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Synthesis, evaluation, and application of new ligands for radiometal based radiopharmaceuticals Price, Eric William 2014

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SYNTHESIS, EVALUATION, AND APPLICATION OF NEW LIGANDS FOR RADIOMETAL BASED RADIOPHARMACEUTICALS  by ERIC WILLIAM PRICE  Hons. B. Sc, co-op, The University of Victoria, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2014  © Eric William Price, 2014 ii  Abstract  Radiometals comprise many useful radioactive isotopes of various metallic elements.  When properly harnessed, these have valuable emission properties that can be used for diagnostic imaging techniques, such as single photon emission computed tomography (SPECT, e.g. 67Ga, 99mTc, 111In, 177Lu) and positron emission tomography (PET, e.g. 68Ga, 64Cu, 44Sc, 86Y, 89Zr), as well as therapeutic applications (e.g. 47Sc, 114mIn, 177Lu, 90Y, 212/213Bi, 212Pb, 225Ac, 186/188Re).  A fundamental critical component of a radiometal-based radiopharmaceutical is the ligand that binds the radiometal ion in a tight stable coordination complex so that it can be properly directed to a desirable molecular target in vivo.  This thesis describes the design, synthesis, and evaluation of novel acyclic ligands based on the versatile picolinic acid moiety.  Acyclic ligands have been selected because facile ambient temperature radiolabeling is an important property when working with heat sensitive molecules such as antibodies, as many currently used ligands require high temperatures for optimal radiolabling performance.  Previous work in the Orvig group has determined the acyclic ligand H2dedpa to possess ideal properties for 67/68Ga radiochemistry.  In light of this success, this thesis has been dedicated to expansion of the H2dedpa molecular scaffold to accommodate larger radiometals with ligand denticities ranging from 8-10.  Once synthesized, new ligands are studied by standard chemical characterization, as well as potentiometric titrations to determine thermodynamic stability parameters, and radiolabeling and in vitro/in vivo stability studies of both “bare” ligands and antibody bioconjugates.  The ligand H4octapa is a highlight of this body of work, and has been found to possess excellent properties with the radiometals 111In and 177Lu, matching or in some cases surpassing the current industry “gold standard” ligand DOTA.  A second highlight is the ligand H6phospa, which is demonstrated to possess enhanced 89Zr radiolabeling properties to H4octapa, showing the best 89Zr radiolabeling performance of any new ligand in several decades, with only DFO retaining superior properties.   iii  Preface Chapter 1 is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Orvig, C., Matching Chelators to Radiometals for Radiopharmaceuticals. Chem. Soc. Rev. 2014, 43 (1), 260-290, Copyright 2014 The Royal Society of Chemistry.  This review article was written by Eric Price, with input and editing from Dr. Chris Orvig.  Chapter 2 is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Cawthray, J. F.; Bailey, G. A.; Ferreira, C. L.; Boros, E.; Adam, M. J.; Orvig, C., H4octapa: An Acyclic Chelator for 111In Radiopharmaceuticals. J. Am. Chem. Soc. 2012, 134 (20), 8670-8683, Copyright 2014 American Chemical Society.  Eric Price performed the synthesis, with some assistance from Gwendolyn Bailey during her summer NSERC USRA term.  Radiochemistry was performed by Eric Price at TRIUMF/Nordion with assistance from Dr. Cara L. Ferreira.  Potentiometric titrations, data fitting, and DFT calculations were performed by Dr. Jacqueline F. Cawthray.  Animal experiments were contracted to the BC Cancer Agency, and the protocol used in these animal studies was approved by the Institutional Animal Care Committee (IACC) of the University of British Columbia (protocol # A10-0171) and was performed in accordance with the Canadian Council on Animal Care Guidelines.  This project was supervised by Dr. Michael J. Adam and Dr. Chris Orvig.  The manuscript was written by Eric Price.  Chapter 3 is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Zeglis, B. M.; Cawthray, J. F.; Ramogida, C. F.; Ramos, N.; Lewis, J. S.; Adam, M. J.; Orvig, C., H4octapa-Trastuzumab: Versatile Acyclic Chelate System for 111In and 177Lu Imaging and Therapy. J. Am. Chem. Soc. 2013, 135 (34), 12707–iv  12721, Copyright 2014 American Chemical Society.  The synthesis work was performed by Eric Price, with some assistance from Caterina F. Ramogida.  Eric traveled to Memorial Sloan-Kettering Cancer Center to work with Dr. Brian M. Zeglis and Dr. Jason S. Lewis to perform radiolabeling, SPECT imaging, and animal experiments.  Nicholas Ramos assisted with dissection and biodistribution experiments when Dr. Zeglis was unavailable.  Dr. Jacqueline F. Cawthray performed potentiometric titrations, data fitting, and DFT calculations.  All animal experiments were performed under an Institutional Animal Care and Use Committee-approved protocol at Memorial Sloan-Kettering Cancer Center, and the experiments followed institutional guidelines for the proper and humane use of animals in research.  Eric Price wrote the manuscript.  Dr. Chris Orvig and Dr. Michael Adam supervised this project. Chapter 4 is an adaption of published work, and is reproduced in part, with permission from Price, E. W.; Ferreira, C. L.; Adam, M. J.; Orvig, C., High denticity ligands based on picolinic acid for In-111 radiochemistry. Can. J. Chem., doi: 10.1139/cjc-2013-0542, 2014.  Synthesis was performed by Eric Price, and radiochemistry was performed by Eric Price with assistance from Dr. Cara L. Ferreira at TRIUMF/Nordion.  The manuscript was written by Eric Price.  The project was supervised by Dr. Chris Orivg and Dr. Michael Adam.  Chapter 5 is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Cawthray, J. F.; Adam, M. J.; Orvig, C. Modular Syntheses of H4octapa and H2dedpa, and Yttrium Coordination Chemistry Relevant to 86/90Y Radiopharmaceutical. Dalton Trans. Doi: 10.1039/C4DT00239C, 2014.  Copyright 2014 The Royal Society of Chemistry.  The synthesis work was performed by Eric Price, and the v  potentiometry was performed by Eric Price with assistance from Dr. Jacqueline F. Cawthray, with data fitting and DFT calculations done by Dr. Jacqueline F. Cawthray.  This project was supervised by Dr. Chris Orvig and Dr. Michael Adam.   Chapter 6 is an adaptation of a manuscript in preparation for publication, Price, E. W.; Zeglis, B. M.; Cawthray, J. F.; Lewis, J. S.; Adam, M. J.; Orvig, C., H4octapa vs H4C3octapa for In-111 and Lu-177 Radiochemistry: the Difference of One Carbon. Expected submission date February-March 2014.  Synthesis was performed by Eric Price, potentiometry was performed by Eric Price and Dr. Jacqueline F. Cawthray, data fitting and DFT calculations were performed by Dr. Jacqueline F. Cawthray.  Ligands were mailed to Dr. Brian M. Zeglis and Dr. Jason S. Lewis at Memorial Sloan-Kettering Cancer Center, and radiolabeling experiments were performed by Dr. Brian M. Zeglis.  The manuscript was written by Eric Price.  Dr. Chris Orvig and Dr. Michael Adam supervised this project.  Chapter 7 is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Zeglis, B. M.; Lewis, J. S.; Adam, M. J.; Orvig, C., H6phospa-Trastuzumab: Bifunctional Methylenephosphonate-based Chelator with 89Zr, 111In and 177Lu. Dalton Trans. 2014, 43, 119-131, Copyright 2014 The Royal Society of Chemistry.  Synthesis was performed by Eric Price, and radiochemistry experiments and SPECT imaging were performed by Eric Price and Dr. Brian M. Zeglis at Memorial Sloan-Kettering Cancer Center with supervision and funding from Dr. Jason S. Lewis.  All animal experiments were performed under an Institutional Animal Care and Use Committee-approved protocol at Memorial Sloan-Kettering Cancer Center, and the experiments followed institutional guidelines for the proper and humane use of animals in research.  Eric Price wrote the manuscript, and Dr. Chris Orvig and Dr. Michael Adam supervised the project.  vi  Chapter 8 is an adaptation of published work, and is reproduced in part, with permission from Bailey, G. A.; Price, E. W.; Zeglis, B. M.; Ferreira, C. L.; Boros, E.; Lacasse, M. J.; Patrick, B. O.; Lewis, J. S.; Adam, M. J.; Orvig, C., H2azapa: a Versatile Acyclic Multifunctional Chelator for 67Ga, 64Cu, 111In, and 177Lu. Inorg. Chem. 2012, 51 (22), 12575-12589, Copyright 2014 The American Chemical Society (*These authors contribued equally to this work).  Gwendolyn A. Bailey was an undergraduate honors student supervised by Dr. Orvig and Eric Price, and with supervision and help from Eric Price she completed a majority of the synthesis in this work, assisted with some of the radiochemistry experiments, and co-wrote the manuscript.  Eric Price assisted with the synthetic work while supervising, and also performed some early synthetic work for this project prior to Gwendolyn beginning.  Eric performed radiochemistry at TRIUMF/Nordion with assistance from Dr. Cara L. Ferreira and Gwendoyln Bailey.  Eric performed radiochemistry, PET imaging, and animal experiments at Memorial Sloan-Kettering Cancer Center with Dr. Brian M. Zeglis under the supervision of Dr. Jason S Lewis.  Eric co-wrote and co-authored this manuscript with Gwendolyn.  No work presented in this chapter was performed by Michael J. Lacasse or Eszter Boros; however, these two had previously attempted synthesis of the H2azapa ligand using a different synthetic (and unfortunately unsuccessful) methodology, and therefore provided valuable intellectual contributions.  Dr. Brian O. Patrick performed X-ray crystallography experiments.  All animal experiments were performed under an Institutional Animal Care and Use Committee-approved protocol at Memorial Sloan-Kettering Cancer Center, and the experiments followed institutional guidelines for the proper and humane use of animals in research.  Dr. Chris Orvig and Dr. Michael J. Adam supervised all of this work.  vii  All animal experiments performed at Memorial Sloan-Kettering Cancer Center in Chapters 3, 7, and 8 were performed according to a protocol approved by Memorial Sloan-Kettering Cancer Center's Institutional Animal Care and Use Committee (#08-07-013).  Animal experiments performed in Chapter 2 used a protocol that was approved by the Institutional Animal Care Committee (IACC) of the University of British Columbia (protocol # A10-0171) and was performed by the BC Cancer Agency in accordance with the Canadian Council on Animal Care Guidelines.    viii  Table of Contents  Abstract	  ............................................................................................................................	  ii	  Preface	  ............................................................................................................................	  iii	  Table	  of	  Contents	  ............................................................................................................	  viii	  List	  of	  Tables	  ..................................................................................................................	  xxii	  List	  of	  Figures	  .................................................................................................................	  xxv	  List	  of	  Schemes	  ...........................................................................................................	  xxxiii	  List	  of	  Symbols	  and	  Abbreviations	  ...............................................................................	  xxxiv	  Acknowledgements	  ....................................................................................................	  xxxix	  Dedication	  ........................................................................................................................	  xl	  Chapter	  1:	  Introduction	  .....................................................................................................	  1	  1.1	   Background	  and	  aims	  .......................................................................................................	  1	  1.2	   Nuclear	  imaging	  and	  therapy	  ............................................................................................	  2	  1.3	   Popular	  radiometal	  isotopes	  .............................................................................................	  4	  1.4	   Radiometal-­‐based	  radiopharmaceutical	  design	  ................................................................	  7	  1.4.1	   Macrocyclic	  versus	  acyclic	  ligands	  ..................................................................................	  10	  1.4.2	   Matching	  ligands	  with	  radiometals	  –	  how	  are	  ligands	  evaluated?	  ................................	  11	  1.4.3	   Thermodynamic	  stability	  ................................................................................................	  13	  1.4.4	   Kinetics	  -­‐	  Acid	  dissociation	  and	  competitive	  radiolabeling	  ............................................	  15	  ix  1.4.5	   In	  vitro	  and	  in	  vivo	  stability	  ............................................................................................	  16	  1.5	   A	  selection	  of	  ligands	  and	  their	  most	  suitable	  radiometal	  companions	  ...........................	  18	  1.5.1	   DOTA	  ..............................................................................................................................	  19	  1.5.2	   DOTA	  Derivatives:	  CB-­‐DO2A,	  3p-­‐C-­‐DEPA,	  TCMC,	  and	  Oxo-­‐DO3A	  ..................................	  24	  1.5.3	   TETA	  ...............................................................................................................................	  25	  1.5.4	   TE2A,	  CB-­‐TE2A,	  CB-­‐TE1A1P,	  CB-­‐TE2P,	  MM-­‐TE2A,	  DM-­‐TE2A	  ..........................................	  27	  1.5.5	   Diamsar	  and	  derivatives	  .................................................................................................	  28	  1.5.6	   NOTA,	  NETA,	  and	  TACN-­‐TM	  ...........................................................................................	  29	  1.5.7	   DTPA,	  1B4M-­‐DTPA,	  and	  CHX-­‐A’’-­‐DTPA	  ...........................................................................	  33	  1.5.8	   TRAP	  (PRP9)	  and	  NOPO	  ..................................................................................................	  37	  1.5.9	   AAZTA	  and	  derivatives	  (DATA)	  .......................................................................................	  39	  1.5.10	   H2dedpa,	  H4octapa,	  H2azapa,	  and	  H5decapa	  ................................................................	  40	  1.5.11	   HBED	  and	  SHBED	  ..........................................................................................................	  43	  1.5.12	   BPCA	  .............................................................................................................................	  44	  1.5.13	   CP256	  ...........................................................................................................................	  45	  1.5.14	   Desferrioxamine	  (DFO)	  ................................................................................................	  46	  1.5.15	   H6phospa	  ......................................................................................................................	  49	  1.5.16	   PCTA	  .............................................................................................................................	  50	  1.6	   Conclusions	  ....................................................................................................................	  50	  Chapter	  2:	  H4octapa:	  an	  acyclic	  ligand	  for	  111In	  radiopharmaceutical	  applications	  ...........	  52	  2.1	   Introduction	  ...................................................................................................................	  52	  2.2	   Results	  and	  discussion	  ....................................................................................................	  55	  2.2.1	   Synthesis	  and	  characterization	  ......................................................................................	  57	  x  2.2.2	   DFT	  structures	  and	  molecular	  electrostatic	  potential	  maps	  ..........................................	  63	  2.2.3	   Radiolabeling	  experiments	  .............................................................................................	  65	  2.2.4	   Thermodynamic	  stability	  ................................................................................................	  67	  2.2.5	   111In	  radiolabeling	  and	  stability	  studies	  ..........................................................................	  69	  2.3	   Conclusions	  ....................................................................................................................	  74	  2.4	   Experimental	  methods	  ...................................................................................................	  76	  2.4.1	   Materials	  and	  methods	  ..................................................................................................	  76	  2.4.2	   Methyl	  6-­‐(hydroxymethyl)picolinate	  (2.1)	  .....................................................................	  77	  2.4.3	   Methyl	  6-­‐(bromomethyl)picolinate	  (2.2)	  .......................................................................	  78	  2.4.4	   N,N’-­‐(Benzyl)ethylenediamine	  (2.3)	  ...............................................................................	  79	  2.4.5	   N,N’-­‐[Benzyl(tert-­‐butoxycarbonyl)methyl]aminoethane	  (2.4)	  .......................................	  80	  2.4.6	   N,N’-­‐[(tert-­‐Butoxycarbonyl)methyl]aminoethane	  (2.5)	  .................................................	  81	  2.4.7	   N,N′-­‐[[(tert-­‐Butoxycarbonyl)methyl]-­‐N,N′-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methylamino]ethane	  (2.6)	  ......................................................................................................	  81	  2.4.8	   H4octapa•4HCl•2H2O,	  N,N′-­‐bis(6-­‐carboxy-­‐2-­‐pyridylmethyl)ethylenediamine-­‐N,N′diacetic	  acid	  ................................................................................................................................	  82	  2.4.9	   Na[In(octapa)]	  ................................................................................................................	  83	  2.4.10	   N,N’’-­‐[Benzyl]diethylenetriamine	  (2.7)	  ........................................................................	  83	  2.4.11	   N,N’’-­‐[[Benzyl]-­‐N,N’,N’’-­‐[(tert-­‐butoxycarbonyl)methyl]]diethylenetriamine	  (2.8)	  ......	  84	  2.4.12	   N,N’,N’’-­‐[[tert-­‐Butoxycarbonyl]methyl]diethylenetriamine	  (2.9)	  ................................	  85	  2.4.13	   N,N’’-­‐[[6-­‐(Methoxycarbonyl)pyridin-­‐2-­‐yl]methylamino]-­‐N,N’,N’’-­‐[(tert-­‐butoxycarbonyl)methyl]]diethylenetriamine	  (2.10)	  ...................................................................	  85	  xi  2.4.14	   H5decapa•5HCl•2.5H2O,	  N,N′,N′′-­‐[(triacetic	  acid]-­‐N,N′′-­‐[6-­‐(carboxy)pyridin-­‐2-­‐yl]methylamino]diethylenetriamine	  ...........................................................................................	  86	  2.4.15	   Na2[In(decapa)]	  ............................................................................................................	  87	  2.4.16	   H2dedpa	  .......................................................................................................................	  87	  2.4.17	   [In(dedpa)]Cl	  ................................................................................................................	  88	  2.4.18	   111In	  Radiolabeling	  studies	  ...........................................................................................	  88	  2.4.19	   Solution	  thermodynamics	  ............................................................................................	  89	  2.4.20	   Molecular	  modeling	  .....................................................................................................	  90	  2.4.21	   Mouse	  serum	  stability	  data	  .........................................................................................	  91	  2.4.22	   Biodistribution	  data	  .....................................................................................................	  92	  Chapter	  3:	  H4octapa-­‐trastuzumab:	  the	  application	  of	  a	  versatile	  acyclic	  ligand	  system	  for	  111In	  and	  177Lu	  imaging	  and	  therapy	  .................................................................................	  93	  3.1	   Introduction	  ...................................................................................................................	  93	  3.2	   Results	  and	  discussion	  ....................................................................................................	  96	  3.2.1	   Synthesis	  and	  characterization	  ......................................................................................	  96	  3.2.2	   Thermodynamic	  stability	  and	  density	  functional	  theory	  structure	  prediction	  ..............	  99	  3.2.3	   Bioconjugation	  and	  in	  vitro	  characterization	  ...............................................................	  102	  3.2.4	   Acute	  biodistribution	  studies	  .......................................................................................	  104	  3.2.5	   Small	  animal	  SPECT/CT	  imaging	  and	  Cerenkov	  luminescence	  imaging	  ........................	  108	  3.3	   Conclusions	  ..................................................................................................................	  110	  3.4	   Experimental	  section	  ....................................................................................................	  112	  3.4.1	   Materials	  and	  methods	  ................................................................................................	  112	  3.4.2	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐1,2-­‐diaminoethane	  (3.1)	  .....................................	  113	  xii  3.4.3	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐N,N′-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1,2-­‐diaminoethane	  (3.2)	  ...........................................................................................................	  114	  3.4.4	   N,N′-­‐[6-­‐(Methoxycarbonyl)pyridin-­‐2-­‐yl]methyl-­‐1,2-­‐diaminoethane	  (3.3)	  ................	  115	  3.4.5	   N,N′-­‐[(tert-­‐Butoxycarbonyl)methyl-­‐N,N′-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1,2-­‐diaminoethane	  (3.4)	  ...........................................................................................................	  116	  3.4.6	   H4octapa,	  N,N′-­‐(6-­‐carboxy-­‐2-­‐pyridylmethyl)-­‐N,N′-­‐diacetic	  acid-­‐1,2-­‐diaminoethane	  (3.5)	   116	  3.4.7	   Na[Lu(octapa)]	  (3.6)	  .....................................................................................................	  117	  3.4.8	   1-­‐(p-­‐Nitrobenzyl)ethylenediamine	  (3.7)	  .......................................................................	  118	  3.4.9	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (3.8)	  .........	  118	  3.4.10	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐N,N’-­‐[(tert-­‐butoxycarbonyl)methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (3.9)	  ......................................................................................	  119	  3.4.11	   N,N’-­‐[(tert-­‐Butoxycarbonyl)methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (3.10)	  ....	  120	  3.4.12	   N,N’-­‐[(tert-­‐Butoxycarbonyl)methyl]-­‐N,N’-­‐[(6-­‐methoxycarbonyl)pyridin-­‐2-­‐yl)methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (3.11)	  ................................................................................	  120	  3.4.13	   p-­‐SCN-­‐Bn-­‐H4octapa,	  N,N’-­‐[(Carboxylato)methyl]-­‐N,N’-­‐[(6-­‐carboxylato)pyridin-­‐2-­‐yl)methyl]-­‐1-­‐(p-­‐benzyl-­‐isothiocyanato)-­‐1,2-­‐diaminoethane	  (3.12)	  ..........................................	  121	  3.4.14	   Solution	  thermodynamics	  ..........................................................................................	  122	  3.4.15	   Molecular	  modeling	  ...................................................................................................	  123	  3.4.16	   [177Lu(chelate)]	  Radiolabeling	  ....................................................................................	  123	  3.4.17	   Trastuzumab	  antibody	  modification/thiourea	  bioconjugation	  ..................................	  124	  3.4.18	   111In-­‐	  and	  177Lu-­‐octapa/DOTA-­‐trastuzumab	  radiolabeling	  .........................................	  124	  3.4.19	   Chelate	  number	  isotopic	  dilution	  assay	  .....................................................................	  125	  xiii  3.4.20	   In	  vitro	  immunoreactivity	  assay	  .................................................................................	  126	  3.4.21	   [177Lu(chelate)]	  Blood	  serum	  competition	  experiments	  ............................................	  127	  3.4.22	   111In-­‐	  and	  177Lu-­‐octapa/DOTA-­‐trastuzumab	  blood	  serum	  competition	  experiments	   127	  3.4.23	   Cell	  culture	  .................................................................................................................	  128	  3.4.24	   SKOV-­‐3	  xenograft	  mouse	  models	  ..............................................................................	  128	  3.4.25	   111In-­‐	  and	  177Lu-­‐octapa/DOTA-­‐trastuzumab	  biodistribution	  studies	  ..........................	  129	  3.4.26	   111In-­‐	  and	  177Lu-­‐octapa/DOTA-­‐trastuzumab	  SPECT/CT	  imaging	  studies	  .....................	  130	  3.4.27	   Cerenkov	  luminescence	  imaging	  (CLI)	  .......................................................................	  131	  3.4.28	   Statistics	  .....................................................................................................................	  131	  Chapter	  4:	  High	  denticity	  ligands	  based	  on	  picolinic	  acid	  for	  111In	  radiochemistry	  ..........	  133	  4.1	   Introduction	  .................................................................................................................	  133	  4.2	   Results	  and	  discussion	  ..................................................................................................	  135	  4.2.1	   Synthesis	  and	  characterization	  ....................................................................................	  135	  4.2.2	   Ligand-­‐metal	  coordination	  ...........................................................................................	  140	  4.2.3	   111In	  radiolabeling	  and	  serum	  stability	  .........................................................................	  144	  4.3	   Conclusions	  ..................................................................................................................	  147	  4.4	   Experimental	  methods	  .................................................................................................	  148	  4.4.1	   Materials	  and	  methods	  ................................................................................................	  148	  4.4.2	   N,N’’-­‐[Benzyl]diethylenetriamine	  (4.1)	  ........................................................................	  148	  4.4.3	   N,N’’-­‐[(Benzyl)-­‐N,N’,N’’-­‐[(6-­‐methoxycarbonyl)pyridin-­‐2-­‐yl)methyl]-­‐diethylenetriamine	  (4.2)	   149	  4.4.4	   Bn-­‐H3nonapa,	  N,N’’-­‐[(Benzyl)-­‐N,N’,N’’-­‐[(6-­‐carboxy)pyridin-­‐2-­‐yl)methyl]-­‐diethylenetriamine	  (4.3)	  ...........................................................................................................	  150	  xiv  4.4.5	   H3nonapa,	  N,N’,N’’-­‐[(6-­‐carboxy)pyridin-­‐2-­‐yl)methyl]-­‐diethylenetriamine	  (4.4)	  ..........	  151	  4.4.6	   N,N’,N’’-­‐Tris[benzyl]ethylamine	  (4.5)	  ...........................................................................	  151	  4.4.7	   N,N’,N’’-­‐Tris[benzyl]-­‐tris[(6-­‐methoxycarbonyl)pyridin-­‐2-­‐yl)methyl]ethyl-­‐amine	  (4.6)	   152	  4.4.8	   Bn-­‐H3trenpa,	  N,N’,N’’-­‐Tris[benzyl]-­‐tris[(6-­‐carboxy)pyridin-­‐2-­‐yl)methyl]	  ethyl-­‐amine	  (4.7)	   153	  4.4.9	   N,N’’-­‐(p-­‐Nitrobenzyl)diethylenetriamine	  (4.8)	  .............................................................	  154	  4.4.10	   N,N’’-­‐[(p-­‐Nitrobenzyl)-­‐N,N’,N’’-­‐[(6-­‐methoxycarbonyl)pyridin-­‐2-­‐yl)methyl]-­‐diethylenetriamine	  (4.9)	  ...........................................................................................................	  155	  4.4.11	   p-­‐NO2-­‐Bn-­‐H3nonapa,	  N,N’’-­‐[(p-­‐Nitrobenzyl)-­‐N,N’,N’’-­‐[(6-­‐carboxy)pyridin-­‐2-­‐yl)-­‐methyl]diethylenetriamine	  (4.10)	  ............................................................................................	  155	  4.4.12	   [In(Bn-­‐nonapa)]	  (4.11)	  ...............................................................................................	  156	  4.4.13	   [In(nonapa)]	  (4.12)	  .....................................................................................................	  157	  4.4.14	   [In(Bn-­‐trenpa)]	  (4.13)	  .................................................................................................	  157	  4.4.15	   111In	  radiolabeling	  studies	  ..........................................................................................	  158	  4.4.16	   Mouse	  serum	  stability	  challenge	  ...............................................................................	  159	  Chapter	  5:	  Modular	  syntheses	  of	  H4octapa	  and	  H2dedpa,	  and	  yttrium	  coordination	  chemistry	  relevant	  to	  86/90Y	  radiopharmaceuticals	  .........................................................	  160	  5.1	   Introduction	  .................................................................................................................	  160	  5.2	   Results	  and	  discussion	  ..................................................................................................	  164	  5.2.1	   Synthesis	  and	  characterization	  ....................................................................................	  164	  5.2.2	   Yttrium	  coordination	  chemistry	  ...................................................................................	  169	  5.2.3	   Density	  functional	  theory/molecular	  electrostatic	  potential	  structure	  prediction	  ......	  177	  5.2.4	   Thermodynamic	  stability	  ..............................................................................................	  179	  xv  5.3	   Conclusions	  ..................................................................................................................	  180	  5.4	   Experimental	  ................................................................................................................	  181	  5.4.1	   Materials	  and	  methods	  ................................................................................................	  181	  5.4.2	   tert-­‐Butyl	  6-­‐(methyl)picolinate	  (5.1)	  ............................................................................	  182	  5.4.3	   tert-­‐Butyl	  6-­‐(bromomethyl)picolinate	  (5.2)	  .................................................................	  183	  5.4.4	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐1,2-­‐diaminoethane	  (5.3)	  .....................................	  184	  5.4.5	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐N,N′-­‐[6-­‐(tert-­‐butoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1,2-­‐diaminoethane	  (5.4)	  ...........................................................................................................	  185	  5.4.6	   N,N′-­‐[6-­‐(tert-­‐Butoxycarbonyl)pyridin-­‐2-­‐yl]methyl-­‐1,2-­‐diaminoethane	  (5.5)	  ............	  185	  5.4.7	   H2dedpa,	  N,N′-­‐[(6-­‐carboxylato)pyridin-­‐2-­‐yl)methyl]-­‐1,2-­‐diaminoethane	  (5.6)	  .........	  186	  5.4.8	   N,N′-­‐[(tert-­‐Butoxycarbonyl)methyl-­‐N,N′-­‐[6-­‐(tert-­‐butoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1,2-­‐diaminoethane	  (5.7)	  .........................................................................................	  187	  5.4.9	   H4octapa,	  N,N’-­‐[(6-­‐carboxylato)pyridin-­‐2-­‐yl)methyl]-­‐N,N′-­‐diacetic	  acid-­‐1,2-­‐diaminoethane	  (5.8)	  .................................................................................................................	  188	  5.4.10	   Na[Y(octapa)]	  (5.9)	  .....................................................................................................	  188	  5.4.11	   1-­‐(p-­‐Nitrobenzyl)ethylenediamine	  (5.10)	  ..................................................................	  189	  5.4.12	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (5.11)	  .....	  190	  5.4.13	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐N,N’’-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (5.12)	  ................................................................................	  190	  5.4.14	   N,N’-­‐[[6-­‐(Methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (5.13)	   191	  5.4.15	   p-­‐SCN-­‐Bn-­‐H2dedpa,	  N,N’-­‐[[(6-­‐carboxylato)pyridin-­‐2-­‐yl]methyl]-­‐1-­‐(p-­‐benzylisothiocyanato)-­‐1,2-­‐diaminoethane	  (5.14)	  ....................................................................	  192	  xvi  5.4.16	   N,N’-­‐[(tert-­‐Butoxycarbonyl)methyl]-­‐N,N’-­‐[[(6-­‐tert-­‐butoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (5.15)	  ...........................................................	  193	  5.4.17	   p-­‐SCN-­‐Bn-­‐H4octapa,	  N,N’-­‐[(Carboxylato)methyl]-­‐N,N’-­‐[[(6-­‐carboxylato)pyridin-­‐2-­‐yl]methyl]-­‐1-­‐(p-­‐benzylisothiocyanato)-­‐1,2-­‐diaminoethane	  (5.16)	  ...........................................	  194	  5.4.18	   Solution	  thermodynamics	  ..........................................................................................	  196	  5.4.19	   Molecular	  modeling	  ...................................................................................................	  196	  Chapter	  6:	  H4octapa	  vs	  H4C3octapa	  for	  111In	  and	  177Lu	  radiochemistry:	  the	  difference	  of	  one	  carbon	  ....................................................................................................................	  198	  6.1	   Introduction	  .................................................................................................................	  198	  6.2	   Results	  and	  discussion	  ..................................................................................................	  202	  6.2.1	   Synthesis	  and	  characterization	  ....................................................................................	  202	  6.2.2	   Variable	  temperature	  NMR	  and	  2D	  NMR	  spectroscopy	  of	  [In(C3octapa)]-­‐	  and	  [Lu(C3octapa)]-­‐	  ..........................................................................................................................	  205	  6.2.3	   Density	  functional	  theory	  structure	  calculations	  .........................................................	  216	  6.2.4	   Thermodynamic	  formation	  constants	  ..........................................................................	  219	  6.2.5	   Trastuzumab	  antibody	  conjugation,	  111In/177Lu	  radiolabeling,	  and	  in	  vitro	  serum	  stability	   219	  6.3	   Conclusions	  ..................................................................................................................	  224	  6.4	   Experimental	  section	  ....................................................................................................	  225	  6.4.1	   Materials	  and	  methods	  ................................................................................................	  225	  6.4.2	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐1,3-­‐diaminopropane	  (6.1)	  ...................................	  226	  6.4.3	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐N,N′-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1,3-­‐diaminopropane	  (6.2)	  .........................................................................................................	  226	  xvii  6.4.4	   N,N′-­‐[6-­‐(Methoxycarbonyl)pyridin-­‐2-­‐yl]methyl-­‐1,3-­‐diaminopropane	  (6.3)	  ..............	  227	  6.4.5	   N,N′-­‐[(tert-­‐Butoxycarbonyl)methyl-­‐N,N′-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1,3-­‐diaminopropane	  (6.4)	  .........................................................................................................	  228	  6.4.6	   H4C3octapa,	  N,N′-­‐(6-­‐carboxy-­‐2-­‐pyridylmethyl)-­‐N,N′-­‐diacetic	  acid-­‐1,3-­‐diaminopropane	  (6.5)	  ...............................................................................................................	  228	  6.4.7	   Na[In(C3octapa)]	  (6.6)	  ..................................................................................................	  229	  6.4.8	   Na[Lu(C3octapa)]	  (6.7)	  .................................................................................................	  230	  6.4.9	   Diethyl-­‐2-­‐(4-­‐nitrobenzyl)malonate	  (6.8)	  ......................................................................	  230	  6.4.10	   2-­‐(4-­‐Nitrobenzyl)malondiamide	  (6.9)	  ........................................................................	  231	  6.4.11	   1,3-­‐Diamino-­‐2-­‐(4-­‐nitrobenzyl)propane	  dihydrochloride	  (2-­‐(p-­‐nitrobenzyl)-­‐1,3-­‐propylenediamine)	  (6.10)	  .........................................................................................................	  232	  6.4.12	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐2-­‐(p-­‐nitrobenzyl)-­‐1,3-­‐diaminopropane	  (6.11)	  ...	  233	  6.4.13	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐N,N’-­‐[(tert-­‐butoxycarbonyl)methyl]-­‐2-­‐(p-­‐nitrobenzyl)-­‐1,3-­‐diaminopropane	  (6.12)	  ..................................................................................	  233	  6.4.14	   N,N’-­‐[(tert-­‐Butoxycarbonyl)methyl]-­‐2-­‐(p-­‐nitrobenzyl)-­‐1,3-­‐diaminopropane	  (6.13)	  ..	  234	  6.4.15	   N,N’-­‐[(tert-­‐Butoxycarbonyl)methyl]-­‐N,N’-­‐[(6-­‐methoxycarbonyl)pyridin-­‐2-­‐yl)methyl]-­‐2-­‐(p-­‐nitrobenzyl)-­‐1,3-­‐diaminopropane	  (6.14)	  ..............................................................................	  235	  6.4.16	   p-­‐SCN-­‐Bn-­‐H4C3octapa,	  N,N’-­‐[(Carboxylato)methyl]-­‐N,N’-­‐[(6-­‐carboxylato)pyridin-­‐2-­‐yl)methyl]-­‐2-­‐(p-­‐benzyl-­‐isothiocyanato)-­‐1,3-­‐diaminopropane	  (6.15)	  ........................................	  235	  6.4.17	   Molecular	  modeling	  ...................................................................................................	  237	  6.4.18	   Trastuzumab	  antibody	  modification	  /	  thiourea	  bioconjugation	  ................................	  237	  6.4.19	   111In-­‐	  and	  177Lu-­‐C3octapa/octapa-­‐trastuzumab	  radiolabeling	  ...................................	  238	  xviii  6.4.20	   111In-­‐	  and	  177Lu-­‐C3octapa/octapa-­‐trastuzumab	  blood	  serum	  competition	  experiments	   239	  Chapter	  7:	  H6phospa-­‐Trastuzumab:	  a	  bifunctional	  methylenephosphonate-­‐based	  ligand	  with	  89Zr,	  111In	  and	  177Lu	  ................................................................................................	  240	  7.1	   Introduction	  .................................................................................................................	  240	  7.2	   Results	  and	  discussion	  ..................................................................................................	  243	  7.2.1	   Synthesis	  and	  characterization	  ....................................................................................	  243	  7.2.2	   Antibody	  modification,	  111In	  and	  177Lu	  radiolabeling,	  and	  in	  vitro	  characterization	  ....	  246	  7.2.3	   89Zr	  Radiolabeling	  .........................................................................................................	  250	  7.2.4	   Small	  animal	  SPECT/CT	  imaging	  ...................................................................................	  252	  7.3	   Conclusions	  ..................................................................................................................	  255	  7.4	   Experimental	  ................................................................................................................	  257	  7.4.1	   Materials	  and	  methods	  ................................................................................................	  257	  7.4.2	   N,N′-­‐[6-­‐(Methoxycarbonyl)pyridin-­‐2-­‐yl]methyl-­‐1,2-­‐diaminoethane	  (7.1)	  ................	  257	  7.4.3	   H6phospa,	  (7.2),	  N,N′-­‐(Methylphosphonate)-­‐N,N′-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1,2-­‐diaminoethane	  ..................................................................................................	  257	  7.4.4	   Na3[Lu(phospa)]	  (7.3)	  ...................................................................................................	  258	  7.4.5	   Na3[In(phospa)]	  (7.4)	  ....................................................................................................	  258	  7.4.6	   1-­‐(p-­‐Nitrobenzyl)ethylenediamine	  (7.5)	  .......................................................................	  259	  7.4.7	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (7.6)	  .........	  259	  7.4.8	   N,N’-­‐(2-­‐Nitrobenzenesulfonamide)-­‐N,N’′-­‐[6-­‐(methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (7.7)	  ..................................................................................	  260	  xix  7.4.9	   N,N’-­‐[6-­‐(Methoxycarbonyl)pyridin-­‐2-­‐yl]methyl]-­‐p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (7.8)	   261	  7.4.10	   N,N’-­‐[Methylphosphonate]-­‐N,N’-­‐[(6-­‐carboxylato)pyridin-­‐2-­‐yl)methyl]-­‐1-­‐(p-­‐nitrobenzyl)-­‐1,2-­‐diaminoethane	  (7.9)	  ......................................................................................	  261	  7.4.11	   p-­‐SCN-­‐Bn-­‐H6phospa,	  N,N’-­‐(methylphosphonate)-­‐N,N’-­‐[(6-­‐carboxylato)pyridin-­‐2-­‐yl)methyl]-­‐1-­‐(p-­‐benzyl-­‐isothiocyanato)-­‐1,2-­‐diaminoethane	  (7.10)	  ..........................................	  262	  7.4.12	   Trastuzumab	  bioconjugation	  .....................................................................................	  263	  7.4.13	   111In-­‐,	  89Zr-­‐,	  and	  177Lu-­‐phospa-­‐trastuzumab	  radiolabeling	  .........................................	  264	  7.4.14	   Chelate	  number	  –	  radiometric	  isotopic	  dilution	  assay	  ..............................................	  265	  7.4.15	   In	  vitro	  immunoreactivity	  assay	  .................................................................................	  265	  7.4.16	   111In-­‐	  and	  177Lu-­‐phospa-­‐trastuzumab	  blood	  serum	  competition	  experiments	  ..........	  265	  7.4.17	   Cell	  culture	  .................................................................................................................	  265	  7.4.18	   SKOV-­‐3	  xenograft	  mouse	  models	  ..............................................................................	  266	  7.4.19	   111In-­‐	  and	  177Lu-­‐phospa-­‐trastuzumab	  SPECT/CT	  imaging	  studies	  ...............................	  266	  7.4.20	   Animal	  protocol	  .........................................................................................................	  267	  Chapter	  8:	  H2azapa:	  a	  click-­‐based	  acyclic	  multifunctional	  ligand	  for	  67/68Ga,	  64Cu,	  111In,	  and	  177Lu	  ..............................................................................................................................	  268	  8.1	   Introduction	  .................................................................................................................	  268	  8.2	   Results	  and	  discussion	  ..................................................................................................	  271	  8.2.1	   Synthesis	  of	  H2azapa	  ....................................................................................................	  271	  8.2.2	   NMR	  Characterization	  ..................................................................................................	  275	  8.2.3	   X-­‐ray	  crystallography	  ....................................................................................................	  278	  8.2.4	   Radiolabeling	  experiments	  ...........................................................................................	  283	  xx  8.2.5	   Blood	  serum	  stability	  studies	  .......................................................................................	  284	  8.2.6	   Biodistribution	  and	  PET	  imaging	  studies	  ......................................................................	  287	  8.3	   Conclusions	  ..................................................................................................................	  292	  8.4	   Experimental	  section	  ....................................................................................................	  294	  8.4.1	   Materials	  and	  Methods	  ................................................................................................	  294	  8.4.2	   Di-­‐tert-­‐butyl	  ethane-­‐1,2-­‐dicarbamate	  (8.1)	  .................................................................	  296	  8.4.3	   N,N’-­‐Propargyl-­‐N,N’-­‐tert-­‐butoxycarbonyl-­‐1,2-­‐diaminoethane	  (8.2)	  ............................	  297	  8.4.4	   N,N’-­‐Propargyl-­‐1,2-­‐diaminoethane	  (8.3)	  ......................................................................	  298	  8.4.5	   N,N’-­‐[6-­‐(Methoxycarbonyl)pyridin-­‐2-­‐yl]methyl-­‐N,N’-­‐propargyl-­‐1,2-­‐diaminoethane	  (8.4)	   298	  8.4.6	   N,N’-­‐[1-­‐Benzyl-­‐1,2,3-­‐triazole-­‐4-­‐yl]methyl-­‐N,N’-­‐[6-­‐(carboxy)pyridin-­‐2-­‐yl]-­‐1,2-­‐diaminoethane	  (H2azapa•2HCl•1.5H2O)	  (8.5•2HCl•1.5H2O)	  ....................................................	  299	  8.4.7	   Metal	  complexation	  experiments:	  general	  procedure	  .................................................	  301	  8.4.7.1	   [Cu(azapa)]	  ..........................................................................................................................	  301	  8.4.7.2	   [Ga(azapa)][ClO4]	  .................................................................................................................	  301	  8.4.7.3	   [In(azapa)(H2O)][ClO4]	  .........................................................................................................	  302	  8.4.7.4	   [Lu(azapa)][ClO4]	  ..................................................................................................................	  302	  8.4.8	   X-­‐Ray	  crystallography	  ...................................................................................................	  303	  8.4.9	   67Ga/111In	  Radiolabeling	  ...............................................................................................	  304	  8.4.10	   177Lu/64Cu	  Radiolabeling	  .............................................................................................	  304	  8.4.11	   67Ga/111In	  Mouse	  serum	  competition	  experiments	  ...................................................	  305	  8.4.12	   177Lu/64Cu	  Blood	  serum	  competition	  experiments	  .....................................................	  306	  8.4.13	   64Cu	  Biodistribution	  studies	  .......................................................................................	  307	  8.4.14	   Small-­‐animal	  64Cu-­‐PET	  imaging	  ..................................................................................	  308	  xxi  Chapter	  9:	  Conclusions	  and	  future	  work	  ........................................................................	  309	  9.1.1	   Overview	  and	  conclusions	  for	  “pa”	  family	  of	  picolinic	  acid-­‐based	  ligands	  ..................	  309	  9.1.2	   General	  direction	  for	  future	  ligand	  design	  ...................................................................	  312	  Bibliography	  .................................................................................................................	  314	  Appendix	  ......................................................................................................................	  331	  Appendix	  A	  Supplementary	  figures	  and	  data	  .........................................................................	  331	   xxii  List of Tables  Table 1.1 Properties of some popular radiometal isotopes, EC = electron capture; some low abundance emissions have been omitted for brevity.6,13-17 ....................................................... 5	  Table 1.2 DOTA and bifunctional derivatives.a ..................................................................... 22	  Table 1.3 DOTA derivatives CB-DO2A, TCMC, 3p-C-DEPA, Oxo-DO3A, and bifunctional derivatives.a ............................................................................................................................. 23	  Table 1.4 TETA, CB-TE2A and derivatives, and bifunctional derivatives. .......................... 26	  Table 1.5 The copper ligands Diamsar, SarAr, AmBaSar, and BaBaSar.a ............................ 29	  Table 1.6 NOTA, NETA, TACN-TM, and bifunctional derivatives.a ................................... 31	  Table 1.7 DTPA, 1B4M, CHX-A’’-DTPA, and bifunctional derivatives. ............................ 35	  Table 1.8 Promising 67/68Ga ligands TRAP (PRP9, TRAP-Pr), AAZTA (DATA), and bifunctional derivatives.a ......................................................................................................... 38	  Table 1.9 H2dedpa, H4octapa, H2azapa, H5decapa, and bifunctional derivatives. ................. 41	  Table 1.10 HBED, SHBED, BPCA, and bifunctional derivatives.a ....................................... 44	  Table 1.11 CP256, Desferrioxamine (DFO), PCTA, H6phospa, and bifunctional derivatives. ................................................................................................................................................. 48	  Table 2.1 Formation constants (log KML) and pMa values for In3+ complexes. ..................... 68	  Table 2.2 Data from mouse serum stability challenges performed at ambient temperature (n=3), evaluated by PD-10 size-exclusion column elution, with stability shown as the percentage of intact 111In complex. ......................................................................................... 69	  Table 2.3 Decay corrected %ID/g values from the biodistribution of 111In-complexes in healthy female ICR mice (6-8 weeks old), n = 4, bold = passed students T-test (p < 0.05). .. 71	  xxiii  Table 3.1 Chemical and in vitro biological characterization data for 111In- and 177Lu-octapa-trastuzumab and 111In- and 177Lu-DOTA-trastuzumab radioimmunoconjugates. ................ 104	  Table 3.2 Biodistribution data of 111In/177Lu-octapa-trastuzumab and 111In/177Lu-DOTA-trastuzumab, performed over a 5 day period in mice bearing SKOV-3 ovarian cancer xenografts, tumour size ~2-3 mm diameter (n = 4 for each time point), showing %ID/g values. ................................................................................................................................... 105	  Table 4.1 Radiolabeling experiments with 111In and the novel acyclic ligands Bn-H3nonapa (4.3), H3nonapa (4.4), Bn-H3trenpa (4.7), and results from the ligands H2dedpa, H4octapa, H5decapa, DOTA, and DTPA (Chapters 2-3).89 ................................................................... 145	  Table 5.1 Formation constants (log KML) and pMa values for In3+, Lu3+, and Y3+ complexes of relevant chelating ligand. .................................................................................................. 179	  Table 6.1 Relevant bond lengths (Å) and angles (°) comparing the DFT-calculated In3+ and Lu3+ complexes of H4octapa and H4C3octapa. ..................................................................... 218	  Table 6.2 Human serum stability challenge performed at 37.5 °C (n = 3), with stability shown as the % intact 177Lu complex, deteremined by PD10 size-exclusion column elution. ............................................................................................................................................... 220	  Table 7.1 Chemical and in vitro biological characterization data for 111In- and 177Lu-phospa-trastuzumab radioimmunoconjugates. .................................................................................. 245	  Table 8.1 Selected bond angles and lengths in the X-ray crystal structure of [In(azapa)(H2O)][ClO4]. ....................................................................................................... 280	  Table 8.2 Selected bond angles and lengths in the X-ray crystal structure of [Cu(azapa)].  Relevant bond angles are compared to the analogous bond angles in the previously reported X-ray crystal structure of [Ga(dedpa)]+ and [Cu(dedpa)].118,273 ............................................ 282	  xxiv  Table 8.3 Stability data collected in mouse blood serum at 25 °C for 67Ga and 111In, and in human blood serum with agitation (550 rpm) at 37 °C for 64Cu and 177Lu, with H2azapa and selected ligand standards.  The % stability shown is the percentage of ligand-bound radiometal, and the error is expressed as standard deviation (n = 3). ................................... 284	  Table 8.4 Biodistribution of [64Cu(azapa)] and [64Cu(DOTA)]2- in healthy nude athymic mice (n=4) showing organ uptake as % ID/g, with the error expressed as standard deviation (SD), at 15 min, 1 h, 4 h, and 24 h time points. .................................................................... 290	   xxv  List of Figures  Figure 1.1 Examples of bioconjugation reactions: (A) standard peptide coupling reaction between a carboxylic acid and a primary amine with a coupling reagent; (B-C) peptide coupling reactions between activated esters of N-hydroxysuccinimide (NHS) or tetrafluorophenyl (TFP) olaand a primary amine; (D) thiourea bond formation between an isothiocyanate and a primary amine; (E) thioether bond formation between a maleimide and thiol; (F) standard Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition (“click” reaction) between an azide and an alkyne; and (G) strain-promoted Diels-Alder “click” reaction between a tetrazine and transcyclooctene. ................................................................................ 7	  Figure 1.2 Illustration of an archetypal radiometal-based radiopharmaceutical agent containing a bifunctional chelator (BFC) conjugated to a targeting vector (e.g. antibody, peptide, nanoparticle) using a variety of conjugation methods (e.g. isothiocyanate-amine coupling (shown), peptide coupling, maleimide-thiol coupling, activated ester amide coupling, click-coupling) and then radiolabeled with a radiometal ion (e.g. 111In3+/177Lu3+/86/90Y3+). ............................................................................................................. 8	  Figure 1.3 Cartoon depiction of metal ion coordination kinetics, enhanced off-rate kinetics in vivo (extremely dilute conditions), and possible routes of radiometal ion loss in vivo. ......... 11	  Figure 2.1 Structures of the 111In-coordinating and industry “gold standard” ligands DTPA/CHX-A’’-DTPA and DOTA, the 68Ga-coordinating ligand H2dedpa, and the novel entrants H4octapa and H5decapa. ............................................................................................ 55	  Figure 2.2 1H NMR spectra in D2O (300 MHz) at ambient temperature of top, H4octapa and [In(octapa)]- showing simple diastereotopic splitting due to minimal isomerization and xxvi  bottom, H5decapa and [In(decapa)]2- showing complicated but sharp splitting arising from multiple static isomers. ........................................................................................................... 60	  Figure 2.3 DFT structure of [In(octapa)]- (solvent = water) showing an 8 coordinate structure (left), and the electrostatic potentials of the complex between octapa4- with In3+ mapped onto the electron density (right). The MEP represent a maximum potential of 0.03 au, and a minimum of -0.25 au, mapped onto electron density isosurfaces of 0.002 e Å-3 (red to blue = negative to positive).  DFT calculations performed by Dr. Jacqueline Cawthray. ................. 65	  Figure 2.4 DFT structure of [In(decapa)]2- (solvent = water) showing an 8 coordinate structure, and the electrostatic potentials of the complex between decapa5- with In3+ mapped onto the electron density (right). The MEP represent a maximum potential of 0.03 au, and a minimum of -0.25 au, mapped onto electron density isosurfaces of 0.002 e Å-3 (red to blue = negative to positive).  DFT calculations performed by Dr. Jacqueline Cawthray. ................. 65	  Figure 2.5 Biodistribution %ID/g values for [111In(DOTA)]-, [111In(octapa)]-, and [111In(decapa)]2-, with error bars plotted as standard deviations (note the y-axis set to 2.0 %ID/g, for clarity of the low activity 4 and 24 hour time points). ......................................... 72	  Figure 3.1 Structures of some common bifunctional ligands used for radiometal chemistry, and H4octapa, the non-bifunctional variant of the ligand p-SCN-Bn-H4octapa used in this work. ....................................................................................................................................... 94	  Figure 3.2 In silico DFT structure predictions. a, 8-coordinate structure of [Lu(octapa)]-; b, 9-coordinate structure of [Lu(octapa)(H2O)]-, as well as the MEP polar-surface area maps (bottom) predicting the charge distribution over the solvent-exposed surface of the metal complexes (red = negative, blue = positive, representing a maximum potential of 0.254 au xxvii  and a minimum of -0.254 au, mapped onto electron density isosurfaces of 0.002 Å-3).  DFT calculations performed by Dr. Jacqueline Cawthray. ........................................................... 101	  Figure 3.3 SPECT/CT imaging of the 111In/177Lu-octapa-trastuzumab and 111In/177Lu-DOTA-trastuzumab immunoconjugates, in female nude athymic mice with subcutaneous SKOV-3 xenografts (identified by arrow at right shoulder, tumour size ~2-3 mm diameter), showing transverse (top) and coronal (bottom) planar images bisecting the tumour, imaged at 24, 48, 72, 96, and 120 h post injection. ........................................................................................... 109	  Figure 3.4 Cerenkov luminescence image (CLI) of 177Lu-octapa-trastuzumab, obtained 24 h post-injection of a female nude athymic mouse bearing an SKOV-3 ovarian cancer xenograft on the right shoulder, indicated by white arrow (2 minute acquisition time). ...................... 110	  Figure 4.1 The new chelating ligands Bn-H3nonapa, H3nonapa, p-NO2-Bn-H3nonapa, and Bn-H3trenpa. ......................................................................................................................... 134	  Figure 4.2 1H NMR spectra of H3nonapa (4.4) (400 MHz, 25 °C, D2O, top) and the In3+ complex In(nonapa) (4.12) (600 MHz, 25 °C, DMSO-d6, bottom). ..................................... 141	  Figure 4.3 1H NMR spectra of Bn-H3nonapa (4.3) (300 MHz, 25 °C, MeOD, top) and the In3+ complex In(Bn-nonapa) (4.11) (600 MHz, 25 °C, DMSO-d6, bottom). ........................ 142	  Figure 4.4 1H NMR spectra of Bn-H3trenpa (4.7) (300 MHz, 25 °C, MeOD, top) and the In3+ complex In(Bn-trenpa) (4.13) (400 MHz, 25 °C, DMSO-d6, bottom). ................................. 143	  Figure 4.5 Mouse serum stability assay of 111In3+ complexes of ligands from Table 4.1, determined after 1 and 24 h by PD-10 size-exclusion column elution. ................................ 146	  Figure 5.1 Ligands synthesized in this chapter, including H2dedpa, H4octapa, and BFC derivatives p-SCN-Bn-H2dedpa and p-SCN-Bn-H4octapa; and the promising new 86/90Y ligand 3p-C-NETA. ............................................................................................................... 161	  xxviii  Figure 5.2 1H NMR spectra of [Lu(octapa)]- in D2O (600 MHz, 25 °C, top, referenced to H-O-D at 4.75 ppm)87, [Y(octapa)]- in D2O (400 MHz, 25 °C, bottom). ................................. 170	  Figure 5.3 Variable temperature (VT) NMR experiments with [Y(octapa)]- (D2O, 400 MHz), with temperature increased to 85 °C in 20 °C increments, with blue arrows identifying fluxional isomers, and red arrows identifying a static (non-fluxional) isomer. .................... 171	  Figure 5.4 1H-1H COSY NMR (600 MHz, D2O, 25 °C) expansion of aromatic signals in spectrum of [Y(octapa)]-, showing no correlations between broad signals arising from a fluxional species (blue arrows), and sharp signals arising from a single static isomer (see Figure A.6 for full spectrum). ............................................................................................... 173	  Figure 5.5 1H-1H COSY NMR (600 MHz, D2O, 25 °C) expansion of alkyl-region signals in spectrum of [Y(octapa)]-, showing no correlations between broad signals arising from a fluxional species (blue arrows, identified by VT-NMR, Figure 5.3), and sharp signals arising from a single static isomer (see Figure A.6 for full spectrum). ............................................ 174	  Figure 5.6 1H-13C HSQC NMR (400/100 MHz, D2O, 25 °C) expansion of aromatic signals in spectrum of [Y(octapa)]-, showing correlations to ~5 unique 13C signals, suggesting the presence of a single static isomer (red arrows), along with a fluxional species (blue arrows) (13C NMR spectra externally referenced to MeOH in D2O) (see Figure A.7 for full spectrum). ............................................................................................................................................... 175	  Figure 5.7 1H-13C HSQC NMR (400/100 MHz, D2O, 25 °C) expansion of alkyl-region signals in spectrum of [Y(octapa)]-, showing correlations to ~4 unique and strong 13C signals from a static isomer (red arrows), and several weak correlations arising from a fluxional species (blue arrows) (13C NMR spectra externally referenced to MeOH in D2O) (see Figure A.7 for full spectrum). .......................................................................................................... 176	  xxix  Figure 5.8 In silico DFT structure predictions: (a) 8-coordinate structure of [Y(octapa)]- (top, left); (b) 9-coordinate structure of [Y(octapa)(H2O)]- (top, right), as well as the MEP polar-surface area maps (bottom) predicting the charge distribution over the solvent-exposed surface of the metal complexes (red = negative, blue = positive, representing a maximum potential of 0.200 au and a minimum of -0.200 au, mapped onto electron density isosurfaces of 0.002 Å-3), ].  DFT calculations performed by Dr. Jacqueline Cawthray. ........................ 178	  Figure 6.1 Structures of the new pyridinecarboxylate-based ligand H4C3octapa (propylene-bridged) and the bifunctional derivative p-SCN-Bn-H4C3octapa, and the popular 111In/177Lu/86/90Y bifunctional ligands DO3A-NHS and DO3A-SCN. ................................. 199	  Figure 6.2 Stacked 1H NMR spectra of the ligand H4C3octapa (D2O, 25 °C, 600 MHz), metal complex [In(C3octapa)]- (D2O, 25 °C, 400 MHz) showing complicated but sharp signals suggesting multiple static isomers, and [Lu(C3octapa)]- (D2O, 25 °C, 300 MHz) showing complicated and broad signals suggesting fluxional isomerization. ...................... 205	  Figure 6.3 Stacked 1H NMR spectra of the ligand H4octapa (D2O, 25 °C, 300 MHz), metal complex [In(octapa)]- (D2O, 25 °C, 600 MHz) showing simple and sharp diastereotopic splitting suggesting the presence of one static isomer, and [Lu(octapa)]- (D2O, 25 °C, 400 MHz) showing complicated and sharp signals suggesting multiple static isomers. ............. 207	  Figure 6.4 1H COSY NMR (400 MHz, D2O, 25 °C) spectrum of [In(C3octapa)]- showing an expansion of the alkyl-region, highlighting two broad signals with red arrows arising from the central –CH2- of the propylene bridge, showing no 1H-1H correlations to each other. .. 208	  Figure 6.5 1H-13C HSQC NMR (400/100 MHz, D2O, 25 °C) expansion of aromatic signals in spectrum of [In(C3octapa)]-, showing correlations to 12 unique 13C signals, with an xxx  additional aromatic signal at 125.32 ppm not shown (13C NMR spectra externally referenced to MeOH in D2O). ................................................................................................................. 210	  Figure 6.6 1H-13C HSQC NMR (600/150 MHz, D2O, 25 °C) expansion of aromatic signals in spectrum of [Lu(C3octapa)]-, showing correlations to 10 unique 13C signals (13C NMR spectra externally referenced to MeOH in D2O). .................................................................. 211	  Figure 6.7 Variable temperature (VT) 1H NMR experiments with [In(C3octapa)]- (D2O, 400 MHz), with the temperature increased to 85 °C in 20 °C increments. .................................. 213	  Figure 6.8 Variable temperature (VT) 1H NMR experiments with [Lu(C3octapa)]- (D2O, 400 MHz), with the temperature increased to 85 °C in 20 °C increments. .................................. 214	  Figure 6.9 In silico DFT structure predictions: (a) 8-coordinate structure of [In(C3octapa)]- (top) from two perspectives; (b) 8-coordinate structure of [Lu(C3octapa)]- (bottom) from two perspectives, with both structures showing overlayed MEP polar-surface area maps predicting the charge distribution over the solvent-exposed surface of the metal complexes (red = negative, blue = positive, representing a maximum potential of 0.254 au and a minimum of -0.254 au, mapped onto electron density isosurfaces of 0.002 Å-3).  DFT calculations performed by Dr. Jacqueline Cawthray. ........................................................... 217	  Figure 6.10 Stability of the immunoconjugates 111In(octapa)-trastuzumab and 111In(C3octapa)-trastuzumab in both phosphate buffered saline (PBS) and human blood serum, evaluated by spotting ~1 μCi of serum competition mixture on silica-embedded paper iTLC plates and eluting with aqueous EDTA (50 mM, pH 5) mobile phase. ...................... 222	  Figure 6.11 Stability of the immunoconjugates 177Lu(octapa)-trastuzumab and 177Lu(C3octapa)-trastuzumab in both phosphate buffered saline (PBS) and human blood xxxi  serum, evaluated by spotting ~1 μCi of serum competition mixture on silica-embedded paper iTLC plates and eluting with aqueous EDTA (50 mM, pH 5) mobile phase. ...................... 223	  Figure 7.1 The current “gold standard” ligand for 89Zr, DFO, and the new entrants H6phospa (7.2) and bifunctional analogue p-SCN-Bn-H6phospa (7.10). .............................................. 242	  Figure 7.2 A) iTLC radiochromatograph of the unpurified radiolabeling mixture of 111In-phospa-trastuzumab (30 minutes, 25 °C, 2.64 mCi/mg); B) iTLC of 111In-phospa-trastuzumab after purification by PD-10 column; C) iTLC trace of the unpurified radiolabeling mixture of 177Lu-phospa-trastuzumab (30 minutes, 25 °C, 2.23 mCi/mg); and D) iTLC trace of 177Lu-phospa-trastuzumab after purification, showing 177Lu transchelated from the weakly-bound 177Lu-phospa-trastuzumab to EDTA4- (~200 µg of antibody conjugate used for each reaction). ............................................................................................................................... 248	  Figure 7.3 The stability of 177Lu/111In-phospa-trastuzumab in human blood serum over 120 h, agitated at 300 rpm and 37 °C, analyzed via radio-iTLC elution with EDTA mobile phase (50 mM, pH 5), compared to previously obtained values for 177Lu/111In-octapa-trastuzumab as a reference.87 ..................................................................................................................... 249	  Figure 7.4 Radiolabeling results of H6phospa-trastuzumab with 89Zr in phosphate buffered saline (pH 7.4), showing results at both room temperature and 37 °C with H6phospa-trastuzumab (3.3 ± 0.1 chelates per antibody) (~300 µg of antibody conjugate used for each reaction and ~1 mCi of 89Zr). ................................................................................................ 251	  Figure 7.5 SPECT/CT images of 111In-phospa-trastuzumab in female nude athymic mice bearing SKOV-3 tumour xenografts in the right shoulder (diameter ~2 mm), imaged at 24, 72, and 120 hrs p.i. (~800-810 mCi injected activity, ~150-200 µg antibody per mouse). .. 253	  xxxii  Figure 7.6 SPECT/CT images of 177Lu-phospa-trastuzumab in female nude athymic mice bearing SKOV-3 tumour xenografts in the right shoulder (diameter ~2 mm), imaged at 24 and 72 hrs p.i. (~575 mCi injected activity, ~150-200 µg antibody per mouse). ................. 254	  Figure 8.1 Structures of selected state-of-the-art “gold standard” 64Cu ligands, and the new ligand H2azapa. ..................................................................................................................... 269	  Figure 8.2 1H NMR spectra (DMSO-d6, 25 °C) of (top) H2azapa, 300 MHz; (middle) [Ga(azapa)]+, 600 MHz; (middle) [In(azapa)]+, 400 MHz; and (bottom, 300 MHz, D2O) [Lu(azapa)]+. ......................................................................................................................... 276	  Figure 8.3 Variable temperature (VT) 1H NMR spectra of [In(azapa)]+ (400 MHz, DMSO-d6) with the temperature increasing from 25 °C to 135 °C. .................................................. 277	  Figure 8.4 ORTEP drawing of the solid-state molecular structure of [In(azapa)(H2O)][ClO4] obtained by X-ray diffraction.  Hydrogen atoms are omitted for clarity. ............................. 279	  Figure 8.5 ORTEP drawing of the solid-state molecular structure of [Cu(azapa)].  Hydrogen atoms are omitted for clarity. ................................................................................................ 282	  Figure 8.6 Biodistribution of [64Cu(azapa)] and [64Cu(DOTA)]2- showing organ and tissue uptake as percent injected dose per gram (% ID/g) obtained over 24 h in healthy athymic nude mice; Y-axis is normalized at 25% ID/g for clarity. .................................................... 286	  Figure 8.7 PET images of two (A and B) healthy female nude athymic mice injected with [64Cu(azapa)] and imaged at 1, 4, and 24 h post injection, with the scales being different for the left images at 20 %ID/g and for the right images being 8 %ID/g. .................................. 287	   xxxiii  List of Schemes  Scheme 2.1 Synthesis of compounds 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 and H4octapaa ..................... 57	  Scheme 2.2 Synthesis of compounds 2.7, 2.8, 2.9, 2.10 and H5decapaa ................................ 59	  Scheme 3.1 Improved synthesis of picolinic acid-based ligands H4octapa (3.5) and p-SCN-Bn-H4octapa (3.12).a ............................................................................................................... 98	  Scheme 4.1 Synthesis of Bn-H3nonapa (4.3) and H3nonapa (4.4). a .................................... 136	  Scheme 4.2 Synthesis of Bn-H3trenpa (4.7). a ...................................................................... 137	  Scheme 4.3 Synthesis of p-NO2-Bn-H3nonapa (4.10). a ...................................................... 138	  Scheme 5.1 Syntheses of H2dedpa (5.6) and H4octapa (5.8) using tert-butyl ester protection chemistrya ............................................................................................................................. 165	  Scheme 5.2 Synthesis of p-SCN-Bn-H2dedpa (5.14), and p-SCN-Bn-H4octapa (5.16) using tert-butyl ester protection chemistrya .................................................................................... 168	  Scheme 6.1 Synthesis of H4C3octapa (6.5) utilizing nosyl protection chemistry.a .............. 203	  Scheme 6.2 Synthesis of p-SCN-Bn-H4C3octapa (6.15) utilizing nosyl protection chemistry.a ............................................................................................................................................... 204	  Scheme 7.1 Synthesis of H6phospa (7.2) and p-SCN-Bn-H6phospa (7.10) a ....................... 244	  Scheme 8.1 Synthesis of click-based ligand H2azapa (8.5) a ............................................... 272	   xxxiv  List of Symbols and Abbreviations  ~  approximate %ID/g  percentage of injected radioactive dose per gram of tissue 2D  two dimensional 3D  three dimensional α  alpha particle Å  Angstrom, 10 · 10-10 m β−  beta particle β+  positron γ  gamma ray δ  delta or chemical shift in parts per million (NMR) Δ  heat µ  micro (10-6) µM  micromolar (10-6 M) AAS  atomic absorption standard AcOH  acetic acid Anal.  analytical ANOVA analysis of variance atm  atmosphere BFC  bifunctional chelate, also means bifunctional ligand biomolecule vector, biovector, targeting vector, (e.g. antibody, peptide) Bn  benzyl t-Boc  tert-butoxycarbonyl Boc2O  di-tert-butyl-dicarbonate br  broad (NMR), e.g. br s (broad singlet) °C  degrees celsius calcd.  Calculated MeCN  acetonitrile xxxv  Ci  Curie CLI  Cerenkov luminescence imaging cm-1  wavenumber CN  coordination number COSY  correlation spectroscopy (1H-1H NMR) CR  Cerenkov radiation CT  computed tomography CV  cyclic voltammetry d  day(s) or doublet (NMR) DCM  dichloromethane DFT  density functional theory (in silico calculations) DFO  desferrioxamine B dien  diethylenetriamine DMF  dimethylformamide DMSO  dimethylsulfoxide DOTA  1,4,7,10-tetraazacyclododecane -N,N',N",N'"-tetraacetic acid DTPA  diethylenetetraaminepentaacetic acid EA  elemental analysis  EDTA  ethylenediaminetetraacetic acid ESI-MS electrospray ionization mass spectrometry EtOAc  ethyl acetate EtOH  ethanol en  ethylenediamine eV  electron volt equiv.  equivalent(s) FDA  Food and Drug Administration (USA) FDG  2-deoxy-2-[18F]fluoro-D-glucose g  gram h  hour(s) Herceptin® see trastuzumab xxxvi  HMBC heteronuclear multiple bond correlation/coherence (1H-13C NMR) HSQC  heteronuclear single bond correlation/coherence (1H-13C NMR) HPLC  high performance liquid chromatography Hz  hertz iTLC  instant thin layer chromatography (typically radioactive) in silico performed on a computer IR  infrared ID  injection dose J  coupling constant (NMR) k  kilo KML  thermodynamic complex stability constant L  litre or ligand LET  linear energy transfer m  milli- or medium or multiplet M  molar (moles/ litre) or mega MeOH  methanol MEP  molecular electrostatic potential min  minute(s) mol  mole MRI  magnetic resonance imaging  MS  mass spectrometry m/z  mass per unit charge n  nano (10-9) or number of unit NBS  N-bromosuccinimide NHE  normal hydrogen electrode NHS  N-hydroxysuccinimide NMR  nuclear magnetic resonance nM  nanomolar (10-9 M) Nosyl  2-nitrobenzenesulfonamide (protecting group) NOTA  1,4,7-triazacyclononane-1,4,7-triacetic acid xxxvii  NRU  National Research Universal (reactor) ORTEP Oak Ridge Thermal Ellipsoid Plot Program PBS  phosphate buffered saline Pd/C  palladium on carbon (10% by weight) PET  positron emission tomography  pH  -log[H3O]+ p.i.  post injection pM   -log[free metal], or picomolar (10-12 M) pn  propylenediamine ppm  parts per million q  quartet (NMR) ®  trademark RCY  radiochemical yield Rf  retention factor RIT  radioimmunotherapy RT  room temperature Rt  retention time s  singlet (NMR) or strong (IR) SD  standard deviation SPECT single photon emission computed tomography t  triplet (NMR) or time tR  retention time (HPLC) t1/2  half-life tBuOH  tert-butanol THF  tetrahydrofuran TFA  trifluoroacetic acid TFP  tetrafluorophenyl TLC  thin layer chromatography trastuzumab HER2/neu targeting antibody TREN  tris(2-aminoethyl)amine xxxviii  UV  ultraviolet V  volt VT-NMR variable temperature NMR w  weak (IR)    xxxix  Acknowledgements   There are many people that I want to acknowledge and thank for their important presence in my life during the ~5 year period while I completed the work presented in this thesis.  My parents Steve P. and Fiona P., who have offered boundless support and encouragement to me since I was a child, without whom the lofty goals to which I’ve aspired may never have been achieved.  My grandmother Marie Carr, and my brothers Kyle P. and Ryan P., who have remained positive and proud through this process, and Ryan’s new family Lindsay P. and their son Wyatt P.  The understanding and love of my partner Rhea D. has helped make this process possible.  The deep friendships of Brandon L. and David S. have helped keep me grounded, and their generous sponsorship of my inclusion into several of their vacations has been phenomenal.  To all of my collaborators and supporters: Eszter B. for helping me get started; Cara F. and Dennis W. for radiochemistry and career inspiration; James I. for mentoring and friendship since my undergraduate days; Kuo-Shyan L. at BC Cancer for peptides and support; and to Brian Z. and Jason L. at Memorial Sloan-Kettering Cancer Center for allowing me to visit and impose on them for 2 months, without whom a huge body of my work would never have been completed.  I also want to thank the support staff at UBC for being crucial to solving many of my problems: Maria E. and Paul X. in the NMR labs; Yun L., Marshall L., Derek S., and Marco Y. in the MS labs; and Brian P. and Anita L. in the X-ray labs.  I want to thank Gwendolyn B. for being the best undergraduate student anyone could ever hope to work with.  I would also like to thank all Orvig group members who have shared the struggles and satisfaction of graduate school with me, for attending countless group meetings and providing a lot of help.  I owe a big thank you to Jacqueline C. for being a huge help to me throughout my PhD and playing a very important role in almost every chapter of this thesis.  Finally I must thank my supervisors Chris O. and Michael A. for endless support and trust, and for encouraging me and funding me to attend international conferences and collaborations, which I could never have participated in without their help, and at which I met many collaborators and friends.  Thank you to my PhD committee Glenn S., Pierre K., and Mike F., because you were available to assist me, and I know you had better things to do than read my verbose 9 chapter thesis.  Thank you to NSERC for CGS-M, CGS-D, and now PDF funding.  To all of these people I owe sincere thanks and the utmost gratitude.    xl  Dedication          For my family, partner, close friends, collaborators, and mentors, for endless support and encouragement.     1 Chapter 1: Introduction  This chapter is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Orvig, C., Matching Chelators to Radiometals for Radiopharmaceuticals. Chem. Soc. Rev. 2014, 43 (1), 260-290, Copyright 2014 The Royal Society of Chemistry.   1.1 Background and aims Radiometals are radioactive isotopes that can be harnessed for applications in medical diagnosis, as well as for cancer therapy.1-9  In order to apply these isotopes to specific biological applications, the “free” radiometal ions must be sequestered from aqueous solution using chelators (ligands) to obviate transchelation and hydrolysis.  Ligands used for this application are typically covalently linked to a biologically active targeting molecule (also called vectors or biomolecules), making an active radiopharmaceutical agent.  The ligand is used to tightly bind a radiometal ion so that when injected into a patient, the targeting molecule can deliver the isotope without any radiometal loss from the radiopharmaceutical, effectively supplying a site-specific radioactive source in vivo for imaging or therapy.  Because some of the most popular targeting vectors are sensitive to elevated temperatures, such as antibodies and antibody fragments, ligands that are capable of quantitatively radiolabeling at ambient temperature (25 °C) are of great interest and utility.  Acyclic ligands generally possess faster radiolabeling properties than do macrocyclic ligands; however, the stability and inertness of acyclic ligands tends to be inferior to macrocycles in vivo.  The specific aims of this body of work are to investigate new acyclic ligands with fast     2 radiolabeling properties with a variety of radiometals, that also possess comparable stability to the currently used macrocyclic ligands.  1.2 Nuclear imaging and therapy Positron emission tomography (PET) imaging is a very accurate and quantitative imaging technique, which utilizes positron particles (β+, anti-electrons) that are emitted from a radioactive isotope upon decay.  After emission, the β+ particle travels a short distance before meeting with an electron and annihilating, which results in two gamma rays (511 keV) ejected at 180 degrees to each other.  These gamma rays are detected by a fixed circular array of coincidence detectors arranged around the subject, where only gamma rays that strike the detector at the same time are registered.  This process allows for 3D images to be constructed that identify the source (radioactive isotope) of β+ emission within the subject, with this source ideally being targeted to a specific location (e.g. cancer cells).  Single photon emission computed tomography (SPECT) operates on a very similar principle to PET.  SPECT imaging differs from PET in that emission of single gamma rays from an isotope is detected, rather than two coincident gamma rays ejected from positron annihilation.  SPECT has decreased image resolution and sensitivity compared to PET, largely because SPECT machines utilize pinhole collimators, whereas PET utilizes a circular array of coincidence detectors and no collimators.  Additionally, gamma ray emission does not occur at the same energy for all isotopes, and many isotopes eject several gamma rays of different energies, which can complicate data collection.  To utilize direct gamma ray emissions for SPECT imaging, several detectors physically rotate around the subject, stopping at many different orientations to perform the lengthy process of acquiring many 2D images, which are used to     3 reconstruct 3D images.  The archetypical PET isotope is 18F, which has a very high positron (β+) abundance of 96%, and a low energy β+ emission of 640 keV (short mean free path from decay location providing high resolution and accuracy).  Many of the more exotic PET radiometal isotopes discussed here have lower positron abundances (low branching ratios, ~20-60% decay by β+) and higher positron energies, which decreases the accuracy of data collection and requires longer image acquisition times and/or higher activity injected dose.10  For β+ annihilation and subsequent detection to occur the β+ must be sufficiently slowed down after emission for it to meet with an electron and annihilate. The size of the spherical radius that a positron travels from its source is dependent on its energy, and higher energy positrons travel a larger radius and therefore decrease spatial resolution. In general, lower energy β+ and γ emissions provide better image quality.  For example, the nuclides 86Y and 89Zr emit a large amount of γ rays relative to the amount of positrons (poor branching ratios).  These additional γ emissions can both complicate PET imaging by interfering with the detection of coincident 511 keV γ rays that originate from β+ emission/annihilation events, and increase the radioactive dose accumulated by patients.11,12  Despite these shortcomings, the PET nuclides discussed here have a multitude of chemical and physical properties that make them attractive for imaging purposes.   Radiotherapeutic effects are derived from the damage that ionizing radiation does to living cells, where gamma rays, β+ particles, and β- particles (high energy electrons) can strip electrons directly from cells, or can create harmful reactive oxygen species from water and other molecules present in living tissue.  Alpha particles (α) are helium nuclei that travel very short distances in living tissue, but that have very high linear energy transfer values (LET), meaning they deposit massive amounts of energy over a very small distance, having     4 the ability to literally blast cells apart.  Radionuclides are typically produced by proton (p,n) or deuteron (d,2n) bombardment via a cyclotron, neutron bombardment via a nuclear reactor (n,xp), or by elution from a generator system (e.g. 68Ge/68Ga, where the parent nuclide in generators must be produced via cyclotron or reactor).  The most common production methods of various isotopes are displayed in Table 1.1.    1.3 Popular radiometal isotopes The availability of a wide range of radiometal ions makes it possible to carefully pick the specific nuclear properties that are needed for a vast number of different applications (Table 1.1).13-16  Some examples of radiometals that can be used for positron emission tomography (PET) imaging are 68Ga, 64Cu, 86Y, 89Zr, and 44Sc, with PET imaging providing sensitive, quantitative, and non-invasive images of a variety of molecular processes and targets.  Single photon emission computed tomography (SPECT) is an older and more ubiquitous imaging modality than PET, and, since its inception in the 1960s, 99mTc has been the workhorse isotope of SPECT.  More recently, the radiometals 67Ga, 111In, and 177Lu have been increasingly used for SPECT imaging in chelator-based radiopharmaceuticals.  For therapy applications, particle emitters such as 111In (Auger electron emitter), 90Y and 177Lu (β-), and 225Ac, 212Pb, and 213Bi (α), are being heavily investigated, typically in conjunction with antibody vectors (immunoconjugates) or peptides.  Each radiometal ion has unique aqueous coordination chemistry properties; these must be properly addressed to if these isotopes are to be safely harnessed for medical applications and use in vivo.        5 Table 1.1 Properties of some popular radiometal isotopes, EC = electron capture; some low abundance emissions have been omitted for brevity.6,13-17  Isotope t1/2 (h) Decay mode E (keV) Production method 60Cu 0.4 β+ (93%) EC (7%) β+, 3920, 3000, 2000 cyclotron, 60Ni(p,n)60Cu 61Cu 3.3 β+ (62%) EC (38%) β+, 1220, 1150, 940, 560 cyclotron, 61Ni(p,n)61Cu 62Cu 0.16 β+ (98%) EC (2%) β+, 2910 62Zn/62Cu generator 64Cu 12.7 β+ (19%) EC (41%) β- (40%) β+, 656 cyclotron, 64Ni(p,n)64Cu 66Ga 9.5 β+ (56%) EC (44%) β+, 4150, 935 cyclotron, 63Cu(α,nγ)66Ga 67Ga 78.2 EC (100%) γ, 93, 184, 300 cyclotron, 68Zn(p,2n)67Ga 68Ga 1.1 β+ (90%) EC (10%) β+, 1880 68Ge/68Ga generator 44Sc 3.9 β+ (94%) EC (6%) γ, 1157 β+, 1474 44Ti/44Sc generator 47Sc 80.2 β- (100%) γ, 159 β-, 441, 600 47Ti(n,p)47Sc 111In 67.2 EC (100%) γ, 245, 172 cyclotron, 111Cd(p,n)111m,gIn 114mIn 114In (daughter) 49.5 d 73 s EC (100%) β- (100%) γ, 190 β-, 1989 cyclotron, 114Cd(p,n)114mIn or 116Cd(p,3n)114mIn 177Lu 159.4 β- (100%) γ, 112, 208 β-, 177, 385, 498 176Lu(n,γ)177Lu 86Y 14.7 β+ (33%) EC (66%) β+, 1221 cyclotron, 86Sr(p,n)86Y 90Y 64.1 β- (100%) β-, 2280 90Zr(n,p)90Y 89Zr 78.5 β+ (23%) EC (77%) β+, 897 cyclotron, 89Y(p,n)89Zr 212Bi 1.1 α (36%) β- (64%) α, 6050 β-, 6089 228Pb/212Pb generator 213Bi 0.76 α (2.2%) β- (97.8%) α, 5549 β-, 5869 228Th/212Pb generator 212Pb (daughter	  is	  212Bi) 10.6 β- (100%) α, 570 224Ra/212Pb generator 225Ac 240 α (100%) α, 5600-5830 (6) 226Ra(p,2n)225Ac n-Capture of 232Th è 233U è 225Ac      6 The major difference between radioactive (“hot”) and non-radioactive (“cold”) metal ion chemistry is that radiochemistry is typically performed under extremely dilute conditions, with radiometal ions typically being utilized at nmol to pmol quantities.18  It is also important to note that several of the elements being discussed have multiple radioactive isotopes that are useful for diagnostic or therapeutic purposes (e.g. 86/90Y, 67/68Ga, 44/47Sc, 60/61/62/64Cu), and all isotopes of a given element have identical chemistry.19-24  This means that a single radiopharmaceutical agent can be radiolabeled with different isotopes of the same element (e.g. 86/90Y), and provide the same charge and physical properties, and therefore the same biological behavior and distribution in vivo.19-24  This class of radiopharmaceutical that utilizes two isotopes of the same element, such as 86Y for PET imaging and 90Y for therapy, has been referred to as a theranostic agent.25,26 Alternatively, 90Y is unique and in some circumstances can be used directly for PET imaging because it emits a very low abundance of positrons (0.003%), which can be used to collect imaging data superior to that obtained by 90Y Bremsstrahlung imaging.27      7  Figure 1.1 Examples of bioconjugation reactions: (A) standard peptide coupling reaction between a carboxylic acid and a primary amine with a coupling reagent; (B-C) peptide coupling reactions between activated esters of N-hydroxysuccinimide (NHS) or tetrafluorophenyl (TFP) olaand a primary amine; (D) thiourea bond formation between an isothiocyanate and a primary amine; (E) thioether bond formation between a maleimide and thiol; (F) standard Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition (“click” reaction) between an azide and an alkyne; and (G) strain-promoted Diels-Alder “click” reaction between a tetrazine and transcyclooctene.   1.4 Radiometal-based radiopharmaceutical design Ligands that are typically used to construct radiometal-based radiopharmaceuticals (not always with 99mTc) are bifunctional chelators (BFCs), which are simply ligands with OHO H2NX Y e.g. DCC, EDCHATU, or HOBT NHOX YOOX NO O H2N Y NHOX Y+ + H2N Y NHOX Y+OOX F FF FX H2N Y+NCS X HN S HN YNO OX HS Y NO OX+ pH ~7pH 8-9NX Y Cu(II) + reducantor Cu(I) X+ NN N NN YS YX N N NN + YOO X N N O YOABCDEFG    8 reactive functional groups that can be covalently coupled (conjugated) to targeting vectors (e.g. peptides, nucleotides, antibodies, nanoparticles).  Common bioconjugation techniques utilize functional groups such as carboxylic acids or activated esters (e.g. N-hydroxysuccinimide NHS-ester, tetrafluorophenyl TFP-ester) for amide couplings, isothiocyanates for thiourea couplings, and maleimides for thiol couplings (Figure 1.1).2,28  Click chemistry is gaining popularity in bioconjugate chemistry, with both the traditional copper(I) catalyzed azide-alkyne Huisgen 1,3-dipolar cycloaddition “click” reaction (forming a 1,2,3-triazole-ring linkage), or newer copper-free reactions such as strain-promoted azide-alkyne cycloadditions (e.g. dibenzocyclooctyne/azide reaction) and Diels-Alder click reactions (e.g. transcyclooctene/1,2,4,5-tetrazine) (Figure 1.1).29  It is interesting to note that the transcyclooctene/1,2,4,5-tetrazine copper-free click coupling displays remarkably fast reaction kinetics, allowing for novel applications such as in vivo pre-targeting, where the click reaction can occur in vivo at very dilute concentrations.30-34  The modular design of BFC systems allows for a theoretically limitless number of different vectors to be conjugated, providing molecular targeting to a constantly increasing number of biological targets.     Figure 1.2 Illustration of an archetypal radiometal-based radiopharmaceutical agent containing a bifunctional chelator (BFC) conjugated to a targeting vector (e.g. antibody, peptide, nanoparticle) using a variety of conjugation methods (e.g. isothiocyanate-amine coupling (shown), peptide coupling, maleimide-thiol coupling, activated ester amide coupling, click-coupling) and then radiolabeled with a radiometal ion (e.g. 111In3+/177Lu3+/86/90Y3+).     9  The structure and physical properties of the radiometal-chelate complex have a large impact on the overall pharmacokinetic properties of a radiopharmaceutical, with many radiometal complexes being very hydrophilic and subsequently leading to rapid renal excretion when attached to small vectors such as peptides and nucleotides (less prominent with large ~150 kDa antibodies).19-24  It has been observed in peptide-conjugates that keeping the radiometal ion and peptide constant, and changing only the ligand can have drastic effects on biodistributions.35  Radiometal-based radiopharmaceuticals contain many synthetically exchangeable components, which can be separated into different modules: the radiometal, which changes the radioactive emission properties and half life (γ for SPECT, β+ for PET, and β-/α particles or Auger electrons for therapy); the ligand, which must be carefully matched with the radiometal for optimal stability; the BFC-vector conjugation method, for different types of bioconjugation reactions and linkages; and the vector/targeting moiety, which allows for the selection of any known molecular target for site-specific delivery of the radioactive “payload” (Figure 1.2).  Each radiometal ion has different chemical demands, including ligand donor atom preferences (e.g. N, O, S, hard/soft), coordination number, and coordination geometry; however, there are many key design considerations that can be applied universally.7  Ligand synthesis should be relatively simple and avoid stereoisomers, and the ligand should ideally be synthesized with modular synthons so that bioconjugation handles and donor atoms can be easily changed.  There is much utility in being able to tune the denticity and physical properties/polarity/charge of the ligand (the degree of polarity can be assessed by octanol-water partition coefficients (log P)), so that biodistribution properties can be adjusted.      10 Before creating new ligands for radiochemistry, it should be understood how to properly evaluate them.  The methods by which ligands are evaluated with different radiometals will be discussed, and the current “gold standard” ligands for each relevant radiometal ion will be identified.  A substantial amount of work has been done in the field of radiometal chelation, and it is prudent to properly introduce the material relevant to the work done in this thesis.2,4-9,15,26,28,36-56   1.4.1 Macrocyclic versus acyclic ligands When designing new ligands, a historical glance at previous work reveals that macrocycles are generally more kinetically inert than acyclic chelators, even if their thermodynamic stabilities have been determined to be very similar.57-61 Macrocyclic chelators require minimal physical manipulation during metal ion coordination, as they possess inherently constrained geometries and partially pre-organized metal ion binding sites, thereby decreasing the entropic loss experienced upon metal ion coordination.62  To contrast this, acyclic chelators must undergo a more drastic change in physical orientation and geometry in solution in order to arrange donor atoms to coordinate with a metal ion, and subsequently they suffer a more significant decrease in entropy than do macrocycles (thermodynamically unfavorable).  The thermodynamic driving force towards complex formation is therefore greater for macrocycles in general, a phenomenon referred to as the macrocycle effect.62  A crucial property where most acyclic chelators excel and most macrocycles suffer is in the coordination kinetics and radiolabeling efficiency.  The ability to quantitatively radiolabel/coordinate with a radiometal in less than 15 minutes at room temperature is a common property of acyclic chelators, whereas macrocycles often require     11 heating to 60-95 °C for extended times (30-90 minutes).63-65  Fast room temperature radiolabeling becomes a crucial property when working with BFC-conjugates of heat sensitive molecules such as antibodies and their derivatives, or when working with short half-life isotopes such as 68Ga, 212/213Bi, 44Sc, and 62Cu.    Figure 1.3 Cartoon depiction of metal ion coordination kinetics, enhanced off-rate kinetics in vivo (extremely dilute conditions), and possible routes of radiometal ion loss in vivo.   1.4.2 Matching ligands with radiometals – how are ligands evaluated? When a new ligand is synthesized for the purpose of radiometal ion sequestration, or an old ligand is repurposed for use with a new radiometal ion, initial screening experiments     12 are usually done by simple radiolabeling to determine a number of factors: whether the ligand can bind the radiometal ion and effectively radiolabel in high yields (quantitative is best), what temperature is required (ambient temperature is best), and what reaction time is required (faster is better).  Short half-life isotopes such as 68Ga are ideally matched with ligands that can radiolabel rapidly (fast radiolabeling kinetics).  Longer half-life isotopes such as 111In and 177Lu allow for extended reaction times, but even if the half-life allows for long reaction times, completing the radiolabeling portion of radiopharmaceutical preparation is most convenient if finished in less than 10 or 15 minutes.  As previously mentioned, room temperature radiolabeling is crucial for sensitive antibody vectors, which are degraded at elevated temperatures.  DOTA inconveniently requires elevated temperatures for radiolabeling with essentially all radiometals (e.g. 44Sc, 111In, 177Lu, 86/90Y, 225Ac) but its abundant application in radiochemistry for decades, its exceptional in vivo stability, and the commercial availability of many different bifunctional DOTA derivatives and vector conjugates are consistent with it being the most commonly used ligand to this day.  Moving forward to the design and testing of new ligands, fast room temperature radiolabeling kinetics should be a priority; however, fast kinetics of radiometal incorporation (on-rate) and consequently low energetic barriers to radiometal-chelate complexation can also mean fast radiometal decorporation (off-rates) and low energetic barriers to radiometal release (Figure 1.3, solid-state structures of ferritin H-chain homopolymer PDB file 1FHA, ceruloplasmin PDB file 2J5W, and apo-transferrin PDB file 2HAV shown).  An arduous balancing act is required to obtain the best set of ligand properties for each application and radiometal ion, requiring the study and availability of a broad selection of different ligands with a variety of properties from which to choose.     13 When a ligand is identified through early screening to possess radiolabeling properties that are suitable for use with a particular radiometal ion, it must then be experimentally determined to be highly thermodynamically stable and kinetically inert.  Further experiments are performed with the specific radiometal-chelate complex under conditions relevant to in vivo translation to judge its potential as the core component of a radiometal-based radiopharmaceutical.  The result of radiometal loss from a radiopharmaceutical in vivo is the non-targeted distribution of the “free” radiometal ion in the body, and its exact fate and distribution in the body depends on the properties and biological behavior of the specific radiometal ion in question (Figure 1.3).  For example, 89Zr and 68Ga are known to accumulate in the bone when released from a BFC, where 64Cu is known to accumulate in the liver.  The fate of these radiometal ions can be tracked using PET/SPECT imaging in the living animal, and/or biodistribution experiments where animals are euthanized at predetermined time points, their organs harvested, and the distribution of radioactivity measured and calculated for the percentage of injected dose of radioactivity per gram of tissue (%ID/g).  Each metal ion has its own unique properties that must be considered when constructing a radiometal-based imaging/therapeutic agent, such as its aqueous hydrolysis chemistry, redox chemistry, and affinity for native biological chelators.   1.4.3 Thermodynamic stability When evaluating and selecting a ligand to match with a specific radiometal ion for use in a radiopharmaceutical, kinetic inertness in vivo is ultimately the most crucial consideration, even beyond that of the absolute thermodynamic stability of the metal-chelate complex.  Thermodynamic stability/formation constants (KML = [ML] / [M][L]) are usually     14 experimentally determined by potentiometric and/or spectrophotometric titrations, but evaluating kinetic inertness under conditions relevant to in vivo applications can be much more problematic.  Thermodynamic stability constants can be a useful metric for preliminary comparisons of various ligand with a particular metal ion, but they do not predict in vivo stability with any level of competence.66,67  A thermodynamic parameter that provides more biologically relevant information than KML values is the pM value (-log[M]Free).68-71  The pM value is the negative log of the concentration of free metal ion uncomplexed by a given ligand under specific conditions, and is essentially the metal scavenging ability of the ligand; the higher the pM value, the lower the concentration of free metal ion.  The pM value is a condition-dependent value that is calculated from the standard thermodynamic stability constant (log KML), accounting for variables and conditions such as ligand basicity, metal ion hydrolysis, (physiological) pH, and ligand:metal ratio.  Stability constants and pM values provide a number for the direction and magnitude of the equilibrium in a metal-chelate coordination reaction under specific conditions, but give no kinetic information (e.g. off-rates for dissociation).72,73  This is a very important factor because the rate of dissociation in vivo is what governs the kinetic inertness of a radiometal complex, regardless of thermodynamic stability, and these off-rates are greatly influenced by the high dilution encountered in vivo when a tiny quantity of radiopharmaceutical (micro- or nano-grams) is diluted into the blood pool (Figure 1.3). Even more complicating is the abundant presence in the body of many strong native biological chelators and competing ions that can transchelate radiometals from BFC-conjugates.  These are often present in higher concentrations than is the radiopharmaceutical, and include transport proteins such as transferrin, ceruloplasmin, and metallothionein, storage proteins such as ferritin, and metal     15 containing enzymes such as superoxide dismutase (Figure 1.3).  This wide range of complicating factors means that a single in vitro assay is typically not accurate in predicting in vivo stability.   1.4.4 Kinetics - Acid dissociation and competitive radiolabeling Acid dissociation experiments can be used to measure and assess the relative kinetic inertness of a metal complex to acidic conditions.  Most complexes are found to de-ligate fairly quickly below pH 2.0,74 and experiments evaluating the rate of dissociation at a constant pH (e.g. 2.0) have been performed to compare and evaluate ligands with a particular metal/radiometal ion.59,74,75  In these acid dissociation experiments, decomplexation can be observed over time with techniques such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and nuclear magnetic resonance (NMR) for diamagnetic metal complexes.  With the exception of copper chelates,72,76 acid dissociation experiments are not commonly performed as they do not provide an accurate prediction of in vivo kinetic inertness, because low pH is not encountered in the blood or most organs (except the stomach) and radiometal dissociation typically occurs via transchelation to serum proteins and enzymes, and is not acid-mediated.73,77-81 Competitive radiolabeling experiments can be performed by adding to a radiolabeling mixture an excess of non-radioactive ions, such as Na+, K+, Ca2+, Mg2+, Cu2+, Zn2+, or Fe3+, followed by addition of a ligand to evaluate the impact of these competing ions on radiolabeling yields.59,82-85  These experiments can reveal the radiolabeling selectivity of a ligand for a specific radiometal ion in the presence of other biologically relevant competing ions.  Alternatively, if the radiometal complex is preformed under standard metal-free     16 radiolabeling conditions, and is then added to a mixture containing these competing ions, stability to transchelation can be assessed.  Because ligand-based radiopharmaceuticals are typically radiolabeled in strictly metal-free conditions (deionized ultrapure water, often passed through a metal-scavenging chelex resin), these experiments may not appear completely relevant for predicting in vivo stability and utility.  Depending on the method of radiometal production and purification, the specific activity of radiometal ions can vary greatly, as can the concentrations of impurity metals ions.18  Radiometals are used in very small quantities and under extremely dilute conditions; therefore, any impurity metal ions present (even if only a few nanomoles) may actually be in excess of the radiometal ion concentration, and competitive binding could be problematic.18,86  The presence and quantity of metal ion impurities in radiometal mixtures depends on the source of the radiometal ion, method of production, and purification.18,86  1.4.5 In vitro and in vivo stability Experiments that are more pertinent to in vivo translation are metal-exchange competitions with biologically relevant mixtures, including blood serum, apo-transferrin, superoxide dismutase, and hydroxyapatite (bone).28,87-92  By incubating radiometallated ligands with these different competition mixtures, the quantity of radiometal that is transchelated from ligand to serum proteins/enzymes can be evaluated over time using size exclusion HPLC, iTLC, or disposable PD10 size exclusion columns.28,87-92  These experiments provide a directly relevant measure of stability and kinetic inertness by competition with the most likely transchelation culprits in vivo; additionally, in these in vitro     17 assays the concentrations of these biological reagents can often be elevated above normal physiological levels to provide a more stringent challenge.  Ultimately the most relevant and practical test of radiopharmaceutical stability is in vivo.  Biodistribution studies in healthy mice can be performed on “naked” radiometal-ligand complexes (no conjugated vectors) to assess clearance and uptake profiles and ensure no abnormal organ distribution or critical instability occurs.  If a radiometal-ligand complex is very stable in vivo, the complex is typically cleared quickly from the animal through the kidneys/bladder/urine or digestive system/liver/feces depending on polarity and metabolism.93  Unstable complexes often demonstrate persistent uptake in organs and tissues where the non-bound radiometal is known to associate (e.g. Zr4+ and lanthanides in the bone, Cu2+ in the liver).93  The major drawback to this type of experiment with “naked” ligand complexes is that highly polar and charged radiometal-ligand complexes are typically cleared very quickly from the body, and therefore do not persist in vivo for long enough to encounter any significant challenge to their structural integrity.88,89  Conversely, highly lipophilic complexes tend to accumulate in the liver and digestive tract, regardless of stability.88,89 To evaluate properly the stability and kinetic inertness of a ligand in vivo, a suitable vector must be attached (e.g. peptide, antibody, nanoparticle) so that the radiometal-ligand complex is made to persist in blood circulation for a substantial period of time, and can be monitored over several hours or days (depending on isotope half-life and the subsequent imaging/therapy window).94  An additional concern with in vivo experiments is the significant normal variance between animals that introduces error; for example, 10 mice of the same sex and breed, procured from the same supplier, will often show significant differences in biodistribution of the same radiopharmaceutical.  Additionally, the specific     18 experimental techniques and methods (e.g. radiopharmaceutical preparation, injection, animal dissection, organ counting) used for these biodistribution experiments can cause large variability between data sets.  Variables such as specific activity of the source radiometal, specific activity of the radiolabeled agent, radiolabeling temperature, purity of the final radiopharmaceutical preparation, injected mass of radiopharmaceutical, and injected volume can make large differences in biodistribution profiles.  The same radiopharmaceutical used at different institutions may offer drastically different tumour uptake and organ distribution values, if the above variables are not tightly controlled, and even then differences between animals and between experimental techniques can introduce large variances.  For these reasons it is crucial to include internal control experiments for every study; for example, evaluation of a new ligand in vivo should be done in parallel with an existing and established “gold standard” ligand so that a direct comparison can be made, because comparison to previously performed studies (even in the exact same animal model and/or cell line) is often not reliable due to the above mentioned complications.83,87,89,95   1.5 A selection of ligands and their most suitable radiometal companions In order to properly design and prioritize experiments towards the discovery and elaboration of new acyclic ligands for radiopharmaceutical applications, a survey of previous work and existing ligands must be executed.  A review of radiometal ligands has been undertaken, with the most relevant and promising examples being discussed.  The most suitable ligand-radiometal matches have been identified, and detailed tables included, with the aim of providing a quick reference for finding the optimal match between ligand and radiometal.  A color-coding scheme has been used, and justification for the assignment of     19 good (green), fair (orange), and poor (red) matches between ligands and radiometals can be found in the text and the provided references.  The color code indicates the general suitability of a ligand for use with a specific radiometal, accounting for factors such as radiolabeling conditions and in vitro/in vivo stability.  An assignment of green may suggest that either a ligand is currently the “gold standard” for use with a given radiometal, or that early work with a new ligand looks very promising and in vivo studies have shown that it works comparably to the current “gold standards”.  Assignment of orange to a ligand-radiometal pair may be made if the combination has been used in vivo successfully, but perhaps in recent years has been surpassed by a new and superior ligand and is no longer the best choice.  Also, an assignment of orange could mean that a new chelator-radiometal ion pair looks very promising, but perhaps a bifunctional derivative has yet to be synthesized, or only preliminary in vivo work has been done and more study is required (e.g. no study of bioconjugates, or no direct comparisons to existing “gold standards” for internal reference).  An assignment of red indicates that the highlighted chelator-radiometal ion pair is an unsuitable match, and generally should not be used due to severe instability or radiolabeling problems.  These color “ratings” were made using the referenced studies and reflect the current trends in radiochemistry and nuclear medicine.  1.5.1 DOTA DOTA is one of the primary workhorse ligands for radiometal chemistry, and is one of the current “gold standards” for a number of isotopes, including 111In, 177Lu, 86/90Y, 225Ac, and 44/47Sc.  DOTA has been extensively used with 67/68Ga, but is widely accepted to be less stable than its more petite macrocyclic counterpart NOTA (Table 1.2). 68Ga-DOTATOC has     20 been shown to exhibit superior in vivo properties to 111In-DOTATOC (Octreoscan) despite non-optimal stability.96  Even though a chelator may be more stable and inert with a given radiometal (e.g. NOTA vs. DOTA for 68Ga), it may not be the optimal choice for a certain application (e.g. a specific peptide vector).  For example, NOTA is widely accepted to form a more stable complex with 68Ga than does DOTA, but due to differences in charge and physical properties (e.g. neutral vs. charged complex), DOTA may provide superior in vivo properties with certain vectors.35,83  This example highlights the complex set of variables one must consider when constructing radiometal-based radiopharmaceuticals. When designing new BFCs or modifying existing ligands to make BFC derivatives, care should be taken not to disrupt the original coordination sphere of the chelator.  Some common bifunctional DOTA derivatives utilize one of the carboxylic acid arms for the site of vector conjugation, effectively blocking one of the coordinating carboxylate arms (amide carbonyl groups may still coordinate, albeit weakly) (e.g. DOTA-NHS, Table 1.2).63  DOTAGA, DOTASA, and various isothiocyanate derivatives of DOTA have been synthesized and solve this problem by conjugating to vectors through the carbon backbone and side-arm functionalization (Table 1.2).63,97-99  These bifunctional DOTA derivatives retain their maximum potential denticity (octadentate) as well as the same thermodynamic stability and kinetic inertness as unadulterated DOTA.63,97,98  Despite its slow radiolabeling kinetics and required heating, DOTA is currently the “gold standard” ligand for use with 111In, 177Lu, and 86/90Y (Table 1.2).  The coordination chemistry and properties of Y3+ and Lu3+ are very similar; they both preferentially form 8-9 coordinate complexes in square antiprismatic or monocapped square antiprismatic geometries, and are hard metal ions with a preference for hard ligand donors such as     21 carboxylate-oxygens and amine-nitrogens.  In3+ has similar properties to Y3+ and Lu3+, but being smaller forms 7-8 coordinate complexes, typically with square antiprismatic geometry.  According to acid dissociation experiments performed at pH 2.0 with the 90Y complexes of DOTA, DTPA (vide infra), and CHX-A’’DTPA, DOTA is the most kinetically inert to acid dissociation by a significant margin, with CHX-A’’-DTPA showing moderate stability, and DTPA decomposing almost immediately.59  The validity of acid dissociation experiments is questionable, as conditions such as these are never encountered in vivo (except for the gastrointestinal tract), but they are a quantifiable metric for measuring off-rate kinetics and further substantiates the place of DOTA as the current “gold standard” ligand for 111In, 177Lu, and 86/90Y.59 Although very little investigation of this isotope has been performed, DOTA is also the current ligand of choice for 44Sc, but the requisite high temperature (90 °C) radiolabeling conditions are not optimal.100-102  As 44Sc has only recently become more popular in the literature, little in vivo work has been performed, and new ligands (preferably with ambient temperature radiolabeling properties) are of strong interest.  Another isotope of recent interest for use with DOTA is the α-emitter 225Ac, a large actinide isotope that possesses no non-radioactive isotopologue, making study of its coordination chemistry difficult. The large macrocycles HEHA and PEPA had previously been used most extensively with 225Ac, but recently DOTA has been shown to have superior in vivo properties.103  The long half-life of 225Ac (10.0 days) lends well to radioimmunotherapy (RIT), and so the high temperatures required for 225Ac radiolabeling with DOTA are very unfavorable for sensitive antibodies.         22 Table 1.2 DOTA and bifunctional derivatives.a   Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.    DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, maximum CN = 8, donor set N4O4 64Cu2+ ~ 25-90 °C, 30-60 min, pH 5.5-6.5 22.2, 22.7 distorted octahedron 88,91,94,104-110 67/68Ga3+ ~ 37-90 °C, 10-30 min, pH 4.0-5.5 21.3 (pM 15.2, 18.5) distorted octahedron 68,96,110-118 44/47Sc3+ ✓ 95 °C, 20-30 min, pH 4.0 27.0 (pM 26.5) square antiprism 100-102  111In3+ ✓ 37-100 °C, 15-60 min, pH 4.0-6.0 23.9 (pM 17.8-18.8) square antiprism? 68,89,97,114,117,119-124 177Lu3+ ✓ 25-100 °C, 15-90 min, pH 4.0-6.0 23.5, 21.6 (pM 17.1) square antiprism 124-130 86/90Y3+ ✓ 25-100 °C, 15-90 min, pH 4.0-6.0 24.3-24.9 square antiprism 59,74,97,124,127,130-132 213Bi3+ ~ 95-100133 °C, 5 min, pH 6.0-8.7 - square antiprism 134,135 212Pb2+ ~ 25-75 °C, 30-60 min, pH 4.0-5.5 - square antiprism 127,136-138 225Ac3+ ✓ 37-60 °C, 30-120 min, pH 6.0 - square antiprism? 139,140   DOTA-NHS-ester141   p-SCN-Bn-DOTA (C-DOTA)99,142,143  DOTAGA, R = amide, NHS ester144  DOTAGA-anhydride145  a highlighting relevant radiometal ions, radiolabeling conditions, thermodynamic stability constants (Log KML), coordination geometry, and color-coded ranking (a green ”✓“ = good/best match, orange “~” = suitable match, or requires more evaluation but shows potential, red ”✗” = poor/unstable match).   The current method used for synthesizing 225Ac-DOTA-antibody conjugates is to radiolabel the BFC p-SCN-Bn-DOTA at 60 °C with 225Ac first, followed by antibody conjugation (thiourea coupling) and purification.139,140  It is important to realize that the isothiocyanate functionality in the BFC p-SCN-Bn-DOTA is very sensitive and degrades rapidly under these conditions.  A study comparing the therapeutic effects of 225Ac, 213Bi, and 90Y using an antibody vector demonstrated 225Ac to be the most effective, with renal toxicity from the daughters 221Fr and 213Bi being the most significant concern.146  A recent clinical trial in humans with 225Ac-DOTA-HuM195 (humanized anti-CD33 monoclonal antibody) has shown promise, with DOTA being the current “gold standard” for 225Ac.140,147  A high NN NN CO2HHO2CHO2C CO2HNN NNHO2CHO2C CO2HOO NOO NN NN CO2HHO2CHO2C CO2H NCS NN NN CO2HHO2CHO2C HO2C NHO R NN NNHO2CHO2C CO2HOO O    23 denticity acyclic ligand (CN = 8-10) that could match the in vivo stability of DOTA and radiolabel at room temperature would be of great utility and would offer a more streamlined radiopharmaceutical preparation than the one outlined above for p-SCN-Bn-DOTA.   Table 1.3 DOTA derivatives CB-DO2A, TCMC, 3p-C-DEPA, Oxo-DO3A, and bifunctional derivatives.a  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   CB-­‐DO2A,	  4,10-­‐bis	  (carboxymethyl)-­‐1,4,7,10-­‐tetraazabicyclo[5.5.2]	  tetradecane,	  N4O2,	  CN	  =	  6	  64Cu2+ ~ 80 °C, 30-60 min, pH 5-7 - distorted octahedron 72,148 67/68Ga3+ ~ - - distorted octahedron 149   TCMC, 1,4,7,1O-tetrakis (carbamoylmethyl)-l,4,7,1O-tetraaza cyclododecane, N4O4, CN = 8 212Pb2+ ✓ 37 °C, 30-60 min, pH 5-6.5 >19 Square antiprismatic 138,150-154    p-SCN-Bn-TCMC153    3p-C-DEPA, N5O5, CN = 10 212/213Bi ✓ 25 °C, 5-10 min, pH 5.5 - Square antiprismatic 155   3p-C-DEPA-NCS155  3p-C-DEPA = 2-[(carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-tris-(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)- amino]acetic acid   p-­‐NH2-­‐Bn-­‐Oxo-­‐DO3A,	  N3O4,	  CN	  =	  7	  64Cu2+ ~ 25 °C, 10 min, pH 5.5 - distorted octahedron? 90,91,156,157 67/68Ga3+ ~ 25 °C, 10 min, pH 4-5 - distorted octahedron? 90,158,159  a highlighting relevant radiometal ions, radiolabeling conditions, thermodynamic stability constants (Log KML), coordination geometry, and color-coded ranking (a green ”✓“ = good/best match, orange “~” = suitable match, or requires more evaluation but shows potential, red ”✗” = poor/unstable match).  NNNN CO2HHO2C NN NN CONH2H2NOCH2NOC CONH2 NN NN CONH2H2NOCH2NOC CONH2 NCSNN NN CO2HHO2CHO2C N CO2HCO2H NO2 NN NN CO2HHO2CHO2C N CO2HCO2H NCSON NN CO2HHO2C CO2H NH2    24 1.5.2 DOTA Derivatives: CB-DO2A, 3p-C-DEPA, TCMC, and Oxo-DO3A CB-DO2A has only been investigated with the radiometal 64Cu for radiochemical use (Table 1.3).72,148 An X-ray crystal structure of CB-DO2A with Ga3+ is available and shows a distorted octahedral coordination geometry, and acid stability experiments have been performed with Ga3+, revealing impressive acid inertness.149  Although very little work has been done with CB-DO2A, studies with the non-bifunctional 64Cu(CB-DO2A) have suggested that it had superior in vivo stability to DOTA and TETA, but inferior stability to CB-TE2A.139   Further comparision of CB-DO2A to CB-TE2A, DOTA, and TETA revealed that CB-TE2A possessed superior acid and electrochemical inertness, which explains why DOTA and its cross-bridged derivative CB-DO2A have been replaced by CB-TE2A and are no longer a prominent subject of current research with 64Cu.56  Another interesting DOTA derivative is Oxo-DO3A, which is a heptadentate N3O4 macrocycle that has shown improved radiolabeling kinetics and stability with 64Cu and 67/68Ga compared to DOTA (Table 1.3). The p-NH2-Bn-Oxo-DO3A derivative is shown in Table 1.3; it can be directly coupled to a peptide via standard peptide coupling reactions, or transformed to a benzyl-isothiocyanate. Although DOTA has been used successfully with 212Pb, its slow radiolabeling kinetics and stability properties were not ideal.160  Replacement of the carboxylic acid donor arms of DOTA with amide arms has produced the ligand TCMC (Table 1.3), which to date is the best ligand available for 212Pb.151,152,161  Chappel et al. have shown TCMC to be superior to C-DOTA (Table 1.2) with 212Pb due to improved radiolabeling kinetics and in vivo stability.153,162  Although not optimal for 212Pb, 3p-C-DEPA (Table 1.3) has been shown to be an excellent ligand for 212/213Bi, with superior properties to C-DOTA.155  The DOTA     25 derivative 3p-C-DEPA has demonstrated greatly improved radiolabeling kinetics with 212/213Bi compared to DOTA, with comparable in vivo stability.155 3p-C-DEPA can be considered to be one of the current “gold standards” for 212/213Bi; however, the NOTA analogue 3p-C-NETA (vide infra) also looks very promising and has not been directly compared with 3p-C-DEPA, and so the superior of the two has yet to be identified.  1.5.3 TETA TETA is an octadentate N4O4 macrocyclic ligand that has only been investigated with 64Cu for radiopharmaceutical applications (Table 1.4).  TETA is generally not used anymore, and has been superseded by newer generation ligands such as NOTA, CB-TE2A, and CB-TE1A1P/CB-TE2P (Table 1.4) that are more stable and kinetically inert in vivo.  Copper is a difficult metal ion to harness, as it is quite labile, has active redox chemistry between Cu1+/Cu2+ in vivo, and Cu2+ has a fast aqua ligand exchange rate of 2 x 108 s-1.44,163  Copper is a first row transition metal with a d9 electron configuration, is best used with hexadentate ligands that saturate its coordination sphere, and has a preference for borderline soft ligand donor atoms such as amines, imines, and thiols.6,44  Despite being made obsolete in recent years, TETA has previously been used successfully for 64Cu imaging with vectors such as octreotide.164,165  TETA can radiolabel with 64Cu at room temperature, but it lacks kinetic inertness and overall stability in vivo.  Surprisingly, the TETA derivative TE2A (Table 1.4) shows enhanced kinetic inertness, despite the fact that TE2A is merely a hexadentate derivative of TETA that is missing two carboxylic acid arms.166  This effect is enhanced even further when the two secondary amines of TE2A are methylated or cross-bridged by an ethylene unit (Table 1.4).148,167  Unlike DOTA (cyclen-based), TETA (cyclam-based) has not     26 been heavily investigated with any radiometals other than 64Cu; however, some structural and physical data is available with other metal ions (Table 1.4).   Table 1.4 TETA, CB-TE2A and derivatives, and bifunctional derivatives.  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   TETA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,-11-tetraacetic acid, N4O4 CN = 8  64Cu2+ ~ 25 °C, 60 min, pH 5-7. 21.9, 21.6. Distorted octahedron 164,168 67/68Ga3+ ✗ - 19.74 (pM 14.1) Distorted octahedron 68 111In3+ ✗ - 21.9 (pM 16.3) Distorted octahedron? 68 177Lu3+ ✗ - 15.3 Distorted dodecahedron? 169 86/90Y3+ ✗ - 14.8 Distorted dodecahedron? 169             BAT (C-TETA derivative)170      p-H2NBn-TE3A171       C-TETA (p-NO2-Bn-TETA)172   CB-­‐TE2A,	  4,11-­‐bis(carboxymethyl)-­‐1,4,8,11-­‐tetraazabicyclo[6.6.2]hexadecane,	  N4O2	  CN	  =	  6	  64Cu2+ ✓ 95 °C, 60 min, pH 6-7 - Distorted octahedron 72,94,106,108,173-178    179                   CB-TE1A1P174,180             CB-TE2P174,180           MM-TE2A167               DM-TE2A167                     TE2A166   NN NN CO2HHO2CHO2C CO2H NN NN CO2HHO2CHO2C CO2HNH O Br NN NN CO2HHO2CHO2C NH2 NN NN CO2HHO2CHO2C CO2HNO2NN NN CO2HHO2C NN NNHO2C OHN R NN NNHO2C O ON OOHO ONN NN POOHOHOHO NN NN PP OOHOHOOHHO NN NHN OOHHOO NN NN OOHHOO NHN NHN CO2HHO2C    27 1.5.4 TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A The cyclam-based macrocycle TETA has spawned a large number of successful derivatives (Table 1.4).  Despite the requisite harsh radiolabeling conditions (95 °C), CB-TE2A has become one of the premier 64Cu ligands, having superior in vivo stability to TETA, TE2A, DOTA, NOTA, and CB-DO2A.148  The stability gains from adding an ethylene cross bridge to TETA/TE2A are significant, despite the removal of two coordinating carboxylic acid arms, and a subsequent decrease in denticity from 8 to 6.  In order to surmount the harsh radiolabeling conditions required for CB-TE2A, several derivatives of CB-TE2A have been synthesized.  Improved radiolabeling kinetics have been achieved by replacement of one (CB-TE1A1P) or both (CB-TE2P) carboxylic acid arms with methylenephosphonate groups, with in vitro and in vivo stability being retained or enhanced compared to the native CB-TE2A (Table 1.4).148,174-177,181  Most importantly, the methylenephosphonate derivatives CB-TE1A1P and CB-TE2P can be radiolabeled with 64Cu at room temperature, although relatively slowly.  Another noteworthy set of derivatives were made by methylating one (MM-TE2A) or two (DM-TE2A) nitrogen atoms of TETA/TE2A, instead of linking them together with an ethylene bridge (Table 1.4).167  Surprisingly, these methylated TE2A derivatives possessed similar radiolabeling kinetics and stability to CB-TE2A.167  This observation suggests that the high stability of CB-TE2A is not a result of steric protection by the cage-like structure created by the cross-bridged ethylene unit, but instead by changing the electronic properties of the nitrogen atoms (secondary to tertiary amines) and their donor properties. Although CB-TE2A has been the most heavily investigated ligand of the above mentioned TETA derivatives, the new methylenephosphonate derivatives CB-TE1A1P and     28 CB-TE2P show the most promise, as they appear to retain the stability of CB-TE2A, while having greatly enhanced radiolabeling kinetics.   1.5.5 Diamsar and derivatives  The radiometal ion 64Cu2+, similar to 68Ga3+, has been extremely popular in recent years due to its favorable positron emission (β+) properties for PET imaging and its intermediate half-life (12.7 hours), which is suitable for use with peptides, antibody fragments, and even whole antibodies.  Unlike 68Ga, 64Cu also undergoes β- emission, making it useful for therapy in addition to PET imaging.  It follows, therefore, that there has been a strong interest in developing new and improved bifunctional chelators to optimize radiolabeling procedures and in vivo performance with isotopes of copper.  In addition to the ubiquitous ligand NOTA (vide infra), new entrants such as CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, and DM-TE2A have recently been determined to be excellent 64Cu ligands (vide supra).  The Sar family of ligands (hexamine sarcophagines) are hexaazamacrobicyclic cage type ligands, and include the ligands SarAr, SarAr-NCS, diamSar, AmBaSar, and BaBaSar (Table 1.5).  One of the major benefits of these nitrogen rich ligands is that they can radiolabel with 64Cu in only a few minutes (5-10 min) at room temperature, which is faster even than the recently devised ligands CB-TE1A1P and CB-TE2P (Table 1.4).44,182-184  Of all the ligands discussed for 64Cu, Sar derivatives have the fastest room temperature radiolabeling kinetics.  In addition to fast kinetics, they demonstrate excellent in vivo stability, and strong resistance to acid dissociation in vitro.182-185  One drawback to nitrogen rich N6 ligands such as the Sar family is lipophilicity, as they contain little or no acid functionality to add negative charge, and as a result form cationic or neutral     29 complexes with 64Cu (depending on the Sar derivative).  Because the half-life of 64Cu (~12 h) pairs exceptionally well with the distrubtion and localization times for peptides, a lipophilic radiometal-ligand complex can have deleterious affects on biodistribution and tumour targeting properties by directing activity to the liver and digestive tract (depending on hydrophilicity of the selected peptide).  The Sar derivative BaBaSar adds two carboxylic acid functional groups to the Sar scaffold, thereby augmenting hydrophilicity.  Animal studies have been promising with the Sar family of ligands, but more work is required for further validation; furthermore, limited commercial availability and challenging synthesis may be a reason why widespread adoption has yet to occur.44,182-185   Table 1.5 The copper ligands Diamsar, SarAr, AmBaSar, and BaBaSar.a  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   Diamsar, N6, CN = 6 64Cu2+ ✓ 25 °C, 5-30 min, pH 5.5  Distorted octahedron or trigonal prism 182-185    1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine (SarAr)182-186  SarAr           R = NH2,     R' = H SarAr-NCS  R = NCS,     R' = H AmBaSar    R = COOH,  R' = H BaBaSar     R = -COOH, R' = -CH2-Ph-COOH   a highlighting relevant radiometal ions, radiolabeling conditions, thermodynamic stability constants (Log KML), coordination geometry, and color-coded ranking (a green ”✓“ = good/best match, orange “~” = suitable match, or requires more evaluation but shows potential, red ”✗” = poor/unstable match).   1.5.6 NOTA, NETA, and TACN-TM NOTA is a hexadentate N3O3 ligand, and is one of the oldest and most successful ligands for use with 67/68Ga and 64Cu (Table 1.6).  NOTA is generally considered to be the N NN NNH HNH2N H HH H NH2 N NN NNH HNHN H HH H NH RR'    30 “gold standard” for Ga3+ chelation, possessing favorable radiolabeling conditions (RT, 30-60 minutes) and excellent in vivo stability.110,117,187,188  Of all the isotopes discussed in this chapter, 68Ga is probably the most popular in recent years.  68Ga has a very favorable positron emission (1880 keV, 90%) for PET imaging, and a short half-life (68 min) suitably matched for imaging with peptide vectors.  The recent development of several 68Ge/68Ga-generator systems with shelf-lives of ~9-12 months has propelled this radiometal into the spotlight of the international research scene.189  Ga3+ is a hard acidic metal ion (pKa 2.6) with an ionic radius of 62 pm (CN = 6), a preference for amine and oxygen donor atoms, and is ultimately a challenging metal ion to chelate because it has a strong affinity for hydroxide anions causing precipitation of gallium hydroxide (Ga(OH)3) between pH 3-7.66,163,190 Because of gallium’s potential clinical utility, many new gallium ligands have been published in the last ~5 years; however, most have not yet amassed the same amount of in vivo data as NOTA.  NOTA has been shown to have superior radiolabeling properties and stability with 64Cu when compared to common ligands such as DOTA, EDTA, DTPA, and TETA.191, 155  NOTA can radiolabel with 64Cu at room temperature in 30-60 minutes, making it compatible with heat sensitive antibody vectors.192  Although many new ligands such as TRAP, AAZTA (DATA), H2dedpa, CP256, and PCTA (vide infra) show great promise for use with 67/68Ga in early experiments, due to commerical availability of BFC derivatives and widespread acceptance, NOTA is still currently the “gold standard” for 67/68Ga.  A similar story can be told for 64Cu, where a plethora of new ligands, including the Sar family (e.g. SarAr, DiamSar), CB-TE2A, and various CB-TE2A derivatives (e.g. CB-TE1A1P, CB-TE2P, MM/DM-TE2A) appear to possess many superior properties, but NOTA is still the practical “gold standard” for 64Cu.     31  Table 1.6 NOTA, NETA, TACN-TM, and bifunctional derivatives.a  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid, CN = 6, N3O3 64Cu2+ ✓ 25 °C, 30-60 min, pH 5.5-6.5 21.6 distorted trigonal prism 191,193 67/68Ga3+ ✓ 25 °C, 30-60 min, pH 4.0-5.5 31.0 (pM 26.4, 27.9) distorted octahedron 110,117,118,187,188,194,195 44/47Sc3+ ~ 95 °C, 20-30 min, pH 4.0 16.5 (pM 19.2) distorted octahedron 102 111In3+ ~ 60-95 °C, 20-30 min, pH 4.0-5.0 26.2 (pM 21.6) distorted octahedron 74,117,188,196-198                                                       p-SCN-Bn-NOTA(C-NOTA)143,196            NODASA, R = NHS ester, amide199              NODAGA, R = NHS ester, amide197   NETA, [2-(4,7-Biscarboxymethyl[1,4,7]triazacyclononan-1-ylethyl)carbonylmethyl-amino]acetic acid,  N4O4, CN = 8 177Lu3+ ✓ 25 °C, 5 min, pH 4.5 - Square antiprism? 127,161,200,201 86/90Y3+ ✓ 25 °C, 5 min, pH 4.0 - Square antiprism? 127,201,202 212/213Bi3+ ✓ 25 °C, 5 min, pH 4.0 - Square antiprism? 161,200,201,203 212Pb2+ ✗ 25 °C, 5 min, pH 4.0 - Square antiprism? 201                  NETA-monoamide202                    C-NE3TA-NCS201               C-NETA-NCS201                            3p-C-NETA127,203,204   TACN-TM, N,N’,N’’, tris(2-mercaptoethyl)l,4,7-triazacyclononane, N3S3 CN6 67/68Ga3+ ✗ 25 °C, 10 min, degassed ethanol 34.2 distorted octahedron 110,205-207  111In3+ ✗ 25 °C, 10 min, degassed ethanol 36.1 distorted octahedron 99, 176, 177, 183  a highlighting relevant radiometal ions, radiolabeling conditions, thermodynamic stability constants (Log KML), coordination geometry, and color-coded ranking (a green ”✓“ = good/best match, orange “~” = suitable match, or requires more evaluation but shows potential, red ”✗” = poor/unstable match).  N NNHO2C CO2HHO2C N NNHO2C CO2HHO2C NCS N NNHO2CHO2C CO2H HNO R N NNHO2CHO2C CO2H OHN RN NNHO2CHO2C N CO2HCO2HN NNHO2CHO2C N NHO RCO2H N NNHO2CHO2C HN CO2HSCN N NNHO2CHO2C N CO2HCO2HSCN N NNHO2CHO2C N CO2HCO2HNCSN NNSHHS SH    32 The modified NOTA ligand NETA (Table 1.6) is an interesting derivative that shows promise with 86/90Y, possessing fast radiolabeling kinetics similar to those of acyclic ligands (5-10 minutes, RT), as well as a high degree of stability and rigidity imparted by the macrocyclic framework.202  A direct comparison between NETA and DOTA has demonstrated greatly enhanced radiolabeling kinetics for NETA with Y3+.202  Biodistribution experiments performed with the 86Y complexes of the non-bifunctional chelators NETA and DOTA have suggested that NETA had comparable clearance and stability properties to DOTA, even showing lower bone accumulation than DOTA.202  NETA has been evaluated with other therapeutic isotopes for radioimmunotherapy (RIT), including 203/212Pb, 212/213Bi, and 177Lu.161,200,201 NETA and bifunctional derivatives C-NETA and C-NE3TA (Table 1.6) were found to be unstable with 203/212Pb isotopes, but looked very stable with 212/213Bi, 90Y, and 177Lu during a period of 11 days in blood serum.201,204,208  Biodistribution studies of C-NETA complexes in healthy mice demonstrated excellent clearance and stability of the 212/213Bi, 90Y, and 177Lu complexes, with the exception of high kidney uptake with the 212/213Bi-C-NETA complex.201  Recent experiments with an isothiocyanate bearing derivative of C-NETA, 3p-C-NETA (Table 1.6) have demonstrated that 90Y and 177Lu radiolabeled immunoconjugates (trastuzumab) had very rapid room temperature radiolabeling kinetics, and excellent in vivo performance, with the potential to surpass DOTA and also DTPA analogues such as CHX-A’’-DTPA.127  3p-C-NETA has been further evaluated with 212/213Bi for RIT applications, and trastuzumab immunoconjugates have been shown to exhibit rapid radiolabeling kinetics and excellent in vitro and in vivo stability, suggesting improved stability over C-NETA, which showed high kidney accumulation of 212/213Bi.203, 201  It is interesting to note that these     33 studies were actually performed with 205/206Bi, as the longer half-lives (15.3/6.2 days, respectively) of these 212/213Bi isotopologues are more amenable to extensive in vitro and in vivo study than are the short half-lives of 212/213Bi (0.76-1.1 hrs).203  Currently it is not clear if 3p-C-DEPA (Table 1.3) is superior to 3p-C-NETA (Table 1.6) for use with 212/213Bi, and a direct comparison would be timely. TACN-TM (Table 1.6) is an interesting thiol-based ligand, and shows extremely high thermodynamic stability constants (log KML) with Ga3+ and In3+ of 34.2 and 36.1, respectively.99, 176, 177, 183  Another interesting thiol TACN derivative is TACN-HSB, which substitutes alkyl-thiols for thiophenol groups.206  Despite the high thermodynamic stabilities, radiolabeling experiments required the use of degassed ethanol due to instability of the thiol groups of TACN-TM/TACN-HSB to air, and ultimately these ligands could not be used for in vivo applications.206  For these reasons, no bifunctional derivatives have been synthesized of these thiol-based TACN/NOTA derivatives, and further study has not been pursued.  1.5.7 DTPA, 1B4M-DTPA, and CHX-A’’-DTPA DTPA is one of the oldest and most pervasive acyclic ligands used in radiochemistry, and like most acyclic ligands it can be radiolabeled with many radiometal ions at room temperature in a matter of minutes (Table 1.7).  As a first generation radiometal ligand, it suffers from stability issues in vivo with many radiometal ions, is universally not as stable as the macrocycles DOTA and NOTA, and has become obsolete in recent years.60  DTPA has been successfully used as the BFC in the FDA-approved SPECT agent OctreoScanTM (111In-DTPA-octreotide), a somatostatin-targeting peptide-conjugate used for imaging neuroendocrine tumours.209,210  DTPA has also been successfully used with radiometals such     34 as 64Cu, 111In, 177Lu, and 86/90Y, but has been made redundant by more stable new ligands such as DOTA, NOTA, and CHX-A’’-DTPA (Table 1.7).60,165  The shortcomings of DTPA have been improved through design of novel derivatives, such as the DTPA derivatives 1B4M-DTPA (Tiuxetan) and CHX-A”-DTPA.60,211  1B4M-DTPA is a bifunctional DTPA derivative that contains a single methyl group on one of its ethylene backbones, and has been successfully incorporated into the FDA-approved 90Y therapeutic immunoconjugate Zevalin (Table 1.7).211-213, 214 Although DTPA and DOTA are the most commonly investigated chelate systems for Y3+, the ligand CHX-A”-DTPA (Table 1.7) shows significantly improved stability over DTPA; however, it is still generally considered to be less stable than DOTA.60  The cyclohexyl backbone of CHX-A”-DTPA makes the ligand more rigid and imposes a degree of preorganization on the metal ion binding site, enhancing kinetic inertness, but retarding radiolabeling kinetics compared to those of DTPA.60,215  Due to the success of antibody vectors, developing acyclic ligands with fast radiometal ion coordination kinetics, as well as high stability and kinetic inertness comparable to that of macrocycles such as DOTA is an important goal.  The work done towards developing CHX-A”-DTPA was particularly interesting because of the 4 possible isomers of this ligand.  The CHX-B”-DTPA isomer was found to be much less stable than the CHX-A’’-DTPA isomer in vivo with 90Y, despite in vitro stability assays suggesting they were very similar.60,216  If CHX-DTPA were to be used as a racemic mixture it would result in significant decomposition of the less stable isomers and would lead to radiodemetallation in vivo, highlighting the importance of enantiopurity.60,216  Different isomers of a BFC-radiometal complex can have different     35 stabilities and off-rates (kinetic inertness), potentially leading to differential biodistributions.49,63   Table 1.7 DTPA, 1B4M, CHX-A’’-DTPA, and bifunctional derivatives.  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   DTPA, Diethylenetriamine pentaacetic acid, N3O5, CN = 8 64Cu2+ ✗ 40 °C, 60 min, pH 6.5 21.4 Distorted octahedron? 119,217 67/68Ga3+ ✗ 25 °C, 30 min, pH 3.5 24.3, 25.5 (pM 20.2) Distorted octahedron? 117,119,218  44/47Sc3+ ~ 25 °C, 10 min, pH 6.0 - Square antiprism? 17 111In3+ ~ 25 °C, 5-10 min, pH 4.5-5.5 29.0 (pM 24.9) Pentagonal bipyramid or square antiprism 117,119,219-221 177Lu3+ ~ 25 °C, 10-20 min, pH 5.5 22.6 Square antiprism? 119,131,222 86/90Y3+ ~ 25 °C, 10-20 min, pH 5.5 21.2, 22.0, 22.5 Monocapped square antiprism 59,131,132,222 89Zr ✗ 25 °C, 60 min, pH 7 (<0.1 % yield) 35.8-36.9 Distorted dodecahedron 119,223 212/213Bi3+ ✗ 25 °C, 10-20 min, pH 5 35.6 Square antiprism 119,224-226   p-SCN-Bn-1B-DTPA215,227   p-SCN-Bn-1B4M-DTPA228-230   CHX-A’’-DTPA, 2-(p-isothiocyanatobenzyl)-cyclohexyldiethylenetriaminepentaacetic acid, N3O5, CN = 8 67/68Ga3+ ✗ 85 °C, 20 min, pH 5.5 - Distorted octahedron? 231 111In3+ ✓ 25-60 °C, 30-60 min, pH 5.5 - Square antiprism 228,231-237 177Lu3+ ✓ 37-75 °C, 30-60 min, pH 5-5.5 - Square antiprism 58,126,128 86/90Y3+ ✓ 37-75 °C, 30-60 min, pH 5-5.5 - Square antiprism 19,20,59,60,216,231,232,238-243 213Bi3+ ~ 25 °C, 10-20 min, pH 5 - Square antiprism 244, 201 245,246   p-SCN-Bn-CHX-A’’-DTPA60,216,247    Although CHX-A’’-DTPA has enhanced radiolabeling kinetics when compared to DOTA, both are still much slower than typical acyclic ligands such as DTPA and H4octapa N N NCO2HHO2C CO2HCO2HHO2CN N NCO2HHO2C CO2HCO2HHO2CSCN N N NCO2HHO2C CO2HCO2HHO2CSCNN N NCO2HHO2C CO2HCO2HHO2C N N NCO2HHO2C CO2HCO2HHO2CSCN    36 (Table 1.9), and often require mild heating (~37-60 °C) and reaction times of 30-60 minutes to achieve reasonable yields.  CHX-A’’-DTPA has been heavily investigated for use with many radiometals, including 86/90Y,19,20,59,60,216,231,232,238-243 177Lu,58,126,128 and 212/213Bi,244, 201 245,246 but work with 111In has comparatively been limited.228,231-237  Studies that have been performed with 111In(CHX-A’’-DTPA) conjugates have shown good in vivo results, but to our knowledge little work has been done comparing it with the “gold standard” 111In ligand DOTA.236  A study comparing the in vivo behavior of the affibody conjugate 114mIn-CHX-A’’-DTPA-ABD-(ZHER2:342)2 to 111In-DOTA-ABD-(ZHER2:342)2 suggests that both ligands have comparable stability and performance, with CHX-A’’-DTPA exhibiting slightly higher tumour uptake, but also slightly higher non-target organ uptake (e.g. kidneys, liver, bone).236  114mIn is a notable γ-emitting isotope because it has identical chemistry to 111In, but a longer 49.5-day half-life and interesting decay scheme (Table 1.1).236  When 114mIn decays it emits a γ-ray for SPECT imaging, but the daughter nuclide 114In has a half-life of 72 seconds and emits high-energy (1989 keV) β- particles useful for therapy.236  When the parent 114mIn radiolabeled affibody internalizes into cells, it acts as an in vivo generator of 114In for intracellular delivery of therapeutic radioactivity (β- particles) to cancer cells.236   Investigations with 212/213Bi have found DTPA to possess fast radiolabeling kinetics, but poor in vivo stability, ultimately rendering the complex unusable.119,224-226  CHX-A’’-DTPA has been shown to possess enhanced stability with 212/213Bi when compared to DTPA and 1B4M-DTPA, and even comparable stability to DOTA, while additionally possessing greatly enhanced radiolabeling kinetics (important for the short half-life of 212/213Bi).225,244,248, 201 245,246  The solid-state structure of the [Bi(CHX-A’’-DTPA)]2- complex suggests that the     37 partially pre-organized binding site offered by the rigid cyclohexyl backbone may contribute to the enhanced stability of the complex (also suggested as the cause for enhanced stability with other isotopes such as 86/90Y and 177Lu).244  Although comparable to DOTA, CHX-A’’-DTPA is still considered to be inferior to 3p-C-NETA and 3p-C-DEPA for use with 212/213Bi.  1.5.8 TRAP (PRP9) and NOPO TRAP (PRP9) is a derivative of the macrocycle NOTA, wherein the traditional carboxylic acid arms have been replaced with phosphinic acid groups (Table 1.8).83-85,249-253  The most successful TRAP derivative, TRAP-Pr, contains an additional ethyl-carboxylate moiety extending distally from the phosphinic acid groups (Table 1.8).83-85,249-253  The most interesting property of the TRAP-Pr ligand is its improved apparent specificity for Ga3+ when compared to NOTA.82,85  This phenomenon is expressed by the difference in thermodynamic stability constants (log KML) of TRAP and NOTA for competing metal ions such as Mg2+, Ca2+, Cu2+, and Zn2+,85 and by competitive radiolabeling experiments done in the presence of an excess of these other ions.82  As a general rule, radiolabeling experiments are performed in strictly metal-free water and so the selectivity of TRAP-Pr for Ga3+ may not appear to be totally relevant, but it does suggest that transchelation by these competing metal ions in vivo should occur to a lesser extent than with NOTA.  Additionally, the enhanced specificity of TRAP-Pr for 68Ga over NOTA may be of benefit if trace-impurities are present in the stock radiometal solution (e.g. 68Ge/68Ga generator eluent, or commercially supplied 64Cu). A similar derivative to TRAP is NOPO, which is a triazacyclononane-triphospinate ligand that has a similar structure to TRAP-Pr, but with two terminal alcohol groups in place of     38 carboxylic acids.78, 248, 250  Like TRAP, NOPO has shown promising properties for 64Cu and 68Ga radiolabeling.78, 248, 250   Table 1.8 Promising 67/68Ga ligands TRAP (PRP9, TRAP-Pr), AAZTA (DATA), and bifunctional derivatives.a  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   TRAP	  (PRP9,	  TRAP-­‐Pr),	  1,4,7-­‐triazacyclononane-­‐1,4,7-­‐tris[methyl(2-­‐	  carboxyethyl)phosphinic	  acid],	  N3O3 CN = 6 (+ distal carboxylates CN = 9?)	  67/68Ga3+ ✓ 95 °C, 5 min, pH 3.2 26.24 Distorted octahedron 83-85,95,249-253    L = Linker, e.g. PEG8, PEG4, Glu, AHX, N3AHX.  P = Peptide, e.g. RGD, DRG.     NOPO82,252,254   AAZTA, and novel DATA derivatives, 1,4-bis (hydroxycarbonyl methyl)-6-[bis (hydroxylcarbonyl methyl)] amino-6-methyl perhydro-1,4-diazepine, N2O4 or N3O3, CN = 6 67/68Ga3+ ~ 25 °C, 1-5 min, pH 4-6.8 - Distorted octahedron 255-258  H3L1  H3L4   The peptide conjugate TRAP(RGD)3 was radiolabeled with 68Ga in 5 minutes at 95 °C (HEPES, pH 3.2), and despite the high reaction temperature was able to radiolabel at lower ligand concentrations than the corresponding DOTA or NOTA conjugates.14,15  RGD (arginine-glycine-aspartic acid tripeptide) is a popular cyclic peptide vector that targets overexpression of integrin αvβ3 receptors on various types of cancer, and is commonly used N NNP OHOO OHPOHOOHO POOH OOH N NNP LOO OHPOHOOL POOH OLP PP N NNPHOO OHPOHOHO POOH OOHN NHO2CN CO2HHO2C CO2H N N OHONMe OOHMe CO2H N N OHONH OOHPh CO2H    39 as a proof-of-principle vector for studying new isotopes and new BFC.38,95,259,260, 14,15  68Ga-TRAP(RGD)3 demonstrated good in vivo tumour targeting properties, showing great promise for use in 68Ga-based PET imaging agents;14,15 however, a recent comparative biodistribution study between 68Ga-TRAP(RGD)3 and 68Ga-NODAGA-RGD revealed higher uptake in several non-target organs for the TRAP-based agent.83,95,250  Despite these apparent shortcomings, TRAP-Pr is a very promising 68Ga ligand, which is a claim substantiated by the work of Dr. Richard P. Baum at Zentralklinik Bad Berka in Germany, who has performed imaging experiments in human patients using 68Ga-TRAP-peptide conjugates.261  1.5.9 AAZTA and derivatives (DATA) The novel AAZTA derivatives H3L1 and H3L4 are new 67/68Ga-ligands that have shown improved radiolabeling and serum stability properties over the parent AAZTA ligand (Table 1.8).  The parent AAZTA ligand was found to form multiple isomers with Ga3+, and so the derivatives H3L1 and H3L4 were crafted such that one major isomer of the Ga3+ complex was kinetically trapped by attachment of bulky alkyl/aryl groups.255-258  These AAZTA derivatives (now called DATA) are promising new Ga3+ ligands with extremely fast radiolabeling kinetics (1-5 minutes at RT) and good preliminary in vitro and in vivo stability results (as non-bifunctional ligands).  These ligands are very recent, as most of this work has just been published in 2013, and so more extensive in vitro and in vivo experimentation will likely follow in the coming years.  Additionally, no bifunctional derivatives (e.g. peptide conjugates) have been published to date, which will be required for proper evaluation and comparison in vivo to existing “gold standards” such as NOTA.      40 1.5.10 H2dedpa, H4octapa, H2azapa, and H5decapa This family of ligands has only been published over the previous 4 years and has been referred to as the “pa family”, as they are all crafted around central picolinic acid (“pa”) binding moieties (Table 1.9).  The body of this thesis discusses the work performed with many of these picolinic acid-based ligands (e.g. H4octapa, H5decapa, H6phospa, H2azapa), and so those that were published at the time of writing have been included in this introduction to provide a broad perspective of where they fit into the current canon of radiometal ligands.  This work originated in ligands made by Rodriguez-Blas and coworkers, originally purposed as contrast agents for magnetic resonance imaging (MRI) and luminescence.262-271  H2dedpa was the first ligand in this family to be investigated, with its acyclic hexadentate scaffold being ideal for 67/68Ga, radiolabeling in less than 10 minutes at room temperature and forming a very symmetrical hexedentate coordination geometry (determined by solid-state X-ray structure).118,266,268,269,271  In vitro analysis of the [67/68Ga(dedpa)]+ complex demonstrated excellent stability and resistance to transchelation by apo-transferrin and blood serum.118  The bifunctional derivative p-SCN-Bn-H2dedpa has been synthesized and conjugated to the cyclic peptide RGD, radiolabeled with 68Ga and 64Cu, and has shown promising results for in vivo tumour targeting and PET imaging.272,273  The H2dedpa ligand has also been functionalized with lipophilic moieties, and the resulting cationic 68Ga complexes have been investigated for cardiac perfusions imaging.274  Although preliminary work looks very promising for H2dedpa, especially towards the production of kit-formulations with its very fast room temperature radiolabeling kinetics, more in vivo validation is required.        41 Table 1.9 H2dedpa, H4octapa, H2azapa, H5decapa, and bifunctional derivatives.  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   H2dedpa,	  1,2-­‐[[6-­‐(carboxy)-­‐pyridin-­‐	  2-­‐yl]methylamino]ethane,	  N4O2	  CN	  =	  6	  64Cu2+ ~ 25 °C, 5-10 min, pH 5.5 19.2 (pM = 18.5) Distorted octahedron 273 67/68Ga3+ ✓ 25 °C, 5-10 min, pH 4.5 28.1 (pM = 27.4) Distorted octahedron 118,272,274                 p-SCN-Bn-H2dedpa118,272    H4octapa,	  N,N′-­‐bis(6-­‐carboxy-­‐2-­‐pyridylmethyl)ethylenediamine-­‐N,N′-­‐di-­‐acetic	  acid,	  N4O4	  CN	  =	  8 111In3+ ✓ 25 °C, 5-10 min, pH 4.5 26.8 (pM = 26.5) Square antiprism 87,89 177Lu3+ ✓ 25 °C, 5-10 min, pH 4.5 20.1 (pM = 19.8) Square antiprism 87                              p-SCN-Bn-H4octapa87       H2azapa,	  N,N′-­‐[1-­‐Benzyl-­‐1,2,3-­‐triazole-­‐4-­‐yl]methyl-­‐N,N′-­‐[6-­‐(carboxy)-­‐	  pyridin-­‐2-­‐yl]-­‐1,2-­‐diaminoethane,	  N6O2	  CN	  =	  8	  64Cu2+ ~ 25 °C, 5-10 min, pH 5.5 - Distorted octahedron? 88 67/68Ga3+ ✗ 25 °C, 5-10 min, pH 4-4.5 - Distorted octahedron? 88 111In3+ ✗ 25 °C, 5-10 min, pH 4.5 - Square antiprism? 88 177Lu3+ ✗ 25 °C, 5-10 min, pH 4.5 - Square antiprism? 88  H5decapa,	  N,N’’-­‐[[6-­‐(Carboxy)pyridin-­‐2-­‐yl]-­‐	  methyl]diethylenetriamine-­‐N,N’,N’’-­‐triacetic	  Acid,	  N5O5	  CN	  =	  10	  111In3+ ✗ 25 °C, 5-10 min, pH 4.5 27.56 (pM = 23.1) Square antiprism 87,89   H2azapa (Chapter 8) was built on a variation of the click-to-chelate approach, where the peptide vector is conjugated to the ligand by click-chemistry, and the resultant triazole-NH HNN NO OH OHO NH HNN NO OH OHONCSN NN NO OH OHOHOO OHO N NN NO OH OHONCSHOO OHON NN NO OH OHONN N N NNN N NN NOHOHOO O OHOHO OHO    42 rings are uniquely positioned so that they are capable of coordinating to radiometal ions in the inner-coordination sphere.40,88,275  The triazole-containing H2dedpa/H4octapa derivative, H2azapa, has been synthesized and evaluated with 64Cu, 68Ga, 111In, and 177Lu (Table 1.9).88  Preliminary results were promising with 64Cu, but in vitro stability assays suggest that complexes with 68Ga, 111In, and 177Lu were unstable.88  Due to the very lipophilic character of the neutral [Cu(azapa)] complex, in vivo evaluation revealed high liver and digestive tract uptake as expected, therefore more hydrophilic conjugates (e.g. peptide conjugates) must be synthesized and evaluated in the future.88  The large decadentate derivative of H4octapa, H5decapa (Chapter 2), was evaluated with 111In in vitro and in vivo, and was found to be significantly less stable than were H4octapa and DOTA (Table 1.9).87,89  H4octapa (Chapters 2-3 and 5-6) is a derivative of H2dedpa, with an additional two carboxylic acid arms increasing the maximum denticity from 6 to 8 (Table 1.9).87,89,266,271  H4octapa has recently been found to exhibit ideal properties for 111In and 177Lu radiochemistry, radiolabeling in quantitative yields at room temperature in less than 10-15 minutes.87,89  Direct comparisons between H4octapa and DOTA with both 111In and 177Lu have demonstrated similar in vivo properties and stability, with H4octapa having greatly enhanced radiolabeling kinetics (significantly faster even than CHX-A’’-DTPA), suggesting that it may be a suitable alternative and improvement to DOTA.87,89 Based on its excellent properties with 177Lu, H4octapa may also be a suitable match for the similar isotopes 86/90Y, although no results have been published towards this goal thus far. Further preclinical work must be performed to validate H4octapa as a suitable alternative to DOTA, although a direct comparison has been made with 111In and 177Lu in vitro and in vivo.87,89      43 It appears that CHX-A’’-DTPA, C-NETA/3p-C-NETA, and H4octapa are the best current alternatives to DOTA for fast radiolabeling kinetics and excellent in vivo stability with 111In for SPECT imaging and dosimetry, and 177Lu and 86/90Y for therapeutic applications.87,89,127  It is currently not clear how C-NETA/3p-C-NETA perform with 111In because, to our knowledge, no studies have been published, and work with 111In and CHX-A’’-DTPA is limited.  If a SPECT imaging surrogate isotope such as 111In is required for pre-therapy imaging and dosimetry, it appears that currently CHX-A’’-DTPA and H4octapa are the most promising alternatives to DOTA.87,89  A direct comparison between all of these ligands with 111In, 177Lu, and 86/90Y in the same animal model at the same time would be of significant value.  1.5.11 HBED and SHBED HBED and its derivative SHBED present an interesting example of ligand development, as the addition of an aromatic para-sulfonate group in SHBED to the phenol functionality of HBED serves to decrease the pKa of the phenolic protons and alter their hard/soft metal-donor properties (Table 1.10).  The original purpose of adding this aryl-sulfonate group to HBED was to increase its negative charge and hydrophilicity/solubility (fully deprotonated SHBED has a 6- charge vs 4- for HBED), but the effect of changing the donor properties of the attached phenolic oxygen highlights an interesting option for tuning other ligands that contain aromatic donor groups.276  Decreasing the pKa of acidic functional groups in ligands can also help to improve radiolabeling efficiency: in this example by making the phenolic protons of SHBED more acidic and easier to deprotonate, and subsequently making SHBED a more effective ligand at lower pH.        44 Table 1.10 HBED, SHBED, BPCA, and bifunctional derivatives.a   Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   HBED, N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid, N2O4, CN = 6 67/68Ga3+ ~ 25 °C, 10-20 min, pH 4-4.5 38.5 (pM 28.6) Distorted octahedron? 117,277-280 44/47Sc3+ ✗ 25 °C, 10 min, pH 6.0 - Square antiprism? 17 111In3+ ✗ 25 °C, 10-20 min, pH 4-7 27.9 (pM 17.9) Distorted octahedron? 117,277,280   HBED-CC279,281   (HBED-CC)TFP279   SHBED, N,N′-bis(2-hydroxy-5-sulfobenzyl ethylenediamine-N,N′-diacetic acid, N2O4, CN = 6 67/68Ga3+ ~ 25 °C, 10-20 min, pH 4-4.5 37.5 (pM 28.3) Distorted octahedron? 117,276,280 111In3+ ~ 25 °C, 10-20 min, pH 4-7 29.4 (pM 20.6) Distorted octahedron? 117,276,280   BPCA, N4O4, CN = 8 111In3+ ~ 25 °C, 60 min, pH 5 - Square antiprism? 282,283  a highlighting relevant radiometal ions, radiolabeling conditions, thermodynamic stability constants (Log KML), coordination geometry, and color-coded ranking (a green ”✓“ = good/best match, orange “~” = suitable match, or requires more evaluation but shows potential, red ”✗” = poor/unstable match).   1.5.12 BPCA BPCA is an interesting ligand that was published in 2010, which is based on a 2,2’-bipyridine backbone, and provides an octadentate N4O4 donor set (Table 1.10).282,283  BPCA was conjugated to a novel cholecystokinin C-terminal tetrapeptide (CCK4), which targets tumours expressing the cholecystokinin receptor subtype 2 (CCK2R), and radiolabeled with N NOHO O OHOH HO N NOHO O OHOH HOHO O OHO N NOHO O OHOH HOHO O OO F F FFN NOHO O OHOH HOO3S SO3N N COOHN COOHHOOC NHOOC COOH    45 111In in high RCY after 1 hour at room temperature.  The novel peptide-conjugate BPCA-(Ahx)2-CCK4 was evaluated in vitro by serum stability transchelation assays, and in vivo by biodistrubtion and planar scintigraphy experiments.  The same (Ahx)2-CCK4 conjugate was made with CHX-A’’-DTPA as an internal reference, and it was demonstrated that BPCA possessed superior stability to CHX-A’’-DTPA in serum (89% vs 45% stable after 2.5 hours, respectively). Biodistribution and planar scintigraphic imaging studies showed that BPCA-based conjugates had higher tumour uptake, lower background organ uptake, and higher tumour/muscle ratios than CHX-A’’-DTPA, suggesting that BPCA is a very promising new ligand for 111In.  A study evaluating BPCA with the 177Lu and 86/90Y would be of great interest.  1.5.13 CP256 CP256 and its bifunctional derivative YM103 are acyclic tripodal tris(hydroxypyridinone) ligands that can rapidly radiolabel with 68Ga in 5 minutes at room temperature (Table 1.11).284  The maleimide derivative YM103 was conjugated to cysteine residues of the protein C2Ac, and in vivo experiments in normal healthy mice (no tumour models) showed rapid clearance through the kidneys with no observable decomposition after 1.5 hours.284  This new ligand was published in 2011, and further studies in tumour bearing mice will help to establish its potential utility.  CP256 has an interesting acyclic tripodal design, similar to L3 (hydroxamate-based), and most interestingly both ligands are oxygen-rich with O6 donor sets, suggesting a potential untapped application as 89Zr ligands (yet to be investigated, to our knowledge).      46 1.5.14 Desferrioxamine (DFO) Desferrioxiamine B (DFO) is a bacterial siderophore that natively binds Fe3+, and has also been used extensively with isotopes of gallium and zirconium (Table 1.11).  DFO is the only competent 89Zr ligand available for radiolabeling and in vivo applications.285  Zr4+ is a highly charged, very hard metal ion with a relatively small ionic radius (84 and 89 pm for CN = 8 and 9, respectively),190 that is prone to forming insoluble polynuclear hydroxide species in aqueous solution under non-acidic conditions, and subsequently is difficult to radiolabel effectively.  DFO is thought to bind 89Zr with its three hydroxamate groups in a hexadentate fashion, although no crystal structures have been obtained.286  For many years the next best ligand for 89Zr has been DTPA, which forms a thermodynamically stable complex with Zr4+ (log KML = 35.8-36.9), but studies have shown that radiolabeling with 89Zr is inefficient and in vivo stability is exceptionally poor.119  A DTPA-antibody conjugate of Zevalin was radiolabeled with 89Zr, and radiochemical yields obtained were < 0.1% after 1 hour at room temperature;223 in contrast, DFO can radiolabel with 89Zr in quantitative yields (> 99%) after 1 hour. The new ligand H6phospa has recently been studied with 89Zr, and has shown greatly improved radiolabeling performance relative to DTPA, but still inferior to DFO (vide infra). No FDA-approved radiopharmaceuticals currently utilize 89Zr, although a number of 89Zr-DFO-antibody conjugates are in clinical trials.212  89Zr has a long half life (78.5 hours), making it ideally paired with antibody vectors, as the biological half-life of an antibody is on the order of 2-3 weeks.45  89Zr-DFO-Zevalin was the first 89Zr antibody conjugate imaged in humans, and was shown to be a suitable PET surrogate for 90Y-Zevalin dosimetry.223  A number of other studies have been performed using 89Zr-DFO-antibody conjugates with success, such as 89Zr-DFO-U36 (anti-CD446 chimeric mAb),212,287 89Zr-DFO-    47 bevacizumab,212,288 89Zr-DFO-J591,285 89Zr-DFO-TRC105,289 and 89Zr-DFO-trastuzumab.212,290,291 The high 4+ charge makes 89Zr challenging to incorporate into BFC systems while still retaining bioequivalence with other common 3+ cationic metal ions (e.g. In3+, Ga3+, Y3+, Lu3+), because the chelate-radiometal charge and polarity is different.  Evaluating the stability of potential BFC for 89Zr can be done in vivo, because 89Zr that is lost from a BFC typically localizes in bone, as demonstrated by the highly unstable 89Zr-DOTA- and 89Zr-DTPA-based antibody conjugates with cetuximab that showed significant 89Zr accretion in the thighbone 72 hours post injection.130  89Zr-chloride and 89Zr-oxalate have also been injected directly into mice, with 89Zr-chloride forming colloids and accumulating in the liver, and the weakly chelated 89Zr-oxalate demonstrating rapid bone uptake.285  Although DFO is an excellent 89Zr ligand, some decomposition can be observed over time in vivo as 89Zr slowly accumulates in bone.36,56 The design of novel ligands for 89Zr with improved solubility, in vivo stability, and chelation properties would be timely, considering there is currently only one option (DFO).  Based on the success of DFO, it would appear that oxygen-rich donors are most suitable for 89Zr chelation, with O6-O8 coordination being preferred, and donor groups including hydroxamates, carboxylates, carbonyls, catechols, and hydroxypyridinones being logical choices.                48  Table 1.11 CP256, Desferrioxamine (DFO), PCTA, H6phospa, and bifunctional derivatives.  Chelator and Common Bifunctional Derivatives Radiometal Ion a Radiolabeling Conditions Log KML  Proposed Geometry Ref.   CP256, O6 CN = 6 67/68Ga3+ ✓ 25 °C, 5 min, pH 6.5 - Distorted octahedron 284  Bifunctional derivative YM103284  CP256 = 4-Acetylamino-4-[2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl]-heptanedioic acid bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-amide]   PCTA,	  3,6,9,15-­‐tetraazabicyclo[9.3.1]pentadeca-­‐1(15),11,13-­‐triene-­‐3,6,9,-­‐triacetic	  acid,	  N4O3	  CN	  =	  7	  64Cu2+ ~ 25 °C, 5 min, pH 5.5 19.1 Distorted octahedron 91,94, 214,292 67/68Ga3+ ✓ 25 °C, 5-10 min, pH 4-5 - Distorted octahedron 90,292-294  p-SCN-Bn-PCTA214   DFO, Desferrioxamine B, O6, CN = 6 67/68Ga3+ ~ 25 °C, 30 min, pH 3.5 28.6 Distorted octahedron? 218,295 89Zr4+ ✓ 25 °C, 60 min, pH 7-7.3. - Distorted octahedron? 56,130,223,285,287-291,296-302   p-SCN-Bn-DFO298 Bifunctional DFO derivatives299,303   H6phospa, N,N’-(methylenephosphonate)-N,N’-[6-(methoxycarbonyl) pyridin-2-yl]methyl]-1,2-diaminoethane, N4O4 CN = 8 89Zr4+ ~ 25 °C, 60 min, pH 7.4 - Square antiprism? 304  Bifunctional derivative p-SCN-Bn-H6phospa    HN OHN ON OHOHN ONOHO HNO NHO O HNO HNON OHOHN ON OHO HN ON OHOHNONOON N NHO2C HO2C CO2HN N NNCO2H CO2HHO2C NCSNNOHO HN O ONHO HN O NO OH NH25 5 5NOHO HN O ONHO HN O NO OH NH5 5 5 NHS NCS NOHO HN O ONHO HN O NO OH5 5 NH R DFOO NO ODFO-Chx-MalDFO O X X = I, BrDFO-IAC, DFO-BAC5N NN NOHOO OH PPOOHHO OOHOH N NN NOHOO OH PP NCSOOHHO OOHOH    49 1.5.15 H6phospa Developing new chelating agents for 89Zr is currently of great interest, as the only ligand available that can radiolabel with 89Zr with any level of proficiency is DFO.  The best alternative ligand to DFO for 89Zr radiolabeling is DTPA; however, with meager radiochemical yields of < 0.1% after 1 hour at room temperature and poor in vivo stability, DTPA is not a viable alternative to DFO.223  DFO can radiolabel with 89Zr in quantitative yields (> 99%) after 1 hour at room temperature, and demonstrates imperfect but acceptable stability in vivo, and no acceptable alternatives have been published to date.  The acyclic ligand H6phospa (Chapter 7, Table 1.11),268,269 and the bifunctional derivative p-SCN-Bn-H6phospa are methylenephosphonate derivatives of H4octapa (Table 1.9) that have been recently studied with 89Zr.304  The antibody conjugate H6phospa-trastuzumab was able to achieve radiochemical yields of ~8% with 89Zr after 1 hour at room temperature (PBS, pH 7.4).304  This work was compared to previous attempts to radiolabel H4octapa-trastuzumab with 89Zr, which performed similarly to DTPA and exhibited essentially no radiolabeling after 1 hour.304  These results demonstrate that replacing the carboxylic acid arms of H4octapa with methylenephosphonate arms in H6phospa improved radiolabeling kinetics with 89Zr, suggesting that methylenephosphonate groups are more suitable than carboxylic acid groups for chelating 89Zr.304  Although a RCY of ~8% is insufficient to supplant DFO as the “gold standard” ligand for radiolabeling with 89Zr, it is a significant improvement over DTPA and H4octapa, and to our knowledge is the highest 89Zr radiolabeling yield behind DFO to be published.      50 1.5.16 PCTA The heptadentate macrocyclic ligand PCTA was originally synthesized by Sherry and coworkers as a potential MRI contrast agent (Table 1.11).292  PCTA has been recently re-purposed and evaluated with 68Ga and 64Cu, and has been shown to possess much faster radiolabeling kinetics than DOTA (5-10 min, RT).90  The serum stability of the 67/68Ga-PCTA complex was found to be superior to DOTA, and comparable to NOTA.90  Additionally, the biodistribution profile of the non-bifunctional 68Ga-PCTA revealed lower kidney retention than 68Ga-NOTA, showing potential improvement over NOTA for use in peptide conjugates.90  Comparisons of RGD conjugates of the bifunctional derivatives of both PCTA and NOTA have demonstrated excellent in vivo stability and comparable biodistribution profiles, with the PCTA-based agent having lower kidney uptake than its NOTA counterpart.294  PCTA has also been investigated with 64Cu, showing greater radiolabeling kinetics and yields to DOTA, and superior in vitro and in vivo stability.75  PCTA has only recently been investigated, but further work may show it to be an excellent ligand for copper and gallium-based radiopharmaceuticals.   1.6 Conclusions With the methods for evaluating ligands with radiometals explained, and the plethora of existing ligands reviewed, we now set out to study novel acyclic ligands for radiochemistry applications.  While synthesizing and evaluating a number of new ligands, factors such as the charge, polarity, denticity, donor atom type (e.g. N, O, S), and kinetics (e.g. macrocyclic vs. acyclic) will be studied and optimized with the goal of achieving maximum in vivo stability.  The following 8 chapters will discuss work on 8 new ligands (as     51 well as bifunctional derivatives) for radiochemistry, covering radiometals such as 64Cu, 68Ga, 111In, 177Lu, and 89Zr.  The synthesis of these oxygen- and nitrogen-rich ligands has been challenging, and much work has been directed towards applying new synthetic methods and protecting group chemistry towards their production.  Once synthesized, ligands and many of their non-radioactive metal complexes were evaluated by standard analytical techniques, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), infrared spectroscopy (IR), elemental analysis (EA), and high-performance liquid chromatography (HPLC).  These ligands, and where possible their bifunctional derivatives, were radiolabeled with a variety of radiometals, and their efficiency and stability were studied using in vitro (e.g. serum stability) and in vivo (e.g. PET/SPECT/Cerenkov imaging, biodistribution) experiments.  A number of the new ligands discussed in this thesis were summarized in the tables presented in Chapter 1 and compared to the entire field, and now their study and elaboration will be discussed in detail.      52 Chapter 2: H4octapa: an acyclic ligand for 111In radiopharmaceutical applications  This chapter is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Cawthray, J. F.; Bailey, G. A.; Ferreira, C. L.; Boros, E.; Adam, M. J.; Orvig, C., H4octapa: An Acyclic Chelator for 111In Radiopharmaceuticals. J. Am. Chem. Soc. 2012, 134 (20), 8670-8683, Copyright 2014 American Chemical Society.   2.1 Introduction  111In is an important isotope in nuclear medicine for SPECT imaging and performing dosimetry for therapeutic chelate-based radiopharmaceuticals.  111In is a cyclotron produced isotope (111Cd(p,n)111In) that decays with a half-life of ~2.8 days via electron capture (100% EC); it emits γ rays (245 and 172 keV) that can be used for imaging, and Auger electrons that can be used for therapy.15  One of the most promising radiotherapeutic isotopes, 90Y (t1/2 = ~2.67 days), is essentially radiographically silent as it only emits β- particles, and therefore must be used in combination with an imaging isotope such as 111In, 89Zr, 86Y, 64Cu, or 67/68Ga to study its organ uptake and perform dosimetry.25,56,305  The generator produced indium isotope 110mIn (t1/2 = 69 min) is a very attractive PET (positron emission tomography) imaging surrogate for 111In, and the two isotopes can be seamlessly interchanged in bifunctional-chelate (BFC) based radiopharmaceuticals because they share identical chemical properties.306  When used with the OctreoScan kit (BFC-peptide conjugate), 110mIn has been shown to significantly improve spatial resolution, dosimetry, and tumour identification when     53 compared to 111In;306 however, the generator parent isotope 110Sn has a half-life of ~4.1 hours, which makes the generator life very short.307  Also, commercial availability of 110mIn is limited, which restricts usage.  There are currently several 111In/90Y-based radiopharmaceuticals FDA-approved for clinical use and many more in clinical trials, demonstrating the importance of these isotopes in nuclear medicine.28,211,214,308-311  The combination of 111In for imaging and dosimetry and 90Y/177Lu for therapy is very effective, but is dependent on having a solid ligand foundation that can bind both isotopes with exceptionally high stability (thermodynamic and kinetic) and that has very similar biological behavior with both isotopes (biologically equivalent).19-24,213,312  The macrocycle DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is the industry “gold standard” for chelation of 111In, 90Y, and 177Lu (Figure 2.1); however, biovector conjugates suffer from the need for elevated temperatures and extended reaction times to achieve quantitative radiolabeling yields (typically 60-95 °C, for 30-120 minutes), which is not optimal for use with heat-sensitive biomolecules such as antibodies.106,177,232,240  Peptide biovectors can often be labeled at elevated temperatures without suffering deleterious effects to their binding integrity, and have also shown excellent tumour targeting and uptake; however, with the current ligand offerings they sometimes demonstrate high and persistent kidney uptake that can disrupt the predictive power of dosimetry calculations and cause unwanted radiation exposure.312,313  Since the properties of the radiometal-chelate complex can strongly influence the biodistribution profile of its biomolecule conjugate, a new ligand with an improved clearance profile and decreased persistent kidney uptake would be a welcome improvement.  Although the influence of ligand properties on the biodistribution of BFC-conjugates is less significant     54 for large biovectors such as antibodies (~150 kDa for an intact antibody) than it is for smaller peptides, the difference can still be significant.213,305,312  Antibody biovectors show exceptionally high tumour uptake and have long biological half-lives (2-3 weeks45), and so are well matched with the long half-lives of 90Y (~2.67 days) and 177Lu (~6.6 days).15  The acyclic ligand DTPA (diethylenetriaminepentaacetic acid) will successfully radiolabel with these isotopes in 10-15 minutes at ambient temperature (Figure 2.1); however, the in vivo stability is less than optimal, showing a greater extent of decomposition and non-target organ uptake than DOTA.60,215,216,312  The DTPA derivative CHX-A’’-DTPA has an improved stability profile with 86/90Y when compared to DTPA, while retaining its low temperature labeling abilities (30-60 minutes); however, the stability and in vivo kinetic inertness are still not as good as DOTA, and its In3+ complexes have been less studied.59,60,215,216,244  Despite the poor in vivo stability, DTPA is the ligand used in the currently available FDA approved 111In-based radiopharmaceuticals.   Our group has recently investigated a series of acyclic ligands based on picolinic acid, resulting in identification of the promising 67/68Ga ligand H2dedpa (Figure 2.1).118,266,272,273  Initial radiolabeling experiments, in vitro apo-transferrin stability experiments, and biodistribution studies in mice have demonstrated H2dedpa to be an ideal ligand for 67/68Ga-based radiopharmaceuticals.274  This success has prompted the investigation of larger acyclic frameworks based on a similar picolinic acid scaffold, supporting higher denticities for accommodating larger radiometal ions such as 111In, 90Y, 177Lu, 89Zr, and 225Ac.  Herein we report the synthesis, characterization, coordination chemistry, thermodynamic stability, radiolabeling, in vitro mouse serum stability, and in vivo biodistribution studies of the 111In complexes of the octadentate ligand N,N’-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-    55 N,N’-diacetic acid (referred to herein as H4octapa) (Figure 2.1),271 and the decadentate analogue H5decapa. DOTA and DTPA were used as benchmarks of the industry “gold standards” for 111In chelation.  The ligands H2dedpa and H4octapa had previously been published for applications as MRI contrast agents, and later as Pb(II), Cd(II), and Zn(II) sequestration agents.266,269-271 	  	  	  Figure 2.1 Structures of the 111In-coordinating and industry “gold standard” ligands DTPA/CHX-A’’-DTPA and DOTA, the 68Ga-coordinating ligand H2dedpa, and the novel entrants H4octapa and H5decapa. 	  	  2.2 Results and discussion  Although the macrocycle DOTA is regarded as the most stable ligand for 111In, it is not best suited for use with antibodies due to its optimal high-temperature radiolabeling, and the excellent match of antibodies with long-lived therapeutic isotopes makes this mismatch especially unfortunate.  DOTA radiolabels with many radiometals at the moderate and antibody-compatible temperature of 37 °C; however, reaction times of 60-180 h are required N NN NOHOHOOO OH OOH N NN N NOHOOHO OHOOHO OHOCHX-A"-DTPANN NNDOTAN N NCO2HHO2C CO2HCO2HHO2C N N NCO2HHO2C CO2HCO2HHO2C DTPA OHO OOHOHO OOHH4octapa H5decapaNH HNN NO OH OOHH2dedpa    56 and radiochemical yields are inconsistent (~50-90%).58-65,94,97,119,127-129,142  This suggests an important and high priority need for new, highly stable acyclic ligands for use with isotopes such as 111In, 90Y, and 177Lu, especially when conjugated to antibodies.  There is a need for ligands that combine the exceptional in vivo stability and kinetic inertness of DOTA with the fast ambient temperature labeling kinetics of DTPA.  These observations suggest that the target for an ideal 111In/90Y/177Lu ligand would have the following properties.  It should 1) be acyclic, or if macrocyclic, be constructed with free-moving appendages to emulate ambient temperature acyclic radiolabeling kinetics, such as the NOTA derivative NETA.202  The coordination complex should 2) be very stable; both thermodynamically and kinetically, but with top priority placed on exceptional in vivo kinetic inertness, particularly to demetallation and/or transchelation.  The ligand should 3) be isomerically pure with preferably only one stable isomer being formed and administered, or such as CHX-A’’-DTPA, the superior isomer of the ligand isolated and administered pure.60,215,216  Finally, it should 4) ideally have similar properties in its ligand/bifunctional-chelate complexes with both of the radiometal ions to be used in an imaging/therapy pair (i.e. 111In/90Y), so that biological clearance and organ uptake are sufficiently similar and that accurate dosimetry information can be obtained (bio-equivalence).  Not every one of these four points must be met for a ligand to be useful as a BFC for radiometal ions, and especially point number four may be hard to achieve, considering the different coordination numbers and geometries preferred by different metal ions such as 111In (6-8 coordinate) and 90Y (8-9 coordinate).6            57 Scheme 2.1 Synthesis of compounds 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 and H4octapaa  a Reagents and conditions: i) NaBH4 (2.4 eq.), CH3OH, 4 h, 0 °C; ii) PBr3 (1.1 eq.), CHCl3, 0 °C, 3.5 h; iii) CH3OH, NaBH4 (4.3 eq.), 8 h; iv) CH3CN, Na2CO3 (excess), 60 °C, Ar (g), 12-16 h, 2.4 = alkylation with tert-butyl bromoacetate, 2.6 = alkylation with 2.2; v) AcOH, Pd/C (10 wt%), H2 (g), 12 h; vi) HCl (6 M), 8 h.   2.2.1 Synthesis and characterization  We have studied a number of novel acyclic ligands, and herein we report our two most promising candidates for use with 111In.  The octadentate ligand N,N’-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N’-diacetic acid (H4octapa) was previously used with various paramagnetic lanthanides to assess their abilities as MRI contrast agents.271  As an expansion of this scaffold, the novel decadentate derivative H5decapa has also been synthesized and evaluated.  The ligands H4octapa and H5decapa were synthesized with a general reaction scheme that follows N-benzyl protection, N-alkylation with an alkyl halide, benzyl deprotection via hydrogenation, a second alkyl halide N-alkylation, and finally deprotection in boiling HCl (6 M) (Schemes 2.1 and 2.2).  Previous methods to synthesize N NN NOHOHOO H4octapaO OH OOH N NN NOOOOO O OO NH NH OOOON N OOOO 2.52.6HO 2.4NH HNH2N NH2 2.3+ (iii) (iv) (v)(iv)(vi)NO OO O NHO OO(i) 2.1 NBr OO2.2(ii)51% >98%49% 93% 87%39%75%2.1 equiv.    58 the similar acyclic ligand H2dedpa utilized reductive amination reactions;118,271 however, this method resulted not only in reduction of the imines, but also hydrolysis/reduction of the picolinic acid methyl ester groups to carboxylates and alcohols, which were very difficult to separate in subsequent steps, resulting in arduous purifications and low yields.  The synthetic scheme presented here circumvents this problem by avoiding the use of sodium borohydride in the presence of the picolinic acid moiety (Schemes 2.1 and 2.2).  The only significant problem encountered with these syntheses is the lability of the picolinate groups to hydrogenation.  Benzylated intermediates that contain picolinate groups undergo unwanted cleavage during hydrogenation, and to minimize this undesirable pathway the reaction order was optimized to perform the debenzylation step only in the presence of the tert-butylacetate groups (Schemes 2.1 and 2.2).  The obvious drawback to this method is that after hydrogenation, the tert-butylacetate-alkylated ethylenediamine/diethylenetriamine compounds (2.5/2.9) are not UV active and must be stained with iodine to be visualized.  Hydrogenation of the diethylenetriamine scaffold during the synthesis of H5decapa resulted in substantial cleavage of the diethylenetriamine backbone, resulting in lower yields than the equivalent hydrogenation of the ethylenediamine scaffold in the H4octapa synthesis.  Decreasing the Pd/C catalyst loading appeared to slow down reaction kinetics but did not improve selectivity for debenzylation over the unwanted side reactions.   The presented synthetic schemes are a substantial improvement over previous attempts, allowing for improved yields and the purification of all intermediates despite the large number of polar functional groups; however, the problem of non-selective debenzylation and ethylene backbone cleavage of diethylenetriamine (H5decapa) is problematic, and with larger scaffolds such as tris(2-aminoethyl)amine (TREN) this problem becomes more severe (see     59 Chapter 4).  These challenges suggest that new protecting group chemistry must be employed to further improve the synthesis of these picolinic acid-based ligands and facilitate the elaboration of larger scaffolds and novel bifunctional derivatives (see Chapter 3).  The relatively simple 5-step synthesis of H4octapa is achieved with a cumulative yield of ~12%, and the 5-step synthesis of H5decapa was completed with a cumulative yield of ~2.5% (yields for both compounds using previous reductive aminations methods were under 1%).  The incorporation of multiple synthons in the synthesis of these ligands will allow for more straightforward synthesis of bifunctional derivatives via (4-nitrobenzyl)ethylenediamine/diethylenetriamine backbone derivatives, which should be an improvement over the difficult and tedious synthesis of bifunctional macrocycles such as p-isothiocyanatobenzyl-DOTA.143   Scheme 2.2 Synthesis of compounds 2.7, 2.8, 2.9, 2.10 and H5decapaa  a Reagents and conditions: (i) CH3OH, NaBH4 (7 eq.), 8 h; (ii) CH3CN, Na2CO3 (excess), 60 °C, Ar (g), 12-16 h, 8 = alkylation with tert-butyl bromoacetate, 2.10 = alkylation with 2.2; (iii) AcOH, Pd/C (5 wt%), H2 (g), 12 h; (iv) HCl (6 M), 8 h.   HOH2N NH +NH2 N N NOOOO OONH N NHOOOO OO2.10N NN N NOOOO OOOO O ON NN N NOHOOHO OHOOHO OHONH NH HN 2.9H5decapa 2.7 2.8(i) (ii) (iii)(ii)(iv)2.1 equiv. 40% 66% 30%45%71%    60  Figure 2.2 1H NMR spectra in D2O (300 MHz) at ambient temperature of top, H4octapa and [In(octapa)]- showing simple diastereotopic splitting due to minimal isomerization and bottom, H5decapa and [In(decapa)]2- showing complicated but sharp splitting arising from multiple static isomers.   In general, ligands/BFC that exhibit minimal isomerization are preferred, although no definitive trend has been identified for this phenomenon.49,63,97  The 1H NMR spectrum of     61 [In(octapa)]- reveals clear and sharply resolved diastereotopic splitting of the protons associated with both picolinic acid moieties (methylene-H, 4.49 ppm, 2J = 16.1 Hz, and 4.34 ppm, 2J = 16.1 Hz), as well as one of the acetic acid arms (3.27 ppm, 2J = 19.3 Hz), suggesting a 7-coordinate solution structure (Figure 2.2).  The coupling constants observed for these metal-bound appendages were 2J = 16.1 Hz for the two picolinic acid methylene groups, and 2J = 19.3 Hz for the acetic acid arm.  The second acetic acid arm appeared as a broad doublet (3.19 ppm) with a much smaller coupling constant of 2J = 8.4 Hz, showing diasterotopic splitting, but suggesting that it is not metal-bound and does not form a 5-membered metallocycle as do the other metal-bound appendages.   The sharp and clear diasterotopic splitting seen for [In(octapa)]- suggests the presence of a single static isomer with no observable fluxional interconversion at ambient temperature (on the NMR timescale).  No change in the number of peaks in the 13C NMR spectrum of the ligand H4octapa was observed upon In3+ coordination, suggesting no chemically distinct isomers were formed (different coordination number/polarity).  The HPLC radiotrace of [In(octapa)]- showed a single sharp peak, affirming that the radiometal-complex exists as a single isomer (Figure A.1).  This analysis depicts a very stable and inert coordination structure present as a single isomer, unlike the [In(DOTA)]- complex which shows multiple isomers with rapid fluxional isomerization at ambient temperature.62,97  The In3+ complex formed with the potentially decadentate ligand H5decapa, [In(decapa)]2-, displayed a more complex 1H NMR splitting (Figure 2.2) and a large number of additional 13C NMR peaks that were not observed for the free ligand H5decapa.  This observation, along with the presence of two peaks in the HPLC radiotrace (tR = 5.4 min (5%), 7.7 min (95%), Figure A.1) suggests that multiple isomers of [In(decapa)]2- are present in     62 solution at ambient temperature.  Considering that In3+ typically forms 7-8 coordinate complexes, the decadentate ligand H5decapa may have several unbound carboxylates, which could give rise to different protonation species in solution and could explain the two peaks observed in the radio-HPLC trace.  Additionally, NMR experiments (D2O) and HPLC experiments were performed at different pH (0.1% TFA as HPLC buffer, pH ~2), which could influence the protonation species present in solution.  [In(DTPA)]2- displays a complicated splitting of the 1H NMR peaks in a similar fashion to [In(decapa)]2-, and also yields sharp splitting patterns suggesting that little or no fluxional behavior between multiple static isomers/diastereomers occurs in solution at ambient temperature (Figure 2.2).314,315  Unlike [In(decapa)]2-, [In(DTPA)]2-  retains a simple 13C NMR spectrum, with the same number of 13C signals being observed as with unbound DTPA.314 These observations for [111In(DTPA)]2- suggest that little or no fluxional behavior between isomers/diastereomers occurs in solution at ambient temperature (on the NMR timescale), although multiple isomers are indeed formed.97,314,315  If minimal isomerization and fluxional behaviour are preferred for stability,49,63,97 then the NMR solution structures of the currently investigated In3+ ligand complexes would have them ranked as [In(octapa)]- > [111In(DTPA)]2- > [In(DOTA)]- >  [In(decapa)]2-.  As previously discussed, DTPA has been shown to be inferior to DOTA in terms of in vivo stability with In3+ and so does not fit with this trend; however, this work largely follows the trend with the in vivo stability of H4octapa being superior to that of DOTA (vide infra), and H5decapa being inferior to both.  It may be the case that acyclic and macrocyclic ligands cannot be compared in this manner, as a fluxional macrocyclic complex is likely to remain more stable than an analogous fluxional acyclic complex, because the metal remains encapsulated inside of the protective macrocyclic framework and is less likely     63 to be released.  The 1H NMR spectrum of the hexadentate [In(dedpa)]+ complex in D2O was very similar to that of [In(octapa)]-, revealing a sharp doublet for each set of methylene protons (4.58 ppm, 2J = 17.3 Hz, and 4.13 ppm, 2J = 17.4 Hz) associated with the picolinic acid moiety (Figure A.2).  The 1H NMR spectrum of the hexadentate [In(dedpa)]+ complex in DMSO-d6 revealed two sharp doublets for each set of methylene protons (4.27 ppm, 2J = 16.6 Hz, 4.26 ppm, 2J = 16.3 Hz, 3.90 ppm, 2J = 16.6 Hz. 3.89 ppm, 2J = 16.6 Hz) attached to the picolinic acid moieties, suggesting clean diastereotopic splitting arising from two separate isomers, most likely from DMSO binding to the open coordination sites of In3+ (Figure A.2).  The sharp peaks observed for [In(dedpa)]+ in D2O and DMSO-d6 suggest no observable fluxional behavior at ambient temperature, and the presence of only one set of peaks in the 13C NMR spectrum (same as unbound H2dedpa) and one peak in the radio-HPLC trace (vide infra) suggest the formation of only one isomer.  In consideration of these results, the very simple and sharp diastereotopic proton splitting observed in the 1H NMR spectrum of [In(octapa)]-, coupled with the single sharp peak observed in the HPLC-radiotrace suggest that this complex displays the least amount of isomerization and fluxional behaviour in solution at ambient temperature out of the currently investigated novel ligands and the industry “gold standards” DTPA and DOTA.  2.2.2 DFT structures and molecular electrostatic potential maps   Although solid-state structures of metal-ligand complexes obtained from X-ray crystallography are useful, they are often not representative of the solution phase structures, and so DFT calculations (modeled in water) and NMR studies in D2O of solution structures     64 are most relevant to a discussion of potential in vivo applications.6,9,97  The DFT structure of [In(octapa)]- (Figure 2.3, left) reveals an 8-coordinate complex with approximately C2v symmetry, showing tight binding of In3+ with slight puckering of the picolinic acid moieties.  The solution structure of [In(octapa)]- as deduced by NMR spectroscopy suggests a 7-coordinate structure with one acetic acid arm unbound; however, it is not certain from the broad doublet (3.22 ppm) and smaller coupling constant (8.4 Hz) of the unbound acetic acid arm whether or not there is transient metal binding.  The DFT structure of [In(decapa)]2- (Figure 2.4, left) shows an 8-coordinate structure, with one picolinic acid group and all 3 acetic acid arms bound to indium (as well as the 3 tertiary backbone nitrogen atoms), and one picolinic acid group unbound and extended away from the metal center into solution.  This unsymmetric coordination sphere has a large impact on the molecular electrostatic potential (MEP) of the complex (Figure 2.4, right), with the MEP distribution revealing the entire molecular surface to be more electronegative than for [In(octapa)]- (Figure 2.3, right).  [In(decapa)]2- shows areas of very high electronegative potential around the unbound picolinic acid group, most likely from the unbound deprotonated carboxylate group (Figure 2.4, right).  The unsymmetric high density of electronegative potential shown on the MEP map of [In(decapa)]2-, in contrast to the symmetric and less electronegative charge distribution of [In(octapa)]-, may result in a higher propensity towards protonation and protein binding in vivo, and may be partially responsible for the poor stability in vivo (vide infra).       65  Figure 2.3 DFT structure of [In(octapa)]- (solvent = water) showing an 8 coordinate structure (left), and the electrostatic potentials of the complex between octapa4- with In3+ mapped onto the electron density (right). The MEP represent a maximum potential of 0.03 au, and a minimum of -0.25 au, mapped onto electron density isosurfaces of 0.002 e Å-3 (red to blue = negative to positive).  DFT calculations performed by Dr. Jacqueline Cawthray. 	  	   Figure 2.4 DFT structure of [In(decapa)]2- (solvent = water) showing an 8 coordinate structure, and the electrostatic potentials of the complex between decapa5- with In3+ mapped onto the electron density (right). The MEP represent a maximum potential of 0.03 au, and a minimum of -0.25 au, mapped onto electron density isosurfaces of 0.002 e Å-3 (red to blue = negative to positive).  DFT calculations performed by Dr. Jacqueline Cawthray.   2.2.3 Radiolabeling experiments  Initial radiolabeling experiments demonstrated the ability of H4octapa to radiolabel quantitatively with 111In at ambient temperature in 10 minutes, showing a single sharp peak     66 in the HPLC radiotrace at tR = 4.7 min (Figure A.1).  Radiolabeling to produce [In(octapa)]- yields specific activities as high as 2.3 mCi/nmol (~5 mCi/µg, 2300 mCi/µmol) in 10 minutes at ambient temperature.  DOTA was radiolabeled with the same activity of 111In (~1 mCi) at the same ligand concentration (10-7 M) used to obtain the specific activity listed above for [In(octapa)]-, and after 10 minutes at ambient temperature less than 40% 111In was radiometallated.  This demonstrates the ability of H4octapa to radiolabel with 111In quantitatively and rapidly in high specific activities at ambient temperature, which is in sharp contrast to the “gold standard” DOTA.  The theoretical maximum 111In specific activity has been calculated to be ~46 mCi/nmol, and specific activities as high as 22 mCi/nmol have been reported for 111In-labeled DOTA-peptide conjugates under ideal conditions; however, temperatures of 100 °C for 30 minutes were required and specific activities that high are not typical with DOTA-conjugates.124  To compare recently reported examples, a 111In-CHX-A’’-DTPA-Re(Arg11)CCMSH peptide conjugate yielded a specific activity of ~0.29 mCi/nmol,231 DTPA-(gp100:154–162mod) (HLA-A.1 binding peptide conjugate) showed a maximum specific activity of ~0.35 mCi/nmol,316 and a DOTA-RGD conjugate obtained a specific activity as high as ~0.050 mCi/nmol;122 these are more typical values and are much lower than the ~2.3 mCi/nmol specific activity obtained for [In(octapa)]- during this study.  It must be considered, however, that the examples listed above are for ligand-peptide conjugates, and the values obtained in this study were for non-conjugated [In(octapa)]-.  DOTA-biovector conjugates typically offer lower specific activities when compared to acyclic ligands such as DTPA and CHX-A’’-DTPA.231  The HPLC radiotrace of [In(decapa)]2- was less promising and revealed two peaks (tR = 5.4 min (5%), 7.7 min (95%)), possibly due to multiple chemically distinct isomers being formed, with specific activities     67 being low at ~0.030 mCi/nmol.  As previously discussed, DOTA required temperatures of 80 °C in a microwave reactor for ~20 minutes to optimally radiolabel with 111In.  2.2.4 Thermodynamic stability  Formation/stability constants (log KML) are well-established measurements of thermodynamic stability for metal-ligand complexes and are typically reported in the literature; however, pM (-log[Mn+free]) values are much more accurate figures for predicting in vivo thermodynamic stability under physiologically relevant conditions.  Values of pM are calculated (at specific conditions, here pH 7.4, [Mn+] = 1 µM, [Lx-] = 10 µM) to take into account all of ligand basicity (ligand pKa values), free metal concentration, ligand-to-metal ratios, pH, and metal hydroxide formation.  The thermodynamic stability of [In(octapa)]- was determined by potentiometric titrations to be log KML = 26.8(1) (pM = 26.5), which is significantly higher than that for DOTA (log KML = 23.9(1), pM = 18.8) and similar to DTPA (log KML = 29.0, pM = 25.7), but possesses the highest pM value of all three In3+ complexes (Table 2.1).  Although CHX-A’’-DTPA has been evaluated with Lu3+/Y3+ to be very stable,238 it has not been thoroughly studied with In3+ and to our knowledge formation constants have not yet been reported for any metal ions.  Despite [In(DOTA)]- being significantly more stable in vivo than is [In(DTPA)],2- 60,215,216,312 the log KML and pM values are much lower for DOTA than DTPA with In3+.  It is interesting to note that the trend of stability constants (log KML) and pM values in Table 2.1 do not correlate well with in vivo stability, which emphasizes that these thermodynamic parameters are not the only factors involved in determining biological stability.  The kinetic parameters of ligand-metal on/off rates are the most important factor, and an example of this is observed by [In(DOTA)]-     68 having one of the lowest pM values but very high stability/kinetic inertness in vivo, and with In(oxine)3 having the highest log KML and pM values (log KML = 35.3, pM = 34.1) shown here, but dissociating very quickly in vivo.311  These values reinforce the concept that in vitro competition experiments such as mouse serum and apo-transferrin assays, and in vivo biodistribution and stability studies are essential to evaluate the practical in vivo kinetic inertness and metabolism of radiometal complexes.  Because the thermodynamic formation constants of the H4octapa, H5decapa, and H2dedpa ligands with In3+ are all very similar, the large differences in pM values must be a result of ligand basicity.  Despite these complications and the lack of predictive power of log KML/pM values for in vivo stability, it is encouraging that [In(octapa)]- has an exceptionally high pM value of 26.9, which is higher than the values determined previously for DOTA (18.8) and DTPA (25.7) with In3+.68,119,317   Table 2.1 Formation constants (log KML) and pMa values for In3+ complexes. Ligand log KML pMa dedpa2- 26.60(4) 25.9 octapa4- 26.8(1) 26.5 decapa5- 27.56(5) 23.1 Oxine (tris) 119 35.4 34.1 DTPA 119,317 29.0 25.7 DOTA 68,119 23.9(1) 18.8 transferrin 318 18.3 18.7  a Calculated for 10 µM total ligand and 1 µM total metal at pH 7.4 and 25 °C.       69 2.2.5 111In radiolabeling and stability studies In consideration of the fact that in vivo kinetic inertness plays a crucial role in determining stability, competition experiments using native biological ligands such as those contained in blood serum (e.g. apo-transferrin, albumin) are useful in vitro assays for predicting the in vivo stability and kinetic inertness of radiometal ion complexes.  During preliminary experiments, mouse serum stability assays were found to transchelate In3+ from ligands more aggressively than were apo-transferrin assays.  These mouse serum competition experiments (incubated at ambient temperature) have demonstrated [In(octapa)]- to have marginally higher stability than [In(DOTA)]- and [In(DTPA)]2- after 24 hours, within error (92.3 ± 0.04%, 89.4 ± 2.2%, 88.3 ± 2.2%, respectively) (Table 2.2).  [In(decapa)]2- also demonstrated exceptional stability against mouse serum transchelation with a stability of 89.1 ± 1.7% at 24 hours.    Table 2.2 Data from mouse serum stability challenges performed at ambient temperature (n=3), evaluated by PD-10 size-exclusion column elution, with stability shown as the percentage of intact 111In complex.  Complex 1 hour stability   24 hour stability [111In(dedpa)]+ 96.1 ± 0.1%   19.7 ± 1.5% [111In(octapa)]- 93.8 ± 3.6%   92.3 ± 0.04% [111In(decapa)]2- 89.7 ± 1.6%   89.1 ± 1.7% [111In(DOTA)]- 89.6 ± 2.1%   89.4 ± 2.2% [111In(DTPA)]2- 86.5 ± 2.2%   88.3 ± 2.2%   To provide a more definitive answer to which of these ligands is most suitable for in vivo radiopharmaceutical applications, mouse biodistribution studies were performed with [111In(octapa)]-,  [111In(DOTA)]-, and [111In(decapa)]2-.  The data summarized in Figure 2.5     70 (y-axis adjusted to 2 %ID/g for visualization of 4 and 24 hour time points) show rapid clearance through the kidneys for [111In(octapa)]- and [111In(DOTA)]-, with radioactivity clearing quickly from all organs.  The clearance of [111In(decapa)]2- was found to be slower, with 111In levels slowly increasing over 24 hours in the liver, spleen, and bone (Table 2.3).  These results are typical of a metal-ligand complex that shows instability in vivo and undergoes demetallation/transchelation, which is surprising because of the high degree of stability of [111In(decapa)]2- in mouse serum competition experiments (Table 2.3).  The most promising result from these animal experiments is that the radiometal complex [111In(octapa)]- has exceptional in vivo stability over 24 hours, with improved clearance compared to [111In(DOTA)]- (Figure 2.5), most notably from the kidneys (0.189 ± 0.019 %ID/g vs 0.664 ± 0.108 %ID/g, respectively), liver (0.0248 ± 0.0030 %ID/g vs 0.0605 ± 0.0022 %ID/g, respectively), and spleen (0.0202 ± 0.0083 %ID/g vs 0.0426 ± 0.0067 %ID/g, respectively) at 24 hours (p < 0.05).  These findings are significant because in vivo instability resulting in demetallation or transchelation via serum proteins typically results in high uptake of “free” 111In3+ metal ion in the liver, spleen, bone, and kidneys (typical of transferrin-bound 3+ metals), with accumulation of radioactivity increasing over time.93  Surprisingly, despite the exceptional mouse serum stability of [In(decapa)]2-, the in vivo stability in mice was found to be sub-optimal, with slower clearance from the blood pool and most notably high persistent kidney uptake (3.68 ± 0.54 %ID/g) and increasing bone uptake (1.95 ± 0.12 %ID/g) after 24 hours; however, all other organs contained less than 1 %ID/g after 24 hours (Figure 2.5, Table 2.3).  Although these values for H5decapa are inadequate when compared to the in vivo stability and clearance of exceptionally stable ligands such as [111In(DOTA)]- and [In(octapa)]-, it is     71 still fair when compared to the very labile 111In-citrate complex, which has shown values of 18.7 ± 3.7 %ID/g in the kidneys after 24 hours.319  Table 2.3 Decay corrected %ID/g values from the biodistribution of 111In-complexes in healthy female ICR mice (6-8 weeks old), n = 4, bold = passed students T-test (p < 0.05).     Often 111InCl3 and 111In-citrate are used to emulate the conditions of an unstable ligand that would undergo complete and rapid decomposition/transchelation in vivo.  One commonly cited study (Ando et al.) has shown 111InCl3 to have higher liver, spleen, and kidney uptake than 111In-citrate;93 however, another study has shown the kidney uptake of 111InCl3 at 24 hours in healthy rats to be 2.58 ± 0.83 %ID/g,320 which is significantly less than the value for 111In-citrate (cited above) of 18.7 ± 3.7 %ID/g,319 and contradicts the observations from Ando et al.93       72  Figure 2.5 Biodistribution %ID/g values for [111In(DOTA)]-, [111In(octapa)]-, and [111In(decapa)]2-, with error bars plotted as standard deviations (note the y-axis set to 2.0 %ID/g, for clarity of the low activity 4 and 24 hour time points).      73  Additionally, the highly anionic 111In-citrate clears much more quickly through the kidneys than 111InCl3, demonstrating that the hypothesis of rapid and complete dissociation upon introduction of these radiometal species into an animal is not accurate.93  Considering these inconsistencies, it is important to utilize internal standards (such as [111In(DOTA)]- in this study), and additionally there is a need for more clear and reliable baseline data for the biodistribution of 111In in its most commonly used forms.93,321,322  In biological systems, transchelated indium(III) is nearly all bound to transferrin, with a maximum stability constant of log KML = 18.3.318  Demetallation and hydroxide formation is a significant concern for acidic metal ions such as Ga3+ and In3+, and a conditional stability constant for transferrin that takes hydrolysis into account has been calculated as being ~10.0 for In3+, which is higher even than Ga3+ at 6.9 and close to Fe3+ at 11.4.318  This demonstrates the strong competition that transferrin holds for In3+ in vivo; however, the ligand exchange kinetics of In3+ are quite slow and it forms a more inert complex with transferrin than do Ga3+ and Fe3+.79,318  It is most likely that due to the high stability and inertness of complexes such as [In(DOTA)]-, and the slow exchange kinetics with transferrin, only gradual in vivo decomplexation is observed over long periods such as > 24 hours.  The higher levels of 111In in the kidneys, liver, and spleen observed for [In(DOTA)]- after 24 hours is most likely a result of this gradual exchange process, and the improved inertness and clearance observed for [111In(octapa)]- correlates well with its lower isomerization and higher pM value of 26.8(1) ([In(DOTA)]- pM = 18.8) (Table 2.1), and suggests superior kinetic inertness.  Additionally, the lower levels of 111In in the kidneys at 24 hours seen for [In(octapa)]-, being roughly a third of that for [In(DOTA)]-, are very promising because persistent kidney uptake is observed with many peptide conjugates of DOTA, CHX-A’’-DTPA, and especially     74 DTPA.60,213,215,216,231,305,312  High residual activity in the kidneys can decrease image quality, obstruct the delineation of tumours for surrounding tissue, decrease the accuracy of dosimetry, and deliver harmful doses of radiation to the healthy and non-targeted kidneys when used with therapeutic isotopes such as 90Y and 177Lu.  2.3 Conclusions  Preliminary investigations of the octadentate acyclic ligand H4octapa (N4O4) with 111In/In3+ have demonstrated it to be a significant improvement on the shortcomings of the current industry gold standards DOTA (N4O4) and DTPA (N3O5).  Four major points were identified in the discussion as guidelines to be used in selecting an ideal chelating agent for radiometal ions, and [In(octapa)]- has been shown to be superior to [In(DOTA)]- and [In(DTPA)]2- for a majority of these points.  We have demonstrated the ability of H4octapa to quantitatively radiolabel with 111In at ambient temperature, which, in contrast to DOTA, allows it to be effectively used with sensitive biovectors such as antibodies.  H4octapa has been radiolabeled with 111In in 10 minutes at ambient temperature with specific activities as high as 2.3 mCi/nmol (97.5% radiochemical yield).  In vitro mouse serum stability assays have demonstrated H4octapa to have slightly improved stability with 111In compared to DOTA and DTPA over 24 hours, within error.  Mouse biodistribution studies have shown that the radiometal complex [111In(octapa)]- has exceptionally high in vivo stability and kinetic inertness, and compared to [111In(DOTA)]-, [111In(octapa)]- has improved stability and improved clearance from the kidneys, liver, and spleen at 24 hours.  1H/13C NMR studies of the [In(octapa)]- complex have revealed a 7-coordinate solution structure, which forms a single isomer and exhibits no observable fluxional behavior at ambient temperature on the     75 NMR timescale and is an improvement on the multiple static isomers observed for [In(DTPA)]2- and the fluxional isomerization of [In(DOTA)]-.49,63,97  Potentiometric titrations have determined the thermodynamic formation constant of the [In(octapa)]- complex to be log KML = 26.8(1) (pM = 26.5), which reveals a higher pM value than those determined for [In(DOTA)]- and [In(DTPA)]2- (18.8 and 25.7, respectively).  The same set of experiments and analyses was performed with the potentially decadentate ligand H5decapa (N5O5) and its [In(decapa)]2- complex; however, the formation of multiple isomers observed via radio-HPLC and NMR studies was not optimal, and unfavorable organ uptake and clearance were observed in biodistribution studies performed in mice.  These results for H5decapa suggest that it is not a suitable candidate for 111In radiopharmaceutical elaboration; however, it may be a suitable candidate for coordinating lanthanide and actinide radiometal ions that can accommodate higher denticities, such as 90Y, 177Lu, and 225Ac.  The relatively simple 5-step synthesis of H4octapa is achieved with a cumulative yield of ~12%, and the 5-step synthesis of H5decapa was completed with a cumulative yield of ~2.5%. Optimization of reaction conditions and/or exploration of alternative protecting groups may improve these yields.  Our initial investigations have revealed the acyclic ligand H4octapa to be a valuable alternative to the macrocycle DOTA and the acyclic ligand DTPA by showing improved stability and kinetic inertness, as well as fast ambient temperature radiolabeling kinetics.  Pending the results of investigations with 90Y and 177Lu, H4octapa could present a very strong alternative to DOTA as an ideal ligand for incorporation into radiometal-based radiopharmaceuticals.  The next step in the evaluation and study of the ligand H4octapa will be the synthesis of a bifunctional derivative that can be conjugated to targeting vectors, such as peptides and antibodies (Chapter 3).  The major shortcoming of in vivo experiments with     76 “bare” and non-bifunctional ligands such as those described in this chapter, is that the highly hydrophilic and charged radiometal complexes are very quickly excreted.  This means that the residency time in blood and tissue is very small, and the time period over which stability can be evaluated is very short (a few hours).  Conjugating a bifunctional derivative of H4octapa to a large biovector such as an antibody will increase the biological half-life from a few hours to a few weeks, allowing for longer experiments to be carried out under conditions that are more realistic for imaging and therapy applications in vivo.   	  2.4 Experimental methods 2.4.1 Materials and methods   All solvents and reagents were purchased from commercial suppliers (TCI America, Sigma Aldrich, Fisher Scientific) and were used as received.  Mouse serum was purchased frozen from Sigma Aldrich. DOTA was purchased from Macrocyclics.  The acyclic ligand H2dedpa was synthesized according to the literature.118,271  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, AV400, or AV600 instruments; the NMR spectra are expressed on the δ scale and were referenced to residual solvent peaks and/or internal tetramethylsilane.  13C NMR experiments run in D2O were externally referenced to a sample of CH3OD/D2O.  Low-resolution mass spectrometry was performed using a Waters ZG spectrometer with an ESCI electrospray/chemical-ionization source, and high-resolution electrospray-ionization mass spectrometry (ESI-MS) was performed on a Micromass LCT time-of-flight instrument at the     77 Department of Chemistry, University of British Columbia. Microanalysis for C, H, and N was performed by UBC MS staff on a Carlo Erba Elemental Analyzer EA 1108.  IR spectra were collected neat in the solid state on a Thermo Nicolet 6700 FT-IR spectrometer.  111In(chelate) mouse serum stability experiments were analyzed using GE Healthcare Life Sciences PD-10 desalting columns (size exclusion for MW < 5000 Da) and counted with a Capintec CRC 15R well counter.  Radiolabeling of DOTA with 111In was performed using a Biotage® Initiator microwave reactor (µW).  The HPLC system used for analysis and purification of cold compounds consisted of a Waters 600 controller, Waters 2487 dual wavelength absorbance detector, and a Waters delta 600 pump.  Phenomenex synergi hydro-RP 80 Å columns (250 x 4.6 mm analytical and 250 x 21.2 mm semi-preparative) were used for purification of several of the deprotected ligands.  Analysis of radiolabeled complexes was carried out using a Waters xbridge BEH130 C18 reverse phase (150 x 6 mm) analytical column using a Waters Alliance HT 2795 separation module equipped with a Raytest Gabi Star NaI (Tl) detector and a Waters 996 photodiode array (PDA) detector.  111InCl3 was cyclotron produced and provided by Nordion as a ~0.05 M HCl solution.  2.4.2 Methyl 6-(hydroxymethyl)picolinate (2.1) Compound 2.1 was synthesized according to a modified literature protocol.323  A suspension of dimethylpyridine-2,6-dicarboxylate (10.0 g, 51.2 mmol) in methanol (400 mL) in a round bottom flask (1000 mL) was cooled in a salted ice bath.  Sodium borohydride (5.56 g, 146.9 mmol, 2.4 eq.) was then added slowly over a period of 1 hour, where the reaction mixture turned pink upon the addition of sodium borohydride.  The reaction mixture was stirred and kept on ice for 4 hours.  After 4 hours, TLC analysis revealed that the     78 reaction mixture contained starting material (Rf : 0.45, TLC in 100% EtOAc), product (Rf : 0.33), and doubly-reduced product (Rf : 0.24).  The reaction mixture was quenched regardless of the remaining starting material in order to prevent over-reduction.  The solution was diluted using dichloromethane (200 mL) and then quenched using saturated NaHCO3 (~200 mL).  The aqueous and organic layers were separated, and much of the methanol from the aqueous phase was evaporated in vacuo.  The aqueous layer was then extracted with chloroform (2 x 100 mL) and ethyl acetate (3 x 100 mL).  The combined organic layers were dried (MgSO4), filtered, and concentrated in vacuo to dryness.  The resulting white solid was purified by column chromatography (column 10”L x 2”W; eluted with a gradient of 3:1 ethyl acetate/petroleum ether to 100% ethyl acetate) to afford the product as a white solid (4.35 g, 51%, Rf: 0.33 in 100% EtOAc).  1H NMR (300 MHz, CDCl3): δ = 7.95 (d, 1H, pyr-H), 7.79 (t, 1H, pyr-H), 7.55 (d, 1H, pyr-H), 4.83 (s, 2H, methylene-H), 4.31 (s, 1H, -OH), 3.92 (s, 3H, methyl-H).  13C NMR (75 MHz, CDCl3): 165.4, 160.6, 146.7, 137.6, 123.9, 123.5, 64.6, 52.7.  HR-ESI-MS calcd. for [C8H9NO3 + H]+: 168.0661; found [M + H]+ 168.0658.  2.4.3 Methyl 6-(bromomethyl)picolinate (2.2)  Compound 2.2 was synthesized according to a modified literature protocol.323  To a solution of 2.1 (4.00 g, 23.9 mmol) in chloroform (100 mL) under argon at 0 °C, phosphorus tribromide (2.50 mL, 26.3 mmol, 1.1 eq.) in chloroform (10 mL) was added dropwise over 15 minutes via dropping funnel.  A white precipitate formed, and then the solution turned bright yellow.  The dropping funnel was rinsed using chloroform (15 mL) and the reaction mixture was stirred at 0 °C and monitored by TLC.  A mini-workup using sodium carbonate     79 in water and ethyl acetate was required to see reaction progress by TLC.  At 3.5 hours TLC indicated the quantitative conversion of alcohol to alkyl halide.  The reaction mixture was quenched using sodium carbonate in water (75 mL) and was extracted with chloroform (4 x 25 mL).  The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to dryness to afford an off-white solid.  The crude product was purified through a short silica plug (2”L x 2”W, 25% EtOAc in petroleum ether) to afford 2.2 as an off-white solid (5.55 g, >99%, Rf : 0.64 in 30% petroleum ether in EtOAc).  1H NMR (400 MHz, CDCl3) δ: 8.01 (d, 1H, pyr-H), 7.84 (t, 1H, pyr-H), 7.65 (d, 1H, pyr-H), 4.61 (s, 2H, methylene-H), 3.96 (s, 3H, methyl-H).  13C NMR (100 MHz, CDCl3) δ: 165.1, 157.2, 147.3, 138.1, 127.0, 124.3, 53.9, 32.9.  HR-ESI-MS calcd. for [C8H8NO279Br + H]+: 229.9817; found [M + H]+: 229.9812.  2.4.4 N,N’-(Benzyl)ethylenediamine (2.3)  To a solution of ethylenediamine (2.78 mL, 41.6 mmol) in dry methanol (distilled over CaH2, 100 mL) was added benzaldehyde (8.48 mL, 83.2 mmol, 2 eq.).  The solution was heated to reflux for 4 hours and then cooled via ice bath.  Addition of NaBH4 (6.77 g, 179 mmol, 4.3 eq.) was performed slowly and in small portions to prevent boiling, and the reaction mixture was stirred for 4 hours until completion.  The solvent was evaporated in vacuo and then saturated NaHCO3 (~50 mL), water (~50 mL), and chloroform (200 mL) were added.  The aqueous layer was extracted twice more with chloroform (100 mL).  The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to afford a waxy yellow solid. Product was purified by silica column chromatography (column 10”L x 2”W; eluted with a gradient of 100% dichloromethane to 25% CH3OH in     80 dichloromethane) to afford 2.3 as yellow oil (4.90 g, 49%, Rf : 0.33 in 20% CH3OH in dichloromethane).  1H NMR (400 MHz, CDCl3) δ: 7.42 (d, 8H, Bn-H), 7.34 (m, 2H, Bn-H), 3.86 (s, 4H, Bn-CH2-N), 2.83 (s, 4H, ethylene-H), 1.72 (s, 2H, amine-H).  13C NMR (100 MHz, CDCl3) δ: 140.2, 127.9, 127.7, 126.4, 53.5, 48.4.  HR-ESI-MS calcd. for [C16H20N2 + H]+: 241.1705; found [M + H]+: 241.1703.  2.4.5 N,N’-[Benzyl(tert-butoxycarbonyl)methyl]aminoethane (2.4) To a solution of 2.3 (1.0 g, 4.2 mmol) and sodium carbonate (1.76 g, 16.6 mmol, 3.9 eq.) in dry acetonitrile (distilled over CaH2, 80 mL) was added dropwise a solution of tert-butyl bromoacetate (1.31 mL, 8.88 mmol, 2.1 eq.).  The reaction mixture was stirred at 60 °C for 16 hours.  TLC (5% CH3OH in dichloromethane) showed quantitative conversion of 2.3 to the tertiary diamine.  Sodium carbonate was removed by suction filtration, and the filtrate was concentrated in vacuo to dryness.  The resulting yellow oil was purified by column chromatography (column 2”L x 2”W; eluted with a gradient of 100% petroleum ether to 10% ethyl acetate in petroleum ether) to afford 2.4 as a yellow solid (1.85 g, 93%, Rf : 0.59 in 5% CH3OH in dichloromethane).  1H NMR (400 MHz, CDCl3) δ: 7.40-7.29 (m, 10H, Bn-H), 3.85 (s, 4H, Bn-CH2-N), 3.30 (s, 4H, (CH3)3CO-C(O)-CH2-N), 2.88 (s, 4H, ethylene-H), 1.52 (s, 18H, (CH3)3CO-C(O)-CH2-N).  13C NMR (100 MHz, CDCl3) δ: 170.8, 139.1, 128.8, 128.1, 126.8, 80.5, 58.3, 55.0, 51.6, 28.1.  HR-ESI-MS calcd. for [C28H40N2O4 + H]+: 469.3066; found [M + H]+: 469.3055.      81 2.4.6 N,N’-[(tert-Butoxycarbonyl)methyl]aminoethane (2.5) To a solution of 2.4 (776.3 mg, 1.656 mmol) in glacial acetic acid (7 mL) was added Pd/C (78 mg, ~10 wt%).  Hydrogen gas was bubbled through the solution for 3 minutes and then the reaction mixture was stirred under a sealed hydrogen atmosphere (balloon) for 16 hours.  The Pd/C was removed by filtration over Celite, rinsing well with methanol, and the filtrate was evaporated to dryness in vacuo.  The crude product was purified by silica gel column chromatography (eluted with a gradient of 100% dichloromethane to 5% methanol in dichloromethane) and identified by TLC using an I2 chamber to stain.  Product fractions were combined and concentrated in vacuo to afford the product 2.5 as a waxy yellow solid (0.51 g, 87%, Rf : 0.20 in 10% CH3OH in dichloromethane).  1H NMR (300 MHz, CDCl3) δ: 3.37 (s, 4H, (CH3)3CO-C(O)-CH2-N), 2.68 (s, 4H, ethylene-H), 2.03 (s, 2H, -NH-), and 1.43 (s, 18H, (CH3)3CO-C(O)-CH2-N).  13C NMR (75 MHz, CDCl3) δ: 171.7, 80.9, 51.4, 48.7, 28.0.  HR-ESI-MS calcd. for [C14H28N2O4 + H]+: 289.2127; found [M + H]+: 289.2126.  2.4.7 N,N′-[[(tert-Butoxycarbonyl)methyl]-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]methylamino]ethane (2.6) To a solution of 2.5 (431.5 mg, 1.496 mmol) and 2.2 (725 mg, 3.142 mmol, 2.1 eq.) in dry acetonitrile (distilled over CaH2, 20 mL) was added sodium carbonate (~300 mg).  The solution was heated for 16 hours at 60 °C under argon.  Sodium carbonate was removed by filtration and rinsed with acetonitrile.  The filtrate was concentrated in vacuo to dryness and the resulting yellow oil was purified by column chromatography (eluted with a gradient of 100% dichloromethane to 5% methanol in dichloromethane) to afford 2.6 as colorless oil (342 mg, 39%, Rf : 0.40 in 10% CH3OH in dichloromethane).  1H NMR (300 MHz, CDCl3)     82 δ: 7.85-7.82 (m, 2H, pyr-H), 7.65-7.63 (m, 4H, pyr-H), 3.85 (s, 4H, Pyr-CH2-), 3.83 (s, 6H, -O-CH3), 3.16 (s, 4H, (CH3)3CO-CO-CH2-N), 2.67 (s, 4H, ethylene-H), 1.28 (s, 18H, (CH3)3CO-C(O)-CH2-N).  13C NMR (75 MHz, CDCl3) δ: 170.1, 165.4, 160.3, 146.7, 137.1, 125.8, 123.2, 80.6, 60.2, 55.9, 52.4, 52.1, 27.7.  HR-ESI-MS calcd. for [C30H42N4O8 + H]+: 587.3081; found [M + H]+: 587.3091.  2.4.8 H4octapa•4HCl•2H2O, N,N′-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N′diacetic acid A portion of 2.6 (277.4 mg, 0.4728 mmol) was dissolved in HCl (6 M) and heated to reflux for 8 hours.  White solid was observed to precipitate from hot HCl (aq) below ~60 °C.  The solution was then cooled in the freezer for 1 hour.  The product was filtered and washed with cold ethanol and diethyl ether to afford the HCl salt H4octapa as a white solid (265 mg, 75% yield using the molecular weight of the HCl salt from elemental analysis).  1H NMR (300 MHz, D2O) δ: 8.12-8.02 (m, 4H, pyr-H), 7.72-7.69 (d, 2H, pyr-H), 4.53 (s, 4H, Pyr-CH2-N), 3.97 (s, 4H, HOOC-CH2-N), 3.57 (s, 4H, ethylene-H).  13C NMR (75 MHz, D2O) δ: 170.6, 165.6, 151.4, 145.4, 142.9, 128.8, 126.3, 58.1, 55.2, 51.5.  IR (neat, ATR-IR): ν = 1720.9 cm-1 (C=O), 1635.9/1618.2 cm-1 (C=C py).  HR-ESI-MS calcd. for [C20H20N4O8 + H]+: 445.1359; found [M + H]+: 445.1368.  Elemental analysis: calcd % for H4octapa•4HCl•2H2O (C20H20N4O8•4HCl•2H2O = 628.283): C 38.23, H 4.81, N 8.92; found: C 38.34, H 4.81, N 8.93.      83 2.4.9 Na[In(octapa)] A portion of H4octapa•4HCl•2H2O (9.9 mg, 0.019 mmol) and In(ClO4)3•8H2O (11.7 mg, 0.021 mmol, 1.1 eq.) were dissolved in HCl (aq) (1 mL, 0.1 M) in a 1 dram screw cap vial.  The pH was adjusted to ~4.5 with NaOH (aq) (0.1 M) while stirring.  The reaction mixture was stirred at 60 °C for 4 h, then evaporated to dryness to afford Na[In(octapa)] as a white solid.  1H NMR (600 MHz, D2O) δ: 8.34-8.32 (m, 2H, pyr-H), 8.23 (d, 2H, pyr-H), 7.85 (d, 2H, Pyr-H), 4.49 (d, 2H, Pyr-CH2-N, 2J = 16.1 Hz), 4.34 (d, 2H, Pyr-CH2-N, 2J = 16.1 Hz), 3.27 (d, 2H, HOOC-CH2-N, 2J = 19.3 Hz), 3.19-3.18 (broad d, 2H, HOOC-CH2-N, 2J = 8.4 Hz), 3.07-3.04 (m, 4H, ethylene-H).  13C NMR (150 MHz, D2O) δ: 177.2, 167.8, 153.7, 148.2, 144.2, 128.3, 124.6, 59.4, 58.3, 55.2.  HR-ESI-MS calcd. for [C20H18115InN4O8 + 2•Na]+: 602.9959; found [M + 2•Na]+:  602.9942.   2.4.10 N,N’’-[Benzyl]diethylenetriamine (2.7) To a solution of diethylenetriamine (5 mL, 46.2 mmol) in dry methanol (distilled over CaH2, 100 mL) was added benzaldehyde (9.43 mL, 96.56 mmol, 2 eq.).  The solution was heated to reflux for 4 hours, and then cooled (0 °C) via ice bath.  Addition of NaBH4 (12.26 g, 323 mmol, 7 eq.) was performed slowly and in small portions to prevent solvent boiling.  The reaction solution was stirred for 4 hours until completion.  The solvent was evaporated in vacuo and then saturated NaHCO3 (~100 mL) and chloroform (200 mL) were added.  The aqueous layer was extracted twice more with dichloromethane (100 mL).  The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to afford a yellow solid.  The crude product was purified by silica gel column chromatography (column 16”L x 3”W; eluted with a gradient of 2 to 10% CH3OH and 2% triethylamine in     84 dichloromethane) to afford 2.7 as yellow oil (5.23 g, 40%, Rf : 0.30 in 20% CH3OH in dichloromethane).  1H NMR (300 MHz, CDCl3) δ: 7.34-7.26 (m, 10H, Bn-H), 3.80 (s, 4H, Bn-CH2-N), 2.75 (s, 8H, ethylene-H), 1.94 (s, 3H, -NH-).  13C NMR (75 MHz, CDCl3) δ: 140.5, 128.6, 128.3, 127.1, 54.1, 49.4, 48.9.  HR-ESI-MS calcd. for [C18H25N3 + H]+: 284.2127; found [M + H]+: 284.2124.  2.4.11 N,N’’-[[Benzyl]-N,N’,N’’-[(tert-butoxycarbonyl)methyl]]diethylenetriamine (2.8) To a solution of 2.7 (673.3 mg, 2.37 mmol) and sodium carbonate (excess, 450 mg) in dry acetonitrile (distilled over CaH2, 20 mL) was added dropwise tert-butyl bromoacetate (1.071 mL, 7.25 mmol, 3.05 eq.) under argon.  The solution was stirred at 60 °C for 20 hours.  Sodium carbonate was removed by suction filtration and the filtrate was concentrated in vacuo to dryness.  The resulting yellow oil was purified by column chromatography (column 10”L x 2”W; eluted with a gradient of 1 CH3OH to 10% CH3OH in dichloromethane) to afford 2.8 as light yellow oil (974.4 mg, 66%, Rf : 0.36 in 10% CH3OH in dichloromethane).  1H NMR (400 MHz, CDCl3) δ: 7.41-7.29 (m, 10H, Bn-H), 3.86 (s, 4H, Bn-CH2-N), 3.39 (s, 2H, (CH3)3CO-CO-CH2-N’), 3.31 (s, 4H, (CH3)3CO-CO-CH2-N/N’’), 2.84 (s, 8H, ethylene-H), 1.54 (s, 18H, (CH3)3CO-CO-CH2-N/N’’), 1.50 (s, 9H, (CH3)3CO-CO-CH2-N’).  13C NMR (100 MHz, CDCl3) δ: 170.8, 170.7, 139.0, 128.8, 128.0, 126.8, 80.4, 80.4, 58.2, 55.9, 55.0, 52.4, 51.9, 28.1, 28.0.  HR-ESI-MS calcd. for [C36H55N3O6 + H]+:  626.4169; found [M + H]+: 626.4166.      85 2.4.12 N,N’,N’’-[[tert-Butoxycarbonyl]methyl]diethylenetriamine (2.9) To a solution of 2.8 (702.6 mg, 1.12 mmol) in glacial acetic acid (10 mL) was added Pd/C (35 mg, ~5 wt%).  Hydrogen gas was bubbled through the solution for 3 minutes and then the reaction mixture was stirred under a sealed hydrogen atmosphere (balloon) for 16 hours.  The Pd/C was filtered over celite, rinsing with methanol, and then the filtrate was evaporated to dryness in vacuo.  The crude product was purified by silica gel column chromatography (0 to 10% methanol in dichloromethane) and identified by TLC using an I2/silica chamber to stain.  Product fractions were combined and concentrated in vacuo to dryness to afford the product 2.9 as colorless oil (150 mg, 30%, Rf : 0.26 in 2.5% CH3OH and 2.5% triethylamine in dichloromethane).  Cleavage of the ethylene bridges was observed during hydrogenation and produced large amounts of byproduct.  1H NMR (300 MHz, CDCl3) δ: 3.28 (s, 2H, (CH3)3CO-CO-CH2-N’), 3.25 (s, 4H, (CH3)3CO-CO-CH2-N/N’’), 2.77-2.73 (m, 4H, ethylene-H), 2.62-2.59 (m, 4H, ethylene-H), 2.14 (s, 2H, -NH-), 1.40 (s, 18H, (CH3)3CO-CO-CH2-N/N’’), 1.39 (s, 9H, (CH3)3CO-CO-CH2-N’). 13C NMR (75 MHz, CDCl3) δ: 170.4, 170.8, 80.7, 80.7, 55.5, 54.0, 51.5, 47.2, 28.0, 27.9.  HR-ESI-MS calcd. for [C22H43N3O6 + H]+: 446.3230; found [M + H]+: 446.3239.  2.4.13 N,N’’-[[6-(Methoxycarbonyl)pyridin-2-yl]methylamino]-N,N’,N’’-[(tert-butoxycarbonyl)methyl]]diethylenetriamine (2.10) To a solution of 2.9 (101.8 mg, 0.228 mmol) and 2.2 (110.4 mg, 0.479 mmol, 2.1 eq.) in dry acetonitrile (distilled over CaH2, 10 mL) was added sodium carbonate (~100 mg).  The solution was heated for 16 hours at 60 °C under argon.  Sodium carbonate was removed by filtration and rinsed with acetonitrile.  The filtrate was concentrated in vacuo to dryness     86 and the resulting yellow oil was purified by column chromatography (100% dichloromethane to 5% CH3OH in dichloromethane) to afford 2.10 as colorless oil (75.7 mg, 45%, Rf : 0.27 in 2.5% CH3OH and 2.5% triethylamine in dichloromethane).  1H NMR (300 MHz, CDCl3) δ: 7.96-7.94 (m, 2H, Pyr-H), 7.82-7.70 (m, 4H, Pyr-H), 3.97 (s, 4H, Pyr-CH2-N), 3.96 (s, 6H, -O-CH3), 3.28 (s, 4H, (CH3)3CO-CO-CH2-N/N’’), 3.23 (s, 2H, (CH3)3CO-CO-CH2-N’), 2.72 (s, 8H, ethylene-H), 1.41 (s, 18H, (CH3)3CO-CO-CH2-N/N’’), 1.37 (s, 9H, (CH3)3CO-CO-CH2-N’).  13C NMR (75 MHz, CDCl3) δ: 170.6, 170.4, 170.4, 165.8, 160.9, 147.0, 137.3, 126.0, 123.4, 80.9, 80.8, 80.6, 60.5, 56.3, 56.3, 55.8, 52.7, 56.7, 55.7, 52.7, 52.7, 52.6, 52.4, 28.1.  HR-ESI-MS calcd. for [C38H57N5O10 + H]+: 744.4184; found [M + H]+: 744.4199.  2.4.14 H5decapa•5HCl•2.5H2O, N,N′,N′′-[(triacetic acid]-N,N′′-[6-(carboxy)pyridin-2-yl]methylamino]diethylenetriamine A portion of 2.10 (76.7 mg, 0.1031 mmol) was dissolved in HCl (6 M) and heated to reflux for 8 hours.  The reaction mixture was concentrated in vacuo to an off-white powder, which was then purified by reverse-phase HPLC (gradient: A: 0.1% TFA (trifluoroacetic acid), B: CH3CN. 0 to 100% B linear gradient 25 min. tR = broad, 13.2-15 min).  The HPLC fractions were combined, 2 mL of HCl (6 M) was added, and the solvent was removed in vacuo to drive off residual trifluoroacetic acid.  Another 2 mL of HCl (6 M) was added, then concentrated in vacuo to afford the HCl salt of H5decapa as a white solid (56 mg, 71% using molecular weight from elemental analysis).   1H NMR (400 MHz, D2O) δ: 8.11-8.04 (m, 4H, Pyr-H), 7.72-7.71 (m, 2H, Pyr-H), 4.55 (s, 4H, Pyr-CH2-N), 3.98 (s, 4H, HOOC-CH2-N/N’’), 3.71 (s, 2H, HOOC-CH2-N’), 3.44 (s, 4H, ethylene-H), 3.28 (s, 4H, ethylene-H).  13C NMR     87 (100 MHz, D2O) δ: 172.5, 170.2, 168.2, 151.1, 145.9, 142.3, 128.8, 126.3, 57.9, 54.9, 54.3, 51.9, 50.8.  IR (neat, ATR-IR): ν = 1727.2 cm-1 (C=O), 1642.7 cm-1 (C=C py).  HR-ESI-MS calcd. for [C24H29N5O10 + H]+: 548.1993; found [M + H]+: 548.1987.  Elemental analysis: calcd % for H5decapa•5HCl•2.5H2O (C24H29N5O10•5HCl•2.5H2O = 774.856): C 37.20, H 5.07, N 9.04; found: C 37.28, H 4.91, N 8.72.  2.4.15 Na2[In(decapa)] A portion of H5decapa (8.6 mg, 0.0112 mmol) and In(ClO4)3•8H2O (6.9 mg, 0.0124mmol, 1.1 eq.) was dissolved in HCl (aq) (1 mL, 0.1 M) in a 1 dram screw cap vial.  The pH was adjusted to ~4.5 with NaOH (aq) (0.1 M) while stirring.  The reaction mixture was stirred at 60 °C for 16 hours, then evaporated to dryness to afford Na2[In(decapa)].  1H NMR (600 MHz, D2O) δ: 8.33-7.71 (m, 6H, Pyr-H), 4.87-2.57 (m, 18H, complex diastereotopic splitting).  13C NMR (150 MHz, D2O) δ: 178.5, 178.3, 178.0, 177.4, 176.6, 168.4, 167.8, 154.0, 153.6, 148.5, 146.6, 145.4, 145.0, 144.2, 143.2, 142.8, 142.5, 128.8, 128.2, 128.1, 128.0, 124.4, 123.6, 123.5, 123.1, 61.3, 59.5, 59.3, 59.1, 58.3, 57.8, 55.7, 55.3, 53.9, 52.5, 52.4, 51.0.  HR-ESI-MS calcd. for [C24H24115InN5O10 + H + 2Na]+: 704.0436; found [M + H + 2Na]+: 704.0430.   2.4.16 H2dedpa The hexadentate ligand H2dedpa was synthesized according to a literature procedure,118,271,274 with a modified purification step performed by reverse-phase HPLC (gradient: A: 0.1% TFA, B: CH3CN. 0 to 100% B linear gradient 25 min. tR = broad, 9.7-11     88 min), followed by a second HPLC purification with a modified gradient (A: distilled deionized water, B: CH3CN. 0 to 100% B linear gradient 25 min. tR = broad, 8.5-10 min).  2.4.17 [In(dedpa)]Cl A portion of H2dedpa (10 mg, 0.020 mmol) and In(ClO4)3•8H2O (13 mg, 0.023 mmol, 1.2 eq.) was dissolved in HCl (aq) (1 mL, 0.1 M) in a 1 dram screw cap vial.  The pH was adjusted to ~4-4.5 with NaOH (aq) (0.1 M) while stirring.  The reaction solution was stirred at 60 °C for 16 hours, then evaporated to dryness to afford [In(dedpa)]Cl as a white solid.  1H NMR (600 MHz, DMSO-d6) δ: 8.19 (t, 2H, pyr-H), 8.07 (d, 2H, pyr-H), 7.71 (d, 2H, pyr-H), 4.40 (s, 2H, -NH-), 4.27/4.26 (two overlapping d, 2H, Pyr-CH2-N, 2J = 16.6/16.3 Hz), 3.90/3.89 (two overlapping d, 2H, Pyr-CH2-N, 2J = 16.6 Hz), 2.63-2.56 (m, 4H, ethylene-H).  1H NMR (400 MHz, D2O) δ: 8.28 (t, 2H, pyr-H), 8.20 (d, 2H, pyr-H), 7.79 (d, 2H, pyr-H), 4.58 (d, 2H, Pyr-CH2-N, 2J = 17.3 Hz), 4.13 (d, 2H, Pyr-CH2-N, 2J = 17.4 Hz), 2.96-2.81 (m, 4H, ethylene-H).  13C NMR (300 MHz, DMSO-d6) δ: 163.7, 151.9, 146.9, 141.3, 124.6, 121.8, 49.5, 44.9.  HR-ESI-MS calcd. for [C16H16115InN4O4]+: 443.0210; found [M]+: 443.0216.   2.4.18 111In Radiolabeling studies  The ligands H4octapa, H5decapa, H2dedpa, DTPA, and DOTA were made up as stock solutions (1 mg/mL, ~10-3 M) in deionized water.  An aliquot of each ligand stock solution was transferred to screw cap mass spectrometry vials and made up to 1 mL with pH 5.5 NaOAc (10 mM) buffer, to a final ligand concentration of ~365 µM for each sample.  A ~10 µL aliquot of the 111InCl3 stock solution (~1 mCi for labeling studies and ~5-7 mCi for     89 mouse serum competitions) was transferred into the vials containing each ligand, allowed to radiolabel at ambient temperature for 10 minutes, and then analyzed by RP-HPLC to confirm radiolabeling and calculate yields.  [111In(DOTA)]- was heated at 80 °C for 20 minutes in a microwave reactor to achieve quantitative radiolabeling yields.  Areas under the peaks observed in the radioactive HPLC trace were integrated to determine radiolabeling yields.  The highest specific activity of 2.3 mCi/nmol obtained for H4octapa was the result of labeling a 990 µL solution of 3.65 x 10-7 M chelate in NaOAc buffer (pH 5.3, 10 mM) with 10 µL of 111In3+ in a 0.05 M HCl solution (1.02 mCi) for 10 minutes at ambient temperature in 97.5% radiolabeling yield.  Elution conditions used for RP-HPLC analysis were gradient: A: 10 mM NaOAc buffer pH 4.5, B: CH3CN. 0 to 100% B linear gradient 20 minutes.  The radiometal complex [111In(dedpa)]+ used a modified HPLC gradient of A: 10 mM NaOAc buffer pH 4.5, B: CH3CN. 0 to 5% B linear gradient 20 min.  [111In(dedpa)]+ (tR = 5.9 min), [111In(octapa)]- (tR = 4.7 min), [111In(decapa)]2- [tR = 5.4 min (5%), 7.7 min (95%)], [111In(DTPA)]2- (tR = 6.5 min), and [111In(DOTA)]- (tR = 3.5 min) were all formed in >99% radiochemical yields.  Overlayed HPLC radiotraces are shown in Figure A.1.  2.4.19 Solution thermodynamics The experimental procedures and details of the apparatus closely followed those of a previous study for H2dedpa with Ga3+.118  As a result of the strength of the binding of the In3+ complex [111In(octapa)]-, the complex formation constant with this ligand could not be determined directly and the ligand-ligand competition method using the known competitor Na2H2EDTA was used.  For H5decapa, the ligand-ligand competition method was not required owing to the presence of a stable MHL species, and instead it was titrated directly     90 with In3+.  Potentiometric titrations were performed by Dr. Jacqueline Cawthray using a Metrohm Titrando 809 equipped with a Ross combination pH electrode and a Metrohm Dosino 800.  Data were collected in triplicate using PC Control (Version 6.0.91, Metrohm).  The titration apparatus consisted of a water-jacketed glass vessel maintained at 25.0 (± 0.1 °C, Julabo water bath).  Prior to and during the course of the titration, a blanket of nitrogen, passed through 10% NaOH to exclude any CO2, was maintained over the sample solution.  Indium ion solutions were prepared by dilution of the appropriate atomic absorption standard (AAS) solution.  The exact amount of acid present in the indium standard was determined by titration of an equimolar solution of In3+ and Na2H2EDTA.  The amount of acid present was determined by Gran’s method.324  Calibration of the electrode was performed prior to each measurement by titrating a known amount of HCl with 0.1 M NaOH.  Calibration data were analyzed by standard computer treatment provided within the program MacCalib325 to obtain the calibration parameters E0.  Equilibration times for titrations were 10 minutes pKa titrations and 15 minutes for metal complex titrations.  Ligand and metal concentrations were in the range of 0.75-1.0 mM for potentiometric titrations.  The data were treated by the program Hyperquad2008.326  The four successive proton dissociation constants corresponding to hydrolysis of In3+aq ion and the indium-chloride stability constants included in the calculations were taken from Baes and Mesmer.163  All values and errors represent the average of at least three independent experiments.  2.4.20 Molecular modeling Calculations were performed by Dr. Jacqueline Cawthray using the Gaussian 09327 and GaussView packages.  Molecular geometries and electron densities were obtained from     91 density functional theory calculations, with the B3LYP functional employing the 6-31+G(d,p) basis set for 1st and 2nd row elements and the ECP basis set, LANL2DZ, was employed for indium.328,329  Solvent (water) effects were described through a continuum approach by means of the IEF PCM as implemented in G09.  The electrostatic potential was mapped onto the calculated electron density surface.  The corresponding harmonic vibration frequencies were computed at the same level to characterize the geometry as a minima.  2.4.21 Mouse serum stability data  The compounds [111In(octapa)]-, [111In(decapa)]2-, [111In(DOTA)]-, [111In(DTPA)]2-, and [111In(dedpa)]+ were prepared with the radiolabeling protocol as described above.  Mouse serum was removed from the freezer and allowed to thaw at ambient temperature for 30 minutes.  In triplicate for each 111In complex listed above, solutions were made in sterile vials with 750 µL mouse serum, 500 µL of 111In-complex (10 mM NaOAc buffer, pH 5.5), 250 µL phosphate buffered saline (PBS), and were left to sit at ambient temperature.  After 1 hour, half of the mouse serum competition mixture (750 µL) was removed from each vial, diluted to a total volume of 2.5 mL with phosphate buffered saline, and then counted in a Capintec CRC 15R well counter to obtain a value for the total activity to be loaded on the PD-10 column.  The 2.5 mL of diluted mouse serum competition mixture was then loaded onto a PD-10 column that had previously been conditioned via elution with 20 mL of PBS.  The 2.5 mL of loading volume was allowed to elute into a 111In waste container, and then the PD-10 column was eluted with 3.5 mL PBS and collected into another sterile vial.  The eluent which contained 111In bound/associated with serum-proteins (size-exclusion for MW < 5000 Da) was counted in a well counter, and then compared to the total amount of activity that was     92 loaded on the PD-10 column to obtain the percentage of 111In that was bound to serum proteins and therefore no longer chelate-bound.  The percent stability values shown in Table 2.2 represent the percentage of 111In that was retained on the PD-10 column and therefore still chelate-bound.  2.4.22 Biodistribution data The protocol used in these animal studies was approved by the Institutional Animal Care Committee (IACC) of the University of British Columbia (protocol # A10-0171) and was performed by the BC Cancer Agency in accordance with the Canadian Council on Animal Care Guidelines.  A total of 16 female ICR mice (20-25 g, 6-8 weeks) were used for the biodistribution study of three radiometal complexes.  [111In(octapa)]-, [111In(DOTA)]-, and [111In(decapa)]2- were prepared as described above, and then diluted in phosphate-buffered saline to a concentration of 100 µCi/mL.  Each mouse was intravenously injected through the tail vein with ∼10 µCi (100 µL) of the 111In complex and then sacrificed by CO2 inhalation 15 min, 1 h, 4 h, or 24 h after injection (n = 4 at each time point).  Blood (500-700 µL) was collected by cardiac puncture with a 25 G needle and placed into an appropriate microtainer tube for scintillation counting.  Urine was collected from the bladder after sacrifice and counted.  Tissues collected included spleen, kidney, intestine, liver, heart, lung, brain, muscle, and femur.  Tissues were weighed and counted with a Capintec CRC 15R well counter, and the counts were decay corrected from the first 15 minute time point and then converted to the percentage of injected dose (%ID) per gram of organ tissue (%ID/g).       93 Chapter 3: H4octapa-trastuzumab: the application of a versatile acyclic ligand system for 111In and 177Lu imaging and therapy  This chapter is an adaptation of published work, and is reproduced in part, with permission from Price, E. W.; Zeglis, B. M.; Cawthray, J. F.; Ramogida, C. F.; Ramos, N.; Lewis, J. S.; Adam, M. J.; Orvig, C., H4octapa-Trastuzumab: Versatile Acyclic Chelate System for 111In and 177Lu Imaging and Therapy. J. Am. Chem. Soc. 2013, 135 (34), 12707–12721, Copyright 2014 American Chemical Society.   3.1 Introduction Radiometallated bioconjugates possess an inherent modularity that can prove extremely useful in the design and construction of agents for nuclear imaging and therapy.  Indeed, they contain a set of four distinct chemical components that can be swapped systematically to tune the properties and functions of the whole: the radiometal can be exchanged to harness isotopes with different decay characteristics; the ligand can be altered to accommodate different radiometals; the nature of the covalent linkage between the ligand and the biomolecule can be changed to take advantage of different types of conjugation chemistry; and, of course, the biomolecule itself can be replaced in order to change the targeting properties or pharmacokinetics of the construct.2,6,7,25,28,36,56  In clinical practice, the use of different isotopes with a single targeting vector becomes especially important in pre-therapy imaging and dosimetry studies, procedures in which an agent bearing an imaging radioisotope is used for scouting scans prior to radiotherapy using the same vector labeled     94 with a therapeutic nuclide.  Two isotopes that are ideally suited for this purpose are 111In, a cyclotron produced radiometal (111Cd(p,n)111In) for SPECT imaging (t1/2 ~2.8 days), and 177Lu, a reactor produced therapeutic radiometal (176Lu(n, γ)177Lu) that emits β- particles as well as	  γ-rays (t1/2 ~6.6 days).15      Figure 3.1 Structures of some common bifunctional ligands used for radiometal chemistry, and H4octapa, the non-bifunctional variant of the ligand p-SCN-Bn-H4octapa used in this work.    As discussed in Chapter 1, antibodies have emerged as extremely promising biomolecules for the delivery of radioactive payloads to cancerous tissues.285,45  However, antibodies are not without their limitations as radiopharmaceutical vectors.  For example, while they are generally considered robust in vivo, antibodies can become damaged or break p-SCN-Bn-H4octapa (3.12)p-SCN-Bn-DOTA N N NSCNHOO OHOOOHO OHOHON NN NOHOO OHH4octapa (3.5) OOHHOO p-SCN-Bn-CHX-A''-DTPAN NN NOHOO OH OOHHOO NCSN N NSCNHOO OHOOOHO OHOHOp-SCN-Bn-DTPANN NNOHO OOHOHO OOHSCN    95 down when subjected to the elevated temperatures required by many radiometal chelation reactions.330  Because macrocycles such as DOTA (Chapter 1 and 2) must be used with elevated temperatures (40-90 °C) and extended reaction times (30-180 min) to achieve quantitative radiolabeling, conditions that can be incompatible with antibodies and other biomolecules, to search for suitable acyclic ligands is important (Figure 3.1).58-65,94,97,119,127-129,142  Acyclic ligands offer improvements in this regard, but not without cost.  The acyclic ligand diethylenetriaminepentaacetic acid (DTPA), for example, exhibits much faster reaction kinetics than DOTA and can radiolabel with a variety of radiometals quantitatively in a matter of minutes at ambient temperature; however, it is not nearly as stable in vivo as its macrocyclic counterparts.60,61,215,216,312 Thus, choosing a radiometal ligand for an antibody conjugate requires a delicate balancing act.  An immunoconjugate bearing a macrocyclic ligand such as DOTA can require extended incubations at elevated temperatures, often at the expense of antibody stability or immunoreactivity, in order to achieve suitable radiochemical yields; however, a radioimmunoconjugate bearing an acyclic ligand such as DTPA risks the unintentional release of the radiometal in vivo, an especially pressing problem considering the long pharmacokinetic half-life of many antibodies.8,10,19-22,26-28  It thus becomes clear that a versatile ligand that combines the thermodynamic stability and kinetic inertness of macrocyclic ligands with the facile radiolabeling of acyclic ligand could prove tremendously valuable in the synthesis and development of novel radioimmunoconjugates.   Our efforts towards these goals have led us to the discovery of H4octapa (Chapter 2).  In this chapter is reported the synthesis, characterization, and in vivo validation of p-SCN-Bn-H4octapa, a bifunctional, versatile, acyclic ligand that can be radiolabeled rapidly at room     96 temperature and exhibits significant thermodynamic stability and kinetic inertness with 111In and 177Lu (Figure 3.1).  Members of the versatile “pa family” of ligands ⎯ including H4octapa, H2dedpa, H5decapa, and H2azapa (Chapter 8) ⎯ cover nearly all of the current medicinally relevant radiometals.88,89,118,266,271-274,331  H4octapa specifically has demonstrated high in vitro and in vivo stability and rapid radiolabeling kinetics with 111In.  Further, comparisons with 111In-DOTA have revealed that H4octapa possesses much more facile radiolabeling kinetics, can be labeled in higher specific activity, undergoes less fluxional isomerization in solution, and exhibits comparable stability in vitro and in vivo.89  A major hurdle during this work, however, has been the cumbersome syntheses of the ligands.88,89,118,272-274,331  As a result, in this chapter we report the synthesis of the bifunctional chelate p-SCN-Bn-H4octapa via a vastly improved synthetic protocol.  More importantly, we have employed the HER2/neu-targeted antibody trastuzumab as a model system for the in vitro and in vivo validation of 111In-octapa- and 177Lu-octapa-based radioimmunoconjugates in a murine model of ovarian cancer, as well as the comparative evaluation of these radioimmunoconjugates to those employing the more traditional macrocyclic ligand DOTA.  3.2 Results and discussion 3.2.1 Synthesis and characterization  In order to properly evaluate the potential of H4octapa for use in biomolecular radiopharmaceuticals, we first sought to develop a highly efficient synthesis of a novel bifunctional variant, p-SCN-Bn-H4octapa (Scheme 3.1), which had never been reported before.  As for other bifunctional chelators (BFC), we have utilized the versatile and enantiopure starting material L-4-nitrophenylalanine.118,215,89  While the N-benzyl-based     97 protecting group chemistry used in Chapter 2 for the synthesis of non-bifunctional H4octapa proved adequate for the construction of the bare ligand, the requisite vigorous hydrogenation conditions resulted in poor yields and were completely incompatible with the synthesis of bifunctional derivatives based on 4-nitrophenylalanine.118,89  Consequently, we revamped the synthesis to utilize the 2-nitrobenzenesulfonamide (nosyl) protecting group, a change that has significantly improved the synthesis of H4octapa (3.5) and has made the synthesis of p-SCN-Bn-H4octapa (3.12) feasible.88,89,118,332-334   Specifically, the switch to the nosyl protecting group has dramatically increased the cumulative yield of H4octapa (3.5) from ~10-12% to ~45-50% over five synthetic steps.89  Further, using this strategy, the bifunctional variant p-SCN-Bn-H4octapa, which was previously inaccessible using N-benzyl protection chemistry (Chapter 2), can now be routinely produced with cumulative yields in 7 steps of ~25-30 %. The 2-nitrobenzenesulfonamide (nosyl) amine-protecting group has been crucial for the synthesis of these chelating ligands.332-336  Compared to the challenging syntheses of bifunctional macrocycles such as p-SCN-Bn-DOTA, this streamlined route to p-SCN-Bn-H4octapa should be of significant utility.  The use of nosyl protecting groups results in a reduced number of purification steps (compounds 3.1, 3.2, and 3.8 can be purified via crystallization), greater yields, and, most relevantly, the accessibility of p-SCN-Bn-H4octapa.  The final 3 steps in the synthesis of p-SCN-Bn-H4octapa are performed sequentially without purification due to the high polarity and difficult separation of the intermediates, and the final molecule is purified via reverse-phase HPLC chromatography.        98  Scheme 3.1 Improved synthesis of picolinic acid-based ligands H4octapa (3.5) and p-SCN-Bn-H4octapa (3.12).a   a (i) THF, NaHCO3, RT, 24 h (ii) DMF, Na2CO3, methyl-6-bromomethylpicolinate (2.2 equiv), 50 °C, 24 h (iii) THF, thiophenol (2.2 equiv), K2CO3, RT, 72 h; (iv) MeCN, Na2CO3, 50 °C, 24 h; (v) 6 M HCl, reflux, 24 h. b) (i) THF, NaHCO3, 50 °C, 24 h; (ii) DMF, Na2CO3, 50 °C, 24 h; (iii) THF, thiophenol (2.2 equiv), K2CO3, RT, 72 h; (iv) CH3CN, Na2CO3, 50 °C, 24 h; (v) 5 mL of (1:1) glacial acetic acid:3 M HCl, Pd/C (20 wt%), H2 (g), RT, 1 h; (vi) 6 M HCl, reflux, 24 h; (vii) thiophosgene in DCM (15 equiv), 3 M HCl, RT, 24 h.      Following synthesis, the HCl salt of pure H4octapa was successfully metallated via reaction with indium perchlorate89 [In(ClO4)3] and lutetium chloride [LuCl3] to form quantitatively coordinated complexes.  Further, all of the new ligands and their metal complexes were characterized using 1H NMR, 13C NMR, HR-ESI-MS, and ATR-IR where H2N NH2H4octapa (3.5)N NN N OHOO OH OOHHOONH HNO2S SO2O2N NO2N NN N OOO O OOOO(i) (ii)(iii)(iv) (v)3.13.23.33.4 OOBrSO2ClO2N Br N OO+NH HNN N OOO O N NN N OOO O O2SO2SNO2 O2N H2N NH2NO2p-SCN-Bn-H4octapa (3.12)N NN N OHOO OH OOHHOO NCSNH HNNO2O2S SO2O2N NO2N NNO2O2S SO2O2N NO2OO OON NN NOOO O OOOO NO2NH NHNO2OO OO(i) (ii)(iii)(iv) (v)(vi)(vii)3.7 3.83.93.103.11SO2Cl NO2+a b87% 74%95% 88%89%91%92% 66% 96% 50%(3 steps)Br N OO OOBr2.2 equiv. 2.2 equiv.2.2 equiv. 2.2 equiv.2.2 equiv.2.2 equiv.    99 appropriate.  Particularly interesting, however, were the 1H NMR spectra of the metallated H4octapa ligand.  Variable temperature NMR experiments with In3+ complexes of DOTA have demonstrated fluxional isomerization in solution, despite the macrocyclic nature of the ligand.89,97,337  In this case, however, the 1H NMR spectra obtained for [In(octapa)]- and [Lu(octapa)]- (3.6) reveal sharp and distinct coupling patterns, indicating little fluxional isomerization in solution at ambient temperature (Figure A.3).89   3.2.2 Thermodynamic stability and density functional theory structure prediction  The thermodynamic stability constants (log KML) for DOTA with Lu3+ have previously been determined by spectrophotometric methods (UV) to be 23.5 and ~25,125,338 and by a competitive potentiometric titration with oxalate to be 29.2 ± 0.2.339  In these studies the only species for which log KML has been documented is for the ML species (no metal hydroxide), and titrations were not done in the presence of NaCl, which is known to lower formation constants values (our titrations are done in 0.15 M NaCl for physiologically relevant conditions).340  These factors, combined with the inconsistencies in the reported log KML values and the methods used, prompted us to perform competitive potentiometric titration experiments of DOTA with EDTA and Lu3+ in order to provide a formation constant that was determined under identical experimental conditions to our own system.  Using potentiometric titrations, we have experimentally determined the thermodynamic stability constant (log KML) for Lu3+ with H4octapa to be 20.08 ±	  0.09	  (pM	  =	  19.8) and with DOTA to be 21.62 ±	  0.10	  (pM	  =	  17.1).  In addition, the log KML value for Lu3+ with DTPA has been reported in the literature to be 22.4, and we have calculated a pM value of 19.1.119  Although the presence of sodium in our experiments can be expected to lower the stability constant of     100 DOTA with Lu3+ from previously reported values, and the different experimental methods used are expected to vary in their results, the low log KML value we have obtained could be inaccurate as a result of the slow complex formation kinetics of DOTA.  During the potentiometric titrations, up to 15 minutes was allowed for stabilization of the electrode reading between each addition of base, which may have been too brief for a proper equilibrium to be established with DOTA.  Previous work has waited over 2 weeks for equilibrium to be reached in this system, which highlights the sluggish formation kinetics.125  Because of this uncertainty, our experimentally determined log KML value for DOTA with Lu3+ cannot be relied on solely or be considered to be more accurate than previously determined values, and therefore all reported formation constants must be considered.  The thermodynamic stability constants of the In3+ complexes of H4octapa, DOTA, and DTPA have been determined to be 26.6 (pM = 26.5), 23.9 (pM = 18.8), and 29.0 (pM = 25.7), respectively.89  It is important to note that thermodynamic stability is generally regarded as an unreliable predictor of in vivo stability on its own,94 and the kinetic inertness of a radiometal-complex is a much more valuable factor.  For example, the [111In(DOTA)]- complex is widely established as being significantly more stable than [111In(DTPA)]2- both in vivo and in vitro, yet the thermodynamic stability constant for In3+ with DTPA is ~5 orders of magnitude higher than with DOTA (29.0 and 23.9, respectively).  In fact, pM values, which are calculated from log KML values while taking into account ligand pKa and metal ion hydroxide formation under specific and physiologically relevant conditions (here pH 7.4, [Mn+] = 1 µM, [Lx-] = 10 µM), are generally considered more useful in predicting in vivo stability than log KML values.  H4octapa has been shown to have high pM values with both     101 In3+ (26.5) and Lu3+ (19.8), further substantiating its promising properties with these metals and their radioactive isotopologues.    Figure 3.2 In silico DFT structure predictions. a, 8-coordinate structure of [Lu(octapa)]-; b, 9-coordinate structure of [Lu(octapa)(H2O)]-, as well as the MEP polar-surface area maps (bottom) predicting the charge distribution over the solvent-exposed surface of the metal complexes (red = negative, blue = positive, representing a maximum potential of 0.254 au and a minimum of -0.254 au, mapped onto electron density isosurfaces of 0.002 Å-3).  DFT calculations performed by Dr. Jacqueline Cawthray.   The coordination geometries of both the 8- and 9-coordinate [Lu(octapa)]- and [Lu(octapa)(H2O)]- complexes were estimated in silico using density functional theory (DFT) calculations, and MEP polar surface area maps were superimposed onto the structures (Figure 3.2).  Both Lu3+ complexes were found to be highly symmetrical and very similar to     102 one another; indeed, that the addition of an H2O ligand results in very little change in metal-ligand bond lengths and angles suggests that water binding bears little influence on the coordination of the ligand.  Importantly, the [Lu(octapa)]- and [Lu(octapa)(H2O)]- DFT structures shown here are quite similar to the DFT structure of the 8-coordinate [In(octapa)]- complex.89  Taken together, the high log KML and pM values, the absence of fluxional behavior in solution, and the structural similarity between the [Lu(octapa)]- and [In(octapa)]- complexes strongly suggest that these two complexes should exhibit similar behavior in vitro and in vivo.  This is especially important when considering 111In as a SPECT imaging surrogate isotope for 177Lu-based radiotherapies, where seamless interchangeability is imperative.  3.2.3 Bioconjugation and in vitro characterization  Synthesis and characterization data aside, the true value of any radiometal ligand ultimately lies in its behavior in vitro and in vivo.  As a result, the next step in our investigation was to assess the in vitro and in vivo performance of H4octapa in a model system based on the HER2/neu-targeting antibody trastuzumab and HER2-expressing SKOV-3 ovarian cancer cells.  In order to provide a basis for comparison, radioimmunoconjugates bearing the ubiquitous ligand DOTA were also constructed and employed in parallel in all in vitro and in vivo experiments.  As mentioned in Chapter 2, the antibody will act as a physiological anchor, imparting a long biological half-life to the conjugated radiometal-ligand complex, and allowing for extensive stability evaluation that was not possible with “bare” H4octapa.   To begin, purified trastuzumab was incubated under slightly basic conditions (pH 8.5-9.0) with 4 equivalents of p-SCN-Bn-H4octapa or p-SCN-Bn-DOTA and purified via size     103 exclusion chromatography (PD-10, GE Healthcare, UK).  Subsequent radiometric isotopic dilution experiments indicated that these modifications yielded 3.03 ± 0.1 chelates per antibody in the case of p-SCN-Bn-H4octapa and 3.40 ± 0.1 chelates per antibody in the case of p-SCN-Bn-DOTA.  H4octapa-trastuzumab was then radiolabeled with either 111In or 177Lu in NH4OAc buffer (pH 5.5, 200 mM) for 15 min at room temperature, rapidly producing quantitatively labeled radioimmunoconjugates (>95% radiochemical yield) in high radiochemical purity (>99% in each case) and specific activity (4.0 ± 0.3 and 3.5 ± 0.4 mCi/mg, respectively) (Table 3.1).  It is important to note that these labile kinetics offer a significant improvement over DOTA-trastuzumab, which required the less favorable reaction conditions of 1 h at 37 °C to achieve highly variable radiochemical yields of ~50-88% and to produce 111In- and 177Lu-labeled radioimmunoconjugates with slightly lower specific activities of 2.0 ± 0.2 and 3.4 ± 0.3 mCi/mg, respectively (Table 3.1).  These results are of practical significance because antibodies and radiometals are very expensive, and so the inconsistent yields provided when radiolabeling DOTA-trastuzumab could waste as much as 50% of the antibody construct and radiometal, greatly increasing cost and making translation to a clinical radiopharmacy setting more challenging where routine and reliable production is require.  The mild radiolabeling conditions afforded by H4octapa-trastuzumab likely contributed to the extremely high immunoreactivity of the immunoconjugates as determined by in vitro cellular assays using SKOV-3 cancer cells: 99.9 ± 0.02% for 111In-octapa-trastuzumab and 98.7 ± 0.8% for 177Lu-octapa-trastuzumab.  These values compared favorably to the slightly lower but statistically different values determined for 111In- and 177Lu-DOTA-trastuzumab: 93.2 ± 0.5 % and 95.2 ± 0.2 %, respectively (p-values of 0.003     104 and 0.02, respectively) (Table 3.1).  In order to assay the stability of the radioimmunoconjugates under biological conditions, all four constructs were incubated in human serum at 37 °C for a period of 5 days.  Over the course of this experiment, the stability of the 111In-octapa- and 111In-DOTA-trastuzumab were both determined to be excellent at 94.9 ± 0.6% and 91.1 ± 0.6%, respectively, and that of the 177Lu-octapa- and 177Lu-DOTA-based conjugates was found to be 92.4 ± 0.6% and 98.6 ± 0.6%, respectively (Table 3.1).  In the future, acid dissociation experiments, and studies probing the ability of H4octapa to radiolabel with 111In and 177Lu in the presence of an excess of other metal ions such as Ca2+, Mg2+, Cu2+, Zn2+, and Fe3+ are of interest.82,85   Table 3.1 Chemical and in vitro biological characterization data for 111In- and 177Lu-octapa-trastuzumab and 111In- and 177Lu-DOTA-trastuzumab radioimmunoconjugates.   a Isotopic dilution assays, n = 3. b Determined immediately prior to in vivo experimentation, n = 6. c Calculated for incubation in human serum at 37 °C for 120 h, n = 3.   3.2.4 Acute biodistribution studies  Biodistribution experiments were performed in order to directly compare the in vivo behavior and pharmacokinetics of the 111In- and 177Lu-based trastuzumab radioimmunoconjugates.  To this end, each of the four radiolabeled antibodies ⎯ 111In-octapa-trastuzumab, 177Lu-octapa-trastuzumab, 111In-DOTA-trastuzumab, and 177Lu-DOTA-trastuzumab ⎯ were injected via tail vein into female nude athymic mice bearing Immunoconjugate Isotope Radiolabeling3conditions3and3yield Chelates3/3mAba Specific3activity3(mCi/mg) Immunoreactive3fraction3(%)b Serum3stability31203h3(%)cH4octapa(trastuzumab 111In 153min,3253°C,394% 3.03±30.1 4.03±30.3 99.93±30.02 94.93±30.6177Lu 153min,3253°C,395% 3.03±30.1 3.53±30.4 99.23±30.8 92.43±30.6DOTA(trastuzumab 111In 603min,3373°C,350(85% 3.43±30.1 2.03±30.2 93.23±30.5 91.13±30.6177Lu 603min,3373°C,350(88% 3.43±30.1 3.43±30.3 95.23±30.2 98.63±30.6    105 subcutaneous SKOV-3 ovarian cancer xenografts in the right shoulder (~30 µCi, ~8-15 µg, in 200 µL of sterile saline; tumour size ~ 2-3 mm diameter).  After 24, 48, 72, 96, or 120 h (n = 4 per time point) the mice were euthanized via CO2 (g) asphyxiation, and 13 organs, including the SKOV-3 tumours, were removed, weighed, and assayed for radioactivity content on a γ-counter.    Table 3.2 Biodistribution data of 111In/177Lu-octapa-trastuzumab and 111In/177Lu-DOTA-trastuzumab, performed over a 5 day period in mice bearing SKOV-3 ovarian cancer xenografts, tumour size ~2-3 mm diameter (n = 4 for each time point), showing %ID/g values.      In all four cases, the biodistribution results showed all the hallmarks of tumour-targeted radioimmunoconjugates, specifically high levels of uptake in the blood pool at early 111In$octapa$trastuzumab 111In$DOTA$trastuzumabOrgan 247h 4877h 727h 967h 1207h Organ 247h 4877h 727h 967h 1207hBlood 6.9(±(5.1 7.6(±(6.5 10.5(±(7.1 6.9(±(6.6 11.4(±(6 Blood 18(±(1.7 17.3(±(2.6 16.8(±(2.2 15(±(3.6 14.3(±(1.6Tumor 57.4(±(22 58.3(±(17.2 57.8(±(14.6 68.7(±(20.5 72.4(±(21.3 Tumor 22.3(±(6.4 28.4(±(5.2 30.6(±(9.1 32.8(±(7.3 31.5(±(3.8Heart 2.6(±(0.5 2.5(±(1.5 2.7(±(1.4 1.9(±(1.4 3.6(±(1.6 Heart 6.1(±(0.9 6.6(±(2 5.6(±(1.3 5.2(±(1.8 5.2(±(0.9Lungs 4.4(±(2.1 3.9(±(2.3 5(±(2.8 3.8(±(3.3 6.3(±(3 Lungs 7.2(±(1.6 6.8(±(3.3 5.2(±(1.4 6.3(±(1.2 5.7(±(2.2Liver 12(±(2.4 10.6(±(1.2 9.1(±(0.8 8.5(±(2 6.6(±(0.6 Liver 6.2(±(0.4 5.5(±(1.4 4.8(±(1.3 4.4(±(1.7 5.2(±(1.2Spleen 8.9(±(2.4 7.6(±(1 8.1(±(0.8 7.4(±(1.1 6.5(±(1.6 Spleen 3.6(±(0.8 3.7(±(1.6 3(±(0.7 2.7(±(0.7 3.4(±(1.4Stomach 1.4(±(0.2 0.8(±(0.2 1.2(±(0.1 0.7(±(0.3 0.7(±(0.2 Stomach 0.7(±(0.1 0.7(±(0.4 0.8(±(0.2 0.3(±(0.2 0.6(±(0.3Lg(Intestine 3.7(±(0.4 2.5(±(0.7 2(±(0.9 1.7(±(0.4 1.2(±(0.1 Lg(Intestine 0.5(±(0.1 0.6(±(0.1 0.7(±(0.1 0.5(±(0.1 0.5(±(0.1Sm(Intestine 3.8(±(0.7 2.1(±(0.4 2.5(±(0.6 2.1(±(0.4 1.5(±(0.1 Sm(Intestine 1.9(±(0.1 1.6(±(0.5 1.7(±(0.1 1.1(±(0.2 1.3(±(0.1Kidney 3.6(±(1.2 4.2(±(2.1 4.3(±(2.1 3.5(±(2 4.3(±(1.6 Kidney 5.3(±(0.5 6.3(±(0.9 5.1(±(1.7 5.8(±(1.7 4.9(±(0.4Muscle 1.3(±(0.5 0.9(±(0.5 1(±(0.5 0.8(±(0.5 0.9(±(0.5 Muscle 0.7(±(0.2 0.8(±(0.3 0.5(±(0.1 0.4(±(0.1 0.3(±(0.1Bone 4.1(±(2 3.5(±(0.4 2.8(±(1 3.3(±(0.5 1.4(±(0.3 Bone 1(±(0.3 0.9(±(0.1 1.1(±(0.4 0.9(±(0.4 0.6(±(0.2Skin 10.8(±(1.4 9.3(±(1.4 7.2(±(2.1 5.4(±(1.2 3(±(1.1 Skin 1.8(±(0.4 2.1(±(0.7 2.6(±(0.1 1.7(±(0.8 2.2(±(0.8177Lu$octapa$trastuzumab 177Lu$DOTA$trastuzumabOrgan 247h 4877h 727h 967h 1207h Organ 247h 4877h 727h 967h 1207hBlood 19.1(±(1.5 9.1(±(9.5 9.7(±(5.2 8.9(±(6.9 7.6(±(5.7 Blood 20.4(±(1.7 17.4(±(1 18.1(±(2.8 15(±(0.8 14.7(±(0.7Tumor 50(±(5.4 63.5(±(15 62.4(±(20 70.4(±(25.8 56.6(±(12.7 Tumor 18.3(±(4.8 31.6(±(2.9 32.1(±(7.4 37(±(9.5 37.4(±(10.3Heart 4.2(±(0.7 3.2(±(1.9 2.5(±(0.9 2.3(±(0.7 2.6(±(1.4 Heart 7.2(±(1 4.9(±(3.3 5.8(±(1.6 4.6(±(1.1 5.4(±(0.6Lung 7.8(±(3.6 3.9(±(3.1 5(±(2.3 4.4(±(2.5 2.7(±(1.8 Lungs 10(±(1.5 6.7(±(4.6 7.1(±(2.3 6.3(±(0.6 6(±(2.1Liver( 10.1(±(1.3 13.2(±(4 9.5(±(3.5 9.8(±(1.7 8(±(2.6 Liver 4.5(±(2 5.7(±(3.6 5.7(±(2.5 2.8(±(0.9 3.7(±(1.9Spleen( 8.8(±(2.3 11.5(±(2.5 7.4(±(2.8 9.2(±(1.2 6.7(±(2.4 Spleen 3.8(±(0.5 3.1(±(2.9 3.2(±(1 3.5(±(0.4 3.8(±(1Stomach 1.4(±(0.3 1(±(0.5 0.9(±(0.4 1.1(±(0.3 0.6(±(0.2 Stomach 0.7(±(0.2 1.3(±(1.9 0.9(±(0.2 0.8(±(0.2 0.7(±(0.2Lg(Intestine 1.3(±(0.3 1(±(0.3 1.1(±(0.5 1.1(±(0.2 0.8(±(0.2 Lg(Intestine 0.6(±(0.1 0.5(±(0.3 0.8(±(0.2 0.5(±(0.1 0.6(±(0.2Sm(Intestine 1.9(±(0.5 2.5(±(1.1 2(±(0.9 2.3(±(0.4 1.6(±(0.4 Sm(Intestine 1.7(±(0.2 1.5(±(1 1.5(±(0.4 1.4(±(0.3 1.2(±(0.1Kidney 4.9(±(0.9 3.8(±(2 3.7(±(1.2 3.8(±(1.8 2.8(±(1.2 Kidney 5.5(±(1.7 7.3(±(0.7 5.1(±(0.8 4.4(±(0.2 7.1(±(5Muscle 0.9(±(0.2 0.7(±(0.5 0.8(±(0.5 0.5(±(0.2 0.4(±(0.2 Muscle 0.7(±(0.2 0.5(±(0.4 0.4(±(0.1 0.5(±(0.2 0.5(±(0.1Bone 3.2(±(0.5 4(±(0.6 3.1(±(0.7 4.2(±(1.5 3.6(±(1.7 Bone 1.2(±(0 1.3(±(0.8 1.3(±(0.3 0.9(±(0.2 1.4(±(0.3Skin 8.4(±(2 6.8(±(2.9 6.9(±(0.5 5.6(±(1.2 4.1(±(2.2 Skin 1.9(±(0.9 2.4(±(1.7 2.1(±(0.2 2.1(±(0.7 3.1(±(1.3    106 time points giving way over time to increasing levels of uptake in the tumour and, ultimately, very high tumour-to-background organ activity ratios.  In all four cases, the level of uptake in non-target organs is roughly similar, with the highest levels of background uptake in the liver, spleen, and kidneys (Tables 3.2).  However, past 24 h, the amount of activity in non-target organs rarely exceeds 5-10 %ID/g; for example, at 96 h post-injection, 177Lu-octapa-trastuzumab displays uptakes of 9.8 ± 1.7 %ID/g, 9.2 ± 1.1 %ID/g, and 3.8 ± 1.8 %ID/g in the liver, spleen, and kidneys, respectively.  A close inspection of Table 3.2 reveals slightly higher uptake of 111In/177Lu-octapa-trastuzumab in the bone when compared to the DOTA-trastuzumab conjugates, but also slightly lower uptake of 111In/177Lu-octapa-trastuzumab in the kidneys.  Far more interesting are the levels of tumour uptake, which are markedly higher with the 111In-octapa- and 177Lu-octapa-based variants compared to their 111In-DOTA- and 177Lu-DOTA-based analogs.  For example, at 96 h post-injection the tumour uptake for 111In-octapa-trastuzumab and 177Lu-octapa-trastuzumab was found to be 68.7 ± 20.5 %ID/g and 70.4 ± 25.8 %ID/g, respectively, compared to 32.8 ± 7.3 %ID/g and 37.0 ± 9.5 %ID/g for the 111In-DOTA-trastuzumab and 177Lu-DOTA-trastuzumab constructs.  Notably, despite the large range in errors, the p-values for these differences are 0.04 for the 111In data sets and 0.05 for 177Lu data sets, indicating statistically significant differences (two-tailed students t-test).  Statistical analysis of tumour uptake by one-way ANOVA provided p-values of 0.02 for the 111In data sets and 0.05 for 177Lu data sets, also indicating statistical significance.  In imaging, tumour-to-tissue activity ratios are arguably even more important measures than absolute uptake values, as these ratios provide quantitative information regarding the contrast that will be observed during imaging.  As a result of the excellent tumour uptake of the     107 H4octapa-based radioimmunoconjugates, the tumour-to-background activity ratios were higher than the corresponding DOTA-based agents for almost all organs.  For example, the tumour-to-blood ratios for 111In-octapa- and 111In-DOTA-trastuzumab at 120 h were found to be 6.4 ± 3.8 and 2.2 ± 0.4, respectively, while for 177Lu-octapa- and 177Lu-DOTA-trastuzumab they were 7.5 ± 5.8 and 2.5 ± 0.7, respectively.  The origin of the elevated tumour uptake values observed for the H4octapa-trastuzumab agents could lie in the enhanced immunoreactivity conferred by the fast and mild radiolabeling conditions.  However, the difference in immunoreactivity was only about 5%, a discrepancy that would not be expected to cause such a change in vivo.  Alternatively, it could be postulated that while the antigen-binding region of the antibody was only slightly adversely affected by temperature in the DOTA-trastuzumab conjugate, a separate region of the immunoglobulin may have suffered bond cleavage or protein structure disruption during radiolabeling, which in turn affected tumour uptake and retention.  Finally, the differences in pharmacokinetic properties from attaching three to four relatively small chelating moieties (H4octapa vs DOTA) to a 150 kD antibody is almost certainly insignificant, but it is not inconceivable that this modification could somehow affect the distribution of the radioimmunoconjugates in vivo.  Because the difference in uptake between the H4octapa- and DOTA-trastuzumab radioimmunoconjugates was not expected and is not easily explained, further experiments in a different cell line (e.g. SkBr3) should be performed in the future.  Ultimately, regardless of the causation, the superior tumour uptake and tumour-to-tissue activity ratios plainly show that 111In-octapa-trastuzumab and 177Lu-octapa-trastuzumab are highly effective and versatile radioimmunoconjugates and, more generally, that H4octapa is a very promising ligand for the construction of biomolecular radiopharmaceuticals.     108 3.2.5 Small animal SPECT/CT imaging and Cerenkov luminescence imaging  Single photon emission computed tomography (SPECT) was used in conjunction with standard helical X-ray CT for in vivo imaging of all 111In and 177Lu labeled immunoconjugates over 5 days, visually demonstrating high uptake and clear delineation of the SKOV-3 ovarian cancer xenografts by 111In- and 177Lu-octapa-trastuzumab as well as 111In- and 177Lu-DOTA-trastuzumab (Figure 3.3).  Images taken at early time points show residual activity in the blood pool at 24 h, as expected; however, at later time points, the background activity in the blood clears and is accompanied by a concomitant increase in the amount of activity in the tumour, culminating at a point at which the tumour is by far the most prominent feature in the image (Figure 3.3).  Overall, the image quality, tumour uptake, and tumour contrast observed are very similar between the H4octapa- and DOTA-based constructs.  In all four cases, high levels of tumour uptake and excellent tumour-to-background contrast are observed at later time points, data which correlate well with the high tumour uptake and tumour-to-tissue ratios revealed in the biodistribution experiments (Tables 3.2).  It is important to note that while the 111In- and 177Lu-octapa-trastuzumab images do not appear qualitatively superior to those produced by the 111In-DOTA- and 177Lu-DOTA variants, the enhanced tumour-to-background activity ratios of the 111In- and 177Lu-octapa-based constructs could potentially be of value in identifying small lesions or metastases in which absolute uptake of radioactivity is limited and for which contrast is crucial.  The use of Cerenkov radiation (CR) for Cerenkov luminescence imaging (CLI) has been emerging as a promising new imaging modality, with applications such as intraoperative radionuclide-guided surgery for the resection of cancers.341  In order to probe the potential of our 177Lu-labeled immunoconjugate, we have successfully imaged 177Lu-octapa-trastuzumab via     109 external optical imaging through the skin 24 h post injection via CLI (Figure 3.4).  To our knowledge, this was the first published example of in vivo CLI imaging of 177Lu (Figure 3.4).87  The bright spots on the head of the mouse in Figure 3.4 are from fluorescence molecules present in the animal’s food.   Figure 3.3 SPECT/CT imaging of the 111In/177Lu-octapa-trastuzumab and 111In/177Lu-DOTA-trastuzumab immunoconjugates, in female nude athymic mice with subcutaneous SKOV-3 xenografts (identified by arrow at right shoulder, tumour size ~2-3 mm diameter), showing transverse (top) and coronal (bottom) planar images bisecting the tumour, imaged at 24, 48, 72, 96, and 120 h post injection.     110  Figure 3.4 Cerenkov luminescence image (CLI) of 177Lu-octapa-trastuzumab, obtained 24 h post-injection of a female nude athymic mouse bearing an SKOV-3 ovarian cancer xenograft on the right shoulder, indicated by white arrow (2 minute acquisition time).     3.3 Conclusions Ultimately, the major benefit of the acyclic H4octapa ligand system over macrocyclic ligands such as DOTA lies in the ability to radiolabel with rapid kinetics at room temperature, a trait which has the potential to shorten radiolabeling times (< 15 min), streamline radiopharmaceutical production, and aid in the retention of antibody integrity and immunoreactivity during radiolabeling.  Trastuzumab is a robust antibody and can generally withstand the radiolabeling conditions required by DOTA.  Although H4octapa provides significant and tangible benefits over DOTA as outlined in this chapter, its use is not strictly required; however, when working with antibodies that are more sensitive than trastuzumab and are less forgiving of elevated temperatures and extended reaction times, a ligand with     111 more facile reaction kinetics such as p-SCN-Bn-H4octapa may become crucial.  The high and reproducible radiochemical yield that H4octapa provides (>95%) is additionally important because antibodies are the most expensive component of these radiolabeled immunoconjugate systems.  The inconsistent yields obtained with DOTA (50-90%) can waste as much as 50% of these expensive biomolecules and radiometals, and could make it difficult to reliably produce specific doses in a clinical radiopharmacy setting.  Yet the advantages provided by H4octapa do not end with facile radiolabeling protocols.  Potentiometric titrations, spectroscopic measurements, and prolonged serum stability assays indicate that this broadly versatile ligand forms highly thermodynamically stable and kinetically inert coordination complexes with isotopes of both Lu3+ and In3+.  Finally, and most importantly, acute biodistribution studies have shown that 111In- and 177Lu-octapa-trastuzumab specifically and selectively accumulate in SKOV-3 ovarian cancer xenografts in vivo to a degree unmatched by analogous 111In-DOTA- and 177Lu-DOTA-trastuzumab radioimmunoconjugates.  Further, SPECT/CT imaging reveals that both 111In- and 177Lu-octapa-trastuzumab are capable of delineating HER2-positive tumours in vivo with excellent contrast and high tumour-to-background activity ratios, producing images that are comparable to those created using 111In- and 177Lu-DOTA-trastuzumab.  Taken together, these data clearly indicate that p-SCN-Bn-H4octapa is a versatile and promising bifunctional ligand for the construction of highly potent and effective 111In- and 177Lu-labeled radiopharmaceuticals.  The last major hurdle for eva