ~98% RCY (RT), while equimolar gave only 76% (111/natIn) and 72% RCY (177/natLu). The results proved a cost-effective and comparable estimation for optimal radiometal-to-chelator ratio without consuming a large amount of radioactivity which is very expensive, while the resulting high apparent molar activity also conveniently obviates post-labeling purification, and thus further enhances their potential in the practical applications. 78   Figure 3.11 (A) Concentration dependent radiolabeling of H4pypa and H4pypa-C7-PSMA617 (10 min, RT) in NH4OAc solution (0.15 M, pH = 7) with 177Lu/111In. (B) Human serum challenge of the radiolabeled complexes over 5-7 d (37 oC).    4 5 6 7 8020406080100RCY %[-log(M)] [177Lu][Lu(pypa)]- [177Lu][Lu(pypa-C7-PSMA617)] [111In][In(pypa)]- [111In][In(pypa-C7-PSMA617)]A0 50 100 150 200020406080100Radiochemical purity %Time (h) [177Lu][Lu(pypa)]- [177Lu][Lu(pypa-C7-PSMA617)] [111In][In(pypa)]-B79   Figure 3.12 RP-rHPLC (A: ACN/0.1% TFA B: H2O/0.1% TFA) of (A) free 177Lu (0-100% A over 30 min, 1 mL/min, tR =  6.44 min); (B) [177Lu][Lu(pypa)] (10-6 M, 0-100% A over 30 min, 1 mL/min, tR =  12.9 min, RCY = 98%); (C) [177Lu][Lu(pypa-C7-PSMA617)] (10-6 M, 0-80% A over 30 min, 1 mL/min, tR =  15.0 min, RCY = >99%); (D) free 111In (0-100% A over 20 min, 1 mL/min, tR =  7.67 min); (E) [111In][In(pypa)] (10-6 M, 0-100% A over 20 min, 1 mL/min, tR =  10.4 min, RCY = 98%); (F) [111In][In(pypa-C7-PSMA617)] (10-6 M, 0-100% A over 20 min, 1 mL/min, tR =  14.1 min, RCY = >99%). A B C D E F 80  3.2.6.   SPECT/CT Imaging, Biodistribution Studies and Binding Affinity       [AE(pypa-C7-PSMA617)] (AE = natIn and natLu) inhibited the binding of 18F-DCFPyL (a urea-based radiotracer targeting PSMA) to PSMA on LNCaP cells in a dose-dependent manner, and their calculated Ki values were 6.41 (1.80) nM and 7.88 (4.34) nM, respectively. For animal studies, 177Lu- and 111In-labeled radiotracers with apparent molar activities of 207 GBq/mol and 459 GBq/mol, respectively, were prepared and then injected into LNCaP-tumor-bearing mice (n=5 per time point). SPECT/CT imaging (Figure 3.13) and ex vivo biodistribution studies (Figure 3.14) showed that both radioactive analogues excreted mainly via the renal pathway. The blood clearances for both radiotracers were fast, measured at 0.89±0.42% injected dose per gram (ID/g) at 4-h post-injection (p.i.) and 1.04±0.34% ID/g at 1-h p.i. for the 177Lu- and 111In-based tracers, respectively. In both cases, uptake in non-specific organs and tissues were low (e.g. fat, intestine, stomach, liver, pancreas, heart, muscle, bone), while high accumulations were observed in PSMA-expressing tissues (e.g. kidney, spleen, adrenal glands, LNCaP tumor).103 For [177Lu][Lu(pypa-C7-PSMA617)], the uptake in kidney, spleen, adrenal glands and tumors were 120±19% ID/g, 4.71±1.68% ID/g, 3.64±1.49% ID/g, 20.6±5.9% ID/g at 1-h p.i., respectively. Except for tumor, in which the accumulation grew to 24.0±7.6% ID/g at 4-h p.i. and then gradually reduced to 12.7±4.2% ID/g after 3 d, the uptake in other organs was rapidly cleared within the first 4 h, resulting in a substantial increase in the tumor-to-background contrast ratio. Moreover, the tumor uptake was more than 60% higher than that of 177Lu-PSMA-617 at both 4-h and 72-h p.i., which was reported with 14.5±1.8% ID/g and 7.80 ±3.69% ID/g, respectively.104 Regarding [111In][In(pypa-C7-PSMA617)], similar to its 177Lu-counterpart, the background organs and tissues uptakes were low and cleared rapidly except in the kidney and tumor. After 24 h, the kidney 81  accumulation was 36.4±18.8% ID/g and the tumor uptake was 8.88±1.92% ID/g, leading to a tumor-to-kidney ratio of 0.24±0.10 compared to 6.70±1.75 for the 177Lu-analogue (24-h post-injection). A DOTA-based construct containing the same PSMA-targeting motif (Glu-urea-Lys) and IRDye800CW was synthesized and radiolabeled with 111In as a dual-modality imaging agent.130 The PC3-PIP tumor uptake was 14.6±1.3% ID/g at 24-h p.i. and the calculated tumor-to-kidney ratio was around 0.3.130  Schottelius et al. published a DOTA-based PSMA I&T which was metalated with 111In, 177Lu and 68Ga.131 In their case, [111In]PSMA-I&T had the highest tumor uptake (~8% ID/g) at 1-h p.i., along with the most kidney (~190% ID/g) and spleen (~46% ID/g) accumulation which was claimed to be CB17 SCID-mice related.131  Figure 3.13 Representative SPECT/CT images (MIP, coronal) of [AE][E(pypa-C7-PSMA617)] [AE=111In (left, 24.9 MBq), 177Lu (right, 44.1 MBq)] in LNCaP-tumor-bearing mice at different p.i. time points.    %ID/g 20 0   1h               4h               24h 50 0 %ID/g   4h               24h            72h 82     Figure 3.14 Ex vivo biodistribution data [AE][E(pypa-C7-PSMA617)] [AE=177Lu (A), 111In (B)] in LNCaP-tumor-bearing mice at selected p.i. time points (n=5 per time point).   bloodurinefatseminaltestesintestinestomachspleenliverpancreasadrenalkidneylunghearttumourmusclebonebrainthyroidsalivarylacrimal02080100120140160180240260% ID/g 1 h p.i. 4 h p.i. 24 h p.i. 72 h p.i.Abloodurinefatseminaltestesintestinestomachspleenliverpancreasadrenalkidneylungshearttumormusclebonebrainthyroidsalivarylacrimal01020304050607080120130% ID/g 1 h p.i. 24 h p.i.B83   3.3.   Conclusions      H4pypa is a potentially nonadentate non-macrocyclic chelator with great affinity for 111In and 177Lu which are excellent radionuclides in cancer diagnosis and therapy. In addition to the quantitative radiolabeling yields at fast complexation kinetics (10 min) under mild conditions (RT, pH = 7) with 111In and 177Lu, the corresponding H4pypa complexes are highly thermodynamically stable and kinetically inert (<1% transmetalation to serum protein over 5-7 d), a benefit of the extra rigidity exerted by the central pyridyl moiety that serves as a cap to further stabilize the whole complex, as shown in the crystal structure of H[Lu(pypa)] where Lu is fully nine-coordinated by H4pypa. Furthermore, the pM values of M3+-H4pypa systems (M=In3+ (30.5), Lu3+ (22.6) and La3+ (19.9)) are much higher than those with DOTA, DTPA, H4octapa, H4octox and H4neunpa. Moreover, inclusion of a p-OH group on the central pyridyl ring also renders H4pypa a functionally versatile chelator. One of the main advantages is that the precursor (compound 3.14) can be synthesized in large scale with excellent stability while any linker of interest can be attached easily, even in milligram scale, for fast screening. Here, an alkyl linker was selected to join the PSMA-targeting pharmacophore and the [AE][E(pypa)] (AE=177Lu, 111In) complexes demonstrating vastly different, but promising pharmacokinetics. In particular, the 177Lu-counterpart shows significantly faster background clearance and higher tumor retention paving the way for potential theranostic applications. Beyond the proof-of-principle PSMA targeting, H4pypa is also highly valuable for other types of cancer treatments, particularly radioimmunotherapy, in which a mild radiolabeling-condition is essential due to the temperature- and pH-sensitive antibody. Current efforts are expanding the applications of H4pypa with other theranostic radionuclides (e.g. 44/47Sc, 86/90Y) and 84  225Ac.  Different bifunctional derivatives have been developed and conjugated to other targeting vectors, particularly to antibodies.   3.4.   Experimental Section 3.4.1.   Materials and Methods      All solvents and reagents were purchased from commercial suppliers (TCI America, Alfa Aesar, AK Scientific, Sigma-Aldrich, Fisher Scientific, Fluka) and were used as received. Human serum was purchased frozen from Sigma-Aldrich. The analytical thin-layer chromatography (TLC) plates used were aluminum-backed ultrapure silica gel 60 Å, 250 μm thickness; the flash column silica gel (standard grade, 60 Å, 32−63 mm) was provided by Silicycle. 1H and 13C 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 were referenced to residual solvent peaks. Low-resolution (LR) mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the Department of Chemistry, University of British Columbia. 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 non-radioactive compounds consisted of a Waters 600 controller, Waters 2487 dual wavelength absorbance detector, and a Waters delta 600 pump. Phenomenex Synergi 4  hydro-RP 80 Å column (250 mm × 21.2 mm semipreparative) was used for purification of deprotected H4pypa and Phenomenex Luna 5 m C18 100 Å LC column (250 mm × 10 mm) was used for purification of deprotected H4pypa-C7-PSMA617. Automated column chromatography 85  was performed using a Teledyne Isco (Lincoln, NE) Combiflash Rf automated system with solid load cartridges packed with Celite and RediSep Rf gold reusable normal-phase silica columns (Teledyne Isco, Lincoln, NE). Analyses of radiolabeled compounds were performed with both Instant TLC (iTLC) plates, impregnated with silicic acid (iTLC-SA) purchased from Agilent Technologies and radio-HPLC. The TLC scanner model was BIOSCAN (system 200 imaging scanner) and the HPLC system was from Agilent Technologies (1200 series). Phenomenex Synergi 4  hydro-RP 80 Å column (250 mm × 4.60 mm) was used for separation of free radioactivity and radio-complex. 111InCl3 was cyclotron-produced and provided by BWX Technologies as a ∼0.05 M HCl solution; 177LuCl3 was purchased from Isotope Technologies Garching (ITG). All the isotopes used were no-carrier added (n.c.a.). Deionized water was filtered through the PURELAB Ultra Mk2 system.  3.4.2.   Synthesis and Characterization Di-tert-butyl pyridine-2,6-dicarboxylate (3.1)       To a stirred suspension of 2,6-pyridinedicarboxylic acid (10.0 g, 59.8 mmol, 1 equiv) in dichloromethane (DCM) (30 mL) was added tert-butyl alcohol (22.6 mL) and 4-dimethylaminopyridine (DMAP) (3.65 g, 29.9 mmol, 0.5 equiv) at room temperature. Then, N,N'-dicyclohexylcarbodiimide (DCC) (27.2 g, 0.132 mol, 2.2 equiv) in DCM (30 mL) was added dropwise using a dropping funnel over 1 h. The mixture was left stirring at room temperature overnight, and then the precipitate was filtered off by vacuum filtration. The filtrate was concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 80 g gold silica column, A: DCM B: methanol (MeOH), 0-5% B). The product 86  fractions were rotary-evaporated to give an off-white solid (8.36 g, 50%).  1H NMR (400 MHz, 298 K, CDCl3): δ 8.18 (d, J = 7.8 Hz, 2H), 7.95 – 7.90 (m, 1H), 1.64 (s, 18H). 13C NMR (75 MHz, 298 K, CDCl3): δ 168.3, 150.0, 138.1, 127.3, 83.2, 27.9.  LR-ESI-MS: calcd for [C15H21NO4 + H]+ 280.1;  found [M + H]+ 280.2 Tert-butyl 6-(hydroxymethyl)picolinate (3.2)       Compound 3.1 (1.40 g, 5.00 mmol, 1 equiv) was dissolved in dry MeOH (150 mL) in a round-bottom flask. NaBH4 (0.189 g, 5.00 mmol, 1 equiv) was added at 0°C. The mixture was stirred at room temperature for 1 h and then another equiv of NaBH4 was added. The reduction continued until the mono-reduced picolinate dominated, as monitored by silica TLC (5% MeOH in DCM). The average reaction time was 3-4 h. After that, the reaction mixture was diluted with DCM (100 mL) and then quenched with saturated NaHCO3 in water (100 mL). The organic phase was separated and the bulk of MeOH in the aqueous phase was removed in vacuo to give an aqueous layer which was then extracted with DCM (100 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration.  The filtrate was concentrated and then purified through a silica column (CombiFlash Rf automated column system, 40 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary evaporated to give an off-white powder (2.25 g, 72%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.88 (d, J = 7.6 Hz, 2H), 7.77 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 4.82 (s, 2H), 1.59 (s, 9H). 13C NMR (75 MHz, 298 K, CDCl3): δ 164.1, 160.3, 148.3, 137.6, 123.6, 123.4, 82.4, 64.5, 28.2. LR-ESI-MS: calcd for [C11H15NO3 + Na]+ 232.1; found [M + Na]+ 232.2  87  Tert-butyl 6-formylpicolinate (3.3)       To a round-bottom flask with a stirred solution of compound 3.2 (4.50 g, 21.5 mmol, 1 equiv) in 1,4-dioxane (50 mL) was added SeO2 (1.19 g, 10.8 mmol, 0.5 equiv). The mixture was refluxed at 100°C overnight. After the reaction completed, the hot mixture was clarified by filtering through a Celite bed and the filtrate was concentrated in vacuo. The crude mixture was purified through a silica column (CombiFlash Rf automated column system, 80 g gold silica column, A: hexanes (Hex) B: ethyl acetate (EtOAc), B: 0-60%) to give a pale yellow solid (2.51 g, 56%). 1H NMR (400 MHz, 298 K, CDCl3): δ 10.19 (s, 1H), 8.25 (d, J = 6.6 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H), 8.01 (t, J = 7.7 Hz, 1H), 1.67 (s, 9H). 13C NMR (75 MHz, 298 K, CDCl3): δ 193.2, 163.3, 152.9, 150.1, 138.3, 128.7, 123.9, 83.2, 28.2. LR-ESI-MS: calcd for [C11H13NO3 - H]+ 206.1; found [M - H]+ 206.1 Tert-butyl 6-(((2-(tert-butoxy)-2-oxoethyl)amino)methyl)picolinate (3.4)       To a round-bottom flask with a stirred solution of compound 3.3 (0.500 g, 2.40 mmol, 1 equiv) in dry MeOH (20 mL) was added tert-butyl glycinate (0.320 g, 2.40 mmol, 1 equiv). The mixture was stirred for one hour at room temperature and then sodium cyanoborohydride (NaBH3CN) (0.31 g, 4.87 mmol, 2 equiv) was added. The reduction reaction was continued for three hours at room temperature before quenching with saturated NaHCO3 in water (10 mL) and then extraction with DCM (20 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was concentrated in vacuo and the residue was purified through a silica column (CombiFlash Rf automated column system, 12 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were combined and rotary-evaporated to give a pale yellow oil (0.55 g, 70%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.76 (d, J = 7.7 Hz, 1H), 7.65 88  (t, J = 7.7 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 3.91 (s, 2H), 3.26 (s, 2H), 1.48 (s, 9H), 1.32 (s, 9H).13C NMR (75 MHz, 298 K, CDCl3): δ 171.1, 163.9, 159.7, 148.6, 137.2, 124.9, 123.0, 81.9, 81.0, 54.2, 51.0, 27.9. LR-ESI-MS: calcd for [C17H26N2O4 + H]+ 323.2;  found [M + H]+ 323.1 2,6-Di(hydroxymethyl)pyridine (3.5)       To a round-bottom flask with a stirred mixture of pyridine-2,6-dicarboxylic acid dimethyl ester (3.00 g, 15.4 mmol, 1 equiv) in dry MeOH (50 mL) at 0oC was slowly added NaBH4 (2.33 g, 61.5 mmol, 4 equiv) in three portions over 15 min. The solution was then stirred at room temperature for 12 h. CHCl3 (25 mL) was added followed by saturated Na2CO3 in water (50 mL) to quench the reaction. The organic phase was separated and the MeOH in the aqueous phase was removed in vacuo to give a concentrated aqueous solution which was then extracted with CHCl3 (100 mL × 10). Multiple extractions were required to recover most of the product. The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was concentrated to give a white solid (1.99 g, 92%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.70 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 7.7 Hz, 2H), 4.79 (s, 4H). 13C NMR (75 MHz, 298 K, MeOD) δ 161.5, 139.2, 120.2, 65.5. LR-ESI-MS: calcd for [C7H9NO2 + H]+ 140.1; found [M + H]+ 140.1  2,6-Bis(bromomethyl)pyridine (3.6)       To a three-neck round-bottom flask with a stirred solution of compound 3.5 (3.00 g, 21.2 mmol, 1 equiv) in dry ACN (acetonitrile)/CHCl3 (30 mL, 50:50 v/v) at 0°C was added PBr3 (6.02 mL, 63.4 mmol, 3 equiv) dropwise using a dropping funnel over 15 min. The mixture was refluxed for 18 h, and then cooled before water (20 mL) was added slowly at 0°C to quench the reaction. After extraction with CHCl3 (50 mL × 3), the combined organic layers were dried over anhydrous 89  MgSO4, and then clarified by filtration. The solvent was removed under reduced pressure and the product was obtained as a pure white solid (4.88 g, 87%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.76 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 4.59 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3) δ 156.8, 138.5, 123.1, 33.3. LR-ESI-MS: calcd for [C7H7Br2N + H]+ 263.9; found [M(79Br) + H]+ 263.9 Di-tert-butyl-6,6'-(((pyridine-2,6-diylbis(methylene))-bis((2-(tert-butoxy)-2-oxoethyl)aza-nedi-yl))bis(methylene))dipicolinate (3.7)       To a round-bottom flask with a stirred solution of compound 3.6 (1.00 g, 3.80 mmol, 1 equiv) in dry ACN (15 mL) was added K2CO3 (1.38 g, 11.4 mmol, 3 equiv), followed by compound 3.4 (2.45 g, 7.60 mmol, 2 equiv) and KI (1.26 g, 7.60 mmol, 2 equiv.). The mixture was stirred at 40°C for 24 h, and then K2CO3 was separated by centrifugation, followed by washing with DCM or ACN (10 mL × 3). The organic phase was concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-8% B). The product fractions were rotary-evaporated to give a yellow oil (1.99 g, 70%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.93 – 7.76 (m, 4H), 7.61 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.4 Hz, 2H), 7.12 (d, J = 7.7 Hz, 2H), 4.23 (s, 4H), 3.97 (s, 4H), 3.18 (s, 4H), 1.32 (s, 18H), 1.24 (s, 18H). 13C NMR (75 MHz, 298 K, CDCl3): δ 169.8, 164.7, 157.3, 157.0, 145.9, 138.1, 137.3, 126., 122.9, 121.8, 80.7, 58.5, 58.1, 53.6, 53.1, 51.9. LR-ESI-MS: calcd for [C41H57N5O8 + Na]+ 770.4; found [M + Na]+ 770.4   90  H4pypa (3.8)       Compound 3.7 (37.6 mg, 5.03 × 10-5 mol) was dissolved in DCM (1 mL) in a round-bottom flask and TFA (1 mL) was added dropwise to the stirred solution using a Pasteur pipette. The mixture was stirred overnight at room temperature and then rotary-evaporated. The residue was redissolved in H2O (2 mL) and then purified through reverse phase HPLC (A: ACN, B: H2O/0.1% TFA, 5-50% A over 30 min, 10 mL/min, tR = 16.8 min) (18.4 mg, 70%). 1H NMR (400 MHz, 298 K, D2O): δ 8.16 – 8.03 (m, 4H), 7.92 (t, J = 7.8 Hz, 1H), 7.74 (d, J = 7.0 Hz, 2H), 7.49 (d, J = 7.9 Hz, 2H), 4.78 (s, 4H), 4.71 (s, 4H), 4.19 (s, 4H). 13C NMR (75 MHz, 298 K, D2O): δ 169.8, 166.3, 150.3, 150.2, 146.3, 141.2, 140.9, 128.2, 125.5, 125.0, 58.1, 58.0, 55.2. HR-ESI-MS: calcd for [C25H25N5O8 + H]+ 524.1781; found [M + H]+ 524.1783. Elemental analysis: calcd % for H4pypa·2TFA·1.7H2O (C29H30.4F6N5O13.7 = 782.1739): C 44.53, H 3.92, N 8.95; found: C 44.86, H 3.59, N 8.63. Na[natIn(pypa)]       Compound 3.8 (9.60 mg, 1.26 × 10-5 mol, 1 equiv) was dissolved in H2O (0.5 mL) in a scintillation vial and 0.1 M NaOH (aq) was added to adjust the pH to 7. In(NO3)3·6H2O (6.17 mg, 1.51  × 10-5 mol, 1.2 equiv) was added. The mixture was stirred at RT for 1 h and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ 8.26 (t, J = 7.7 Hz, 1H), 8.20 – 8.15 (m, 2H), 8.02 – 7.94 (m, 2H), 7.88-7.83 (m, 2H), 7.50 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 4.44 (d, J = 17.4 Hz, 1H), 4.10 (d, J = 17.4 Hz, 1H), 3.91 (d, J = 17.1 Hz, 1H), 3.78 (d, J = 17.1 Hz, 1H), 3.22 (d, J = 17.5 Hz, 1H), 2.94 (d, J = 17.7 Hz, 1H). 13C NMR (100 MHz, 298 K, D2O): δ 176.7, 176.2, 168.3, 168.2, 154.0, 153.9, 153.2, 152.9, 145.1, 144.9, 142.5, 142.4, 142.2, 91  128.0, 127.7, 124.99, 123.48, 123.0, 117.8, 61.0, 60.7, 59.6, 57.4, 57.3, 54.8. HR-ESI-MS: calcd for [C25H21115InN5O8 + 2Na]+ 680.0224; found [M + 2Na]+ 680.0223. Na[natLu(pypa)]       Compound 3.8 (13.6 mg, 1.79 × 10-5 mol, 1 equiv) was dissolved in H2O (0.5 mL) in a scintillation vial and 0.1 M NaOH (aq) was added to adjust the pH to 7. Lu(NO3)3·6H2O (9.23 mg, 1.97 x 10-5 mol, 1.1 equiv.) was added. The mixture was stirred at RT for 1 h and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ 8.21 (t, J = 7.8 Hz, 2H), 8.05 (d, J = 7.6 Hz, 2H), 7.85 (t, J = 7.8 Hz, 1H), 7.80 (d, J = 7.8 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 4.66 (d, J = 2.6 Hz, 4H), 4.39 (d, J = 14.7 Hz, 2H), 4.07 (d, 2J = 14.7 Hz, 2H), 3.98 (d, 2J = 16.6 Hz, 2H), 3.49 (d, 2J = 17.0 Hz, 2H). 13C NMR (100 MHz, 298 K, D2O): δ 179.1, 173.1, 156.5, 152.7, 150.5, 141.6, 140.0, 125.5, 124.0, 123.0, 64.6, 64.5, 63.4. HR-ESI-MS: calcd for [C25H21175LuN5O8 + 2H]+ 696.0954; found [M + 2H]+ 696.0956. Na[natLa(pypa)]       Compound 3.8 (13.5 mg, 1.79 × 10-5 mol, 1 equiv) was dissolved in H2O (0.5 mL) in a scintillation vial and 0.1 M NaOH (aq) was added to adjust the pH to 6. La(ClO4)3·6H2O (10.7 mg, 1.97 x 10-5 mol, 1.1 equiv.) was added. The mixture was stirred at RT for 1 h and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ 8.07 (t, J = 7.7 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.80 (s, 2H), 7.75 (t, J = 7.7 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.17 (s, 1H), 4.62 (d, J = 16.2 Hz, 1H), 4.50 (d, J = 14.0 Hz, 1H), 4.22 (d, J = 16.0 Hz, 1H), 4.06 (d, J = 18.6 Hz, 2H), 3.85 (d, J = 14.1 Hz, 4H), 3.64 (d, J = 16.3 Hz, 1H), 3.09 (s, 2H). 13C NMR (100 MHz, 298 K, D2O): δ 180.2, 173.4, 156.6, 156.1, 150.0, 92  145.3, 140.4, 126.5, 125.0, 123.7, 123.6, 123.3, 123.2, 63.5, 63.4, 62.5, 62.2. HR-ESI-MS: calcd for [C25H21139La N5O8 + 2Na]+ 704.0249; found [M + 2Na]+ = 704.0251. Dimethyl 4-hydroxypyridine-2,6-dicarboxylate (3.9)       Thionyl chloride (SOCl2) (9.50 mL, 0.130 mol, 5 equiv) was added slowly using a syringe to a stirred suspension of chelidamic acid monohydrate (5.28 g, 26.2 mmol, 1 equiv) in MeOH (60 mL) in a two-neck round-bottom flask at 0°C. The mixture was stirred at RT for 24 h and then refluxed for an additional 2 h. The solvent was removed under reduced pressure gently at RT and then D.I. water was added at 0°C. The mixture was neutralized with 1 M K2CO3 in water solution and the precipitate was filtered by vacuum filtration, and then washed with 50% MeOH in water solution (~10 mL). The white precipitate was dried under reduced pressure to give a white solid (5.54 g, >99%). 1H NMR (400 MHz, 298 K, (CD3)2SO): δ 6.74 (s, 2H), 3.72 (s, 6H). 13C NMR (75 MHz, 298 K, (CD3)2SO): δ 165.7, 149.2, 116.6, 52.7. LR-ESI-MS : calcd for [C9H9NO5 + Na]+ 234.0; found [M + Na]+ 234.2 Dimethyl 4-(benzyloxy)pyridine-2,6-dicarboxylate (3.10)       To a round-bottom flask with a stirred solution of compound 3.9 (1.65 g, 7.82 mmol, 1 equiv) in dry ACN was added anhydrous K2CO3 (2.19 g, 15.8 mmol, 2.02 equiv) and benzyl bromide (1.02 mL, 8.60 mmol, 1.1 equiv). The reaction mixture was refluxed overnight at 60°C. K2CO3 was filtered out by vacuum filtration and then washed with DCM. The filtrate was concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to give a white powder (1.51 g, 64%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.90 (s, 2H), 7.44-7.38 93  (m, 5H), 5.23 (s, 2H), 4.01 (s, 6H). 13C NMR (75 MHz, 298 K, CDCl3): δ 150.0, 129.0, 128.9, 127.9, 115.0, 71.0, 53.4. LR-ESI-MS: calcd for [C16H15NO5 + Na]+ 324.1; found [M + Na]+ 324.1 (4-(Benzyloxy)pyridine-2,6-diyl)dimethanol (3.11)       To a round-bottom flask with a stirred solution of compound 3.10 (8.74 g, 29.0 mmol, 1 equiv) in dry MeOH (90 mL) was added NaBH4 (3.29 g, 87.1 mmol, 3 equiv) in three portions over 30 min at 0°C. The reaction mixture was stirred at RT. After 24 h, the mixture was diluted with CHCl3 (50 mL) and then quenched with saturated aqueous NaHCO3 (50 mL). The organic phase was separated and the bulk of MeOH in the aqueous layer was removed in vacuo to give an aqueous solution which was extracted with CHCl3 (50 mL × 4). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to give a white solid (5.86 g, 82%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.42-7.35 (m, 5H), 6.79 (s, 2H), 5.12 (s, 2H), 4.70 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 184.4, 166.5, 162.7, 160.6, 149.6, 135.6, 128.9, 128.6, 127.6, 117.2, 111.8, 107.7, 106.5, 106.1, 105.2, 70.2, 64.5. LR-ESI-MS: calcd for [C14H15NO3 + Na]+ 268.1; found [M + Na]+ 268.2 4-(Benzyloxy)-2,6-bis(bromomethyl)pyridine (3.12)       Compound 3.11 (1.76 g, 12.6 mmol, 1 equiv) was suspended in dry ACN/dry CHCl3 (40 mL, 50:50 v/v) in a three-neck round-bottom flask. PBr3 (3.60 mL, 37.9 mmol, 3 equiv) in CHCl3 (5 mL) was added dropwise using a dropping funnel to the stirred solution of compound 3.11 at 0°C over 15 min. The reaction mixture was stirred at 60oC for 18 h and then saturated aqueous Na2CO3 was added slowly to quench the reaction at 0°C. The aqueous phase was extracted with CHCl3 (50 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by 94  filtration. The filtrate was rotary-evaporated to yield a colorless oil which later solidified to a white solid (3.28 g, 70%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.43 (m, 5H), 7.36 (s, 2H), 5.37 (s, 2H), 4.95 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 170.9, 154.5, 133.2, 129.5, 129.3, 128.3, 113.2, 73.0, 25.3. LR-ESI-MS: calcd for [C14H1379Br2NO + H]+ 369.9; found [M(79Br) + H]+ 369.9 Di-tert-butyl-6,6'-((((4-(benzyloxy)pyridine-2,6-diyl)bis(methylene))bis((2-(tert-butoxy)-2-oxoethyl)azanediyl))bis(methylene))dipicolinate (3.13)       Compound 3.12 (0.400 g, 1.30 mmol, 1 equiv), K2CO3 (595 mg, 4.31 mmol, 3.3 equiv) and KI (434 mg, 2.61 mmol, 2 equiv) were added sequentially to the stirred solution of compound 3.4 (0.837 g, 2.60 mmol, 2 equiv) in dry ACN (15 mL) in a round-bottom flask. The mixture was stirred at 30°C for 24 h. K2CO3 was removed by centrifugation and then washed with DCM/ACN (10 mL × 3). The combined supernatants were concentrated in vacuo and then purified with a silica column (CombiFlash Rf automated column system, 12 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to give a pale-yellow oil (0.67 g, 73 %). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.92-7.61 (m, 6H), 7.52-7.30 (m, 5H), 7.12 (s, 2H), 5.11 (s, 2H), 4.03 (s, 4H), 3.86 (s, 4H), 3.33 (s, 4H), 1.57 (s, 18H), 1.43 (s, 18H). 13C NMR (75 MHz, 298 K, CDCl3): δ 170.5, 166.2, 164.0, 160.2, 148.6, 137.2, 136.1, 128.5, 128.1, 127.7, 125.6, 123.3, 123.0, 107.8, 81.8, 80.9, 69.7, 64.4, 59.8, 56.1, 53.4, 28.0. LR-ESI-MS: calcd for [C48H63N5O9 + Na]+ 876.5; found [M + Na]+ 876.6   95  Di-tert-butyl-6,6'-((((4-hydroxypyridine-2,6-diyl)bis(methylene))bis((2-(tert-butoxy)-2-oxoethyl)azanediyl))bis(methylene))dipicolinate (3.14)       Compound 3.13 (0.170 g, 0.200 mmol) was dissolved in dry MeOH (20 mL) in a three-neck round-bottom flask, saturated with N2(g). Pd/C (10 % w/w, 0.1 equiv) was added under a stream of N2(g). The flask was purged with N2(g), followed by H2(g) from a balloon.  The mixture was stirred vigorously at room temperature overnight under H2 atmosphere, and then Pd/C was filtered off through a Celite bed, washed with MeOH (10 mL × 5). The filtrate was rotary-evaporated to a pale-yellow oil (0.150 g) and used without purification. LR-ESI-MS: calcd for [C41H57N5O9 + H]+ 764.4; found [M + H]+ 764.6 Benzyl 8-bromooctanoate (3.15)       8-Bromooctanoic acid (2.00 g, 8.96 mmol, 1 equiv), benzyl alcohol (0.930 ml, 8.96 mmol, 1 equiv) and a catalytic amount of DMAP (0.1-0.2 equiv) were dissolved sequentially in dry DCM (20 mL) in a round-bottom flask. DCC (2.04 g, 9.86 mmol, 1.1 equiv) in DCM (10 mL) was added dropwise using a dropping funnel over 1 h. The mixture was stirred at RT for 24 h. The white precipitate was filtered off by filtration and then the solvent was evaporated in vacuo. The residue was purified with a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to yield a colorless oil (2.51 g, 89%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.35-7.32 (m, 5H), 5.12 (s, 2H), 3.39 (t, J = 6.8 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H), 1.83 (p, J = 6.9 Hz, 2H), 1.69 – 1.61 (m, 2H), 1.44-1.40 (m, 2H), 1.32 (dt, J = 7.3, 3.5 Hz, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 173.7, 136.2, 128.7, 128.3, 66.3, 34.4, 34.0, 32.8, 29.0, 28.5, 28.1, 24.9. LR-ESI-MS: calcd for [C15H21BrO2 + Na]+ 335.1; found [M + Na]+ 335.1 96  Di-tert-butyl-6,6'-((((4-((8-(benzyloxy)-8-oxooctyl)oxy)pyridine-2,6-diyl)bis-(methylene))bis-((2-(tert-butoxy)-2-oxoethyl)azanediyl))-bis(methylene))-di-picolinate (3.16)       To a round-bottom flask with a stirred solution of compound 3.14 (152 mg, 0.200 mmol, 1 equiv) in dry tetrahydrofuran (THF) (4 mL) was added anhydrous K2CO3 (82.6 mg, 0.600 mmol, 3 equiv). The mixture was stirred for 1 h before the addition of compound 3.15 (65.6 mg, 0.210 mmol, 1.05 equiv). The mixture was stirred for 24 h at 30°C, followed by separation of K2CO3 by centrifugation. The isolated salt was washed with DCM twice (~5 mL each) while the combined organic phases were concentrated in vacuo to a yellow oil. The product crude was characterized by MS and NMR, and then used directly in the next step (0.195 g, 90%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.85 – 7.80 (m, 4H), 7.72 (t, J = 7.7 Hz, 2H), 7.34-7.27 (m, 5H), 6.98 (s, 2H), 5.09 (s, 2H), 4.02 (s, 4H), 3.96 (t, J = 6.4 Hz, 2H), 3.84 (s, 4H), 3.32 (s, 4H), 2.34 (t, J = 7.5 Hz, 2H), 1.77 – 1.70 (m, 2H), 1.66-1.58 (m, 24H), 1.42 (s, 18H), 1.34 – 1.32 (m, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 173.6, 170.6, 166.7, 164.2, 160.4, 160.2, 148.7, 137.3, 136.1, 128.6, 128.2, 125.7, 123.1, 107.7, 82.0, 81.0, 67.9, 66.1, 64.3, 59.9, 56.2, 34.3, 29.1, 28.1, 25.9, 25.4, 24.9. LR-ESI-MS: calcd for [C56H77N5O11 + H]+ 996.6; found [M + H]+ 996.7 8-((2,6-Bis(((2-(tert-butoxy)-2-oxoethyl)((6-(tert-butoxycarbonyl)pyridin-2-yl)methyl)-amino)methyl)pyridin-4-yl)oxy)octanoic acid (3.17)       Compound 3.16 (94.8 mg, 0.0952 mmol) was dissolved in dry MeOH (7 mL) in a three-neck round-bottom flask, saturated with N2(g). Pd/C (10 % w/w) was added under a stream of N2. The flask was purged with N2(g) again, followed by H2(g) from a balloon.  The mixture was stirred vigorously at room temperature overnight under H2 atmosphere, and then Pd/C was filtered off through a Celite bed, washed with MeOH (10 mL × 5). The filtrate was concentrated in vacuo to 97  a pale-yellow oil (75.70 mg, 88%) and used without purification. LR-ESI-MS: calcd for [C49H71N5O11 + Na]+ 928.5; found [M + Na]+ 928.7  tBu4pypa-C7-NHS (3.18)       To a two-neck round-bottom flask with a stirred solution of compound 3.17 (75.7 mg, 0.0837 mmol, 1 equiv) in dry ACN (2 mL) was added N-hydroxysuccinimide (10.6 mg, 0.0922 mmol, 1.1 equiv) and EDCl (19.2 mg, 0.101 mmol, 1.2 equiv.) under N2 (g). The mixture was stirred at room temperature under an inert atmosphere overnight. Then, the solvent was removed in vacuo and the residue was redissolved in DCM (10 mL), and then washed with water (10 mL × 3) and brine (10 mL × 2). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to yield a yellow oil (71.70 mg, 86%) which was used in next step without further purification. 1H NMR (400 MHz, 298 K, CDCl3): δ 7.92 – 7.77 (m, 4H), 7.77 – 7.67 (m, 2H), 6.98 (s, 2H), 4.11 – 3.92 (m, 6H), 3.83 (s, 4H), 3.30 (s, 4H), 2.79 (s, 4H), 2.59 – 2.54 (m, 2H), 1.76 – 1.69 (m, 4H), 1.56 (s, 18H), 1.40 (s, 24H). 13C NMR (75 MHz, 298 K, CDCl3): δ 173.7, 170.7, 169.3, 168.7, 164.2, 160.4, 148.8, 137.4, 128.7, 128.3, 125.8, 123.2, 82.1, 81.1, 77.4, 66.2, 60.0, 56.4, 31.0, 29.8, 29.0, 28.8, 28.3, 28.2, 25.9, 25.7, 24.6. HR-ESI-MS: calcd [C53H75N6O13 + H]+ 1003.5392 found [M + H]+ 1003.5358  3.4.3.   Solid-Phase Peptide Coupling      Solid-phase synthesis of H4pypa-C7-PSMA617 was modified from literature procedures.86 Fmoc-Lys(ivDde)-Wang resin (0.046 mmol, 0.61 mmol/g loading) was suspended in dimethylformamide (DMF) for 30 min. Fmoc was then removed by treating the resin with 20% 98  piperidine in DMF (3 × 8 min). The isocyanate derivative of di-t-butyl ester of glutamate (0.138 mmol, 3 equiv) was prepared according to literature procedures86 and added to the lysine-immobilized resin to react for 16 h. After washing the resin with DMF, the ivDde-protecting group was removed with 2% hydrazine in DMF (5 × 5 min), followed by coupling of Fmoc-2-Nal-OH and Fmoc-tranexamic acid to the side chain of Lys using Fmoc-protected amino acid (0.138 mmol, 3 equiv), N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (0.138 mmol, 3 equiv), hydroxybenzotriazole (HOBt) (0.138 mmol, 3 equiv) and N,N-diisopropylethylamine (DIPEA) (0.368 mmol, 8 equiv). Afterwards, the chelator tBu4pypa-C7-NHS (0.138 g, 0.138 mmol) was coupled to the peptide-bound resin by using DIPEA (0.460 mmol, 10 equiv) in DMF overnight. The peptide was then deprotected and simultaneously cleaved from the resin by treating with 95/5 TFA/triisopropylsilane (TIS) for 2 h at RT. After filtration, the peptide was precipitated by adding cold diethyl ether to the TFA solution. The crude peptide was purified by semi-preparative HPLC (32% acetonitrile in water containing 0.1% TFA at a flow rate of 4.5 mL/min, tR = 8.8 min). The eluates containing the desired peptide were collected, pooled, and lyophilized (5.16 mg, 3.91 mol, 8.5%) HR-ESI-MS: calcd [C66H82N10O19 + H]+ 1319.5836; found [M+H]+ 1319.7376.   3.4.4.   X-ray Crystallography      Single orange colored rhombic-shaped crystals of H[Lu(pypa)] were obtained by the slow evaporation of 1:1 LuCl3 and H4pypa solutions in water after adjustment of pH to 2. A suitable crystal 0.15×0.05×0.01 mm was selected and mounted on a suitable support on a Bruker APEX-II CCD diffractometer. The crystal was kept at a steady T = 90(2) K during data collection. The 99  structure was solved with the ShelXT132 structure solution program using the dual solution method and by using Olex2133 as the graphical interface. The model was refined with version 2018/1 of ShelXL132 using Least Squares minimization.  3.4.5.   Solution Thermodynamics      All potentiometric titrations were carried out with a Metrohm Titrando 809 and a Metrohm Dosino 800 with a Ross combined electrode. A 20 mL and 25ºC thermostated glass cell with an inlet-outlet tube for nitrogen gas (purified through a 10% NaOH solution to exclude any CO2 prior to and during the course of the titration) was used as a titration cell. The electrode was daily calibrated in hydrogen ion concentration by direct titration of HCl with freshly prepared NaOH solution and the results were analyzed with Gran procedure134 in order to obtain the standard potential Eº and the ionic product of water pKw at 25°C and 0.16 M NaCl as a supporting electrolyte. Solutions were titrated with carbonate-free NaOH (0.16 M) that was standardized against freshly recrystallized potassium hydrogen phthalate. The first seven protonation equilibria of the ligand were studied by titrations of an acidified solution containing H4pypa 1.06  10-3 M at 25ºC and 0.16 M NaCl ionic strength using a joined potentiometric-spectrophotometric procedure.85 Spectra were recorded in the 200–450 nm spectral range with a 0.2 cm path length optic dip probe connected to a Varian Cary 60 UV/Vis spectrophotometer. The last two ligand protonation equilibria were studied via UV-Vis spectrophotometry on a set of solutions at the same ligand concentration ([H4pypa] = 1.07  10-4 M) containing different amounts of standardized HCl and NaCl to set the ionic strength constant at 0.16 M when possible. The equilibrium H+ concentration in this last UV in batch titration procedure at low pH solutions (2 ≥ pH ≤ 0) was calculated from 100  solution stoichiometry, not measured with a glass electrode. For the solutions of high acidity, the correct acidity scale H0 was used.135 In the study of complex formation equilibria, the determination of the stability constants of M(Hpypa) species (M3+ = In3+, Lu3+ and La3+) was carried out by two different methods. The first method used UV-Vis spectrophotometric measurements on a set of solutions containing 1:1 metal to ligand molar ratio ([H4pypa] = [M]3+ ~ 1.33  10-4 M) and different amounts of HCl in the spectral range 200-400 nm at 25°C and 1 cm path length. The molar absorptivities of all the protonated species of H4pypa calculated with HypSpec2014124 from the protonation constant experiments were included in the calculations. The second method used competition pH-potentiometric titrations with ttha6- and edta4- as ligand competitors and the composition of the solutions was [M]3+ = In3+or Lu3+~ 6.69  10 -4 M, [H4pypa] ~ 2.23  10 -4 M and [ttha6-] ~ 4.47  10 -4 M at 25 °C and I = 0.16 M NaCl and [M]3+ = La3+~ 1.31  10 -3 M, [H4pypa] ~ 5.96  10 -4 M and [edta4-] ~ 5.96  10 -4 M at 25 °C and I = 0.16 M NaCl. Protonation constants of the ttha6- ligand were previously reported127 and metal stability constants of the complexes formed with In(III) and Lu(III) metal ions were determined by pH-potentiometric titration [M]3+ (In3+or Lu3+) = [ttha6-] ~ 6.84  10-4 M at 25°C and I = 0.16 M, while the stability constants for the complexes formed by H4edta and La3+ were taken from literature.136 Direct pH-potentiometric titrations of the M3+-H4pypa systems were also carried out. Metal solutions were prepared by adding the atomic absorption (AA) standard metal ion solutions to a H4pypa solution of known concentration in the 1:1 metal to ligand molar ratio for In(III) and Lu(III) metal ions, while additional 1:2 metal to ligand molar ratios were also carried out for La(III) metal ion. Ligand and metal concentrations were in the range of 0.6-0.8  10-4 M. The exact amount of acid present in the atomic standard metal solutions standards was determined by Gran’s method134 titrating equimolar solutions of either In(III), Lu(III) or La(III) and Na2H2-EDTA. Each titration consisted 101  of 100-150 equilibrium points in the pH range 1.6-11.5, equilibration times for titrations were 2 min for pKa titrations and up to 5 min for metal complex titrations. Three replicates of each titration were performed for each system. Relying on the stability constants for the species M(Hpypa) obtained by the two different methods, the fitting of the direct potentiometric titrations was possible and yielding the stability constants in Table 3.3. All the potentiometric measurements were processed using the Hyperquad2013125 software while the obtained spectrophotometric data were processed with the HypSpec2014124 program. Proton dissociation constants corresponding to hydrolysis of In(III), Lu(III) and La(III) aqueous ions included in the calculations were taken from Baes and Mesmer.137 The overall equilibrium (formation) constants log β referred to the overall equilibria: pM + qH + rL ⇆ MpHqLr (the charges are omitted), where p might also be 0 in the case of protonation equilibria and q can be negative for hydroxide species. Stepwise equilibrium constants log K correspond to the difference in log units between the overall constants of sequentially protonated (or hydroxide) species. The parameter used to calculate the metal scavenging ability of a ligand towards a metal ion, pM, is defined as –log [Mn+]free at [ligand] = 10 mM and [Mn+] = 1 M at pH = 7.4.128   3.4.6.   Radiolabeling Studies       (Concentration-dependent) An aliquot of the ligand solution (H4pypa or H4pypa-C7-PSMA617) (25 L) in NH4OAc solution (0.15 M, pH = 7) was mixed with around 2 MBq of 177Lu/111In (final volume = 250 L). The reactions were incubated at ambient temperature. 5 L of the mixture was spotted on an iTLC-SA plate and developed in EDTA (50 mM, pH = 5.2) buffer. The TLC plate was read by a TLC reader, showing the free metal migrated to the solvent front 102  while the complex stayed at the baseline. The areas of both peaks were used to calculate RCY% which was also confirmed with RP-rHPLC (A: H2O/0.1% TFA B: ACN/0.1%TFA): [177Lu][Lu(pypa)] (100-0% A over 20 min, tR = 12.9 min); [177Lu][Lu(pypa-C7-PSMA617)] (100-20% A over 30 min, tR = 15.0 min); [111In][In(pypa)] (100-0% A over 20 min, tR = 10.4 min); [111In][In(pypa-C7-PSMA617)] (100-0% A over 20 min, tR = 14.1 min).        (Ratio-dependent: example for ~1:1 L/M ratio) The study was following a revised protocol published and it should be noted that this is a close estimation of the L/M ratio.129 Firstly, the specific activity of the applied radioisotopes (i.e. 177Lu and 111In) were calculated at the time of radiolabeling and the amount of Lu or In (mol) was calculated from the specific activity data. The amount of ligand required is determined from the desired L/M ratio in the reaction mixture. In the case of Lu, natLu solution (5.70 × 10-5 M, 3.5 L, 1.97 × 10-10 mol) was added to NH4OAc solution (0.15 M, pH = 7, 167 L), followed by n.c.a. 177LuCl3 (2 MBq, 10 L, 3.22 × 10-12 mol) from a stock solution with a specific activity of 3510 GBq/mg measured at the formulation time. Finally, the ligand solution (10-5 M, 20 L, 0.2 nmol) was added and the mixture was incubated at RT for 15 min. The studies with 111In was similar. natIn solution (8.71 × 10-5 M, 2.3 L, 1.98 × 10-10 mol) was added to NH4OAc solution (0.15 M, pH = 7, 168 L), followed by n.c.a. 111InCl3 (2 MBq, 10 L, 2.17 × 10-12 mol) from a stock solution with a specific activity of 8295.4 GBq/mg. Finally, the ligand solution (10-5 M, 20 L, 0.2 nmol) was added and the mixture was incubated at RT for 15 min.  103  3.4.7.   In Vitro Human Serum Challenge      Ligand solution (10-3 M, 50 L) was added into NH4OAc solution (0.15 M, pH = 7, 400 L), followed by 177LuCl3/111InCl3 in HCl (2 MBq, 50 L). The volume was divided into triplicates. To each sample, an equal volume (167 L) of human serum was added. The mixture was incubated at 37°C and 5 L aliquots was collected at desired time points (1 h, then once daily over 8 d). The aliquot was spotted onto iTLC-SA plate and developed in EDTA solution (50 mM, pH = 5.2). The TLC plate was read by a TLC reader. The free metal migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate % RCY.  3.4.8.   In Vitro Competition Binding Assays      LNCaP prostate cancer cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37°C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO2. Cells grown to 80-90% confluence were then washed with sterile phosphate-buffered saline and trypsinization. The collected cells number was counted manually with Bal Supply (Sylvania, OH) 202C laboratory counter. Cells (400,000/well) were plated onto a 24-well poly-D-lysine coated plate for 48 h. Growth media was removed and replaced with HEPES buffered saline (50 mM HEPES, pH = 7.5, 0.9% sodium chloride) and allowed to incubate with the cells for 1 h at 37°C. 18F-DCFPyL (0.1 nM) was added to each well (in triplicate) containing various concentrations (0.5 mM – 0.05 nM) of tested compounds (natLu-/natIn-pypa-C7-PSMA617). Non-specific binding was determined in the presence of non-radiolabeled DCFPyL (10 µM). The assay mixtures were further incubated 104  for 1 h at 37°C with gentle agitation. Then, the buffer and hot ligand were removed, and cells were washed twice with cold HEPES buffered saline. Trypsin solution (400 µL, 0.25%) was then added to each well to harvest the cells. Radioactivity was measured on the gamma counter. Data analyses of Ki were performed using the nonlinear regression algorithm of GraphPad Prism 7 software.   3.4.9.   Radiotracer Preparation for Biodistribution Studies       [177Lu][Lu(pypa-C7-PSMA617)] : An aliquot of H4pypa-C7-PSMA617 solution in MQ water (<5% DMSO) (4.83x10-5 M, 20 L, 0.966 nmol) was added to NH4OAc solution (0.15 M, pH 7, 170 L), followed by 177Lu solution in ~0.04 M HCl (200 MBq, 10 L). The mixture was incubated at RT for 10 min before measuring the RCY % (>98%) with iTLC-SA plate and rHPLC as described above.         [111In][In(pypa-C7-PSMA617)] : An aliquot of H4pypa-C7-PSMA617 solution in MQ water (<5% DMSO) (2.18x10-5 M, 20 L, 0.436 nmol) was added to NH4OAc solution (0.15 M, pH 7, 170 L), followed by 111In solution in ~0.04 M HCl (200 MBq, 10 L). The mixture was incubated at RT for 10 min before measuring the RCY % (>98%) with iTLC-SA plate and rHPLC as described above.    3.4.10.   Biodistribution Studies      Imaging and biodistribution experiments were performed using NODSCID IL2RγKO male mice. The mice were maintained, and the experiments were conducted in according to the 105  guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. Mice were anesthetized by inhalation with 2% isoflurane in oxygen and implanted subcutaneously with LNCaP cells (1×107) behind left shoulder. Mice were imaged or used in biodistribution studies when the tumor grew up to reach 5-8 mm in diameter during 5-6 weeks. The mice were injected with the radiotracer (2-4 MBq) through the tail vein under anesthesia (2% isoflurane in oxygen). The mice were allowed to recover and roam freely in their cages. At predetermined time points (1, 4, 24 or 72 h p.i.), the mice were euthanized by CO2 inhalation. Blood was withdrawn immediately from the heart, and the organs/tissues of interest were collected. The collected organs/tissues were weighed and counted using a Perkin Elmer (Waltham, MA) Wizard2 2480 automatic gamma counter.   3.4.11.   SPECT/CT Imaging Studies      SPECT/CT imaging experiments were conducted using the MILabs (Utrecht, The Netherlands) U-SPECT+/CT scanner. Each tumor bearing mouse was injected with 111Lu/111In labeled H4pypa-C7-PSMA617 (44.1 MBq for 177Lu and 24.9 MBq for 111In) through the tail vein under anesthesia of 2% isoflurane in oxygen. The mice were allowed to recover and roam freely in their cage and imaged at 1, 4, 24, and 72 h after injection.  At each time point, the mice were sedated again with 2% isoflurane in oxygen and positioned in the micro scanner. Body temperature was maintained via a heating pad and vitals monitored throughout the scan.  A baseline CT scan was obtained for localization and attenuation correction with voltage setting at 60 kV and current at 615 µA followed by a 60 min static emission scan acquired in list mode using an extra ultra-high sensitivity multi-pinhole big mouse (2mm pinhole size) collimator.  Data was reconstructed using MILabs 106  reconstruction software centered on the 208 keV (177Lu), 173 and 247 keV (111In) photopeaks.  Reconstruction parameters used similarity regulated ordered subset expectation maximization (128 subsets, 3 iterations) and a post-processing filter (Gaussian blurring) of 0.6 mm.   Scatter correction was performed using the automatic triple energy window setting and a calibration factor was applied generating images in MBq/ml.  Images were decay corrected to injection time and divided by the injected activity in PMOD (PMOD Technologies, Switzerland) to obtain quantitative images expressed as the percentage of the injected dose per gram of tissue (%ID/g).   Data was then converted to DICOM for visualization using Inveon Research Workplace (Siemens Medical Solutions USA, Inc.), a Guassian filter of 0.4 mm was applied and maximum intensity projection images were generated.         107  Chapter 4. [nat/44Sc(pypa)]- : Characterization and Evaluation of Thermodynamic Stability, Radiolabeling and Biodistribution of a Prostate-Specific-Membrane-Antigen-Targeting Conjugate  This chapter contains an adaptation of published work, and is reproduced in part from Li, L.; de G. Jaraquemada-Peláez, M.; Aluicio-Sarduy, E.; Wang, X.; Jiang, D.; Sakhie, M.; Kuo, H.-T.; Barnhardt, T. E.; Cai, W.; Radchenko, V.; Schaffer, P.;  Lin, K.-S.; Engle, J. W.; Bénard, F.; Orvig, C. [nat/44Sc(pypa)]- : Thermodynamic Stability, Radiolabeling and Biodistribution of a Prostate-Specific-Membrane-Antigen-Targeted Conjugate.  Inorg. Chem. 2020, 59, 1985-1995.  4.1.   Introduction      Decay properties of radionuclides can be harnessed for cancer diagnosis and therapy. In nuclear medicine, there are two principal imaging techniques – Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET). Compared to SPECT imaging, PET generally has higher sensitivity and spatial resolution (2-4 mm v.s. 6-8 mm).138 In PET imaging, the location of the radiotracer in the body is identified through the co-incident detection of two back-to-back (~180o) -rays (511 keV each) produced upon the annihilation between the positron (+) emitted and the electron (e-) encountered.1,5,6 The travel distance of the positron before the annihilation event correlates to the positron energy thereby determining the image resolution.6  108       An ideal PET isotope should possess low positron energy, high positron branching ratio and long enough half-life for radiolabeling and probing biological events.139 Unlike the more “organic radionuclides” (carbon-11, nitrogen-13, oxygen-15, fluorine-18), which are mostly limited by short half-lives,139 a much broader decay spectrum can be found in radiometals such as yttrium-86 (t1/2 = 14.7 h), copper-61/64 (t1/2 = 3.33 h/12.7 h), scandium-44 (t1/2 = 3.97 h), zirconium-89 (t1/2 = 78.5 h), gallium-68 (t1/2 = 1.13 h), etc.16,140 Their much longer half-lives allow for production in distant cyclotrons which facilitate the expansion of PET diagnosis beyond large medical centers. Gallium-68 is one of the most used clinical positron emitters due to its high positron branching ratio (I+ = 89%) and the widely available germinium-68/gallium-68 generator,27,141,142 but the application is restricted by its rather short half-life, plus the breakthrough of germinium-68 and other metal-ion impurities present in the eluate complicates the separation and radiolabeling procedures.76,139,143 In this regard, scandium-44 offers several advantages, including its almost quadrupled longer half-life, higher positron fraction (I+ = 94.3%) with lower energy (E+avg) = 632 keV), compared to gallium-68 (E+avg = 830 keV), which enables more favorable spatial resolution.77,143 Furthermore, a human PET/CT imaging study conducted by Singh et al. using [44Sc][Sc(DOTATOC)] proved prolonged imaging up to 23.5 h post-injection feasible,144 while 68Ga-labeled tracers generally have limitation to image beyond 4 h.145,146 Hence, scandium-44 is considered a better surrogate marker for long-lived therapeutic isotopes such as lutetium-177 and scandium-47 in pretherapeutic dosimetry study.147–149 Currently, scandium-44 can be produced via either a titanium-44/scandium-44 generator or cyclotron irradiation of a calcium-44 target (44Ca(p,n)44Sc).150,151 109       Incorporating a metallic radionuclide into a radiopharmaceutical entails a bifunctional chelator that can secure the radiometal ion. To date, macrocyclic DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (Figure 4.1) and its derivatives are the workhorses for scandium-44 complexation, but harsh radiolabeling conditions (>95oC) are a drawback.16,143 Therefore, alternatives are sought (Figure 4.1), including acyclic DTPA (diethylenetriamine-pentaacetic acid) and mesocyclic AAZTA (6-[bis(hydroxycarbonyl-methyl)amino]-1,4-bis(hydroxycarbonylmethyl)-6-methylperhydro-1,4-diazepine).29,148 AAZTA offers radiolabeling at room temperature, but it is heavily time-dependent (~80% radiochemical yield in 30 min).29       Considering the above-mentioned factors, we set out to explore the potential of H4pypa to scandium-44 as a chelating agent for pharmaceutical purposes. H4pypa previously displayed excellent affinity for lutetium-177 (-, , t1/2 ~ 6.64 d);17 therefore, its chelation with scandium-44 can pave the way for this “matched pair” in the theranostic applications.  Herein, studies of H4pypa and the conjugate of Glutamate-urea-Lysine-based PSMA-targeting pharmacophore, H4pypa-C7-PSMA617 (Figure 4.1), for prostate-cancer (PCa) targeting are reported with respect to its suitability for Sc3+ ion. The targeting vector was selected for its excellent affinity and selectivity to PSMA which overexpresses in nearly all stages of PCa, the most common cancer in men in the United State. 31,86,94,95,152 Both syntheses of H4pypa and H4pypa-C7-PSMA617 were reported in our first report.17 Herein, the non-radioactive [Sc(pypa)]- complex was reported and characterized with HR-ESI-MS and different NMR spectroscopic techniques. Variable-temperature 1H NMR spectroscopy revealed the co-existence of two structural isomers, the geometries of which were estimated with DFT calculation. Spectrophotometric and potentiometric titrations were conducted to determine the thermodynamic stability of the complex while the radiolabeling experiments were 110  performed to evaluate the complexation efficiency in extremely diluted solutions. To assess the biological stability of the complex and the impacts of injected molar activity on the pharmacokinetics, biodistribution studies were performed on PSMA-xenograft-bearing mice using tracers with two different apparent molar activities.     Figure 4.1 Chemical structures of the discussed chelators.   111  4.2.   Results and Discussion 4.2.1.   Metal Complexation and Characterization      The complex of H4pypa (Figure 4.1) with Sc3+ was fully characterized with NMR spectroscopy and HR-ESI-MS (Figure 4.2). 1H NMR spectrum revealed the co-existence of two geometric isomers of the complex (major and minor) which interconverted in a temperature-dependent manner (Figure 4.3). As shown in the VT-NMR spectra, from 25˚C to 85˚C, some minor isomers converted to the major one and the conversion was reversible as the temperature cooled down. The interconversion could be seen from the significantly reduced signal intensity of the minor 1H peaks, and therefore, the corresponding correlations in both 1H-13C HSQC (Figures B4 and B9) and COSY (Figures B6 and B10) spectra became invisible at higher temperatures. Although the characterization of the minor isomer by NMR techniques was impossible due to excessive overlapping of the 1H signals, the major isomer could be identified by performing 1H-13C HSQC and HMBC, as well as COSY and NOESY at 70˚C, at which temperature the intervening cross-peaks from the minor isomer were minimized (Figures B4-B7). The assignment of 1H and 13C signals is depicted in Figure 4.2 and the labels can be referred to Figure 4.3 (1H, a-i) and Figures B1-B3 (13C, 1-23).       The asymmetry in the Sc-pypa complex could be seen from the 12 aliphatic and 9 aromatic 1H-13C couplings in HSQC (Figure B4), as well as the 6 pairs of diastereotropic methylene-H atoms (Figure B6). The J3 coupling between the ortho-H (g(g᾽), i(i᾽), h(h᾽)) and the neighboring para-H (g’’, i’’, h’’) confirmed the 3 distinct pyridyl groups in the complex. With reference to the chemical shift, g’’ (triplet) was assigned to the para-H in the central pyridine while two overlapping triplets (i’’ and h’’) belonged to those in the picolinate arms. Ha,a’ and Hb,b’ were assigned to the methylene 112  groups in two acetate arms due to the strong interaction with the adjacent CC=O (C22 and C23, respectively, Figure B5). CHa,a’ (C1) correlated to He’ and Hd’, while Hd’ also coupled to CHg’ (C8) and the quaternary C18 (Figure B5). Therefore, Hd,d’ and He,e’ were close to Ha,a’, and were assigned to the methylene groups adjacent to the central pyridine and the pyridine in the picolinate arm, respectively. Similarly, Hc,c’ correlated to both CHb,b’ (C4) and CHf,f’ (C5) (Figure B5), while Hf spatially coupled to Hi (Figure B7) and Hc,c’ correlated to CHg (C6) (Figure B5). Thus, Hc,c’ and Hf,f’ were assigned to the methylene groups neighboring the middle pyridine and that in the second picolinate arm, respectively. The assignment could be further confirmed by the 1H - 13C (quaternary) interactions (i.e. Hc,c’ and Hg,g’’ with C19; Hf’ and Hi,i’’ with C16; Hd’ and Hg’,g’’ with C18; He’ and Hh’’ with C17) (Figure B5). The only missing assignments are the carbonyl carbons (C20 and C21) in both picolinate arms due to the absence of observable correlation with the adjacent H. Additionally, the fluxionality in the [Sc(pypa)]- complex was reflected by the exchange of the pendent arms in space (between two acetate arms and between two picolinate arms), indicated by the strong exchange cross-peaks (blue) in the NOESY spectrum at 70oC (a & b’, a’ & b, c & d’, f & e’, f’& e, g & g’, h’’ & i’’, h’ & i’, Figure B7). The spatial exchange of the chelating arms suggested that the fluxionality possibly involved the chelate-ring-opening and reclosing. The broader 1H peaks at room temperature, particularly in the aliphatic region, indicated a slower chelating-arm exchange, which was further confirmed by the absence of the exchange cross-peaks (blue) in the NOESY spectrum at room temperature (Figure B11). The intrinsic fluxional behavior of the [Sc(pypa)]- complex can be justified by the small size of the Sc3+ ion (ionic radii = 0.75-0.87 Å, coordination number= 6-8) relative to the 9-coordinating binding cavity of H4pypa.123 Nonetheless, the splitting of the methylene-1H signals upon coordination implied a 9-coordinated [Sc(pypa)]- complex.  113   Figure 4.2 [Sc(pypa)]- 1H and 13C NMR assignments (See Figures 2 and S2(A-C) for labels).      Coexisting in equilibrium, two [Sc(pypa)]- isomers could not be distinguished with HPLC (Figure 4.7), perhaps due to the similar chemical properties. Admittedly, the isomerism could be a concern for the radiopharmaceutical applications; however, it should not be a limitation since more important considerations in this regard should be the biological stability and properties of the [Sc(pypa)]- complex in both isomeric forms, which can only be evaluated by biodistribution studies. Furthermore, the biological properties of a metal complex are heavily dependent on the conjugated targeting vector. For example, when conjugated to a larger targeting vector such as antibody or antibody fragment, the influences from the metal-chelate on the biodistributions would be significantly reduced, as long as the complex is stable in vivo. As a result, despite the co-existence of both isomers at room temperature, it is still important to evaluate the in vivo behavior of the [Sc(pypa)]- complex as a bioconjugate.  114   Figure 4.3 [Sc(pypa)]- variable temperature 1H NMR (A) upfield and (B) downfield spectra (400 MHz, D2O, pH 7) (25, 35, 45, 65, 85˚C from bottom to top).   4.2.2.   DFT Calculations      DFT calculations were carried out to study the structure of the [Sc(pypa)]− anion in solution. Two stable isomeric structures, A and B (Figure 4.4), were identified, featuring different bond configurations around the pyridyl backbone. In structure B, both acetate arms were on the same side of the plane with respect to the central pyridyl moiety, while they were opposite in structure a a’ b b' c c’ d e f d' f' e' g g' h i i' g" h' i" h" A B 115  A, similar to the geometry of the [Lu(pypa)]- complex, which was previously identified as nine-coordinated by crystallography.17 Lu3+ is slightly larger than Sc3+ (ionic radii = 0.87 vs. 0.98 Å, coordination number = 8),123 and the common coordination number for Sc3+ is between 6 and 8 vs. 8 and 9 for Lu3+. However, upon comparing the bond lengths between the structure A and the [Lu(pypa)]- complex, the average M-N bonds were similar while the Sc-O bonds were shorter than the Lu-O bonds, suggesting that the [Sc(pypa)]− complex in this geometry was also nine-coordinated. In fact, this geometry was also energetically more favorable than structure B. With longer average bond distances between the donor atoms and the Sc3+ ion (Table 4.1), structure B was about 22.4 kJ/mol less stable than was structure A. Nonetheless, the small energy gap reasonably justifies the co-existence of two diastereoisomers, with structure A dominating.  As seen in the VT-NMR spectra (Figure 4.3), the isomeric equilibrium position further shifted towards the major one as the temperature elevated but reversed to the initial position as soon as the temperature cooled down. On the basis of the DFT calculation, the transformation could possibly happen by switching the positions of O3 and N4 in structure B. One possible mechanism could involve breaking the Sc-O3 bond, followed by rearrangement of the Sc-N4 bond and finally re-formation of the Sc-O3 bond on the opposite side. Due to the particularly long bond distance between N4 and the Sc3+ ion (2.8345 Å, 0.2684 Å longer than that in the structure A), it is also possible that both the Sc-O3 and Sc-N4 bonds were broken, rearranged and then reformed. Either way, the transformation to structure A resulted in significantly stronger interactions between the metal center and the backbone nitrogen atoms (i.e. N2-N4), and thus a more stable complex. 116    Figure 4.4 Two isomeric species of the [Sc(pypa)]- anion simulated using DFT calculations.  Table 4.1 Comparison of DFT calculated Sc-O and Sc-N bond lengths in structures A and B.   Structure A Structure B Atom1 Atom2 Length (Å) Atom1 Atom2 Length (Å) Sc N1 2.3500 Sc N1 2.4174 Sc N2 2.5647 Sc N2 2.6676 Sc N3 2.4960 Sc N3 2.5960 Sc N4 2.5661 Sc N4 2.8345 Sc N5 2.3495 Sc N5 2.4198 Sc O1 2.1925 Sc O1 2.1915 Sc O2 2.2149 Sc O2 2.2190 Sc O3 2.2115 Sc O3 2.1436 Sc O4 2.1872 Sc O4 2.2279    Structure A Structure B  N1 N2 N3 N4 N5 N1 N2 N3 N4 N5 O1 O1 O2 O2 O3 O3 O4 O4 Sc3+ Sc3+ 117  4.2.3.   Solution Thermodynamics      Due to the competition between the metal ion and protons for the binding sites of a chelator, the basicity of different ionizable and non-ionizable protons is an essential consideration when evaluating the metal complexation. In our previous publication, we reported that H4pypa possessed a total of nine protonation sites and all the protonation constants were determined using combined potentiometric-spectrophotometric titrations and UV in-batch spectrophotometric titrations.17 H4pypa was found to be a moderately basic chelator with the highest pKa being 7.78, assigned to one of the protonated tertiary amines in the pyridyl backbone,17 suggesting that at physiological pH (~7.4) the competition from protons is not a significant concern, as compared to more basic chelators, such as H4octox (Figure 4.1, highest pKa = 10.65).85 Furthermore, the complex formation equilibria of H4pypa with In3+, Lu3+ and La3+ were studied,149 and the complex stability (pM) followed the order of  In3+ (30.5) > Lu3+ (22.6) > La3+ (19.9), opposite their ionic radii (In3+< Lu3+ < La3+).123 From this observation, we concluded that the binding cavity of H4pypa was best suited to fit the smaller In3+ ion.       In this paper, Sc3+ is the subject metal ion of interest with an even smaller ionic radius compared to that of In3+ (0.87 Å vs. 0.92 Å, 8-coordinated).123 To investigate the complexation equilibria of the Sc3+-H4pypa system, similar studies were conducted to determine the thermodynamic stability constant (log KSc(pypa)). Because the complexation between Sc3+ and H4pypa was complete at pH ≤ 2, direct determination of log KSc(pypa) by potentiometric titration was not feasible. Alternative approaches using a ligand-ligand competition method with an EDTA competitor (Figure 4.1), as well as acidic in-batch UV spectrophotometric titrations (Figure 4.5A), were required to first determine the stability constant of the protonated species ([Sc(Hpypa)]). Once log KSc(Hpypa) was 118  known, direct potentiometric methods were used to determine the stability constants of the [Sc(pypa)]- and [Sc(OH)(pypa)] species. Potentiometric and spectrophotometric experimental data were refined using the Hyperquad2013125 and HypSpec2014124 programs. The thermodynamic stability constant of [Sc(pypa)]- (log K[Sc(pypa)]-) was finally calculated to be around 26.98(1) (0.16 M NaCl, 25˚C) (Table 4.2), which is similar to that of [Sc(AAZTA)]- (log KSc(AAZTA) = 27.69(4), 0.1 M KCl, 25˚C)29 and around 3.8 units lower than that of [Sc(DOTA)]- (log KSc(DOTA) = 30.79, 0.1 M Me4NCl, 25˚C)36. In order to accurately compare the stability with other complexes involving different chelators, a parameter that accounts for not only the stability constant, but also the basicity and the denticity of the chelator is necessary. Therefore, the pSc value (-log [M]free at [L] = 10 M, [M] = 1 M and pH = 7.4) was adopted for a comprehensive measurement of the metal sequestering ability. The calculated pSc value of the Sc3+-H4pypa system was 27.1 (Table 4.2), which is more than 2 units higher than that of AAZTA (24.7) and more than 3 units higher than that of DOTA (23.9).29,148 The exceptionally high log KSc(pypa) and pSc values unequivocally manifest the superiority of H4pypa over both DOTA and AAZTA to form more thermodynamically stable complex with the Sc3+ ion at physiological pH (7.4). Unexpectedly, as depicted in Figure 4.5B, [Sc(pypa)]- was an exception in the otherwise size- dependent pM value of the M3+-H4pypa systems (M3+ = Sc3+, In3+, Lu3+ and La3+). It could be interpreted that the size of In3+ ion is the most suitable for the binding cavity of H4pypa among the four metal ions studied. Nonetheless, all of them, once again, exhibited superior metal ion scavenging ability over complexes with the comparison chelators. 119         Figure 4.5 A) Representative spectra of the in-batch UV-titration of the Sc3+-H4pypa system as the pH is raised. [L] = [Sc3+] = 1.33  10-4 M at 25˚C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. B) pM vs. ionic radius123 for M3+ ions of interest and different ligands in discussion.   Figure 4.6 Distribution diagram of the Sc3+-pypa system calculated with stability constants in Table 4.2, [L] = [Sc3+] = 8.53  10-4 M Dashed line indicates physiological pH (7.4).   250 275 300 3250.000.250.500.751.001.251.501.75iso = 283 nmH -0.24  -0.24 -0.17 -0.09 0.10 0.22 0.36 0.45 0.59 0.81 1.45 1.86 2.27AbsorbanceWavelength (nm)pH 2.27iso = 273 nmA0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.2014161820222426283032 H4pypa DOTA H4octapa DTPA H4neunpa H4octox AAZTASc3+La3+Lu3+pM values (-log [M] free)Ionic radius (CN = 8) In3+B0 2 4 6 8 10 120102030405060708090100[Sc(OH)L]2-[ScL]-Sc3+% Formation relative to Sc3+pHASc(HL)120  Table 4.2 Stepwise stability constants (log K) of H4pypa with Sc3+ ion.  Equilibrium Reaction log K M3+ + L ⇆ ML 26.79(2) (a); 26.98(1)(b) ML+ H+ ⇆ MHL 3.74(1)(c), 3.55(2)(b) M(OH)L + H+ ⇆ ML 10.34(3)(a) pMd 27.1 a) from direct UV-potentiometric titration method at 25°C, I = 0.16 M NaCl; b) from EDTA potentiometric competition titration method at 25 °C, I = 0.16 M NaCl; c) from direct UV-acidic competition titration method at 25 °C, I ≠ 0.16 M NaCl. d) pM is defined as -log [M]free at [L] = 10 M, [M] = 1 M and pH = 7.4.  4.2.4.   Radiolabeling and Mouse Serum Challenge Experiment      All the radiolabeling experiments with H4pypa and H4pypa-C7-PSMA617 presented here were performed at room temperature and the samples were agitated during incubation. The radiochemical yield was determined with instant thin layer chromatography plates impregnated with silicic acid (iTLC-SA), developed in sodium citrate (0.4 M, pH = 4.5) and the radiocomplexes were confirmed with radioactive high-performance liquid chromatography (radio-HPLC) (Figure 4.7). Initial concentration-dependent radiolabeling at room temperature demonstrated that both H4pypa and H4pypa-C7-PSMA617 radiolabeled scandium-44 (2.9 MBq) efficiently in 5 min (0.5 M NH4OAc buffer, pH = 5.5) (Figure 4.8A), with apparent molar activities (end-of-bombardment (EoB)-corrected) 45 GBq/mol and 44 GBq/mol, respectively. The calculated apparent molar activity increased when the radiolabeling mixtures were incubated longer (48 GBq/mol (30 min) and 53 GBq/mol (60 min) for H4pypa; 47 GBq/mol (30 min) and 52 GBq/mol (60 min) for H4pypa-C7-PSMA617). In order to investigate the effects of pH on the radiolabeling efficiency, pH-dependent radiolabeling experiments with the same amount of radioactivity (i.e. 2.9 MBq) 121  were carried out, showing that the radiolabeling of H4pypa with scandium-44 was the most efficient at pH = 2 (98±0% at 10-5 M, 94±1% at 10-6 M, 15 min), and gradually deteriorated as the pH increased (77±7% at 10-5 M, 10±5% at 10-6 M, pH = 7, 15 min) (Figure 4.8B). This phenomenon could be explained by metal ion hydrolysis, supported by studies which proved that different scandium(III) hydrolysis products or the dimeric hydrolysis complex ([Sc2(-OH)2(H2O)10]4+) could form at pH ≥ 1, contingent on the concentration and ionic strength.137,153 Nonetheless, in our radiolabeling study, decent yields were still attained at pH = 4 and 5.5 (95±2% and 96±2% at 10-5 M; 82±1% and 89±2% at 10-6 M, respectively, in 15 min).  Figure 4.7 Radio-HPLC chromatographs of (A) [44Sc][Sc(pypa)]- (tR = 13.41 min) (B) [44Sc][Sc(pypa-C7-PSMA617)] (tR = 23.67 min)  (A: H2O/0.1%TFA B:ACN/0.1%TFA. 5-65% B over 32 min, 1 mL/min.    temperature in NH4OAc buffer (0.5 M, pH=5.5) over 5 min. (B) Mouse serum stability of the corresponding complexes  over 24 h at 37 oC.     -505001 Sc-pypa_new_10-6mV1-2.016.02 Sc-pypa_new_10-6mAU2WVL:254 nm0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0-1.09.03 Sc-pypa_new_10-6mAUmin3WVL:280 nm-505001 Sc-pypa_new_10-6mV1-2.016.02 Sc-pypa_new_10-6mAU2WVL:254 nm0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0-1.09.03 Sc-pypa_new_10-6mAUmin3WVL:280 nm-503001 Sc-pypa-C7-PSMA617 10-6MmV1-2.012.02 Sc-pypa-C7-PSMA617 10-6MmAU2WVL:254 nm0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0-1.010.03 Sc-pypa-C7-PSMA617 10-6MmAUmin3WVL:280 nmA B 122                        Figure 4.8 (A) Concentration-dependent radiolabeling studies of H4pypa and H4pypa-C7-PSMA617 with 44Sc at room temperature in NH4OAc buffer (0.5 M, pH=5.5) over 5 min. (B) Concentration-dependent radiolabeling studies of H4pypa with 44Sc at room temperature in NH4OAc solution (0.5 M, pH = 2, 4, 5.5, 7) over 15 min. (C) Mouse serum stability of the corresponding complexes over 24 h at 37 oC.       These radiolabeling results are in sharp contrast with that of the “gold standard” DOTA, reported in the recent [44Sc][Sc(AAZTA)] work (~0% RCY in 15 min, 0.2 M NH4OAc buffer pH = 4, 0.1 MBq 44Sc, [DOTA] = 0.1 M).29 Under the same conditions as DOTA, ~60% RCY was obtained for AAZTA,29 which is similar to [44Sc][Sc(pypa)]- (50±8% RCY, RT, 15 min, pH = 4, 0.1 M); however, more 44Sc was used in labeling H4pypa (~2.9 MBq). Also, similar pH-dependent radiolabeling was observed with AAZTA which possessed the highest affinity for scandium-44 in the pH range 3 - 4, and declined as the pH was raised (0.3 M, 0.1 MBq, 95oC, 5 min); DOTA, however, yielded only ~15% RCY under the same labeling conditions even at its 4 5 6 7020406080100RCY (%)Concentration [-log(M)] H4pypa H4pypa-C7-PSMA6174 5 6 7020406080100RCY (%)Concentration [-log(M)] pH 2 pH 4 pH 5.5 pH 7A            B                                           0 5 10 15 20 25020406080100Radiochemical purity (%)Time (hour) H4pypa H4pypa-C7-PSMA617C                                           123  optimal pH of 3.29 Another study with DOTATOC reported by Pruszyński et al., demonstrated that a temperature as high as 115˚C was necessary for >90% radiolabeling (15 min, pH = 4).154      After the successful radiolabeling studies, the stability of both [44Sc][Sc(L)] (L = pypa and pypa-C7-PSMA617) was challenged with mouse serum. The complexes remained intact in the presence of the mouse serum proteins for at least six half-lives of scandium-44 with <1% transchelation (Figure 4.8C), which is extremely encouraging, considering the efficient radiolabeling at room temperature that H4pypa offers.  4.2.5.   PET/CT Imaging, Biodistribution Studies and Binding Affinity      The binding affinities of H4pypa-C7-PSMA617 to PC3-PIP (PSMA+) and PC3-Flu (PSMA-) cell lines were compared using a series of diluted H4pypa-C7-PSMA617 solutions in the presence of a small amount of [44Sc][Sc(pypa-C7-PSMA617)]. The study was performed under the assumptions that the radiolabeling would not affect the binding of the PSMA ligands to the cell surface receptors, and due to the small portions of the radiolabeled tracers, its influence on overall binding was omitted. The results revealed the PSMA-specific binding of H4pypa-C7-PSMA617 with high affinity (KD = 2.96±0.81 nM and Bmax = 7.84±0.37 fmol/mg).        For in vivo study, two batches of radiotracers were prepared with two different apparent molar activities (7.4 GBq/mol and 74 GBq/mol), and then injected into nude mice (n = 3) bearing both PSMA-positive (PC3-PIP, left shoulder) and PSMA-negative (PC3-Flu, right shoulder) tumors to investigate the targeting specificity of the tracer, as well as the effects on pharmacokinetics associated with the injected molar activity. Both PET/CT images (Figure 4.9) and ex vivo 124  biodistribution study (Figures 4.10 and 4.11) showed that the 44Sc-labeled H4pypa-C7-PSMA617 was excreted renally and collected through the bladder, while the tumor accumulation was PSMA-specific. The absence of abnormal liver and bone radioactivity suggests the biological stability of the radiotracers. In both experiments, the biodistributions of the radiotracers were largely similar, and the tumor uptake was rapid. After 30 min, the tumor radioactivity had already reached 15.1±2.4% ID/g (% injected dose/gram) (7.4 GBq/mol) and 22.1±1.9% ID/g (74 GBq/mol). In both cases, the accumulation peaked at 4-hour post-injection (p.i.) (20.9±3.4% and 24.0±2.8% ID/g, respectively) and gradually reduced over time. Nonetheless, the tumor retention was effective at 14.9±1.7% ID/g (7.4 GBq/mol) and 20.0±0.6% ID/g (74 GBq/mol) after ~4 half-lives of scandium-44. Unexpectedly, comparing the two profiles, although lower apparent molar activity contributed to slightly reduced tumor uptake, it significantly brought down the kidney accumulation by more than half. Consequently, with 7.4 GBq/mol, the tumor-to-kidney ratio increased considerably from 0.39 (30 min p.i.) to 0.57 (16 h p.i.), as opposed to around 0.28 throughout the study when using 74 GBq/mol (Figure 4.12). Besides the kidney, the background radioactivity generally decreased in tandem with the injected molar activity, leading to much higher tumor-to-background ratios. The contrasts were particularly prominent in blood (113 vs. 63), bone (91 vs. 61), heart (194 vs. 83), lung (85 vs. 18), liver (113 vs. 37), spleen (34 vs. 17) and pancreas (194 vs. 83) (16 h p.i.). This phenomenon, however, should not be generalized to all PSMA-targeting radiotracers. The results reported by Fendler et al. showed that [177Lu][Lu(DOTA-PSMA617)] with high molar activity was related to higher tumor-to-background ratio.99 Therefore, it is crucial to evaluate the effect associated with the molar activity independently for each radiotracer to attain an optimal tumor-to-background contrast.  125       Despite different tumor models being used, the pharmacokinetics of [44Sc][Sc(pypa-C7-PSMA617)] resembled those for the published 111In-analog, especially the strong kidney retention, but were drastically different from the 177Lu-counterpart where the clearance from the background organs/tissues were much faster, including the kidney (~2% ID/g at 24 h p.i.), leaving the tumor with major radioactivity accumulations (~14% ID/g at 24 h p.i.).17 The observed differences in the biological behavior of 44Sc- and 177Lu-labeled H4pypa-C7-PSMA617 were unexpected since it was contrary to that of 44Sc- and 177Lu-labeled DOTA-PSMA617, which appeared to have similar biodistributions.77 In the same tumor models (PC-3 PIP/Flu), both 44Sc- and 177Lu-labeled DOTA-PSMA617 were rapidly cleared from the background tissues/organs, including the kidney (~2-3 % ID/g at 6 h p.i.), while their tumor (PSMA+) uptakes and the tumor-to-kidney ratios were comparable (~52-55% ID/g and 17-23, respectively, at 6 h p.i.).77 The similarity renders [44Sc][Sc(DOTA-PSMA617)] a promising pre-therapeutic imaging agent for the 177Lu-counterpart, while the discrepancies in [44Sc][Sc(pypa-C7-PSMA617)] and [177Lu][Lu(pypa-C7-PSMA617)] suggested that the biochemical properties of 44Sc- and 177Lu-labeled pypa complexes are different. Nonetheless, the biodistribution of a radiotracer is heavily dependent on the targeting molecule and therefore, the differences observed in [44Sc][Sc(pypa-C7-PSMA617)] and [177Lu][Lu(pypa-C7-PSMA617)] cannot be generalized to other bioconjugates with different targeting vectors. What is important from this animal study is the demonstration of the exceptional in vivo stability of the [Sc(pypa)]- complex, which can be radiolabeled with a simple and robust procedure in 5 min at room temperature, compatible with the temperature-sensitive biomolecules such as antibody and antibody-fragment. These advantages would be particularly valuable for imaging at late time points and could be exploited when using scandium-47 (t1/2 = 3.35 d) or lutetium-177 as the therapeutic agents for targeted radioligand/radioimmuno-therapy. 126    Figure 4.9 Representative PET/CT images (MIP, coronal) of [44Sc][Sc(pypa-C7-PSMA617)] (7.4 (top) and 74 (bottom) GBq/mol)] in PC3-PIP (left shoulder) and PC3-Flu (right shoulder) tumor-bearing mice at different p.i. time points.           Figure 4.10 Biodistribution data of [44Sc][Sc(pypa-C7-PSMA617)] (7.4 (left) and 74 (right) GBq/mol)] in PC3-PIP-tumor-bearing mice at selected p.i. time points (n = 3 per time point).  0.5 h 2 h 4 h 8 h 16 h 0 35 %ID/g 127    Figure 4.11 Ex vivo biodistribution data of [44Sc][Sc(pypa-C7-PSMA617)] (7.4 and 74 GBq/mol)] in PC3-PIP-tumor-bearing mice at selected p.i. time points (n = 3 per time point).   Figure 4.12 Tumor (PSMA+)-to-kidney ratio over time.  0 2 4 6 8 10 12 14 16 180.30.40.50.6Tumor (PSMA+) / Kidney RatioTime (Hour p.i.) 7.4 Gbq/mol 74 GBq/mol128  4.3.   Conclusions      The potentially nonadentate non-macrocyclic chelator, H4pypa, which previously demonstrated excellent affinities for both In3+ and Lu3+ ions,17 has also presented highly stable chelation with the Sc3+ ion. The complex was synthesized and characterized by HR-ESI-MS and a range of NMR spectroscopic techniques. On the basis of the 1H NMR spectrum, [Sc(pypa)]- in aqueous solution (pH = 7) existed as two isomers (major and minor) in an equilibrium further shifted towards the major isomer as the temperature was raised (>25˚C). The structures of both isomers were predicted by DFT calculation with a small energy difference of 22.4 kJ/mol, rendering their co-existence reasonable. In addition, the formation constant (log KSc(pypa)) and pSc value of the [Sc(pypa)]- complex were calculated to be around 26.98(1) and 27.1, respectively, which were significantly higher than those of [Sc(DOTA)]- and [Sc(AAZTA)]-. Furthermore, radiolabeling experiments with scandium-44 confirmed efficient radio-metalation at room temperature in 5-15 min at a chelator concentration as low as 10-6 M (pH = 2 - 5.5), resulting in a highly stable complex upon mouse serum challenge over at least six decay half-lives.  Lower pH also led to better radiolabeling (i.e. higher radiochemical yield at lower concentration), possibly due to the facile Sc(III) hydrolysis. To further evaluate the biological applicability of [Sc(pypa)]-, PSMA-targeting [44Sc][Sc(pypa-C7-PSMA617)] was  injected into tumor-bearing nude mice in two molar activities, demonstrating PSMA-dependent accumulation and excellent long-term stability. To our surprise, considerable influences from the apparent molar activity on the pharmacokinetic profile were observed where lower apparent molar activity (7.4 GBq/mol) substantially increased the tumor-to-background ratio. This finding is important for radiotracer formulation to optimize the biodistribution profile. Most importantly, the long-term in vivo stability presented H4pypa as a 129  promising chelator for scandium-44, and even scandium-47, as a theranostic radiopharmaceutical which can be exploited for immuno-PET imaging and targeted radiotherapy as well.   4.4.   Experimental Section 4.4.1.   Materials and Methods      All solvents and reagents were purchased from c ommercial suppliers (TCI America, Alfa Aesar, AK Scientific, Sigma-Aldrich, Fisher Scientific, Fluka) and were used as received. 1H, 13C, 1H-13C HSQC, 1H-13C HMBC, COSY and NOESY NMR spectra were recorded at ambient and elevated temperature on Bruker AV400 instruments, as specified; the NMR spectra are expressed on the δ scale and were referenced to residual solvent peaks. Low-resolution (LR) mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray-ionization mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the Department of Chemistry, University of British Columbia. Analyses of radiolabeled compounds were performed with both iTLC-silicic acid impregnated plates (iTLC-SA) purchased from Agilent Technologies and radio-HPLC. The TLC scanner model was BIOSCAN (system 200 imaging scanner) and the HPLC system was from Agilent Technologies (1200 series). DIONEX Acclaim C18 5 m 120 Å column (250 mm × 4.60 mm) was used for separation of free radioactivity and radio-complex. 44Sc was provided by UW-Madison Cyclotron Lab in 0.1 M HCl solution (~259 MBq). Deionized water was filtered through the PURELAB Ultra Mk2 system. 130  4.4.2.   Na[natSc(pypa)] Complexation Reaction      H4pypa · 2 TFA· 2 H2O (10.5 mg, 1.27 × 10-5 mol, 1 equiv) was dissolved in H2O (0.5 mL) in a scintillation vial and 0.1 M NaOH (aq) was added to adjust the pH to 7. ScCl3 · 6H2O (4.93 mg, 1.91 × 10-5 mol, 1.5 equiv) was added. The mixture was stirred at room temperature for 1 h and the complexation was confirmed by LR-ESI-MS. The reaction mixture was dried in vacuo and then re-dissolved in D2O for NMR spectroscopic characterizations. 1H NMR (400 MHz, 343 K, D2O): δ 8.73-8.66 (m, 2H), 8.49 (d, J = 7.8 Hz, 1H), 8.42 (t, J = 7.8 Hz, 1H), 8.38 (d, J = 7.7 Hz, 1H), 8.36 – 8.31 (m, 2H), 7.98 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 5.66 (d, J = 15.1 Hz, 1H), 5.47 (d, J = 15.3 Hz, 1H), 5.39 (d, J = 14.9 Hz, 1H), 5.32 (d, J = 15.4 Hz, 1H), 5.21 (d, J = 15.3 Hz, 1H), 5.16-5.11 (m, 2H), 4.64 (d, J = 17.5 Hz, 1H), 4.46 (d, J = 16.7 Hz, 1H), 4.34 (d, J = 16.9 Hz, 1H), 4.03 (d, J = 17.6 Hz, 1H), 3.82 (d, J = 17.6 Hz, 1H). 13C NMR (100 MHz, 343 K, D2O): δ 179.0, 178.2, 176.8, 173.0, 159.2, 158.6, 158.2, 157.8, 150.0, 149.5, 142.9, 142.6, 142.3, 127.1, 126.8, 123.5, 123.0, 122.4, 63.1, 62.9, 62.2, 61.4, 60.1 HR-ESI-MS: calcd for [C25H21N5O845Sc + 2H]+ 566.1106; found [M + 2H]+ 566.1102.  4.4.3.   DFT Calculations      All DFT simulations were performed as implemented in the Gaussian 09 revision D.01 suite of ab initio quantum chemistry programs (Gaussian Inc., Wallingford, CT). B3LYP functional105,106 and the effective core potentials LanL2DZ basis sets for Sc155–157 were applied to optimize the structural geometry in the presence of water solvent (IEF PCM as implemented in G09) without the use of symmetry constraints. Normal self-consistent field (SCF) and geometry convergence 131  criteria were conducted for all the calculations. The calculated structures were visualized using Mercury 4.1.  4.4.4.   Solution Thermodynamics      All potentiometric titrations were carried out with a Metrohm Titrando 809 and a Metrohm Dosino 800 with a Ross combined electrode. A 20 mL and 25°C thermostated glass cell with an inlet-outlet tube for nitrogen gas (purified through a 10% NaOH solution to exclude any CO2 prior to and during the course of the titration) was used as a titration cell. The electrode was daily calibrated in hydrogen ion concentration by direct titration of HCl with freshly prepared NaOH solution and the results were analyzed with the Gran procedure134 in order to obtain the standard potential Eº and the ionic product of water pKw at 25°C and 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. In the study of complex formation equilibria, the determination of the stability constants of Sc(Hpypa) species was carried out by two different methods. The first method used UV-Vis spectrophotometric measurements on a set of solutions containing 1:1 metal to ligand molar ratio ([H4pypa] = [M]3+ = 1.33  10-4 M) and different amounts of standardized HCl and NaCl to set the ionic strength constant at 0.16 M when possible. The equilibrium H+ concentration in this UV in-batch titration procedure at low pH solutions (2 ≥ pH ≤ 0) was calculated from solution stoichiometry, not measured with a glass electrode. For the solutions of high acidity, the correct acidity scale H0 was used.135 The spectral range was 200-400 nm at 25°C and 1 cm path length. The molar absorptivities of all the protonated species of H4pypa calculated with HypSpec2014124 from the protonation constant experiments17 132  were included in the calculations. The second method used competition pH-potentiometric titrations with EDTA as a ligand competitor and the composition of the solutions was [M]3+ = Sc3+ ~ 1.54  10 -3 M, [H4pypa] ~ 7.07  10 -4 M and [EDTA] ~ 1.55  10 -3 M at 25˚C and I = 0.16 M NaCl. The stability constants for the complexes formed by EDTA and Sc3+ were taken from literature.136 Direct pH-potentiometric titrations of the Sc3+-H4pypa systems were also carried out. Sc3+ metal ion solution was prepared by adding the atomic absorption (AA) standard solution to a H4pypa solution of known concentration in the 1:1 metal to ligand molar ratio. Ligand and metal concentrations were in the range of 8.40-8.53  10-4 M. The exact amount of acid present in the AA standard solution was determined by Gran’s method134 titrating equimolar solutions of Sc(III) and Na2H2-EDTA. Each titration consisted of 100-150 equilibrium points in the pH range 1.6-11.5, equilibration time for titrations was up to 5 min for metal complex titrations. Three replicates of each titration were performed. Relying on the stability constants for the species Sc(Hpypa) obtained by the two different methods, the fitting of the direct potentiometric titrations was possible and yielding the stability constants in Table 4.2. All the potentiometric measurements were processed using the Hyperquad2013 software125 while the obtained spectrophotometric data were processed with the HypSpec2014124 program. Proton dissociation constants corresponding to hydrolysis of Sc(III) aqueous ions included in the calculations were taken from Baes and Mesmer.137 The overall equilibrium (formation) constants log β referred to the overall equilibria: pM + qH + rL ⇆ MpHqLr (the charges are omitted), where p might also be 0 in the case of protonation equilibria and q can be negative for hydroxide species. Stepwise equilibrium constants log K correspond to the difference in log units between the overall constants of sequentially protonated (or hydroxide) species. The parameter used to calculate the metal scavenging ability of 133  a ligand towards a metal ion, pM, is defined as –log [Mn+]free at [ligand] = 10 mM and [Mn+] = 1 M at pH = 7.4.128  4.4.5.   Production and Radiochemical Isolation of Scandium-44      Scandium-44 was cyclotron-produced using natCa[p,n]4xSc nuclear reactions on pressed targets of metallic calcium (300-350 mg). Target preparation was performed in air, and rapidly mounted in the cyclotron to reduce calcium oxidation. Irradiations were performed at 20 µA for 1 h with direct water cooling, and a 12.7 µm Nb foil was used to degrade the beam energy from the nominal 16 MeV to 14.1 MeV. Under these conditions, a scandium-44  production yield of 0.4 mCi/Ah was obtained through the reaction natCa(p,n)44Sc. Isolation of the produced scandium-44  was carried out by single column extraction chromatography using a N,N,N’,N’-tetrakis-2-ethylhexyldiglycolamide functionalized resin (DGA-branched, Eichrom).155 The target was dissolved in 9 M HCl (10 mL) and passed through a 1 mL fritted solid phase extraction (SPE) tube filled with the DGA resin (~120 mg), loading the scandium-44  and eluting bulk Ca2+. Remaining Ca2+ was removed by rinsing the column with 4 M HCl (20 mL). Next, a 12 mL wash with 1 M HNO3 was performed to elute possible trace metal contaminants such as Zn, Fe and Cu. Finally, scandium-44 was eluted in a small volume using 0.1 M HCl (4 x 500 L fractions). The radionuclidic and chemical purity were confirmed by high purity germanium (HPGe) gamma spectrometry and microwave plasma atomic emission spectroscopy (MP-AES), respectively.  134  4.4.6.   Radiolabeling Studies      An aliquot of ligand solution (H4pypa or H4pypa-C7-PSMA617) (10 L) in NH4OAc solution (88 L, 0.5 M, pH = 2, 4, 5.5, 7) was mixed with around 2.9 MBq of scandium-44 (2 L). The reactions were agitated at ambient temperature over the desired period.  The mixture (3 L) was spotted on an iTLC-SA plate, and then developed in sodium citrate buffer (0.4 M, pH = 4.5). The TLC plate was read by a TLC reader, showing the free metal ion migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate RCY %. The complex was also confirmed with radio-HPLC (A: H2O/0.1% TFA B: ACN/0.1%TFA, 5% - 65% B over 32 min, 1 mL/min): [44Sc][Sc(pypa)] (tR = 13.41 min); [44Sc][Sc(pypa-C7-PSMA617)] (tR = 23.67 min); free 44Sc (tR = 3.50 min).  4.4.7.   In vitro Mouse Serum Challenge      To the radiolabeled sample, an equal volume (100 L) of mouse serum was added. The mixture was incubated at 37°C and 5 L aliquots was collected at desired time points (0.5 h, 4 h, 8 h, 24 h). The aliquot was spotted onto iTLC-SA plate next to the control spot (free scandium-44 in serum without chelator) and developed in sodium citrate solution (0.4 M, pH = 4.5). The TLC plate was read by TLC reader. The free metal ion migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate RCY %.  135  4.4.8.   In vitro Competition Binding Assays      To determine the binding affinity of H4pypa-C7-PSMA617 (in the presence of small amount of [44Sc][Sc(pypa-C7-PSMA617)]) for prostate cancer cells, a receptor saturation assay was performed as previously described.158 Briefly, 1 x 105 cells (either PC3-PIP or PC3-FLU) were plated into filter-bottom 96-well plates (Corning), and various concentrations of PSMA ligand solutions (0.03-100 nM) were incubated with the cells for 2 h. Amount of H4pypa-C7-PSMA617 ligands in nmol and corresponding radioactivity in μCi can be calculated by using the labeling efficiency (μCi/nmol) to allow accurate measurement of binding affinity. After the incubation period, the wells were rinsed, dried, and the filter papers were counted in an automated gamma counter to determine the amount of bound tracer. GraphPad Prism was utilized to plot saturation binding isotherms, and the affinity constant (KD) and maximum specific binding (Bmax) were determined.  4.4.9.   Radiolabeling of Conjugates for in vivo Study      For in vivo study, radiolabeling with scandium-44 was performed by mixing 111-185 MBq (3-5 mCi) of radiometal ion with 15-25 nmol of H4pypa-C7-PSMA617 (7.4 or 74 GBq/mol) in 0.5 M NaOAc buffer solution (pH = 5.5). The reactions were incubated for 30 min at room temperature under agitation (300 rpm). Next, the radio-conjugate was purified by reverse phase chromatography using Sep Pak C18 cartridges (Waters), eluted in absolute ethanol, dried at 70oC in N2 stream, and reconstituted in PBS. Quantitative radiochemical yields (>95%) and high 136  radiochemical purities (>95%) were confirmed by radio-TLC using EDTA solution (50 mM, pH=5.5) as mobile phase, and radio-HPLC.  4.4.10.   PET/CT Imaging Studies      All animal experiments were performed under the approval of the University of Wisconsin Institutional Animal Care and Use Committee. Male nude mice (6-8 weeks) were xenografted with 1.5-2.0 × 106 PC3-PIP (PSMA+) cells (left shoulder) and 1.5-2.0 × 106 PC3-FLU (PSMA-) cells (right shoulder) suspended in Matrigel (1:1). Tumor bearing mice were employed for in vivo imaging, when tumors reached a volume of 400-500 mm3. For non-invasive in vivo PET/CT imaging, mice (n=3) were administered 5.5-11.1 MBq (150-300 µCi) of [44Sc][Sc(pypa-C7-PSMA617)] via tail vein injection. Following isoflurane anesthesia, mice were placed into a microPET/microCT scanner (Inveon, Siemens) and 40 millions coincidence event static PET scans were acquired at different timepoints (0.5 h, 2 h, 4 h, 8 h and 16 h post-injection, p.i.). CT scans were taken prior to each PET acquisition for attenuation correction and anatomical reference. Quantitative analysis of images was performed by manually drawing volumes-of-interest over the tumor and other organs of interest, and data was reported as percent injected dose per gram of tissue (% ID/g), mean ± SD.  4.4.11.   Ex Vivo Biodistribution Studies      Ex vivo tissue distribution studies were performed to validate the in vivo imaging quantification results. After the last PET/CT scan, mice were sacrificed, and select organs were harvested, 137  weighed, and counted in an automatic gamma counter (Wizard 2, Perkin Elmer). Uptake in each tissue is reported as % ID/g (mean ± SD).  138  Chapter 5. [89Zr][Zr(pypa)]- : Nonmacrocyclic  Picolinate-Based Chelator for 89Zr-PET Imaging.  5.1.   Introduction      Monoclonal antibodies (mAbs) are high affinity molecules that enable the specific delivery of diagnostic and therapeutic drugs to cell surface molecules.159 Their targeting specificity can combine a radioactive isotope with appropriate decay characteristics for non-invasive disease imaging, particularly in oncology. In this regard, immuno-positron emission tomography (immuno-PET) has shown great potential because of the high image resolution, sensitivity and accurate image quantification.138,160       Due to the long plasma half-life (days to weeks) of a full Immunoglobulin G (IgG), PET imaging should be performed one to three days after injection when the radioactivity in the blood has been cleared to maximize the tumor-to-background contrast.161 This necessitates long-lived radionuclides, such as 124I, (t1/2 = 4.2 days) making it conducive to label a full antibody for prolonged imaging. However, the large portion of high-energy -rays and the high-energy + particle (Eavg = 826 keV) not only increase the radiation dose to the patients, but also degrade the image quality.162,163 Besides, iodinated antibodies are prone to significant dehalogenation reactions in vivo which further reduce the tumor-to-background ratio.164 These factors have spurred the need for alternatives. With t1/2 = 3.3 d and a relatively low-energy positron (Eavg = 396 keV), zirconium-89 has garnered significant attention in antibody-labeling.6 Importantly, zirconium-89 is a much 139  more residualizing radionuclide than is 124I, which results in enhanced tumor retention, and consequently, higher tumor-to-background ratio.45 Although the decay characteristics of a radiometal ion are important considerations, translating it into a radioimmunoconjugate is impossible without a reliable chelating agent that can stably sequester the metal ion under antibody-compatible conditions (i.e. room temperature and pH ~ 7).1,16 The most common chelator for zirconium-89 is the linear desferrioxamine B (DFO, Figure 5.1). From an inorganic chemistry perspective, the hexadentate DFO is far from adequate to satisfy the osteophilic, octa-coordinate zirconium-89, with only three bidentate hydroxamate groups. In vivo, zirconium-89 eventually leaches out of DFO and mineralizes into the skeleton.165,166 Not only does this cause significant radiation dose to the bone marrow, but also hampers the detection of bone metastases and thus impairs its ability as a PET-imaging surrogate for therapeutic radiometal ions (e.g. lutetium-177 and yttrium-90).167  For these reasons, oxygen-based moieties, specifically hydroxamate and hydroxypyridinonate, have been exploited for zirconium-89-chelation.41,42,161,168,169,170,171 While the mainstream focuses on oxygen-enriched ligands, Pandya et al., unusually employed the traditional macrocyclic DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, Figure 5.1) to scavenge the oxophilic zirconium-89 with extraordinary stability, but under harsh radiolabeling conditions (i.e. 95˚C, 1 h).172 Additionally, 89ZrCl4 has to be prepared from the commercially available [89Zr(oxalate)4]4- and was necessary for good radiochemical yield, perhaps due to the weaker competition from the chloride anion in binding zirconium-89.172 Although the labeling temperature is suboptimal for 89Zr-immuno-PET constructs, the study is undoubtedly a vital contribution to the ill-defined Zr(IV)-coordination chemistry. 140       Knowing that the macrocyclic polyamino-backbone significantly enhances the overall stability of [89Zr][Zr(DOTA)], but concomitantly worsens the labeling conditions, our group posits that a non-macrocyclic chelator with a somewhat pre-organized binding pocket could be useful to coordinate zirconium-89 under mild conditions without compromising the complex stability. Herein, we propose a nonadentate chelator, H4pypa, with a N5O4 donor set, for zirconium-89 chelation. H4pypa has previously demonstrated excellent affinities for scandium-44, indium-111 and lutetium-177 at room temperature (Chapters 3 and 4). The built-in central pyridyl ring not only allows facile bifunctionalization, but also rigidifies the whole construct. As a result, it could add value to the realm of 89Zr-immuno-PET.  Investigation of Zr4+-chelation with H4pypa commenced with studying the non-radioactive metal complexation reaction. [Zr(pypa)] was fully characterized with high-resolution electrospray mass spectrometry (HR-ESI-MS) and different nuclear magnetic resonance (NMR) spectroscopy techniques. The complex geometry was estimated by density functional theory (DFT) calculations. Further, a series of concentration-dependent radiolabeling studies were performed with the commercially available [89Zr(oxalate)4]4- at both room temperature and 40˚C, pH = 5.5 and 7 to determine the optimal radiolabeling conditions. Finally, challenge studies with excess EDTA, DFO and human serum were performed to investigate the kinetic inertness of the [89Zr][Zr(pypa)] complex. 141   Figure 5.1 Chemical structures of selected chelators.  5.2.   Results and Discussion 5.2.1.   Metal Complexation and Characterization      The [Zr(pypa)] complex was prepared from the ZrCl4 salts in deionized water at pH = 7. The complex was identified with HR-ESI-MS and fully characterized by different NMR techniques (Figure 5.2-5.5) at ambient temperature. Figure 5.2A shows the [Zr(pypa)] complex with 1H and 13C NMR assignments. The sharp and well-defined 1H signals suggest the presence of potentially one stereoisomer in solution without observable fluxionality within the NMR timescale (Figure 5.2B). In the 1H aromatic region, two triplets at 8.09 ppm (Hf, 1H, J = 7.8 Hz) and 8.38 ppm (Hh, 2H, J = 7.8 Hz), along with three doublets at 8.13 (Hg), 8.00 (He) and 7.70 (Hd) ppm, are observed 142  which is a pattern consistent with the free ligand (Figure 5.2B), implying a [Zr(pypa)] complex symmetric about the central pyridine, further supported by the absence of extra 13C signal (Figure 5.3). The diastereotropic behavior of the methylene-H is realized through the 2J – couplings between Ha and Ha’, Hb and Hb’, Hc and Hc’ (superimposed with the water signal) (Figure 5.4), which indicates that the Zr4+ cation is fully coordinated by the four pendent arms of H4pypa and secured by the pyridyl cap in aqueous solution (pH = 7), generating a neutral complex which is useful in radiopharmaceutical applications.  To further characterize the complex, 1H-13C{1H} HSQC and HMBC experiments were performed at room temperature (Figure 5.5). The ortho-H (Hd) in the central pyridyl ring strongly interacts with the nearby quaternary carbon, C10, which simultaneously couples with the neighboring methylene-Hb(b’). C12 and C13 belong to the carbonyl carbons on the picolinate and acetate arms, respectively, and since the latter correlates to Ha(a’), Ha(a’) is assigned to the methylene-H atoms on the acetate arms. Hc(c’) is allocated to that in the picolinate arms, evidenced by the coupling with the nearby quaternary carbon (C11). Finally, the intense correlation with C11 differentiates the picolinate ortho-He, from -Hg, and therefore, the quaternary C9 is identified.   143     Figure 5.2 A) [Zr(pypa)] 1H and 13C NMR assignment (See Figures 5.2B and 5.3 for labels). B) 1H NMR spectra of [Zr(pypa)] (top) and H4pypa (bottom).   Figure 5.3 [Zr(pypa)] 13C{1H} NMR spectrum (100 MHz, 298 K, D2O). h      gfe      d                                                                 c’c    b’    b a’          a  13 12 11  13 10  13 9  13 8  13 7  8  13 6  8  13 5  8  13 4  13 3  8  13 2  8  13 1  8  13 A B 144   Figure 5.4 [Zr(pypa)] COSY spectrum (400 MHz, 298 K, D2O).  Figure 5.5 [Zr(pypa)] 1H-13C HMBC (red)/ HSQC (green-blue) spectra overlap (400/100 MHz, 298 K, D2O) 145   5.2.2.   DFT Calculations      The geometry of [Zr(pypa)] complex was estimated using DFT calculation (Figure 5.6). On the basis of the calculated Zr-donor bonds, all the bond lengths (Table 5.1) were shorter than the corresponding distances in the [Lu(pypa)]- crystal structure reported previously, while both of them adopted the same stable geometries (i.e. distorted capped square antiprism),17 indicating a nine-coordinating [Zr(pypa)] complex. Furthermore, the N-Zr-N bond angles  (Table 5.2) were also very close to the ideal N-M-N bond angle (~69˚) in a five-membered NCCNM chelate ring,97,98,173 suggesting a favorable geometry adopted by [Zr(pypa)].    Figure 5.6 DFT-calculated geometry for [Zr(pypa)].    N1 N2 N3 N4 N5 O1 O2 O3 O4 Zr 146  Table 5.1 DFT-calculated metal-donor bond lengths for [Zr(pypa)]. Donor atom [Zr(pypa)] Å Backbone pyr-N N1 2.4100 Backbone-N N2 2.4611 Backbone-N N3 2.4613 Picolinate-N N4 2.3438 Picolinate-N N5 2.3437 Acetate-COO O1 2.2250 Acetate-COO O2 2.2233 Picolinate-COO O3 2.2051 Picolinate-COO O4 2.2051  Table 5.2 DFT-calculated X-Zr-Y bond angles for [Zr(pypa)]. X Y Zr4+ Backbone pyr-N Backbone-N 67.46˚ Backbone pyr-N Backbone-N 67.42˚ Picolinate-N Backbone-N 68.94˚ Picolinate-N Backbone-N 68.86˚  5.2.3.   Radiolabeling and Challenge Experiments       A concentration-dependent radiolabeling study was performed to evaluate the radiolabeling efficiency of H4pypa with zirconium-89 by adding a constant amount of radioactivity (~2 MBq) to a series of diluted chelator solutions (10-4 – 10-7 M). The radiochemical yield (RCY) was analyzed with instant-thin layer chromatography plates impregnated with silicic acid (iTLC-SA), developed in ethylenediaminetetraacetic acid solution (EDTA, pH = 5.5, 50 mM), and the radiocomplex was identified with radioactive high-performance liquid chromatography (radio-HPLC) (Figures 5.7 and 5.8). Although room temperature is ideal for radiolabeling an antibody, 40˚C is still tolerable to preserve the targeting specificity of biomacromolecules, thus experiments 147  were conducted at both room temperature and 40˚C. The radiolabeling mixtures were buffered with either 0.2 M ammonium acetate solution (NH4OAc) at pH = 5.5 or 0.2 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid solution (HEPES) at pH = 7, showing significantly different radiolabeling performances. As depicted in Figures 5.9A and 5.9B, the metalation was far more efficient at neutral pH; upon incubation of a 10 µM chelator solution containing ~2 MBq [89Zr][Zr(oxalate)]4- at 40˚C, quantitative radiolabeling was reached within 2 h, compared to 58±2% at pH = 5.5. However, without warming only 26±4% RCY was achieved (pH = 7) (Figure 5.9C). The radiochemical yield could not be determined via radio-HPLC since the zirconium-89 did not elute as a single, sharp peak; however, the complex was successfully identified at 8.27 min as one single sharp peak (Figure 5.7). The results indicated a significant breakthrough for the picolinate-based non-macrocyclic chelators as our group previously reported two multidentate chelators, H4octapa and H4phospa (Figure 5.1), with low zirconium-89 radiolabeling yields.174 The difference between H4octapa and H4pypa is the extra pyridyl moiety in the latter that contributes to a larger and more rigid binding cavity, and perhaps significantly enhanced labeling efficiency with zirconium-89. For comparison, the same radiolabeling experiment was performed with DFO at 40˚C and pH = 7, showing that 10 M was also the minimum chelator concentration for quantitative radiolabeling within an hour (Figure 5.9D).       After the successful radiolabeling of [89Zr][Zr(pypa)], challenge studies with excess EDTA (100-fold), DFO (100- and 1000-fold), and human serum were performed to test the kinetic inertness (Figure 5.9E), showing <1% zirconium-89 was transchelated after 7-day incubation at 37˚C in all the challenge experiments. In contrast, although [89Zr][Zr(DFO)]+ appeared to be stable in human serum, almost 40% zirconium-89 leached out of DFO in the presence of 100-fold excess 148  EDTA (Figure 5.8F). This clearly demonstrated the superior inertness of [89Zr][Zr(pypa)] over the analogous DFO complex.    Figure 5.7 Radio-HPLC chromatograph of free zirconium-89 (A: H2O/0.1% TFA B: ACN/0.1% TFA, 0-70% B over 20 min, 1 mL/min)    Figure 5.8 HPLC chromatograph of [89Zr][Zr(pypa)] (Top : 10-5 M, bottom : 10-6 M) (A: H2O/0.1% TFA B: ACN/0.1% TFA, 0-70% B over 20 min, 1 mL/ min). 149                             Figure 5.9 Concentration-dependent radiolabeling of zirconium-89 with H4pypa at A) 40˚C, pH = 5.5, B) RT, pH = 7, C) 40°C, pH = 7; D) zirconium-89 with DFO at 40˚C, pH = 7. Challenge studies of E) [89Zr][Zr(DFO)]+ with EDTA (100×) and F) [89Zr][Zr(pypa)] with EDTA (100×) and DFO (100× and 1000×). 4 5 6 7020406080100[89Zr][Zr(pypa)]40 oC, pH 5.5RCY %-log[M] 1 h 2 h 22 h4 5 6 7020406080100[89Zr][Zr(pypa)]40 oC, pH 7RCY %-log[M] 1 h 2 h 22 h4 5 6 7020406080100RCY %-log[M] 1 h 2 h 22 h[89Zr][Zr(pypa)]RT, pH 74 5 6 7020406080100[89Zr][Zr(DFO)]2-40 oC, pH 7RCY %-log[M] 1 h 2 h40 80 120 160020406080100[89Zr][Zr(pypa)], 37 oC% IntactHour EDTA (100x) DFO (100x) DFO (1000x)40 80 120 160020406080100% IntactHour EDTA (100x)[89Zr][Zr(DFO)]2-, 37 oCB C D      E F    A 150  5.3.   Conclusions      Zirconium-89 possesses a favorable decay half-life for immuno-PET imaging. In this work, we presented the coordination chemistry of [Zr(pypa)] complex. On the basis of the 1H NMR spectrum, [Zr(pypa)] possesses a 2-fold rotational symmetry about the central pyridine and exists as a single isomer in aqueous solution. Furthermore, DFT calculations showed a nine-coordinating complex with favorable N-Zr-N bond angles very close to the ideal N-M-N bond angle (~69˚) in a five-membered NCCNM chelate ring. The concentration-dependent radiolabeling studies demonstrated a temperature- and pH-dependent radiometalation where 40°C, pH = 7 and 2 h are the optimal labeling conditions with 10 M H4pypa. This high radiolabeling efficiency was unprecedented compared to previously tested picolinate-based chelators H4octapa and H4phospa.174 Most importantly, the resulting complex was also highly inert upon challenges with excess EDTA (100-fold), DFO (100- and 1000-fold) and human serum.    5.4.   Experimental Section 5.4.1.   Materials and Methods      All solvents and reagents were purchased from commercial suppliers (TCI America, Alfa Aesar, AK Scientific, Sigma-Aldrich, Fisher Scientific, Fluka) and were used as received. 1H, 13C, 1H-13C HSQC, 1H-13C HMBC and COSY NMR spectra were recorded at ambient temperature on Bruker AV400 instruments, as specified; the NMR spectra are expressed on the δ scale and were referenced to residual solvent peaks. Low-resolution (LR) mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-151  resolution electrospray-ionization mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the Department of Chemistry, University of British Columbia. Analyses of radiolabeled compounds were performed with both iTLC-silicic acid impregnated plates (iTLC-SA) purchased from Agilent Technologies and radio-HPLC. The TLC scanner model was Amersham Typhoon biomolecular imagers and the HPLC system was from AZURA (P 6.1 L and DAD 6.1 L). Phenomenex Synergi 4-micron Hydro/HP 80 A 250 x 4.6 mm was used for separation of free radioactivity and radio-complex. [89Zr(oxalate)4]4- was purchased from ITG Isotope Technologies Garching in 1 M oxalic acid solution. Deionized water was filtered through the PURELAB Ultra Mk2 system.  5.4.2.   [natZr(pypa)] Complexation and Characterization      H4pypa·2TFA·2H2O (20.7 mg, 26.2 mmol, 1 equiv) was dissolved in D2O (0.5 mL) in a scintillation vial and 0.1 M NaOD (aq) was added to adjust the pH to 7. ZrCl4 (7.30 mg, 31.5 mmol, 1.2 equiv) was added. The mixture was stirred at 40˚C for 1 h and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ 8.38 (t, J = 7.8 Hz, 2H), 8.13 (d, J = 7.6 Hz, 2H), 8.09 (t, J = 7.8 Hz, 1H), 8.00 (d, J = 7.9 Hz, 2H), 7.70 (d, J = 7.8 Hz, 2H), 5.16 – 5.07 (m, 2H), 5.01 (d, J = 16.2 Hz, 2H), 4.50 (d, J = 15.0 Hz, 2H), 4.41 (d, J = 17.4 Hz, 2H), 3.89 (d, J = 17.4 Hz, 2H). 13C NMR (100 MHz, 298 K, D2O): δ 178.3, 172.5, 155.9, 151.6, 148.4, 143.5, 141.9, 126.5, 124.9, 124.2, 66.2, 66.1, 65.3. HR-ESI-MS: calcd for [C25H21N5O845Zr + H]+ 610.0515; found [M + H]+ 610.0513. 152  5.4.3.   DFT Calculations       All DFT simulations were performed as implemented in the Gaussian 09 revision D.01 suite of ab initio quantum chemistry programs (Gaussian Inc., Wallingford, CT). B3LYP functional,105,106 and the effective core potentials LanL2DZ basis sets for zirconium175 were applied to optimize the structural geometry in the presence of water solvent (IEF PCM as implemented in G09) without the use of symmetry constraints. Normal self-consistent field (SCF) and geometry convergence criteria were conducted for all the calculations. The calculated structures were visualized using Mercury 4.1.  5.4.4.   Radiolabeling Experiments      An aliquot of ligand solution (H4pypa or DFO) (10 L) in either NH4OAc buffer solution (0.2 M, pH = 5.5) or HEPES solution (0.2 M, pH = 7) (89 L) was mixed with ~2 MBq of zirconium-89 (1  L). The reactions were incubated at ambient temperature or 40˚C over the desired period (1 h, 2 h, 22 h). The reaction mixture (3 L) was spotted on an iTLC-SA plate, which was developed in EDTA buffer (50 mM, pH = 5.5). The TLC plate was analyzed by a TLC reader, showing the free metal ion migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate RCY%. The complex was also confirmed with radio-HPLC (A: H2O/0.1% TFA B: ACN/0.1%TFA, 0 - 70% B over 20 min, 1 mL/min): [89Zr][Zr(pypa)] (tR = 8.267 min).  153  5.4.5.   EDTA (100-fold) Challenge Experiments       To a quantitatively labeled [89Zr][Zr(pypa)] or [89Zr][Zr(DFO)]+  solution (100 L, 10-4 M) was added HEPES buffer solution (0.5 M, pH = 7, 100 L), followed by EDTA dissolved in water (0.012 M, 83 L). The reactions were agitated at 37˚C over the desired period (1 h, 1 d, 2 d, 5 d, 6 d, 7 d). The reaction mixture (3 L) was spotted on an iTLC-SA plate, and then developed in EDTA buffer (50 mM, pH = 5.5). The TLC plate was analyzed by a TLC reader, showing the transchelated zirconium-89 migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate the intact %.  5.4.6.   DFO (100- and 1000-fold) Challenge Experiments       To a quantitatively labeled [89Zr][Zr(pypa)] solution (100 L, 10-4 M) was added HEPES buffer solution (0.5 M, pH = 7, 100 L), followed by DFO dissolved in water (8.37 mM, 12 L (100×);  120 L (1000×)). The reactions were agitated at 37˚C over the desired period (1 h, 1 d, 2 d, 5 d, 6 d, 7 d). The reaction mixture (3 L) was spotted on an iTLC-SA plate next to the [89Zr][Zr(DFO)]+ control, and then developed in EDTA buffer (50 mM, pH = 5.5). The TLC plate was analyzed by a TLC reader, showing the transchelated 89Zr migrated to the level of [89Zr][Zr(DFO)]+ control while the complex stayed at the baseline. The areas of both peaks were used to calculate the intact %.  154  5.4.7.   In vitro Human Serum Challenge       To a quantitatively labeled [89Zr][Zr(pypa)] or [89Zr][Zr(DFO)]+   solution (100 L, 10-4 M) was added seven parts of the human serum (700 L). The mixture was incubated at 37˚C and 5 L aliquots were collected at desired time points (1 d, 2 d, 4 d, 7 d). An aliquot (3 L) was spotted onto an iTLC-SA plate and then developed in EDTA buffer (50 mM, pH = 5.5). The TLC plate was analyzed by a TLC reader, showing the transchelated 89Zr migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate the intact %.    155  Chapter 6. Coordination Chemistry of [Y(pypa)]- and Comparison Immuno-PET Imaging of 44Sc- and 86Y-pypa-phenyl-TRC105       Chapter 6 contains an adaptation of an accepted manuscript: Li, L.; Jaraquemada-Peláeza, M. G.; Aluicio-Sarduy, E.; Wang, X.; Barnhart, T. E.; Cai, W.; Radchenko, V.; Schaffer, V.; Engle, J. W.; Orvig, C. Coordination Chemistry of [Y(pypa)]- and Comparison Immuno-PET Imaging of 44Sc- and 86Y-pypa-phenyl-TRC105. Dalton Trans. 2020, 49, 5547-5562.  6.1.   Introduction      Medical imaging is a vital part of drug discovery in oncology. Advancing at a rapid pace, Single-Photon Computerized Tomography (SPECT) and Positron-Emission Tomography (PET) provide information complementary to the anatomical images generated by X-Ray Computed Tomography (CT), thereby allowing non-invasive imaging of the tumour microenvironment with excellent sensitivity.176 Both rely on the detection of the photons emitted (SPECT,  100-250 keV) or indirectly generated (PET,  511 keV) during the radioactive decay,1,16 but the latter generally has higher sensitivity and spatial resolution (< 2-3 mm vs. 6-8 mm).138,160,177       Positron-emitting radionuclides are either non-metallic or metallic; the former is incorporated into the carrier via a covalent linkage,139 while the latter is coordinated to a bifunctional multidentate chelating ligand, or chelator, conjugated to a targeting molecule (i.e. antibody, antibody fragment, peptide, etc).1,5,16 The combination of a radionuclide and a bio-vector requires an appropriate match of the radioactive and biological half-lives. Monoclonal antibodies (mAb) 156  are a promising targeting vector with high binding selectivity and recognition of the cellular targets, and thus, ideal for immuno-PET imaging when combined with an appropriately long-lived positron-emitting radionuclides.178,179 Two attractive PET isotopes are scandium-44 (t1/2 = 3.97 h) and yttrium-86 (t1/2 = 14.7 h).143,180 Scandium-44 has a very high positron-branching ratio (I+ = 94%, E+avg = 632 keV)77,143 and can be produced by either a titanium-44/scandium-44 generator or cyclotron irradiation of a calcium-44 target (44Ca(p,n)44Sc), allowing for large quantity production to address clinical needs.150,151  Furthermore, both preclinical and clinical studies have proposed scandium-44 as a better imaging surrogate for lutetium-177 (t1/2 ~ 6.64 d).77,181,182 Although lutetium-177 emits an imageable -ray, using it in low-dose dosimetry study can be unreliable as high therapeutic dose can follow different pharmacokinetics.183 Most importantly, the therapeutic isotopologue, scandium-47 (t1/2 = 3.35 d, E-avg=162 keV), possesses highly favorable decay characteristics for therapy.140 These two constitute the chemically equivalent theranostic pair that permits direct translation of the imaging data to the radiation doses of the therapeutic version.184,185,186 As for yttrium-86, the high energy +-particles and -rays are the major concerns since it impacts the image resolution and complicates the logistics.187 Also, the emission of a plethora of -rays increases the gamma-coincidences, and subsequently the quantitative errors, which are further amplified in tissues with high attenuation coefficient, such as bone, posing difficulties in detection of bone metastases.184,188 Nonetheless, yttrium-86 is still useful in pre-therapy “scout” imaging for the extensively used therapeutic yttrium-90 (t1/2 = 2.67 d, -).5,83,189      Free scandium-44 and yttrium-86 are rapidly taken up in bone and liver; therefore, a carefully tailored coordination chemistry approach for a chelator is required for specific delivery of the 157  radiation dose to the tumor site.190,191  An appropriate ligand not only should bind the radiometal ion with great thermodynamic stability, but also should result from a facile synthetic scheme with high functional versatility since the chemical properties of the linker can significantly alter the properties of the whole radiotracer, and consequently the biodistribution. When a temperature- and pH-sensitive monoclonal antibody is involved, the radiolabeling conditions are limited to ~RT and pH = 6 - 7.192 The tetracarboxylato-macrocyclic DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Figure 6.1) is incompatible with these conditions, albeit dominating the chelation for a myriad of metal ions, including those of scandium-44 and yttrium-86.16,138,5 In this regard, chelators that combine fast radiolabeling kinetics at room temperature and high stability are desired. The recently reported nonadentate H4pypa demonstrated promising coordination chemistry with scandium-44, with the advantage of RT-radiolabeling.100 As for yttrium-86, although CHX-A”-DTPA (Figure 6.1) often replaces DOTA in radioimmunoconjugates due to more efficient radiolabeling at ambient temperature, 40,191,193 it was shown that the 88Y-labeled p-nitrobenzyl derivatives of CHX-A”-DTPA steadily dissociated in serum over time, while yttrium-88 gradually accumulated in the cortical bone of femurs after the labeled antibody-conjugates was injected into the mice.194 Another human serum stability study with 90Y-labeled CHX-A”-DTPA-Trastuzumab also proved transchelation of radioactivity with time.40 Others such as H4octapa, the NETA- and the pyclen-based chelators (Figure 6.1) have each been investigated to a different extent.24,40,195, 196,197,198,199,200 Despite a few promising findings, some of these chelators were derived from rather complicated synthetic routes with limited functional variations, hindering large scale (kg) production and clinical translations.198 Further, it would be advantageous to employ the same chelator-conjugate for both imaging and therapeutic radionuclides since the resulting radiotracers 158  are more probable to have similar chemical properties. For example, the combination of the 44Sc- and 177Lu-labeled H4pypa can be useful in theranostic applications.17,100       Considering the above-mentioned factors, the goal of this work is to explore and evaluate the possibility of employing H4pypa in immuno-PET imaging using scandium-44 and/or yttrium-86. Since H4pypa possesses high affinity for Lu3+ at room temperature,17 while Y3+ and Lu3+ ions have rather similar physical properties and chelator selectivity,191 it was anticipated that H4pypa would be a suitable chelating agent for yttrium-86 as well. In order to conjugate H4pypa to an antibody (TRC105, an anti-CD105 monoclonal antibody that inhibits angiogenesis and tumor growth, was selected herein),201,202 a N-isothiocyanate (NCS)-bifunctionalized H4pypa (H4pypa-phenyl-NCS) was developed via a facile synthetic route and labeled with both scandium-44 and yttrium-86 after conjugation. Biodistribution and PET/CT imaging studies were performed to compare their pharmacokinetics. Prior to the animal studies, the basic coordination chemistry of the [Y(pypa)]- complex anion was investigated, including the thermodynamic stability via a series of potentiometric and spectrophotometric titrations. Furthermore, density-functional theory (DFT) calculation predicted the complex geometry, while the radiolabeling efficiency and the stability in mouse serum were demonstrated with yttrium-86.   159   Figure 6.1 Chemical structures of subject ligands.  6.2.   Results and Discussion 6.2.1.   Synthesis and Characterization      A N-hydroxysuccinimide (NHS)-functionalized pypa (tBu4pypa-alkyl-NHS) has previously been reported for peptide conjugation on solid-phase with high coupling efficiency.17 However, because tert-butyl carboxylate groups are supposed to be hydrolyzed after bioconjugation using trifluoroacetic acid (TFA), it is not compatible with antibody-conjugation. Fortunately, the para-hydroxyl (p-OH) group in the central pyridine in compound 6.7 renders H4pypa highly functionally versatile. Theoretically, any spacer with a leaving group can be integrated through nucleophilic 160  substitution with the p-OH moiety, engendering a much simpler linker-switching procedure because the bifunctional precursor (compound 6.7) can be produced in bulk and repeatedly used for attachment of different linkers which is particularly helpful for preliminary screening. Herein, with an intention to incorporate a reactive N-isothiocyanate (NCS) group into H4pypa for antibody-coupling, commercially available N-boc-2-(4-aminophenyl)ethanol was selected as the starting material of the spacer (Scheme 6.1), and tosylated with p-tosyl chloride in tetrahydrofuran (THF) and 6 M sodium hydroxide (NaOH) aqueous solution at room temperature (compound 6.8, 71%).  The p-tosyl leaving group allowed the linker to be coupled to compound 6.7 (reproduced following published protocol17). The linker was used in 0.1-0.2 equivalent in excess, stirred with compound 6.7 and anhydrous potassium carbonate (K2CO3) (4 equiv) at room temperature for 24-48 h to ensure complete alkylation. Due to the instability of compound 6.9 on silica columns, isolation was not performed, and indeed, not required, if all compound 6.7 was alkylated because the excess linkers would not interfere the following reactions and could be removed during the HPLC purification in the later step.  An important key for this reaction is that the chelator should be stirred with the base vigorously for about an hour prior to the addition of the linker. After that, the tert-butyl groups and the tert-butyloxycarbonyl (boc) group in compound 6.9 were simultaneously removed with TFA in dichloromethane (DCM) (1:1) at room temperature overnight to give H4pypa-phenyl-NH2 (compound 6.10, 50%), which was isolated using the reverse-phase high-performance liquid chromatography (HPLC) as a single peak at tR = 17.8 min (A: H2O/0.1% TFA B: acetonitrile (ACN)/0.1% TFA, 5-60% B over 40 min, 10 mL/min). The purified aniline compound was finally activated with thiophosgene (CSCl2) in a vigorously stirred biphasic  161   Scheme 6.1 Reagents and conditions. i) SOCl2, MeOH, RT-60 oC, 26 h, >99%; ii) BnBr, ACN, K2CO3, 60 oC, overnight, 64%; iii) NaBH4, dry MeOH, RT, overnight, 82%; iv) PBr3, dry CHCl3/dry ACN, 60 oC, overnight, 70%; v) K2CO3, dry ACN, RT, 24 h, 73%; vi) Pd/C (10% w/w), H2 (g), MeOH, RT, overnight; vii) K2CO3, dry THF, RT, 24-48 h, 90%; viii) p-TsCl, THF, 6 M NaOH, 71%; ix) TFA/DCM, overnight, 50%; x) CSCl2, 1 M HCl/ glacial AcOH/CHCl3, RT, 24 h, 30%   solution of hydrochloric acid (5%, aq)/glacial acetic acid (4:1) and chloroform (CHCl3) at room temperature overnight. The resulting product, H4pypa-phenyl-NCS (compound 6.11), was isolated 162  with HPLC using the gradient above (tR = 35.0 min), and then lyophilized to a fluffy white solid in 30% yield.  6.2.2.   Complexation and Characterization      Non-bifunctional H4pypa was synthesized following the published protocol,17 and then complexed with yttrium(III) perchlorate hexahydrate (Y(ClO4)3 · 6 H2O, 1.1 equiv) in water (pH ~ 7) at room temperature for 1 h to form [Y(pypa)]-. The complex was characterized by different nuclear magnetic resonance (NMR) spectroscopic techniques at ambient temperature (Figures C1-C3) and high-resolution electrospray-ionization mass spectrometry (HR-ESI-MS). Characterizations of the [Sc(pypa)]- complex were reported previously and two isomeric species were identified in a temperature-dependent equilibrium.100 As the temperature increased from 25˚C to 85˚C, the equilibrium position further shifted towards the major isomer with sharper 1H NMR peaks.100 This geometric isomerism, however, was not observed with [Y(pypa)]- which formed a single species in neutral aqueous solution without observable fluxionality on the NMR timescale, indicated by the sharp and well-defined 1H NMR signals (Figure 6.2). Furthermore, the [Y(pypa)]- complex appeared to have a 2-fold rotational symmetry about the central pyridine, evidenced by five chemically distinct aromatic 1H signals in a pattern concordant with the symmetric ligand (two triplets: δ 8.23 (Hh) and δ 7.87 (Hf) ppm; three doublets: δ 8.08 (Hg), 7.80 (He), and 7.45 (Hd) ppm). Also, there was no additional 13C NMR peaks compared to H4pypa (Figure C1). The sharp diastereotopic coupling peaks in the aliphatic region confirmed the metal coordination (Figure 6.2) and the correlations between the diastereotropic 1H were realized in COSY (Figure C3), showing 2J – couplings between Ha and Ha’, Hb and Hb’, as well as between 163  the two peaks merged in Hc. Hf (t, 1H) represented the para-H atom in the central pyridyl ring and underwent 3J – coupling with the ortho-Hd. Similarly, the para-Hh atoms (t, 2H) in two chemically equivalent picolinate arms showed the expected 3J – correlations with both neighboring ortho-Hg and -He atoms. Hb and Hc signals were assigned to the methylene-H atoms on the pyridyl backbone and both picolinate arms, respectively, confirmed by the intense 4J – coupling with the neighboring ortho-H in the pyridines. The most upfield methylene-Ha(a’) signals were assigned to the acetic arms.       Upon comparing the 1H NMR spectra of [M(pypa)]-, where M = Sc, Lu and Y (Figure 6.2), the coordination environment of the Y3+-complex was more similar to that of Lu3+, which also exhibited a 2-fold rotational symmetry about the central pyridine.17 The major difference between the two was the diastereotropic behavior of the 1H signals at δ ~ 4.66 ppm which was significantly weaker in [Lu(pypa)]- than in [Y(pypa)]-. Nonetheless, both Lu3+- and Y3+-pypa complexes were vastly different from the Sc3+ analogue, which existed in solution as two asymmetric isomers with broader 1H NMR signals at room temperature. Even at 85˚C, the [Sc(pypa)]- complex was not isomerically pure. These observations could be due to the size similarity between the Lu3+ and Y3+ ions, while both are larger than the Sc3+ ion (Sc3+ (0.87 Å) < Lu3+ (0.98 Å) < Y3+ (1.02 Å), 8-coordinated).123  164   Figure 6.2 1H NMR spectra (400 MHz, D2O) of H4pypa (RT), [Y(pypa)]- (RT), [Lu(pypa)]- (RT) and [Sc(pypa)]- (RT & 85˚C).  6.2.3.   DFT Calculations      The geometry of the [Y(pypa)]- anion (Figure 6.3A) was calculated by DFT and compared to those of [Sc(pypa)]- and [Lu(pypa)]-. The calculated bond lengths suggested nine-coordination for all three complexes, consistent with the 1H NMR spectra where diastereotropic splitting of the methylene-H atoms were observed (Figure 6.2). All three pypa-complexes shared the same stable geometry of distorted capped square antiprism (Figure 6.3B). As reported previously, the smaller Sc3+ ion also complexed with H4pypa in another geometry ~22.4 kJ/mol less stable than the distorted capped square antiprism.100 The small energy difference allowed both [Sc(pypa)]- [Sc(pypa)]- (85 oC) [Y(pypa)]- H4pypa (RT) [Lu(pypa)]- (RT) [Sc(pypa)]- (RT)   h  g   f e      d                                                                 c    b’      b a’       a H4pypa (RT)  [44Sc][Sc(pypa)]- (RT)  [44Sc][Sc(pypa)]- (85˚C)  [177Lu][Lu(pypa)]- (RT)  [86Y][Y(pypa)]- (RT)  165  isomers to co-exist in solution and interconvert in a temperature-dependent manner, as confirmed in the 1H NMR spectra (Figure 6.2).100 The average bond lengths of the less stable [Sc(pypa)]- geometry were expectedly longer than the more stable one.100 In order to directly compare the structural information of [M(pypa)]- (M = Sc, Lu and Y), the less stable [Sc(pypa)]- conformer was not considered due to its completely different geometry, and only the bond lengths (Table 6.1) and the X-M-Y bond angles (Table 6.2) of the shared geometry were concerned. As shown in Table 6.1, the bond distances between the metal ion and the donor atoms increased in tandem with the ionic radii (i.e. Sc3+ < Lu3+ < Y3+), indicating weaker coordination bonds in the [Y(pypa)]- complex among all,123 while the X-M-Y bond angles were smaller for the bigger metal ions (Table 6.2).  The observations were consistent with the trend reported by Bazargan et al. based on the Cambridge Structural Database search.203 The flexibility and the relationship between the bond length and the bond angle allow the binding cavity of the ligand to match itself with different metal ions.203 Based on that the ideal M-N bond length is ~2.5  Å and the ideal N-M-N bond angle is ~69˚ in a five-membered NCCNM chelate ring, which is preferred by the larger metal ions (i.e. ionic radius ~1 Å),97,173,98 the coordination bonds between the metal ion and the N atoms of two tertiary amines are slightly more favorable in [Sc(pypa)]- (~2.56 Å) compared to [Y(pypa)]- (~2.64 Å) (Table 6.1), so as the N-M-N bond angles (Table 6.2). Despite the differences, all three metal ions demonstrated strong preferences for the oxygen donor atoms (i.e. significantly shorter M-O bonds) due to their hard nature.  166                                    Figure 6.3 (A) DFT calculated geometry for the [Y(pypa)]- anion. (Same geometries calculated for the [Sc(pypa)]- and [Lu(pypa)]- anions). (B) [Y(pypa)]- coordination environment showing only Y3+ and the donor atoms (A perspective looking through the central pyridine).  Table 6.1 DFT-calculated metal-donor bond lengths for the [M(pypa)]- (M = Sc, Lu, Y) anions.   Donor atom Bond length (Å) [Sc(pypa)]- [Lu(pypa)]- [Y(pypa)]- Backbone pyr-N N1 2.4960 2.5150 2.5405 Backbone-N N2 2.5661 2.6058 2.6354 Backbone-N N3 2.5647 2.6057 2.6353 Picolinate-N N4 2.3495 2.4495 2.4803 Picolinate-N N5 2.3500 2.4501 2.4793 Acetate-COO O1 2.2115 2.3514 2.3723 Acetate-COO O2 2.2149 2.3494 2.3753 Picolinate-COO O3 2.1872 2.3341 2.3647 Picolinate-COO O4 2.1925 2.3375 2.3618    B A 167  Table 6.2 DFT-calculated X-M-Y bond angles for the [M(pypa)]- (M = Sc, Lu, Y) anions.   X Y M Sc3+ Lu3+ Y3+ Backbone pyr-N Backbone-N 65.89˚ 65.49˚ 64.76˚ Backbone pyr-N Backbone-N 65.93˚ 65.36˚ 64.93˚ Picolinate-N Backbone-N 67.74˚ 66.69˚ 66.02˚ Picolinate-N Backbone-N 67.67˚ 66.59˚ 66.19˚ Backbone-N Acetate-COO 70.83˚ 68.99˚ 68.28˚ Backbone-N Acetate-COO 70.75˚ 69.03˚ 68.17˚ Picolinate-N Picolinate-COO 70.38˚ 67.55˚ 66.73˚ Picolinate-N Picolinate-COO 70.30˚ 67.42˚ 66.82˚  6.2.4.   Solution Thermodynamics      The stability constant of a metal complex is an important thermodynamic parameter, despite the lack of predictability of the kinetic inertness. Since the metal complexation reaction always occurs in competition with the protonation equilibria of the ligand, knowledge of the protonation constants of the ligand is mandatory to determine the stability constant of its metal complexes. H4pypa was previously determined to possess nine protonation sites, the most basic of which deprotonated with a pKa = 7.78 (protonated tertiary amines in the pyridyl backbone), suggesting that at physiological pH (~7.4), almost all the donor atoms are free for chelation. Complex formation equilibria with more basic chelators, such as DOTA (Figure 6.1, highest pKa = 12.60204) and CHX-A”-DTPA (Figure 6.1, 12.30205) will occur with higher competition between the protons and the basic sites on the ligand. In light of this, a more comprehensive parameter that allows for a better comparison of the affinity of different chelators for a metal ion is pM. The pM value is 168  widely adopted for this purpose and is defined under physiologically relevant conditions (-log [M]free at [L] = 10 M, [M] = 1 M and pH = 7.4). It accounts not only for the stability of the metal complexes, but also for the ligand basicity (pKa values) and denticity, and can provide a more accurate prediction of the in vivo thermodynamic stability and speciation at physiological pH = 7.4.39        To investigate the complexation equilibria of Y3+-H4pypa system, a series of titration studies were conducted. Since the complexation between the Y3+ ion and H4pypa was complete at pH ≤ 2, below the electrode threshold (Figure 6.4), direct determination of the thermodynamic stability constant of [Y(pypa)]- (log KY(pypa)) was not feasible. Instead, a ligand-ligand competition method with EDTA competitor (Figure 6.1) and acidic in-batch UV spectrophotometric titrations (Figure 6.5) were applied to determine the formation constant of the protonated species ([Y(Hpypa)]) prior to those of [Y(pypa)]- and [Y2(OH)(pypa)2]3- species using direct potentiometric titration. Potentiometric and spectrophotometric experimental data were refined using the Hyperquad2013125 and HypSpec2014124 programs. The thermodynamic stability constant of [Y(pypa)]- (log K[Y(pypa)]-) was finally calculated to be 21.60(1) (0.16 M NaCl, 25˚C) (Table 6.3). Although it is 2.49 and 3.10 units lower than those of the DOTA- and CHX-A”-DTPA- complexes, respectively,195,205 the pY value (22.0), a more accurate assessment, as described above, was ≥3 units higher.195,205 The difference was partially a result of the drastically lower basicity of H4pypa. When comparing to other M3+-H4pypa systems (M3+ = Sc3+, In3+, Lu3+ and La3+),17,100 pY value was consistent with the inverse correlation between the size of the metal ion and the metal sequestering ability of H4pypa (except the Sc3+-complex).17,100 With similar ionic radii (Y3+ = 1.075 Å vs. Lu3+ = 1.032 Å, 9-coordinated),123 pY is also very close to pLu, implying similar 169  thermodynamic stability in both systems (Figure 6.6). Another interesting observation is that with larger metal ions such as Y3+ and La3+, H4pypa tends to form a 2:2 metal:ligand hydroxide species under basic conditions, rather than the 1:1 complex as seen in the systems with the Sc3+, In3+ and Lu3+ ions (Table 6.3).  Figure 6.4 Distribution diagram of the Y3+-H4pypa system calculated with stability constants in Table 6.3, [L] = [Y3+] = 1  10-3 M. Dashed line indicates physiological pH (7.4).    0 2 4 6 8 10 120255075100[Y2(OH)(L2)]3-[Y(L)]-Y3+% Formation relative to Y3+pHY(HL)170     Figure 6.5 (A) and (B) Representative spectra of the in-batch UV-titration of the Y3+-pypa system as the pH is raised. [L] = [Y3+] = 1.33  10-4 M at 25 °C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl.    Figure 6.6 pM vs. ionic radius123 for M3+ and ligands of interest.    250 275 300 3250.000.250.500.751.001.251.501.75pH 0.98 -0.01 0.04 0.09 0.15 0.22 0.30 0.39 0.59 0.96 0.98AbsorbanceWavelength (nm)pH -0.01A250 275 300 3250.000.250.500.751.001.251.501.75pH 0.98 0.98 1.07 1.19 1.26 1.35 1.41 1.46 1.64 1.89 2.36AbsorbanceWavelength (nm)pH 2.36B0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.2016182022242628303234 H4pypa DOTA H4octapa DTPA H4neunpa H4octoxSc3+La3+Y3+Lu3+pM values (-log [M] free)Ionic radius (CN = 8) In3+B171  Table 6.3 Stepwise stability constants (log K) of H4pypa with Sc3+, Lu3+, Y3+ and La3+ ions. Equilibrium reaction Sc3+  Lu3+ Y3+ La3+ M3+ + L ⇆ ML 26.98(1)100 22.20(2)17 21.60(1) (a) 19.74(3)17 ML + H+ ⇆ MHL 3.55(2)100 3.60(6)17 2.14(5)(b) 2.29(c) 3.24(5)17 M(OH)L + H+ ⇆ ML 10.34(3)100 10.77(8)17 - - M2L2(OH) + H+ ⇆ M2L2 - - 37.23(a) 34.40(6)17 pM(d) 27.1100 22.617 22.0 19.917 a) from direct UV-potentiometric titration method at 25 °C, I = 0.16 M NaCl; b) from EDTA potentiometric competition titration method at 25 °C, I = 0.16 M NaCl; c) from direct UV-acidic competition titration method at 25 °C, I ≠ 0.16 M NaCl; d) pM is defined as -log [M]free at [L] = 10 M, [M] = 1 M and pH = 7.4.  6.2.5.   Radiolabeling of [86Y][Y(pypa)]- and Mouse Serum Challenge Experiments      Since the radiotracer is usually formulated with very low chelator concentrations (<10-5 M), it is important to ascertain the affinity of a chelator for a radiometal ion in diluted conditions. In this regard, one common and useful study is the concentration-dependent radiolabeling experiment in which a constant amount of radioactivity is added to a series of diluted chelator solutions and then incubated for a desired period. With an intention to use H4pypa in immuno-PET imaging, the labeling experiment of H4pypa with yttrium-86 was performed under antibody-compatible conditions (pH = 7 and ambient temperature) using 0.2 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer and ~4.1 MBq of yttrium-86. The mixtures were agitated at room temperature for 7, 15, 30 and 60 min, and then the radiochemical yield percentage (RCY%) of [86Y][Y(pypa)]- was determined with silica aluminum-backed thin layer chromatography (TLC) plates, developed in diethylenetriamine-pentaacetic acid (DTPA) buffer (0.1 M, pH = 5.5). Attempts to identify the complex by the radioactive high-performance liquid chromatography 172  (HPLC) failed as [86Y][Y(pypa)]- decomplexed in the HPLC column, probably due to the fast acid-assisted dissociation kinetics at low concentrations of the metal complex in the acidic mobile phases (ACN/H2O/0.1% TFA solution, pH ~ 2). Regarding the radio-metalation, the efficiency was concentration-dependent (96±1% at 10-5 M vs. 88±3 at 10-6 M after 15 min) (Figure 6.7 and Table 6.4)  and that at the lower concentrations (i.e. 10-6 M and 10-7 M) gradually improved with time (83±2%, 7 min vs. 93±1, 60 min at 10-6 M; 15±2%, 7 min vs. 26±2, 60 min at 10-7 M) (Table 6.4). Furthermore, the apparent molar activity of the [86Y][Y(pypa)]- complex (the radioactivity was corrected to the end-of-bombardment (EoB)) significantly increased with time, from 79 GBq/mol at 7 min to 105 GBq/mol at 60 min, meaning that more radioactivity could be used to achieve quantitative RCY if the complexation time is longer. The efficient radiolabeling at room temperature is an advantage of non-macrocyclic/acyclic chelator and was also seen with CHX-A”-DTPA. A radiolabeling study of yttrium-90 and CHX-A''-DTPA-DUPA-Pep (a peptide conjugate) conducted by Benjamin et al. obtained ~98% RCY in 5 min at room temperature (25 g of peptide, pH = 5.5 and 3.7-3.8 MBq).206 On the other hand, quantitative radiolabeling with DOTATOC in 10 min required heating at 100˚C (pH = 4),30 which is not compatible with the monoclonal antibody, but expected for a constrained macrocycle. The radiolabeling results for [44Sc][Sc(pypa)]- were previously reported,100 and similar to [86Y][Y(pypa)]-, the radiochemical yields were both concentration- and time-dependent (95±0% at 10-5 M vs. 90±2 at 10-6 M after 5 min; 9±3%, 5 min vs. 21±1, 60 min at 10-7 M, pH = 5.5, 2.9 MBq). In spite of the similar radiolabeling behavior, only [44Sc][Sc(pypa)]- could be isolated in the acidic HPLC system as a single sharp peak at 13.4 min (same stationary and mobile phases as [86Y][Y(pypa)]- were used), implying a much slower acid-assisted dissociation kinetics at pH ~ 2 compared to the 86Y-analog.100 Nevertheless, the efficient 173  radiolabeling of H4pypa at room temperature with both radionuclides renders it a promising alternative to the “gold standard” chelator, DOTA, in building immuno-constructs.30,154        After the successful radiolabeling results, it was deemed prudent to test the stability of the [86Y][Y(pypa)]- complex in mouse serum. In the study, the labeled complex was incubated in mouse serum at 37˚C over 48 h, and the intact percentage of the complex was determined by silica aluminum-backed TLC plates, developed in DTPA buffer (0.1 M, pH = 5.5). To validate the experiment, a control sample containing only yttrium-86 in the labeling buffer and mouse serum was prepared, showing only one radioactivity spot at the solvent front on the developed TLC plate, which confirmed that both the free and the transchelated yttrium-86 were moved by DTPA. The study showed that ~97% of [86Y][Y(pypa)]- remained intact after 2 days which is encouraging (Figure 6.7B).      Figure 6.7 (A) Concentration-dependent radiolabeling studies of H4pypa with 44Sc in NH4OAc buffer (0.5 M, pH=5.5) and 86Y in HEPES buffer (0.5 M, pH=7) at room temperature over 15 min. (B) Mouse serum stability of [86Y][Y(pypa)]-over 48 h at 37˚C  4 5 6 7020406080100RCY (%)[-log(M)] [44Sc][Sc(pypa)]- [86Y][Y(pypa)]- A0 10 20 30 40 50020406080100Radiochemical purity (%)Time (hour) [86Y][Y(pypa)]- B174  Table 6.4 Radiochemical yield% of [86Y][Y(pypa)]- at pH=7. Final concentration [-log(L)] RCY % (n = 3) 7 min 15 min 30 min 60 min Avg. St.dev. Avg. St.dev. Avg. St.dev. Avg. St.dev. 4 95 3 98 0 97 1 97 0 5 96 0 96 1 97 1 97 1 6 83 2 88 3 92 1 93 1 7 15 2 16 5 23 2 26 2 ESA (EoB) GBq/mol 79 85 98 105  6.2.6.   PET/CT Imaging and Biodistribution Studies      H4pypa-phenyl-TRC105 was prepared for biodistribution studies using 10:1 chelator:antibody ratio for the reaction at pH = 9 (0.1 M Na2CO3 buffer solution). The bioconjugate was purified with a size-exclusion column and PBS buffer solution, before radiolabeling. The number of chelators per antibody was not determined in this study.      Following the injection of 44Sc- and 86Y-labeled H4pypa-phenyl-TRC105 (~18 and 11 MBq/mouse, respectively) into the 4T1-xenograft-bearing mice (n = 3), serial PET/CT images (maximum intensity projection, MIP) were acquired at 30 min, 4 h, 8 h and 18 h post-injection (p.i.) for the 44Sc-labeled tracer, as well as 30 min, 4 h, 24 h and 48 h p.i. for the 86Y-counterpart (Figure 6.8). A higher dose of the 44Sc-labeled radiotracer was injected in order to acquire the late-timepoint images. Radioactivity uptakes of selected organs were analyzed with quantitative regions-of-interest (ROI) imaging and plotted in Figure 6.9 in % injected-dose-per-gram (% ID/g). The ex vivo biodistribution results at the last timepoint was shown in Figure 6.10 which confirmed the accuracy of the image-based ROI quantification. Both radioimmunoconjugates showed expected uptakes in the blood, tumor, liver, kidney and spleen due to the antibody circulation, 175  metabolism and excretion. High uptake in lung was seen in other reported TRC105-constructs, perhaps due to the prolonged residence in the blood pool.35,89,207 The tumor was lineated shortly after 4 h post-injection (p.i.) with 10.2±1.6% ID/g and 8.1±0.6% ID/g for 44Sc- and 86Y-constructs, respectively. In the case of 44Sc, the tumor uptake increased by ~6% ID/g from 4 to 18 h p.i. (16.1±1.9% ID/g), but only ~1% ID/g for [86Y][Y(pypa-phenyl-TRC105)] between 4 and 24 h p.i, and then stabilized over the following 24 h (9.1±0.9% ID/g at 48 h p.i.). Additionally, for the 44Sc-tracer, the radioactivity in blood decreased in tandem with that in the liver without significant change in the bone uptake throughout the course of study, indicating the long-term in vivo stability of the [44Sc][Sc(pypa)]- complex. Unfortunately, for the 86Y-counterpart, despite the clearance in the blood pool, the bone uptake jumped by nearly 3-fold from 30 min (2.1±0.3% ID/g) to 48 h p.i. (5.7±0.5% ID/g), which was almost double compared to that of the [86Y][Y(DTPA-TRC105)] (~3% ID/g at 48 h p.i.).35 The liver activity also increased by ~2.9% ID/g within the first 4 h. Taken together, it was concluded that [86Y][Y(pypa-phenyl-TRC105)] was not stable in vivo. The in vivo transchelation of the 86Y-construct could also explained the significantly lower tumor accumulation compared to the 44Sc-counterpart. This outcome was not expected as [86Y][Y(pypa)]- appeared to be stable in mouse serum, with high thermodynamic stability comparable to the Lu3+-H4pypa system which was stable in vivo.17 A possible explanation could be that the conjugation changed the coordination environment of the [86Y][Y(pypa)]- complex. Furthermore, since the radiopharmaceutical is usually injected in a very low concentration and is further diluted as it is distributed in the blood volume, the rate of dissociation of the complex, rather than the thermodynamic equilibrium, will dictate the in vivo stability.16,21,208 This is further enhanced when there are orders-of-magnitude more concentrated endogenous ligands (e.g. transferrin) competing with the chelator-conjugate in binding the radiometal ions.16 Therefore, the disappointing 176  pharmacokinetics of [86Y][Y(pypa-phenyl-TRC105)] was likely a result of the insufficient kinetic inertness and metabolic stability of the complex. Despite the unsatisfactory result with 86Y, that of [44Sc][Sc(pypa-phenyl-TRC105)] was deemed a great success. The tumor was visualized at the highest contrast at 18 h p.i. (Figure 6.8A). Although the studies of 44Sc-labeled TRC105 with other chelators were incompatible with direct comparison, the biodistribution pattern of [44Sc][Sc(pypa-phenyl-TRC105)] closely resembled that of [86Y][Y(DTPA-TRC105)] with major uptakes in blood, lung, liver, kidney, spleen and tumor, and no abnormal bone and liver retentions which manifest signs of in vivo stability (Figure 6.9A),35 evincing the aptness of H4pypa in scavenging 44Sc.    Figure 6.8 PET/CT MIP Images of (A) [44Sc][Sc(pypa-phenyl-TRC105)] (B) [86Y][Y(pypa-phenyl-TRC105)] at different post-injection time points.  0.5 h %ID/g 0 4 h 8 h 18 h 25  0.5 h 4 h 24 h 48 h 15  %ID/g 0 (A) (B) 177         Figure 6.9 Quantitative ROI analysis of the in vivo PET imaging data of (A) [44Sc][Sc(pypa-phenyl-TRC105)] (B) [86Y][Y(pypa-phenyl-TRC105)] at different post-injection time points.   Figure 6.10 Ex vivo biodistribution data of [44Sc][Sc(pypa-phenyl-TRC105)] (18 h p.i.) and [86Y][Y(pypa-phenyl-TRC105)] (48 h p.i.).  blood / heart bone liver Tumor0102030% ID/g 0.5 h 4 h 8 h 18 hAblood / heart bone liver Tumor0102030% ID/g 0.5 h 4 h 24 h 48 hBBloodSkinMuscleBoneHeartLungLiverKidneySpleenPancreasStomachIntestine4T1 tumorBrain051015202530% ID/g [44Sc][Sc(pypa-phenyl-TRC105)] [86Y][Y(pypa-phenyl-TRC105)]178  6.3.   Conclusions      The focus of this work was to evaluate the possibility to use H4pypa in immuno-PET imaging with scandium-44 and yttrium-86, which eventually could be useful for theranostic applications with long-lived therapeutic radionuclides such as lutetium-177. The chelator previously demonstrated favorable radiolabeling properties and complex stability with scandium-44 and lutetium-177.17,100 Due to the similar chemical properties between Lu3+ and Y3+ ions,191 H4pypa was anticipated to hold promise for the Y3+ ion as well. The [Y(pypa)]- complex presented a single symmetric species with the DFT-calculated bond lengths longer than both [Sc(pypa)]- and [Lu(pypa)]- complexes. Although the Y3+-H4pypa system was thermodynamically favorable (pY value of 22.0, more than 3 units higher than those of the DOTA- and CHX-A”-DTPA-complexes195,205), and the [86Y][Y(pypa)]- complex appeared to be stable in mouse serum as well, when [86Y][Y(pypa-phenyl-TRC105)] was injected into a cohort of 4T1-xenograft mice (n = 3), progressive bone accumulation was observed due to the undesirable release of free yttrium-86 in the biological system. On the other hand, the pharmacokinetics of [44Sc][Sc(pypa-phenyl-TRC105)] were highly favorable with long-term in vivo stability. Although the previously reported [44Sc][Sc(pypa-C7-PSMA617)] showed promising biological stability as well,100 this finding is important because, firstly, H4pypa-phenyl-NCS was a new bifunctional chelator which could lead to different metal coordination properties. In addition, antibody-conjugates possess much longer blood half-life (1-3 weeks) which prolonged the interactions between the radio-complex and the blood serum proteins, while the PSMA-conjugates tend to be excreted very quickly;118 therefore, the in vivo stability, particularly at late timepoints, would be more accurate when examined with an antibody-construct.  The long-term in vivo stability of [44Sc][Sc(pypa)]- encouraged the future 179  immuno-PET applications using 44Sc-labeled pypa-phenyl-antibody, especially in the dosimetry study for the long-lived therapeutic radionuclides such as scandium-47 and lutetium-177, which can be conveniently chelated with the same bioconjugate to attain more comparable biodistributions.  6.4.   Experimental Section 6.4.1.   Materials and Methods      All solvents and reagents were purchased from commercial suppliers (TCI America, Alfa Aesar, AK Scientific, Sigma-Aldrich, Fisher Scientific, Fluka) and were used as received. Deionized water was filtered through the PURELAB Ultra Mk2 system. 1H, 13C{1H}, 1H-13C{1H} HSQC and COSY NMR spectra were recorded at ambient temperature on Bruker AV400 instruments, as specified; the NMR spectra are expressed on the δ scale and were referenced to residual solvent peaks. Low-resolution (LR) mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray-ionization mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the Department of Chemistry, University of British Columbia. Analyses of radiolabeled compounds were performed with both thin layer chromatography (TLC) (i.e. silica aluminum-backed TLC plates and iTLC-impregnated with silica gel (iTLC-SG) strips) purchased from Agilent Technologies. The radio-TLC scanner model was the Cyclone Storage Phosphor System (Cyclone Plus) from Perkin Elmer and the HPLC system was Dionex Ultimate 3000. DIONEX Acclaim C18 5 m 120 Å column (250 mm × 4.60 mm) was used for separation of free 180  radioactivity and radio-complex. 44Sc and 86Y were provided by the UW-Madison Cyclotron Lab as 0.1 M HCl solutions.    6.4.2.   Synthesis and Characterization Dimethyl 4-hydroxypyridine-2,6-dicarboxylate (6.1)       Thionyl chloride (SOCl2) (9.50 mL, 0.130 mol, 5 equiv) was added slowly using a syringe to a stirred suspension of chelidamic acid monohydrate (5.28 g, 26.2 mmol, 1 equiv) in methanol (60 mL) in a two-neck round-bottom flask at 0˚C. The mixture was stirred at room temperature for 24 h and then refluxed for an additional 2 h. The solvent was removed under reduced pressure gently at room temperature and then deionized water was added at 0˚C. The mixture was neutralized with 1 M aqueous K2CO3 solution and the precipitate was filtered by vacuum filtration, and then washed with 50% MeOH in water (~10 mL). The white precipitate was dried under reduced pressure to give a white solid (5.54 g, >99%). 1H NMR (400 MHz, 298 K, (CD3)2SO): δ 6.74 (s, 2H), 3.72 (s, 6H). 13C NMR (100 MHz, 298 K, (CD3)2SO): δ 165.7, 149.2, 116.6, 52.7. LR-ESI-MS: calcd. for [C9H9NO5 + Na]+ 234.0; found [M + Na]+ 234.2 Dimethyl 4-(benzyloxy)pyridine-2,6-dicarboxylate (6.2)       To a round-bottom flask with a stirred solution of compound 6.1 (1.65 g, 7.82 mmol, 1 equiv) in dry acetonitrile (ACN) was added anhydrous K2CO3 (2.19 g, 15.8 mmol, 2.02 equiv) and benzyl bromide (1.02 mL, 8.60 mmol, 1.1 equiv). The reaction mixture was refluxed overnight at 60˚C. 181  K2CO3 was filtered out by vacuum filtration and then washed with DCM. The filtrate was concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to give a white powder (1.51 g, 64 %). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.90 (s, 2H), 7.44-7.38 (m, 5H), 5.23 (s, 2H), 4.01 (s, 6H). 13C NMR (100 MHz, 298 K, CDCl3): δ 150.0, 129.0, 128.9, 127.9, 115.0, 71.0, 53.4. LR-ESI-MS: calcd. for [C16H15NO5 + Na]+ 324.1; found [M + Na]+ 324.1 (4-(Benzyloxy)pyridine-2,6-diyl)dimethanol (6.3)       To a round-bottom flask with a stirred solution of compound 6.2 (8.74 g, 29.0 mmol, 1 equiv) in dry MeOH (90 mL) was added sodium borohydride (NaBH4) (3.29 g, 87.1 mmol, 3 equiv) in three portions over 30 min at 0˚C. The reaction mixture was stirred at room temperature. After 24 h, the mixture was diluted with CHCl3 (50 mL) and then quenched with saturated aqueous NaHCO3 solution (50 mL). The organic phase was separated and the bulk of MeOH in the aqueous layer was removed in vacuo to give an aqueous solution which was extracted with CHCl3 (50 mL × 4). The combined organic phases were dried over anhydrous sodium sulfate (Na2SO4), and then clarified by filtration. The filtrate was rotary-evaporated to give a white solid (5.86 g, 82%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.42-7.35 (m, 5H), 6.79 (s, 2H), 5.12 (s, 2H), 4.70 (s, 4H). 13C NMR (100 MHz, 298 K, CDCl3): δ 184.4, 166.5, 162.7, 160.6, 149.6, 135.6, 128.9, 128.6, 127.6, 117.2, 111.8, 107.7, 106.5, 106.1, 105.2, 70.2, 64.5. LR-ESI-MS: calcd. for [C14H15NO3 + Na]+ 268.1; found [M + Na]+ 268.2  182  4-(Benzyloxy)-2,6-bis(bromomethyl)pyridine (6.4)       Compound 6.3 (1.76 g, 12.6 mmol, 1 equiv) was suspended in dry ACN/CHCl3 (40 mL, 50:50 v/v) in a three-neck round-bottom flask. Phosphorus tribromide (PBr3) (3.60 mL, 37.9 mmol, 3 equiv) in CHCl3 (5 mL) was added dropwise using a dropping funnel to the stirred solution of compound 6.3 at 0˚C over 15 min. The reaction mixture was stirred at 60˚C for 18 h and then saturated aqueous Na2CO3 solution was added slowly to quench the reaction at 0˚C. The aqueous phase was extracted with CHCl3 (50 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to yield a colorless oil which later solidified to a white solid (3.28 g, 70 %). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.43 (m, 5H), 7.36 (s, 2H), 5.37 (s, 2H), 4.95 (s, 4H). 13C NMR (100 MHz, 298 K, CDCl3): δ 170.9, 154.5, 133.2, 129.5, 129.3, 128.3, 113.2, 73.0, 25.3. LR-ESI-MS: calcd. for [C14H1379Br2NO + H]+ 369.9; found [M(79Br) + H]+ 369.9 Tert-butyl 6-(((2-(tert-butoxy)-2-oxoethyl)amino)methyl)picolinate (6.5)       To a round-bottom flask with a stirred solution of tert-butyl 6-formylpicolinate17 (0.500 g, 2.40 mmol, 1 equiv) in dry MeOH (20 mL) was added tert-butyl glycinate (0.320 g, 2.40 mmol, 1 equiv). The mixture was stirred for 1 h at room temperature and then sodium cyanoborohydride (NaBH3CN) (0.310 g, 4.87 mmol, 2 equiv) was added. The reduction reaction was continued for 3 h at room temperature before quenching with saturated NaHCO3 in water (10 mL) and then extraction with DCM (20 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was concentrated in vacuo and the residue was purified through a silica column (CombiFlash Rf automated column system, 12 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were combined and rotary-evaporated 183  to give a pale yellow oil (0.550 g, 70%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.76 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 3.91 (s, 2H), 3.26 (s, 2H), 1.48 (s, 9H), 1.32 (s, 9H).13C NMR (100 MHz, 298 K, CDCl3): δ 171.1, 163.9, 159.7, 148.6, 137.2, 124.9, 123.0, 81.9, 81.0, 54.2, 51.0, 27.9. LR-ESI-MS: calcd. for [C17H26N2O4 + H]+ 323.2;  found [M + H]+ 323.1 Di-tert-butyl-6,6'-((((4-(benzyloxy)pyridine-2,6-diyl)bis(methylene))bis((2-(tert-butoxy)-2-oxoethyl)azanediyl))bis(methylene))dipicolinate (6.6)       Compound 6.4 (0.400 g, 1.30 mmol, 1 equiv), K2CO3 (595 mg, 4.31 mmol, 3.3 equiv) and KI (434 mg, 2.61 mmol, 2 equiv) were added sequentially to the stirred solution of compound 6.5 (0.837 g, 2.60 mmol, 2 equiv) in dry ACN (15 mL) in a round-bottom flask. The mixture was stirred at 30˚C for 24 h. K2CO3 was removed by centrifugation and then washed with DCM (10 mL × 3). The combined supernatants were concentrated in vacuo and then purified with a silica column (CombiFlash Rf automated column system, 12 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to give a pale-yellow oil (0.670 g, 73%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.92-7.61 (m, 6H), 7.52-7.30 (m, 5H), 7.12 (s, 2H), 5.11 (s, 2H), 4.03 (s, 4H), 3.86 (s, 4H), 3.33 (s, 4H), 1.57 (s, 18H), 1.43 (s, 18H). 13C NMR (100 MHz, 298 K, CDCl3): δ 170.5, 166.2, 164.0, 160.2, 148.6, 137.2, 136.1, 128.5, 128.1, 127.7, 125.6, 123.3, 123.0, 107.8, 81.8, 80.9, 69.7, 64.4, 59.8, 56.1, 53.4, 28.0. LR-ESI-MS: calcd. for [C48H63N5O9 + Na]+ 876.5; found [M + Na]+ 876.6  184  Di-tert-butyl-6,6'-((((4-hydroxypyridine-2,6-diyl)bis(methylene))bis((2-(tert-butoxy)-2-oxoethyl)azanediyl))bis(methylene))dipicolinate (6.7)       Compound 6.6 (0.170 g, 0.200 mmol) was dissolved in dry MeOH (20 mL) in a three-neck round-bottom flask, saturated with N2(g). Pd/C (10% w/w, 0.1 equiv) was added under a stream of N2(g). The flask was purged with N2(g), followed by H2(g) from a balloon.  The mixture was stirred vigorously at room temperature overnight under H2 atmosphere, and then Pd/C was filtered off through a pre-wet (MeOH) Celite bed, washed with MeOH (10 mL × 5). The filtrate was rotary-evaporated to a pale-yellow oil (0.150 g) and used without further purification. LR-ESI-MS: calcd. for [C41H57N5O9 + H]+ 764.4; found [M + H]+ 764.6 4-((Tert-butoxycarbonyl)amino)phenethyl 4-methylbenzenesulfonate (6.8)       N-boc-2-(4-aminophenyl)ethanol (1.97 g, 8.28 mmol, 1 equiv) was dissolved in THF (12 mL) and cooled to 0˚C with an ice-water bath. NaOH aqueous solution (6 M, 11.9 mL) was added, followed by dropwise addition of para-tosyl chloride (3.16 g, 0.0169 mol, 2 equiv) in THF (24 mL) under N2(g). After stirring at 0˚C for 1 h, the reaction mixture was warmed to room temperature and further stirred overnight. The mixture was extracted with DCM (30 mL × 3). The combined organic phases were washed with 1 M NaOH aqueous solution (40 mL × 2) and deionized water (40 mL × 2), and then dried over magnesium sulfate (MgSO4). The mixture was clarified with filtration, evaporated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to give a white solid (2.30 g, 71%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.68 (d, J = 8.3 Hz, 2H), 7.25 (dd, J = 13.4, 8.7 Hz, 4H), 7.01 (d, J = 8.4 Hz, 2H), 6.45 (s, 1H), 4.16 (t, J = 7.0 Hz, 2H), 2.89 (t, J = 7.0 Hz, 2H), 2.43 (s, 3H), 1.51 (s, 185  9H).13C NMR (100 MHz, 298 K, CDCl3): δ 152.8, 144.8, 137.3, 133.1, 130.7, 129.9, 129.6, 128.0, 118.8, 80.7, 70.8, 34.8, 28.5, 21.7. LR-ESI-MS: calcd. for [C20H25NO5S + H]+ 392.1; found [M + H]+ 392.1 Tetramethyl 6,6',6'',6'''-((((4-(4-((tert-butoxycarbonyl)amino)phenethoxy)pyridine-2,6-diyl)bis(methylene))bis(azanetriyl))tetrakis(methylene))tetrapicolinate (6.9)       To a round-bottom flask with a stirred solution of compound 6.7 (72.1 mg, 0.0944 mmol, 1 equiv) in dry THF (1 mL) was added anhydrous K2CO3 (52.1 mg, 0.378 mmol, 4 equiv). The mixture was stirred vigorously for 1 h at 25˚C before the addition of compound 6.8 (42.1 mg, 0.108 mmol, 1.14 equiv). The mixture was stirred for 48 h at 25˚C when compound 6.7 was completely consumed. The solvent was evaporated in vacuo, and the residue was resuspended in DCM (6 mL). K2CO3 was removed by centrifugation and washed with DCM twice (~5 mL each). The combined organic phases were washed with saturated NaHCO3 in water (10 mL × 2), water (10 mL × 2) and brine (10 mL × 2), and then dried over anhydrous MgSO4. The drying agent was filtered off and the filtrate was concentrated in vacuo to a yellow oil. The product was confirmed by MS and then used without further purification in the next step. LR-ESI-MS: calcd. for [C54H74N6O11 + Na]+ 1005.5; found [M + K]+ 1005.9 6,6',6'',6'''-((((4-(4-aminophenethoxy)pyridine-2,6-diyl)bis(methylene))bis(azane-triyl))tetra-kis(methylene))tetrapicolinic acid (6.10)       Compound 6.9 (166 mg, 0.169 mmol, 1 equiv) was dissolved in TFA/DCM (1:1) (4 mL). The mixture was stirred overnight vigorously at room temperature, and then concentrated to dryness in vacuo. The crude product was re-dissolved in deionized water, and then purified through reverse 186  phase HPLC (A: ACN/0.1% TFA, B: H2O/0.1% TFA, 5-60% A over 40 min, 10 mL/min, tR = 17.8 min). The combined product fractions were dried in vacuo to give a yellow oil (55.7 mg, 50%).1H NMR (400 MHz, 298 K, D2O): δ 8.23 (t, J = 7.9 Hz, 2H), 7.97 (d, J = 7.8 Hz, 2H), 7.91 (d, J = 7.7 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 6.91 (s, 2H), 4.49 (s, 4H), 4.34 (t, J = 5.8 Hz, 2H), 4.28 (s, 4H), 3.82 (s, 4H), 3.17 (t, J = 5.8 Hz, 2H). 13C NMR (100 MHz, 298 K, D2O): δ 13C NMR (101 MHz, D2O) δ 174.1, 169.6, 164.9, 154.1, 152.6, 146.5, 144.3, 139.5, 131.0, 128.7, 128.4, 125.2, 123.3, 110.7, 70.6, 57.8, 57.2, 33.9.. LR-ESI-MS: calcd. for [C33H34N6O9 + H]+ 659.7; found [M + H]+ 659.4.  H4pypa-phenyl-NCS (6.11)       Compound 6.10 (68.0 mg, 0.103 mmol, 1 equiv) was dissolved in 1 M HCl/ glacial acetic acid (2 mL, 4:1 v/v) in a round-bottom flask. Then, thiophosgene (CSCl2) (119 L, 1.55 mmol, 15 equiv) in CHCl3 (2 mL) was added dropwise using a Pasteur pipette to the stirred mixture of the starting material. The resulting mixture was stirred vigorously at room temperature overnight. After the reaction completed, CHCl3 was removed with a Pasteur pipette. The aqueous phase was further washed with CHCl3 (1 mL) which was removed by a Pasteur pipette. The process was repeated 4 times and then the residue was injected onto reverse phase HPLC (A: ACN/0.1% TFA, B: H2O/0.1% TFA, 5-60% A over 40 min, 10 mL/min, tR = 35 min). The product fractions were combined and lyophilized to give a fluffy white solid (21.7 mg, 30%). 1H NMR (400 MHz, 298 K, CD3CN:D2O 1:1):  δ 8.62 – 8.50 (m, 4H), 8.27 (d, J = 7.3 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.50 (s, 2H), 4.90 (s, 6H), 4.31 (s, 4H), 3.65 (t, J = 6.3 Hz, 2H).  13C NMR (100 MHz, 298 K, CD3CN:D2O 1:1): δ 173.5, 165.9, 155.0, 142.2, 131.2, 131.2, 128.7, 126.5, 187  126.5, 125.4, 111.2, 58.7, 56.6, 56.6, 56.2, 56.2. HR-ESI-MS: calcd. for [C34H32N6O9S+H]+ 701.2030; found [M+H]+ 701.2028  Na[natY(pypa)]       H4pypa · 2 TFA· 2 H2O (21.8 mg, 4.16 × 10-5 mol, 1 equiv) was dissolved in D2O (1 mL) in a scintillation vial and 0.1 M NaOD solution (aq) was added to adjust the pH to 7. Y(ClO4)3 · 6 H2O (45.4 L, 4.58 × 10-5 mol, 1.1 equiv) was added. The mixture was stirred at room temperature for 1 h and the complexation was confirmed by LR-ESI-MS. 1H NMR (400 MHz, 298 K, D2O): δ 8.23 (t, J = 7.8 Hz, 2H), 8.08 (d, J = 7.7 Hz, 2H), 7.87 (t, J = 7.8 Hz, 1H), 7.80 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 7.8 Hz, 2H), 4.71 – 4.58 (m, 4H), 4.40 (d, J = 14.7 Hz, 2H), 4.05 (d, J = 14.8 Hz, 2H), 3.97 (m, J = 17.0 Hz, 2H), 3.47 (d, J = 17.0 Hz, 2H).. 13C NMR (100 MHz, 298 K, D2O): δ 178.7, 172.5, 156.7, 152.9, 151.1, 141.8, 140.2, 125.6, 124.1, 123.0, 64.6, 63.2. HR-ESI-MS: calcd. for [C25H21N5O889Y + 2Na]+ 654.0244; found [M + 2Na]+ 654.0251.  6.4.3.   DFT Calculations      All DFT simulations were performed as implemented in the Gaussian 09 revision D.01 suite of ab initio quantum chemistry programs (Gaussian Inc., Wallingford, CT). B3LYP functional,105,106 and the effective core potentials LanL2DZ basis sets for scandium155,157  and yttrium105,107,108  were applied to optimize the structural geometry in the presence of water solvent (IEF PCM as implemented in G09) without the use of symmetry constraints. Normal self-consistent field (SCF) and geometry convergence criteria were conducted for all the calculations. The calculated structures were visualized using Mercury 4.1. 188   6.4.4.   Solution Thermodynamics       All potentiometric titrations were carried out with a Metrohm Titrando 809 and a Metrohm Dosino 800 with a Ross combined electrode. A 20 mL and 25˚C thermostated glass cell with an inlet-outlet tube for nitrogen gas (purified through a 10% NaOH solution to exclude any CO2 prior to and during the course of the titration) was used as a titration cell. The electrode was calibrated daily in hydrogen ion concentration by direct titration of HCl (aq) with freshly prepared NaOH aqueous solution and the results were analyzed with the Gran procedure134 in order to obtain the standard potential Eº and the ionic product of water pKw at 25˚C and 0.16 M NaCl (aq) used as a supporting electrolyte. Solutions were titrated with carbonate-free NaOH (aq, 0.16 M) that was standardized against freshly recrystallized potassium hydrogen phthalate aqueous solution. In the study of complex formation equilibria, the determination of the stability constants of the [Y(Hpypa)] species was carried out by two different methods. The first method used UV-Vis spectrophotometric measurements on a set of solutions containing 1:1 metal to ligand molar ratio ([H4pypa] = [Y]3+ = 1.33  10-4 M) and different amounts of standardized HCl (aq) and NaCl (aq) to set the ionic strength constant at 0.16 M when possible. The equilibrium H+ concentration in this UV in-batch titration procedure at low pH solutions (2 ≥ pH ≤ 0) was calculated from solution stoichiometry, not measured with a glass electrode. For the solutions of high acidity, the correct acidity scale H0 was used.135 The spectral range was 200-400 nm at 25˚C and 1 cm path length. The molar absorptivities of all the protonated species of H4pypa calculated with HypSpec2014124 from the protonation constant experiments17 were included in the calculations. The second method used competition pH-potentiometric titrations with EDTA as a ligand competitor and the 189  composition of the solutions was [Y]3+ = Y3+ ~ 1.51  10-3 M, [H4pypa] ~ 6.88  10-4 M and [EDTA] ~ 1.51  10-3 M at 25˚C and I = 0.16 M NaCl (aq). The stability constants for the complexes formed by EDTA and Y3+ were taken from the literature.136 Direct pH-potentiometric titrations of the Y3+-H4pypa systems were also carried out. The Y3+ metal ion solution was prepared by adding the atomic absorption (AA) standard solution to a H4pypa solution of known concentration in the 1:1 metal to ligand molar ratio. Ligand and metal concentrations were in the range of 8.24  10-4 M. The exact amount of acid present in the AA standard solution was determined by Gran’s method134 titrating equimolar solutions of Y(III) and Na2H2-EDTA. Each titration consisted of 100-150 equilibrium points in the pH range 1.6-11.5, equilibration time for titrations was up to 5 min for metal complex titrations. Three replicates of each titration were performed. Relying on the stability constants for the species [Y(Hpypa)] obtained by the two different methods, the fitting of the direct potentiometric titrations was possible and yielding the stability constants in Table 6.3. All the potentiometric measurements were processed using the Hyperquad2013125 software while the obtained spectrophotometric data were processed with the HypSpec2014124 program. Proton dissociation constants corresponding to hydrolysis of Y(III) aqueous ions included in the calculations were taken from Baes and Mesmer.137 The overall equilibrium (formation) constants log β referred to the overall equilibria: pM + qH + rL ⇆ MpHqLr (the charges are omitted), where p might also be 0 in the case of protonation equilibria and q can be negative for hydroxide species. Stepwise equilibrium constants log K correspond to the difference in log units between the overall constants of sequentially protonated (or hydroxide) species. The parameter used to calculate the metal scavenging ability of a ligand towards a metal ion, pM, is defined as –log [Mn+]free at [ligand] = 10 mM and [Mn+] = 1 M at pH = 7.4.128 190   6.4.5.   Production and Radiochemical Separation of Yttrium-86      Yttrium-86 (t1/2 = 14.7 h, 34% β+, Eβ+max = 3.2 MeV) was produced in a 16 MeV GE PETtrace biomedical cyclotron using enriched [86Sr][SrCO3] targets of pressed powder. Following irradiation, the radiochemical isolation of the yttrium-86 was performed by single column extraction chromatography, as previously described.209 Briefly, the target material was dissolved in 5 mL of 9 M HCl (aq), and loaded onto a column filled with a resin functionalized with N,N,N′,N′-tetrakis-2-ethylhexyldiglycolamide (branched DGA, Eichrom). Subsequent washes were carried out using 9 M HCl (aq, 15 mL) and 0.5 M HNO3 (aq, 15 mL) to remove bulk strontium and other trace metal contaminants. No-carrier-added yttrium-86 was eluted with 0.1 M HCl (aq, 4 × 0.3 mL). The activity was assayed in a Capintec CRC-15R dose calibrator (setting #850/2).  6.4.6.   Production and Radiochemical Separation of Scandium-44      Scandium-44 was cyclotron-produced using natCa[p,n]4xSc nuclear reactions on pressed targets of metallic calcium (300-350 mg). Target preparation was performed in air, and rapidly mounted in the cyclotron to reduce calcium oxidation. Irradiations were performed at 20 µA for 1 h with direct water cooling, and a 12.7 µm Nb foil was used to degrade the beam energy from the nominal 16 MeV to 14.1 MeV. Under these conditions, a scandium-44 production yield of 0.4 mCi/Ah was obtained through the reaction natCa(p,n)44Sc. Isolation of the produced scandium-44 was carried out by single column extraction chromatography using a N,N,N’,N’-tetrakis-2-ethylhexyldiglycolamide functionalized resin (DGA-branched, Eichrom).155 The target was 191  dissolved in 9 M HCl (aq, 10 mL) and passed through a 1 mL fritted solid phase extraction (SPE) tube filled with the DGA resin (~120 mg), loading the scandium-44 and eluting bulk Ca2+. Remaining Ca2+ was removed by rinsing the column with 4 M HCl (aq, 20 mL). Next, a 12 mL wash with 1 M HNO3 (aq) was performed to elute possible trace metal contaminants such as Zn, Fe and Cu. Finally, the scandium-44 was eluted in a small volume using 0.1 M HCl (aq, 4 x 500 L fractions). The radionuclidic and chemical purity were confirmed by high purity germanium (HPGe) gamma spectrometry and microwave plasma atomic emission spectroscopy (MP-AES), respectively.  6.4.7.   Radiolabeling Studies      An aliquot of ligand solution (H4pypa) (10 L) was mixed with HEPES solution (0.5 M, pH = 7, 87 L), followed by [86Y][YCl3] in HCl (aq) (4.14 MBq, 3 L). The reactions were incubated at ambient temperature over the desired time period. The mixture (3 L) was spotted on a silica aluminum-backed TLC plate, and then developed in DTPA buffer (0.1 M, pH = 5.5). The TLC plate was read by a TLC reader, showing the free metal ion migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate RCY%.  6.4.8.   In Vitro Mouse Serum Challenge      To the radiolabeled sample (100 L, 1.4 MBq), an equal volume (100 L) of the mouse serum was added. The mixture was incubated at 37˚C and 5 L aliquots was collected at desired time points (0.5 h, 4 h, 24 h, 48 h). The aliquot was spotted onto a silica aluminum-backed TLC plate 192  next to the control spot (yttrium-86 in buffer with serum), and then developed in DTPA buffer (0.1 M, pH = 5.5). The TLC plate was read by the TLC reader. The free metal migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate % intact.  6.4.9.   Bioconjugation of H4pypa-phenyl-TRC105 and Radiolabeling with Yttrium-86 and Scandium-44      TRC105 (TRACON Pharmaceuticals) was prepared for radiolabeling through conjugation with H4pypa-phenyl-NCS. The pH of the TRC105 solution was adjusted to ~9.0 with 0.1 M sodium carbonate buffer and H4pypa-phenyl-NCS chelator was added in a 10:1 (chelator:antibody) molar ratio. After reacting for 2 h at room temperature, H4pypa-phenyl-TRC105 was purified by size exclusion chromatography (PD-10, GE-Healthcare) using phosphate-buffered saline (PBS) as the mobile phase.      For animal studies, 2.7 μg of H4pypa-phenyl-TRC105 per MBq of yttrium-86 (or scandium-44) was added to the activity solution in sodium acetate buffer (0.1 M, pH = 5.5) and incubated at 37˚C for 30 min. After labeling, [AE][E(pypa-phenyl-TRC105)] (AE = yttrium-86 and scandium-44) were purified by size exclusion chromatography using PBS as the mobile phase. Radiochemical yields (RCY) were measured via iTLC-SG plates developed in DTPA buffer (0.1 M, pH = 5.5) for the 86Y-conjugate and sodium citrate buffer (0.4 M, pH = 4.5) for the 44Sc-conjugate.  The radiochemical yields were consistently above 90% for both radiotracers.  193  6.4.10.   PET/CT Imaging and Biodistribution Studies       All animal studies were conducted under a protocol approved by the University of Wisconsin Institutional Animal Care and Use Committee. 4T1 cells were cultured in RPMI 1640 growth medium (Invitrogen) with a 10% fetal bovine serum (FBS) supplement. During culturing, cells were incubated at 37˚C with 5% CO2. Tumors were grafted in four-week-old female Balb/c mice by subcutaneous injection of 2-3 × 106 cells, suspended in 100 μL of 1:1 mixture of RPMI 1640 and Matrigel (BD Biosciences).       For imaging studies, mice bearing 4T1 murine breast cancer tumors were injected via the tail vein with 11-18 MBq of either [86Y][Y(pypa-phenyl-TRC105)]  or [44Sc][Sc(pypa-phenyl-TRC105)]. PET scans of 20 million coincidence events per mouse were obtained using an Inveon PET/CT scanner (Siemens) at different post-injection timepoints. Measurements of regions-of-interest (ROI) in PET images were performed using the Inveon Research Workspace (Siemens).      Following the final imaging timepoint, mice were euthanized via CO2 asphyxiation, and major organs were excised, wet-weighed, and radioactive content was measured using a gamma counter (PerkinElmer). Results of PET ROI analysis and biodistribution studies are presented as % ID/g.       194  Chapter 7. 225Ac-H4py4pa for Targeted-Alpha-Therapy  This chapter contains an adaptation of an accepted manuscript: Li, L.; Rousseau, J.; de G. Jaraquemada-Peláez, M.; Wang, X.; Robertson, A.; Radchenko, V.; Schaffer, P.; Lin, K.-S.; Bénard, F.; Orvig, C. 225Ac-H4py4pa for Targeted-Alpha-Therapy. Bioconjugate Chem. In press (03/2020)  7.1.   Introduction      Targeted radionuclide therapy (TRT) is a rapidly developing therapeutic modality for cancer, effected by -particles, -particles and Auger electrons. In particular, targeted alpha therapy (TAT) is a highly potent, localized radiation treatment. By virtue of their high linear energy transfer (LET : ~80 keV/m), a measure of energy deposition from radiation ionization per unit length of travel in tissue, -particles have a short effective range of approximately less than 10 cell diameters (40-100 m) compared to several hundred for -particles.1,5 This short distance makes -emitters more suitable for the treatment of isolated cells or micrometastases.210 With greater energy deposition, -emitters are prone to generate DNA double-strand breaks which are more difficult to repair, as opposed to single-strand breaks induced by β-emitters. In addition, the radiobiology of α-particles depends on neither the dose rate nor the oxygenation of the irradiated tissue.211 The associated radiation damage seems to be impervious to resistance mechanisms.212 As a consequence, TAT is considered more efficient and less toxic than targeted β-therapy.        Chan et al. reported high therapeutic effects using [213Bi][Bi(DOTATATE)] which was 6-time more effective in cell killing than the 177Lu-counterpart.213,214 Furthermore, Friesen et al. demonstrated the potency of the bismuth-213 (, 45.6 min)-radiolabeled antibody ([213Bi][Bi-anti-195  CD45]) to overcome the chemoresistance and radioresistance of leukemia cells by inducing irreparable DNA damages and apoptosis.215 However, the short half-life not only poses challenges for radiolabeling and drug administration, but also significantly limits the timeframe for circulation in target accumulation.216 To circumvent such shortcomings, an alternative is to adopt the parent, actinium-225 (225Ac, t1/2 ~ 9.92 d), where the long half-life matches the prolonged circulation of the antibody (biological half-life of 1-3 weeks), rendering it more suitable for radioimmunotherapy (RIT).5 Moreover, the generation of four net alpha particles through its progeny makes it extremely tumoricidal when delivered to, and ideally internalized into the cancer cells where the decay products are confined.138,217,218  The enhanced efficacy by the favorable decay characteristics were further evinced in a comparative in vitro cytotoxicity study conducted by McDevitt et al. in which they proved that the lethal dose (LD50) of the 225Ac-labeled antibody-construct was two- to four-orders-of-magnitude lower than that of 213Bi.217 Besides, a labeled anti-CD33 antibody showed anti-leukemic responses with either 225Ac or 213Bi, while 225Ac offers advantages to overcome many logistical difficulties due to the much shorter half-life of 213Bi and the need for preparing the 225Ac/213Bi generator.219 Recently, impressive responses in mCRPC (metastatic castration-resistant prostate cancer) patient were obtained using [225Ac][Ac(PSMA617)], even if they were refractory to 177Lu-radioligand therapy.31,220       Despite the tremendous potential of 225Ac in RIT, its widespread application is largely deterred by its limited availability and the lack of a stable chelating agent that can radiolabel efficiently under mild conditions (RT, pH ~ 7) to accommodate the pH- and temperature-sensitive antibody-constructs.16,5 Efforts are ongoing to increase the worldwide production of this attractive radionuclide, which is currently limited to approximately 63 GBq/year.221 For chelation, 196  macrocyclic DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Figure 7.1) remains the state-of-the-art chelator, primarily due to the high stability of the resulting complex and its high affinity for many diagnostic radionuclides. 30,77,180,207 These are the major advantages of DOTA over most acylic chelators (e.g. citrate, ethylenediaminetetraacetic acid (EDTA) and cyclohexane-1,2-diamine-pentaacetic acid (CHX-A”-DTPA), Figure 7.1) which were shown to cause unspecific accumulation of radioactivity in both liver and bone due to the release of 225Ac in vivo that could lead to non-targeted toxicity.222,223,224 However, the inertness of the [225Ac][Ac(DOTA)]- complex comes at the expense of slow complexation kinetics, particularly at ambient temperature, leading to low radiochemical yields and posing challenges in antibody-radiolabeling.225,226,227 That and an intrinsic preference for the smaller metal ions,228 make DOTA a notably imperfect “gold-standard chelator” for actinium. In light of this, efforts have been dedicated to exploring the alternatives. Deal et al. have reported improved chelation using HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid, Figure 7.1), another macrocycle demonstrating improved 225Ac-coordination when compared to DOTA;222 but unfortunately, complex instability precluded the future use of this chelator.229,93 Another 18-membered macrocycle, H2bp18c6 (N,N′-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6, Figure 7.1), was reported by Roca-Sabio et al. showing unprecedented preference towards larger lanthanides,230 and the selectivity was further elaborated with other metal ions by the same group.231,232 The application of the same chelator with 225Ac in nuclear medicine was first described by Thiele et al. who renamed as H2macropa.28      Apart from mild radiolabeling conditions and high complex stability, a practical and useful chelator for radiopharmaceutical applications should be synthetically friendly and functionally 197  versatile as the chemical properties of the spacer (i.e. hydrophilicity, hydrophobicity, charges) often exert dramatic influences on the overall pharmacokinetics.95,86 For example, a hydrophobic linker can increase the biological half-life of the radiotracer.95 With that in mind, we designed a non-macrocyclic undecadentate chelator with a partially pre-organized binding cavity surrounded by four picolinate arms and a pyridyl cap, H4py4pa (Figure 7.1), for 225Ac-chelation. The central pyridyl moiety was adopted to allow for facile bifunctionalization by incorporating a free para-hydroxyl moiety that could be alkylated with a variety of linkers. An N-isothiocyanate (NCS)-activated H4py4pa (i.e. H4py4pa-phenyl-NCS, Figure 7.1) was used for antibody-coupling. With no stable actinium isotope, we adopted the La3+ ion as a non-radioactive surrogate of actinium-225 to permit a range of fundamental chemical studies with H4py4pa and to provide vital insights into its coordination chemistry with large trivalent metal ions. The selection was rationalized by the size similarity (Ac3+ = 1.12 Å, La3+ = 1.03 Å, 6-coordinated),123 despite the small difference in the absolute chemical hardness (Ac3+ = 14.4 eV, La3+ = 15.4 eV).233 In this work, we detail the syntheses and characterizations of both chelators, as well as the [La(py4pa)]- complex. The geometry of the La3+-complex was further calculated by density-functional theory (DFT), while its thermodynamic stability was evaluated with a series of spectrophotometric and potentiometric titrations. After successful preliminary studies with the La3+ ion, the investigation proceeded with the radiochemistry of [225Ac][Ac(py4pa)]-. Finally, we employed the FDA-approved monoclonal antibody, Trastuzumab, known to target the human epidermal growth factor receptor 2 (HER2), for validating the in vitro and in vivo stability of the complex in an ovarian-cancer-xenografts murine model as compared to the more traditional [225Ac][Ac(DOTA-benzyl-Trastuzumab)].  198   Figure 7.1 Chemical structures of selected chelators.  7.2.   Results and Discussion 7.2.1.   Synthesis and Characterization      The syntheses of H4py4pa and H4py4pa-phenyl-NCS were outlined in Scheme 7.1-7.3. H4py4pa is structurally related to H4pypa, a nonadentate chelator previously reported for 111In and 177Lu.17 Both chelators have four pendent arms connected by a pyridyl bridge for chelation, but the tetra-picolinate H4py4pa possesses two additional N-donor atoms to scavenge larger lanthanides and actinides. The structural similarity suggested a parallel synthetic strategy to be adopted, wherein the dibromo-pyridyl backbone (7.6) and the dipicolinate arm (7.4) were synthesized independently and then assembled in one convergent step (Scheme 7.1). Compound 199  7.6 was prepared following a similar method reported for H4pypa,17 while different synthetic strategies were adopted for the synthesis of the dipicolinate arm (7.4), which consists of two picolinate moieties tethered to a secondary amine. Initial attempts with one-pot Schiff-base synthesis and reduction using the aldehyde and amine derivatives of the picolinate arms (7.9 and 7.10) failed due to the unsuccessful reduction of the stable Schiff base intermediate (7.11), even upon reflux, using either excess sodium cyanoborohydride (NaBH3CN) in methanol (MeOH) or sodium triacetoxyborohydride (NaBH(OAc)3) in 1,2-dichloroethane (DCE) (Scheme 7.2). Replacing the picolinate amine (7.10) with a benzyl amine did not help. Attempts to protect the picolinate amine (7.10) with 2-nitrobenzenesulfonyl chloride failed in purifying the desired product (7.13). Finally, the dipicolinate arm (7.4) was successfully synthesized in excellent yield by full alkylation of the benzyl amine with the bromo-picolinate 2,6-substitute (7.2). Compound 7.2 was derived from the commercially available 2,6-picolinate dimethyl ester by mono-reduction with sodium borohydride (NaBH4, 1.5 equiv) at room temperature for 3-4 h, monitored with silica aluminum-backed TLC plate (5% MeOH in dichloromethane, DCM) (7.1, 60%), followed by bromination with phosphorus tribromide (PBr3) to give compound 7.2 (80%). Two equivalents of 7.2 were then reacted with benzyl amine in the presence of diisopropylethylamine (DIPEA) at room temperature overnight to give the benzyl-protected dipicolinate compound (7.3) in high yield (91%). The benzyl group was subsequently removed by palladium/carbon (Pd/C, 10% w/w)-catalyzed hydrogenation in glacial acetic acid for 3-4 h to yield compound 7.4 (83%). Two equiv of compound 7.4 were coupled to the dibromo-pyridyl backbone (7.6) via SN2 nucleophilic substitution in the presence of DIPEA and potassium iodide (KI) at room temperature overnight to give the methyl-protected py4pa (70%) which was eventually hydrolyzed with lithium hydroxide (LiOH, 10 equiv) in tetrahydrofuran (THF)/ water (2:1) mixture to yield H4py4pa (7.8,  200   Scheme 7.1 Reagents and conditions: i) NaBH4, dry DCM/dry MeOH, 0 oC-ambient temp, 4 h, 60%; ii) PBr3, dry ACN, 0 oC- ambient temp, 6 h, 80%; iii) DIPEA, dry ACN, ambient temp, 24 h, 91%; iv) Pd/C, H2(g), glacial AcOH, ambient temp, 3-4 h, 83%; v) NaBH4, dry MeOH, 0 oC-ambient temp, 12 h, 92%; vi) PBr3, dry CHCl3/ACN, 0-60 oC, 18 h, 87%; vii) DIPEA, KI, dry ACN, ambient temp, 24 h, 70%; viii) LiOH, H2O/THF, ambient temp, 24 h, 50%  50%) after purification with the reverse-phase high-performance liquid chromatography (HPLC, 5-40% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) in water over 30 min, 10 mL/min, tR = 22.1 min). Conveniently, both non-bifunctional and bifunctional H4py4pa share the same pendent arm (7.4), while the bifunctional pyridyl backbone (7.17) was reproduced with the protocol reported for the bifunctional H4pypa.17 Compound 7.17 was coupled with 2 equiv of 201   Scheme 7.2 Unsuccessful dipicolinate arm (7.4) synthetic routes.  dipicolinate arms (7.4) to give Bn-O-Me4py4pa (7.18, 71%) which was subjected to Pd/C (10% w/w)-catalyzed O-debenzylation to yield the bifunctional py4pa precursor (7.19, 78%). To the free para-hydroxyl group, the tosylated boc-protected aniline (7.20, 71%) derived from the hydroxyl precursor was incorporated as a linker (7.21). The synthesis was completed with deprotection and activation. First, the methyl esters in compound 7.21 were hydrolyzed with LiOH (10 equiv) in THF/H2O (2:1) solution overnight at room temperature. After that, the mixture was dried with rotary-evaporator, and then the crude mixture was acidified and stirred in TFA/DCM (1:1) for an additional 24 h to eliminate the boc-group. The resulting mixture was purified with reverse-phase HPLC (5-60% ACN/0.1%TFA in H2O over 40 min, 10 mL/min, tR = 20.7 min) to give compound 7.22 (50%). Lastly, the primary amine was activated with thiophosgene (CSCl2) in a vigorously stirred mixture of hydrochloric acid (1 M, aq)/ glacial acetic acid/ chloroform overnight at room temperature. The final product (7.23) was isolated with reverse-phase HPLC (20-70% ACN/0.1%TFA in H2O over 30 min, 10 mL/min, tR = 22.3 min) and then lypophilized to a white fluffy solid in 30% yield. 202   Scheme 7.3 Reagents and conditions: i) SOCl2, MeOH, ambient temp-65 oC, 26 h, >99%; ii) BzBr, K2CO3, dry ACN, 60 oC, 24 h, 64%; iii) NaBH4, dry MeOH, ambient temp, 24 h, 82%; iv) PBr3, dry ACN/CHCl3, 0-60 oC, 70%; v) DIPEA, KI, dry ACN, ambient temp, 24 h, 71%; vi) Pd/C, H2(g), dry MeOH, ambient temp, 24 h, 78%; vii) TsCl, 6 M NaOH, THF, 0 oC-ambient temp, 24 h, 71%; viii) K2CO3, dry ACN, ambient temp, 48 h; ix) 1. LiOH, THF/D.I. H2O, ambient temp, 24 h 2. TFA/DCM, ambient temp, 24 h, 50 %; x) CSCl2, 1 M HCl/glacial AcOH/CHCl3, ambient temp, 24 h, 30%  203  7.2.2.   Metal Complexation and Characterizations      To ascertain that the binding cavity of H4py4pa could effectively coordinate large metal ions (e.g. Ac3+), we conducted a complexation study with non-radioactive La(III) which is the largest lanthanide that enables a series of solution studies, including different NMR characterization techniques of the py4pa complex. Although the result cannot be directly translated to the corresponding 225Ac-complex, it unequivocally demonstrated the capability of H4py4pa to accommodate large metal ions. The complexation was performed at ambient temperature and characterized with 1H, 13C{1H}, COSY and HSQC NMR spectroscopies (Figures 7.1-7.5). Based on the 1H NMR spectrum (Figure 7.1), the La-coordination with H4py4pa was confirmed by the sharp and well-defined diastereotopic splitting of the methylene-H atoms. Upon complexation, the four chemically equivalent picolinate arms in the free chelator were separated in two chemically distinct environments.  The 1H NMR results suggested a two-fold rotational symmetry axis about the central pyridine ring of the complex, supported by three pairs of disastereotopic methylene-H atoms (δ (ppm) = 3.60, overlapping; 3.82 and 4.29, J = 14.2 Hz; 4.00 and 5.62, J = 14.5 Hz) (Figure 7.4). Each pair of diastereotropic methylene groups were associated with the chemically identical carbons (13C at 62.5 ppm coupled with H at 3.82 and 4.29 ppm; 63.1 ppm with H at 3.60 ppm; 65.1 ppm with H at 4.00 and 5.62 ppm) (Figure 7.5). Further evidences of the symmetry could be seen in the aromatic region which showed three triplets corresponding to the para-Hpyr in the central pyridine (δ 7.59 ppm, 1H), as well as those in each pair of the picolinate arms (δ (ppm) = 7.44 (2H) and 7.92 (2H)) (Figure 7.2). The neighboring meta- and ortho-Hpyr of each para-Hpyr were identified via COSY (δ (ppm) = 7.44 (t) with 6.76 (d) and 7.52 (d); 7.59 (t) with 7.12 (d); 7.92 (t) with 7.64 (d) and 7.68 (d)) (Figure 7.4). Taken together, the La-complexation with 204  H4py4pa was confirmed and the results suggested that the binding cavity of H4py4pa potentially can fit the metal ions of similar size.  Figure 7.2 1H NMR spectra of H4py4pa (bottom) and [La(py4pa)]- (top) (400 MHz, 298 K, D2O).   Figure 7.3 Na[La(py4pa)] 13C NMR spectrum (100 MHz, 298 K, D2O).  205   Figure 7.4 Na[La(py4pa)] COSY NMR spectrum (400 MHz, 298 K, D2O).   Figure 7.5 Na[La(py4pa)] 1H-13C HSQC NMR spectrum (400/100 MHz, 298 K, D2O).  206  7.2.3.   DFT Calculations      DFT calculations were carried out to study the geometry of the anion [La(py4pa)]− in solution (Figure 7.6) and the calculated bond lengths were listed in Table 7.1. It is clearly seen from the predicted structure that the complex was 11-coordinated wherein the La3+ ion occupied the binding cavity of H4py4pa, capped by the central pyridyl moiety. Consistent with the 1H NMR result, the four chemically equivalent picolinate arms in H4py4pa divided into two chemically inequivalent pairs upon complexation. Within each pair of the picolinate, the metal-donor bond lengths were very similar (La-N2 vs. La-N7, La-O1 vs. La-O8, La-N5 vs. La-N6, La-O4 vs. La-O5), so as the bond lengths between the La3+ ion and two tertiary amines on the backbone (N3 and N4), suggesting a “pseudo-symmetry” in the complex .  However, between two pairs of picolinate, the bond lengths were quite different with the bottom pair (La with N5, N6, O4 and O5) closer to the La3+ ion relative to the upper pair (La with N2, N7, O1 and O8) and two tertiary amines. The variations in the bond lengths between two pairs of picolinate arms led to two chemically distinct picolinate environments. Overall, the calculated structure provides a vital insight into the coordination environment of the [La(py4pa)]− complex reflecting a 2-fold symmetry about the central pyridine, which was also seen in the 1H NMR spectroscopic results. 207    Side view Bottom view Figure 7.6  DFT calculated structure of [La(py4pa)]- anion.  Table 7.1 Bond lengths in [La(py4pa)]- calculated by DFT. Bond Length (Å) N1-La 2.7175 N2-La 3.1412 O1-La 2.6605 N3-La 3.0519 N4-La 3.0926 O4-La 2.5479 N5-La 2.7592 N6-La 2.7735 O5-La 2.5718 O8-La 2.6626 N7-La 3.2408 N1 N3 N2 N6 N4 N7 N5 O1 O5 O8 O4 208  7.2.4.   Solution Thermodynamics      The basicity of different ionizable and non-ionizable protons governs the extent of the competition between the metal ion and the protons for the binding sites of the chelator during metal complexation. Therefore, knowing protonation constants is essential in evaluating a chelator for metal complexation. H4py4pa possesses in total eleven protonation sites, and in this work, we determined the acidity constants for the first seven protonation equilibria using combined potentiometric-spectrophotometric titrations to follow pH-dependent spectral changes in the absorption band of the picolinate chromophores. Figures 7.7A-7.7C shows the pH-dependent absorption spectra for the protonation equilibria mentioned above, while Table 7.2 presents the protonation constants calculated from the experimental data using the HypSpec2014124 and Hyperquad2013125 programs. Figure 7.8 presents one of the titration curves of an acidified solution of H4py4pa·4TFA·H2O and it shows that 8 equivalents of base (NaOH) were consumed in the titration. The speciation plots of different species of H4py4pa in Figure 7.7D were calculated from the protonation constants in Table 7.2 with the Hyss software.126      The pKa values for the four most acidic protons in H4py4pa were not determined due to the extremely low pKa and they could be attributed to the deprotonation of the four protonated pyridinium nitrogen atoms in the picolinate moieties. Following that, H7L3+, H6L3+, H5L3+ and H4L3+ which deprotonates with pK5 = 2.31 (4), pK6 = 2.55 (4), pK7 = 2.71 (3) and pK8 = 3.58 (2), respectively, could be assigned to the deprotonation of the carboxylic acid groups in the picolinate arms. The two most basic protons, H2L2- and HL3- (pK10 = 6.07 (1) and pK11 = 6.96 (1)), could be reasonably allocated to the protonated tertiary amines on the backbone. Not surprisingly, the additional picolinate moieties caused a decrease in basicity of these two protonated amines in 209  H4py4pa compared to H4pypa (pK8 = 6.78 (1) and pK9 = 7.78 (1)).17 Finally, the protonated pyridinium nitrogen in the center deprotonated at pK9 = 4.06 (2).    Figure 7.7 (A, B and C) Representative spectra of the combined UV-potentiometric titration of H4py4pa at [L] = 4.05  10-4 M at 25°C, l = 0.2 cm and I = 0.16 M NaCl. (D) Speciation plot of H4py4pa calculated with protonation constants on Table 7.2. [H4pypa] = 1  10-3 M. Dashed line indicates pH 7.4.   250 275 300 3250.000.250.500.751.001.251.501.75pH 2.18 2.18 2.40 2.60 3.13AbsorbanceWavelength (nm)pH 3.13A250 275 300 3250.000.250.500.751.001.251.501.75pH 5.21 3.13 3.42 3.62 3.93 4.28 5.21AbsorbanceWavelength (nm)pH 3.13B250 275 300 3250.000.250.500.751.001.251.501.75pH 9.62  5.21 5.63 5.97 6.23 6.48 6.72 6.96 7.26 7.74 8.86 9.62AbsorbanceWavelength (nm)pH 5.21C2 4 6 8 100102030405060708090100L4-HL3-H2L2-H3L-H4LH5L+H6L2+% Formation relative to LigandpHDH7L3+210   Figure 7.8 Normalized curve of the basic titration curve of H4py4pa · 4TFA · H2O. [H4py4pa] = 4.05  10-4 M at 25°C and I = 0.16 M NaCl.  Table 7.2 Protonation constants of H4py4pa at 25°C, I = 0.16 M (NaCl). The numbers in parentheses indicated the standard deviation of the last digit.  Equilibrium Reaction log β log K L4- + H+ ⇆ HL3- 6.96 (1) 6.96 (1) HL3- + H+ ⇆ H2L2- 13.03 (1) 6.07 (1) H2L2- + H+ ⇆ H3L- 17.09 (2) 4.06 (2) H3L- + H+ ⇆ H4L 20.67 (2) 3.58 (2) H4L + H+ ⇆ H5L+ 23.38 (3) 2.71 (3) H5L+ + H+ ⇆ H6L2+ 25.93 (4) 2.55 (4) H6L2+ + H+ ⇆ H7L3+ 28.24 (4) 2.31 (4)  0 1 2 3 4 5 6 7 8 9 10 11 1224681012pHmol NaOH/mol H4py4paA211       The complex formation equilibria of H4py4pa with La(III) were determined by different methods. Since the metal complexation was complete at pH ≤ 2, direct determination of the stability constant for the [ML]- species by potentiometric titration of H4py4pa with La3+ ion was not feasible. Instead, the stability constant of the protonated species of the metal complex, [La(Hpy4pa)], was found with both a competition method using EDTA as a ligand competitor and an acidic in-batch UV spectrophotometric titration for the La3+-H4py4pa system (Figure 7.9). Once log K[La(Hpy4pa)] was known, the direct potentiometric method was used to determine the stability constants for the [La(py4pa)]- and [La(OH)(py4pa)]2- species. Potentiometric and spectrophotometric experimental data were refined using the HypSpec2014124 and Hyperquad2013125 programs and the stability constants are presented in Table 7.3.      Depending on the methods, the stability constant of the [La(py4pa)]- species, log K[La(py4pa)]-, was found to be 20.37(2) or 20.33(3), >5 units higher than that of the H2macropa complex (log K[La(macropa)]- = 14.99, 25°C, I = 0.1 M (KCl)),230 but ~ 4 units lower than that of the DOTA complex (log K[La(DOTA)]- = 24.25, 25°C).228 However, the thermodynamic stability constant alone is insufficient when comparing the metal sequestering ability of ligands with different basicity as the complexation reaction is always in competition with the protonation processes. In this regard, the pM value which is defined as –log [Mn+]free at [ligand] = 10 M,  [Mn+] = 1 M at pH = 7.4, takes into account the stability, the basicity and the denticity of the chelator, and allows for a much better comparison. The pM value for the La3+-H4py4pa system was 21.0, which was higher than that of DOTA (19.2) and significantly higher than that of H2macropa (~8.5), indicating a promising ability of H4py4pa to effectively bind larger metal ions like La3+ and Ac3+.230 212    Figure 7.9 (A) and (B) Representative spectra of the in-batch UV-titration of the La3+-py4pa system as the pH is raised. [L] = [La3+] = 7.86  10-5 M at 25°C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by addition of different amounts of NaCl. (C) Distribution diagram of the La3+-py4pa system calculated with stability constants in Tables 7.2 and 7.3. [L] = [La3+] = 1  10-3 M. Dashed line indicates physiological pH (7.4).  Table 7.3 Stepwise stability constants (log K) of H4py4pa complexes with La3+. The numbers in parentheses indicated the standard deviation of the last digit.  Equilibrium reaction La3+ - H4py4pa M3+ + L ⇆ ML 20.37(2)a; 20.33(3)b ML + H+ ⇆ MHL 3.78(4)a; 4.01(5)c M(OH)L + H+ ⇆ ML 9.94(3)a; 9.96(4)b  pMd 21.0 a) ligand-ligand potentiometric competition with EDTA at I = 0.16 M (NaCl) and 25°C; b) potentiometric titrations at I = 0.16 M (NaCl) and 25°C; c) in-batch acidic spectrophotometric competition at 25°C, not evaluated at constant I = 0.16 M (NaCl); d) pM is defined as -log [M]free at [L] = 10 M, [M] = 1 M and pH = 7.4. Charges are omitted for clarity. 250 275 300 3250.000.250.500.751.001.251.50H -0.24 -0.24 -0.09 0.0 0.23 0.36 0.45 0.81AbsorbanceWavelength (nm)pH 0.81A250 275 300 3250.000.250.500.751.001.251.50pH 1.99 0.81 1.20 1.36 1.47 1.63 1.99AbsorbanceWavelength (nm)pH 0.81B0 2 4 6 8 10 120102030405060708090100[LaL]-La3+[La(OH)L]2-% Formation relative to La3+pHC La(HL)213  7.2.5.   Radiolabeling H4py4pa and In vitro Serum Stability of [225Ac][Ac(py4pa)]-      Concentration-dependent radiolabeling (i.e. 10-4-10-7 M of a 100-L solution) of H4py4pa with 225Ac (65 kBq) was undertaken, in parallel with DOTA (i.e. 10-4-10-6 M of a 100-L solution), to study the radiolabeling efficiency of the chelating agents in extremely low concentrations. With an interest in 225Ac-radioimmunotherapy, the radiolabeling experiments were performed under antibody-compatible conditions: ambient temperature and pH = 7 using ammonium acetate solution (1 M) (Figure 7.10). It is important to note that the radiochemical yields (RCY) were measured 6 h after the radio-iTLC plates were prepared to ensure that the short-lived daughters of 225Ac had decayed and therefore, the radiolabeling of the daughters were not seen in the results.   At ambient temperature, H4py4pa was able to chelate 225Ac efficiently with ~97% RCY in 30 min using 10-6 M chelator concentration (6.5 MBq/nmol). The results irrefutably demonstrated advantages of H4py4pa over DOTA in radio-tagging immuno-constructs because under the same conditions (RT, pH = 7, 30 min), even with 100-fold higher concentration of DOTA, only ~75% of 225Ac was radiolabeled, as anticipated for a macrocyclic chelator with which high-temperature radiolabeling is necessary for quantitative RCY.       In addition, being an octadentate chelator with a constrained binding cavity, DOTA has an intrinsic preference for smaller metal ions,228 and therefore does not provide the best accommodation for Ac3+ ion.123 Thiele et al., in the recent 225Ac-macropa report, also demonstrated inefficient radiolabeling of DOTA with 225Ac at RT with only 11% RCY (reaction conditions: 5.9 × 10-5 M DOTA, 10-26 kBq, NH4OAc buffer pH = 6, 30 min).28 In the same report, they further demonstrated that the 18-membered macrocycle, H2macropa, possessed much stronger affinity for 225Ac than DOTA under the same reaction conditions (~97% RCY, 5.9 × 10-7 M), with 214  insignificant transchelation/decomplexation in vivo using the PSMA (prostate-specific membrane antigen)-targeting small peptidomimetic as a targeting vector.28   Figure 7.10 Concentration-dependent radiolabeling of H4py4pa and DOTA at room temperature, pH = 7 (NH4OAc, 1 M) in 30 min.        Lastly, with the successful radiolabeling results for the [225Ac][Ac(py4pa)]- complex, it was deemed prudent to determine the stability of the complex in serum proteins, in parallel with [225Ac][Ac(DOTA)]-. For this experiment, both labeled complexes were challenged with 7-part volume of mouse serum at 37°C. The % intact was determined by iTLC plates demonstrating high stability over 9 days (<1% transchelation) (Table 7.4).     215  Table 7.4 Mouse serum stability results of [225Ac][Ac(L)]- (L = DOTA and py4pa) at 37 oC over 9 d (n = 3). Day [225Ac][Ac(py4pa)]-  [225Ac][Ac(DOTA)]- 1 99 (0) 99 (0) 2 99 (0) 99 (0) 5 99 (0) 99 (0) 7 99 (0) 99 (0) 9 99 (0) 99 (0)  7.2.6.   In vitro Characterization of [225Ac][Ac(py4pa-phenyl-Trastuzumab)]      The rapid and high yielding radiolabeling efficiency of H4py4pa with 225Ac was obtained at ambient temperature, neutral pH and non-reducing conditions, which are mandatory parameters to maintain the structure of the antibody. To verify that H4py4pa will also allow efficient radiolabeling of a proper monoclonal antibody with 225Ac, the well-established FDA-approved anti-HER2 Trastuzumab was used. Trastuzumab was conjugated by targeting free amines with either the bifunctional H4py4pa-phenyl-NCS or the p-NCS-Bn-DOTA with different chelator:antibody molecular ratios from 3:1 to 20:1. After purification, various buffers were tested to optimize labeling conditions in addition to multiple temperature settings. As a standard practice, the radiochemical yield was measured 6 h after the radio-iTLC plates were prepared to ensure that the short-lived daughters of 225Ac had decayed and therefore, the radiolabeling of the daughters were not seen in the results.  For the DOTA-benzyl-Trastuzumab, the best results were obtained using 0.1 M tris(hydroxymethyl)aminomethane (TRIS) buffer at pH = 9. While increasing temperature from ambient to 37 and 50°C slightly improved the radiolabeling efficiency, the increase of the molecular ratio significantly increased the RCY with 4.9, 12.9, 56.7 and 79.9% for 216  3:1, 5:1, 10:1 and 20:1 respectively. A two-step radiolabeling protocol was also tested with first 225Ac chelation by the p-NCS-benzyl-DOTA followed by conjugation of 225Ac-DOTA-benzyl-NCS to Trastuzumab.234 Unfortunately, this strategy was not efficient under the conditions tested (data not shown). For H4py4pa-phenyl-Trastuzumab, all conditions tested resulted in desired product with the best RCY (98.7±1.2% regardless of the temperature or the molecular ratio) obtained with 0.15-0.2 M ammonium acetate solution (pH = 7). After purification, size-exclusion HPLC showed that the labeled antibody contained no aggregates (Figure 7.11). For 1 mg of each immunoconjugate, a higher specific activity was obtained for the [225Ac][Ac(py4pa-phenyl-Trastuzumab)] even when using 1.5-fold more 225Ac to label the DOTA construct: 0.60  vs. 0.21 kBq/μg, respectively, which are suitable for in vivo experiments (87.0 vs. 30.5 MBq/mol)). When conjugated to Trastuzumab, H4py4pa is still easier to use and more efficient for radiolabeling with 225Ac at RT using both low and high number of chelators per antibody than DOTA. These results make H4py4pa a powerful chelator for labeling with 225Ac with reproducibly high RCY. The efficient labeling at RT eases the overall procedure as compared to DOTA. Lysine-based bioconjugation methods are the most prevalent that lead to the development of clinically-approved radioimmunoconjugates for either imaging or therapy, but can be associated with a decrease of the binding of the labeled antibody.44 The ability to label immunoconjugates with a few chelators can be an asset for those that present significant number of lysines in the antigen binding site. In this case, a low chelator:antibody ratio is recommended to maintain the immunoreactivity.      Immunoreactive fractions were therefore determined prior to in vivo experiment to select an appropriate chelator:antibody molecular ratio. Labeling was performed using the conditions previously determined, i.e 0.15 M ammonium acetate pH = 7 at RT for the H4py4pa-phenyl-217  Trastuzumab and 0.1 M TRIS pH 9 at 45°C for the DOTA-benzyl-Trastuzumab, with 5 times more 225Ac for DOTA conjugates than H4py4pa analogues. The radioimmunoconjugates were then purified resulting in radiochemical purities >99% for all tested constructs with specific activities of 0.7-4.0 kBq/μg (102-580 MBq/mol). Ratios of 5:1 and 10:1 for the DOTA-benzyl-Trastuzumab and 5:1 for the H4py4pa-phenyl-Trastuzumab showed immunoreactive fractions of 88, 99 and 61%, respectively. Ratios of 10:1 and 5:1 were therefore selected for further experiments for DOTA-Trastuzumab and H4py4pa-Trastuzumab, respectively. The stability of the 225Ac chelation was also assessed for both immunoconjugates in vitro in mouse plasma at 37°C using iTLC-SA plates. Free 225Ac in the labeling buffer and mouse serum was prepared as the control experiment. De-metalation was observed with 91-93% from day 1 to day 6 for [225Ac][Ac(DOTA-benzyl-Trastuzumab)] and only 86% remained on day 11, while [225Ac][Ac(py4pa-phenyl-Trastuzumab)] showed intact fraction values of 97-99% over time (Figure 7.12). 218   Figure 7.11 Size-exclusion HPLC profiles of (A) [225Ac][Ac(DOTA-benzyl-Trastuzumab)] and (B) [225Ac][Ac(py4pa-phenyl-Trastuzumab)]. For each panel, the top trace shows the 225Ac radioactive peak and the bottom one the protein UV280nm peak. For both products, an expected peak at 8.3-8.4 min post-injection that corresponds to an intact IgG is observed. For the radiotrace, the additional small peak around 12 min corresponds to the 225Ac daughters released from each chelators after 225Ac decay. The time difference between the UV and the radioactive peaks for each compound is due to the time needed for the antibody to travel from the absorbance detector to the scintillation detector.  Figure 7.12 Mouse serum challenge of 225Ac labeled DOTA- and H4py4pa-phenyl-Trastuzumab at 37 oC over 11 days (n=3 per time points). 0 5 1 0 1 5051 01 5mVA8.812.60 5 1 0 1 502 04 06 08 0mVB8.912.30 5 1 0 1 501 0 02 0 03 0 04 0 05 0 0T im e  (m in )mAu8.30 5 1 0 1 501 0 02 0 03 0 04 0 05 0 0T im e  (m in )mAu8.40 2 4 6 8 10020406080100% IntactDay DOTA-benzyl-Trastuzumab H4py4pa-benzyl-TrastuzumabB219  7.2.7.   In vivo Biodistribution Studies      To compare the in vivo pharmacokinetics of [225Ac][Ac(DOTA-benzyl-Trastuzumab)] and [225Ac][Ac(py4pa-phenyl-Trastuzumab)], biodistribution experiments were performed in female NRG mice bearing subcutaneous SKOV-3 ovarian cancer xenografts that express HER2 antigen.  The same amount of antibody (50.3±3.6 μg) and similar injected activities (10.1±0.7 kBq) were injected for each construct. After intravenous injection of each radioimmunoconjugate, mice were euthanized at day 1, 3, 6 and 10 post-injection.  Tumors and organs of interest were collected for 225Ac quantification at least 6 h post-mortem to allow for short-lived daughters to decay prior measurement. Both radioimmunoconjugates showed expected radioactivity in the blood, tumor, liver and spleen due to the antibody circulation, metabolism and excretion (Figure 7.13 and Table 7.5).235 Blood radioactivity concentration decreased similarly for both radiotracers from day 1 to 3, from 15.9±2.0% ID/g to 2.4±0.4% ID/g for [225Ac][Ac(DOTA-benzyl-Trastuzumab)], and 17.2±2.0 to 0.4±0.3% ID/g for [225Ac][Ac(py4pa-phenyl-Trastuzumab)]. Besides, liver and bone uptakes were comparable over time for both constructs, indicating similar in vivo stabilities. Significant tumor uptake was obtained for both [225Ac][Ac(DOTA-benzyl-Trastuzumab)] (22.2±5.5% ID/g at day 1 and 29.1±11.0% ID/g at day 10), and [225Ac][Ac(py4pa-phenyl-Trastuzumab)] (23.1±8.8 and 36.9±11.1% ID/g at day 1 and 6, respectively, followed by a decrease to 17.7±9.3% ID/g at day 10). Nevertheless, for both constructs, none of the time variations observed in the tumor uptake were significantly different as compared to day 1 (p>0.05). In addition, at each timepoint, no significant difference was noticed for the tumor uptake between the two radioimmunoconjugates (p>0.05). The only significant differences were the radioactivity concentrations within the ovaries and the spleen. A significant variation was noticed over time in 220  the ovaries from day 1 to day 3 for both radioimmunoconjugates (p<0.001). When comparing the two constructs at each time point, differences were also noticed at day 1 and day 10 (p<0.05). These variations observed for the ovaries between the different timepoints or between the two tracers can be explained by the different stages of the ovarian cell cycles between mouse-cages. For the spleen, significant time variations were also noticed at day 6 for [225Ac][Ac(py4pa-phenyl-Trastuzumab)] and at both day 6 and day 10 for the [225Ac][Ac(DOTA-benzyl-Trastuzumab)] (p<0.01 as compared to day 1). At day 10, a higher spleen uptake was observed for [225Ac][Ac(DOTA-benzyl-Trastuzumab)] than [225Ac][Ac(py4pa-phenyl-Trastuzumab)] (83.29±23.7 % ID/g  vs. 27.4±8.4 % ID/g)  (p<0.001). A decrease in the spleen size was noticed with 25.6±3.2 mg for the mice euthanized at day 1 vs. 17.2±4.0 mg for those sacrificed at day 3 on average considering both radioimmunoconjugates. This decrease can indicate toxicity. Interestingly, at day 10 the spleen was significantly smaller for the group of [225Ac][Ac(DOTA-benzyl-Trastuzumab)] group than that of [225Ac][Ac(py4pa-phenyl-Trastuzumab)] (6.5±1.1 vs. 19.1±5.8 mg). Nevertheless, the mice did not show any external signs of sickness during the course of the experiment. This suggests that under the same injection conditions, the [225Ac][Ac(DOTA-benzyl-Trastuzumab)] could be more toxic to the spleen than the py4pa analog. Regardless, a reduction in spleen mass can explain the higher % ID/g values.  221   Figure 7.13 Biodistribution of [225Ac][Ac(DOTA-benzyl-Trastuzumab)] (black) and [[225Ac][Ac(py4pa-phenyl-Trastuzumab)]  (grey) at (A) day 1, (B) day 3, (C) day 6 and (D) day 10 post-injection in an ovarian cancer xenografts model (SKOV-3). Data are presented as percentage of the injected dose per gram of tissue (% ID/g) for the tumor and the main organs (mean ± SD, n=3-4) and statistical differences between the two tracers are highlighted with * p<0.01 and ** p<0.001. NA: not available as the counts were in the background, i.e very low radioactivity concentration that cannot be quantified.   222  Table 7.5 Biodistribution data of the 225Ac-labeled DOTA and H4py4pa conjugated Trastuzumab in SKOV-3 tumor bearing mice at 1, 3, 6 and 10 days post-injection. Data are presented as mean±SD %ID/g (n=3-4). Mice were injected with similar amount of antibody (50.3±3.6 μg) and similar injected activities (10.1±0.7 kBq). Significant difference between [225Ac]Ac-py4pa-Trastuzumab and the DOTA construct are highlighted: * p<0.01 and ** p<0.001. NA: not available as counts were in the background which did not allow for proper quantification.   225Ac-labeled Trastuzumab  Day 1 Day 3 Day 6 Day 10  DOTA H4py4pa DOTA H4py4pa DOTA H4py4pa DOTA H4py4pa Blood 15.9±2.0 17.4±2.0 2.4±4.2 0.4±0.2 2.2±0.5 3.0±1.2 NA 0.2±0.2 Fat 0.5±0.2 0.9±0.1 11.6±10.6 11.5±4.2 0.4±0.1 0.3±0.1 0.7±0.4 0.3±0.1 Uterus 10.8±2.1 11.2±2.7 6.5±8.8 0.3±0.1 18.6±3.2 21.4±8.2 17.9±8.0 14.1±7.1 Ovaries 8.2±1.0 14.1±1.8* 120.8±94.3 101.5±61.1 14.7±11.0 15.8±6.2 32.8±43.9 5.1±3.3* Small int 3.2±0.2 4.0±0.4 1.2±2.3 0.1±0.1 4.2±0.4 2.9±0.5 2.9±1.0 1.4±0.5 Large int 1.5±0.2 3.5±0.1 1.9±0.3 1.8±0.5 1.5±0.2 2.2±0.4 0.9±0.6 1.1±0.3 Spleen 22.7±3.1 24.2±3.9 19.0±24.6 14.5±3.0 90.2±71.7 60.9±23.3 83.2±23.7 27.4±8.4** Liver 12.5±1.1 13.5±1.1 7.1±3.6 5.3±0.4 11.2±0.2 11.1±2.2 13.5±2.1 12.0±1.6 Pancreas 1.9±0.5 1.7±0.3 7.5±4.1 28.7±5.5 1.1±0.2 1.2±0.5 1.2±0.6 1.0±0.3 Stomach 2.8±0.1 3.2±0.4 2.8±0.1 2.9±0.4 2.6±0.4 2.2±0.3 1.4±.2 1.3±0.3 Kidneys 5.0±1.0 7.5±0.6 1.4±1.3 0.5±0.1 1.9±0.1 4.8±0.9 1.3±0.3 2.7±0.6 Lungs 8.0±0.8 8.9±1.4 4.5±1.1 4.0±0.6 2.9±0.3 2.9±0.7 1.2±0.5 1.6±0.8 Heart 4.0±0.5 4.8±0.5 1.1±1.2 0.7±0.2 1.5±0.1 1.7±0.8 1.0±0.4 1.0±0.4 SKOV-3  22.2±5.5 23.1±8.8 27.9±27.1 35.9±24.1 27.5±8.7 36.9±11.1 29.1±11.0 17.7±9.3 Muscle 0.8±0.1 1.2±0.1 3.9±2.3 5.8±0.2 0.4±0.2 0.6±0.3 0.4±0.1 NA Bone 4.9±0.2 4.3±0.2 4.6±0.8 5.0±1.0 5.5±1.3 9.0±1.2 6.1±1.1 7.7±2.4 Brain 0.4±0.1 0.4±0.1 7.5±5.7 7.4±2.9 0.2±0.1 0.2±0.1 1.7±0.0 0.2±0.1   7.3.   Conclusions       A undecadentate non-macrocyclic chelator H4py4pa, has been synthesized and characterized. Its capability to coordinate large trivalent metal ions was demonstrated with the La3+ ion. According to 1H NMR and the structure predicted by the DFT calculations, [La(py4pa)]- is symmetric about the central pyridine. The La-H4py4pa system also had a superior thermodynamic stability (pM = 21.0) compared to DOTA and H2macropa (pM = 19.2 and ~8.5, respectively). The promising results with La3+ ion translated with 225Ac. Concentration-dependent radiolabeling study demonstrated quantitative radiochemical yield of [225Ac][Ac(py4pa)]- at room temperature in 30 min at a chelator concentration as low as 10-6 M, resulting in a highly stable complex over at least 223  9 days of in mouse serum. To further evaluate its biological applicability, a short phenyl-NCS linker was incorporated into H4py4pa via nucleophilic substitution through the p-OH site on the central pyridyl bridge in the bifunctional precursor (compound 7.19). H4py4pa-phenyl-NCS was further conjugated to Trastuzumab, which did not impede its 225Ac scavenging, nor did it reduce the in vitro serum stability. Higher RCY and specific activity was obtained with H4py4pa-phenyl-Trastuzumab using less 225Ac than for the DOTA-benzyl-Trastuzumab. The immunoreactive fraction appeared to be lower for [225Ac][Ac(py4pa-phenyl-Trastuzumab)]. Nevertheless, the biodistribution data of [225Ac][Ac(py4pa-phenyl-Trastuzumab)] when compared with the DOTA analogue, demonstrated similar in vivo stability profile (i.e. low bone and liver uptakes) and expected tumor accumulations. The spleen uptake in the case of DOTA was 3-fold higher than that of the py4pa counterpart on day 10 and seemed to be associated with higher spleen toxicity. Nonetheless, the overall results were a propitious start for translating non-macrocyclic H4py4pa to 225Ac-based targeted alpha therapy.  7.4.   Experimental Section 7.4.1.   Materials and Methods      All solvents and reagents were purchased from commercial suppliers (TCI America, Alfa Aesar, AK Scientific, Sigma-Aldrich, Fisher Scientific, Fluka) and were used as received. Deionized water was filtered through the PURELAB Ultra Mk2 system. The S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) was purchased from Macrocyclics (Plano, TX, USA) and Trastuzumab (Herceptin) from Genentech (San Francisco, CA, USA). The analytical thin-layer chromatography (TLC) plates used were aluminum-backed 224  ultrapure silica gel 60 Å, 250 μm thickness; the flash column silica gel (standard grade, 60 Å, 32−63 mm) was provided by Silicycle. 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. Low-resolution (LR) mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the Department of Chemistry, University of British Columbia. 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 non-radioactive compounds consisted of a Waters 600 controller, Waters 2487 dual wavelength absorbance detector, and a Waters delta 600 pump. Phenomenex Synergi 4  hydro-RP 80 Å column (250 mm × 21.2 mm semipreparative) was used for purification of H4py4pa and H4py4pa-phenyl-NCS. Automated column chromatography was performed using a Teledyne Isco (Lincoln, NE) Combiflash Rf automated system with solid load cartridges packed with Celite and RediSep Rf gold reusable normal-phase silica columns (Teledyne Isco, Lincoln, NE). Analyses of radiolabeled compounds, including the radioimmunoconjugates were performed with instant TLC plates, impregnated with silicic acid (iTLC-SA) or silica gel (iTLC-SG) purchased from Agilent Technologies. The TLC scanner model was BIOSCAN (system 200 imaging scanner). Radiochemical purities and specific activities of the final 225Ac-labeled antibodies were determined by using gamma-spectrometry performed with a GR1520 (Canberra Industries, Meriden, CT, USA) high purity Germanium detector (HPGe) in addition to size-exclusion column (Phenomenex, BioSep-SEC-s-3000) on an Agilent HPLC system equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at 280 nm), and a Bioscan 225  (Washington, DC) NaI scintillation detector (the radiodetector was connected to a Bioscan B-FC-1000 flow-count system, and the output from the Bioscan flow-count system was fed into an Agilent 35900E interface, which converted the analogue signal to a digital signal). For labeling of the chelators, actinium-225 in 0.05 M nitric acid was produced by TRIUMF via Th-232 spallation.236 For antibody labeling for either in vitro or in vivo experiments, 225AcCl3 was purchased from Isotope Technologies Garching (ITG, Germany) as a solution in dilute HCl. For antibody purification, PD-10 desalting columns (Sephadex G-25M, 50kDa, GE Healthcare) and/or centrifugal filter devices (Amicon Ultra 4 Centrifugal Filtration Units, 50 kDa cutoff, Millipore Corp.) were used.   7.4.2.   Synthesis and Characterization Methyl 6-(hydroxymethyl)picolinate (7.1)       To a round-bottom flask with a stirred mixture of dimethyl 2,6-pyridinedicarboxylate (10.0 g, 51.3 mmol, 1 equiv) in dry dichloromethane (DCM) (150 mL) was added dry methanol (MeOH) (50 mL). The solution was stirred at 0°C in an ice-water bath and then sodium borohydride (NaBH4) (2.91 g, 76.6 mmol, 1.5 equiv) was added in six portions over 1 h. The mixture was further stirred at room temperature for another 3-4 h and the reaction progress was monitored with silica-aluminum backed TLC plates (5% MeOH in DCM) every 30 min.  When the mono-reduced product dominated, saturated sodium carbonate (Na2CO3) in water (100 mL) was added to quench the reaction. The organic phase was separated and the MeOH in the aqueous phase was removed in vacuo to give a concentrated aqueous solution which was then extracted with chloroform (CHCl3) (100 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate 226  (Na2SO4), and then clarified by filtration. The filtrate was concentrated and purified through a silica column (CombiFlash Rf automated column system, 80 g gold silica column, A: DCM B: MeOH, 0-5% B). The product fractions were rotary-evaporated to give a white solid (5.15 g, 60%). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.06 – 7.95 (m, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 4.85 (s, 2H), 3.97 (s, 3H). LR-ESI-MS: calcd for [C8H9NO3 + H]+ 168.2; found [M + H]+ 168.2 Methyl 6-(bromomethyl)picolinate (7.2)       Compound 7.1 (2.69 g, 16.1 mmol, 1 equiv) was dissolved in dry acetonitrile (ACN, 60 mL) under Ar(g). Phosphorus tribromide (PBr3) (2.27 mL, 24.1 mmol, 1.5 equiv) was added dropwise to the stirred mixture at 0°C over 15 min via a syringe. The mixture was stirred at RT for 6 h, monitored by silica aluminum-backed TLC plate (hexanes:ethyl acetate 1:1). After the reaction was finished, saturated Na2CO3 in water (150 mL) was added to quench the reaction, and then the bulk of ACN was removed in vacuo to give an aqueous layer which was extracted with CHCl3 (80 x 4 mL). The combined organic phases were dried over anhydrous magnesium sulfate (MgSO4), and then clarified by filtration. The filtrate was rotary-evaporated to give a white powder (2.95 g, 80%). If necessary, the product can be further purified through a silica column (CombiFlash Rf automated column system, 40 g gold silica column, A: hexanes B: ethyl acetate, 0-60% B). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.07 (d, J = 7.7 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 4.65 (s, 2H), 4.02 (s, 3H). LR-ESI-MS : calcd for [C8H8Br79NO2+H]+ 230.0; found [M + H]+ 230.1  227  Dimethyl 6,6'-((benzylazanediyl)bis(methylene))dipicolinate (7.3)       To a stirred solution of compound 7.2 (1.00 g, 4.35 mmol, 2.05 equiv) in dry ACN (10 mL) was added diisopropylethylamine (DIPEA) (1.11 mL, 6.36 mmol, 3 equiv) and benzyl amine (231.6 L, 2.12 mmol, 1 equiv). The mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was evaporated and the product mixture was purified through a silica column (CombiFlash Rf automated column system, 40 g gold silica column, A: hexanes B: ethyl acetate, 0-60% B) to yield a yellow oil (0.781 g, 91%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.99 (d, J = 6.0 Hz, 2H), 7.82 (s, 4H), 7.39 (d, J = 6.5 Hz, 2H), 7.33 – 7.19 (m, 3H), 3.98 (s, 6H), 3.93 (s, 4H), 3.70 (s, 2H). 13C NMR (75 MHz, 298 K, CDCl3) δ 165.9, 160.5, 147.5, 138.7, 137.5, 129.0, 128.5, 127.4, 126.0, 123.8, 59.9, 58.8, 53.0. LR-ESI-MS : calcd for [C23H23N3O4+H]+ 406.2; found [M + H]+ 406.2 Dimethyl 6,6'-(azanediylbis(methylene))dipicolinate (7.4)       Compound 7.3 (0.799 g, 1.97 mmol, 1 equiv) was dissolved in glacial acetic acid (20 mL) in a three-necked round-bottom flask, saturated with N2(g). Pd/C (10% w/w, 0.1 equiv) was added under a stream of N2(g). The flask was purged with N2(g) again, followed by H2(g) from a balloon.  The mixture was stirred vigorously at room temperature for 3-4 h under H2 atmosphere. The reaction was monitored with silica aluminum-backed TLC plate (hexanes:ethyl acetate, 1:1) and MS until the starting material was completely consumed. Then, Pd/C was filtered off through a Celite bed, washed with MeOH (10 mL × 5). The filtrate was rotary-evaporated to a pale-yellow oil (0.514 g, 83%) and used without purification. 1H NMR (400 MHz, 298 K, CDCl3): δ 7.93 (d, J = 7.7 Hz, 2H), 7.74 (t, J = 7.7 Hz, 2H), 7.57 (d, J = 7.8 Hz, 2H), 4.01 (s, 4H), 3.91 (s, 6H). 13C 228  NMR (100 MHz, 298 K, CDCl3) δ 166.1, 160.7, 147.8, 137.8, 126.0, 123.9, 54.9, 53.1. LR-ESI-MS: calcd for [C16H17N3O4 + H]+ 316.1; found [M + H]+ 316.2 2,6-Di(hydroxymethyl)pyridine (7.5)       To a round-bottom flask with a stirred mixture of dimethyl 2,6-pyridinedicarboxylate (3.00 g, 15.4 mmol, 1 equiv) in dry MeOH (50 mL) at 0°C was slowly added NaBH4 (2.33 g, 61.5 mmol, 4 equiv) in three portions over 15 minutes. The solution was then stirred at room temperature for 12 h. CHCl3 (25 mL) was added followed by saturated Na2CO3 in water (50 mL) to quench the reaction. The organic phase was separated and the MeOH in the aqueous phase was removed in vacuo to give a concentrated aqueous solution which was then extracted with CHCl3 (100 mL × 10). Multiple extractions were required to recover most of the product. The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was concentrated to give a white solid (1.99 g, 92%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.71 (t, J = 7.7 Hz, 1H), 7.21 (d, J = 7.7 Hz, 2H), 4.79 (s, 4H). 13C NMR (75 MHz, 298 K, MeOD) δ 161.5, 139.2, 120.2, 65.5. LR-ESI-MS: calcd for [C7H9NO2 + H]+ 140.1; found [M + H]+ 140.1  2,6-Bis(bromomethyl)pyridine (7.6)       To a three-necked round-bottom flask with a stirred solution of compound 7.5 (3.00 g, 21.2 mmol, 1 equiv) in dry ACN/CHCl3 (30 mL, 50:50 v/v) at 0°C was added PBr3 (6.02 mL, 63.4 mmol, 3 equiv) dropwise using a dropping funnel over 15 min. The mixture was refluxed for 18 h, and then cooled before water (20 mL) was added slowly at 0°C to quench the reaction. After extraction with CHCl3 (50 mL × 3), the combined organic layers were dried over anhydrous MgSO4, and then clarified by filtration. The solvent was removed under reduced pressure and the 229  product was obtained as a pure white solid (4.88 g, 87%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.76 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 4.59 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3) δ 156.8, 138.5, 123.1, 33.3. LR-ESI-MS: calcd for [C7H7Br2N + H]+ 263.9; found [M(79Br) + H]+ 263.9 Tetramethyl-6,6',6'',6'''-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetrakis-(methylene))tetrapicolinate (7.7)       To a round-bottom flask with a stirred solution of compound 7.4 (0.208 g, 0.658 mmol, 2.05 equiv) in dry ACN (2 mL) was added DIPEA (115 L, 0.658 mmol, 2.05 equiv), followed by compound 7.6 (85 mg, 0.312 mmol, 1 equiv) and KI (109 mg, 0.658 mmol, 2.05 equiv). The mixture was stirred at 25°C for 24 h, and then KI was separated by centrifugation, followed by washing with DCM or ACN (5 mL × 3). The combined organic phases were concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-8% B). The product fractions were rotary-evaporated to give a yellow oil (0.183 g, 80%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.95 (d, J = 7.6 Hz, 4H), 7.85 (d, J = 7.7 Hz, 4H), 7.77 (t, J = 7.7 Hz, 4H), 7.60 (d, J = 8.2 Hz, 1H), 7.38 (d, J = 7.7 Hz, 2H), 3.99 (s, 8H), 3.95 (s, 12H), 3.85 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 165.9, 160.2, 158.5, 147.5, 137.6, 137.0, 126.2, 123.8, 121.6, 60.2, 60.0, 53.0. LR-ESI-MS: calcd for [C39H39N7O8 + K]+ 772.3; found [M + K]+ 772.2 H4py4pa (7.8)       Compound 7.7 (183 mg, 0.249 mmol) was dissolved in tetrahydrofuran (THF) (2 mL), and then lithium hydroxide (LiOH) (60 mg, 2.49 mmol, 10 equiv) in water (2 mL) was added dropwise 230  using a Pasteur pipette. The mixture was stirred vigorously at room temperature overnight. THF was removed in vacuo and the residue was diluted with water, and then purified with reverse phase HPLC (A: ACN/0.1% TFA, B: H2O/0.1% TFA, 5 - 40 % A over 30 min, 10 mL/min, tR = 22.1 min). The combined product fractions were lyophilized to give a white fluffy solid (84.3 mg, 50%).1H NMR (400 MHz, 298 K, D2O): δ 7.92 (t, J = 7.8 Hz, 1H), 7.79 (d, J = 4.4 Hz, 8H), 7.53 (d, J = 7.8 Hz, 2H), 7.43 (t, J = 4.1 Hz, 4H), 4.88 (s, 4H), 4.76 (s, 8H). 13C NMR (100 MHz, 298 K, D2O): δ 166.8, 150.6, 150.1, 146.8, 140.1, 139.8, 128.0, 125.6, 125.1, 59.6, 58.5. HR-ESI-MS: calcd for [C35H31N7O8 + H]+ 678.2312; found [M + H]+ 678.2333. Elemental analysis: calcd % for H4py4pa · 4TFA · 2H2O (C35H31N7O8 · 4C2HF3O2 · 2H2O = 1169.2162): C 44.15, H 3.36, N 8.38; found: C 43.93, H 3.18, N 8.31. Na[La(py4pa)]       Compound 7.8 (8.75 mg, 12.9 mol, 1 equiv) was dissolved in H2O (1 mL) in a scintillation vial. Lanthanum(III) nitrate (La(NO3)3) AAS standard solution (7.19 mM, 1.86 mL, 13.6 mol) was added, followed by 4.2 M sodium hydroxide solution (NaOH, aq) to bring the pH to 7. Then, the mixture was stirred at room temperature for 1 h and complexation was confirmed by MS. 1H NMR (400 MHz, 298 K, D2O): δ 7.92 (t, J = 7.7 Hz, 2H), 7.68 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 7.7 Hz, 2H), 7.59 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 7.5 Hz, 2H), 7.44 (t, J = 7.7 Hz, 2H), 7.12 (d, J = 7.7 Hz, 2H), 6.76 (d, J = 7.5 Hz, 2H), 5.62 (d, J = 14.5 Hz, 2H), 4.29 (d, J = 14.2 Hz, 2H), 4.00 (d, J = 14.5 Hz, 2H), 3.82 (d, J = 14.2 Hz, 2H), 3.60 (q, J = 15.9 Hz, 4H). 13C NMR (75 MHz, 298 K, D2O): δ 172.9, 171.9, 163.4, 163.0, 158.9, 158.1, 151.6, 151.4, 139.9, 139.8, 125.8, 123.6, 123.4, 123.2, 121.8, 118.0, 115.1, 100.1, 65.0, 63.1, 63.1, 62.5. HR-ESI-MS: calcd for [C35H27LaN7O8 + 2H]+ 814.1141; found [M + 2H]+ 814.1128. 231  Methyl 6-formylpicolinate (7.9)       To a round-bottom flask with a stirred solution of compound 7.1 (4.50 g, 26.9 mmol, 1 equiv) in 1,4-dioxane (50 mL) was added selenium dioxide (SeO2) (1.50 g, 13.5 mmol, 0.5 equiv). The mixture was refluxed at 100°C overnight. After the reaction completed, the hot mixture was clarified by filtering through a Celite bed and the filtrate was concentrated in vacuo. The crude mixture was purified through a silica column (CombiFlash Rf automated column system, 80 g gold silica column, A: hexanes B: ethyl acetate, 0-60% B) to give a pale yellow solid (2.49 g, 56%). 1H NMR (400 MHz, 298 K, CDCl3): δ 10.20 (s, 1H), 8.36 (d, J = 8.7 Hz, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.06 (t, J = 7.7 Hz, 1H), 4.07 (s, 3H). LR-ESI-MS : calcd for [C8H7NO3 + H]+ 166.0; found [M + H]+ 166.3 Methyl 6-(aminomethyl)picolinate (7.10)       To a stirred solution of compound 7.2 (2.22 g, 9.64 mmol, 1 equiv) in dry ACN (19 mL) was added potassium phthalimide (1.96 g, 10.6 mmol, 1.1 equiv). The mixture was stirred at room temperature for 12 h, and then concentrated in vacuo. The white residue was re-dissolved in DCM (~50 mL) and then washed with H2O (20 mL × 2) and brine (20 mL × 2). The organic phase was dried over anhydrous MgSO4, and then clarified by filtration. The filtrate was evaporated in vacuo and the crude product was purified through a silica column (CombiFlash Rf automated column system, 40 g gold silica column, A: DCM B: MeOH, 0-5% B) to give product in a white powder form (2.22 g, 78%). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.02 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.83 – 7.73 (m, 3H), 7.36 (d, J = 7.8 Hz, 1H), 5.14 (s, 2H), 3.96 (s, 3H). 13C NMR (75 MHz, 298 K, CDCl3): δ 168.2, 156.4, 148.0, 138.0, 134.4, 132.3, 124.2, 123.8, 53.1, 43.3. LR-ESI-MS : calcd for [C16H12N2O4 + K]+ 335.1; found [M + K]+ 335.1 232  To the stirred solution of the product above in ethanol (EtOH) (4 mL) at 70oC was added hydrazine monohydrate (50 L, 1.02 mmol, 3 equiv) using an autopipette. The resulting mixture was stirred at 70°C for 4 h and then the white precipitate formed was filtered through a filter paper. The filtrate was concentrated in vacuo and then re-dissolved in minimal EtOH (1-2 mL). If more precipitate formed, the filtration should be repeated (2-3 times). Finally, after filtration, the filtrate was dried in vacuo to give a light-yellow oil as a product which was used in the next step immediately. LR-ESI-MS : calcd for [C8H10N2O2 + H]+ 167.1; found [M + H]+ 167.3 Dimethyl 4-hydroxypyridine-2,6-dicarboxylate (7.14)       Thionyl chloride (SOCl2) (9.50 mL, 0.130 mol, 5 equiv) was added slowly using a syringe to a stirred suspension of chelidamic acid monohydrate (5.28 g, 26.2 mmol, 1 equiv) in MeOH (60 mL) in a two-neck round-bottom flask at 0°C. The mixture was stirred at room temperature for 24 h and then refluxed for an additional 2 h. The solvent was removed under reduced pressure gently at room temperature and then deionized water was added slowly at 0°C. The mixture was neutralized with 1 M aqueous potassium carbonate (K2CO3) solution and the precipitate was filtered out by vacuum filtration, and then washed with 50% aqueous MeOH solution (~10 mL). The white precipitate was dried under reduced pressure to give a white solid (5.54 g, >99%). 1H NMR (400 MHz, 298 K, (CD3)2SO): δ 6.74 (s, 2H), 3.72 (s, 6H). 13C NMR (75 MHz, 298 K, (CD3)2SO): δ 165.7, 149.2, 116.6, 52.7. LR-ESI-MS : calcd for [C9H9NO5 + Na]+ 234.0; found [M + Na]+ 234.2   233  Dimethyl 4-(benzyloxy)pyridine-2,6-dicarboxylate (7.15)       To a round-bottom flask with a stirred solution of compound 7.14 (1.65 g, 7.82 mmol, 1 equiv) in dry ACN was added anhydrous K2CO3 (2.19 g, 15.8 mmol, 2.02 equiv) and benzyl bromide (1.02 mL, 8.60 mmol, 1.1 equiv). The reaction mixture was refluxed overnight at 60°C. K2CO3 was filtered out by vacuum filtration and then washed with DCM. The filtrate was concentrated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-5 % B). The product fractions were rotary-evaporated to give a white powder (1.51 g, 64%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.90 (s, 2H), 7.44-7.38 (m, 5H), 5.23 (s, 2H), 4.01 (s, 6H). 13C NMR (75 MHz, 298 K, CDCl3): δ 150.0, 129.0, 128.9, 127.9, 115.0, 71.0, 53.4. LR-ESI-MS : calcd for [C16H15NO5 + Na]+ 324.1; found [M + Na]+ 324.1 (4-(Benzyloxy)pyridine-2,6-diyl)dimethanol (7.16)       To a round-bottom flask with a stirred solution of compound 7.15 (8.74 g, 29.0 mmol, 1 equiv) in dry MeOH (90 mL) was added NaBH4 (3.29 g, 87.1 mmol, 3 equiv) in three portions over 30 min at 0°C. The reaction mixture was stirred at room temperature. After 24 h, the mixture was diluted with CHCl3 (50 mL) and then quenched with saturated sodium bicarbonate aqueous solution (NaHCO3) (50 mL). The organic phase was separated and the bulk of MeOH in the aqueous layer was removed in vacuo to give an aqueous solution which was extracted with CHCl3 (50 mL × 4). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to give a white solid (5.86 g, 82%).1H NMR (400 MHz, 298 K, CDCl3): δ 7.42-7.35 (m, 5H), 6.79 (s, 2H), 5.12 (s, 2H), 4.70 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3): 166.5, 160.6, 135.6, 128.9, 128.6, 127.6, 106.1, 70.2, 64.5. LR-ESI-MS: calcd for [C14H15NO3 + Na]+ 268.1; found [M + Na]+ 268.2 234  4-(Benzyloxy)-2,6-bis(bromomethyl)pyridine (7.17)       Compound 7.16 (1.76 g, 12.6 mmol, 1 equiv) was suspended in dry ACN/dry CHCl3 (40 mL, 50:50 v/v) in a three-necked round-bottom flask. PBr3 (3.60 mL, 37.9 mmol, 3 equiv) in CHCl3 (5 mL) was added dropwise using a dropping funnel to the stirred solution of compound 7.16 at 0°C over 15 min. The reaction mixture was stirred at 60°C for 18 h and then saturated aqueous Na2CO3 (~100 mL) was added slowly to quench the reaction at 0°C. The aqueous phase was extracted with CHCl3 (50 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, and then clarified by filtration. The filtrate was rotary-evaporated to yield a colorless oil which later solidified to a white solid (3.28 g, 70%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.43 (m, 5H), 7.36 (s, 2H), 5.37 (s, 2H), 4.95 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 170.9, 154.5, 133.2, 129.5, 129.3, 128.3, 113.2, 73.0, 25.3. LR-ESI-MS: calcd for [C14H1379Br2NO + H]+ 369.9; found [M(79Br) + H]+ 369.9 Tetramethyl 6,6',6'',6'''-((((4-(benzyloxy)pyridine-2,6-diyl)bis(methylene))bis(azane-triyl))-tetrakis(methylene))tetrapicolinate (7.18)       DIPEA (568 L, 3.26 mmol, 4.1 equiv), compound 7.17 (0.295 g, 0.796 mmol, 1 equiv) and KI (0.264 g, 1.592 mmol, 2 equiv) were added sequentially to the stirred solution of compound 7.4 (0.514 g, 1.63 mmol, 2.05 equiv) in dry ACN (6 mL) in a round-bottom flask. The mixture was stirred at 25°C for 24 h. KI was removed by centrifugation and then washed with DCM or ACN (5 mL × 3). The combined supernatants were concentrated in vacuo and re-dissolved in DCM (30 mL). The DCM phase was washed vigorously with saturated NaHCO3 aqueous solution (20 mL × 2), water (20 mL × 2) and brine (20 mL × 2). The organic phase was dried over anhydrous MgSO4, filtered and then concentrated in vacuo to give compound 7.18 as a yellow oil which, based on the 235  1H NMR spectrum, was pure enough for the next step without further purification. (0.476 g, 71%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.92 (d, J = 8.2 Hz, 4H), 7.74 (d, J = 7.3 Hz, 8H), 7.40 – 7.30 (m, 5H), 7.06 (s, 2H), 5.09 (s, 2H), 3.95 (s, 8H), 3.89 (s, 12H), 3.77 (s, 4H). 13C NMR (75 MHz, 298 K, CDCl3): δ 166.1, 165.7, 160.1, 159.9, 147.4, 137.5, 136.0, 128.7, 128.3, 127.7, 126.2, 123.6, 108.2, 69.9, 60.0, 59.9, 52.8. LR-ESI-MS: calcd for [C46H45N7O9 + H]+ 840.3; found [M + H]+ 840.5 Tetramethyl 6,6',6'',6'''-((((4-hydroxypyridine-2,6-diyl)bis(methylene))bis(azane-triyl))te-trakis(methylene))tetrapicolinate (7.19)       Compound 7.18 (0.476 g, 0.568 mmol, 1 equiv) was dissolved in dry MeOH (20 mL) in a three-necked round-bottom flask, saturated with N2(g). Pd/C (10% w/w, 0.1 equiv) was added under a stream of N2(g). The flask was purged with N2(g), followed by H2(g) from a balloon.  The mixture was stirred vigorously at room temperature overnight under H2 atmosphere, and then Pd/C was filtered off through a Celite bed (pre-wet with MeOH), washed with MeOH (10 mL × 5). The filtrate was rotary-evaporated to a pale-yellow oil (0.330 g, 78%) and used without purification. 1H NMR (400 MHz, 298 K, CDCl3): δ 7.86 (d, J = 7.5 Hz, 4H), 7.70 (t, J = 7.7 Hz, 4H), 7.62 (d, J = 7.6 Hz, 4H), 6.60 (s, 2H), 3.87-3.85 (m, 20H), 3.78 (s, 4H). 13C NMR (100 MHz, 298 K, CDCl3): δ 165.3, 158.4, 147.0, 137.8, 137.1, 126.9, 123.9, 122.3, 115.1, 58.9, 54.8, 52.8.  LR-ESI-MS: calcd for [C39H39N7O9 + H]+ 750.3; found [M + H]+ 750.4 4-((Tert-butoxycarbonyl)amino)phenethyl 4-methylbenzenesulfonate (7.20)       N-boc-2-(4-aminophenyl)ethanol (1.97 g, 8.28 mmol, 1 equiv) was dissolved in THF (12 mL) and cooled to 0°C with an ice-water bath. 6 M NaOH aqueous solution (11.9 mL) was added, 236  followed by dropwise addition of p-tosyl chloride (3.16 g, 0.0169 mol, 2 equiv) in THF (24 mL) under N2(g). After stirring at 0°C for 1 h, the reaction mixture was warmed to room temperature and again stirred overnight. The mixture was extracted with DCM (30 mL × 3). The combined organic phases were washed with 1 M NaOH aqueous solution (40 mL × 2) and deionized water (40 mL × 2), and then dried over MgSO4. The mixture was clarified with filtration, evaporated in vacuo and then purified through a silica column (CombiFlash Rf automated column system, 24 g gold silica column, A: DCM B: MeOH, 0-5 % B). The product fractions were rotary-evaporated to give a white solid (2.30 g, 71%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.68 (d, J = 8.3 Hz, 2H), 7.25 (dd, J = 13.4, 8.7 Hz, 4H), 7.01 (d, J = 8.4 Hz, 2H), 6.45 (s, 1H), 4.16 (t, J = 7.0 Hz, 2H), 2.89 (t, J = 7.0 Hz, 2H), 2.43 (s, 3H), 1.51 (s, 9H).13C NMR (100 MHz, 298 K, CDCl3): δ 152.8, 144.8, 137.3, 133.1, 130.7, 129.9, 129.6, 128.0, 118.8, 80.7, 70.8, 34.8, 28.5, 21.7. LR-ESI-MS: calcd for [C20H25NO5S + H]+ 392.1; found [M + H]+ 392.1 Tetramethyl 6,6',6'',6'''-((((4-(4-((tert-butoxycarbonyl)amino)phenethoxy)pyridine-2,6-diyl)bis(methylene))bis(azanetriyl))tetrakis(methylene))tetrapicolinate (7.21)       To a round-bottom flask with a stirred solution of compound 7.19 (124 mg, 0.165 mmol, 1 equiv) in dry ACN (1 mL) was added anhydrous K2CO3 (91.4 mg, 0.661 mmol, 4 equiv). The mixture was stirred vigorously for 1 h at 25°C before the addition of compound 7.20 (77.6 mg, 0.300 mmol, 1.2 equiv). The mixture was stirred for 48 h at 25°C when compound 7.19 was completely consumed. The solvent was evaporated in vacuo, and the residue was resuspended in DCM (6 mL). K2CO3 was removed by centrifugation and washed with DCM twice (~5 mL each). The combined organic phases were washed with saturated NaHCO3 in water (10 mL × 2), H2O (10 mL × 2) and brine (10 mL × 2), and then dried over anhydrous MgSO4. The drying agent was 237  filtered off and the filtrate was concentrated in vacuo to a yellow oil. The product was confirmed by MS and then used without isolation in the next step. LR-ESI-MS: calcd for [C52H56N8O11 + K]+ 1007.4; found [M + K]+ 1007.7 6,6',6'',6'''-((((4-(4-aminophenethoxy)pyridine-2,6-diyl)bis(methylene))bis(azanetriyl))tetra-kis(methylene))tetrapicolinic acid (7.22)       Compound 7.21 (166 mg, 0.171 mmol, 1 equiv) was dissolved in THF (2 mL), and then LiOH (41 mg, 1.71 mmol, 10 equiv) in water (1 mL) was added dropwise using a Pasteur pipette. The mixture was stirred vigorously at room temperature overnight. THF was removed in vacuo and the residue was acidified with TFA/DCM (1:1) (10 mL). The mixture was stirred overnight vigorously at room temperature, and then concentrated to dryness in vacuo. The crude product was re-dissolved in deionized water, and then purified through reverse phase HPLC (A: ACN/0.1% TFA, B: H2O/0.1% TFA, 5-60% A over 40 min, 10 mL/min, tR = 20.7 min). The combined product fractions were dried in vacuo to give a yellow oil (69.5 mg, 50%).1H NMR (400 MHz, 298 K, D2O): δ 7.79-7.70 (m, 8H), 7.50 (d, J = 6.9 Hz, 4H), 7.36 – 7.26 (m, 4H), 6.81 (s, 2H), 4.60-4.56 (m, J = 17.4 Hz, 12H), 4.11 (s, 2H), 2.98 (s, 2H).13C NMR (100 MHz, 298 K, D2O): δ 167.5, 166.0, 151.8, 150.5, 146.0, 140.4, 139.2, 130.5, 128.2, 125.2, 123.0, 111.8, 69.4, 58.7, 33.6. LR-ESI-MS: calcd for [C43H40N8O9 + H]+ 813.3; found [M + H]+ 813.5.  H4py4pa-phenyl-NCS (7.23)       Compound 7.22 (171 mg, 0.210 mmol, 1 equiv) was dissolved in 1 M HCl (aq)/ glacial acetic acid (2 mL, 4:1 v/v) in a round-bottom flask. Then, thiophosgene (CSCl2) (323 L, 4.21 mmol, 20 equiv) in CHCl3 (2 mL) was added dropwise using a Pasteur pipette to the stirred mixture of the 238  starting material. The resulting mixture was stirred vigorously at room temperature overnight. After the reaction completed, the CHCl3 and the aqueous phases were separated by centrifugation. The CHCl3 phase was carefully removed with a Pasteur pipette without collecting the precipitate, while the aqueous phase and the white precipitate were washed with additional CHCl3 (1 mL). The phases were separated with centrifugation. The CHCl3 and the aqueous layers were removed by a Pasteur pipette sequentially, while the precipitate (suspended in some CHCl3) was dissolved in ACN/0.1%TFA and water (1:1, 2mL) which was centrifuged again. The aqueous layer was extracted. The combined CHCl3 was washed with another portion of water (2-3 mL) and the aqueous layer was collected. The combined aqueous layers were injected into reverse phase HPLC (A: ACN/0.1% TFA, B: H2O/0.1% TFA, 20%-80% A over 30 min, 10 mL/min, tR = 22.3 min). The product fractions were combined and lyophilized to give a fluffy white solid (53.9 mg, 30%). 1H NMR (400 MHz, 298 K, CD3CN:D2O 1:1): δ 7.90-7.84 (m, 8H), 7.53 (d, J = 7.0 Hz, 4H), 7.28 (d, J = 7.5 Hz, 2H), 7.20 (d, J = 7.4 Hz, 2H), 6.88 (s, 2H), 4.44-4.39 (m, 12H), 4.20 (overlapped with D2O peak), 3.02 (t, J = 5.5 Hz, 2H). 13C NMR (100 MHz, 298 K, CD3CN:D2O 1:1): δ 166.9, 155.1, 154.4, 148.2, 141.0, 139.1, 139.0, 131.4, 128.7, 126.7, 125.4, 112.3, 70.5, 58.5, 35.0. HR-ESI-MS: calcd for [C44H38N8O9S+H]+ 855.2561; found [M+H]+ 855.2562  7.4.3.   DFT Calculations      All DFT calculations were performed as implemented in the Gaussian 09 revision D.01 suite of ab initio quantum chemistry programs (Gaussian Inc., Wallingford, CT) and visualized using Avogadro 1.2.237,238 The structure geometry was optimized using the B3LYP functional105,106 and the effective core potentials LanL2DZ basis sets for La,156,157,239 in the presence of water solvent 239  (IEF PCM as implemented in G09) without the use of symmetry constraints. Normal self-consistent field (SCF) and geometry convergence criteria were conducted for all the calculations.  7.4.4.   Solution Thermodynamics      All potentiometric titrations were carried out with a Metrohm Titrando 809 and a Metrohm Dosino 800 with a Ross combined electrode. A 20 mL and 25ºC thermostated glass cell with an inlet-outlet tube for nitrogen gas (purified through a 10% NaOH solution to exclude any CO2 prior to and during the course of the titration) was used as a titration cell. 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 procedure134 in order to obtain the standard potential Eº and the ionic product of water pKw at 25°C and 0.16 M NaCl as a supporting electrolyte. Solutions were titrated with carbonate-free NaOH (0.16 M) that was standardized against freshly recrystallized potassium hydrogen phthalate. The first seven protonation equilibria of the ligand were studied by titrations of an acidified solution containing H4py4pa 8.97  10-4 M at 25ºC and 0.16 M NaCl ionic strength using a joined potentiometric-spectrophotometric procedure.85 Spectra were recorded in the 200–450 nm spectral range with a 0.2 cm path length optic dip probe connected to a Varian Cary 60 UV/Vis spectrophotometer. In the study of complex formation equilibria, the determination of the stability constant of [La(Hpy4pa)] species was carried out by two different methods. The first method used UV-Vis spectrophotometric measurements on a set of solutions containing 1:1 metal to ligand molar ratio ([H4py4pa] = [M]3+ ~ 7.86  10-5 M) and different amounts of HCl in the spectral range 200-400 nm at 25°C and 1 cm path length. The molar absorptivities of all the protonated species of H4py4pa calculated with HypSpec2014124 from 240  the protonation constant experiments were included in the calculations. The second method used competition pH-potentiometric titrations with EDTA as a ligand competitor and the composition of the solutions was [La]3+ = 1.37  10 -3 M, [H4py4pa] = 5.77  10-4 M and [EDTA] = 8.20  10 -4 M at 25°C and I = 0.16 M NaCl. The stability constants for the complexes formed by H4edta and La3+ were taken from literature.136 Direct pH-potentiometric titrations of the La3+-H4pypa system were also carried out. Metal solution was prepared by adding the atomic absorption (AA) standard metal ion solution to a H4py4pa solution of known concentration in the 1:1 metal to ligand molar ratio for La(III). Ligand and metal concentrations were ~ 4.17  10-4 M. The exact amount of acid present in the atomic standard metal solutions standards was determined by Gran’s method134 titrating equimolar solutions of La(III) and Na2H2-EDTA. Each titration consisted of 100-150 equilibrium points in the pH range 1.6-11.5, equilibration times for titrations were 2 min for pKa titrations and up to 5 min for metal complex titrations. Three replicates of each titration were performed for each system. Relying on the stability constant for the species [La(Hpy4pa)] obtained by the two different methods, the fitting of the direct potentiometric titrations was possible and yielding the stability constants in Table 7.3. All the potentiometric measurements were processed using the Hyperquad2013 software125 while the obtained spectrophotometric data were processed with the HypSpec2014124 program. Proton dissociation constants corresponding to hydrolysis of La(III) aqueous ion included in the calculations were taken from Baes and Mesmer.137 The overall equilibrium (formation) constants log β referred to the overall equilibria: pM + qH + rL ⇆ MpHqLr (the charges are omitted), where p might also be 0 in the case of protonation equilibria and q can be negative for hydroxide species. Stepwise equilibrium constants log K correspond to the difference in log units between the overall constants of sequentially protonated (or hydroxide) 241  species. The parameter used to calculate the metal scavenging ability of a ligand towards a metal ion, pM, is defined as –log [Mn+]free at [ligand] = 10 mM and [Mn+] = 1 M at pH = 7.4.128  7.4.5.   225Ac Quantification      For all the following procedures using 225Ac, the presence of the radionuclide was measured based on the emission of its daughters: 211Fr and/or 213Bi. All measures of 225Ac (iTLC-SG, gamma-spectrometry or gamma-counter measures) were therefore performed after reaching equilibrium with the daughters: 40 min for 211Fr and 6 h for 213Bi. For accurate determination of the specific activities and the biodistribution data of the radioimmunoconjugates, the 213Bi peak was used. We chose this peak as it would not have any downscatter contributions from the 211Fr one which has a lower energy.  All measures were decay-corrected based on the half-life of 225Ac.  7.4.6.   Radiolabeling of H4py4pa and DOTA      An aliquot of a ligand solution (10-3 – 10-6 M, 10 L) of desired concentration was mixed with ammonium acetate solution (1 M, pH = 7, 88 L), and then 225Ac in 0.05 M nitric acid (65 kBq, 2 L) was added. The final solution (10-4 – 10-7 M) was confirmed at pH ~ 7 by pH papers and then incubated at room temperature for 30 min. The radiochemical yield (RCY) was determined with iTLC-SA plate developed with EDTA solution (50 mM, pH 5.2) and read by a TLC reader. The areas corresponding to the free metal (that migrated to the solvent front) and the labeled complex (that stayed at the baseline) were integrated and used to calculate the RCY%. 242  7.4.7.   Antibody Conjugation with the Bifunctional Chelators      Trastuzumab (0.5-1 mg) was conjugated with either H4py4pa-phenyl-NCS or p-SCN-benzyl-DOTA at 37°C for 1-3 h with a chelator:mAb molecular ratio of either 3:1, 5:1, 10:1 or 20:1 in PBS pH 8.9-9.1 with a final concentration of 2 mg/mL of antibody. Conjugated antibodies were then purified using centrifugal filter units with a 50 kDa molecular weight cutoff and washed once with the buffer then used for radiolabeling. The concentration of the solution containing the purified immunoconjugates was determined by a Bradford assay according to the manufacturer’s recommendations (Sigma-Aldrich).  7.4.8.   Immunoconjugate Radiolabeling with 225Ac      For studies using the conjugated antibodies, i.e, either optimization of antibody labeling, plasma stability or biodistribution studies, 225Ac solution was purchased from ITG. For radiolabeling, the immunoconjugates (0.1-1.0 mg) and the 225Ac solution (1.1-39.6 µL that correspond to 0.1-3.0 MBq) were added to different reaction buffers including 0.15-0.2 M ammonium acetate buffer (pH = 5-8), 0.1 M tris(hydroxymethyl)aminomethane (TRIS) buffer (pH = 9) and 2 M tetramethyl ammonium acetate (TMAA) buffer pH 5-6. The resulting solution was incubated for 30-60 min either at RT, 37, 45 or 50 °C. The radiochemical yields (RCY) were determined using iTLC-SG plate with EDTA solution (50 mM, pH = 7) as a solvent and read 6 h post-running which is the time that is needed for 225Ac to reach equilibrium with both 211Fr and 213Bi. For the following methods, 0.15-0.2 M ammonium acetate solution (pH = 7) at RT for H4py4pa-phenyl-Trastuzumab and 0.1 M TRIS buffer (pH = 9) at 45 °C for DOTA-benzyl-Trastuzumab were used. The 225Ac-labeled antibodies were purified by PD-10 desalting columns and concentrated using 50 kDa 243  molecular weight cut off filters. The chemical purity was determined by iTLC-SG plate and the specific activities were calculated based on the quantity of 225Ac determined by gamma-spectrometry and the associated quantity of Trastuzumab determined by size-exclusion HPLC. The HPLC buffer was an isocratic gradient of 0.1 M sodium phosphate monobasic dihydrate, 0.1 M sodium phosphate dibasic dodecahydrate, 0.1 M sodium azide and 0.15 M sodium chloride (pH 6.2–7.0).   7.4.9.   Antibody Immunoreactivity      The immunoreactivity fractions of the 225Ac-labeled antibodies were estimated according to the Lindmo cell-binding method using the human ovarian cancer cell line SKOV-3 as previously described.175 Briefly, 0.45 µg of the labeled antibody, diluted in 1% PBS-BSA, was added to different cell concentrations in duplicate and incubated 1 h at 37°C. The bound and unbound antibody fractions were determined using the WIZARD2 gamma counter (PerkinElmer). Linear regression of total/bound activity against 1/[cell concentration] was used to estimate the remaining immunoreactivity of the labeled antibodies.  7.4.10.   Mouse Serum Challenge Experiments      For [225Ac][Ac(py4pa)]- and [225Ac][Ac(DOTA)]- complexes, 100 L of the labeled complex (65 kBq) was added to 700 L mouse serum. For the immunoconjugates, 3 μL of purified 225Ac-labeled antibodies (16.4±0.9 μg), that corresponded to 24.5 and 19.6 kBq of 225Ac for [225Ac][Ac(DOTA-benzyl-Trastuzumab)] and [225Ac][Ac(py4pa-phenyl-Trastuzumab)], 244  respectively, were added to 500 μL of mouse serum. In each case, the mixture was incubated at 37oC and an aliquot of the mixture was spotted on iTLC-SA ([225Ac][Ac(py4pa)]- and [225Ac][Ac(DOTA)]-) or iTLC-SG (immunoconjugates) plate at desired time-points, and then developed in EDTA solution (50 mM, pH = 5.2). The TLC plate was read by a TLC reader. The free metal migrated to the solvent front while the complex stayed at the baseline. The areas of both peaks were used to calculate RCY%.  7.4.11.   Assessment of Radiopharmaceutical Biodistributions      All animal experiments were performed at the Animal Resource Centre of the BC Cancer Research Centre in accordance with the institutional guidelines of the University of British Columbia Animal Care Committee (Vancouver, British Columbia, Canada) and under the supervision of authorized investigators. Female immunodeficient NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) mice (obtained from an in-house breeding colony) were subcutaneously injected with 6 × 106 SKOV-3 cells in 1:1 mixture with matrigel (BD Bioscience, Mississauga, Ontario, Canada). Two weeks after tumor cell inoculation, when tumors reached 100 mm3 in average, mice were injected with 10.1±0.7 kBq of 225Ac that corresponded to 50.3±3.6 μg of labeled Trastuzumab for both constructs. At different time points post-injection (Day 1, 3, 6 and 10), mice were euthanized by inhalation of isoflurane-O2 mixture (3%, 2 mL/min) followed by CO2. Blood was withdrawn by intracardiac puncture, tumors and organs of interest were then harvested, washed in PBS, blotted dry and weighed. For each sample, activity was measured by the WIZARD2 gamma counter by measuring the 213Bi at least 6 h post-mortem, with background 245  and decay correction based on the 225Ac half-life. The activity uptake was expressed as a percentage of the injected dose per gram of tissue (% ID/g) of 3-4 mice per group.  7.4.12.   Data Analysis      All statistical analyses were performed using GraphPad Prism 7 software. Differences of the radioimmunoconjugates in vivo uptake were calculated using a two-way ANOVA and values <0.05 were considered significantly significant.     246  Chapter 8: Other Work    8.1.   Improved Synthesis of H2dedpa-benzyl-NCS and H4octapa-benzyl-NCS  8.1.1.   Results and Discussion  8.1.1.1.   Synthesis       H2dedpa-benzyl-NCS and H4octapa-benzyl-NCS were previously synthesized and coupled to acyclic peptide c(RGDyK) which targets αvβ3 integrin, and a monoclonal antibody Trastuzumab, which targets the human epidermal growth factor receptor type 2 (HER2), respectively, showing promising biodistribution results.26,37,196 The old synthetic scheme adopted either benzyl or nosyl N-protection, which made the later N-alkylation very challenging.37,196 For example, the addition of picolinate or acetate arms to the nosyl-protected secondary amine led to very low yields even after extended reaction time. As a result, nosyl deprotection was not continued. Also, the reduction of p-NO2-benzyl-tBu4octapa (compound 8.8) using Pd/C catalyzed hydrogenation in a mixture of 3 M HCl and glacial acetic acid,196 resulted in fragmentation of the compound with free picolinate arm observed in TLC and MS. This, however, was not observed if MeOH was used as solvent. Interestingly, the fragmentation was not as obvious in the case of H2dedpa. In addition, the previous protocol proposed the sequence of N-isothiocyanate (NCS) conversion as (1) reduction of -NO2 to -NH2 (2) deprotection of picolinate (3) conversion of -NH2 to -NCS.37,196 This required 3M HCl/ glacial AcOH as the solvent in the last step, which was highly unfavorable to the subsequent -NH2/thiophosgene (CSCl2) reaction, where the amine should theoretically remain 247  nucleophilic to attack the partial positive sulfur atom in CSCl2. Consequently, the reaction consumed excessive CSCl2 (15 equiv) and extended reaction time.196       To overcome the discussed challenges, a new synthetic scheme is presented herein (Scheme 8.1). First, the p-nitrobenzyldiamine (compound 8.2) was synthesized according to a modified literature-procedure.240 The commercially available (L)-4-nitrophenylalanine methyl ester was used as starting material and converted to the amide (compound 8.1) using 7N NH3 in MeOH, followed by amide reduction to compound 8.2 with borane-tetrahydrofuran (BH3-THF) with an overall yield of 65%.240 Without any N-protecting group, the p-NO2-benzyl-dedpa scaffold (compound 8.4) was constructed directly by coupling this diamine backbone with tert-butyl 6-formylpicolinate (compound 8.3, synthesized according to published protocol17) through the “one-pot” Schiff base synthesis and reduction. Not only did this new route shorten the synthesis from three challenging steps, which required one to two weeks including purifications, to one step that finished in less than two days, but it also significantly improved the overall yield. To yield H2dedpa-benzyl-NCS (compound 8.7), the nitro group was reduced by Pd/C catalyzed hydrogenation in MeOH, followed by picolinate deprotection (compound 8.6) and finally NCS conversion using CSCl2 at room temperature overnight. The final NCS analog was purified through reverse phase HPLC, lyophilized to give a white solid as product.       For the synthesis of NCS-bifunctional octapa, compound 8.4 was alkylated with tert-butyl bromoacetate at 60°C for 24-48 h to give compound 8.8 in an average yield of ~40%, followed by nitro-reduction to compound 8.9 as in the case of H2dedpa-benzyl-NCS. Instead of first hydrolyzing the picolinate arm, the p-benzyl amino group was converted to N-isothiocyanate using 1.1 equivalent of CSCl2 and ethyl acetate as solvent. Facile conversion to compound 8.10 was 248  observed in 3 h. The crude mixture was concentrated and used without further purification. The advantages of tBu4octapa-benzyl-NCS is the versatility that it can either be deprotected with TFA/DCM at room temperature overnight to afford H4octapa-benzyl-NCS (compound 8.11) with an overall yield of 70%; or first be coupled to small peptides or molecules, 4-(2-amino-ethyl)benzenesulfonamide (compound 8.12, Carbonic Anhydrase-IX-targeting) and PSMA-ureido (compound 8.13, PSMA-targeting), in this report, before deprotection. The former would be useful for the synthesis of radio-immunoconjugates, which is not compatible with strong acid; while the latter prevents the potential interferences to the conjugations from the free carboxylic acid groups. H4octapa-benzyl-NCS has been characterized by 1H and 13C{1H} NMR spectroscopies, as well as HR-ESI-MS while the bioconjugates have been confirmed by HR-ESI-MS.      In the future, the radiolabeling efficiency and in vitro stability of H4octapa-benzyl-PSMA-ureido (compound 8.13) and H4octapa-benzyl-sulfonamide (compound 8.12) will be assessed; if promising, further biological studies with tumor models will be planned because it will be valuable to compare the performances with the acetate arm functionalization (Chapter 2).   249                        Scheme 8.1 Reagents and conditions: i) 7N NH3 in MeOH, 85 %; ii) BH3-THF, Dry THF, N2(g), 77%; iii) 1. MeOH 2. NaBH3CN, MeOH, sat. NaHCO3, 64%; iv) Pd/C, H2, MeOH; v) TFA/DCM, o/n; vi) CSCl2, CHCl3, glacial AcOH, 3M HCl, RT, o/n 30%; vii) tert-butyl bromoacetate, K2CO3, Dry ACN, 48 h, 40%; viii) Pd/C, H2, MeOH; ix) CSCl2, CHCl3, EtOAc, RT, 4h; x) TFA/DCM, o/n, RT, 70%; xi) 1. 4-(2-aminoethyl)-benzenesulfonamide, DIEA, dry ACN, RT, o/n, 32%; 2. TFA/DCM, RT, o/n; xii) 1. tBu-(Lys-CO-Glu), DIEA, dry ACN, RT, o/n; 2. TFA/DCM, RT, o/n, 65% 250  8.1.2.   Experimental Procedures 8.1.2.1.    Synthesis and Characterization 2-Amino-3-(4-nitrophenyl)propenamide (8.1)       Triethylamine (TEA) (9.95 mL, 71.3 mmol) was added to a slurry of methyl 4-nitro-L-phenylalaninate hydrochloride (6.64 g, 25.5 mmol) in anhydrous MeOH (40 mL), followed by diethyl ether (130 mL), and the sealed solution was cooled at -10°C for 2 h for precipitation. The precipitate was filtered off by vacuum filtration, and the filtrate was collected and rotary-evaporated to a limpid yellow oil. The crude mixture was then dissolved in 7N NH3 in MeOH and stirred at room temperature for 4 d. The solvent was evaporated in vacuo to yield a dark yellow oil (2.20 g, 85%). 1H NMR (400 MHz, 298 K, MeOD): δ 8.19 (d, 3J = 8.8 Hz, 2H), 7.51 (d, 3J = 8.7 Hz, 2H), 3.65 (dd, 3J = 7.4, 6.3 Hz, 1H), 3.17-3.12 (m, 1H), 3.00-2.95 (m, 1H). LR-ESI-MS: calcd for [C9H11N3O3 + H]+ 210.2;  found [M+H]+ 210.2 3-(4-Nitrophenyl)propane-1,2-diamine (8.2)       In a three-neck round-bottom flask, compound 8.1 (1.36 g, 6.51 mmol) was dissolved in dry THF, sealed and purged with Ar(g) while submerged in an ice-water bath. BH3-THF (1 M, 32.5 mL, 32.5 mol) was added through a syringe to the stirred mixture at 0°C and the reaction mixture was stirred at 0°C for 3 h before refluxing for 48 h at 65°C. Upon completion, the mixture was cooled to -10°C before H2O (12 ml) was added slowly to quench the reaction (color changed from cloudy pale pink to clear orange). About 50% volume of the solvent was evaporated in vacuo, then 18% HCl (aq) (16-17 mL) was added. The mixture was stirred overnight at room temperature and then 80°C for 15 min. The mixture was cooled in an ice-water bath for 1 h while stirring. The mixture was filtered, and then sufficient concentrated ammonium hydroxide solution (~100 mL) 251  was added to bring the pH to 12. The aqueous layer was extracted with CHCl3 (75 mL × 5), and the combined aqueous layers were dried over anhydrous MgSO4, filtered and evaporated in vacuo to yield a brown oil (0.98 g, 77%). 1H NMR (400 MHz, 298 K, MeOD): δ 8.19 (d, 3J = 8.0 Hz, 2H), 7.50 (d, 3J = 8.3 Hz, 2H), 3.14 – 2.99 (m, 1H), 2.96-2.91 (m, 1H), 2.77-2.70 (m, 2H), 2.59-2.54 (m, 1H). 13C {1H} NMR (75 MHz, 298 K, MeOD): δ 130.2, 123.9, 55.0, 48.3, 42.3 LR-ESI-MS: calcd for [C9H13N3O2 + H]+ 196.2; found [M+H]+ 196.3 Di-tert-butyl-6,6'-(((3-(4-nitrophenyl)propane-1,2-diyl)bis-(azanediyl))-bis-(methylene))-(S)-dipicolinate (8.4)       To a round-bottom flask with a stirred solution of compound 8.2 (130 mg, 0.666 mol) in dry MeOH (4 mL) was added compound 8.3 (0.220 g, 1.34 mmol, 2 equiv). The mixture was stirred for 6 h at room temperature. NaBH3CN (0.310 g, 4.87 mmol) was added after confirmation of the Schiff base by MS, and the reduction reaction proceeded overnight at room temperature. The reaction was then diluted in DCM (10 mL) and quenched with saturated NaHCO3 in water (10 mL). The organic phase was separated and MeOH in the aqueous phase was removed in vacuo to give an aqueous solution which was extracted with DCM (20 mL x 3). The combined organic phases were dried over anhydrous Na2SO4, and clarified by filtration. The organic phase was concentrated in vacuo and purified through a neutral alumina column (CombiFlash Rf automated column system, 12 g gold silica column, A: DCM B: MeOH, 0-5 % B). The product fractions were rotary-evaporated to give a yellow oil (0.25 g, 64%).1H NMR (400 MHz, 298 K, CDCl3): δ 8.06 (d, 3J = 8.1, 2H), 7.84 (d, 3J = 7.6 Hz, 2H), 7.71 (t, 3J = 8.9 Hz, 2H), 7.50 – 7.27 (m, 4H), 4.05-3.88 (m, 4H), 2.99-2.96 (m, 2H), 2.85-2.81 (m, 1H), 2.69-2.66 (m, 1H), 2.55-2.51 (m, 1H), 1.57 (s, 18H). 13C {1H} NMR (75 MHz, 298 K, CDCl3): δ 164.0, 160.4, 160.2, 148.7, 148.6, 147.4, 252  146.5, 137.4, 137.3, 130.3, 125.3, 123.5, 123.2, 82.16, 58.3, 54.9, 53.5, 52.3, 51.9, 39.3, 28.1. LR-ESI-MS: calcd for [C31H39N5O6 + H]+ 578.7; found [M+H]+ 578.2  Di-tert-butyl-6,6'-(((3-(4-aminophenyl)propane-1,2-diyl)bis(azanediyl))bis-(methylene))(S)-dipicolinate (8.5)       Compound 8.4 (100 mg, 0.173 x − mol) was dissolved in dry MeOH (7 mL) in a three-neck round-bottom flask, saturated with N2(g). Pd/C (10 % w/w) was added under a stream of N2(g). The mixture was stirred for 6 h at room temperature under an inert atmosphere. Then, Pd/C was filtered off through a Celite bed and washed with MeOH ad libitum. The filtrate was concentrated to a yellow oil and used without purification. (S)-6,6'-(((3-(4-aminophenyl)propane-1,2-diyl)bis(azanediyl))bis(methylene))-dipicolinic acid (8.6)       Compound 8.5 (74.2 mg, 0.160 mmol) was dissolved in TFA/DCM (2 mL, 1:1 v/v) in a round-bottom flask. The mixture was stirred vigorously at room temperature overnight, and then rotary-evaporated and used without purification. LR-ESI-MS: calcd for [C23H25N5O4 + H]+ 436.5; found [M + H]+ 436.1 H2dedpa-benzyl-NCS (8.7)       Compound 8.6 (40.0 mg, 0.919 mol) was dissolved in 3 M HCl/glacial AcOH (2 mL, 1:1 v/v) in a round-bottom flask. CSCl2 (107 l, 1.38 mmol) in CHCl3 (4 mL) was added dropwise using a Pasteur pipette over 2 min to the stirred mixture of the starting material. The resulting mixture was stirred vigorously at room temperature overnight. Upon reaction completion, CHCl3 was removed 253  with a Pasteur pipette and the aqueous phase was washed with CHCl3 (1 mL x 5). The resulting aqueous layer was diluted with H2O (2 mL) and purified by reverse phase HPLC (A: ACN, B: H2O/0.1% TFA, 5-60 % A over 40 min, 10 mL/min, tR = 26 mins. The product fractions were combined and lyophilized to give a white solid (13.1 mg, 30 % over 3 steps). 1H NMR (400 MHz, 298 K, MeOD): δ 8.15-8.05 (m, 4H), 7.73-7.70 (m, 2H), 7.33-7.31 (m, 2H), 7.23-7.20 (m, 2H), 4.83-4.62 (m, 4H), 3.89 (m, 1H), 3.62-3.56 (m, 1H), 3.45-3.42 (m, 2H), 3.89 (m, 1H). 13C {1H} NMR (75 MHz, 298 K, MeOD- d4): δ 167.7, 167.5, 154.5, 153.3, 148.3, 148.3, 140.8, 140.7, 135.6, 132.2, 132.5, 137.6, 127.3, 126.4, 126.4, 59.3, 50.7, 36.0  HR-ESI-MS: calcd for [C24H23N5O4S+H]+ 478.1549; found [M+H]+ 478.1556 Elemental analysis: calcd % for H2dedpa-benzyl-NCS·2TFA·1MeOH (C24H24N5O4S·  2TFA·1MeOH = 737.6274): C 47.16 H 4.09 N 9.48; found %: C 47.1 H 4.03 N 9.45  Di-tert-butyl-6,6'-((2,2,13,13-tetramethyl-7-(4-nitrobenzyl)-4,11-dioxo-3,12-di-oxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))(S)-dipicolinate (8.8)       Compound 8.4 (90.8 mg, 0.160 mmol) was dissolved in dry ACN in a round-bottom flask, and then tert-butylbromoacetate (81.5 L, 0.550 mmol) and K2CO3 (127 mg, 0.920 mmol) were added sequentially. The reaction mixture was stirred at 60°C for 48 h. K2CO3 was removed by centrifugation and then washed with ACN/DCM ad libitum. The combined supernatants were concentrated in vacuo and purified with a neutral alumina column. (CombiFlash Rf automated column system; neutral alumina, A: Hex B: EtOAc, 0-100% B). The product fractions were rotary-evaporated to yield a yellow oil (51.6 mg, 40%). 1H NMR (400 MHz, 298 K, CDCl3): δ 7.97 (d, 3J = 8.7 Hz, 2H), 7.78 (t, 3J = 6.5 Hz, 2H), 7.58 (t, 3J = 7.7 Hz, 1H), 7.53-7.43 (m, 3H), 7.24 (d, 3J = 8.6 Hz, 2H), 4.00 – 3.89 (m, 4H), 3.35 – 3.17 (m, 4H), 3.01 – 2.90 (m, 2H), 2.80-2.79 (m, 2H), 254  2.47-2.42 (m, 1H), 1.58 (2s, 18H), 1.38 (2s, 18H). 13C {1H} NMR (75 MHz, 298 K, CDCl3): δ 170.7, 170.6, 164.1, 164.0, 160.3, 160.2, 148.8, 148.7, 148.3, 146.2, 130.3, 126.2, 125.8, 123.3, 123.2, 123.1, 82.1, 82.0, 81.1, 81.0, 61.5, 60.9, 57.0, 56.7, 54.2, 53.5, 52.4, 35.7, 28.2, 28.2, 28.1. LR-ESI-MS: calcd for [C43H59N5O10 + H]+ 806.4; found [M+H]+ 806.4 Di-tert-butyl 6,6'-((7-(4-aminobenzyl)-2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-dia-zatetradecane-6,9-diyl)bis(methylene))-(S)-dipicolinate (8.9)       Compound 8.8 (60.4 mg, 0.910 mol) was dissolved in dry MeOH (7 mL) in a three-neck round-bottom flask, saturated with N2(g). Pd/C (10% w/w) was added under a stream of N2(g). The mixture was then stirred for 6 h at room temperature under an inert atmosphere before Pd/C was filtered off through a Celite bed and washed with MeOH ad libitum. The filtrate was concentrated in vacuo to a yellow oil and used without purification. tBu4octapa-benzyl-NCS (8.10)       Compound 8.9 (40 mg, 0.52 mol) was dissolved in EtOAc (1 mL), followed by the addition of CSCl2 (0.800 L, 1.04 mol). The mixture was stirred vigorously at room temperature for 4 h, and then the solvent was rotary-evaporated to a brown oil. The crude mixture was used without further purification. LR-ESI-MS: calcd for [C44H59N5O8S + H]+ 818.4; found [M+H]+ 818.5  H4octapa-benzyl-NCS (8.11)       Compound 8.10 (40 mg, 0.77 mol) was dissolved in TFA/DCM (2 mL, 1:1 v/v), and then stirred vigorously at room temperature overnight. DCM was then removed in vacuo and the residue was diluted in water (2 mL), and purified by reverse phase HPLC (A: ACN, B: H2O/0.1% TFA, 5-60 % A over 40 min, 10 mL/min, tR = 32.42 min). The product fractions were lyophilized to give 255  a white solid (26.14 mg, 70 %). 1H NMR (400 MHz, 298 K, CDCl3): δ 8.02-7.89 (m, 4H), 7.60-7.55 (m, 2H), 7.21-7.19 (m, 2H), 7.13-7.11 (m, 2H), 5.10-5.07 (m, 2H), 4.92-4.89 (m, 2H), 4.70 (broad s, 2H), 4.51-4.47 (m, 1H), 4.14 (broad s, 1H), 4.01-3.94 (m, 2H), 3.68-3.60 (m, 2H), 3.24-3.20 (m, 1H), 2.68-2.62 (m, 1H). 13C{1H} NMR (75 MHz, 298 K, CDCl3): δ 174.3, 169.1, 167.3, 167.0, 161.4, 160.7, 152.6, 149.0, 148.5, 139.9, 139.8, 138.8, 137.1, 131.5, 131.1, 127.8, 126.9, 126.0, 125.2, 61.0, 55.5, 34.6. HR-ESI-MS: calcd for [C28H27N5O8S +Na]+ 594.1659; found 594.1652 H4octapa-benzyl-sulfonamide (8.12)       To a solution of compound 8.10 (27.7 mg, 32.4 mol) in dry ACN (1 mL) was added 4-(2-aminoethyl)benzenesulfonamide (7.14 mg, 35.7 mol) and TEA (5.00 L, 35.7 mol). The mixture was stirred at 60°C overnight, followed by rotary-evaporation of the solvent. The crude mixture was redissolved in DCM (1 mL) and TFA (1 mL), and then stirred overnight at room temperature again. Upon completion, DCM was removed in vacuo and the mixture was diluted with water (2 mL), and then purified with reverse phase HPLC (A: ACN/ 0.1%TFA, B: H2O/0.1% TFA, 5-80 % A over 40 min, 10 mL/min, tR = 22.10 min). The product fractions were lyophilized to give a white solid (8.3 mg, 32 %). HR-ESI-MS: calcd for [C36H39N7O10S2 +Na]+ 794.2278; found 794.2288 H4octapa-benzyl-PSMA-ureido (8.13)       To a solution of compound 8.10 (18.8 mg, 23.0 mol) in dry ACN (1 mL) was added di-tert-butyl((6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)glutamate (12.3 mg, 25.3 mol) and TEA (3.53 L, 25.28 mol). The mixture was stirred at 60°C overnight, followed by rotary-256  evaporation of the solvent. The crude mixture was redissolved in DCM (1 mL) and TFA (1 mL), and then stirred overnight at room temperature again. Upon completion, DCM was removed in vacuo and the mixture was diluted with water (2 mL) and purified by reverse phase HPLC (A: ACN/0.1% TFA B: H2O/ 0.1% TFA, 5–80 % A over 40 min, 10 mL/min, tR = 19.72 min). The product fractions were lyophilized to give yellowish solid (13.7 mg, 65%). HR-ESI-MS: calcd for [C40H48KN8O16+H]+ 935.2858; found 935.2860   257  8.2.   Comparison of 177Lu-labeled H4pypa-phenyl-Panitumumab and H4pypa-PEG4-benzyl-Panitumumab  8.2.1.   Results and Discussion 8.2.1.1.   Synthesis of H4pypa-PEG4-benzyl-NCS   Scheme 8.2 Reagents and conditions: i) 2 M NaOH, p-TsCl, THF, 0˚C, 3h, 51%; ii) potassium phthalimide, dry ACN, 60˚C, 18 h, 72%; iii) TEA, DMAP, p-TsCl, 0˚C-RT, 48%   Scheme 2 outlines the synthesis of the poly(ethyleneglycol) (PEG4) linker (compound 8.16). The synthesis began with the selective tosylation of one of the terminal hydroxyl groups in tetraethylene glycol with para-tosyl chloride (compound 8.14, 51%). The tosyl group then underwent nucleophilic substitution with potassium phthalimide in dry ACN (compound 8.15, 72%). Finally, the remaining hydroxyl group was tosylated (48%) to give compound 8.16 which was used in the synthesis of the bifunctional pypa precursor (Scheme 8). 258   Scheme 8.3 Reagents and conditions: iv) K2CO3, dry THF, 25˚C, 48 h; v) hydrazine, EtOH, 60˚C, overnight; vi) NHS, EDC, dry ACN, N2(g), RT, overnight, 86%; vii) DIPEA, DCM,  N2(g), RT, overnight; viii) TFA/DCM, RT, overnight, 65%; ix) CSCl2,1 M HCl/ glacial acetic acid/CHCl3, 24% 259  Compound 8.18 was not purified with column chromatography due to its instability on silica/neutral alumina columns. The synthesis proceeded to the deprotection of the phthalimide group using hydrazine monohydrate (compound 8.19) in ethanol and the product was coupled to compound 8.20 to give compound 8.21 in the presence of DIPEA under an inert atmosphere. The tert-butyl ester groups and the boc-protecting group in compound 8.21 were removed with TFA in DCM (1:1) at room temperature, and then the product was purified with reverse-phase HPLC (10-30% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) in water over 30 min, 10 mL/min, tR = 20.7 min) (compound 8.22). Finally, the aniline group in compound 8.22 was activated using thiophosgene in a vigorously stirred biphasic solution of hydrochloric acid (1 M, aq)/ glacial acetic acid/ chloroform overnight at room temperature. The final product (8.23) was isolated with reverse-phase HPLC (28-50% ACN/0.1%TFA in H2O over 40 min, 10 mL/min, tR = 19.0 min) and then lyophilized to a white fluffy solid in 24% yield (Figure 8.1). 260   Figure 8.1 1H NMR spectrum of H4pypa-PEG4-benzyl-NCS (400 MHz, CD3CN, 298K).  8.2.1.2.   Radiolabeling and Human Serum Stability of [177Lu][Lu(pypa-phenyl-Panitumumab)] and [177Lu][Lu(pypa-PEG4-benzyl-Panitumumab)]      Both H4pypa-phenyl-NCS (S) and H4pypa-PEG4-benzyl-NCS (L) were coupled to the monoclonal antibody, Panitumumab, which targets epidermal growth factor receptor (EGFR), in both 5:1 and 20:1 chelator:antibody reaction ratios (i.e. S5, S20, L5, L20). The lower the chelator:antibody ratio, the less modifications are made on the antibody and the better the antibody retains its immunoreactivity. However, if the ratio is too low, the radiolabeling yield could be significantly compromised. Therefore, two acceptable ratios were tested in this experiment. 261       Each conjugate (1 nmol) was radiolabeled with [177Lu]LuCl3 in 0.04 M HCl solution (10 MBq) for 1 h at room temperature in NH4OAc solution (0.2 M, pH = 7). The samples of [177Lu]Lu-Panitumumab (Panitumumab) and free lutetium (Lu) in the labeling buffer were prepared as the control experiments. The radiochemical yield % was measured with instant thin-layer chromatography impregnated with silica gel (iTLC-SG), developed in an EDTA buffer (50 mM, pH = 5.6). As shown in Figure 8.2, all the conjugates were radiolabeled quantitatively (99%) within an hour at room temperature, which means that the conjugations did not hamper the radiolabeling efficiency. The radiolabelin