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Acyclic chelates for imaging with radiometals Boros, Eszter 2011

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ACYCLIC CHELATES FOR IMAGING WITH RADIOMETALS by Eszter Boros B.Sc., Universität Zürich, 2006 M. Sc., Universität Zürich, 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2011 © Eszter Boros, 2011  Abstract This thesis investigates acyclic chelates containing pyridine, carboxylate and amino groups and their fast and efficient and stable coordination of the (radio-)metals 99m  Tc, Re, Cu,  64  Cu, Ga,  67/68  Ga. The radiometals mentioned are all of significant interest  for SPECT (single photon emission computed tomography) and PET (positron emission tomography) imaging, two of the most important non-invasive diagnostic tools in the clinic. The design, synthesis, radiolabelling, evaluation of stability, and in vivo investigation are presented for three variably charged, novel, short C8 chain derivatized chelates for the [M(CO)3]+ core (M =  99m  Tc or Re). Distinct localization of these small  amphiphilic molecules in vivo was studied via biodistribution and imaging and was found to be dependent on the charge of the polar moiety. In an attempt to identify a universal chelate applicable to the [M(CO)3]+ core and  64  Cu, the synthesis of the dipin bifunctional  chelate system, capable of fast coordination of Cu, its radionuclide +  [M(CO)3] core (M =  99m  64  Cu, as well as the  Tc, Re) is described. The versatility of the dipin ligand system is  based on its ability to adopt a variety of binding modes, tridentate when protected, tetradentate when deprotected. Based on this small, tetradentate scaffold, a series of acyclic chelates with varying bite angle and denticity were synthesized and screened for their ability to coordinate radioisotopes of Ga and Cu under mild conditions within a short period of time. It was found that the linear N4O2 chelate H2dedpa coordinates 67/68  Ga quantitatively to form [67Ga(dedpa)]+ after 10 min at room temperature with  exceptional kinetic inertness towards human apo-transferrin. The corresponding  64  Cu  complex maintains inertness against serum proteins within 2 h, but shows 23 % transchelation to serum proteins after 24 h. Both potential small molecule perfusion agents, as well as peptide conjugated versions as applications of the H2dedpa scaffold are reported. Both types of derivatives maintained the labelling and radiolabelled complex (67/68Ga and  64  Cu) stability properties found with the non-derivatized versions, further  indicating the H2dedpa scaffold is a useful platform for the development of clinically applicable radiopharmaceuticals.  ii  Preface Chapter 2 is based on work conducted at UBC, TRIUMF, the McMaster Institute of Applied Radiation Sciences (McIARS) and the McMaster Centre for Preclinical and Translational Imaging (MCPTI). Radiochemical synthesis and HPLC purification were done in the laboratory of Prof. John Valliant, under the instruction of Dr. Andrea Armstrong. Imaging of the animals on their SPECT/CT scanner was done by Troy Farncombe, Chantal Saab, and Rod Rhem. Biodistribution and subsequent evaluation of the imaging and biodistribution data was done by Prof. Urs O. Häfeli. This work has also been published and reproduced in part with permission from: Boros, Eszter; Häfeli, Urs O.; Patrick, Brian O.; Adam, Michael J.; Orvig, Chris. Design, Synthesis, and Imaging of Small Amphiphilic Rhenium and 1002-1009.  99m  Technetium Tricarbonyl Complexes. Bioconjugate Chem. (2009), 20(5), Copyright  2009  American  Chemical  Society.  http://pubs.acs.org/doi/pdf/10.1021/bc900022c. I conducted all synthetic experiments, radiochemistry and wrote most of the manuscript with the editorial help of Chris Orvig. Urs O. Häfeli generated the bar graphs of the biodistribution, curves of all  99m  Tc-compounds’  levels in selected organs taken from the SPECT/CT images and sagittal and coronal plane SPECT/CT images. The solid state structure determination was done by Dr. Brian O. Patrick. The work in Chapter 3 has been published: Boros, Eszter; Lin, Yi-Heng S.; Ferreira, Cara L.; Patrick, Brian O.; Häfeli, Urs O.; Adam, Michael J.; Orvig, Chris. One to chelate them all: investigation of a versatile, bifunctional chelate for 64Cu, 99mTc, Re and Co. Dalton Trans., 2011 (23), 6253-6259. Reproduced by permission of The Royal Society of Chemistry (RSC). http://pubs.rsc.org/en/content/articlepdf/2011/dt/c0dt01458c. Model chelates H2dipin, Me2dipin, Cu complexes thereof and precursors for the functionalization were synthesized first by Yi-Heng Scott Lin under my instruction and supervision and were included in his Bachelor Honours thesis. Remaining synthetic work on conjugation with biotin, radiochemistry and all of the [Re(CO)3]+ work was done by myself. I was responsible for writing the manuscript with the editorial help of Cara L. Ferreira and Chris Orvig. The solid state structure determination was done by Dr. Brian O. Patrick.  iii  Parts of Chapter 4 have been published and are reproduced in part with permission from: Boros, Eszter; Ferreira, Cara L.; Cawthray, Jacqueline F.; Price, Eric W.; Patrick, Brian O.; Wester, Dennis W.; Adam, Michael J.; Orvig, Chris. Acyclic Chelate with Ideal Properties for 68Ga PET Imaging Agent Elaboration. J. Am. Chem. Soc. (2010), 132(44), 15726-15733.  Copyright  2010  American  Chemical  Society.  http://pubs.acs.org/doi/pdf/10.1021/ja106399h. I conducted all synthetic experiments, radiochemistry, generated the bar graphs of the biodistribution and wrote most of the manuscript with the editorial help of Cara L. Ferreira and Chris Orvig. The diamino precursor of H2dp-bb-NO2 was synthesized by Eric W. Price. Biodistribution data were collected by Dr. Dawn Waterhouse of the BC Cancer Agency. Potentiometric measurements were conducted by Dr. Jacqueline F. Cawthray. The solid state structure determination was done by Dr. Brian O. Patrick. The solid state structure determination in Chapter 5 was done by Dr. Brian O. Patrick. Glenn Bremner (Wolf group) assisted with Cyclic Voltammetry measurements. Work presented in Chapter 7 has been accepted for publication: "New Ga Derivatives of the H2dedpa Scaffold with Improved Clearance and Persistent Heart Uptake", Boros, E.; Ferreira, Cara L.; Patrick Brian O.; Adam, Michael J.; Orvig ,Chris. Nucl. Med. Biol. (2011) in press. Copyright 2011 Elsevier. Biodistribution on the 67Ga complexes reported in Chapter 7 were carried out by Dr. Dawn Waterhouse of the BC Cancer Agency. The solid state structure determination was done by Dr. Brian O. Patrick.  iv  Table of Contents Abstract.................................................................................................................................... ii  Preface..................................................................................................................................... iii  Table of Contents .................................................................................................................... v  List of Tables .......................................................................................................................... xi  List of Figures....................................................................................................................... xiii  List of Schemes................................................................................................................... xviii  List of Symbols and Abbreviations .................................................................................... xix  Acknowledgements ............................................................................................................ xxiii  Dedication ........................................................................................................................... xxiv  Chapter 1: Introduction ......................................................................................................... 1  1.1  Medicinal Inorganic Chemistry ................................................................................... 1  1.2  Medicinal Imaging ....................................................................................................... 2  1.3  Therapy in Nuclear Medicine ...................................................................................... 5  1.4  Radioisotope Production and Generators..................................................................... 7  1.5  Isotope Supply Problems ............................................................................................. 9  1.6  Chelates for the Purpose of Radiometal Coordination .............................................. 10  1.7  Coordination Chemistry of 99mTc .............................................................................. 11  1.8  Coordination Chemistry of 64Cu ................................................................................ 13  1.9  Coordination Chemistry of 67/68Ga............................................................................. 14  1.10  Thesis Overview ...................................................................................................... 16  Chapter 2: Small Amphiphilic Rhenium and 99mTechnetium Tricarbonyl Complexes . 19  2.1  Introduction................................................................................................................ 19  2.2  Experimental .............................................................................................................. 22  2.2.1  Materials and Methods........................................................................................ 22  2.2.2  Ligand Synthesis................................................................................................. 23  2.2.3  Synthesis of Re(CO)3+ Complexes ..................................................................... 28  2.2.4  X-ray Diffraction Analysis of [Re(CO)3L1] ....................................................... 30  2.2.5  Synthesis of 99mTc(CO)3 Complexes .................................................................. 30  2.2.6  Cysteine and Histidine Stability Challenges....................................................... 31   v  2.2.7  Octanol-Water-Distribution Coefficients ........................................................... 31  2.2.8  Imaging and Biodistribution ............................................................................... 31  2.3  Results and Discussion .............................................................................................. 32  2.3.1   Ligand Preparation............................................................................................ 32   2.3.2  Preparation of Re Complexes ............................................................................. 33  2.3.3  X-Ray Diffraction Structural Characterization of [Re(CO)3L1] ........................ 36  2.3.4  Radiolabelling..................................................................................................... 37  2.3.5  In Vitro Stability and log Po/w ............................................................................. 39  2.3.6  In Vivo Evaluations............................................................................................. 40  2.4  Conclusions................................................................................................................ 42  Chapter 3: Investigation of the H2dipin Scaffold as Chelate with Adjustable Denticity for 64Cu, Re and 99mTc .......................................................................................................... 44  3.1  Introduction: Multifunctional Chelate Systems ......................................................... 44  3.1.1  Prior Art on H2dipin Type Chelates.................................................................... 45  3.2  Experimental .............................................................................................................. 47  3.2.1  Materials and Methods........................................................................................ 47  3.2.2  Synthesis of Ligands ........................................................................................... 48  3.2.3  Coordination Chemistry...................................................................................... 52  3.2.4  X-Ray Diffraction Structural Characterization................................................... 54  3.2.5  Radiolabelling with 99mTc................................................................................... 55  3.2.6  Cysteine and Histidine Challenge....................................................................... 56  3.2.7  Radiolabelling with 64Cu .................................................................................... 56  3.2.8  Serum Challenge................................................................................................. 56  3.3  Results and Discussion .............................................................................................. 56  3.3.1  Chelate Synthesis................................................................................................ 56  3.3.2  Coordination Complexes .................................................................................... 59  3.3.3  X-Ray Diffraction Structural Characterization of [Cu(dipin-NO2)H2O] and [Re(CO)3(Me2dipin-NO2)]+ ................................................................................ 64  3.3.4  Radiolabelling and Stability of 99mTc Complexes .............................................. 66  3.3.5  Radiolabelling and Stability of 64Cu Complexes ................................................ 67  3.4  Conclusions................................................................................................................ 68   vi  Chapter 4: A Novel Chelate for 68Ga Imaging Agent Elaboration ................................. 70  4.1  Introduction: Prior Art on 67/68Ga Chelates................................................................ 70  4.2  Pyridine Carboxylate Chelates and Their Relevance for Chelation of Ga(III).......... 72  4.3  Experimental .............................................................................................................. 74  4.3.1  Materials and Methods........................................................................................ 74  4.3.2  General Synthesis Procedures............................................................................. 74  4.3.3  Ligand Synthesis................................................................................................. 75  4.3.4  Coordination Chemistry with Ga ........................................................................ 82  4.3.5  Labelling with 67/68Ga ......................................................................................... 84  4.3.6  In Vitro Stability: Transferrin Challenge and Challenge against NOTA............ 85  4.3.7  X-Ray Diffraction Structural Characterization of [Ga(dedpa)][ClO4] and [Ga(dpN-NO2)] [ClO4]................................................................................................... 86  4.3.8  Biodistribution of [67Ga (dedpa)]+, [67Ga(dp-bb-NO2)]+ and [67Ga(dp-N-NO2)]+ ............................................................................................................................ 86  4.4  Results and Discussion .............................................................................................. 87  4.4.1  Ligand Synthesis................................................................................................. 87  4.4.2  Screening with 67Ga ............................................................................................ 89  4.4.3  In Vitro stability of Select Ligand Candidate ..................................................... 91  4.4.4  Coordination Chemistry with Non-radioactive Ga and X-Ray Diffraction Structural Characterization of [Ga(dedpa)][ClO4].............................................. 92  4.4.5  Potentiometric Measurements on [Ga(dedpa)]+.................................................. 94  4.4.6  Model Functionalization of the H2dedpa Scaffold ............................................. 94  4.4.7   67  Ga Labelling of the Model Bifunctionals H2dp-bb-NO2 and H2dp-N-NO2 and  Transferrin Challenge Experiments of [67Ga(dp-bb-NO2)]+ and [67Ga(dp-NNO2)]+ ................................................................................................................. 96  4.4.8  Coordination Chemistry of the Model Bifunctionals with Cold Ga and X-Ray Diffraction Structural Characterization of [Ga(dp-N-NO2)][ClO4].................... 97  4.4.9  Biodistribution of 67Ga Complexes of (dedpa)2- and the Model Bifunctionals 100  4.5  Conclusions.............................................................................................................. 101  Chapter 5: Viability of H2dedpa for Labelling with 64Cu ............................................... 103  5.1  Introduction: Prior Art in 64Cu Chelates .................................................................. 103   vii  5.2  Experimental ............................................................................................................ 105  5.2.1  Materials and Methods...................................................................................... 105  5.2.2  Coordination Chemistry with Non-radioactive Cu ........................................... 105  5.2.3  Labelling with 64Cu and In Vitro Stability........................................................ 106  5.2.4  X-Ray Diffraction Structural Characterization of [Cu(dedpa)]·3H2O.............. 106  5.2.5  Cyclic Voltammetry Measurements on [Cu(dedpa)]........................................ 107  5.3  Results and Discussion ............................................................................................ 108  5.3.1  Cu(II) Coordination Chemistry and X-Ray Diffraction Structural Characterization of [Cu(dedpa)]·3H2O ............................................................. 108  5.3.2  Cyclic Voltammetry.......................................................................................... 110  5.3.3  Labelling with 64Cu and In Vitro Stability........................................................ 112  5.4  Conclusions.............................................................................................................. 112  Chapter 6: Functionalization of the H2dedpa Scaffold (1): Bioconjugation and Multifunctionality ............................................................................................................... 114  6.1  Introduction.............................................................................................................. 114  6.1.1  Modes of Bioconjugation.................................................................................. 114  6.1.2  Convenient Biomolecules for Proof of Principle.............................................. 117  6.2  Experimental ............................................................................................................ 120  6.2.1  Synthesis of Fragments for Coupling ............................................................... 120  6.2.2  Thiourea Coupling with [c(RGDyK)]............................................................... 127  6.2.3  Coordination Chemistry with Ga ...................................................................... 128  6.2.4  Radiolabelling with 67/68Ga, and Transferrin Stability...................................... 128  6.2.5  Radiolabelling with 64Cu, and Serum Stability................................................. 129  6.2.6  Avidin binding .................................................................................................. 130  6.3  Results and Discussion ............................................................................................ 131  6.3.1  Synthesis of H2dp-bb-NH2, H2dp-N-NH2, H2dp-bb-NCS and H2dp-N-NCS ... 131  6.3.2  Amide Coupling with Biotin............................................................................. 135  6.3.3  Coordination Chemistry of Ga with H2dp-N-Biotin......................................... 136  6.3.4  Radiolabelling of H2dp-N-Biotin with 67Ga and Avidin Binding..................... 137  6.3.5  Thiourea Coupling with [c(RGDyK)]............................................................... 138  6.3.6  Radiolabelling and Stability of RGD Conjugates with 67/68Ga ......................... 139   viii  6.3.7  Radiolabelling and Stability of RGD Conjugates with 64Cu ............................ 140  6.4  Conclusions.............................................................................................................. 141  Chapter 7: Functionalization of the H2dedpa Scaffold (2): Small Lipophilic Cations for Potential Imaging of Myocardial Bloodflow..................................................................... 142  7.1  Introduction: Cardiac Imaging Agents..................................................................... 142  7.2  Experimental ............................................................................................................ 144  7.2.1  Materials and Methods...................................................................................... 144  7.2.2  Chemical Synthesis........................................................................................... 144  7.2.3  Synthesis of Lipophilic Ligand Systems .......................................................... 145  7.2.4  Coordination Chemistry.................................................................................... 154  7.2.5  X-Ray Diffraction Structural Characterization of [Ga(6EtO·2MeO)][ClO4] ... 157  7.2.6  Radiolabelling with 67/68Ga, Log Po/w Measurements and Transferrin Stability .......................................................................................................................... 158  7.2.7  In Vivo Evaluation ............................................................................................ 159  7.3  Results and Discussion ............................................................................................ 160  7.3.1  Ligand Synthesis............................................................................................... 160  7.3.2  Non-radioactive Complexes with Ga................................................................ 161  7.3.3  X-Ray Diffraction Structural Characterization of [Ga(6EtO·2MeO)][ClO4] ... 163  7.3.4  Radiolabelling with 67/68Ga and In Vitro Stability ............................................ 164  7.3.5  Log Po/w Measurements .................................................................................... 165  7.3.6  Biodistribution .................................................................................................. 166  7.4  Conclusion ............................................................................................................... 169  Chapter 8: Conclusions and Future Work ....................................................................... 170  8.1  Further Enhancing the H2dedpa Technology........................................................... 170  8.1.1  Extension of the Denticity for Other Metals of Interest ................................... 170  8.1.2  H2dedpa Peptide Derivatives of Relevance for Clinical Applications.............. 171  8.2  Small Molecule Imaging Agents ............................................................................. 172  8.2.1  New Lipophilic Ligand Systems for Imaging of Myocardial Bloodflow......... 172  8.2.2  Alternative H2dedpa Derivatives as Small Molecule Imaging Agents............. 172  References............................................................................................................................ 174  Appendices........................................................................................................................... 182   ix  Appendix A Crystallographic Data................................................................................... 182  Appendix B Biodistribution Data ..................................................................................... 184  B.1  Biodistribution of Compounds [67Ga(dedpa)]+, [67Ga(dp-N-NO2)]+ , [67Ga(dp-bbNO2)]................................................................................................................. 184  B.2  Biodistribution of Compounds [67Ga(0MeO)]+, [67Ga(2MeO)]+ , [67Ga(4MeO)]+ , [67Ga(6MeO)]+ , [67Ga(8MeO)]+ , [67Ga(6EtO)]+ , [67Ga(6EtO·2MeO)]+. ....... 186   x  List of Tables Table 1.1 Widely used and investigated radionuclides for imaging......................................... 4 Table 1.2 Widely used and investigated radionuclides for therapy. ......................................... 6 Table 1.3 Widely used and investigated radionuclides and their production.15........................ 8 Table 2.1 Selected bond lengths in [Re(CO)3L1] ([Å]).......................................................... 36 Table 2.2 Summarized experimental properties of the three 99mTc(CO)3 complexes............. 39 Table 2.3 Biodistribution, % ID/g ± SD (n = 4) of all complexes at the 4 h time point......... 42 Table 3.1 Summarized relevant IR stretching frequencies for Cu complexes and the corresponding ligands. ............................................................................................................ 60 Table 3.2 Summarized relevant IR stretching frequencies for Re complexes and the corresponding ligands. ............................................................................................................ 61 Table 3.3 Selected bond lengths in [Cu(dipin-NO2)( H2O)] ([Å]).......................................... 65 Table 3.4 Selected bond lengths in [Re(CO)3(Me2dipin-NO2)]+ ([Å])................................... 66 Table 4.1 Summary of some investigated chelates for labelling of 67/68Ga ............................ 71 Table 4.2 Summarized results from initial labeling experiments with the synthesized chelates. HPLC gradients include: A: 0 to 5% in 10 mM NaOAc (pH 4.5), 20 min. B: 0 to 100% CH3OH in 10 mM NaOAc (pH 4.5), 20 min.......................................................................... 90 Table 4.3 Selected bond lengths in [Ga(dedpa)][ClO4] , ([Å])............................................... 93 Table 4.4 Formation constants (log KML) and pMa of Ga(III) complexes. a Calculated for 10 μM total ligand and 1 μM total metal at pH 7.4 and 25°C. b Conditional constant for log KML from Ref 57. ............................................................................................................................. 94 Table 4.5 Selected bond lengths in [Ga(dp-N-NO2)][ClO4] (bold), compared to selected bond lengths in [Ga(dedpa)][ClO4], ([Å]). ...................................................................................... 99 Table 5.1 Summary of relevant properties of previously investigated chelates for labelling of 64  Cu ....................................................................................................................................... 104  Table 5.2 Selected bond lengths in [Cu(dedpa)] (bold), compared to selected bond lengths in [Ga(dedpa)][ClO4], ([Å]). ..................................................................................................... 109 Table 6.1 Summary of frequently used target molecules for imaging and therapy with radiometals............................................................................................................................ 118  xi  Table 7.1 Selected bond lengths in [Ga(6EtO·2MeO)][ClO4] (bold), compared to selected bond lengths in [Ga(dedpa)][ClO4], ([Å]). ........................................................................... 163 Table 7.2 Log Po/w values and corresponding HPLC retention times of the seven new compounds. ........................................................................................................................... 165 Table 7.3 Heart/blood, heart/lung and heart/liver ratios for compounds [67Ga(8MeO)]+, [67Ga(6EtO)]+, [67Ga(6EtO·2MeO)]+ .................................................................................... 168  xii  List of Figures Figure 1.1 The author’s view of the field of bioinorganic chemistry. ...................................... 1 Figure 1.2 Cisplatin, Auranofin and Cardiolite (left to right)................................................... 1 Figure 1.3 Schematic description of imaging techniques mentioned in Chapter 1.2. .............. 3 Figure 1.4 Schematic production pathway for radiopharmaceuticals....................................... 7 Figure 1.5 Scheme of two complexes following two different methodologies for radiometal incorporation while targeting the same receptor..................................................................... 10 Figure 1.6 Four of the most commonly used 99mTc synthons; Tc(oxo) with a MAG3-type coordination environment, [Tc(CO)3]+ coordinated by a typical tridentate ligand, Tc(nitrido) with an N4-xSx type chelate and Tc(HYNIC) with a typical coordination environment. All chelates can be derivatized through the variable R functionality. .......................................... 11 Figure 1.7 Summary of desired properties for the optimal chelate for 64Cu, shown on the example of the 64Cu complex with a derivatized version of the cross-bridged macrocycle CB-TE2A. ............................................................................................................................... 13 Figure 1.8 Summary of desired properties for the optimal chelate for 68Ga, shown on the example of the 68Ga complex with a derivatized version of the macrocycle NOTA. ........... 15 Figure 1.9 DOTANOC, a somatostatin analogue currently used with 68Ga in clinical trials. 16 Figure 1.10 H2dedpa. .............................................................................................................. 18 Figure 2.1 Examples of the integrated (left) and tagged (right) approach on steroid scaffolds. ................................................................................................................................................. 19 Figure 2.2 Examples of commercial surfactants with different polar head groups (denoted in red): neutral, anionic and zwitterionic (from top to bottom). ................................................. 20 Figure 2.3 Previously reported [Tc(CO)3]+ complexes bearing long aliphatic chains.74,76..... 21 Figure 2.4 1H NMR spectra (300 MHz, 298 K) of HL1 (a) and [Re(CO)3L1] (b), * denotes residual solvent peak, while characteristic diastereotopic protons are highlighted with their assignments. ............................................................................................................................ 34 Figure 2.5 Complexes described within this chapter. ............................................................. 35 Figure 2.6 ORTEP view of [Re(CO)3L1], showing the atom numbering scheme (50% thermal ellipsoids)................................................................................................................... 36 Figure 2.7 γ-HPLC traces of the crude reaction mixtures of [99mTc(CO)3L1] and [99m Tc(CO)3L2]-. ........................................................................................................................... 37 xiii  Figure 2.8 γ-HPLC traces of the crude reaction mixtures of [99mTc(CO)3L3], SM denotes starting material, P denotes product and SP stands for side products..................................... 38 Figure 2.9 Activity (counts/pixel) levels vs. time curves for each of the evaluated compounds in select organs taken from the planar scintigraphy images. .................................................. 40 Figure 2.10 Representative µSPECT/CT pictures taken 15 min after injection of A) [99mTc(CO)3L1], B) [99mTc(CO)3L2]- and C) [99mTc(CO)3L3]. Transverse (left) and coronal (right) planes shown in each case. Hot spots within the images are the liver in A, bladder and some residual activity in the kidneys in B and the gall bladder with liver uptake and bladder in C.......................................................................................................................................... 41 Figure 3.1 Examples of the previously reported ligand systems DpK (SAAC approach46) and bifunctional, multi-purpose ATSE.94 ...................................................................................... 45 Figure 3.2 Multifunctional ligand system dip described in this chapter; the variable denticity is indicated by the arrows. ...................................................................................................... 46 Figure 3.3 Biotin. .................................................................................................................... 47 Figure 3.4 1H NMR spectra (300 MHz, CDCl3/CD3OD, 298 K) of the benzylnitro derivatized compounds synthesized, * denotes residual solvent peaks..................................................... 62 Figure 3.5 1H NMR spectra (300 MHz, CD3OD, 298 K) of the biotin derivatized compounds synthesized, * denotes residual solvent peaks. ....................................................................... 63 Figure 3.6 ORTEP drawing of [Cu(dipin-NO2)(H2O)], showing the atom numbering scheme (50% thermal ellipsoids). ........................................................................................................ 64 Figure 3.7 ORTEP drawing of [Re(CO)3(Me2dipin-NO2)]+, with co-crystallized counter ion [Re(CO)3Br3]2- shown is the atom numbering scheme (50% thermal ellipsoids). ................. 65 Figure 3.8 TLC labelling traces of [99mTc(CO)3(Me2dipin-NO2)]+ (left, spot 1) and [99mTc(CO)3(Me2dipin-biotin)]+ (right, spot 2); bottom spots indicate unreacted [99mTc(CO)3(H2O)3]+ (bottom, spot 3). ................................................................................... 66 Figure 3.9 Labelling traces of 64Cu copper complex [t0] and trace after 1h serum challenge experiment [t1h]. Left shows Cu complex with [64Cu(dipin-NO2)], right shows Cu complex with [64Cu(dipin-biotin)], ° denotes transchelation of 64Cu to serum proteins. ..................... 67 Figure 4.1 Structures of some previously investigated chelate systems for 68Ga................... 70 Figure 4.2 Previously reported pyridyl containing ligand systems......................................... 73  xiv  Figure 4.3 1H NMR spectrum (300 MHz, CD3OD, 298 K) of a propyl bridged chelate, * denotes residual solvent peaks. ............................................................................................... 88 Figure 4.4 Labelling traces from transferrin challenge experiment (left) and NOTA versus H2dedpa challenge (right). ...................................................................................................... 91 Figure 4.5 ORTEP drawing of [Ga(dedpa)][ClO4]. Shown is the atom numbering scheme (50% thermal ellipsoids). ........................................................................................................ 92 Figure 4.6 1H NMR spectra (300 MHz, d6-DMSO/CD3OD, 298 K) of H2dedpa and [Ga(dedpa)][NO3], * denotes residual solvent peaks.............................................................. 93 Figure 4.7 Stacked HPLC monitoring traces of [67Ga(dp-N-NO2)]+ (left) and [67Ga(dp-bbNO2)]+ (right) over 2 h stability experiment against apo-transferrin...................................... 96 Figure 4.8 1H NMR spectra (300 MHz, CD3OD, 298 K) of H2dp-N-NO2 and [Ga(dp-NNO2)][NO3]. * denotes residual solvent peaks. ...................................................................... 97 Figure 4.9 1H NMR spectra (300 MHz, CD3OD, 298 K) of H2dp-bb-NO2 and [Ga(dp-bbNO2)][NO3]. * denotes residual solvent peaks. ...................................................................... 98 Figure 4.10 ORTEP drawing of [Ga(dp-N-NO2)][ClO4] (50% thermal ellipsoids); H atoms and the (ClO4) counter ion are omitted for clarity. ................................................................. 99 Figure 4.11 Biodistribution of [67Ga(dedpa)]+, [67Ga(dp-N-NO2)]+ and [67Ga(dp-bb-NO2)]+ in female ICR mice over 4h; complete data of the latter two compounds for urine is shown also in separate diagrams to the right.................................................................................... 100 Figure 5.1 Structures of selected, previously investigated chelate systems for labelling with 64  Cu. ...................................................................................................................................... 104  Figure 5.2 ORTEP drawing of [Cu(dedpa)]; shown is the atom numbering scheme (50% thermal ellipsoids) and hydrogen atoms within the complex structure are omitted for clarity. ............................................................................................................................................... 108 Figure 5.3 Side-on view of [Cu(dedpa)] (right), compared to selected bond lengths in [Ga(dedpa)]+ (left), H atoms and (ClO4)- counter ion of Ga complex omitted for clarity. Graphic generated using Mercury 2.2.150.............................................................................. 109 Figure 5.4 Side-on view of [Cu(dedpa)] (right), compared to compared to selected bond lengths in [Ga(dedpa)]+ (left), H atoms and (ClO4)- counter ion of Ga complex omitted for clarity. Graphic generated using Mercury 2.2.150 ................................................................. 110  xv  Figure 5.5 Cyclic voltammogram of [Cu(dedpa)], -1.25 and 0.5 V (solid line). Also shown is the blank voltammogram with solvent only (dotted, -1.25 – 1.5 V). Potential values are given with reference electrode Ag/AgCl (sat.). ................................................................... 111 Figure 5.6 [Cu(dedpa)], -0.5 and 0.5 V(solid, black line), overlayed with full range voltammogram. Note, how the oxidation peak at 0.02 V is not observed, therefore it could be related to observed reduction peak at -1.12 V. Potential values are given with reference electrode Ag/AgCl (sat.). ...................................................................................... 111 Figure 6.1 Structures of frequently used target molecules for imaging and therapy with radiometals............................................................................................................................ 119 Figure 6.2 Hypothetical, postulated building blocks for functionalization of the H2dedpa scaffold.................................................................................................................................. 131 Figure 6.3 1H NMR spectra (300 MHz, CD3OD, 298 K) of H2dp-bb-NCS and H2dp-N-NCS, * denotes residual solvent peaks. .......................................................................................... 134 Figure 6.4 Desired and observed products from coupling of biotin-TFP and Me2dp-bb-NH2. ............................................................................................................................................... 135 Figure 6.5 1H NMR spectra (300 MHz, d6-DMSO, 298 K) of H2dp-N-Biotin and [Ga(dp-NBiotin)]+, * denotes residual solvent peaks........................................................................... 136 Figure 6.6 Stacked HPLC monitoring traces of transferrin challenge experiment for [67Ga(dpN-Biotin)]+ . .......................................................................................................................... 137 Figure 6.7 HPLC monitoring traces of labelling traces for [67Ga(dp-bb-RGD)]+ (left) and [67Ga(dp-N-RGD)]+ (right). .................................................................................................. 139 Figure 6.8 Radioabelling traces for [64Cu(dp-bb-RGD)]+ (left) and [64Cu(dp-N-RGD)]+ (right). ................................................................................................................................... 140 Figure 7.1 Some commercially approved radiopharmaceuticals for heart imaging. ............ 142 Figure 7.2 Previously investigated Ga-based compounds for heart imaging. ...................... 143 Figure 7.3 1H NMR (300 MHz, CD3OD, 298 K) spectra of 6EtO and [Ga(6EtO)][NO3]. * denotes residual solvent peaks. ............................................................................................. 162 Figure 7.4 1H NMR spectra (300 MHz, CD3OD, 298 K) of 6EtO·2MeO and [Ga(6EtO·2MeO)][NO3]. * denotes residual solvent peaks. ................................................ 162  xvi  Figure 7.5 ORTEP representation of [Ga(6EtO·2MeO)][ClO4], with corresponding molecular drawing. Shown is the atom numbering scheme (50% thermal ellipsoids). Hydrogen atoms are omitted for clarity............................................................................................................ 164 Figure 7.6 Biodistribution of [67Ga(0MeO)]+, [67Ga(2MeO)]+, [67Ga(4MeO)]+, [67Ga(6MeO)]+, [67Ga(8MeO)]+, [67Ga(6EtO)]+and [67Ga(6EtO·2MeO)]+ in female ICR mice over 2h. ................................................................................................................................. 167 Figure 8.1 Proposed octadentate model compounds based on the H2dedpa scaffold........... 171 Figure 8.2 Nitroimidazole derivatives of NOTA (left) and H2dedpa (right). ....................... 173  xvii  List of Schemes Scheme 1.1 Preparation of 18FDG and 99mTc-Sestamibi........................................................... 5  Scheme 2.1 Synthetic methods to furnish ligand systems HL1, H2L2, and H2L3. (i) Boc2O, neat (ii) Octanoyl 1-chloride, EDMA, toluene, (iii) TFA/ CH2Cl2 (1:1) (iv) tert-Butyl bromoacetate, Na2CO3, CH2Cl2 (v) Pyridine-2-aldehyde, EtOH. .......................................... 32  Scheme 2.2 Sample synthesis scheme for 99mTc(CO)3 complexes, here shown on [99mTc(CO)3L1]. (v) NaHCO3 (0.1 M) (vi) [99mTc(H2O)3(CO)3]+ , 90°C, 25-45 min. ........... 37  Scheme 3.1 Synthetic scheme for the synthesis of 2 and 3 (i) 0.5 eq 4-Nitrobenzylamine, NaBH(OAc)3, DCE, reflux, overnight (ii) 4 eq LiOH, in THF/H2O. ..................................... 57  Scheme 3.2 Synthetic scheme for the synthesis of 5 and 6 (iii) NEt3, DMF, 60 °C, overnight (iv) 5 eq LiOH, THF/ H2O / CH2Cl2 (1:2:1)........................................................................... 58  Scheme 4.1 Schematic overview of synthesis methodology used to synthesize chelates of library (i) 1. CH3OH, reflux, 30 min 2. NaBH4, CH3OH, 0°C, 2 h (ii) tert-butyl bromoactetic acid, Na2CO3, CH3CN, 70 °C, o.n. (iii) TFA/ CH2Cl2, 30 min (iv) LiOH, THF/ H2O, 30 min – 2 h......................................................................................................................................... 87  Scheme 4.2 Syntheses of H2dp-N-NO2 and H2dp-bb-NO2: (i) 1. CH3OH, reflux, 2h, 2. NaBH4, 0°C, 2h (ii) 4-nitrobenzyl bromine, Na2CO3, CH3CN, 18 h (iv) LiOH, THF/water (3:1), 45 min............................................................................................................................ 95  Scheme 6.1 Scheme illustrating pre- and post-labelling approach based on H2dedpa. ........ 115  Scheme 6.2 Possible conjugation strategies using H2dedpa to couple a bifunctional chelate with a targeting molecule...................................................................................................... 116  Scheme 6.3 Synthesis scheme for compounds 1, 2 and 3. (i) Na2CO3, CH3CN, 70 °C, 18 h (ii) CH2Cl2/TFA, 30 min (iii) 1. LiOH (4 eq), THF/H2O (3:1), 2. HCl (3 M), 2 h, 3. SCCl2 , 18 h........................................................................................................................................ 132  Scheme 6.4 Synthesis scheme for compounds 4, 5, 6 and 7. (iv) 1. EtOH, 0 °C, 2 h, 2. NaBH4, 0°C, 2 h (i) Na2CO3, MeCN, 70 °C, 18 h (v) Pd/C, AcOOH, 18 h (vi) 1. LiOH (4 eq), THF/H2O (3:1), 2. HCl (3 M), SCCl2, 18 h. .................................................................. 133  Scheme 6.5 Synthesis scheme for compounds H2dp-bb-RGD and H2dp-N-RGD. (vii). 0.1 M NaHCO3 (pH 9 -9.5)/ CH3OH (3:1), room temperature, under light exclusion, 18 h. ......... 138  Scheme 7.1 General synthetic scheme for all compounds synthesized and reported in this chapter................................................................................................................................... 160  xviii  List of Symbols and Abbreviations ~  approximate  2D  two dimensional  3D  three dimensional  6SS  N,N'-bis(2,2-dimethyl-2-mercaptoethyl)ethylenediamine-N,N'diacetic acid  α  alpha or alpha particle  Å  angstrom, 10 · 10-10 metre  A  ampère  β  beta  β−  beta particle  β+  positron  γ  gamma ray  δ  delta or chemical shift in parts per million (NMR)  ε  epsilon  μ  micro (10-6)  Ac  acetyl  AcOH  acetic acid  Anal.  analytical  atm  atmosphere  Bn  benzyl  t-Boc  N-tert-butoxycarbonyl  Boc2O  di-tert-butyl-dicarbonate  °C  degrees celsius  calcd.  calculated  CB-DO2A  4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane  CB-TE2A  4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane  Ci  Curie  cm-1  wavenumber  xix  CT  computed tomography  CV  cyclic voltammetry  d  day(s) or doublet (NMR)  DIAMSAR  1,8-diamino-3,6,10,13,16,19- hexaazabicyclo(6,6,6)eicosane  DMF  dimethylformamide  DMSO  dimethylsulfoxide  DOTA  1,4,7,10-tetraazacyclododecane -N,N',N",N'"-tetraacetic acid  EA  elemental analysis  EC  N,N'-ethylene-di-L-cysteine  EDMA  ethyldimethylamine  ESI-MS  electrospray ionization mass spectrometry  EtOAc  ethyl acetate  EtOH  ethanol  eV  electron volt  eq  equivalent(s)  f  fission  fac  facial  FDA  Food and Drug Administration (USA)  FDG  18  F-2-deoxy-2-fluoro-D-glucose  FMISO  18  F-fluoromisonidazole  g  gram  h  hour(s)  HMBC  heteronuclear multiple bond coherence  HSQC  heteronuclear single bond coherence  HPLC  high performance liquid chromatography  HYNIC  hydrazinopyridine-3-carboxylic acid  Hz  hertz (s-1)  IR  infrared  ID  injection dose  J  coupling constant (NMR)  k  kilo  xx  KML  thermodynamic complex stability constant  L  litre or ligand  m  milli- or medium or multiplet  M  molar (moles/ litre) or mega  MeOH  methanol  min  minute(s)  mol  mole  MAG3  mercaptoacetyltriglycine  MRI  magnetic resonance imaging  MS  mass spectrometry  m/z  mass per unit charge  n  nano (10-9) or number of unit  NHE  normal hydrogen electrode  NMR  nuclear magnetic resonance  n. o.  not observed  n. d.  not determined  NOTA  1,4,7-triazacyclononane-1,4,7-triacetic acid  NRU  National Research Universal (reactor)  ORTEP  Oak Ridge Thermal Ellipsoid Plot Program  PBS  phosphate buffered saline  PCTA  3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9triacetic acid  Pd/C  palladium on carbon (10% by weight)  PET  positron emission tomography  pH  -log[H3O]+  pM  -log[free metal]  ppm  parts per million  q  quartet (NMR)  ®  trademark  Rf  retention factor  Rt  retention time  xxi  s  singlet (NMR) or strong (IR)  SD  standard deviation  SPECT  single photon emission computed tomography  t  triplet (NMR) or time  t1/2  half-life  TAME-Hex  tris(aminomethyl)- ethane-N,N,N’,N’,N’’,N’’-hexaacetic acid  THF  tetrahydrofuran  TFA  trifluoroacetic acid  TLC  thin layer chromatography  UV  ultraviolet  V  volt  w  weak (IR)     xxii  Acknowledgements What a crazy ride this has been. First and foremost, I must thank my dear husband and best friend, Labros. You are always there for me, make me laugh, listen to my complaints and encourage me to keep pushing for my dreams. My parents, for supporting their beloved, only child in moving across the world to pursue a career as a scientist instead of becoming a globetrotting WTA tennis player. The Bosses Chris and Mike are greatly acknowledged. Your persistence and belief in the Ga project for almost one decade prior to my arrival was the great basis for what followed. I was lucky to be at the right place at the right time. Thank you for all the support and the freedom to pursue all the other scientific interests I had, even if sometimes they were far off my “main” main group metal... It’s all a young, aspiring scientist could wish for. Cara, without you, this would not all have been possible. Thank you for your scientific help and your friendship. The girls: Ashlee, Danielle, Jen, Meryn, Tulin, it was so good to have you around, more girls nights out, please. Eric, balling! Scott, thank you for all your hard work night and day (literally) on the Cu/Tc project. “Little” Mike, for all your good work during the summer of 2010 and the introduction to Canadian rock music from the last century. The rest of the Orvig group for being around, always busy and hard working, making fabulous group meeting treats. All collaborators, essential to my work: Dr. Urs O. Häfeli (both of my Tc projects), Dr. Dawn Waterhouse and Dr. Brent Sutherland (animal studies), Dr. Janet McIntosh (for eluting the  68  Ga generator on oddest dates and times) Dr. Jacqueline  Cawthray (Potentiometry) and Dr. Dennis Wester (for supporting our collaborative work with NORDION). Dr. Brian O. Patrick for all the solid state structure elucidation, even when some of the crystals I brought down to the X-ray lab were incredibly minuscule. The MS Staff (Marshall, Derek, David, Yun) and the NMR staff, especially Maria, Jason and Paul for all their help. UBC is acknowledged for awarding me a Four Year Fellowship, NSERC and NORDION for the generous support of my research. I also want to thank some stellar scientists for motivating me. Roger: I will never forget how you told me “you are good, but slow”. I am working on that… I am grateful for everything you taught me. Peter, Thomas, Suzy, Tom, your enthusiasm and devotion to the science and to our field has and will continue to truly inspire me. xxiii  Dedication  To my grandfather, who never ceases to ask about my science, supports and encourages me.  xxiv  Chapter 1: Introduction 1.1  Medicinal Inorganic Chemistry  Metalloenzymes  Bioinorganic Chemistry  “What does nature do with metals?“  Medicinal Inorganic Chemistry “What can we do with metals?“  Figure 1.1 The author’s view of the field of bioinorganic chemistry.  Bioinorganic chemistry is a broad field, and can be generally split into two subfields: metalloenzyme research and medicinal inorganic chemistry (Figure 1.1).1 Metalloenzyme research tries to understand how metals are transported, stored and used in catalysis of essential biological processes. The area of interest of the Orvig group is medicinal inorganic chemistry. Paul Ehrlich, who succeeded in finding a treatment for syphilis by screening a library of organoarsenates in 1909, is regarded as one of the pioneers of the field.2 Since then, a myriad of coordination compounds has been investigated for therapeutic and diagnostic applications. Anticancer agents based on Pt such as Cisplatin,3 antirheumatic agents based on Au such as Auranofin,4 or perfusion imaging agents based on  99m  Tc such as  Cardiolite5 are some of the most widely applied metal-based drugs worldwide (Figure 1.2).  Figure 1.2 Cisplatin, Auranofin and Cardiolite (left to right).  1  The vast difference in structure and composition of these complexes, and the metals they contain, exemplifies the great variety of metals in medicine.  1.2  Medicinal Imaging Non-invasive imaging serves as one of the most important diagnostic tools in the  clinic. Besides X-ray images, which can only show the contrast between the heavier elements found for example in bone (Ca, P) versus the lighter elements found in soft tissues (C, H, O, N), other more sophisticated imaging techniques have been developed and are in use in the clinic.6 Magnetic resonance imaging (MRI) has become an increasingly important technique in the armamentarium of the modern clinician. This fairly young technology, originally introduced by Lauterbur in 1973,7 is a powerful tool to visualize the most abundant molecule in biological tissue, water. Compared to single photon emission computed tomography (SPECT) and positron emission tomography (PET), MRI has the advantages of no ionizing radiation and enhanced spatial resolution increasing the attractiveness of this technology.8 The ability of the magnetic moments of water protons to interact with an applied magnetic field, depending on the local environment of the water molecule, can be used to identify different tissues due to their characteristic consistency and water content.9 This effect can be greatly enhanced in the presence of paramagnetic contrast agents containing Gd(III).10 On its own, Gd(III) is highly toxic due to its similar size to Ca(II); therefore, Gd(III) is chelated to form thermodynamically stable complexes in order to decrease toxicity and improve clearance. Most contrast agents in current clinical applications are non-target specific agents11 and require high concentrations (≥ 0.1 mM) to achieve a detectable change in MRI signal.12 In the case of non-target specific, extracellular, commercial contrast agents with a relaxivity of about 4 mM-1 s-1, a concentration of approximately 125 μM is necessary to achieve robust tissue contrast.  2  MRI  SPECT γ  H2O  99mTc  Gd  PET γ β+ 68  Ga  γ  Detector Imaged object  Figure 1.3 Schematic description of imaging techniques mentioned in Chapter 1.2.  These criteria currently render MRI a somewhat limited technique and, for many applications inferior to techniques, based on radioactive elements, such as SPECT and PET (schemes provided in Figure 1.3). Radioactive elements have unstable nuclei, which decay by emitting one or more types of ionizing radiation: alpha particles (equivalent to a helium nucleus, α), positrons (β+), high energy electrons (β−) or gamma radiation (γ). α and β− are found to have less kinetic energy due to their increased mass; therefore, they have only a short range. The smaller the space over which the energy is transferred, the more damaging the interaction will be to its surroundings.13 This renders them useful for therapeutic applications, where large amounts of dose are delivered specifically to a certain area in order to damage that area of interest. The emission relevant for SPECT is a single photon, also denoted as γ. These light particles have great kinetic energy and will interact only weakly with tissue. The γ rays can be detected by a γ camera, consisting of a multihole lead collimator, a scintillating medium and an array of photomultipliers. After collection of an array of such 2D images, a tomographic algorithm is used to reconstruct the corresponding 3D image. As an added complication, the energy of the γ can greatly vary depending on the radionuclide used.14 This can represent a problem with radionuclides other than  99m  Tc, since most SPECT cameras are tuned for the emission properties of this  specific nuclide. PET provides immediate, higher resolution 3D information on the location of the 3  radiation source. PET is based on positron emission, denoted as β+. Being antimatter, any β+, regardless of its emission energy, always annihilates with an electron in the immediate vicinity of the area of decay and produces two 511 keV γ rays oriented at 180° to one another. Simultaneous detection of both γ rays allows for precise localization of the β+ source. This makes PET a desireable imaging technique. Both SPECT and PET have the great advantage of being highly sensitive techniques; the presence of a radionuclide at concentrations of 100 pM and lower eliminates any possible inherent pharmacological side effects of these compounds. This also reduces the “toxicity” of this type of imaging as the radiation exposure is about the same dose as a chest X-ray. In Table 1.1, the most widely used and investigated radionuclides for imaging purposes are summarized, together with their emission properties.15 Table 1.1 Widely used and investigated radionuclides for imaging.  radionuclide 11  t1/2  used in clinic?  imaging modality (relevant particle)  B  clinical trials16  PET (β+)  O  yes  PET (β+)  Ni  clinical trials16  PET (β+)  decays to  20.4 min  11  F  109.7 min  18  64  Cu  12.8 h  64  67  Ga  78 h  67  Zn  yes  SPECT (γ, 184 keV)  68  Ga  68 min  68  Zn  clinical trials16  PET (β+)  yes  SPECT (γ, 140.5 keV)  yes  SPECT (γ, 171 keV)  yes  SPECT (γ, 157 keV)  C  18  99m  Tc  6.01 h  99  111  In  67.4 h  111  13.22 h  123  123  I  Covalent incorporation of  11  C or  Tc Cd Te  18  F requires sequential synthetic techniques,  often even after the labelling step, while radiometals can be incorporated using simple coordination chemistry without additional chemical modifications post-labelling. This makes radiometals especially attractive for kit-formulations, where the nuclear medicine technician can synthesize the ready-to-use radiopharmaceutical by simple addition and  4  mixing  of  the  radionuclide  radiopharmaceutical.  Scheme  with 1.1  the  shows  non-radioactive the  preparations  radiopharmaceuticals FDG (18F-2-deoxy-2-fluoro-D-glucose) and  components of 99m  of  routinely  the used  Tc-Sestamibi. This  thesis focuses on fast and facile incorporation of a variety of radiometals into potential radiopharmaceuticals.  Scheme 1.1 Preparation of 18FDG and 99mTc-Sestamibi.  1.3  Therapy in Nuclear Medicine Another advantage of the use of radionuclides in the clinic includes the therapy  aspect. Some radionuclides emit α or β− particles upon decay, rendering them suitable for therapeutic applications. The mode of action of therapeutic radionuclides is the specific killing of cells by emitting a particle with a characteristic energy within a small area.15 A long half-life is preferred for a prolonged treatment time, as well as localization at the target site before most of the dose is decayed in order to keep the exposure to nontarget tissue as low as possible. β− emitters deliver particles with average ranges of 0.2 to 4 mm. α emitters are found to have a one order of magnitude shorter range, which could facilitate more target specific agent delivery without harming the surrounding non-target tissue; however, no α emitting radiopharmaceuticals are yet in use in the clinic.17 By conjugation of a targeting molecule such as an antibody, peptide or small 5  molecule that targets a specific type of tissue or tumor, to a radionuclide with imaging properties one can image the area of interest. By subsequent use of the same targeting molecule incorporating a radionuclide with therapeutic emissions, one can provide therapeutic value simply by switching the radionuclide. Radionuclides fullfilling these criteria are referred to as “imaging and therapy pairs”. An overview of radionuclides of current interest for therapeutic applications, as well as corresponding pairs is summarized in Table 1.2.15 Table 1.2 Widely used and investigated radionuclides for therapy.  radionuclide  t1/2  decays to  use in clinic?  emitted particle  avg. energy emitted  range*  imaging  [mm]  pair?  64  Cu  12.8 h  64  Zn  clinical trials16  β−  573 keV  ~2.1  64  Cu (β+)  67  Cu  61.9 h  67  Zn  no  β−  141 keV  0.26  64  Cu (β+)  Y  64.1 h  90  yes  β−  932 keV  4.0  186  Re  3.8 d  186  Os  yes  β−  349 keV  1.1  188  Re  17 h  188  Os  yes  β−  764 keV  3.1  99m  177  Lu  6.7 d  177  clinical trials16  β−  133 keV  0.23  177  7.2 h  207  clinical trials16  α  6.79 MeV  0.06  86 90  211  At  Zr  Hf  Bi  Y (β+)  (111In)18 99m  Tc19 (γ)  Tc (γ)  Lu (γ)  * mean range in H2O 64  Cu is an intrinsic imaging and therapy pair and therefore of great interest to the  radiopharmaceutical community. Chapters 3, 5 and 6 evaluate new chelate systems for this radionuclide, as well as a multifunctional chelate for use with 64Cu, Re and 99mTc.  6  1.4  Radioisotope Production and Generators  Cyclotron p+  Chemical processing  Example: 18FDG  Elute off target  Reactor Example: Chemical processing  n0  99mTc-Cardiolite  Generator system  Figure 1.4 Schematic production pathway for radiopharmaceuticals.  While many radionuclides have favourable properties for imaging and therapy, the ease of production can be a greatly limiting factor for both clinical applications and research. Radioisotopes can be produced in two main facilities: cyclotrons or reactors. Briefly, reactor-produced isotopes are furnished by irradiation of a nonradioactive target with a neutron flux in a specific energy range. Subsequently, the radioactive product is extracted from the target, processed and ready for incorporation into the actual radiopharmaceutical. Cyclotron-produced isotopes are synthesized by irradiation of a non-radioactive target with a beam consisting of protons, deuterions or alpha nuclei. The subsequent processing is analogous to reactor-produced isotopes (Figure 1.4). These types of immediate radiopharmaceutical production after generation of the radionuclide are dependent on the presence of a production facility (reactor or cyclotron) in the immediate vicinity. This is especially true for isotopes with relatively short halflives such as  18  F and  11  C. Despite the current commercial availability of cyclotrons that  7  are virtually 1/10 of the size of the cyclotrons manufactured in the 1980s, the need for isotope production in close proximity to the patient remains a greatly limiting factor for the application of radionuclides, which depends on this production pathway, such as  18  F  11  and C. Generator systems alleviate this spatial problem because they serve as portable radionuclide dispensaries. In order to furnish a generator system, the parent nuclide must have a reasonably long half-life, must be produced at a reactor or cyclotron facility, and should be immobilized on a sorbent. The parent nuclide then decays to produce the daughter nuclide, which can be conveniently eluted off the sorbent material and subsequently chemically processed to afford the radiopharmaceutical. Due to their small size, generators can be shipped and used at clinics far removed from cyclotron or reactor facilities. Cyclotron produced radionuclides and generator systems relevant to work presented in this thesis, together with production method and decay properties, are summarized in Table 1.3. Table 1.3 Widely used and investigated radionuclides and their production.15 daughter nuclide  parent nuclide  t1/2 parent  production of parent  nuclide  235 99m  Tc  99  188  Re  188  186  Re  -  68  Ga  67  Ga  64  Cu  Mo  68  W  66 h  98  Mo [n, γ] (reactor)  69.4 d  186  -  185  Re [n, γ] (reactor)  -  Ga[p, 2n] (cyclotron)  clinical trials16  -  -  68  -  -  Zn[p, 2n] (cyclotron)  64  99  yes no  69  The relative ease of production of  generator?  W [n, γ] (reactor)  271 d  Ge  U [n, f] (reactor)  clinically used  Ni[p, n] (cyclotron)  -  Mo for the past four decades, and the  favourable decay properties of both parent and daughter nuclide has led to domination of 8  the  imaging  field  by  radiopharmaceuticals are  the 99m  SPECT  nuclide  99m  Tc.  Hence,  the  majority  of  Tc-based agents, used in 85% of the millions of scans  performed annually in nuclear medicine.20  1.5  Isotope Supply Problems Due to aging reactor facilities that produce the global supply of  NRU in Chalk River, Ontario, the supply of  99  99  Mo such as the  Mo to all of North America, parts of South  America and Asia has been impacted by reactor shut-downs. This looming problem became a full-blown isotope crisis when the only operating reactor, NRU, was shut down unexpectedly in 2007, and the failure of the replacement reactors MAPLE 1 and 2 tore a large hole in the global isotope supply. Solutions to this isotope supply problem include two different, strongly contrasting approaches. One is to find a convenient route to produce parent nuclide  99  Mo via the increasingly available cyclotron facilities,  21  99m  Tc and/or its  and the other is  to investigate other radionuclides with favourable properties to replace  99m  radiopharmaceuticals. Due to the multiple decade “reign” of the market by  Tc-based  99m  Tc, the  development of other radionuclides with suitable properties for imaging has not been vigorously pursued. Now, in light of this recent crisis, alternatives are being seriously re-evaluated. The criteria for selection include: cyclotron production, optimal decay properties for PET or SPECT and suitably long half-life, with generator availability being strongly preferred. The radioisotopes  64  Cu and  68  Ga are amongst the most suitable candidates.  64  Cu is of  interest because of its dual β-/ β+ emission and reasonably long half-life which could allow shipping and applications for antibodies and other slowly localizing targeting vectors.  68  Ga is attractive because it can be eluted from a  Compared to the  99m  Tc-based system, the  68  68  Ge-based generator.  Ga-generator would also have a much longer  lifetime in the clinic. The positron emission would allow for higher resolution scans and the short half-life of is eluted as  99m  68  Ga would limit the exposure for patients. In addition, while  99m  Tc  TcO4- (oxidation state VII) from the generator and must be reduced in  most cases in order to have the capability to form coordination compounds, 68Ga is eluted  9  as its only stable and ready to chelate oxidation state III. While the original concept for the century ago,22 the problem of for several decades.  23  68  68  Ga-generator was developed over half a  Ge bleed-through upon column elution remained an issue  Only recently, due to rekindled interest in alternative generator  technologies, attempts have been made to solve this problem, but the current commercially available generators are designated “for research purposes only”.24 The generator system is not FDA approved, hence the number of current routinely used  68  Ga  based imaging agents in the clinic is zero. A clinical phase IV study on 68Ga-DOTA-NOC is recruiting patients currently,16 which is promising; however, the sophisticated labelling and purification procedure necessary adds an additional hurdle for the simple translation of this technology into the clinic.  1.6  Chelates for the Purpose of Radiometal Coordination Integrated approach Chelate  Tagged approach Both derivatives bind target  Chelate  Radionuclide  Radionuclide  Linker Targeting molecule  Targeting molecule  Target  Figure 1.5 Scheme of two complexes following two different methodologies for radiometal incorporation while targeting the same receptor.  Radiometals by themselves generally have no targeting ability. Specific targeting of tissues or organs can be achieved by coordination of the radiometal to specific scaffolds. This can be done by using two distinct methodologies: integration or tagging  10  (Figure 1.5). In the case of the integrated approach, the metal complex is part of the moiety relevant for target binding. For the tagged approach, the metal complex is not essential for binding of the target and is usually attached to the targeting moiety by a linker. Both methodologies require a chelate system to be present which binds the metal strongly in order to avoid metal loss by decomplexation in vivo. The chelate itself can strongly influence clearance or localization of the radiopharmaceutical.  1.7  Coordination Chemistry of 99mTc As the only naturally radioactive second row transition metal, Tc has no stable  isotope.  99  Tc is the most readily available isotope with a half-life of 2.13 x 105 years.25  While most Tc chemists were accustomed to working with this long lived radioisotope several decades ago, nowadays, strict regulations for work with extremely long lived isotopes has limited this practice. Now, the heavier, group 7 congener Re is frequently used to study the corresponding coordination complexes on a macroscopic level. Despite small differences in redox chemistry ([ReO4]- is somewhat harder to reduce then [TcO4]-) and ligand preference (Re is softer than Tc), the similarity between the two metals and their main, stable oxidation states is great enough for the validation of Re complexes as macroscopic placeholders for the corresponding microscopic 99mTc complexes.  Figure 1.6 Four of the most commonly used 99mTc synthons; Tc(oxo) with a MAG3-type coordination environment, [Tc(CO)3]+ coordinated by a typical tridentate ligand, Tc(nitrido) with an N4-xSx type chelate and Tc(HYNIC) with a typical coordination environment. All chelates can be derivatized through the variable R functionality.  11  Because of the extremely small quantity of the radiochemical complexes (~10-10 M), the most frequently used characterization tool is measurement of retention time on radio-HPLC or radio-TLC; the retention time of the fully characterized Re complex can be found on the corresponding UV-HPLC trace for comparison. Due to the extensive investigation of  99m  Tc over multiple decades and the variety  of oxidation states available, a great number of successfully used coordination environments have been developed. The different cores are preferentially coordinated by multidentate ligand systems, which can be modulated for their pharmacokinetic behaviour. The oxo core is among the earlier developments of  99m  Tc chemistry. Some of  the most frequently used coordinating ligands are of the N4-xSx type, first developed by Davison, Orvig and coworkers.26 Mercaptoacetyltriglycine (MAG3) remains one of the predominant ligand systems used in the clinic as a small molecule imaging agent,27 but it has also been widely explored as a bifunctional chelate.28-30 Drawbacks of this core are the harsh reduction conditions required using SnCl2. The reduction must be done in the presence of either the MAG3 ligand or with an intermediate easily exchangable ligand system such as glucoheptonate or gluconate, in order to avoid rapid re-oxidation or the formation of syn and anti isomers,31 which can exhibit different in vivo behaviour.32 Subsequently, the field of Tc chemistry endeavoured to find a new  99m  Tc core which  would obviate the need for harsh reducing agents and solve the isomer problem. Among two earlier solutions are the nitrido [TcN]2+-core33 and the Tc(HYNIC)core.34 While both systems exhibit interesting coordination chemistry and can produce stable complexes, the multi-step labelling procedure and difficulties in characterizing the chemical structure of the complexes formed render these systems incompatible with kit formulations. Abandoning the high oxidation state Tc(V)oxo-core however constituted a different approach; Alberto and coworkers first published a kit formulation, which produces the fac-[99mTc(CO)3]+ core35 with three labile aquo ligands. The compound [99mTc(CO)3(H2O)3]+ is stable and soluble in aqueous solution and the water ligands can be readily exchanged using bi- and tri-dentate ligand systems (Figure 1.6).36 The lowspin, d6 system exhibits increased inertness compared to the Tc(V)oxo-core (d2), slowing ligand exchange rates and providing a highly versatile, stable coordination environment, even with ligand systems such as the cyclopentadienyl moiety37 and truncated, nido-  12  carboranes.38 This facilitates simple modification of the pharmacokinetic behaviour by adaptation of the coordinating ligand system and has led to a wide variety of applications for small molecule imaging agents39-41 including bioconjugates of small molecules42,43 and a plethora of peptide conjugates.44-47 One aspect of the broad ligand tolerance of the [99mTc(CO)3]+ core is showcased in this thesis in Chapter 2.  1.8  Coordination Chemistry of 64Cu 64  Cu (t1/2 = 12.7 h, β+ 17.4 %, Emax = 0.656 MeV, β- 39 %, Emax = 0.573 MeV) is a  cyclotron-produced radionuclide of interest for both positron emission tomography (PET) and radiotherapy.48 Unlike Tc, Cu has naturally occurring stable isotopes, which serve as a convenient tool for the investigation and characterization of the coordination complexes formed, both micro- and macroscopically. The most relevant oxidation states present in aqueous solution are Cu(I) and Cu(II). As Cu(I), the metal ion has a d10 configuration with a preference for rather soft ligands, forming a variety of fairly labile complexes.49 Under normal, oxidative conditions the predominant oxidation state is Cu(II), where the metal ion has a d9 configuration with preference for borderline hard ligand systems. Predominant complex geometries with tetra- to hexadentate environments are square planar, trigonal bipyramidal, square pyramidal and distorted octahedral. 64Cu  Preferred donors and ligand system: COOH, R3N, NR2H, hexadentate  Overall charge: -1, 0, +1  Uptake in liver  64Cu  Transchelation of 64Cu  Serum proteins  Preferred labelling procedure: T < room temperature, t < 30 min.  64Cu  Point of derivatization with a targeting moiety available  Figure 1.7 Summary of desired properties for the optimal chelate for 64Cu, shown on the example of the 64Cu complex with a derivatized version of the cross-bridged macrocycle CB-TE2A.  The latter is prone to Jahn-Teller distortion, manifested in axial elongation or 13  equatorial compression of bonds in the corresponding complexes. Due to rapid ligand exchange kinetics, the stable coordination of Cu(II) is a difficult task, but important in radiochemical applications. Rapid coordination of the radiometal under mild conditions is an advantage (especially if sensitive biomolecules are present) but not essential. If dissociated from the coordinating ligand in vivo,  64  Cu rapidly associates with serum  proteins and subsequently accumulates in the liver, impacting clearance and possibly imaging.50 The overall charge of the complex must be -1, 0 or +1 in order to facilitate clearance of the complex in vivo. Cross-bridged macrocyclic and cage-like hexadentate ligand  systems  such  as  4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]-  hexadecane (CB-TE2A) are among the current front runners for stable coordination with minimal metal ion loss even after multiple days of incubation under physiologically relevant conditions (Figure 1.7). Acyclic chelates often fall victim to rapid decomplexation kinetics, resulting in the loss of the radiometal. Chapter 5 gives a more elaborate review of current literature on Cu(II) chelates for the purpose of radiolabelling and investigates an unusual acyclic chelate for the purpose of stable Cu(II) chelation. Work described in Chapter 3 attempts to bridge the vastly different 99mTc and 64Cu technologies.  1.9  Coordination Chemistry of 67/68Ga Although Ga is a main group metal, it has many similarities to transition metals  with respect to its coordination chemistry. Because of its low redox potential, Ga(III) is the only relevant oxidation state in aqueous media, with its valence electrons in a d10 configuration. While tetradentate to hexadentate coordination is observed with geometries ranging over tetrahedral,51 square pyramidal52 and distorted octahedral,53-56 many Ga coordination complexes tend to be rather labile. In order to be suitable for applications as a radiopharmaceutical, the Ga(III) ion must be coordinated in a stable fashion; preventing the radiochemical complex from in vivo hydrolysis to water soluble [Ga(OH)4]- (the predominant species at physiological conditions) or transchelation by the iron scavenging protein transferrin leading to subsequent bone uptake (Figure 1.8).57  14  68Ga  Overall charge: -1, 0, +1  Preferred donors and ligand system: COOH, R3N, NR2H, SH, hexadentate  Uptake in bone 68  68Ga  Ga  Transchelation of 68Ga  Transferrin Preferred labelling procedure: T < room temperature, t < 15 min. Point of derivatization with a targeting moiety available  Figure 1.8 Summary of desired properties for the optimal chelate for 68Ga, shown on the example of the 68Ga complex with a derivatized version of the macrocycle NOTA.  Ga(III) is present as the hydrated cation at pH < 3, so coordination must happen under acidic conditions. Paired with the short half-life of  68  Ga (68 minutes), these  limitations make the coordination of this radiometal non-trivial. The Ga(III) cation prefers hexadentate chelation using hard ligand systems much like a redox innocent Fe(III). The similarity to Fe(III) in charge and ionic radius explains the high affinity of Ga(III) for apo-transferrin. Few ligand systems have established themselves as reliable bifunctional  chelates  for  radiopharmaceutical  applications.  These  ligands  are  predominantly macrocylic, and form complexes with Ga(III) with an overall charge of -1, 0 or +1 in order to facilitate clearance of the complex in vivo. Due to the slow kinetics of most macrocycles compared to acyclic chelates,58 only one routinely used functionalized chelate, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), is capable of coordinating 68  Ga at room temperature within less then 15 minutes and holding on to the metal in a  stable fashion in vivo.58 The only  68  Ga-based radiopharmaceutical in clinical studies,  68  Ga-DOTANOC  (Figure 1.9)59 relies on a complex pre- and post-labelling purification system, because the macrocyclic chelate used for this conjugate is 1,4,7,10-tetraazacyclododecane N,N',N",N'"-tetraacetic acid (DOTA), a chelate with less than favourable labelling properties.60 A kit formulation, where the generator is directly eluted into a vial containing the ligand system, forming a ready-to-inject radiopharmaceutical and  15  obviating the need for lengthy purification procedures could be a major advantage for a compound with potential for clinical applications. Rapid coordination is an attractive property for kit formulations, thus acyclic chelates may have great and so far underestimated potential if fast decomplexation can be impeded.  Figure 1.9 DOTANOC, a somatostatin analogue currently used with 68Ga in clinical trials.  The introduction to Chapter 4 completes this small overview given above, while the experimental part elaborates on the screening of acyclic chelates containing amino, pyridine and carboxylate ligands for the fast and efficient coordination of  68  Ga at room  temperature. This represents both a continuation of, but also a somewhat new spin on, the Orvig group’s two decade long investigation of group 13 metal complexes.  1.10 Thesis Overview This thesis focuses on exploring the synthesis of ligand systems and their corresponding coordination chemistry with metals of great relevance for nuclear medicine applications. The ultimate goal in each chapter is the investigation of the radioactive complex and the corresponding non-radioactive coordination complex, but great detail of the actual organic syntheses of ligand systems is included, as the ligand systems are integral to the development of potential imaging agents. All systems investigated are acyclic ligands containing pyridine and/or carboxylate donor groups. Chapter 2 focuses on the synthesis, chemistry and biological behaviour of three amphiphilic complexes of Re and  99m  Tc(CO)3 with different coordination spheres while  carrying the same lipophilic aliphatic “tail”. The multi-step synthesis is a key focus of  16  this chapter, which also includes the coordination compounds, radiochemistry and contrasting biodistributions of the three radiolabelled complexes. Chapter 3 attempts to bridge the contrasting coordination chemistry of the [M(CO)3]+ core (M = Re,  99m  Tc) with  64  Cu by designing and synthesizing both a model  compound and a biotin conjugate of a flexible, acyclic ligand scaffold capable of fast coordination of both radiometals. While the basic ligand structure had been reported previously,61 the functionalization has not been previously attempted. The tetradentate ligand system investigated in Chapter 3 provides the basis for the small library of acyclic ligands with various bite sizes and denticities investigated for Ga(III) radionuclide coordination in Chapter 4. The various acyclic ligand systems were compared with respect to radiolabelling efficiency and the stability of the resulting complex. The most suitable ligand system (termed H2dedpa, Figure 1.10) was found to be surprisingly stable against apo-transferrin. Subsequently, two model compounds for possible functionalization and conjugation to targeting molecules were also synthesized and investigated for coordination properties with radioactive and non-radioactive Ga. Chapter 5 investigates the viability of the H2dedpa ligand system for the coordination of radioactive and non-radioactive isotopes of Cu. Redox properties were also investigated; these are relevant for comparison to other frequently used ligand systems for 64Cu. Chapter 6 describes the functionalization of the H2dedpa ligand system with targeting moieties, following the tagged approach (Figure 1.5). This chapter has a significant synthetic focus and culminates in the successful syntheses of two conjugates of the small, cyclic peptide RGD that targets angiogenesis.62 Subsequently, both conjugates were tested for their ability to coordinate the radionuclides  67/68  Ga and  64  Cu,  and yield results similar to those obtained for the corresponding, non-functionalized model compounds investigated in Chapters 4 and 5. Chapter 7 investigates functionalization of the ligand system following the integrated approach (Figure 1.5). Because H2dedpa forms a cationic complex upon coordinating Ga(III), attempts were made to mimic the routinely used SPECT agents for myocardial perfusion imaging, which are cationic, lipophilic structures. These extensive synthetic efforts culminated in seven different H2dedpa derivatives exhibiting a range of  17  lipophilicity. The biodistribution of these compounds was also investigated to evaluate their potential for cardiac imaging. Finally, Chapter 8 summarizes and concludes the work showcased in this thesis and gives some perspectives for future work with the H2dedpa scaffold.  Figure 1.10 H2dedpa.  18  Chapter 2: Small Amphiphilic Rhenium and 99mTechnetium Tricarbonyl Complexes* 2.1  Introduction Because of its availability and low cost,  99m  Tc has been a radionuclide of interest in  radiopharmaceutical chemistry and nuclear medicine for the past four decades.63 Tc also has ideal physical properties (t1/2 =  6 h, γ =  141 keV) for imaging with single photon emission computed tomography (SPECT).64 As a result, its chemistry has been extensively researched and the vast majority of nuclear medicine scans utilize this isotope. While many compounds in the clinic still rely on high oxidation state  99m  Tc cores65 demanding harsh labelling  conditions, more recent research has focused on the versatile and highly stable fac[99mTc(CO)3(H2O)3]+  complex developed by Alberto et al.35 The fac-[99mTc(CO)3(H2O)3]+  complex has a 99mTc(I) metal center in a d6 low-spin electron configuration, rendering it inert toward a broad range of experimental conditions. It can be prepared in high yields via a commercially available kit (Isolink, Covidien, Mansfield, MA) under aqueous conditions using the boranocarbonate anion as the reducing agent and in situ CO source.35 Simple exchange of the three water molecules can be achieved with suitable chelates in a fac coordination mode,66,67 shown previously in examples of small molecule constructs as potential perfusion agents, or in targeted agent bioconjugates such as derivatized amino acids, carbohydrates, peptides, and nucleosides.36,68,69 These mild labelling conditions are more applicable for use with potential highly sensitive biomolecules.  Figure 2.1 Examples of the integrated (left) and tagged (right) approach on steroid scaffolds. *  Work presented in this chapter has been published: "Design, Synthesis, and Imaging of Small Amphiphilic Rhenium and 99mTechnetium Tricarbonyl Complexes", E. Boros, U. O. Häfeli, B. O. Patrick, M. J. Adam, C. Orvig , Bioconjugate Chem 20 , 1002-1009 (2009).  19  Two distinct methodologies can be followed to incorporate  99m  Tc into novel imaging  agents: the so-called “tagged” approach.70 and the “integrated” or “technetium-based” approach.71 Examples are represented in Figure 2.1 from the work of Katzenellenbogen and coworkers, who have found ways of using both methodologies on the same targeting moiety. The work in this chapter describes efforts towards obtaining a simple, small, and non-target specific complex following the “integrated” approach. It was the aim to explore the in vivo properties of novel, amphiphilic metal complexes of the [99mTc(CO)3]+ core. In an attempt to achieve this, the synthesis incorporated a mimic of the charge distribution of molecules highly abundant and essential to cell membranes, aiming for an in vivo distribution of the resulting complexes comparable to other small amphiphilic molecules such as cocamidopropyl betaine (CAPB, zwitterionic), sodium dodecylsulfate (SDS, anionic),72 and glyceryl laurate (neutral) (Figure 2.2). These compounds, also known as surfactants, are known to integrate into the lipid bilayer before their concentration (much larger than the concentration range imaging agents usually operate at) exceeds the saturation concentration and solubilizes the membrane.73 By utilizing significantly shorter lipophilic chains, it is proposed that a large fraction of liver uptake can be obviated, opposed to complexes bearing longer aliphatic carbon chains.74  Figure 2.2 Examples of commercial surfactants with different polar head groups (denoted in red): neutral, anionic and zwitterionic (from top to bottom).  20  Various entities were examined, including a neutral, anionic and amphiphilic complex. Bidentate attachment onto the [99mTc(CO)3]+ core has been widely explored by previous members of the Orvig group and has been found to have decreased in vitro stability, hence tridentate chelates were chosen.40 Two ligand systems investigated in this work have been explored previously and their stability and coordination mode have been found to have excellent properties.36 Each lipophilic residue was incorporated by attachment of a short, saturated C8-chain in a consistent fashion via a simple ester bond joined with an ethanolamine linker piece. By forming the coordination complex, compounds with an overall structure analogous to commercially available surfactants (Figure 2.2) are furnished. This differs strongly from the previously used tagged approach with long-chain fatty acids used as lipiodol surrogates for liver targeting,74 as well as from conventional fatty acid type labelling for the purpose of cardiac imaging,75,76 where carboxylic acids are not used for coordination but for potential oxidation by the fast metabolizing heart muscle (Figure 2.3). 77  Figure 2.3 Previously reported [Tc(CO)3]+ complexes bearing long aliphatic chains.74,76  21  2.2  Experimental 2.2.1  Materials and Methods  Re(CO)5Br is commercially available (Strem). The analytical thin-layer chromatography (TLC) plates were aluminum-backed ultrapure silica gel 250 μm; the flash column silica gel (standard grade, 60 Å, 32‐63 mm) was acquired from Silicycle. 1H and  13  C NMR spectra  were recorded on Bruker AV300 or AV400 instruments at ambient temperature; the NMR spectra are expressed on the δ scale and referenced to residual solvent peaks or internal tetramethylsilane. Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded on a Micromass LCT instrument at the Department of Chemistry, University of British Columbia. High-performance liquid chromatography (HPLC) analysis of cold compounds was done on a Phenomenex Synergi 4 μm Hydro-RP 80A column (250 mm × 4.6 mm) on a Waters WE 600 HPLC system equipped with a 2478 dual-wavelength absorbance UV detector run using the Empower software package. IR spectra were collected neat in the solid or liquid state on a Thermo Nicolet 6700 FT-IR spectrometer. TLC analysis of radiolabeled compounds was performed on a Bioscan-System 200 Imaging scanner equipped with a Bioscan-Autochanger 100. Radio-TLCs were prepared with acetonitrile as the mobile phase. HPLC analyses of radiolabeled complexes were performed on a Knauer Wellchrom K-1001 HPLC equipped with a K-2501 absorption detector and a Capintec radiometric well counter. A Phenomenex Hydro- Synergi 4 μm C18 RP analytical column with dimensions 250 × 4.6 mm was used for both analysis and purification. HPLC solvents consisted of 0.1% trifluoroacetic acid in water (solvent A) and methanol (solvent B). Samples were analyzed and purified with a linear gradient method (100% solvent A to 100% solvent B over 30 min). For biodistributions, organ samples were counted in a Packard Cobra II autogamma counter one to two days after administration. μSPECT and CT images were recorded on an XSPECT instrument from Gamma Medica (Northridge, CA).  22  2.2.2  Ligand Synthesis  (2-Hydroxyethyl)carbamic acid tert-butyl ester (1). Boc2O (2.18 g, 10 mmol) was added to ethanolamine (610 mg, 10 mmol) at 0 °C and allowed to stir for 1 h. A colorless precipitate formed immediately with the evolution of heat. Column chromatography (hexanes/ethyl acetate, silica, 6:1) with subsequent evaporation of the solvent yields 1.45 g (9.9 mmol, yield 90 %, Rf 0.1) of the desired product as a colourless oil. 1H NMR (300 MHz, CDCl3, δ): 3.55 (t, 3J1,2 = 5.8 Hz, H1, 2H), 3.14 (t, 3J1,2 =5.8 Hz, H2, 2H), 1.33 (t, H5, 9H). 13C NMR (75 MHz, CDCl3, δ): 155.6 (C3), 79.3 (C4), 61.6 (C1), 42.9 (C2), 28.1(C5). IR (cm‐1): 1682 (m), 1517 (m), 1164 (m). HR-ESI-MS calcd. for C7H15NNaO3: 184.1887, found: 184.1874 [M +  Na]+.  Octanoic acid 2-tert-butoxycarbonylaminoethyl ester (2). (2-Hydroxyethyl)carbamic acid tert-butyl ester (1, 1.45 g, 9.9 mmol) was dissolved in toluene (50 mL)  to  afford  a  (ethyldimethylamine,  clear 20  solution.  mL)  and  EDMA octanoyl  chloride (1.908 g,11.78 mmol, 1.3 eq) were added dropwise simultaneously. A cloudy precipitate formed immediately, and the reaction solution was stirred at room temperature for another 1.5 h, after which time the solvent and a large fraction of the added EDMA were evaporated under reduced pressure. The residue was redissolved in H2O (30 mL) and extracted into ether (30 mL) twice. The organic phase was collected and the solvent evaporated under reduced pressure to afford the crude product as a yellow oil which was purified by column chromatography (silica, hexanes/ethyl acetate, 1:1, Rf =  0.6) to yield octanoic acid 2-tert-butoxycarbonylaminoethyl ester (1.505 g, 5.45 mmol, yield 53 %) after solvent evaporation. 1H NMR (400 MHz, CDCl3, δ): 4.07 (t, H7, 2H), 3.33 (t, 3J1, 2 = 7.6 Hz, H2, 2H), 2.27 (t, 3J1, 2 =7.6 Hz, H1, 2H), 1.57 (m, CH2, 2H), 1.40 (s, H5, 9H), 1.24 (m, CH2, 8H), 0.83 (t, H8, 3H).  13  C NMR (100 MHz, CDCl3, δ): 173.5 (C6), 155.7 (C3), 79.6 (C4),  63.2 (C1), 39.55 (C2), 33.9, (C7), 31.5, (CH2), 28.7 (CH2), 28.2 (C5), 24.7 (CH2), 22.4  23  (CH2), 13.9 (C8). IR (cm‐1): 1684 (m), 1631 (m), 1482 (w). HR-ESI-MS calcd. for C15H29KNO4: 326.1734, found: 326.1731 [M + K]+.  Octanoic acid 2-aminoethyl ester (3). 2-tert-Butoxycarbonylaminoethyl ester (2, 1.505 g, 5.45 mmol) was dissolved in dichloromethane (4 mL). Trifluoroacetic acid (4 mL) was added, and the reaction solution was stirred for 20 min after which time a slow change of colour to light yellow was observed. TFA and CH2Cl2 were removed in vacuo, and the oily residue was purified by column chromatography (CH2Cl2/ CH3OH, 4:1, Rf = 0.15) yielding 2-aminoethyl ester octanoic acid (444 mg, 2.37 mmol, yield 43 %) after rotary evaporation as a colourless oil which solidifies upon standing. 1H NMR (300 MHz, CD3OD, δ): 3.55 (t, 3J1, 2 = 5.8 Hz, H1,2H), 3.25 (t, 3J1, 2 = 5.8 Hz, H2, 2H), 2.18 (t, 3J4, CH2 = 6.01 Hz, H4, 2H), 1.57 (m, CH2, 2H), 1.28 (m, CH2, 8H), 0.86 (t, CH3, 3H). 13C NMR (75 MHz, CD3OD, δ): 173.7 (C3), 62.3 (C1), 43.4 (C2), 37.6‐23.9 (six signals, CH2), 14.7 (CH3). IR (cm‐1): 3312 (w), 2954 (s), 2924 (s), 2855 (s), 1637 (s),1552 (m), 1464 (m). HR-ESI-MS calcd. for C10H21NNaO2: 210.1470, found: 210.1481 [M + Na]+.  Octanoic acid 2-[(pyridin-2-ylmethyl)amino]ethyl ester (4). Octanoic acid 2-aminoethyl ester (3, 0.51 g, 2.7 mmol) was dissolved in dry ethanol (15 mL). Na2CO3 (10 equiv, 2.86 g) was added to afford a turbid solution. Pyridine-2-aldehyde (0.27 g, 237 μL, 0.95 eq) was added, and the reaction mixture was stirred overnight at room temperature. After formation of the intermediate imine was confirmed by TLC, NaBH4 (95 mg, 2.51 mmol, 0.98 eq) was added and the reaction mixture was subsequently stirred for another 1 h. CH2Cl2 (20 mL) was then added and extracted twice with a saturated solution of NaHCO3 (20 mL). The organic fractions were collected, and the solvent was evaporated. The crude product was purified by column chromatography (silica, CH2Cl2/ CH3OH, 85:15, Rf = 0.55) yielding octanoic acid 2-[(pyridin-2-ylmethyl)amino]ethyl ester (361 mg, 1.3 mmol, yield 48 %) as a colourless oil with low viscosity, after evaporation of the solvent. 1H NMR (300  24  MHz, CDCl3, δ): 8.48 (m, 3J6, 7 = 5.6 Hz, H7, 1H), 7.66 (m, 3J4, 5 = 7.7 Hz , H5, 1H), 7.32 (d, 3  J4, 5 = 7.7 Hz, H4, 1H), 7.17 (t, 3J6, 7 = 5.6 Hz, H6, 1H), 3.91 (s, H3, 2H), 3.63 (m, H2, 2H),  2.14 (t, 3J9,  CH2  = 6.01 Hz, H9, 2H), 1.56 (m, CH2, 2H), 1.22 (m, CH2, 8H), 0.82 (t, 3J10,  CH2  =  13  6.3 Hz, H10, 3H). C NMR (75 MHz, CDCl3, δ): 174.5 (C8), 149.0 (C7), 148.3 (C5), 136.8 (C4), 122.3 (C6), 120.7 (C7), 64.3 (C3), 61.6 (C2), 54.1 (C1), 42.1-22.5 (six signals, CH2), 13.9 (C10). IR (cm-1): 3294 (br), 2925 (s), 2855 (s), 1632 (s), 1595 (s), 1549 (m). HR-ESIMS calcd. for C17H30N2NaO2: 317.2205, found: 317.2188 [M + Na]+. Octanoic acid 2-(tert-butoxycarbonylmethylpyridin-2-ylmethylamino)ethyl ester (5). Octanoic acid 2-[(pyridin-2-ylmethyl)amino]ethyl ester (4, 361 mg, 1.3 mmol) was dissolved in dry CH2Cl2 (20 mL). Na2CO3 (about 10 eq, 2.86 g) was added to afford a turbid solution. tert-Butyl bromoacetate (1.4 eq, 354 mg, 1.8 mmol) was added and the mixture was stirred for 2 days. The solvent was evaporated after confirmation of product presence by ESI-MS. The crude product was purified by column chromatography (silica, CH2Cl2/ CH3OH, 95:5, Rf = 0.8) and yielded the final product (109 mg, 0.27 mmol, yield 21%) after rotary evaporation of all solvents as a light yellow oil. 1H NMR (300 MHz, CDCl3, δ): 8.52 (d, 3J7, 8 = 4.1 Hz, H8, 1H), 7.68 (m, 3J5, 6 = 5.2 Hz, H6, 1H), 7.56 (d, 3J5, 6 = 5.2 Hz, H5, 1H), 7.17 (m, 3J7, 8 = 4.1 Hz, H7, 1H), 4.16 (t, 3J1, 2 = 5.7 Hz, H1, 2H), 4.03 (s, H4, 2H), 3.40 (s, H3, 2H), 3.00 (t, 3J1, 2 = 5.7 Hz, H2, 2H), 2.26 (t, 3J12, CH2 = 7.6 Hz, H12, 2H), 1.56 (m, CH2, 2H), 1.46 (s, H10, 9 H), 1.26 (m, CH2, 8H), 0.87 (t, 3J13, CH2 = 6.0 Hz, H13, 3H). 13C NMR (75 MHz, CDCl3, δ): 184.6 (C9), 173.7 (C11), 148.6 (C8), 136.8 (C6), 122.9 (C5), 122.1 (C7), 62.5 (C1), 60.3 (C4), 56.2 (C3), 52.5 (C2), 34.2‐22.5 (six signals, CH2) 28.2 (C10), 14.0 (C13). HR-ESI-MS calcd. for C22H36N2NaO4: 415.2573, found: 415.2584 [M + Na]+.  25  Octanoic acid 2-(bis-tert-butoxycarbonylmethylamino)ethyl ester (6). 2-tert-butoxycarbonylaminoethyl ester (3, 0.1 g, 0.53 mmol) was dissolved in dry CH2Cl2 (10 mL). Na2CO3 (1 g) was added to afford a turbid solution. tert-Butyl bromoacetate (209 mg, 179 μL, 1.06 mmol, 2 eq) was added, and the mixture was stirred for 2 days. After the presence of the product was confirmed by ESI-MS, the solvent was evaporated and the crude mixture was purified by column chromatography (silica, hexanes/ethyl acetate, 1:1, Rf = 0.8) to yield the desired product (60 mg, 0.144 mmol, yield 27 %) after rotary evaporation of all solvents. 1H NMR (75 MHz, CDCl3, δ): 4.13 (t, 3  J1, 2 = 4.6 Hz, H1, 2H), 3.69 (s, H3, 4H), 3.45 (t, 3J1, 2 = 4.6, Hz, H2, 2H), 2.95 (t, 3J8,  CH2  =  5.4 Hz, H8, 2H), 1.55 (m, CH2, 2H), 1.41 (s, H6, 18 H), 1.21 (m, CH2, 8H), 0.84 (t, H9, 3H). 13  C NMR (75 MHz, CDCl3, δ): 170.2 (C7), 166.0 (C4), 62.7 (C1), 58.2 (C3), 52.4 (C2), 34.1‐  22.4 (six signals, CH2), 27.6 (C6), 13.9 (C9). HR-ESI-MS calcd. for C22H41NNaO6: 317.2832, found: 317.2844 [M + Na]+.  Bis-tert-butoxycarbonylmethyl(2-octanoyloxyethyl)pyridin-2-ylmethylammonium bromide (7). This compound is afforded as a side product of the synthesis of N-N-(tert-butylcarboxyethyl)pyridinemethyl-2-aminoethyl ester octanoic acid and separated from the main product 5 by column chromatography (silica, CH2Cl2/ CH3OH, 95:5, Rf = 0.35) yielding the product (37 mg, 0.072 mmol, yield 5.6 %) after solvent evaporation as a light orange oil. 1H NMR (300 MHz, CDCl3, δ): 9.59 (d, 3J7, 8 = 4.8 Hz, H8, 1H), 8.48 (t, 3J5, 6 = 7.7 Hz, H6, 1H), 8.21 (d, 3J5, 6 = 7.7 Hz, H5, 1H), 8.05 (t, 3J7, 8 = 4.8 Hz, H7, 1H), 4.38 (t, H1, 2H), 4.10 (s, 3J1, 2 = 5.4 Hz, H4, 2H), 3.71 (s, H3, 2H), 2.92 (t, 3J1, 2 = 5.4 Hz, H2, 2H), 2.26 (t, 3J12, CH2 = 7.5 Hz, H12, 2H), 1.56 (m, CH2, 2H), 1.47 (s, H10, 18 H), 1.23 (m, CH2, 8H), 0.83 (t, 3J13, CH2 = 5.9 Hz, H13, 3H). 13C NMR (75 MHz, CDCl3, δ): 187.2 (C9), 173.7 (C11), 169.5 (C8), 164.7 (C6), 155.4 (C5), 128.8 (C7), 64.7 (C2), 61.7, (C4) 55.3 (C3), 52.65  26  (C12), 34.04‐22.42 (six signals, CH2) 27.25 (C10), 13.91 (C13). HR-ESI-MS calcd. for C28H47N2O6+: 507.3429, found: 507.3411 [M]+.  Octanoic acid 2-(carboxymethylpyridin-2-ylmethylamino)ethyl ester (HL1). Octanoic  acid  2-(tert-butoxycarbonylmethyl  pyridin-2-ylmethylamino)ethyl ester (5, 109 mg, 0.27 mmol) was dissolved in dry CH2Cl2 (1 mL). TFA (1 mL) was added, and the reaction mixture was stirred for 30 min at room temperature. Xylene (1 mL) was added, and all the solvents were evaporated under reduced pressure to yield the product HL1 (45 mg, 0.133 mmol, yield 49 %) after solvent evaporation. 1H NMR (400 MHz, CD3OD, δ): 8.76 (d, 3J7, 8 = 5.4 Hz, H8, 1H), 8.41 (t, 3J5, 6 = 7.6 Hz, H6, 1H), 7.93 (d, 3  J5, 6 = 7.6 Hz, H5, 1H), 7.86 (t, 3J7, 8 = 5.4 Hz, H7, 1H), 4.48 (s, H3, 2H), 4.22 (t, 3J1, 2 = 5.1  Hz, H2, 2H), 3.78 (s, H4, 2H), 3.23 (t, 3J1, 2 = 5.1 Hz, H1, 2H), 2.28 (t, 3J11, CH2 = 7.6 Hz, H11, 2H), 1.56 (m, CH2, 2H), 1.29 (m, CH2, 8H), 0.9 (t, 3J12, CH2 = 6.8 Hz, H12, 3H).  13  C NMR  (100 MHz, CD3OD, δ): 184.2 (C9), 171.4 (C10), 156.1 (C8), 146.1 (C6), 144.4 (C5), 127.0 (C7), 84.2 (C3), 63.1 (C4), 57.8 (C2), 55.3 (C11), 35.2-23.9 (six signals, CH2), 13.9 (C12). IR (cm-1): 2929 (w), 2858 (w), 1727 (m), 1668 (m). HR-ESI-MS calcd. for C18H29N2O4: 337.2127, found: 337.2120 [M + H]+.   Octanoic acid 2-(bis-carboxymethylamino)ethyl ester (H2L2). The exact same procedure as for HL1 was followed with octanoic acid 2-(bis-tert-butoxycarbonylmethylamino)ethyl ester (6, 120 mg, 0.288 mmol) to yield H2L2 (56 mg, 0.184 mmol, yield 64 %). 1H NMR (400 MHz, CD3OD, δ): 4.36 (t, 3J1,  2  = 5.2 Hz, H1,  2H), 3.85 (s, H3, 4H), 3.33 (t, 3J1, 2 = 5.2 Hz, H2, 2H), 2.35 (t, 3J6, CH2 = 7.5 Hz, H6, 2H), 1.61 (m, CH2, 2H), 1.32 (m, CH2, 8H), 0.89 (t, 3J7, CH2 = 6.0 Hz, H7, 3H). 13C NMR (100 MHz, CD3OD, δ): 183.61 (C4), 177.18 (C5), 62.45 (C3), 57.55 (C2), 55.64 (C1), 38.33 - 24.10 (six signals, CH2), 16.22 (C7). IR (cm-1): 2928 (w), 2857 (w), 1737 (m), 1682 (m). HR-ESI-MS calcd. for C14H26N2O6: 304.1760, found: 304.1739 [M + H]+.  27  Bis-carboxymethyl-(2-octanoyloxy-ethyl)-pyridin-2-ylmethylammonium trifluoroacetate (H2L3)(TFA). The exact same procedure as for HL1 was followed with bis-tert-butoxycarbonylmethyl(2-octanoyloxy -ethyl)pyridin-2-ylmethylammonium chloride (7, 37 mg, 0.072 mmol) to afford H2L3 (12 mg, 0.0303 mmol, yield 42 %). 1H NMR (400 MHz, CD3OD, δ): 8.95 (d, 3J7, 8 = 5.2 Hz, H8, 1H), 8.65 (t, 3J5, 6 = 8.8 Hz, H6, 1H), 8.30 (t, 3J5, 6 = 8.8 Hz, H5, 1H), 8.11 (t, 3J7, 8 = 5.2 Hz, H7, 1H), 4.14 (t, 3J1, 2 = 5.2 Hz, H1, 2H), 4.07 (s, H4, 2H), 3.91 (s, H3, 4H), 3.91 (t, 3J1, 2 = 5.2 Hz, H2, 2H), 2.19 (t, 3J11, CH2 = 7.5 Hz, H10, 2H), 1.60 (m, CH2, 2H), 1.31 (m, CH2, 8H), 0.90 (t, 3J12, CH2 = 6.7 Hz, H11, 3H). 13C NMR (100 MHz, CD3OD, δ): 186.22 (C9), 172.1 (C10), 169.48 (C8), 163.94 (C6), 128.20 (C5), 65.31 (C4), 61.79 (C2) 56.65 (C3), 52.65 (C11), 37.26-23.81 (six signals, CH2), 14.54 (C12). IR (cm-1): 2927 (w), 2857 (w), 1731 (m), 1630 (m). HR-ESI-MS calcd. for C20H31N2O6+: 395.2177, found: 395.2197 [M]+.  2.2.3  Octanoic  acid  Synthesis of Re(CO)3+ Complexes  2-(carboxymethylpyridin-2-ylmethylamino)ethyl  ester  tricarbonyl  rhenium(I) [Re(L1)(CO) 3]. A solution of [NEt4]2[ReBr3(CO)3] (40 mg, 0.052 mmol) and HL1 (17 mg, 0.052 mmol) in CH3OH (5 mL) was refluxed overnight. The solution flask was placed in the freezer for 2 h to afford the product as a white solid (21 mg, 0.034 mmol, yield 66 %) after filtration. Small, colourless crystals were obtained by slow diffusion of the product dissolved in CH2Cl2 into diethyl ether. HPLC-Rt= 21.3 minutes. 1H NMR (300 MHz, CDCl3, δ): 8.84 (d, 3J9, 10 = 5.1 Hz, H10, 1H), 7.79 (m, 3J9, 10 = 5.1 Hz, H9, 1H), 7.56 (d, 3J7, 8 = 7.6 Hz, H7, 1H), 7.46 (t, 3J7, 8 = 7.6 Hz, H8, 1H), 4.55 (dd, 2J3, 3’ = 14.4 Hz, H3, 2H), 4.48 (m, 3J1, 2 = 7.2 Hz , H2, 2H), 3.89 (s, H5, 2J5, 5’ = 16.8 Hz, 2H), 3.78 (q, 3J1, 28  2  = 7.2 Hz, H1, 2H), 2.40 (t, 3J12, CH2 = 7.7 Hz, H12, 2H), 1.54 (m, CH2, 2H), 1.30 (m, CH2,  8H), 0.89 (t, 3J13, CH2 = 6.9 Hz, H13, 3H). 13C NMR (125 MHz, CDCl3, δ): 196.8 (CO), 196.1 (CO), 195.1 (CO), 178.1 (C10), 172.8 (C9), 157.5 (C10), 152.5 (C9), 139.6 (C7), 125.7 (C8), 122.8 (C6), 84.2 (C3), 68.3 (C5), 67.4 (C1), 61.5 (C2), 69.6 (C12) 35.2-23.9 (six signals, CH2), 8.5 (C13). IR (cm-1): 2020 (m) (νCO), 1905 (w) (νCO), 1870 (m) (νCO). HR-ESI-MS calcd. for C21H28N2O7185Re: 605.1426, found: 605.1410 [M + H]+.   Sodium octanoic acid 2-(bis-carboxymethylamino)ethyl ester tricarbonylrhenate Na[Re(L2)(CO) 3]. A solution of [NEt4]2[ReBr3(CO)3] (40 mg, 0.052 mmol) and H2L2 (16 mg, 0.052 mmol) in CH3OH (5 mL) was allowed to reflux overnight. The solvent was evaporated, and the oily residue was purified by preparative HPLC to afford the product as a white solid (5 mg, 0.0087 mmol, yield 15 %) after rotary evaporation of all solvents. HPLC-Rt= 14.01 minutes. 1H NMR (300 MHz, CD3OD, δ): 4.38 (s, 2J3, 3’ = 16.2 Hz, H3, 4H) 3.67 (t, 3J1, 2 = 6.0 Hz , H2, 2H), 3.78 (t, 3J1, 2 = 6.0 Hz, H1, 2H), 2.39 (t, H6, 2H), 1.62 (m, CH2, 2H), 1.31 (m, CH2, 8H), 0.90 (t, 3J7, CH2 = 6.7 Hz, H7, 3H).  13  C NMR (125 MHz, CD3OD, δ): 196.8  (CO), 195.7 (CO), 177.5 (C4), 172.9 (C5), 66.5 (C3), 62.5 (C1), 62.1 (C2), 60.16 (C6) 33.0 21.8 (six signals, CH2), 12.5 (C7). IR (cm-1): 2017 (m) (νCO), 1881 (m, br) (νCO), 1606 (w). HR-ESI-MS calcd. for C17H24N2O9185Re: 570.0902, found: 570.0914 [M]‐.     Bis-carboxymethyl-(2-octanoyloxyethyl)pyridin-2-ylmethylammonium tricarbonylrhenium [Re(L3)(CO) 3]. A solution of [NEt4]2[ReBr3(CO)3] (4 mg, 0.0052 mmol) and H2L3 (1.6 mg, 0.0052 mmol) in CH3OH (5 mL) was allowed to reflux overnight. The solvent was evaporated, and the oily residue was purified by preparative HPLC to afford the product as a  29  yellow solid (3 mg, 0.0045 mmol, yield 86 %). HPLC-Rt= 18.7 minutes. 1H NMR (400 MHz, CD3OD, δ): 8.96 (d, H10, 1H), 8.66 (t, H8, 1H), 8.30 (d, H7, 1H), 8.08 (t, H9, 1H), 5.71 (s, H3, 4H), 4.36 (t, H5, 2H), 4.14 (s, H2, 2H), 3.97 (t, H1, 2H), 2.32 (H12, 2H), 1.54 (m, CH2, 2H), 1.30 (m, CH2, 8H), 0.90 (t, H13, 3H).  13  C NMR (125 MHz, CD3OD, δ): 197.2 (CO),  195.5 (CO), 176.5 (C4), 173.1 (C11), 151.1 (C9), 137.9 (C7), 127.1 (C8), 121.6 (C6), 72.5 (C3), 68.1 (C2), 64.5 (C1), 62.1 (C12) 38.0 - 22.2 (six signals, CH2), 11.7 (C13). IR (cm-1): 2018 (s) (νCO), 1894 (m) (νCO), 1874 (m), 1661 (w). HR-ESI-MS calcd. for C23H30N2O9185Re: 663.1481, found: 663.1483.  2.2.4  X-ray Diffraction Analysis of [Re(CO)3L1]  Colourless crystals of [Re(CO)3L1] were obtained by slow diffusion of the product dissolved in CH2Cl2 into diethyl ether. The sample was mounted on a glass fiber and cooled to 173 K. X-ray data were collected and processed using a Bruker X8 APEX II diffractometer using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) to a maximum 2θ value of 48.0°. Data were collected and integrated using the Bruker SAINT software package78 and corrected for Lorentz and polarization effects, as well as for absorption effects, using the multiscan technique (SADABS).79 The X-ray structure was solved using direct methods (SIR92)80 and expanded using Fourier techniques.81 All calculations were performed using the SHELXTL crystallographic software package.82 The collection and analysis of the data was done by Dr. Brian O. Patrick.  2.2.5  Synthesis of 99mTc(CO)3 Complexes  The organometallic precursor [99mTc(H2O)3(CO)3]+ was prepared from a saline solution of Na[99mTcO4] (1 mL, 5.4 mCi) using the Isolink kit. A solution of Na[99mTcO4] (1 mL) was added to the Isolink kit, and the vial was heated to reflux for 25 min. Upon cooling, 0.1 M HCl solution (1 mL) was added to adjust the pH to 7-8. Labelling was achieved by mixing an aliquot (0.2 mL) of the [99mTc(H2O)3(CO)3]+ precursor with a 0.1 mM aqueous solution of L1-L3 (pH 7-7.5, 0.1 M NaHCO3 solution, 1 mL) at 90 °C for 25 (L1), 35 (L2), or 45 (L3) min. Analysis was performed by HPLC and TLC. HPLC solvents consisted of 30  0.1% trifluoroacetic acid in water (solvent A) and neat methanol (solvent B). Samples were analyzed with a linear gradient method (100% solvent A to 100% solvent B over 30 min). TLC’s were developed with acetonitrile as the mobile phase.  2.2.6  Cysteine and Histidine Stability Challenges  To a solution of either cysteine (0.1 M, 0.9 mL) or histidine (0.1 M, 0.9 mL) in PBS (1 mM, pH 7.4) was added a solution of the  99m  Tc complex (0.1 mL, final ligand  concentration 10-5 M). The samples were incubated at 37 °C and aliquots analyzed after 24 h by analytical TLC and HPLC.  2.2.7  Octanol-Water-Distribution Coefficients  The log Po/w values of complexes [99mTc(CO)3L1], Na[99mTc(CO)3L2], and [99mTc(CO)3L3] were determined by extraction with equivalent amounts of octanol and aqueous solutions of the  99m  Tc complex.83 Octanol (1 mL) was mixed with an equivalent  amount of the aqueous reaction mixture and stirred for 1 min. Subsequently, the two phases were partitioned, and the activity of both was determined by the measurement of activity of 5 μL droplets of each solution on a radio-TLC-plate.  2.2.8  Imaging and Biodistribution  Animals were used in accordance with the regulations on the protection of animals in Canada. Female C57Bl/6 mice were obtained from Sarah Reid, McMaster University. Each mouse had a 4 mm tumor in its neck, grown from a primary cell line at passage six derived from mammary tumors of a PyVMT transgenic mouse.  99m  Tc complexes were prepared as  described above. While two complexes [99mTc(CO)3L1] and Na[99mTc(CO)3L2], were injected without any further purification because of their high radiochemical yield, [99mTc(CO)3L3] was purified by preparative HPLC before injection. The solvent was removed from the collected fraction in vacuo, and [99mTc(CO)3L3] was redissolved in 0.9 % NaCl saline solution. Aliquots (100 μL) of complex solutions of [99mTc(CO)3L1], Na[99mTc(CO)3L2], and [99mTc(CO)3L3] were injected into groups of four mice via their tail  31  vein. After injection of [99mTc(CO)3L1] and Na[99mTc(CO)3L2], dynamic imaging was carried out during the first 15 min. All animals were imaged under isoflurane anesthesia in the supine position during the first 15 min, and then 1, 2, and 4 h after injection for 15 min each. After the last time point, animals were sacrificed by pentobarbital administration, and samples of blood, liver, kidney, muscle, spleen, heart, brain, lung, gallbladder, intestine, stomach, bladder and tumor were removed, weighed, and counted in a gamma counter. Results are expressed as the percentage of the injected dose per gram of tissue (% ID/g). Imaging of the animals on their SPECT/CT scanner was done by Troy Farncombe, Chantal Saab, and Rod Rhem. Biodistribution and subsequent evaluation of the imaging and biodistribution data was done by Prof. Urs O. Häfeli.  2.3  Results and Discussion  2.3.1  Ligand Preparation  Scheme 2.1 Synthetic methods to furnish ligand systems HL1, H2L2, and H2L3. (i) Boc2O, neat (ii) Octanoyl 1-chloride, EDMA, toluene, (iii) TFA/ CH2Cl2 (1:1) (iv) tert-Butyl bromoacetate, Na2CO3, CH2Cl2 (v) Pyridine-2-aldehyde, EtOH.  32  The tridentate chelate moieties of HL1 and H2L2 were selected for this study because they have been previously shown to form highly stable complexes with the metals of interest.36 The chelation entity of H2L3 is novel, arising from the synthesis of HL1 where its tert-butyl-protected form appears as a byproduct in the synthesis of tert-butyl protected HL1. All three ligand systems were synthesized from ethanolamine. Intermediate 1 (Scheme 2.1) was synthesized through protection of the free amine of ethanolamine as the tert-butyl carbamate or Boc, in order to prevent reaction of the acid chloride with the much more reactive amine in the subsequent reaction step. Compound 1 was then esterified using the acid chloride of octanoic acid, forming 2. Subsequent deprotection of the amine yields 3, which served as a synthon to furnish all three ligand systems. For ligand systems HL1 and H2L3, 3 was reacted with one equivalent pyridine-2aldehyde to form an imino intermediate, which was then reduced to afford 4 in a one pot reaction (Scheme 2.1). Subsequent alkylation of 4 yields both protected ligand systems 5 and 7. Due to the added ionicity of the quaternary amine on 7, separation of the two products is easily achieved with column chromatography, as both compounds had easily distinguishable Rf values. Deprotection to yield HL1 and H2L3 proceeds through acid catalyzed saponification, cleaving exclusively the tert-butyl protection group.84 In order to synthesize the H2L2 system, intermediate 4 was alkylated with two equivalents of tert-butyl bromoacetate. Double alkylation and subsequent purification with column chromatography yields the protected ligand 6. Analogous to deprotection of 5 and 7, the tert-butyl protection groups are removed using acid catalysis, leaving the aliphatic ester unaltered.84 The identity of all ligands was confirmed by characterization through 1H and  13  C  NMR spectroscopy, as well as mass spectrometry and IR spectroscopy. All compounds synthesized have the appearance of thick oils, characteristic of compounds bearing aliphatic chains of significant length. 2.3.2  Preparation of Re Complexes  [Re(CO)3]+ complexes were synthesized as macroscopic models for the  99m  Tc(CO)3  complexes by mixing a methanolic solution of ligand with [NEt4]2[ReBr3(CO)3] previously dissolved in H2O, and stirring the mixture at elevated temperatures overnight.85 While the  33  neutral complex [Re(CO)3L1] precipitates from the reaction solution, solutions of the complexes Na[Re(CO)3L2] or [Re(CO)3L3] turn slightly yellow in color without precipitation of the product. Na[Re(CO)3L2] and [Re(CO)3L3] were isolated and purified via preparative HPLC. A sample of characteristic 1H spectra, documenting the change of environment of specific protons upon coordination is shown (Figure 2.4) for HL1 and [Re(CO)3L1].  Figure 2.4 1H NMR spectra (300 MHz, 298 K) of HL1 (a) and [Re(CO)3L1] (b), * denotes residual reference solvent peak, while characteristic diastereotopic protons are highlighted with their assignments (water and silicone grease, at 1.56 and 0.07 are also observed in both spectra as minor impurities).  The CH2 groups bearing H3 and H4 appear as singlet peaks in HL1 and H2 appears as a triplet, coupling to H1. In [Re(CO)3L1], the same protons, formerly homotopic protons, now appear as two diastereotopic protons with different chemical shifts showing strong 2J coupling to each other. Both H3 and H4 within [Re(CO)3L1] appear as a doublet of doublets,  34  while the highly structured conformation of the complex causes H2 to appear as two distinct multiplet signals due to coordination of the neighbouring amine, which constitutes one of the donor atoms within the coordination complex (Figure 2.4).69 The  13  C NMR spectra of the  complexes show other characteristic features such as the diagnostic peaks for the nonequivalent carbonyls. In the case of [Re(CO)3L1], three non-equivalent peaks are observed between 195.1 and 196.8 ppm, mirroring the different chemical environment of each carbonyl of this complex.86 In both Na[Re(CO)3L2] and [Re(CO)3L3], however, only two peaks are observed for the carbonyls on the fac-[Re(CO)3]+ core, since both these complexes are found to have a mirror plane. Observation of all carbonyl peaks in the region of 194 – 197 ppm is characteristic for these types of complexes.87 Another useful characterization tool is IR spectroscopy, where intense, diagnostic CO bands between 2020 and 1875 cm-1 can be assigned to the fac- [Re(CO)3L]-products. While in [Re(H2O)(CO)3]+ the characteristic stretching frequencies can be found at 2000 and 1868 cm-1, a shift to higher wave numbers is observed upon coordination.87 These carbonyl stretching frequencies can be easily distinguished from the other carbonyl stretching frequencies present, which originate from the two types of carbonyls present in the ligand systems. The carbonyl linking chelate and aliphatic residue are only mildly affected by the event of metal coordination, while the coordinating carbonyl is affected in all cases. Due to interaction with the metal center, the IR frequency is slightly decreased.  Figure 2.5 Complexes described within this chapter.  Mass spectrometry also gave conclusive results on identifying the desired complexes (Figure 2.5). Due to two stable Re isotopes present (187Re 62.6 %,  185  Re 37.4 %) the  corresponding ([Re(CO)3L1] + H)+, [Re(CO)3L2]- and ([Re(CO)3L3] + H)+ appear with the diagnostic isotope distributions of Re. It is interesting to note that due its zwitterionic nature, the overall charge of [Re(CO)3L3] is indeed neutral. This is also confirmed by the [M + H]+ observed. 35  2.3.3  X-Ray Diffraction Structural Characterization of [Re(CO)3L1]  The [Re(CO)3L1] complex (Figure 2.6, Table 2.1) displays a nearly perfect octahedral coordination sphere, with the three carbonyl ligands facially coordinated at approximately 90° to one another. The bond lengths between the individual CO ligands are in accordance with other Re(CO)3 complexes reported.88 The Re-CO bond lengths are all close to 1.9 Å and therefore shorter than the Re-O bond length (2.142 Å) and the Re-N bond lengths (2.184 Å, 2.227 Å). The Re-N2 bond proves to be the longest because of the nondirected lone pair on the tertiary amine nature of the nitrogen atom N2.  Figure 2.6 ORTEP view of [Re(CO)3L1], showing the atom numbering scheme (50% thermal ellipsoids). Table 2.1 Selected bond lengths in [Re(CO)3L1] ([Å]) and angles ([°]). bond  length [Å]  angle  degree[°]  C(9)-Re(1)  1.928(12)  O(2)-Re(1)- N(1)  78.2(3)  C(10)-Re(1)  1.909(12)  N(1)-Re(1)- N(2)  77.2(3)  C(11)-Re(1)  1.918(11)  N(2)-Re(1)- C(11)  97.0(4)  N(1)-Re(1)  2.184(8)  C(9)-Re(1)- C(11)  88.6(5)  N(2)-Re(1)  2.227(8)  C(10)-Re(1)- C(11)  88.8(5)  O(2)-Re(1)  2.142(6)  C(10)-Re(1)- C(9)  87.1(5)  C(10)-O(4)  1.158(13)  C(10)-Re(1)- O(2)  97.5(4)  C(11)-O(5)  1.168(14)  C(10)-Re(1)- N(1)  96.8(4)  36  2.3.4  Radiolabelling  Scheme 2.2 Sample synthesis scheme for 99mTc(CO)3 complexes, here shown on [99mTc(CO)3L1]. (v) NaHCO3 (0.1 M) (vi) [99mTc(H2O)3(CO)3]+ , 90°C, 25-45 min.  Figure 2.7 γ-HPLC traces of the crude reaction mixtures of [99mTc(CO)3L1] (left ) and [99m Tc(CO)3L2]- (right).  The complexes [99mTc(CO)3L1] and [99mTc(CO)3L2]- were prepared in high radiochemical yield through reaction of fac-[99mTc(CO)3(H2O)3]+ with the ligands HL1 and H2L2. Due to residual acidity after HPLC purification of the ligands using an acidic aqueous mobile phase, the ligands were initially deprotonated with 0.1 NaHCO3 (2 molar eq) and added to the technetium tricarbonyl solution with final concentrations of approximately 3 × 10-4 M. After a reaction time of 25-35 min at 90 °C, the chelation reaction was 95% complete as verified by HPLC and radio-TLC analysis. No major by-products were detected by HPLC. A sample reaction scheme is shown (Scheme 2.2) as well as γ-HPLC traces of the crude reaction mixtures (Figure 2.7). The retention times mirror the difference between charged and neutral complexes well.  37  Figure 2.8 γ-HPLC traces of the crude reaction mixtures of [99mTc(CO)3L3], SM denotes starting material, P denotes product and SP stands for side products.  [99mTc(CO)3L3] was produced in only 32 % yield after a 45 min reaction time. Several by-products were visible in the γ-trace of the crude HPLC spectrum from coordination with decomposition fragments of H2L3, resulting from the high-temperature reaction conditions (Figure 2.8). Cleavage of one of the chelate arms to afford one of the other ligand systems is unlikely, since the retention time of the decomposition product does not match any of the other retention times observed. Since the reaction proceeds in the presence of considerable amounts of base, a reaction mechanism similar to a Hofmann type E2 elimination, which typically proceeds in the presence of heat and base, seems plausible. The observed, shorter retention time of the side product would be in accordance with such a process; however, synthetic investigations on the corresponding cold complexes were not undertaken. The peak of the desired product was assigned by comparison of the retention time of the fully characterized Re complex isolated by HPLC purification. The investigated properties described in this subchapter are outlined in Table 2.2.  38  Table 2.2 Summarized experimental properties of the three 99mTc(CO)3 complexes.  Complex  Radiochemical yield [%]  t [min]  T [°C]  Ligand conc. (M)  tR [min] 99m  Log Po/w  Rf  [99mTc(CO)3L1]  > 95%  25  90  3x10-4  20.5 (21.3)  -0.034  0.49  [99mTc(CO)3L2]-  > 95%  35  90  3.3x10-4  15.6 (14.01)  -1.014  0.39  [99mTc(CO)3L3]  32%  45  90  1x10-3  18.1 (18.7)  -0.38  0.46  2.3.5  Tc (Re)  In Vitro Stability and log Po/w  To investigate the complex stability toward the naturally occurring amino acids cysteine and histidine (both can act as potential tridentate ligands),88 the  99m  Tc-complexes  were incubated in 10-3 M cysteine/histidine solutions in PBS (pH 7.4, 37 °C) for 24 h. In the subsequent HPLC analyses of the solutions, no noticeable decomposition could be detected for any of the three complexes, rendering them stable in vitro. The lipophilicity of the complexes was determined by evaluation of water/octanol partition coefficients. [99mTc(CO)3L2]- should be the most ionic compound by virtue of its full -1 charge, followed by the zwitterionic [99mTc(CO)3L3] and then the neutral [99mTc(CO)3L1]; this was confirmed by the order of lipophilicity log Po/w values ranging from [99mTc(CO)3L1] > [99mTc(CO)3L3] > [99mTc(CO)3L2]- (listed in the order from highest to lowest lipophilicity, Table 2.2). This was also confirmed by order of HPLC retention times, where the most lipophilic complex will be retained for the longest time on the column. Radio-TLC measurements match in a similar manner, since the most lipophilic compound will have the greatest retention factor, whereas the most polar will have the smallest. It is important to note however that these compounds, especially the charged complex, are considerably lipophilic despite the presence of a fairly large polar fraction. The [99mTc(CO)3]+ core contributes strongly to this lipophilicity.  39  2.3.6  In Vivo Evaluations  Imaging of all three compounds was performed at 15 min, 1 h, 2 h, and 4 h after injection. Biodistribution studies were performed at the 4 h time point after injection (Table 2.3). This tumor-bearing model was used to evaluate the imaging potential of [99mTc(CO)3L1], [99mTc(CO)3L2]-, and [99mTc(CO)3L3]. Data from the imaging is displayed  in Figure 2.9. Figure 2.9 Activity (counts/pixel) levels vs. time curves for each of the evaluated compounds in select organs taken from the planar scintigraphy images.  Imaging screenshots of mice 15 min postinjection are shown in Figure 2.10. All compounds show very distinct biodistribution behaviour. The in vivo distribution of [99mTc(CO)3L1] was followed from the onset of injection for the first 15 minutes. Very fast localization of the complex in the liver was observed, with clearance through the intestines, which is confirmed through the biodistribution data at the 4 h time point. The compound  40  showed large uptake of activity in liver, intestines, and feces. This behaviour is not surprising considering that this complex has no overall charge and a barely negative log Po/w value. Compound [99mTc(CO)3L2]- was also followed after injection with dynamic imaging. Here, fast processing of the compound through the kidneys, within the first 5 - 10 min after injection, and consequent localization in the bladder, followed by excretion, was observed. The excretion of large portions of the dose is confirmed by the small fraction of the originally injected dose in the biodistribution measurements at the 4 h time point. The negative log Po/w value of this compound, due to its localized negative charge, determines its fate to be excreted through the renal tract.89 Finally, for [99mTc(CO)3L3] after 1 h after injection, high localization in the gall bladder was observed, paired with slow clearance through the intestines. The biodistribution profile shows some similarity to the profile of [99mTc(CO)3L1], but with faster excretion through the intestines and the renal system (high percentage of dose in the feces, elevated organ/blood ratio in kidneys), which may be a consequence of the increased polarity of the complex (compared to [99mTc(CO)3L1]). No significant heart, brain, or tumor uptake was observed for any of the investigated compounds.  Figure 2.10 Representative µSPECT/CT pictures taken 15 min after injection of A) [99mTc(CO)3L1], B) [99mTc(CO)3L2]- and C) [99mTc(CO)3L3]. Transverse (left) and coronal (right) planes shown in each case. Hot spots within the images are the liver in A, bladder and some residual activity in the kidneys in B and the gall bladder with liver uptake and bladder in C.  41  Table 2.3 Biodistribution, % ID/g ± SD (n = 4) of all complexes at the 4 h time point.  2.4  Organ  [99mTc(CO)3L1]  [99mTc(CO)3L2]-  [99mTc(CO)3L3]  Blood  0.73 ± 0.17  0.42 ± 0.12  0.68 ± 0.24  Heart  0.42 ± 0.05  0.20 ± 0.04  0.48 ± 0.14  Tumor  0.27 ± 0.03  0.16 ± 0.06  0.18 ± 0.02  Liver  7.37 ± 1.35  2.34 ± 0.49  6.20 ± 1.46  Kidney  3.43 ± 0.37  1.75 ± 0.18  4.14 ± 1.34  Lung  0.68 ± 0.12  0.45 ± 0.08  0.65 ± 0.18  Intestines  1.50 ± 0.49  0.37 ± 0.20  1.69 ± 0.75  Brain  0.02 ± 0.01  0.02 ± 0.01  0.02 ± 0.01  Bladder  1.57 ± 1.57  0.74 ± 0.66  3.07 ± 2.59  Muscle  0.20 ± 0.07  0.03 ± 0.02  0.26 ± 0.26  Spleen  0.36 ± 0.04  0.38 ± 0.12  0.38 ± 0.18  Feces  65.34 ± 43.03  9.14 ± 4.49  132.37 ± 86.29  Gallbladder  52.52 ± 40.40  24.83 ± 18.58  13.28 ± 14.74  Conclusions For the first time, small amphiphilic  99m  Tc(CO)3 complexes, one with a localized  negative charge, one zwitterionic complex, and one neutral molecule, have been synthesized, fully characterized, and investigated for their lipophilicity and behavior in vivo. For the zwitterionic complex, a new type of chelation entity was incorporated, forming eightmembered rings between the metal center and the ligand sphere. Growing crystals of this complex in order to determine the coordination environment was attempted for an extended period of time but failed due to the “sticky” nature of this complex, arising from its zwitterionic, amphiphilic molecular structure. This novel chelate proved to be slower coordinating than the other two systems investigated, but still have high stability toward cysteine and histidine challenges. Lower reaction yields arose from the instability of the ligand toward prolonged heating at 90 °C, and the formation of by-products which could be separated by HPLC. The products afforded by this decomposition were capable of coordinating to the metal center making an additional purification step inevitable.  42  For a more efficient mimic of phospholipid-type molecules, a technetium-tagged approach may be more suitable, such as for example attachment of a small neutral  99m  Tc  complex to the aliphatic residue of the corresponding amphiphilic molecule. In order to increase the impact of the lipophilic tail on the biodistribution of the entire molecule, the aliphatic chain could be further elongated. Changing the charge of a small amphiphilic molecule resulted in highly contrasting biodistribution behaviour in vivo. This is somewhat surprising considering the negligible impact of the lipophilic residue. This may have importance in the future design of small molecule imaging agents using the [99mTc(CO)3]+ core.  43  Chapter 3: Investigation of the H2dipin Scaffold as Chelate with Adjustable Denticity for 64Cu, Re and 99mTc†  3.1  Introduction: Multifunctional Chelate Systems The development of new radiopharmaceuticals for imaging and therapy of disease is a  constantly burgeoning field.60,90 Due to the availability and properties of the 99mTc generator over the past 3 decades, the development of novel imaging agents has centred around this particular isotope20 and has been strongly affected by the recent shortage of its parent 99Mo. The Orvig group, like many other research groups, has recognized a need for closer investigation of potential imaging agents based on alternative isotopes60,91 and has longstanding experience with stable and radioactive transition and main group metals.92 Of specific importance is the ability to choose isotopes according to their properties such as t1/2, mode and energy of decay, range of emitted particle, or even availablity.93,94 The need for different chelates specific to each radiometal, or particular leaving groups for the incorporation of radiohalogens or  11  C, imposes a non-negligible additional difficulty and  often obviates the fast and simple synthesis of analogous targeting vectors for labelling with a different radionuclide. In this chapter, the design and synthesis of a versatile, universal chelate system, able to label a variety of metals of different oxidation states and varying coordination environment preferences, is described. All metals investigated in this chapter have radioisotopes that are of considerable interest for either imaging or therapy. 64  Cu (t1/2=12.7 h, β+ 17.4%, Emax=0.656 MeV, β- 39%, Emax= 0.573 MeV) is of  strong current interest for both positron emission tomography (PET) and radiotherapy.48 The longer half-life is more applicable to developing PET agents with larger biomolecules, such as affibodies, that may require longer circulation times before imaging to achieve optimal target uptake.95  †  Work presented in this chapter has been published: "One to Chelate Them All: Investigation of a Versatile, Bifunctional Chelate for 64Cu, 99mTc, Re and Co", E. Boros, Y.-H. S. Lin, C. L. Ferreira, B. O. Patrick, U. O. Häfeli, M. J. Adam, C. Orvig , Dalton Trans. 40 , 6253-6259 (2011).  44  99m  Tc is the most commonly used isotope in nuclear medicine, partly due to its near  ideal physical properties (t1/2 = 6 h, γ = 140 keV).64 The [99mTc(CO)3]+ core can be conveniently prepared by the Isolink kit developed by Alberto and coworkers96 and serves as a scaffold that can be easily chelated through exchange of the three labile H2O molecules to form highly stable complexes with tridentate ligands.36 The stable isotopes of Re serve as cold congeners for radioactive Tc and are used to optimize non-radioactive chemistry and perform standard characterization techniques, while  186/188  Re is attractive for therapeutic β-  applications. 186Re (t1/2 = 90 h, β = 1.07 MeV, max. range in tissue = 5 mm, γ = 137 keV) is suitable for treating smaller tumours, whereas 188Re (t1/2 = 17 h, β = 2.1 MeV, max. range in tissue = 11 mm, γ = 155 keV) is suitable for treating larger tumours. produced (neutron irradiation of activity from a  188  185  Re), while  188  186  Re is reactor  Re can be furnished with high specific  W/188Re generator system in the form of [188ReO4]–.97,98 Due to great  similarities with the group 7 congener Tc, ligand systems used with the latter are usually adequate for stable and radioactive Re isotopes. All (radio-)metals described have a preference for being coordinated by polyaminocarboxylate ligands of a variety of denticities.90  3.1.1  Prior Art on H2dipin Type Chelates  Figure 3.1 Examples of the previously reported ligand systems DpK (SAAC approach46) and bifunctional, multi-purpose ATSE.94  Despite the fact that most acyclic ligands have low stability with Cu(II),99 select examples  such  as  the  thiosemicarbazone  H2ATSM  (diacetyl-bis(N-4-  45  methylthiosemicarbazone)) still are a viable option for the stable chelation of this metal (Figure 3.1).100 This chelate has been used to synthesize potential small molecule imaging agents for hypoxia, as well as the bifunctional chelate ATSE/A (Diacetyl-2-(4-N-ethyl-3thiosemicarbazone)-3-(4-N-amino-3-thiosemicarbazone),94 which was shown to chelate both  64  Cu and  99m  Tc. However, the identity of the Tc complex could never be fully  assigned and both radiochemical complexes exhibited only moderate serum stability.94 The versatile SAAC (single amino acid chelate) approach of Valliant, Babich, Zubieta and coworkers42 has been used to successfully introduce a chelate for the [99mTc(CO)3]+ core into peptide sequences, however the tridenticity of this approach limits applications for other metals. There is a limited set of examples of multifunctional chelation systems seen in the literature that incorporate both the group 7 metals, as well as other commonly used trivalent radiometals.55,101  Figure 3.2 Multifunctional ligand system dip described in this chapter; the variable denticity is indicated by the arrows.  By introduction of two additional carboxylate arms, the dipicolyl amine chelation entity can be enhanced in denticity to form the “dip” (di-picolyl-carboxylate) ligand system which contains two pyridine carboxylate moieties joined by a benzyl amine functionality for derivatization. The chelate system itself was previously investigated for Pb2+ and found to bind this particular metal ion in a pentagonal, planar fashion.61 In this chapter, different chelation modes of dip are investigated, namely tetradentate, square planar coordination of the the divalent metal Cu2+ and fac-tridentate for the [M(CO)3]+ cores (M = Re,  99m  Tc) (Figure 3.2). A model bifunctional chelate  system was developed and the viability of the system for simple functionalization was illustrated through synthesis and coordination of biotin conjugates.  46  Biotin is a water soluble molecule also known as vitamin H (Figure 3.3).102 In vivo, biotin acts as a coenzyme involved in metabolism and in the production of important biomolecules such as fatty acids and antibodies. At the same time, it has a very high affinity towards avidin and streptavidin (Kd ~ 10-15M),103 which has led to many biochemical applications that are dependent on this strong affinity.104 The biotin-avidin interaction has also been investigated in the area of targeted cancer radiotherapy.105 Conjugation of the bifunctional chelate described in this chapter to biotin is therefore highly relevant, and serves as a convenient proof of principle for the feasibility of the ligand design.  Figure 3.3 Biotin.  3.2  Experimental  3.2.1  Materials and Methods  Refer to Chapter 2.2 for general information. A Phenomenex Jupiter 5 μm C18 RP analytical column with dimensions 100 ×  4.6 mm was used to purify [Re(CO)3(Me2dipinbiotin)]Br. HPLC solvents consisted of 0.1% trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B). Samples were analyzed and purified with a linear gradient method (100% solvent A to 100% solvent B over 30 min).  64  Cu was obtained as a dilute HCl  solution (MDS Nordion); it was commercially available from Nordion at the time of the 64  Cu experiments. Product specification reports specific activity of the  64  Cu to be >  5000Ci/g with < 0.2 micrograms of Cu per mCi. The HPLC system used for analysis of the  64  Cu labelled compounds consisted of a Waters Alliance HT 2795 separation module  equipped with a Raytest Gabbistar NaI detector and a Waters 996 photodiode array (PDA) detector. The analyses of 64Cu labelled compounds and their serum stabilities were analyzed on a Waters XBridge BEH130 4.6 mm × 150 mm column. [99mTcO4]- was provided by Vancouver Coastal Health UBC hospital. Reaction monitoring of  99m  Tc 47  experiments was performed by TLC (see above), using a mobile phase mixture of methanol and dichloromethane (1:4). Radioactive TLCs were measured using a phosphor imager (Cyclone storage phosphor imager with (20 × 25) cm2 phosphor screen, PerkinElmer,  Waltham,  MA,  USA)  and  analyzed  using  OptiQuest  software.  The  [99mTc(H2O)3(CO)3]+ precursor was synthesized using the Isolink kit (Covidien, St. Louis,  MO).  hydrochloride  4-(Aminomethyl)-N-(9-fluorenylmethoxycarbonyl)  phenylamine  and biotin-TFP were prepared according to literature steps.104,106 4-  (Aminomethyl)-N-(9-fluorenylmethoxycarbonyl)phenylamine  hydrochloride  and  4-  (aminomethyl)-N-(9-fluorenylmethoxycarbonyl)phenylamino-di-pyridine-2-carboxylic acid methyl ester was synthesized by Scott Lin as part of his bachelor thesis.107  3.2.2  Synthesis of Ligands  6-Formylpyridine-2-carboxylic acid methyl ester (1). 6-Hydroxymethylpyridine-2-carboxylic acid methyl ester (2.333 g, 13.95 mmol) and SeO2 (1.421 g, 12.80 mmol) were refluxed in 1, 4dioxane (80 mL) for 2.5 hours. The reaction mixture was cooled to room temperature, and the black solid was filtered off. Dioxane was removed from the filtrate by rotary evaporation. A mixture of white and red solids remained. The crude product was purified by column chromatography (silica, chloroform) as an offwhite solid (1.107 g, 6.70 mmol, yield 48 %). 1H NMR (300 MHz, CDCl3, δ): 10.20 (s, H8, 1H), 8.35 (dd, 3J4, 5 = 7.5 Hz, 4J4, 6 = 1.1 Hz, H4, 1H), 8.15 (d, 3J5, 6 = 7.8 Hz, 4J4, 6 = 1.1 Hz, H6, 1H), 8.04 (t, 3J4, 5 = 7.5 Hz, 3J5, 6 = 7.8 Hz, H5, 1H), 4.08 (s, H1, 3H).  13  C NMR (75  MHz, CDCl3, δ): 192.8 (C8), 165.0 (C2), 152.9 (C7), 148.7 (C3), 138.6 (C5), 129.2 (C4), 124.5 (C6), 53.5 (C1). HR-ESI-MS calcd. for C8H8NO3: 166.0504, found: 166.0501 [M + H]+.  48  6-[(4-Nitrobenzylamino)methyl]-di-pyridine-2-carboxylic acid methyl ester (2, Me2dipin-NO2). 4-Nitrobenzylamine·HCl (0.457 g, 2.42 mmol) was stirred in dichloromethane (7 mL). NaOH (1 M, 3.5 mL) and H2O (2 mL) were added to the suspension, and the mixture was stirred for 30 minutes until all solids dissolved. The organic layer was separated from the aqueous layer, which was extracted with dichloromethane (5 x 5 mL). The combined organic layer was reduced by rotary evaporation to afford an orange oil. To this orange oil was added 1 (0.800 g, 4.84 mmol, 2 eq) and NaBH(OAc)3 (2.567 g, 12.11 mmol, 5 eq), and the mixture was refluxed for 25 hours in 1,2-dichloroethane (80 mL). The reaction was quenched with saturated aqueous Na2CO3 (40 mL) and extracted with dichloromethane (5 x 15 mL). The combined organic layers were dried over anhydrous MgSO4. The solvent was removed to give a brown oil, which was recrystallized with 4:1 CH3OH / CH2Cl2. Brown crystals were collected (0.580 g, 1.29 mmol, yield 53 %). 1H NMR (300 MHz, CDCl3, δ): 8.17 (d, 3J9, 10 = 8.7 Hz, H10, 2H), 8.02 (d, 3J8, 9 = 7.5 Hz, H8, 2H), 7.85 (t, 3J8, 9 = 7.5 Hz, 3J9, 10 = 8.7 Hz, H9, 2H), 7.78 (d, 3J2, 3 = 8.4 Hz, H2, 2H), 7.61 (d, 3J2, 3 = 8.4 Hz, H3, 2H), 4.01 (s, H13, 6H), 3.96 (s, H6, 4H), 3.83 (s, H5, 2H).  13  C NMR (75 MHz, CDCl3, δ): 165.9 (C12), 159.6 (C7), 147.7  (C11), 147.4 (C1), 146.8 (C4), 137.7 (C9), 129.7 (C3), 126.3 (C8) , 124.0 (C10), 123.8 (C2), 60.0 (C6), 57.9 (C5), 53.1 (C13). IR (neat, cm-1): 1717 (s), 1516 (w), 1337 (m). HR-ESIMS calcd. for C23H22NaN4O6: 473.1437, found: 473.1433 [M + H]+ .  6-[(4-Nitrobenzylamino)methyl]-di-pyridine-2-carboxylic acid (3, H2dipin-NO2). 6-[(4-Nitrobenzylamino)methyl]-di-pyridine-2-carboxylic  acid  methyl ester (2, 102 mg, 0.227 mmol) was dissolved and stirred in THF (16 mL). LiOH (22 mg, 0.908 mmol, 4 eq) was added and the solution turned light yellow. The mixture was stirred for 2 hours, after which time the solvent was removed by rotary evaporation. The crude mixture was then washed with dichloromethane to yield a yellow solid. This was then redissolved in H2O and acidified with conc. HCl to afford the corresponding HCl salt.  1  H  49  NMR (300 MHz, D2O, δ): 8.03 (d, 3J9, 10 = 9.2 Hz, H10, 2H), 7.68 (d, 3J8, 9 = 8.7 Hz, H8, 2H), 7.61 (t, 3J8, 9 = 8.7 Hz, 3J9, 10 = 9.2 Hz, H9, 2H), 7.53 (d, 3J2, 3 = 6.9 Hz, H2, 2H), 7.44 (d, 3J2, 3 = 6.9 Hz, H3, 2H ), 3.83 (s, H6, 4H), 3.79 (s, H5, 2H).  13  C NMR (75 MHz, D2O, δ): 172.6  (C12), 157.2 (C7), 152.2 (C11), 146.2 (C1), 146.0 (C4), 137.6 (C9), 129.6 (C3), 125.2 (C8) , 122.8 (C10), 121.6 (C2), 60.0 (C6), 58.2 (C5). IR (neat, cm-1, of acidified species): 1739 (s), 1523 (m), 1340 (s, br). HR-ESI-MS calcd. for C21H17N4O6: 421.1148, found: 421.1145 [M – H] -. Elemental Analysis calcd. (found) for C21H18N4O6·HCl·H2O: C, 52.70 (52.13); H, 4.44 (4.16); N, 11.75 (11.43).  6-[(4-Aminobenzylamino)methyl]-di-pyridine-2-carboxylic acid methyl ester (4, Me2dipin-NH2). 4-(aminomethyl)-N-(9-fluorenylmethoxycarbonyl)phenylamino-dipyridine-2-carboxylic acid methyl ester (40 mg, 0.0623 mmol) was stirred in 20% piperidine/DMF (4 mL) for 30 minutes at room temperature. A small volume of CH2Cl2 was added, and the mixture was extracted three times with aqueous saturated NaHCO3 solution (15 mL). Basic extraction with NaHCO3 was repeated (3 x 5 mL). The organic layer was evaporated and then triturated with hexane. Rotary evaporation to remove hexane afforded the product as a yellow solid (24 mg, yield 92 %).  1  H NMR (300  MHz, CDCl3, δ): 7.99 (d, 3J9, 10 = 9.0 Hz, H10, 2H), 7.81 (m, 3J8, 9 = 7.5 Hz, H8, H3, 4H), 7.18 (t, 3J8, 9 = 7.5 Hz, 3J9, 10 = 9.0 Hz, H9, 2H), 6.65 (d, 3J2, 3 = 8.4 Hz, H2, 2H), 3.99 (s, H13, 6H), 3.92 (s, H6, 4H), 3.58 (s, H5, 2H).  13  C NMR (75 MHz, CDCl3, δ): 165.1 (C12), 160.9  (C7), 147.5 (C11), 145.8 (C1), 137.5 (C4), 130.3 (C9), 126.9 (C3) , 126.1 (C8), 123.7 (C10), 115.2 (C2), 59.8 (C6), 58.5 (C5), 53.1 (C13). HR-ESI-MS calcd. for C23H24NaN4O4: 443.1686, found: 443.1695 [M + Na]+.  50  6-[(4-Amino- benzylamido-biotinyl)-methyl]-di-pyridine-2-carboxylic acid methyl ester (5, Me2dipin-biotin). 6-[(4-Amino-benzylamino)-methyl]-di-pyridine2-carboxylic acid methyl ester (4, 173 mg, 0.4 mmol) and triethylamine (173 μL, 1.245 mmol) were stirred in DMF (6 mL), biotinTFP (181 mg, 0.461 mmol) was added, and the mixture was stirred overnight at 60°C.  The  solvent was removed and the product was precipitated with THF as a white solid (105 g, 0.163 mmol, yield 40 %) which was collected by filtration. 1H NMR (300 MHz, CD3OD, δ): 7.95 (m, H8, H9, H10, H11, 6H), 7.54 (d, 3J2, 3 = 8.4 Hz, H2, 2H), 7.35 (d, 3J2, 3 = 8.4 Hz, H3, 2H), 4.30 (m, Hβ, 1H), 4.15 (m, Hχ, 1H), 3.87 (s, H13, 6H), 3.77 (s, H6, 4H), 3.60 (m, H5, 4H), 2.9 (dd, 3Jε, CH2 = 5.1 Hz, Hε, 2H), 2.72 (m, Hδ, 2H), 2.28 (m, CH2, 2H), 1.85 – 1.40 (m, 6H).  13  C NMR (75  MHz, CD3OD, δ): 174.4 (C14), 165.3 (C12), 162.7 (Cα), 159.7 (C7), 146.7 (C11), 137.9 (C1), 129.2 (C10), 126.2 (C4), 123.3 (C3), 118.9 (C8), 67.0 (C6), 61.1 (Cχ), 59.2 (Cβ), 55.4 (C5), 52.4 (C13), 33.5, 28.2, 28.1, 25.1, 24.5. IR (neat, cm-1): 1697 (w), 1428 (m), 1313 (w). HR-ESI-MS calcd. for C33H39N6O6S: 647.2652, found: 647.2645 [M + H]+.  6-[(4-Aminobenzylamidobiotinyl)methyl]-di-pyridine-2-carboxylic acid (6, H2dipinbiotin). 5 (8 mg, 0.012 mmol) was dissolved in THF/ H2O / CH2Cl2 (1:2:1, 4 mL). LiOH (2 mg, 0.08 mmol) was added and the reaction was monitored by TLC (20% CH3OH in CH2Cl2). After the reaction was found to be complete, the solvent was removed in vacuo to afford a yellow solid which was redissolved in CH3OH and filtered to remove insoluble impurities. The product was isolated as a light yellow solid, (6.8 mg, 0.011 mmol, yield 91 %) 1H NMR 51  (300 MHz, d6-DMSO, δ): 8.06 (d, 3J9, 10 = 7.7 Hz, H10, 2H), 7.87 (d, 3J9, 10 = 7.7 Hz, 3J8, 9 = 7.7 Hz, H9, 2H), 7.53 (d, 3J2, 3 = 8.5 Hz, H2, 2H), 7.41 (d, 3J8, 9 = 7.7 Hz, H8, 2H), 7.32 (d, 3J2, 3 = 8.5 Hz, H3, 2H), 4.44 (m, Hβ, 1H), 4.27 (m, Hχ, 1H), 3.73 (s, H6, 4H), 3.60 (m, H5, 2H), 3.19 (m, Hε, 1H), 2.88 (dd, Hδ, 2H), 2.65 (m, CH2, 2H), 2.13 (m, CH2, 2H), 1.51 (m, 6H)  13  C NMR (75 MHz, CD3OD, δ): 183.0 (C12), 172.4 (C13), 166.3 (Cα), 158.7 (C7),  139.5 (C11), 136.5 (C1), 131.0 (C9), 127.6 (C4), 123.8 (C3), 121.5 (C8), 115.9 (C2), 63.4 (C6), 61.8 (C5), 41.2 (Cχ), 39.1 (Cβ), 30.31 (Cδ), 29.9, 29.7, 27.7. IR (neat, cm-1): 1682 (w, amide), 1636 (w), 1567 (w), 1429 (m). HR-ESI-MS calcd. for C31H32Li3N6O6S: 623.2264, found: 623.2274 [M – 2H + 3 Li]+. 3.2.3  Coordination Chemistry  [Re(CO)3(2)]Br ([Re(CO)3(Me2dipin-NO2)]Br). 2  (44  mg,  0.098  mmol)  was  dissolved  in  dichloromethane (5 mL). [Re(CO)3(Br)3][N(Et)4]2 (76 mg, 0.98 mmol) was dissolved in CH3OH (5 mL) and added dropwise to the ligand solution. The reaction mixture was heated for 18 h at 40°C. Subsequently, the solvent was removed and the crude mixture was triturated with dichloromethane (to remove unreacted ligand) and extracted with CH3OH (to precipitate most of the [N(Et)4]Br) to afford the product after removal of the solvent in vacuo as a white solid (32 mg, 0.043 mmol, yield 44 %). 1H NMR (300 MHz, CD3OD, δ): 8.41 (d, 3J9, 10 = 8.7 Hz, H10, 2H), 8.09 (d, 3J8, 9 = 8,7 Hz, 3J9, 10 = 8.7 Hz, H9, 2H), 8.01 (d, 3J8, 9 = 8.7 Hz, H8, 2H), 7.79 (d, 3J2, 3 = 7.8 Hz, H2, 2H), 7.69 (d, 3J2, 3 = 7.8 Hz, H3, 2H), 5.16-5.11 (m, H6, 4H), 4.57 (d, H5, 2H), 4.06 (s, H13, 6H). 13C NMR (75 MHz, CD3OD, δ): 195.7 (CO), 191.6 (CO), 163.4 (C12), 142.7(C7), 140.8 (C11), 139.6 (C6), 135.0 (C9), 131.4 (C4), 126.8 (C3), 125.2 (C8), 124.7 (C2), 70.7 (C6), 64.1 (C5), 59.7 (C13). IR (neat, cm-1): 2034 (s, νCO) 2011 (s, νCO), 1895 (s, νCO), 1867 (m), 1728 (w). HR-ESI-MS calcd. for C26H22N4O9185Re: 719.0917, found: 719.0906 [M + H]+.  52  [Re(CO)3(5)]Br ([Re(CO)3(Me2dipin-biotin)]Br). 5 (5 mg, 0.007 mmol) was dissolved in CH3OH (2 mL). [Re(CO)3(Br)3][N(Et)4]2 (6 mg, 0.007 mmol) was added to the ligand solution. The reaction mixture was  heated  for  18  h  at  60°C.  Subsequently, the solvent was removed and the crude product was purified by reversed phase HPLC. The solvent was removed and the product was afforded as a colourless solid (1 mg, 0.001 mmol, yield 14 %). 1H NMR (300 MHz, CD3OD, δ), 8.09 (m, H10, 2H), 7.79 (m, H9,H8, 4H), 7.65 (m, H2, H3, 4H), 5.11-4.94 (m, H6, 4H), 4.60 (d, H5, 2H), 4.48 (m, Hβ, 1H), 4.30 (m, Hχ, 1H), 3.21 (m, Hε, 1H), 2.93 (dd, Hδ, 2H), 2.69 (m, CH2, 2H), 2.21 (m, CH2, 2H), 1.78-1.41 (m, 6H).  13  C NMR (75 MHz, CD3OD,  δ): 195.5 (CO), 191.8 (CO), 177.9 (C), 174.1, 166.9 (C12), 166.5 (Cα), 163.9 (C11), 155.0 (C6), 142.9 (C9), 134.5 (C4), 134.5 (C3), 126.9 (C8), 121.8 (C2), 67.1 (C6), 63.7 (C5), 57.4 (C13), 44.3 (Cχ), 41.5 (Cβ), 35.1 (Cδ), 30.2, 29.9, 26.9, 26.4, 25.9. IR (neat, cm-1): 2033 (s, νCO), 2011 (s, νCO), 1722 (m), 1698 (w). HR-ESI-MS calcd. for C36H38N6O9185Re 32S: 915.1951, found: 915.1964 [M]+.  [Cu(3)]·EtOH ([Cu(dipin-NO2)]·EtOH). 3 (5 mg, 0.0126 mmol) was stirred in CH3OH /H2O (1:1). The pH was adjusted with 0.1 M HCl to 2-3. Cu(C2H3O2)·H2O (2.5 mg, 0.0126 mmol, in 2:1 CH3OH /H2O) was added, and the solution turned clear green. The pH was raised to 7-8 with 0.1 M NaOH, and a solid precipitated. The mixture was left to stir for 3 hours, and the solvent was removed by rotary evaporation to give a green solid (5 mg, 0.007 mmol, yield 58 %). Small green plates suitable for X-ray diffraction were afforded through slow evaporation of a solution of the complex in a water-methanol mixture (1:3) in order to remove salts, which precipitate together with the product; the crude product is  53  purified using a C18 cartridge. The cartridge is rinsed thoroughly with H2O before the product is eluted using EtOH. IR (neat, cm-1): 1639 (m,br), 1513 (s), 1344 (s). HR-ESIMS calcd. for C21H1663CuN4O6: 506.0264, found: 506.0272 [M]+. Elemental Analysis calcd. (found) for C21H16CuN4O6·EtOH: C, 52.12 (52.04); H, 4.18 (4.30); N, 10.57 (10.36). [Cu(6)]·2 H2O ([Cu(dipin-biotin)]·2H2O). The biotinylated complex was afforded through the same synthetic procedure as [Cu(dipinNO2)]·EtOH but using 6 (6 mg, 0.0097 mmol) as the starting material. The product is eluted using CH3OH. The product was afforded as a light green solid (4 mg, 0.0058 mmol, 89 %). IR (neat, cm-1): 1684 (s), 1636 (w), 1551 (m), 1410. HR-ESI-MS calcd. for C31H3263CuN6NaO6S: 702.1398, found: 702.1406 [M + Na]+. Elemental Analysis calcd. (found) for C31H34CuN6O7·2H2O: C, 51.98 (51.73); H, 5.07 (5.28); N, 11.63 (11.15).  3.2.4  X-Ray Diffraction Structural Characterization  For [Cu(dipin-NO2)]·EtOH, a green plate crystal of C21H18CuN4O7 having approximate dimensions of 0.02 x 0.15 x 0.25 mm was mounted on a glass fiber. All measurements were made on a Bruker DUO APEX II diffractometer with graphite monochromated Mo-Kα radiation. The data were collected at a temperature of -183.0 ± 0.1°C to a maximum 2θ value of 45.2°. Data were collected in a series of φ and ω scans in 0.50° oscillations with 60.0-second exposures. The crystal-to-detector distance was 40.00 mm. Of the 6190 reflections that were collected, 2560 were unique (Rint = 0.072); equivalent reflections were merged. Data were collected and integrated using the Bruker SAINT78 software package. The linear absorption coefficient μ for Mo-Kα radiation is 11.55 cm-1. Data were corrected for absorption effects using the multi-scan technique (SADABS79), with  54  minimum and maximum transmission coefficients of 0.730 and 0.977, respectively. The data were corrected for Lorentz and polarization effects. For [Re(CO)3(2)]Br, a colourless blade crystal of C26H22N4O9Re.½Re(CO)3Br3 having approximate dimensions of 0.02 x 0.10 x 0.40 mm was mounted on a glass fiber. All measurements were made on a Bruker APEX DUO diffractometer with graphite monochromated Mo-Kα radiation. The data were collected at a temperature of -183.0 ± 0.1 °C to a maximum 2θ value of 58.4°. Data were collected in a series of φ and ω scans in 0.5 oscillations using 20.0-second exposures. The crystal-to-detector distance was 40.00 mm. Of the 50217 reflections that were collected, 8033 were unique (Rint = 0.048); equivalent reflections were merged. Data were collected and integrated using the Bruker SAINT78 software package. The linear absorption coefficient, μ, for Mo-Kα radiation is 81.83 cm-1. Data were corrected for absorption effects using the multi-scan technique (SADABS79), with minimum and maximum transmission coefficients of 0.611 and 0.849, respectively. The data were corrected for Lorentz and polarization effects. The collection and analysis of the data were done by Dr. Brian O. Patrick.  3.2.5  Radiolabelling with 99mTc  The organometallic precursor [99mTc(H2O)3(CO)3]+ was prepared from a saline solution of Na[99mTcO4] (1 mL, 2.7 mCi) using the Isolink kit. A solution of Na[99mTcO4] (1 mL) was added to the Isolink kit, and the vial was heated to reflux for 40 min. Upon cooling, 0.1 M HCl solution (1 mL) was added to adjust the pH to approximately 7. [99mTc(H2O)3(CO)3]+ (10 MBq, in 0.2 mL) was added to a 10-4 M solution of ligand (0.8 mL) in a sealed vial and heated to reflux for 40 min. TLCs were developed with a 4:1 mixure of dichloromethane and methanol as mobile phase.  55  3.2.6  Cysteine and Histidine Challenge  To evaluate complex stability, an aliquot (0.5 mL) of 99mTc-complex was challenged with a 0.1 M solution of cysteine and histidine (0.5 mL). The mixture was incubated at 37°C and analyzed by TLC after 24 h.  3.2.7 A solution of  64  Radiolabelling with 64Cu  Cu (non-carrier added, 0.4 – 0.7 mCi, in 50 μL) was added to a 10-5 M  solution (pH 5, sodium acetate buffer, 0.95 mL) of ligand. The reaction mixture was analyzed after 10 minutes at room temperature by HPLC (gradient: A: H2O, 0.1 % TFA, B: CH3CN. 5100% B linear gradient 30 min). Rt of [64Cu(dipin-NO2)] radiolabelled product on HPLC: 10.9 minutes. Stability versus serum (1 h; in % complex intact): 22. Rt of [64Cu(dipinbiotin)] radiolabelled product on HPLC: 8.1 minutes. Stability versus serum (1 h; in % complex intact): 48.  3.2.8  Serum Challenge  To evaluate complex stability, an aliquot (500 μL) of the reaction was added to a solution of mouse serum (pH 5 buffer, 500 μL). The mixture was incubated at 37°C and analyzed by HPLC (gradient: A: H2O, 0.1 % TFA, B: CH3CN. 5-100% B linear gradient 30 min) after 1h.  3.3  Results and Discussion  3.3.1  Chelate Synthesis  A double reductive amination reaction was chosen to join the two functional ends of the proposed bifunctional chelate. Commercially available dipicolinic acid can be converted into 1 using a three-step literature procedure.108,109  56  Scheme 3.1 Synthetic scheme for the synthesis of 2 and 3 (i) 0.5 eq 4-Nitrobenzylamine, NaBH(OAc)3, DCE, reflux, overnight (ii) 4 eq LiOH, in THF/H2O.  Aldehyde 1 can be reacted with a benzylamine derivative and undergoes a double reductive amination to form the framework of the intended chelate.42 For the model chelate, a benzyl nitro derivative is used, which was initially supposed to act also as an intermediate to prepare an easy-to-couple phenyl amine. This synthetic strategy involved use of a 4-nitrobenzylamine fragment to form 2 (Me2dipin, Scheme 3.1). Sodium triacetoxyborohydride is routinely used as a mild reducing agent for double reductive amination, strong enough to reduce the imine intermediate, but not too strong to reduce the methyl ester. Since 4-nitrobenzylamine was purchased as the hydrochloride salt, basifying the salt with sodium hydroxide was initially performed to form the corresponding amine. The reductive amination reaction was performed overnight and quenched with saturated aqueous Na2CO3. Since the product was minimally soluble in methanol, it was found that recrystallization of the crude product mixture was possible with a 4:1 mixture of CH3OH / CH2Cl2, affording the pure product 2 as brown needle-like crystals. Subsequent deprotection of the methyl ester protection group with LiOH afforded the desired model compound 3 (H2dipin) quantitatively.  57  Scheme 3.2 Synthetic scheme for the synthesis of 5 and 6 (iii) NEt3, DMF, 60 °C, overnight (iv) 5 eq LiOH, THF/ H2O / CH2Cl2 (1:2:1).  To afford the easily derivatized phenylamine, however, reducing the nitro group of 2 to form 4 proved to be unsuccessful. Initially, reduction using hydrazine with Raney nickel as the catalyst was attempted. Formation of the product was evidenced by ESI-MS, but the 1H NMR spectrum showed broadened peaks, suggesting possible coordination of the chelate to Ni(II). The same reduction reaction was attempted again using H2 with Pd/C catalyst. Low to moderate pressure of H2 did not drive the reaction to a significant yield, whereas higher pressure (70 psi) led to cleavage of the nitrobenzylamine fragment, as suggested by both 1H NMR spectroscopy and ESI-MS. Hence, the reduction method for this specific nitro group was concluded to be ineffective. The  second,  more  successful  strategy  involved  Fmoc-protected  4-  aminobenzylamine as the starting material. 4-Aminobenzylamine can be selectively protected by Fmoc at the phenylamine position to produce 4-(aminomethyl)-N-(9fluorenylmethoxycarbonyl)phenylamine as a hydrochloride salt.106 Basic extraction was then performed to obtain the corresponding free amine. This was reacted with 2 equivalents of 1 to form the Fmoc-protected version of 4 via a double reductive  58  amination reaction. The product was purified by column chromatography. Finally, deprotection with 20% piperidine in DMF afforded the intended product 4 in 92 %. To synthesize the biotin-conjugated fragment, the terminal carboxylic acid of commercially available D(+)-biotin was first activated using a tetrafluorophenyl ester following literature conditions.104 The strong electron-withdrawing ability of the tetrafluorophenyl (TFP) group transforms the -OH into O-TFP, which serves as a convenient leaving group, making the carbonyl particularly susceptible to nucleophilic attack by the benzyl amine functionality of 4. Once biotin-TFP was prepared, it was reacted with 4 in DMF (Scheme 3.2). Triethylamine was added to maintain the basic condition that would be ideal for the coupling reaction (Scheme 3.2).104 After overnight stirring at 60°C, the product was identified by ESI-MS; the crude solid was triturated with THF according to the literature42 to remove impurities and to afford 5 (Me2dipin-biotin) as a yellow solid. Compound 6 (H2dipin-biotin) was prepared through LiOH deprotection of the methyl esters and extracted with CH3OH, since the protected starting material and the salts present have low solubility in this solvent. Removal of the solvent in vacuo yielded the desired, deprotected product 6.  3.3.2  Coordination Complexes  To synthesize the copper and rhenium complexes, the chelate was mixed with the corresponding precursor metal complex to form the desired product complex via a ligand substitution reaction. Copper(II) acetate was used as the starting material of copper complex formation. Copper(II) acetate is a dark green crystalline solid. To synthesize copper complexes, 3 or 6 was acidified in a CH3OH/ H2O mixture. The initial acidic condition prevents the hydrolysis of copper to form insoluble copper hydroxides. The solution was then neutralized to pH 7-8 after addition of complex precursors. Upon addition of the chelate, an immediate colour change was observed, and the colour of the solution intensified as the pH was increased. When 3 was added to the chelate solution, the product complex precipitated, forming a cloudy suspension. Evaporation of the solvent gave the light green copper complex mixed with NaCl. In order to remove excess  59  salt and confirm bulk composition of the complex by elemental analysis, both copper complexes were purified using C18 cartridges. [Cu(dipin-biotin)] readily elutes using MeOH and can be confirmed as the dihydrate salt. In the case of [Cu(dipin-NO2)], a large amount of EtOH is used to elute the sparingly soluble complex from the cartridge. Presence of the complexes in solution was confirmed with high resolution mass spectrometry, which shows the characteristic isotope pattern for copper with corresponding masses [M + H]+ and [M + Na]+ for both Cu(II) complexes. Table 3.1 Summarized relevant IR stretching frequencies for Cu complexes and the corresponding ligands. IR stretching frequency of diagnostic functional groups [cm-1] Compound  Amido  Nitro  Carboxylate  H2dipin-NO2  -  1340  1739  H2dipin-biotin  1684  -  1710  [Cu(dipin-NO2)]  -  1344  1639  [Cu(dipin- biotin)]  1682  -  1636  Table 3.1 summarizes diagnostic IR frequencies observed. While the nitro group on the H2dipin-NO2 ligand system remains virtually uninfluenced by the coordination of the Cu ion, similar to the amido group on the H2dipin-biotin ligand system, the stretching frequency of the carboxylate functional group strongly decreases upon coordination. Compounds 2 and 5 were used for the coordination of the [Re(CO)3]+ core.110,111 While the carboxylates remain protected and could only act as poor binding groups, the two pyridyl nitrogens together with the aliphatic nitrogen form the widely explored and popular dpa (dipicolylamine) ligand system. To afford [Re(Me2dipin-NO2)(CO)3]Br, equimolar amounts of [Re(CO)3(H2O)3]Br and ligand were stirred over night at 60°C in a 1:1 methanol/dichloromethane mixture. Excess ligand was removed from the crude product through trituration with dichloromethane to give the corresponding tridentate rhenium complex. In the case of [Re(Me2dipin-biotin)(CO)3]Br, reactants were stirred in methanol overnight at 60°C. The crude reaction mixture was purified using preparative HPLC.  60  Table 3.2 Summarized relevant IR stretching frequencies for Re complexes and the corresponding ligands. IR stretching frequency of diagnostic functional groups [cm-1] Compound  Amido  Ester  Re-Carbonyl  Me2dipin-NO2  -  1717  -  Me2dipin-biotin  1698  n. o.  -  [Re(CO)3(Me2dipin-NO2)]Br  -  1728  2034, 2011  [Re(CO)3(Me2dipin- biotin)]Br  1687  n. o.  2033, 2011  Again, IR spectroscopy served as a good tool for confirming which donor atoms were involved in coordination (Table 3.2). The amide bond in both ligand and complex of the biotin conjugate remains unchanged, confirming the great distance from the coordination sphere for this specific functional group. The ester functionality is not observed in the case of the biotin conjugate due to overlap with the stronger amido peak, but for the nitro derivatized compounds a slight increase in wave number is observed. The characteristic metal bound carbonyl frequencies are observed and confirm coordination of identical ligand spheres in both cases. Metal ion coordination of the tridentate ligands could be further verified by shifts in peaks in both the 1H and  13  C NMR spectra. The summarized 1H NMR spectra of  ligands including the Re complexes are shown in Figures 3.4 and 3.5.  61  Figure 3.4 1H NMR spectra (300 MHz, CDCl3/CD3OD, 298 K) of the benzylnitro derivatized compounds synthesized, * denotes residual solvent peaks, presence of residual N(Et)4Br is observed for the Re complex (1.2 and 3.2 ppm).  As clearly recognizeable from the presented spectral data (Figures 3.4, 3.5), the coordination of the metal leads to observation of diasterotopic protons around the coordination sphere. The methylene groups 6 show a characteristic splitting pattern observed for coordination of the fac-[Re(CO)3]+ core, which was already described in Chapter 2. Due to coordination of the tertiary amine, methylene group 5 becomes fixed in its confirmation and splitting of the formerly singlet peak into two distinct peaks is observed; however, methyl group 13 maintains its free rotation, appearing as a defined singlet peak in both ligand and complex systems.  62  Figure 3.5 1H NMR spectra (300 MHz, CD3OD, 298 K) of the biotin derivatized compounds synthesized, * denotes residual solvent peaks.  The mass spectrometry data confirmed the predicted coordination product by appearance of the mass of complex cations [M+] with the characteristic isotope splitting pattern observed for Re.  63  3.3.3  X-Ray Diffraction Structural Characterization of [Cu(dipin-  NO2)H2O] and [Re(CO)3(Me2dipin-NO2)]+  Figure 3.6 ORTEP drawing of [Cu(dipin-NO2)(H2O)], showing the atom numbering scheme (50% thermal ellipsoids).  The pale green solid [Cu(dipin-NO2)] was dissolved in a CH2Cl2/CH3OH mixture, and crystals were obtained by slow evaporation (Figure 3.6). The complex has a strongly distorted square pyramidal geometry and the four donor atoms from the chelate are sitting on the distorted square plane. The bond length between Cu and any of the 4 donor atoms of the chelate is roughly the same; however, the Cu-N bond lengths are slightly longer than the Cu-O bond lengths. The bond length between Cu and the apical water oxygen atom is longer than the other coordinate bonds, suggesting that the water molecule may be loosely coordinated to the metal centre. The axial elongation is typical of the Jahn Teller distortion observed for Cu(II) complexes and is also in agreement with previously reported square pyramidal Cu(II) complexes. The axially coordinated water molecule could be another indicator for the instability of this particular ligand system in vitro, since it could be easily displaced by serum proteins facilitating the transchelation  64  of the metal out of the dipin ligand (vide infra). Table 3.3 Selected bond lengths in [Cu(dipin-NO2)( H2O)] ([Å]) and angles ([°]). bond  length [Å]  angle  degree [°]  N(1) – Cu(1)  2.005(10)  O(3)- Cu(1)- O(1)  90.7(3)  N(2) – Cu(1)  2.061(9)  O(1)- Cu(1)- N(1)  81.3(3)  O(1) – Cu(1)  1.988(7)  O(3)- Cu(1)- N(2)  82.9(4)  O(3) – Cu(1)  1.927(8)  O(3)- Cu(1)- O(7)  86.5(3)  O(7) – Cu(1)  2.155(8)  N(1)- Cu(1)- O(7)  90.1(4)  For the structure of [Re(CO)3(Me2dipin-NO2)]+ (Figure 3.7), the colorless crude solid was dissolved in CH3OH and crystals were obtained by slow evaporation. The complex cocrystallized with [Re(CO)3Br3]2- as the counter ion for two molecules of [Re(CO)3(2)]+.  Figure 3.7 ORTEP drawing of [Re(CO)3(Me2dipin-NO2)]+, with co-crystallized counter ion [Re(CO)3Br3]2shown is the atom numbering scheme (50% thermal ellipsoids).  The structure displays a distorted octahedral geometry. With all Re-N bond lengths close to 2.2 Å (Table 3.4), which is characteristic for bipyridyl complexes of the [Re(CO)3]+ core.112 This is one indication that the stable coordination of this scaffold is not influenced by the two adjacent methyl ester functionalities.  65  Table 3.4 Selected bond lengths in [Re(CO)3(Me2dipin-NO2)]+ ([Å]) and angles ([°]). bond  length [Å]  angle  degree [°]  N(1) – Re(1)  2.201(2)  C(26)- Re(1)- C(2)  90.62(12)  N(2) – Re(1)  2.209(2)  C(24)- Re(1)- N(1)  95.68(11)  N(3) – Re(1)  2.240(2)  C(25)- Re(1)- N(2)  100.24(11)  C(24) – Re(1)  1.929(3)  C(26)- Re(1)- N(2)  89.26(11)  C(25) – O(8)  1.161(4)  N(1)- Re(1)- N(2)  84.06(9)  C(25) – Re(1)  1.902(3)  C(26)- Re(1)- N(3)  96.51(11)  C(26) – O(9)  1.153(4)  C(24)- Re(1)- N(3)  98.66(11)  C(26) – Re(1)  1.915(3)  N(1)- Re(1)- N(3)  78.85(9)  3.3.4  Radiolabelling and Stability of 99mTc Complexes  The structural investigation of the Re complexes provided insight into the radiochemical complexes formed with  99m  Tc. In order to synthesize the  99m  Tc complexes,  the organometallic precursor [99mTc(H2O)3(CO)3]+ was synthesized via heating, using the Isolink kit. When the mixture was cool, the pH was adjusted to approximately 7. A 10-4 M solution of ligand was mixed with [99mTc(H2O)3(CO)3]+ in a sealed vial and heated to reflux for 40 min. Reaction monitoring was performed by radio-TLC (Figure 3.8). Labeling with 2 was performed in a 1:1 acetonitrile/water mixture due to poor solubility of the ligand in protic solvents, yielding 72% product after reaction, with Rf = 0.8.  1  2  3  Figure 3.8 TLC labelling traces of [99mTc(CO)3(Me2dipin-NO2)]+ (left, spot 1) and [99mTc(CO)3(Me2dipinbiotin)]+ (right, spot 2); bottom spots indicate unreacted [99mTc(CO)3(H2O)3]+ (bottom, spot 3).  66  Due to increased solubility of 5 in protic solvents, labeling for this ligand was performed in a 3:7 methanol/water mixture, yielding 85% product with an Rf of 0.57. Both complexes were stirred in 0.1 M solution of histidine and cysteine at 37°C for 24 h, and were found to be fully stable against decomposition.  3.3.5 A solution of  Radiolabelling and Stability of 64Cu Complexes 64  Cu was added to a 10-4 M solution of ligand (H2dipin-NO2 or  H2dipin-biotin, both in aqueous solutions) at pH 5. The reaction was analyzed by HPLC after 10 minutes at room temperature. [64Cu(dipin-NO2)] was found to form in a 95 % yield with Rt = 10.9 min. [64Cu(dipin-biotin)] was found to form in a 91% yield with Rt = 8.1 min (Figure 3.9). To evaluate complex stability, an aliquot of the reaction was added to a solution of mouse serum. The mixture was incubated at 37°C and analyzed by HPLC after 1h. For [64Cu(dipin-NO2)], 78% serum binding was found after 1h, while in the case of [64Cu(dipin-biotin)], 52% activity was found to be bound to serum after the same incubation time.  °  °  t0  t0  t1h  t1h 0  2  4  6  8  time [min]  10  12  14  0  2  4  6  8  10  12  14  time [min]  Figure 3.9 Labelling traces of 64Cu copper complex [t0] and trace after 1h serum challenge experiment [t1h]. Left shows Cu complex with [64Cu(dipin-NO2)], right shows Cu complex with [64Cu(dipin-biotin)], ° denotes transchelation of 64Cu to serum proteins.  67  The HPLC labelling peak observed for the two complexes correlate with their polarity. Due to increased water solubility of the biotinylated complex, the retention time is shorter than the retention time of the model complex. [64Cu(dipin-biotin)] appears as multiple peaks, suggesting formation of different coordination modes in solution. This was not observed in the cold coordination reaction, hence it could be an effect of the great excess of ligand used for the radiochemical coordination reaction. 3.4  Conclusions Successful synthesis of the model ligands Me2dipin-NO2 and H2dipin-NO2 as well as  the corresponding conjuguate versions Me2dipin-biotin and H2dipin-biotin are outlined in this chapter. Corresponding cold complexes with copper(II) and tricarbonylrhenium(I) were prepared in satisfying yields; the intended mode of coordination was confirmed by IR and NMR spectroscopy (for diamagnetic Re(I)) as well as solid state structural elucidation of both benzyl-nitro derivatives with the corresponding metal. In the case of the Cu complex, the coordination of an apical H2O molecule is observed, which indicates potential destabilization of the chelation scaffold. Radiolabelling, while fast and efficient with  64  Cu,  confirms this worry, since neither [64Cu(dipin-NO2)] nor [64Cu(dipin-biotin)] proves to withstand serum competition tests. This renders the dipin ligand system unstable and not worthy of further investigations for the purpose of labelling with  64  Cu. The acyclic,  tetradentate coordination mode of the dip ligand system was shown to be a non ideal chelation scaffold, despite the use of strong donor atoms as ligands. It is also suggested that the formation of an 8-membered ring upon coordination leads to destabilization of this complex in solution, also caused by the enhanced mobility and twisting of the chelate arms.113 [99mTc(CO)3]+ complexes with both Me2dipin-NO2 and Me2dipin-biotin were synthesized under standard labelling conditions, showing satisfactory stability of the [99mTc(CO)3]+ complexes. The solid state structure of [Re(CO)3(Me2dipin-NO2)]+ confirms feasibility and compares well to other complexes of similar make-up. It appears to be the case that the methyl ester functionalities do not influence the coordination chemistry in a negative way. This approach could bring opportunities to further functionalize SAAC type  68  chelates, as outlined similarly by Babich and coworkers,114 where pyrazolyl ligands were functionalized with a carboxylic acid moiety to enhance the clearance properties for the otherwise quite lipophilic [99mTc(CO)3]+ core.  69  Chapter 4: A Novel Chelate for 68Ga Imaging Agent Elaboration ‡ 4.1  Introduction: Prior Art on 67/68Ga Chelates The disruptions in the supply chain of  daughter isotope  99m  99  Mo, the parent of the clinically important  Tc, has had worldwide effects on diagnostic nuclear medicine and  has turned many researchers’ attention to other generator-produced isotopes.91 Among attractive alternatives to  99  Mo/99mTc is the  68  Ge/68Ga generator system,22 which has the  potential to be eluted for up to one year due to the long half-life (t1/2 = 271 d) of the parent radionuclide  68  Ge. The  68  Ga generator has been used to prepare  68  Ga  radiopharmaceuticals for clinical imaging in Europe for several years,115 but the development of a generator with regulatory approval for human use would further facilitate the transition of  68  Ga based agents into the clinic in North America. One of the  most sophisticated commercially available generator systems is based on titanium dioxide (Cyclotron Co., Ltd., Obninsk, Russia) and is currently used worldwide.  Figure 4.1 Structures of some previously investigated chelate systems for 68Ga.  ‡  Parts of this chapter have been published: "Acyclic Chelate with Ideal Properties for 68Ga PET Imaging Agent Elaboration", E. Boros, C. L. Ferreira, J. F. Cawthray, E. W. Price, B. O. Patrick, D. W. Wester, M. J. Adam, C. Orvig , J. Am. Chem. Soc. 132 , 15726-15733 (2010).  70  The advantages of this particular generator include the use of a nontoxic packing material and, most importantly, the elution of free, cationic  68  Ga with low acid  concentration of 0.1 M HCl, allowing universal application for radiopharmaceutical preparations. Other advantages include low  68  Ge breakthrough, which is reported to be  between 0.001 and 0.005%.60 Ga(III) forms stable complexes with many multidentate ligands,116 and the radionuclide  68  Ga has suitable properties for high-quality positron-  emission tomography (PET) imaging including a short half-life (t1/2 = 68 min), decay by 89% positron emission and a maximum positron energy of 1.899 keV.60,117,118 While Ga(I) is only stable in non aqueous media, under physiological conditions Ga(III) is the only stable and relevant oxidation state. The free hydrated Ga(III) cation is only stable under acidic conditions, while above pH 4 formation of hydroxide species Ga(OH)3 (pH 4 – 7) and [Ga(OH)4]- (> pH 7) occurs.119 To make the metal ion available for ligand systems that coordinate preferentially at pH < 5, a pre-labelling strategy uses a weakly coordinating ligand such as citrate, which is then easily replaced by a stronger ligand system.120 This strategy is not compatible with the short t1/2 of  68  Ga, hence a direct labelling strategy is preferred,  which can be executed at a pH compatible with biomolecules. An additional nonnegligible factor includes kinetic stability of the formed complex towards the iron sequestering protein transferrin, which is capable of transchelating weakly bound Ga(III) due to the similarity between the charge to size ratio of Ga(III) and Fe(III).57 Table 4.1 Summary of some investigated chelates for labelling of 67/68Ga Ligand  Log K ML  Labelling conditions (yield)  DOTA  21.33121  5 min, 80°C (95 %)58  NOTA  30.98122  10 min, room temperature, (> 98 %)58  6SS  41.060  60 min, 90 °C (> 90 %)60  EC  31.551  5 min, room temperature, (> 90 %)51  TAME-Hex  n. d.  10 min, 100 °C (99 %)60  PCTA  19.37123  10 min, room temperature, (> 98 %)58  apo-transferrin  20.357  71  The efficient and strong chelation of radiometals of Ga(III) has been investigated for the past 40 years with high hopes for potentially useful complexes in radiopharmacy.124,125 The sustained efforts of Martell and Welch over several decades126 have been succeeded by the significant progress of Maecke and co-workers in the late 1990s. Bifunctional versions of the tri- and tetra-aza-based aminocarboxylate macrocyclic chelates 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were able to deliver highly promising results not only for the isotopes  67/68  Ga and  111  In but also for a wide range of  radiolanthanides.55 Many attempts to find chelates of comparable kinetic and thermodynamic stability for  68  Ga have been less successful.51,127,128 The macrocyclic  chelates themselves are challenging to synthesize as selectively functionalized analogues, thus most research groups in need of bifunctional derivatives of NOTA or DOTA are supplied by a commercial source. An array of previously investigated chelates for Ga(III), including thermodynamic stability constants and optimal labelling conditions, is summarized in Table 4.1 and Figure 4.1. In order to furnish the ideal bifunctional chelate for  68  Ga, rapid coordination under mild conditions (pH > 4, room temperature, labelling  within 15 minutes), high kinetic stability (stable versus apo-transferrin challenge and in vivo conditions) and simple synthesis and functionalization (for applications for peptide labelling) are key. A thermodynamic stability constant log KML above the value found for the main in vivo competitor, transferrin (log KML = 20.3) is strongly desired. The 3+ charge is preferentially reduced to 1+, 1- or neutral in order to avoid high kidney uptake and accelerated excretion. In this chapter, the synthesis of a series of acyclic chelates with pyridyl and carboxylate donor arms is described, as well as screening for their capability to label 67/68  4.2  Ga under the ideal conditions outlined above.  Pyridine Carboxylate Chelates and Their Relevance for Chelation of Ga(III) Ga(III) is often classified as hard Lewis acid because of its high charge density and  small ionic radius (0.62 Å), having a preference for ligands of highly ionic, non-polarizable hard Lewis base nitrogen and oxygen donors (mainly as in aliphatic amines and  72  carboxylates). However, it was also found that much softer donors such as thiols and pyridyls (a softer type of nitrogen donor atom) have been used in order to form stable complexes with Ga(III).120,129 This observation leads to the conclusion that a combination of hard and soft donor atoms within one ligand system could result in a rather successful ligand design for Ga(III). Phenolates are less attractive, since the deprotonation of the phenolic proton requires a pH at which Ga(III) preferentially forms insoluble Ga(OH)3, while thiols have a strong tendency to form disulfide bonds in the presence of O2 as an oxidant. Pyridine-containing ligand systems however have no obvious drawbacks. The directed lone pair of the nitrogen serves as a convenient donor atom and a wide variety of chelates has been explored with pyridines as donors; a variety of building blocks can be synthesized or is even commercially available. PCTA, a macrocyclic chelate bearing one pyridyl group has been shown to chelate Ga(III) efficiently.58  Figure 4.2 Previously reported pyridyl containing ligand systems.  Some of the compounds outlined in this chapter have been investigated before. The acyclic, tripodal chelate H2bped has been investigated for its chelation properties with Ga(III) previously in the Orvig group and found to have moderate thermodynamic stability (log KML = 19.9).130 A permutation of this system is H2bpce (in this work referred to as H2dedpa), where all donors are aligned in a linear manner (Figure 4.2).131 This chelate has not been investigated previously for its coordination properties with Ga(III). An advantage of these acyclic systems is the simple synthesis. Extension of denticity or bite angle is easily achieved, as opposed to more sophisticated macrocyclic systems which have been widely explored and are assembled over many synthetic steps. Macrocyclic chelates are typically kinetically slow to bind, while acyclic chelates  73  coordinate fast. This is another key feature of successful chelate design for a short-lived radioisotope such as 68Ga.118 4.3  Experimental  4.3.1  Materials and Methods  All solvents and reagents were from commercial sources and were used as received unless otherwise indicated. Human serum apo-transferrin was purchased from Sigma-Aldrich (St. Louis, MO). Refer to Chapter 2.2 for general information. The HPLC system used for analysis consisted of a Waters Alliance HT 2795 separation module equipped with a Raytest Gabbistar NaI detector and a Waters 996 photodiode array (PDA) detector.  67  Ga was obtained as a 0.1 M HCl solution, and  68  Ga (5-10 mCi/mL)  (both from Nordion Inc.) was obtained from a generator constructed of titanium dioxide sorbent that was charged with  68  Ge and eluted with aqueous HCl (0.1 M).132 The  generator has been previously used for radiolabelling NOTA- and DOTA-based chelate systems and the resulting radiochemical yields and specific activities achievable for these chelates using this generator have been reported.58  4.3.2  General Synthesis Procedures  (i) Reductive amination: Two equivalents of the aldehyde were dissolved in CH3OH (80 mL). The solution was heated to reflux and one equivalent of the corresponding diamine was added dropwise into the hot reaction solution and refluxing continued for 30 minutes. Subsequently, the reaction was filtered hot and cooled to 0°. NaBH4 (1.2 eq) was slowly added into the solution. The mixture was then stirred for 2 h at 0°C and subsequently quenched with a saturated solution of NaHCO3 and extracted three times with CH2Cl2. (ii) Alkylation: One equivalent of diamine was dissolved in CH3CN (100 mL). Two equivalents of tert-butyl bromoacetic acid were added dropwise into the solution, as well as 5-10 equivalents of Na2CO3. The white slurry was then stirred overnight at 70 °C. The reaction mixture was then cooled, filtered through a filter paper and the solvent was  74  removed in vacuo to afford the crude product as an oil, which was subsequently purified with column chromatography (silica, 10% CH3OH in CH2Cl2). (iii) Acid-catalyzed deprotection: The starting material was dissolved in a 1:1 mixture of CH2Cl2 and TFA (total volume 10 mL) and stirred until no more starting material was visible by TLC. The solvent of the reaction mixture was subsequently removed in vacuo to afford the product. (iv) Base-catalyzed deprotection: The starting material was dissolved in a 3:1 mixture of THF and water (total volume 10 mL). LiOH (2-4 eq) was added and the reaction was monitored by TLC until no more starting material was detectable (30 min – 2 h). The solvent was removed in vacuo to afford the product as a Li+ salt. In order to afford the HCl salt, 12 M HCl was added to the solid dissolved in minimal amount of water and the dihydrochloride salt was produced as a precipitate and collected by filtration.  4.3.3  Ligand Synthesis  6-Propylaminomethyl-di-pyridine-2-carboxylic acid methyl ester. N-propylamine (7 mg, 10 μL, 0.119 mmol) was dissolved in CH2Cl2, together with 2 equivalents of 6-bromomethyl-pyridine-2-carboxylic acid methyl ester and 5 equivalents of Na2CO3. The mixture was stirred overnight and subsequently filtered. The solvent was removed in vacuo and the product was purified using column chromatography (silica, 20% CH3OH in CH2Cl2) to afford the product as a yellow oil which crystallizes upon standing (27 mg, 0.08 mmol, yield 64 %). Rf = 0.72. 1H NMR (400 MHz, CDCl3, δ): 7.87 (m, H3, H4, 4H), 7.78 (d, 3J4, 5 = 5.2 Hz, H5, 2H), 3.87 (s, H11, 6H), 3.83 (s, H7, 4H), 2.43 (t, 3J8, 9 = 7.2 Hz, H8, 2H), 1.44 (qt, 3J8, 9 = 7.2 Hz, 3J9, 10 = 7.3 Hz, H9, 2H), 0.75 (t, 3J9, 10 = 7.3 Hz, H10, 3H).  13  C NMR (100 MHz, CDCl3, δ):  165.8 (C1), 160.8 (C6), 147.2 (C2), 137.4 (C4), 124.4 (C5), 121.1 (C3), 60.4 (C7), 56.7 (C8), 52.8 (C11), 20.4 (C9), 11.8 (C10). (HR-ESI-MS calcd. for C19H23N3NaO4: 380.1586, found: 380.1589 [M + Na]+.  75  6-Propylaminomethyl-di-pyridine-2-carboxylic acid. 6-Propylaminomethyl-di-pyridine-2-carboxylic acid methyl ester was dissolved in a 3:1 mixture of THF and water, following procedure (iv) to afford the product quantitatively (83 mg, 0.25 mmol). 1H NMR (300 MHz, CD3OD, δ): 8.04 (d, 3J3, 4 = 7.5 Hz, H3, 2H), 7.9 (m, 3J3, 4 = 7.5 Hz, 3J4, 5 = 7.5 Hz, H4, 2H), 7.56 (d, 3J4, 5 = 7.5 Hz, H5, 2H), 3.83 (s, H7, 4H), 2.51 (t, 3J8, 9 = 7.6 Hz, H8, 2H), 1.63 (qt, 3J8, 9 = 7.6 Hz, 3J9,  10  = 7.2 Hz, H9, 2H), 0.93 (t, 3J9,  10  = 7.2 Hz, H10, 3H)  13  C NMR (75 MHz,  CD3OD, δ): 172.6 (C1), 156.7 (C6), 151.9 (C2), 137.5 (C4), 125.7 (C5), 121.6 (C3), 58.9 (C7), 56.9 (C8), 18.4 (C9). 10.6 (C10). IR (neat, cm-1): 1620 (m), 1586 (s), 1397 (m). HR-ESI-MS calcd. for C17H17LiN3O4: 334.1379, found: 334.1378 [M + Li]+.  N-N’-Bis(2-pyridylmethyl)ethylenediamine-N-N’-diacetic acid (H2bped). Using the original reference,130 the compound was synthesized affording the reported compound. 1H NMR (300 MHz, CD3OD, δ): 8.73 (d, 3J1, 2 = 5.5 Hz, H1, 2H), 8.36 (m, 3J3, 4 = 7.5 Hz, 3J3, 4 = 8.2 Hz, H3, 2H), 7.96 (d, 3J3, 4 = 8.2 Hz, H4, 2H), 7.82 (m, 3.83 3  J1, 2 = 5.5 Hz, H2, 2H), 4.58 (s, H6, 4H), 3.91 (s, H8, 4H), 3.45 (s, H7, 4H).  13  C NMR  (75 MHz, CD3OD, δ): 171.7 (C9), 151.0 (C5), 147.3 (C1), 144.5 (C4), 129.5 (C2), 128.1 (C3), 56.4 (C8), 55.2 (C6), 52.9 (C7). IR (neat, cm-1): 1719 (s), 1633 (m), 1408 (m). HRESI-MS calcd. for C18H22NaN4O4 : 381.1539, found: 381.1532 [M + Na]+.  N-N’-Bis(2-pyridylmethyl)propylenediamine-N-N’-diacetic acid tert-butyl ester. Using methods (i) and (ii) in sequence, the title compound was afforded from 1,3-diaminopropane, 2-pyridinecarboxaldehyde and tert-butyl bromoacetate after column chromatography (20 mg, 0.041 mmol, yield 12 %). 1H NMR (300 MHz, CDCl3, δ): 8.38 (d, 3J1, 2 = 4.8 Hz, H1, 2H), 7.49 (m, 3J3, 4 = 7.8 Hz, H3, 2H), 7.25 (d, 3J3, 4 = 7.8 Hz, H4, 2H), 7.00 (m, 3J1, 2 = 4.8 Hz, H2, 2H), 3.76 (s, H6, 4H), 3.14 (s, H9, 4H), 2.57 (t, 3J7, 8 = 7.2 Hz, H7, 4H), 1.57 (quin, 3J7, 8 = 7.2 Hz, H8, 2H),  76  1.32 (s, H12, 18H).  13  C NMR (75 MHz, CDCl3, δ): 170.3 (C10), 159.6 (C5), 148.6 (C1),  136.1 (C3), 122.6 (C2), 121.6 (C4), 80.42 (C11), 59.9 (C6), 55.6 (C7), 51.83 (C9), 27.9 (C12), 25.5 (C8). HR-ESI-MS calcd. for C27H41N4O4: 485.3128, found: 485.3131 [M + H]+.  N-N’-Bis(2-pyridylmethyl)propylenediamine-N-N’-diacetic acid. Using method (iii), the title compound was afforded quantitatively  from  N-N’-bis(2-pyridylmethyl)  propylenediamine-N-N’-diacetic acid tert-butyl ester (13 mg, 0.035 mmol). 1H NMR (300 MHz, CD3OD, δ): 8.70 (d, 3J1, 2 = 5.3 Hz, H1, 2H), 8.21 (m, 3J3, 4 = 7.9 Hz, H3, 2H), 7.76 (d, 3J3, 4  = 7.9 Hz, H4, 2H), 7.71 (m, 3J1, 2 = 5.3 Hz, H2, 2H), 4.48 (s, H6, 4H), 3.92 (s, H9, 4H),  3.15 (t, 3J7, 8 = 7.5 Hz, H7, 4H), 2.02 (quin, 3J7, 8 = 7.5 Hz, H8, 2H).  13  C NMR (75 MHz,  CD3OD, δ): 170.2 (C10), 152.5 (C5), 145.8 (C1), 141.7 (C4), 125.4 (C2), 124.9 (C3), 56.7 (C9), 54.2 (C6), 52.8 (C7) 21.8 (C8). IR (neat, cm-1): 1674 (s), 1434 (w), 1202 (s). HR-ESI-MS calcd. for C19H25N4O4: 373.1876, found: 373.1877 [M + H]+.  1,2-[{6-(Carboxylato)pyridin-2-yl}methylamino]ethane (H2dedpa). Protected  precursor  1,2-[{6-(methoxycarbonyl)pyridin-2-yl}  methylamino]ethane was synthesized according to the literature.108 Deprotection of the precursor was achieved using method (iv) to afford the product as a white solid (24 mg, 0.059 mmol, yield 59 %). 1  H NMR (d6-DMSO, 300 MHz): 8.12-8.09 (m, 3J4, 5 = 6.8 Hz, 4J3, 5 =  1.8 Hz, H3, H4, 4H), 7.78 (dd, 3J4, 5 = 6.8 Hz, 4J3, 5 = 1.8 Hz, H5, 2H), 4.52 (s, H7 ,2H), 3.51 (d, H8, 2H). 13C NMR (d6-DMSO, 150 MHz, δ): 166.1 (C1), 153.5 (C6), 148.1 (C2), 139.7 (C4), 127.2 (C5), 124.9 (C3), 50.7 (C7), 43.8 (C8). IR (neat, cm-1): 1761 (s), 1749 (s), 1599 (m). HR-ESI-MS calcd. for C16H19N4O4: 331.1406, found: 331.1329 [M + H]+. Elemental analysis: calcd. (found) for H2dedpa·2HCl: C 47.65 (47.30), H 5.00 (5.11), N 13.81 (13.38).  77  1,2-[{6-(Methoxycarbonyl)pyridin-2-yl}methylamino]propane. The product was synthesized using method (i) using 6-formylpyridine-2-carboxylic acid methyl ester and 1,3-diaminopropane. The procedure afforded the product as a colourless oil (69 mg, 0.18 mmol, yield 17 %).  1  H NMR (300 MHz, CDCl3, δ): 7.98  (m, 3J3, 4 = 8.2 Hz, H3, 2H), 7.78 (m, 3J4, 5 = 7.6 Hz, 3J3, 4 = 8.2 Hz, H4, 2H), 7.55 (d, 3J4, 5 = 7.6 Hz, H5, 2H), 3.96 (s, H7, 4H), 3.94 (s, H10, 6H), 2.75 (t, 3J8, 9  = 6.8 Hz, H8, 4H), 2.02 (quin, 3J8, 9 = 6.8, H9, 2H). 13C NMR (75 MHz, CDCl3, δ) δ: 161.2  (C1), 160.5 (C6), 148.0 (C2), 137.9 (C4), 126.2 (C5), 124.0 (C3), 55.73 (C10), 53.4 (C7), 48.6 (C8), 29.8 (C9). HR-ESI-MS calcd. for C19H24NaN4O4: 395.1695, found: 395.1699 [M + Na]+.  1,2-[{6-(Carboxylato)pyridin-2-yl}methylamino]propane. Using method (iv), the title compound was afforded quantitatively  from  1,2-[{6-(Methoxycarbonyl)pyridin-2-  yl}methylamino]propane after deprotection (62 mg, 0.18 mmol).  1  H NMR (300 MHz, CD3OD, δ): 7.92 (m, 3J3, 4 = 8.2  Hz, H3, H4, 4H), 7.51 (m, 3J4, 5 = 7.4 Hz, 4J3, 5 = 1.5 Hz, H5, 2H), 3.88 (s, H7, 4H), 2.61 (t, 3J7, 8 = 6.9 Hz, H8, 4H), 1.75 (quin, 3J8, 9 = 6.9, H9, 2H). 13C NMR (125 MHz, CD3OD, δ): 170.7 (C1), 159.5 (C6), 152.8 (C2), 137.1 (C4), 121.8 (C5), 121.7 (C3), 63.3 (C7), 53.1 (C8), 27.3 (C9). IR (neat, cm-1): 1614 (m), 1433 (m), 1383 (m). HR-ESI-MS calcd. for C17H20LiN4O4: 351.1645, found: 351.1637 [M + Li]+. N,N’-Bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N’-diacetic acid Using the original reference,108 the desired compound was synthesized. 1H NMR (300 MHz, CD3OD, δ): 8.12 (d, 3J3, 4 = 7.8 Hz, H3, 2H), 7.97 (m, 3J3, 4 = 7.8 Hz, 3J4, 5 = 7.5 Hz, H4, 2H), 7.78 (d, 3J4, 5 = 7.5 Hz, H5, 2H), 4.7 (s, H8, 4H), 4.10 (s, H7, 4H), 3.68 (s,  78  H9, 4H). HR-ESI-MS calcd. for C20H23N4O8: 447.1516, found: 447.1511 [M + H]+. N,N’-Bis(6-carboxy-2-pyridylmethyl)propylenediamine-N,N’-diacetic acid tert-butyl ester 1,2-[{6-(Methoxycarbonyl)pyridin-2-yl}  methylamino]propane  was alkylated using method (ii) to afford the product after column chromatography (24 mg, 0.04 mmol, yield 8 %).  1  H  NMR (300 MHz, CDCl3, δ): 7.98 (m, H4, 2H), 7.76 (m, H3, H5, 4H), 3.99 (s, H14, 6H), 3.97 (s, H7, 4H), 3.25 (s, H10, 4H), 2.67 (t, 3J8, 9 = 7.2 Hz, H8, 4H), 2.02 (quin, 3J8, 9 = 7.2, H9, 2H), 1.44 (s, H13, 18H). HR-ESI-MS calcd. for C31H44N4NaO8: 623.3057, found: 623.3057 [M + Na]+. N,N’-Bis(6-carboxy-2-pyridylmethyl)propylenediamine-N,N’-diacetic acid The product was afforded from 1,3-propylenediamine and methyl 6-formylpyridine-2-carboxylate using methods (iii) sequentially (11 mg, 0.023 mmol, yield 57 %).  1  and  (iv)  H NMR (300  3  MHz, CD3OD, δ): 8.17 (d, J3, 4 = 7.2 Hz, H3, 2H), 8.08 (m, 3J3, 4 = 7.2 Hz, 3J4, 5 = 7.8 Hz, H4, 2H), 7.72 (d, 3J4, 5 = 7.56 Hz, H5, 2H), 4.78 (s, H10, 4H), 4.27 (s, H7, 4H), 3.55 (t, 3J8, 9 = 7.2 Hz, H8, 4H), 2.43 (quin, 3J8, 9 = 7.2, H9, 2H). 13C NMR (125 MHz, CD3OD, δ): 178.2 (C11), 171.0 (C1), 157.5 (C6), 152.7 (C2), 137.8 (C4), 123.3 (C5), 121.1 (C3), 69.3 (C10), 58.9 (C7), 52.5 (C8), 25.7 (C9). IR (cm-1): 1679 (w), 1417 (s). HR-ESI-MS calcd. for C21H25N4O8: 461.1672, found: 461.1662 [M + H]+. (1,2-[N,N’-{p-Nitrobenzyl}methyl]-N,N’-[6-{methoxycarbonyl}-pyridin-2-yl] methylamino)ethane (Me2dp-N-NO2). N,N’-[6-{Methoxycarbonyl}-pyridin-2-yl]methyl amino)ethane  was  synthesized  according  to  the  literature.108 4-Nitrobenzyl bromide (135 mg, 0.625 mmol) was dissolved in 20 mL acetonitrile together with 1,2-[{6-(methoxycarbonyl)pyridin-2-yl}methyl  amino]  ethane (105 mg, 0.293 mmol). Na2CO3 (400 mg) was added into the solution and the reaction 79  was stirred overnight at 70 °C. Subsequently, the suspension was filtered and the solvent removed in vacuo. The resulting orange oil was purified by column chromatography (silica, CH2Cl2); the product was eluted with 5% CH3OH, and isolated as an orange oil (60 mg, 0.095 mmol, 33%, Rf: 0.6). 1H NMR (300 MHz, CDCl3, δ): 8.01 (d, 3J11, 12 = 8.9 Hz, H11, 4H), 7.98 (d, 3J3, 4 = 7.7 Hz, H3, 2H), 7.76 (m, 3J3, 4 = 7.7 Hz, , 3J4, 5 = 7.8 Hz, H4, 2H), 7.59 (d, 3J4, 5 = 7.8 Hz, H5, 2H), 7.46 (d, 3J11, 12 = 8.9 Hz, H12, 4H), 4.98 (s, H14, 6H) 3.83 (s, H7, 4H), 3.69 (s, H9, 4H), 2.71 (s, H8, 4H).  13  C NMR (75 MHz, CDCl3, δ): 165.8 (C1), 160.1  (C6), 147.6 (C2), 147.3 (C13), 137.6 (C10), 129.3 (C4), 125.9 (C5), 123.8 (C3), 123.7 (C11), 60.6 (C7), 58.7 (C9), 53.1 (C14), 52.3 (C8). IR (neat, cm-1): 1734 (s), 1515 (s), 1342 (s). HRESI-MS calcd. for C32H32NaN6O8: 651.2179, found: 651.2289 [M + Na]+.  (1,2-[N,N’-{p-Nitrobenzyl}methyl]-N,N’bis-[6-carboxy-2-pyridylmethyl] ethylene diamine (H2dp-N-NO2). The starting material (14, 27 mg, 0.042 mmol) was dissolved in 4 mL of a 3:1 mixture of THF and water. LiOH (5 mg, 0.21 mmol) was added to the solution resulting in an immediate colour change of the solution. The reaction was monitored by TLC and found to be complete after 45 min. The solvent was removed in vacuo to afford a white solid (25 mg, 0.041 mmol, 97%). 1H NMR (400 MHz, CD3OD, δ): 8.12 (d, 3J3, 4 = 8.7 Hz, H3, 2H), 8.10 (d, 3J4, 5 = 7.8 Hz, H5, 2H), 7.91 (t, 3J3, 4 = 8.7 Hz, 3J4, 5 = 7.8 Hz, H4, 2H), 7.42 (d, 3J11, 12 = 7.8 Hz, H11, 4H), 7.36 (d, 3J11, 12 = 7.8 Hz, H12, 4H), 3.88 (s, H7, 4H), 3.56 (s, H9, 4H), 2.41 (s, H8, 4H).  13  C NMR (100 MHz, CD3OD, δ): 172.5 (C1), 159.2 (C6), 155.0 (C2),  148.78 (C13), 145.0 (C10), 139.9 (C4), 132.2 (C5), 125.9 (C3), 124.4 (C11), 123.4 (C12), 61.0 (C7), 57.6 (C9), 31.1 (C8). IR (neat, cm-1): 1621 (w), 1512 (s), 1345 (s). HR-ESI-MS calcd. for C30H27N6O8: 599.1890, found: 599.1887 [M – H]-.  80  2-(p-Nitrobenzyl)-N,N’-[6-{methoxycarbonyl}-pyridin-2-yl]methylamino)ethane (Me2dp-bb-NO2). 3-(4-Nitrophenyl)-propane-1,2-diaminemethane and 6-formylpyridine-2-carboxylic acid methyl ester were synthesized according to the literature.108,133 To a mixture of 3-(4nitrophenyl)-propane-1,2-diaminemethane (0.46 g, 2.36 mmol) in methanol (50 mL), 6-formyl-pyridine-2-carboxylic acid methyl ester (0.78 g, 4.72 mmol) was added. The mixture was refluxed for 2 h and then cooled to 0 °C in an ice bath. After cooling, NaBH4 (0.139 g, 3.67 mmol) was added slowly and stirred at 0 °C for 2 h. Saturated aqueous NaHCO3 was then added (150 mL) and the mixture stirred for 15 min, followed by extraction with dichloromethane (5 x 80 mL). The combined dichloromethane fractions were dried over MgSO4 and evaporated to give 0.96 g of crude yellow oil. Subsequent purification of a 50 mg aliquot with column chromatography (10% CH3OH in dichloromethane) afforded the product as a colourless oil (5 mg, 0.012 mmol, 8%, Rf: 0.05). 1H NMR (300 MHz, CDCl3, δ): 8.12 (d, 3J11, 12 = 8.0 Hz, H12, 2H), 7.99 (m, H3, H3’, 2H), 7.78 (m, H4, H4’, 2H), 7.54 (m, H5, H5’, 2H), 7.35 (d, 3J11, 12 = 8.0 Hz, H13, 2H), 4.06 – 4.03 (m, H7, H7’, 4H), 3.97 (s, H15, H15’, 6H), 3.04 (m, H8’,1H), 2.97 (m, H9, H9’, 2H), 2.60 (m, 2J10, 10’ = 12.0 Hz, H10, H10’, 2H).  13  C NMR (75 MHz, CDCl3, δ): 165.9 (C1,  C1’), 160.8 (C6, C6’), 147.7 (C2, C2’), 147.6 (C14), 147.4 (C4, C4’), 146.8 (C5, C5’), 137.7 (C11), 130.4 (C3, C3’), 125.9 (C12), 123.8 (C13), 58.5 (C7, C7’), 53.1 (C15, C15’), 52.6 (C8), 52.1 (C9), 39.6 (C10). HR-ESI-MS calcd. for C25H28N5O6: 494.2040, found 494.2049 [M + H]+.  81  2-(p-Nitrobenzyl)-(1,2-[N,N’-{p-nitrobenzyl}methyl]-N,N’bis-[6-carboxy-2-pyridyl methyl] ethylenediamine (H2dp-bb-NO2). 2-(p-Nitrobenzyl)-N,N’-[6-{methoxycarbonyl}-pyridin-2-yl] methylamino)ethane (5 mg, 0.012 mmol) was dissolved in 2 mL of a 3:1 mixture of THF and water. LiOH (1 mg, 0.04 mmol) was added into the solution. The reaction was monitored by TLC and found to be complete after 30 minutes. The solvent was removed in vacuo to afford a light yellow solid (4 mg, 0.01 mmol, 83%). 1  H NMR (400 MHz, CD3OD, δ): 8.15 (d, 3J12, 13 = 8.0 Hz, H13,  2H), 7.99 (d, 3J3, 4 = 7.9, H3, H3’, 2H), 7.88 (m 3J3, 4 = 7.9, H4, H4’, 2H), 7.41 (m, H12, H5, H5’,4H), 4.12 - 3.91 (m, 2J7, 7 = 16.0, H7, H7’, 4H), 2.75 - 2.17 (m, H8’, H9, H9’, H10, H10’, 5H).  13  C NMR (100 MHz, CD3OD, δ): 165.0 (C1, C1’), 160.2 (C6, C6’), 155.2 (C2, C2’),  149.0 (C4, C4’), 148.1 (C14), 139.5 (C5, C5’), 131.6 (C11), 125.6 (C3, C3’), 124.6 (C12), 123.4 (C13), 59.5 (C7, C7’), 51.9(C9), 39.9 (C8), 39.7 (C10).  HR-ESI-MS calcd. for  -  C23H22N5O6: 464.1570; found: 464.1581 [M – H] .  4.3.4  Coordination Chemistry with Ga  1,2-[{6-(Carboxylato-)pyridin-2-yl}methylamino]ethane  gallium(III)  perchlorate  ([Ga(dedpa)][ClO4]). H2dedpa·2HCl (21 mg, 0.052 mmol) was dissolved in a CH3OH-water mixture (1:2, total volume 5 mL). Ga(ClO4)3·6H2O (24 mg, 0.052 mmol) was added and the pH was adjusted to 4.5 by addition of 0.1 M NaOH. The reaction mixture was heated for 30 minutes and then set aside in the fumehood for slow evaporation. After 72 h, rhombic colorless crystals suitable for X-ray diffraction had precipitated in quantitative yield. 1  H NMR (300 MHz, d6-DMSO, δ): 8.59 (d, 3J4, 5 = 7.6 Hz, H5, 2H), 8.29 (d, 3J3, 4 = 3J4, 5 =  7.6 Hz, H4, 2H), 8.09 (d, 3J3, 4 = 7.6 Hz, H3, 2H), 4.60-4.32 (dd, 2J7, 7’ = 17.4 Hz, H7, H7’, 4H), 3.06 (dd, 2J8, 8’ = 10.0 Hz, H8, 2H), 2.4 (dd, 2J8, 8’ = 10.0 Hz, H8’, 2H). 13C NMR (75  82  MHz, d6-DMSO, δ): 162.0 (C1), 150.4 (C6), 145.2 (C4), 144.1 (C2), 126.5 (C5), 122.0 (C3), 48.2 (C7), 46.0 (C8).  IR (neat, cm-1): 1695, 1664, 1606. HR-ESI-MS calc. for  C16H1669GaN4O4: 397.0427, found: 397.0431 [M]+.  (1,2-[N,N’-{p-Nitrobenzyl}methyl]-N,N’bis-[6-carboxy-2-pyridylmethyl]ethylene diamine gallium(III) nitrate ([Ga(dp-N-NO2)][NO3]). H2dp-N-NO2 (7 mg, 0.011 mmol) was dissolved in a CH3OH-water mixture (1:2, total volume 2 mL). Ga(NO3)3·6H2O (4 mg, 0.011 mmol) was added and the pH was adjusted to 4.5 by addition of 0.1 M NaOH. The reaction mixture was stirred at 60 °C for 2 h. The solvent was removed in vacuo to afford a white solid in quantitative yield. The solid was redissolved in a mixture of water and methanol (1:2). Colourless plates suitable for Xray diffraction were obtained by slow evaporation of the solvent mixture. 1H NMR (400 MHz, CD3OD, δ): 8.71 (m, 3J3, 4 = 7.6 Hz, 3J4, 5 = 7.9 Hz, H4, 2H), 8.49 (d, 3J3, 4 = 7.6 Hz, H3, 2H), 8.32 (d, 3J11, 12 = 8.5 Hz, H11, 2H), 8.18 (d, 3J4, 5 = 7.9 Hz, H5, 2H), 7.72 (d, 3J11, 12 = 8.5 Hz, H12, 2H), 5.03-4.34 (dd, 2J7, 7’ = 16.8 Hz, H7, H7’, 4H), 4.18-3.87 (dd, 2J8, 8’ = 17.4 Hz, H8, H8, 4H), 3.13 (dd, 2J7, 7’ = 17.4 Hz, H9 H9’, 4H).  13  C NMR (150 MHz, CD3OD, δ):  165.1 (C1), 152.0 (C6), 150.3 (C4), 148.3 (C13), 145.7 (C10), 138.1 (C2), 134.5 (C5), 129.8 (C11), 125.6 (C3), 124.8 (C12), 57.7 (C7), 55.4 (C9), 48.1 (C8). IR (neat, cm-1): 1607 (w), 1519 (s), 1346 (s). HR-ESI-MS calcd. for C30H26N6O869Ga: 667.1068, found: 667.1075 [M]+.  83  2-(p-Nitrobenzyl)-(1,2-[N,N’-{p-nitrobenzyl}methyl]-N,N’bis-[6-carboxy-2-pyridyl methyl] ethylenediamine gallium(III) nitrate ([Ga(dp-bb-NO2)][NO3]). H2dp-bb-NO2 (2.5 mg, 5.3  μmol)  was  dissolved in water (1 mL). Ga(NO3)3·6H2O (2 mg, 5.5 μmol, in 0.5 mL water) was added and the pH was adjusted to 5 by addition of 0.1 M NaOH. The reaction mixture was stirred at 60 °C for 2 h. The solvent was removed in vacuo to afford an off-white solid in quantitative yield. 1H NMR (300 MHz, CD3OD, δ): 8.62 (m, 3  J3, 4 = 7.5 Hz, H3, 2H), 8.36 (d, 3J12, 13 = 8.7 Hz, H12, 2H), 8.20 - 8.09 (d, 3J3, 4 = 7.5 Hz, H4,  H5, 4H), 7.53 (d, 3J12, 13 = 8.7 Hz, H13, 2H), 4.81 - 4.39 (dd, 2J7, 7’ = 17.4/ 16.8 Hz, H7, H7’, 4H), 3.59 - 2.21 (m, 2J8, 8’ = 12.3 Hz, , 3J8, 9 = 3 Hz, H8, H8’, H9, H10, H10’, 5H).  13  C NMR  (150 MHz, CD3OD, δ): 165.5 (C1), 165.4 (C1’), 152.1 (C6), 151.6 (C6’), 148.8 (C14), 147.4 (C4), 147.3 (C4’), 146.1 (C10), 145.9 (C2), 145.3 (C2’), 131.8 (C5), 129.1 (C5’), 129.0 (C13), 125.1 (C3), 124.7 (C3’), 124.6 (C12), 58.9 (C7, C7’), 53.7 (C9), 51.5 (C8), 37.6 (C10). HR-ESI-MS calcd. for C23H2169GaN5O6: 532.0748, found: 532.0743 [M]+.  4.3.5 67  Labelling with 67/68Ga  GaCl3 (100 μL, 1 mCi) or 68Ga3+(100 μL, 1 mCi) in a 0.1 M HCl solution was added to 900  μL of a 10-4 M solution of ligand in 10 mM NaOAc solution (pH 4.5) and left for 10 minutes at RT. The reaction progress was monitored by analytical HPLC to evaluate yield of the coordination reaction. [67/68Ga(dedpa)]+: tR on HPLC: 6.1 minutes (gradient: A: 10 mM NaOAc buffer, pH 4.5, B: CH3OH. 0-5% B linear gradient 20 min). Ligand concentrations of up to 10-7 M are capable of coordinating the radionuclide to above 96% (under the same labeling conditions, 10 min, room temperature). The high specific activity of 9.8 mCi/nmol was achieved with 900 μL of a 10-7 M solution of H2dedpa in 10 mM NaOAc solution (pH 4.5) and 100 μL of  68  Ga3+ in a 0.1 M HCl solution (0.98 mCi) under standard labelling  conditions (10 min, room temperature). Analysis of radiolabelled complexes was done using reversed phase HPLC, on a Phenomenex Hydrosynergy RP C18 4.6 × 150 mm analytical  84  column ([Ga(dedpa)]+), Phenomenex Jupiter 5u C18 300 A 4.6 × 100 mm analytical column (transferrin challenge with [Ga(dedpa)]+, NOTA versus H2dedpa challenge, H2dedpa challenge of Ga(NOTA), retention time of  67  Ga-Tf: 10.7 min) and Waters XBridge BEH130  +  4.6 × 150 mm ([Ga(dp-bb-NO2)] , [Ga(dp-N-NO2)]+, as well as the transferrin challenges thereof, retention time of  67  Ga-Tf: 2.5 min).  Rt of [67/68Ga(dedpa)]+ on HPLC: 5.5 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH3OH. 0-5 % B linear gradient 20 min). Stability versus transferrin (10 min/1 h/ 2 h; in % complex intact): 99/ 99/ 99. Rt of [67/68Ga(dp-N-NO2)]+ on HPLC: 10.8 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH3OH. 0-100 % B linear gradient 20 min). Stability versus transferrin (10 min/1 h/ 2 h; in % complex intact): 88/ 69/ 51. Rt of [67/68Ga(dp-bb-NO2)]+ on HPLC: 7.7 minutes (gradient: A: NaOAc buffer, pH 4.5, B: CH3OH. 0-100 % B linear gradient 20 min). Stability versus transferrin (10 min/1 h/ 2 h; in % complex intact): 98/ 97/ 97.  4.3.6  In Vitro Stability: Transferrin Challenge and Challenge against  NOTA Analysis of radiolabelled complexes Both the NOTA versus H2dedpa challenge, H2dedpa challenge of Ga(NOTA), retention time of  67  Ga-Tf: 10.7 min) and ([Ga(dp-bb-  NO2)]+, [Ga(dp-N-NO2)]+, as well as the transferrin challenges thereof, retention time of 67  Ga-Tf: 2.5 min) were done using reversed phase HPLC, using a Waters XBridge BEH130  4.6 × 150 mm analytical column. For apo-transferrin competition, 67GaCl3 was added to a 10-4 M solution of ligand in 10 mM NaOAc solution (pH 4.5). Complex formation was checked by HPLC. A 400 μL aliquot was added to 1 mg/mL apo-transferrin in a NaHCO3 solution (10 mM, 600 μL) and incubated at 37° C (water bath). Complex stability was checked at time points 10 minutes, 1 h and 2 h via analytical HPLC. 67  GaCl3 was added to 10-4 M solution of both NOTA and H2dedpa in 10 mM NaOAc  solution (pH 4.5). After a reaction time of 10 minutes at room temperature the reaction  85  mixture was checked for the formed complex by analytical HPLC. Over 98% of the  67  Ga-  dedpa complex was detected opposed to 0.2 % Ga-NOTA.  4.3.7  X-Ray Diffraction Structural Characterization of  [Ga(dedpa)][ClO4] and [Ga(dp-N-NO2)] [ClO4] Data for compound [Ga(dedpa)][ClO4] were collected with graphite-monochromated Mo-Kα radiation (0.71073 Å) at -17˚C on a Bruker X8 APEX II diffractometer. The structure was solved using direct methods using SIR-97134 and refined using SHELXL-97.135 All non-hydrogen atoms were refined anisotropically. All N-H hydrogen atoms were located in a difference map and refined isotropically. All other hydrogen atoms were placed in calculated positions and refined using a riding model.  Data for compound [Ga(dp-N-  NO2)][ClO4] were collected with graphite-monochromated Mo- Kα radiation at -183˚C on a Bruker APEX DUO diffractometer. The structure was solved using direct methods using SIR-97134 and refined using SHELXL-97.135 The material crystallizes with two crystallographically independent moieties in the asymmetric unit. One perchlorate anion is disordered and was modeled in two orientations, with restraints used to maintain reasonable geometries. Finally, CH3OH solvent was found in the lattice. Two molecules of solvent were located and modeled, however one region within the asymmetric unit had residual electron density that could not be properly modeled. The SQUEEZE136 program was used to generate a data set free of residual electron density in that region. All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were placed in calculated positions and refined using a riding model. The collection and analysis of the data was done by Dr. Brian O. Patrick.  4.3.8  Biodistribution of [67Ga (dedpa)]+, [67Ga(dp-bb-NO2)]+ and  [67Ga(dp-N-NO2)]+ The protocol used for the animal studies was approved by the Institutional Animal Care Committee of the University of British Columbia and was performed in accordance with the Canadian Council on Animal Care Guidelines. A total of 16 female ICR (20-30 g)  86  mice were used for the animal study of each of the three compounds. [67Ga(dedpa)]+, [67Ga(dp-bb-NO2)]+ and [67Ga(dp-N-NO2)]+ were each prepared as described above and then diluted in phosphate buffered saline to a concentration of 100 μCi/mL. Each mouse was i.v. injected with ~10 μCi (100 μL) of the  67  Ga complex and then sacrificed by CO2  inhalation at 30 min, 1 h, 2 h, or 4 h after injection (n=4 at each time point). Blood was collected by cardiac puncture and plasma was separated from whole blood by centrifuging (2500 rpm, 15 min). Urine was collected from the bladder. Tissues collected included kidney, liver, spleen, femur, muscle, heart, lung, intestine and brain. Tissues were weighed and counted on a gamma counter and the counts were converted to % injected dose/gram (%ID/g). The collection of the data was done by Dr. Dawn Waterhouse.  4.4  Results and Discussion  4.4.1  Ligand Synthesis  In order to find a novel chelate for fast, clean and mild labelling with the radioisotope  68  Ga, a library of chelates with the same set of donor atoms was first  established. The compounds outlined in this chapter have denticities reaching from tetradentate to octadentate, while bearing permutations of pyridyl, aliphatic amino- and carboxylato donor atoms. Bridging units can be varied in a simple manner and reach from no bridging unit to three carbon bridges.  Scheme 4.1 Schematic overview of synthesis methodology used to synthesize chelates of library (i) 1. CH3OH, reflux, 30 min 2. NaBH4, CH3OH, 0°C, 2 h (ii) tert-butyl bromoactetic acid, Na2CO3, CH3CN, 70 °C, o.n. (iii) TFA/ CH2Cl2, 30 min (iv) LiOH, THF/ H2O, 30 min – 2 h.  87  The assembly of these chelates was simple. The primary amines are commercially available and were reacted with the pyridyl unit using reductive amination to afford the pyridyl functionalized secondary amine. This approach afforded either an already protected chelate scaffold, or provided a protected intermediate, which could be further functionalized by alkylation with additional carboxylate arms. In all cases, deprotection of the carboxylate functionalities constituted the last synthetic step furnishing a selection of simple chelation scaffolds. The facile assembly of the chelate library compounds is shown with the general reaction Scheme 4.1. Some chelates were synthesized using a variation of this methodology, such as direct alkylation with the pyridyl fragment. Syntheses of a variety of fragments, such as N-N’-bis(2-pyridylmethyl) ethylenediamineN-N’-diacetic acid,130 1,2-[{6-(carboxylato-)pyridin-2-yl}methylamino] ethane131 and N,N’-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N’-diacetic acid108 have been reported previously. All chelates included in this chapter have an even number of donor atoms. While some heptadentate chelates were afforded as byproducts of the alkylation reaction, none of them was found to have particularly favourable characteristics. mirror plane  *  H9 H10 H8  H7  *  H10  H7  H8  H9  Figure 4.3 1H NMR spectrum (300 MHz, CD3OD, 298 K) of a propyl bridged chelate, * denotes residual solvent peaks.  88  All final compounds were characterized using 1H and  13  C NMR spectroscopy, mass  spectrometry and IR. Previously synthesized compounds and protected intermediates were characterized using  1  H NMR spectroscopy and mass spectrometry. Characterisic  spectroscopic features include aromatic 1H NMR peaks in the low field region between δ = 7 - 8.2, as well as peaks of bridging methylene groups neighboring secondary and tertiary amines, which appear as singlets, either due to the lack of vicinal protons or due to the mirror plane present in these ligand systems. The only multiplets observed in the non-aromatic region were found for propyl bridged ligand systems and for the aliphatic chain on the low denticity ligand 6-propylaminomethyl-di-pyridine-2-carboxylic acid. An example of an 1H NMR spectrum of a propyl bridged ligand, N-N’-bis(2-pyridylmethyl) propylenediamineN-N’-diacetic acid, is shown in Figure 4.3.  4.4.2  Screening with 67Ga  After establishing the ligand library, the ligands were screened for radiolabelling properties. Since the most successful chelate systems for the coordination of the radioisotope  67  Ga will label at room temperature, pH 4.5 and within 10 minutes,58 the  chelate systems were tested under these labelling conditions as well. Reaction monitoring was performed with analytical HPLC. Chelates yielding below 95 % were additionally heated in order to drive the coordination reaction to completion. In all cases where heating was employed, additional side product formation was observed. The results from the screening procedure are summarized in Table 4.2. The ligand concentration was kept at 10-4 M in all cases. One lead compound was clearly identified. 1,2-[{6(Carboxylato)pyridin-2-yl}methylamino]ethane (H2dedpa) shows excellent labelling yields of 99 % and shows quantitative, clean labelling under the mild conditions outlined above with no residual free metal detectable. Concentration dependent coordination of H2dedpa at concentrations as low as 10-7 M with both  68  conversion to the desired product. When coordinating to  Ga and  67  Ga showed quantitative  68  Ga, high specific activities (as  high as 9.8 mCi nmol-1) were obtainable without any purification steps. This is the highest specific activity measured for any chelate with  68  Ga when neither heating nor  68  Ga pre-  137  purification is used.  89  Table 4.2 Summarized results from initial labeling experiments with the synthesized chelates. HPLC gradients include: A: 0 to 5% in 10 mM NaOAc (pH 4.5), 20 min. B: 0 to 100% CH3OH in 10 mM NaOAc (pH 4.5), 20 min. Chelate  Retention time (Gradient)  8.9 min (B)  Temp.  RT Heating  7.7 min (B)  RT Heating  5.3 min (B)  RT Heating  Purity  73% mixture of products  70% 88%,impurities  92% 72%,impurities  6.19 min (A)  RT  99%  7.6 min (A)  RT  44 %  Heating  44%  6.29 min (B)  RT  95 %  7.5 min (B)  RT  95%  90  4.4.3  In Vitro stability of Select Ligand Candidate  After identification of H2dedpa as the clear lead chelate, further investigations were made to examine the potential of the chelate as a viable alternative to the gold standard NOTA. To investigate the stability of the [67Ga(dedpa)]+ radiochemical complex, a 2 h competition experiment was conducted in the presence of excess human apo-transferrin, the iron sequestering/transport protein that has very high affinity for Ga(III).57 [67Ga(dedpa)]+ was fully intact after 2 h, suggesting that it should have very high in vivo stability, similar to that reported for Ga(NOTA) complexes.58  The  corresponding labelling traces are shown in Figure 4.4. In order to compare the radiolabelling efficiency of NOTA and H2dedpa, a direct competition for chelation of  67  Ga was done using equal concentrations (10-4 M) of both  NOTA and H2dedpa, over 96% was coordinated by (dedpa)2-, less than 1 % by NOTA, demonstrating the expected faster Ga complexation with the acyclic H2dedpa compared with the macrocyclic NOTA.  [67Ga(dedpa)]+  [67Ga(dedpa)]+  [67Ga(dedpa)]+ 67  Ga-Tf  0h 15 min 67GaCl  1h  3 67  Ga(NO [67Ga(NOTA)]  67  GaCl3  2h Reference  0  2.5  5  7.5  10  12.5  15  17.5  time [min]  Figure 4.4 Labelling traces from transferrin challenge experiment (left) and NOTA versus H2dedpa challenge (right).  91  4.4.4  Coordination Chemistry with Non-radioactive Ga and X-Ray  Diffraction Structural Characterization of [Ga(dedpa)][ClO4] In  order  to  characterize  the  formed  Ga  complex  macroscopically,  [Ga(dedpa)][ClO4] was prepared. Due to formation of insoluble Ga(OH)3 at pH > 4.5, which can be observed by characteristic cloudiness of an aqueous solution, the ligand and the Ga(ClO4)3 salt were mixed at pH ~ 2 in a 1:1 solution of water and CH3OH. The pH was subsequently raised slowly by dropwise addition of 0.1 M NaOH to pH 4.2 – 4.5 and then stirred at 70 °C for 30 minutes to guarantee complex formation. The solution was set aside in the fume hood for slow evaporation of solvent, which led to the formation of crystals suitable for X-ray diffraction structural characterization. [Ga(dedpa)][NO3] is formed using the analogous procedure, affording a salt which is more soluble in protic solvents and therefore more suitable for spectroscopic characterization; however, the crystals formed from this salt were never suitable for X-ray diffraction. The solid state Xray crystal structure (Figure 4.5) provides significant insight into the coordination environment of [Ga(dedpa)]+. In comparison with the crystallized Ga complexes of NOTA55 and DOTA,138 which have widely dispersed metal-to-ligand bond distances, [Ga(dedpa)]+ has a more equally distributed array of bond lengths (Table 4.3), suggesting that the unusually high stability of the complex is due to a near-perfect fit with the Ga3+ ion.  Figure 4.5 ORTEP drawing of [Ga(dedpa)][ClO4]. Shown is the atom numbering scheme (50% thermal ellipsoids).  92  Table 4.3 Selected bond lengths in [Ga(dedpa)][ClO4] , ([Å]) and angles ([°]). bond  length [Å]  angle  degree [°]  N1-Ga N2-Ga N3-Ga N4-Ga O1-Ga O2-Ga  1.9866(16) 1.9902(16) 2.1115(16) 2.1132(16) 1.9708(13) 1.9828(13)  O(1)- Ga(1)- O(2) O(1)- Ga(1)- N(1) O(2)- Ga(1)- N(1) O(1)- Ga(1)- N(2) O(2)- Ga(1)- N(3) N(1)- Ga(1)- N(3)  101.39(6) 80.14(6) 94.02(6) 94.72(6) 94.78(6) 77.82(6)  Another characteristic that clearly differentiates [Ga(dedpa)]+ from complexes with the macrocyclic chelates, is the C2 rotational axis, which has also been confirmed in solution through  13  C NMR spectroscopy, where only one signal is observed per type of carbon in  the ligand molecule. Other information that can be easily gained from spectroscopy is the conversion from homotopic protons on the non-metal bound ligand to diastereotopic protons with individual shifts and large geminal coupling constants within the complex. This is a phenomenon observed and described also in previous chapters and, along with the shift of aromatic protons to lower field upon metal binding to the pyridyl unit, it gives a good indication of completion of the coordination reaction (Figure 4.6).  Figure 4.6 1H NMR spectra (300 MHz, d6-DMSO/CD3OD, 298 K) of H2dedpa and [Ga(dedpa)][NO3], * denotes residual solvent peaks.  93  4.4.5  Potentiometric Measurements on [Ga(dedpa)]+  Solution thermodynamic investigations of the corresponding cold complex [Ga(dedpa)]+ provided a complex stability constant of log KML = 28.11(8),139 obtained by ligand-ligand competition with EDTA using potentiometric titration (performed by Dr. J. Cawthray). A more relevant indicator of the extent to which a metal complex is stable in solution is given by pM (-log[free M]), which considers the influence of ligand basicity and chelate hydrolysis. The values of log KML and pM of the Ga(III) complexes of dedpa2- and other relevant multidentate ligands are shown in Table 4.4. The high value of log KML and pM for [Ga(dedpa)]+ confirms the high affinity of dedpa2- for Ga(III) as well as high thermodynamic stability.139 Table 4.4 Formation constants (log KML) and pMa of Ga(III) complexes. a Calculated for 10 μM total ligand  and 1 μM total metal at pH 7.4 and 25°C. b Conditional constant for log KML from Ref 57.  4.4.6  Ligand  log KML  pM  dedpa2-  28.11  27.4  EDTA140  21.7  18.3  DOTA121  21.33  18.5  NOTA122  30.98  27.9  Transferrinb  20.3  21.3  Model Functionalization of the H2dedpa Scaffold  While the close investigation of the chelation scaffold H2dedpa was able to show very promising results, the derivatization of H2dedpa to allow conjugation to a potential biomolecule had not yet been explored. To investigate different modes of functionalization analogous to the bifunctional versions of the macrocyclic chelates, two model compounds H2dp-N-NO2 and H2dp-bb-NO2 were synthesized (Scheme 4.2).  94  H2dp-N-NO2 displays derivatization through the two aliphatic nitrogens, affording a scaffold capable of carrying two targeting molecules, while compound H2dp-bb-NO2 is derivatized through the backbone of the ethylenediamine (en) component of the basic ligand structure, retaining the original coordination environment more closely, but only allowing the attachment of one targeting molecule.  Scheme 4.2 Syntheses of H2dp-N-NO2 and H2dp-bb-NO2: (i) 1. CH3OH, reflux, 2h, 2. NaBH4, 0°C, 2h (ii) 4nitrobenzyl bromine, Na2CO3, CH3CN, 18 h (iv) LiOH, THF/water (3:1), 45 min.  Both H2dp-N-NO2 and H2dp-bb-NO2 incorporate the nitrobenzyl functionality, which can be converted easily into the corresponding amino- or isothiocyanato-benzyl coupling moieties frequently employed for conjugation to target molecules via a free carboxylate or primary amine, respectively.141,142 H2dp-N-NO2 is synthesized from 1,2[{6-(methoxycarbonyl)pyridin-2-yl}methylamino]ethane,108 which is then subsequently alkylated with 4-nitrobenzyl bromide. Intermediate Me2dp-N-NO2 is purified and the carboxylates are deprotected under standard conditions to afford the clean product H2dpN-NO2 as a white solid, characterized using 1H and  13  C NMR spectroscopy, mass  spectrometry and IR. Compound H2dp-N-NO2 is furnished through a different route; 3(4-nitro-phenyl)-propane-1,2-diaminemethane is derived from 4-nitro-L-phenylalanine,133 while 6-formyl-pyridine-2-carboxylic acid methyl ester is afforded through a four step  95  synthesis from 2,6-pyridinedicarboxylic acid.108 A one-pot reductive amination process produces Me2dp-bb-NO2 in moderate yields, along with a mixture of impurities, which can be separated from the product through column chromatography. The subsequent deprotection leads to H2dp-bb-NO2 as a light orange solid characterized using 1H and 13C NMR spectroscopy, and mass spectrometry. Since only a very small amount of compound H2dp-bb-NO2 was isolated, no IR spectroscopy was performed.  4.4.7  67  Ga Labelling of the Model Bifunctionals H2dp-bb-NO2 and  H2dp-N-NO2 and Transferrin Challenge Experiments of [67Ga(dp-bbNO2)]+ and [67Ga(dp-N-NO2)]+ Coordination of H2dp-N-NO2 to  67  Ga or  68  Ga affords the complex within 10  minutes at room temperature in 98 % radiochemical yield. The subsequent apo-transferrin challenge experiment revealed that 51 % of the radiolabeled complex remained intact after 2 h in the presence of excess apo-transferrin, a stability inferior to that of [Ga(dedpa)]+, but comparable to that of Ga-DOTA.58 In the case of H2dp-bb-NO2, the 67  Ga and  68  Ga complexes are formed within 10 minutes at room temperature in 97 %  radiochemical yield. The subsequent apo-transferrin challenge experiment reveals a stability comparable to that of [Ga(dedpa)]+, with over 97 % of the complex remaining intact after 2 h (Figure 4.7). Concentration-dependent coordination to 2-  68  Ga showed that  2-  both (dp-N-NO2) and (dp-bb-NO2) are capable of coordinating under mild conditions at concentrations as low as 10-6 M. 98% 88%  98% 98% 97%  69%  97%  51%  0h  0h  10 min  10 min  1h  1h 2h  2h 67  Figure 4.7 Stacked HPLC monitoring traces of [ Ga(dp-N-NO2)]+ (left) and [67Ga(dp-bb-NO2)]+ (right) over 2 h stability experiment against apo-transferrin.  96  4.4.8  Coordination Chemistry of the Model Bifunctionals with Cold Ga  and X-Ray Diffraction Structural Characterization of [Ga(dp-NNO2)][ClO4] The complex with cold Ga is formed within 2 h at pH 4-5 under gentle heating and again the C2 rotational axis was confirmed in both the solid-state structure and the solution NMR. The same procedure as for [Ga(dedpa)][ClO4] was employed. For both model compounds, the complex was formed with (NO3)- as the counter ion, since this salt provides better solubility in protic solvents. In the case of  [Ga(dp-N-NO2)][NO3],  information that can be easily extracted from 1H NMR spectroscopy includes the conversion from homotopic protons on the non-metal bound ligand to diastereotopic protons with individual shifts and large geminal coupling constants upon complexation of Ga(III) within the complex, analogous to the previously described [Ga(dedpa)]+ complex (Figure 4.8).  Figure 4.8 1H NMR spectra (300 MHz, CD3OD, 298 K) of H2dp-N-NO2 and [Ga(dp-N-NO2)][NO3]. * denotes residual solvent peaks.  For H2dp-bb-NO2 however, where all protons appear already as diastereotopic  97  protons within the ligand due to the asymmetry of this ligand system, the coordination reaction can be traced using the 1H NMR spectrum mainly through the shift of the aromatic protons and bridging methylene protons to lower field, as well as increased splitting between geminal protons upon coordination (Figure 4.9).  Figure 4.9 1H NMR spectra (300 MHz, CD3OD, 298 K) of H2dp-bb-NO2 and [Ga(dp-bb-NO2)][NO3]. * denotes residual solvent peaks.  In order to afford crystals suitable for X-ray diffraction structural characterization of the Ga complex with H2dp-N-NO2, the complex with (ClO4) - as the counter ion was prepared and, after slow evaporation, crystals suitable for X-ray diffraction were formed. The solid state structure (Figure 4.10) shows similar bond lengths as for [Ga(dedpa)][ClO4], however the N3-Ga and the N4-Ga bond lengths appear to be slightly elongated compared to the non-derivatized structure, which could be an indication as to why the corresponding radiochemical complex exhibited decreased stability in the apotransferrin challenge experiment.  98  Table 4.5 Selected bond lengths in [Ga(dp-N-NO2)][ClO4] (bold), compared to selected bond lengths in [Ga(dedpa)][ClO4], ([Å]). bond N1-Ga N2-Ga N3-Ga N4-Ga O1-Ga O2-Ga  length [Å] 1.992(5) 1.981(5) 2.188(5) 2.159(5) 1.967(4) 1.976(4)  length [Å] 1.9866(16) 1.9902(16) 2.1115(16) 2.1132(16) 1.9708(13) 1.9828(13)  Figure 4.10 ORTEP drawing of [Ga(dp-N-NO2)][ClO4] (50% thermal ellipsoids); H atoms and the (ClO4) counter ion are omitted for clarity.  99  Biodistribution of 67Ga Complexes of (dedpa)2- and the Model  4.4.9  Bifunctionals The biodistribution study in mice (Figure 4.11, appendix Tables B) indicated that [67Ga(dedpa)]+ cleared from the background tissue, such as muscle, within the first 30 min and was excreted mainly through the kidneys. 67  The in vivo stability of  +  [ Ga(dedpa)] was supported by the low uptake in bone, which is known to be a site of increasing accumulation for weakly chelated  67  Ga.143 The overall biodistribution profile  compares well to macrocyclic chelates evaluated in a similar study,58 also exhibiting the low uptake in liver and intestines characteristic of ionic compounds such as [67Ga(dedpa)]+. The persistent high uptake in the blood serum was not confirmed with the  derivatized  compounds,  suggesting  that  added  functionality  influences  biodistribution.  [67Ga(dedpa)]+  [67Ga(dp-N-NO2)]+  [67Ga(dp-bb-NO2)]+  Figure 4.11 Biodistribution of [67Ga(dedpa)]+, [67Ga(dp-N-NO2)]+ and [67Ga(dp-bb-NO2)]+ in female ICR mice over 4h; complete data of the latter two compounds for urine is shown also in separate diagrams to the right.  100  In the biodistribution studies for [67Ga(dp-N-NO2)]+and  [67Ga(dp-bb-NO2)]+,  whole blood was collected instead of serum, and urine was collected as an additional data point (Figure 4.11, Table A.1). Both [67Ga(dp-N-NO2)]+and [67Ga(dp-bb-NO2)]+ exhibit improved clearance from all organs, low bone uptake (indicator for complex stability) and excretion through urine. Despite lower in vitro stability, the biodistribution of [67Ga(dp-N-NO2)]+ suggests high stability in vivo and shows better clearance from blood and kidneys than either [67Ga(dp-bb-NO2)]+ or [67Ga(dedpa)]+. It is possible that compounds containing secondary amines associate more strongly with blood serum proteins and kidney tissue; however, the difference in biodistribution of [67Ga(dp-bbNO2)]+and [67Ga(dedpa)]+ shows that added functionalities, such as peptides or other targeting vectors, should have a great impact on the interaction of these compounds with in vivo systems.  4.5  Conclusions A set of easily assembled acyclic chelates was synthesized and screened for their  ability to chelate  67/68  Ga under mild conditions (pH 4.5, room temperature) and short  reaction time. The hexadentate H2dedpa ligand complexes quickly with Ga, forming complexes of very high stability, comparing well to the widely used macrocyclic chelate NOTA and exceeding the properties of DOTA. The mild conditions used, combined with the high radiochemical yields make this ligand system an ideal scaffold for applications with more sensitive biomolecules such as peptides and antibodies. The direct labelling, high radiochemical yield and high specific activity of the products could obviate the need for time consuming HPLC purification, a major advantage when using the short lived isotope  68  Ga. Two model derivatives were synthesized in order to predict the most  optimal mode of conjugation to a biomolecule. While the backbone derivatization gave rise to a complex with higher stability, the N-derivative complex showed slightly decreased in vitro stability. The biodistributions of [67Ga(dedpa)]+, [67Ga(dp-N-  101  NO2)]+and [67Ga(dp-bb-NO2)]+ confirmed the stability of the complexes measured in vitro with general clearance rendering these frameworks a good basis for elaboration of new Ga bioconjugates. It is important to note that many of the advantageous properties described for H2dedpa have been observed previously with only one macrocyclic chelate (NOTA), and they are unexpected for an acyclic system. Chapter 6 elaborates further on the functionalization of the H2dedpa scaffold with biomolecules, as well as the properties of the corresponding bioconjugates.  102  Chapter 5: Viability of H2dedpa for Labelling with 64Cu 5.1  Introduction: Prior Art in 64Cu Chelates  A wide variety of Cu isotopes applicable to diagnostic imaging and radiotherapy has been the subject of extensive research. 64Cu (t1/2=12.7 h, β+ 17.4 %, Emax = 0.656 MeV, β- 39 %, Emax = 0.573 MeV) is of high current interest for both positron emission tomography (PET) and radiotherapy.48 The longer half-life is more applicable to developing PET agents with larger biomolecules, such as monoclonal antibodies that may require longer circulation times before imaging to achieve optimal target uptake.95 While not available as a generator produced isotope, the longer half-life allows for shipment of the isotope to remote areas where no cyclotron is available.118 As opposed to the non-redox active Ga(III), three oxidation states (+1, +2, +3) are accessible for copper in aqueous solution. The d9 Cu(II) configuration is predominant in aqueous solution and prefers square planar and Jahn-Teller distorted octahedral geometries. The ligand exchange kinetics of Cu(II) are particularly rapid, therefore kinetically inert ligand systems with strong crystal field stabilization for complexation are preferred for incorporation into radiopharmaceuticals.49 Cu(II) is a borderline soft metal center, with preferences for donor atoms similar to the preferences of Ga(III): amines, imines, pyridines and carboxylates.143 Among the bifunctional ligand systems investigated and previously reported for the purpose of targeted delivery of 64Cu, a strong preference for hexadentate cage-like poly-aza macrocyclic ligands can be found. Preferred properties for good  64  Cu bifunctional chelates  include: complex stability against serum over the course of 24 h, and in vivo inertness toward transchelation, which can be monitored by liver uptake, negative complex reduction potentials well below -0.4 V (NHE),49 as well as reversibility of the reduction reaction without loss of ligand.144 The metal ion 2+ charge is preferentially reduced to a 1+, -1 or neutral charge of the complex in order to minimize high kidney uptake and accelerate excretion. Mild labelling conditions are preferred, despite the long half-life, since heat- and pH- sensitive biomolecules such as antibodies are used preferentially with this isotope.  103  Thermodynamic stability is a consideration when designing Cu chelates; however, it does not necessarly correlate with in vivo complex stability.49 In Figure 5.1 and Table 5.1, a selection of important chelate structures and some of their relevant characteristics such as labelling conditions, reduction potential and thermodynamic stability constant log KML are shown.  Figure 5.1 Structures of selected, previously investigated chelate systems for labelling with 64Cu. Table 5.1 Summary of relevant properties of previously investigated chelates for labelling of 64Cu Ligand system  Labelling conditions  log KML  Ered (vs NHE)  NOTA145  30 min, room temp.  19.8, 21.6  -0.7 (irrev.)  DOTA146  5 min, room temp.  22.7  -0.74 (irrev.)  Cyclam147  10 min, room temp.  27  -0.48 (irrev.)  DIAMSAR148  1h, room temp.  n. d.  -0.90 (irrev.)  CB-TE2A149  1 h, 95 °C  n. d.  -0.88 (q-rev.)  CB-DO2A149  30 min, 80 °C  n. d.  -0.72 (irrev.)  104  5.2  Experimental  5.2.1  Materials and Methods  Refer to Chapter 2.2 for general information. H2dedpa was prepared according to Chapter 4. A Phenomenex Synergi 4 μm Hydro-RP 80A column (250 × 4.6 mm) was used to monitor decomplexation in 6 M HCl at 90 °C. HPLC solvents consisted of 0.1% trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B). Samples were analyzed with a linear gradient method (100 % solvent A to 100 % solvent B over 30 min).  64  Cu was obtained as a dilute HCl solution (Nordion); it was commercially  available from Nordion at the time of the reported the specific activity of the  64  64  Cu experiments.  Product specification  Cu to be > 5000Ci/g with < 0.2 micrograms of Cu  per mCi. The HPLC system used for analysis of the  64  Cu labelled compounds consisted  of a Waters Alliance HT 2795 separation module equipped with a Raytest Gabbistar NaI detector and a Waters 996 photodiode array (PDA) detector.  64  Cu labelled compounds  and their serum stability were analyzed on a Waters XBridge BEH130 4.6 mm × 150 mm column. Samples were analyzed with a linear gradient method (95 % solvent A to 100 % solvent B over 30 min).  5.2.2  Coordination Chemistry with Non-radioactive Cu  1,2-[{6-(Carboxylato)pyridin-2-yl}methylamino]ethane  copper(II)  trihydrate  [Cu(dedpa)]·3H2O. H2dedpa·2 HCl (5.8 mg, 0.023 mmol) was dissolved in 1 mL H2O. Cu(Cl)2·H2O (2.4 mg, 0.023 mmol) is dissolved in a 1:1 mixture of CH3OH / H2O (2 mL total volume) and added dropwise to the ligand solution. The color of the mixture intensified to a greenish blue indicating coordination of the ligand. The pH was adjusted to 6-7 by dropwise addition of an aqueous solution of 0.1 M NaOH. Subsequently, the solvent was removed and the blue crude residue is redissolved  105  in minimal amounts of H2O and loaded onto a C18 Waters sep-pak column cartridge. The column was flushed with 20 mL H2O and the complex was eluted with EtOH as a clear blue solution, which was set aside for slow evaporation in the fumehood. IR (neat, cm-1): 1621 (w), 1574 (s, br), 1410 (m), 1362 (m). HR-ESI-MS calcd. for C16H16CuN4NaO4: 414.0365, found: 414.0374 [M + Na]+.  Elemental analysis: calcd. (found) for  Cu(dedpa)·3H2O: C 43.0 (42.58), H 4.97 (4.67), N 12.56 (12.04).  5.2.3 64  Labelling with 64Cu and In Vitro Stability  Cu2+ in dilute HCl (100 μL, 0.7 mCi) was added to 900 μL of a 10-5 M solution  of H2dedpa in 10 mM NaOAc solution (pH 5.5) and left to react for 10 minutes at RT. The reaction progress was monitored by analytical HPLC to evaluate yield of the coordination reaction. [64Cu(dedpa)]. tR on HPLC: 5.2 minutes (gradient: A: 0.1 % TFA in H2O, B: CH3CN 5-100% B linear gradient 30 min), yield: 99 %.  Ligand  concentrations as low as 10-6 M were capable of coordinating the radionuclide with a 63 % yield (under these labelling conditions). To evaluate complex stability, an aliquot (500 μL) of the reaction was added to a solution of mouse serum (500 μL). The mixture was incubated at 37°C and analyzed by PD-10 column filtration after 2 h and 24 h. Aliquots of reaction mixtures were loaded onto the pre-rinsed PD-10 columns (total loading volume: 2.5 mL). Subsequently, the column was rinsed with 3.5 mL PBS. The resulting eluate was collected and measured for radioactivity. The activity eluted corresponds to serum-bound 64Cu. Stability versus serum (2 h/ 24 h; in % complex intact): 97/ 77.  5.2.4  X-Ray Diffraction Structural Characterization of  [Cu(dedpa)]·3H2O A blue prism crystal of C16H16Cu N4O4·3H2O having approximate dimensions of 0.11 x 0.15 x 0.21 mm was mounted on a glass fiber. All measurements were made on a Bruker APEX DUO diffractometer with graphite monochromated Mo-Kα radiation. Data were collected and integrated using the Bruker SAINT78 software package. The linear absorption  106  coefficient, μ, for Mo-Kα radiation is 12.07 cm-1. Data were corrected for absorption effects using the multi-scan technique (SADABS79), with minimum and maximum transmission coefficients of 0.808 and 0.876, respectively. The data were corrected for Lorentz and polarization effects. The structure was solved by direct methods. The material crystallizes with three molecules of H2O in the asymmetric unit. All non-hydrogen atoms were refined anisotropically. All hydroxyl hydrogen atoms and N-H hydrogen atoms were located in difference maps and refined isotropically. The hydrogen atoms of each water molecule were disordered in three orientations, each orientation taking advantage of a possible hydrogen bond interaction. The excellent quality of the data made it possible to refine the positions and occupancies of the hydrogen fragments on each water molecule; however, the isotropic thermal parameters were constrained to be 1.5 times the thermal parameter of its associated oxygen. All other hydrogen atoms were placed in calculated positions. Finally, the material crystallizes as a racemic twin, in an approximately 80:20 ratio between the two twin volumes. The collection and analysis of the data was done by Dr. Brian O. Patrick.  5.2.5  Cyclic Voltammetry Measurements on [Cu(dedpa)]  Cyclic voltammetry was carried out using an Autolab potentiostat. A glassy carbon electrode (MF 2012, 3 mm diameter) was used as the working electrode, the reference electrode was Ag/AgCl (sat.), and the counterelectrode was platinum mesh. Cyclic voltammetry was carried out in 0.1 M NaOAc, pH 7 (adjusted with glacial acetic acid), with a 0.1 M solution of [Cu(dedpa)] at scan rates 100 mV/s and 10 mV/s. A 100 mV/s scan was applied to a solution of solvent between 2 and -2 V in order to establish solvent reduction and oxidation. This was found to be 0.5 to -1.25 V and should not lead to solvent redox behaviour. Subsequently, the ligand or complex was dissolved in the previously measured solvent solution and the cyclic voltammogram was measured within a variety of electrochemical potential windows (0.5 to -1.25 V, as well as 0.5 to -0.5 V). It was found that the one electron reduction of the copper complex could be observed at 1.12 V (normal hydrogen electrode, NHE: 0.92 V) with re-oxidation at 0.02 V (NHE: 0.22). The afforded voltammogram is reproducible upon cycling.  107  5.3  Results and Discussion  5.3.1  Cu(II) Coordination Chemistry and X-Ray Diffraction Structural  Characterization of [Cu(dedpa)]·3H2O Formation of the copper complex with H2dedpa can be followed by the strong, obvious color change of the reaction mixture upon mixing of ligand and metal solution. A variety of copper salts was evaluated as starting material for complex formation: Cu(OAc)2·H2O, CuCl2·2H2O, as well as Cu(ClO4)2·6H2O. The resulting complex is only soluble in protic solvents despite its neutral nature, hence the product cannot be precipitated or extracted. Separation from salts was done with purification using C18 reversed phase cartridge. The paramagnetic nature of the Cu(II) metal center hinders NMR spectroscopic investigations, hence the product was characterized through elemental analysis, IR spectroscopy and mass spectrometry. IR spectral data show a characteristic decrease of carboxylate stretching frequencies upon metal coordination. In the mass spectrum, diagnostic [M + Na]+ peaks were easily identified and displayed the characteristic isotope distribution of 63Cu/65Cu.  Figure 5.2 ORTEP drawing of [Cu(dedpa)]; shown is the atom numbering scheme (50% thermal ellipsoids) and hydrogen atoms within the complex structure are omitted for clarity.  108  The product crystallizes forming small blue prism crystals suitable for X-ray diffraction. The complex co-crystallizes with three water molecules in the lattice (Figure 5.2). Bond length comparisons between the metal complex and the ligand provides insight into the coordination environment (Table 5.2). Table 5.2 Selected bond lengths in [Cu(dedpa)] (bold), compared to selected bond lengths in [Ga(dedpa)][ClO4], ([Å]). Select bond angles are given [°].  bond  length [Å] (M = Cu)  length [Å] (M = Ga)  angle  degree [°]  N1-M N2-M N3-M N4-M O1-M O2-M  2.0008(12) 2.3171(13) 2.1364(13) 1.9386(13) 2.3014(11) 2.0430(10)  1.992(5) 1.981(5) 2.188(5) 2.159(5) 1.967(4) 1.976(4)  N(4)- Cu(1)- O(3) N(4)- Cu(1)- N(3) N(1)- Cu(1)- N(3) N(4)- Cu(1)- O(1) N(1)- Cu(1)- O(1) N(1)- Cu(1)- N(2)  81.17(5) 78.65(5) 104.83(5) 97.86(5) 76.23(4) 76.82(5)  Most bond lengths in [Cu(dedpa)] are between 1.93 – 2.04 Å, except N2-Cu and O1-Cu, which at 2.30 Å show a Jahn-Teller type distortion, typical for octahedral Cu(II) complexes and similarly observed for [Cu(CB-TE2A)].144 In order to illustrate the elongation of the axial bonds, a side-on view in Figure 5.3 is provided, with comparison to [Ga(dedpa)]+.  2.30  1.97  2.32  1.99  Figure 5.3 Side-on view of [Cu(dedpa)] (right), compared to selected bond lengths in [Ga(dedpa)]+ (left), H atoms and (ClO4)- counter ion of Ga complex omitted for clarity. Graphic generated using Mercury 2.2.150  109  By looking at another side-on view of both complexes, the degree of distortion of both complexes is illustrated. The Cu(II) complex appears to be closer to octahedral geometry; however, both complexes are strongly distorted. The H2dedpa ligand system shows great flexibility in terms of metal ion tolerance. A possible reason for this could be the enhanced intrinsic rigidity of the carboxylate subunit, which is fixed in conformation with regard to the pyridine ring (Figure 5.4), while the aliphatic units of the chelate regulate the bite angle with which it wraps around the metal center.  101.39°  95.99°  Figure 5.4 Side-on view of [Cu(dedpa)] (right), compared to compared to selected bond lengths in [Ga(dedpa)]+ (left), H atoms omitted for clarity. Graphic generated using Mercury 2.2.150  5.3.2  Cyclic Voltammetry  In order to evaluate the capability of the complex to withstand bio-reductive conditions without reduction of Cu(II) to Cu(I) and subsequent decomplexation, a cyclic voltammogram was measured. A reduction process was observed at -1.12 V (-0.92 V vs. NHE, Figure 5.5), and an oxidation process was observed at 0.02 V (0.22 V vs. NHE, Figure 5.5). The cyclic voltammogram, while irreversible, was found to be reproducible over multiple cycles without decrease of intensities of either peak. In order to show that the two distinct, dissimilar peaks observed are related, a smaller electrochemical potential window was chosen, which excludes the reduction process found at -1.12 V. Subsequently, the oxidation peak at 0.02 was not observed (Figure 5.6); hence, the two peaks are assumed to be related. Due to dissimilar peak shape, it is likely that the coordination mode of dedpa2- changes upon reduction.  110  0.00005 0.00005 0.00004 0.00004  0.02 V Cu(I)/ Cu(II)  A current A (A)  0.00003 0.00003 0.00002 0.00002 0.00001 0.00001 0.00000 0.00000 -0.00001 -0.00001 -0.00002 -0.00002 -0.00003 -0.00003 -1.5  -0.00004 3 -1.5  - 1.12 -1.0  V -0.5 Cu(II)/ Cu(I) -1.0  -0.5  0.0  0.5  1.0  1.5  V 0.0  0.5  1.0  1.5  V (V) potential  Figure 5.5 Cyclic voltammogram of [Cu(dedpa)], -1.25 and 0.5 V (solid line). Also shown is the blank voltammogram with solvent only (dotted, -1.25 – 1.5 V). Potential values are given with reference electrode Ag/AgCl (sat.).  current (A)  0.00001  0.00000  -0.00001 -0.5  -0.0  0.5  potential (V)  Figure 5.6 [Cu(dedpa)], -0.5 and 0.5 V(solid, black line), overlayed with full range voltammogram. Note, how the oxidation peak at 0.02 V is not observed, therefore it could be related to observed reduction peak at -1.12 V. Potential values are given with reference electrode Ag/AgCl (sat.).  111  Previously reported CV data on Cu(II) complexes with ligands DOTA, TETA, CB-DO2A and CB-TE2A indicate that complexes, which are found to dissociate in vivo are also found to produce irreversible voltammograms indicating loss of the metal upon reduction (TETA, CB-DO2A).149 This was not observed for dedpa2-, hence no copper is lost in the course of multiple reduction and re-oxidation cycles. The reduction potential well below the -0.4 V (NHE) threshold for bioreductants, suggests that [Cu(dedpa)] could have high complex stability in vivo.118  5.3.3  Labelling with 64Cu and In Vitro Stability  Labelling of 64Cu with H2dedpa proceeds quantitatively with ligand concentrations of 10-5 M in ten minutes at room temperature. The stability of the radiochemical complex was measured by a competition experiment in mouse serum. While the complex remains relatively stable over the first two hours (only 3 % transchelation), over the course of 24 h, 23 % of the  64  Cu previously coordinated to dedpa2- is associated with serum. It cannot  be determined if actual transchelation has happened, or if the complex is still intact but associated with serum proteins as a whole entity. In a related study, the thermodynamic stability constant was found to be log KML = 20.43.151 This value is found to be much lower than values determined for stronger chelates such as cyclam, indicating that a transchelation event after 24 h is plausible.  5.4  Conclusions Evidently H2dedpa has many promising properties for the coordination of Cu(II)  and its radionuclide of interest,  64  Cu. The solid state structure shows similarities to the  CB-TE2A coordination sphere. Investigations on redox properties of [Cu(dedpa)] yielded a reproducible, irreversible cyclic voltammogram with a reduction event at -0.92 V and oxidation at 0.22 V (vs. NHE). While the two events are related but show dissimilar peak shape, it is likely that the ligand system changes its coordination sphere according to the predominant redox species. Labelling of  64  Cu proceeds within 10 minutes at room  temperature, unlike some of the established ligand systems that require heat and  112  prolonged reaction times. In a related study, the thermodynamic stability constant was found to be log KML = 20.76.151 Together with the reasonable kinetic stability over 2 h in the presence of serum (less than 3 % transchelated), this chelate is likely to have favourable properties for coordination and target specific delivery of  64  Cu when  conjugated to a targeting molecule. Chapter 6 investigates bifunctional versions of H2dedpa and its coordination to  64  Cu. Once target specific chelates are evaluated in vivo,  the liver uptake (indicative of transchelated  64  Cu) will provide insight into the in vivo  stability of this scaffold.  113  Chapter 6: Functionalization of the H2dedpa Scaffold (1): Bioconjugation and Multifunctionality  6.1  Introduction  6.1.1  Modes of Bioconjugation  Chapters 4 and 5 have given a detailed description of the required criteria for an optimal metal chelate with radiometals. In order to achieve target specific delivery of a radionuclide, an actual targeting moiety must be attached to the radiochemical complex in a covalent manner (for receptor mediated delivery) or the chemical and physical properties must be modified to direct the biodistribution of the radiochemical complex. This chapter focuses on conjugation of the H2dedpa scaffold to targeting biomolecules. Many biomolecules have been successfully conjugated to chelate systems and have been used to deliver a radioactive payload; examples are  111  In-DTPA-Octreotide and  Ibitumomab Tiuxetan (Zevalin®), both FDA approved agents.  62 68  90  Y-  Ga-DOTANOC is  currently in clinical trials.152 The main difficulty in conjugating a biomolecule to a chelate system lies in the selective coupling of functional groups, which are non-essential to the coordination chemistry, without interfering with functional groups necessary for coordination. One solution is to coordinate the metal first and conjugate the targeting moiety second. This pre-labelling approach has been explored for isotopes that require reduction using strong agents prior to conjugation, such as not an option for short-lived radionuclides such as  68  99m  Tc and  188  Re.153,154 This is  Ga. In order to design kit-  formulations suitable for clinical applications, which can be used with a variety of radioisotopes, the post-labelling approach is more desirable. The two approaches are summarized in Scheme 6.1 with a hypothetical H2dedpa derivative as the chelate example.  114  Since post-labelling was selected as the more viable option, orthogonal protection of functional groups must be carried out to promote controlled reaction of specific functional groups. Selective amide coupling (Scheme 6.1) is difficult to control when multiple free carboxylate functionalities are present on the same molecule.  Scheme 6.1 Scheme illustrating pre- and post-labelling approach based on H2dedpa.  As an added difficulty, the final deprotection step cannot interfere with potentially sensitive functional groups on the targeting biomolecule, and must be done either before coupling of the latter or under conditions mild enough to preserve the native form of the biomolecule. Scheme 6.2 elaborates on possible combinations of functional groups and their corresponding protecting group chemistry combined with different coupling methodologies relevant for H2dedpa-type chelate systems. Approach a is a frequently employed coupling method,155 which requires a tert-butyl protection group on the pyridine carboxylate subunit. The synthesis of this fragment failed since this protection group was found to be too labile. Method b was used with slight modifications in Chapter 3 and is further elaborated in this chapter.42  115  Scheme 6.2 Possible conjugation strategies using H2dedpa to couple a bifunctional chelate with a targeting molecule.  116  Method c has been used previously for conjugation of antibodies to macrocyclic chelates.156 Because the backbone derivatized H2dedpa derivative contains secondary amines, which could be easily alkylated, this method appears to be a less viable possibility. Method d entails the currently very popular, Cu(I) catalyzed, Huisgen 1,3dipolar cycloaddition forming 1,2,3-triazoles. This reaction has been used successfully to furnish a wide variety of chelates, especially for the coordination of the [99mTc(CO)3]+ core.157 The synthesis of a corresponding derivatized alkyne was successfully realized; however, the affinity of the H2dedpa/ Me2dedpa scaffold to both Cu(I) and Cu(II) species impedes a clean coupling reaction. A subsequent treatment with highly acidic conditions, in order to remove Cu(II) is not in accordance with the presence of a sensitive biomolecule, hence this route was also abandoned. Method e has been widely explored and is employed among others, for the formation of Zevalin®.158 Isothiocyanates are somewhat more shelf stable then NHS-esters, while they display a strong preference for reaction with primary amines to form the thiourea functional group in aqueous media with pH 9-9.5.159 The successful synthesis and coupling of H2dedpa-isothiocyanato derivatives  is described in this chapter. Method f is limited to targeting molecules  containing thiols; however, since optimal reaction conditions are found at pH 7 this is the coupling condition of choice for molecules sensitive to pH > 8.160  6.1.2  Convenient Biomolecules for Proof of Principle  In order to prove that a bifunctional chelate is able to deliver its radioactive payload successfully, the chelate must be attached to a targeting molecule with a known target and evaluated for maintained binding efficiency. A variety of benchmark targeting molecules are used for this purpose (Table 6.1), with corresponding assays and animal models readily available. Biotin was also used as bioconjugation proof of principle in Chapter 3. Due to its small size and chemical robustness, it is a convenient moiety for preliminary, simple proof of principle. The corresponding target (avidin, Kd ~ 10-15) is readily available and inhibitory assays are, especially in the presence of a radioactively labelled species, straightforward.42  117  For evaluation of targeting molecules with potential applications for tumor imaging, small polypeptides such as c(RGDyK) provide a convenient tool. c(RGDyK) (further referred to as RGD) is a cyclic pentapeptide targeting the vitronectin receptor αvβ3, a membrane protein which has been linked to tumor growth and metastatic potential, since it is highly expressed in tissues undergoing angiogenesis.161 A variety of in vitro assays are available, as are animal models where angiogenesis can be easily induced by artery ablation.162 Since RGD exhibits fast localization and excellent organ clearance, it is well suited for shorter lived radioisotopes such as  68  Ga and serves as a  convenient tool to investigate chelate induced effects such as poor clearance or organspecific uptake. Conjugation of a chelate molecule to RGD can be done conveniently using either of methods a or e via the primary amine of the Lys residue, because it is not essential for binding to the integrin membrane protein. Other RGD derivatives such as cRGDfD (Asp instead of Lys in the non-binding position) are available, but require additional synthetic steps for conjugation due to necessary orthogonal protection of two Asp residues.142 Other frequently used target molecules, together with target, structural features and preferred conjugation method are summarized in Table 6.1. While not investigated in this thesis, they might become relevant for future work with the H2dedpa chelate system. Table 6.1 Summary of frequently used target molecules for imaging and therapy with radiometals.  Target molecule  In vivo target  Features  Conjugation method  Biotin  Avidin  Small molecule  a,104 b,42 d, e163  RGD  αvβ3 integrin  Peptide (5 mer)  a, 142 b,142 e162  Bombesin  GRP-receptor  Peptide (8-14 mer)  a,164 d165  Somatostatin receptor  Peptide (14 mer)  a166, e167  Somatostatin (OC, TATE, Octreotide)  118  Target molecule EGF, hEGF Cetuximab HER-2/neu Fab4D5  In vivo target  EGF-receptor  HER-2/neu receptor  Features Peptide (53 mer) Antibody Antibody Antibody-fragment  Conjugation method  a168, b, e, f  a169, e, f  Figure 6.1 Structures of frequently used target molecules for imaging and therapy with radiometals.  119  6.2  Experimental  6.2.1  Synthesis of Fragments for Coupling  Materials and Methods: Refer to Chapter 2.2 for general information. HPLC analysis or purification of non-radioactive compounds was done on a Phenomenex Jupiter 5μ C18 300 Å (100 x 4.6 mm) in a Waters WE 600 HPLC system equipped with a 2478 dual wavelength absorbance UV detector controlled by the Empower software package. The HPLC system used for analysis of the radiochemical complexes consists of a Waters Alliance HT 2795 separation module equipped with a Raytest Gabbistar NaI detector and a Waters 996 photodiode array (PDA) detector. Analysis of radiolabeled complexes was done on a Waters XBridge BEH130 4.6 × 150 mm (retention time of  67  Ga-Tf: 2.5 min). If not  mentioned otherwise, the Rf values are measured on standard TLC plates with 10% CH3OH in CH2Cl2 as the mobile phase. Common  starting  materials  such  as  the  protected  precursor  1,2-[{6-  (methoxycarbonyl)pyridin-2-yl}methylamino]ethane108 were synthesized according to the literature. 6-Bromomethylpyridin-2-carboxylic acid methyl ester was synthesized according to the literature.170 The synthesis of biotin-TFP has been reported,104 as has the synthesis of the precursor 3-(4-nitro-phenyl)-propane-1,2-diamine.133 General procedure for synthesis of non-radioactive Ga complexes: the ligand was dissolved in a CH3OH-water mixture (1:2) and acidified to pH 2 by addition of 0.1 M HCl. Ga(NO3)3·6H2O (1 eq) was added and the pH was adjusted by addition of 0.1 M NaOH to 4.5. The reaction mixture was stirred at 60 °C for 2 h. The solvent was removed in vacuo to afford the complex as a white solid in quantitative yield.  120  (1,2-[N, N’-{p-Benzylamino-tert-butyl carbamato} methyl]-N, N’-[6-{methoxycarbonyl} -pyridin-2-yl] methylamino)ethane (1). 1,2-[{6-(Methoxycarbonyl)pyridin-2yl}methylamino]ethane mmol)  and  (23  mg,  0.064  (4-bromomethyl-phenyl)-  carbamic acid tert-butyl ester (38 mg, 0.135 mmol, 2.1 eq) were dissolved in acetonitrile (80 mL). Na2CO3 (400 mg) was added into the reaction mixture and it was stirred at 60°C over night (Scheme 6.3). The resulting milky solution was filtered and the solvent was removed in vacuo to afford the crude product as an oil, which was subsequently purified by column chromatography (10% CH3OH in CH2Cl2) to afford 1 as a very viscous, colourless oil (19 mg, 0.024 mmol, 38 % Rf: 0.5). 1H NMR (CDCl3, 400 MHz, δ): 7.97 (d, 3J4, 5 = 7.1 Hz, H5, 2H), 7.70 (d, 3J4, 5 = 7.1 Hz, H4, 2H), 7.24 – 7.14 (m, 3J11, 12  = 7.7 Hz, H3, H11, H12, 10H), 6.58 (s, CO-NH, 2H), 3.98 (s, H14, 6H), 3.81 (s, H7,  4H), 3.49 (s , H9, 4H), 2.62 (s, H8, 4H), 1.52 (s, H16, 18H).13C NMR (CDCl3, 100 MHz, δ): 163.1 (CO-NH), 161.5 (C1), 158.3 (C6), 153.0 (C2), 147.3 (C13), 137.5 (C10), 129.5 (C4), 118.8 (C11), 58.7 (C7), 58.6 (C9), 53.6 (C14), 53.1 (C15), 28.6 (C16). IR (neat, cm-1): 1716 (s), 1525 (m), 1313 (s), 1232 (s), 1157 (s). HR-ESI-MS: m/z calcd. for C42H52N6NaO8: 791.3744, found: 791.3730 [M + Na]+. Elemental Analysis: calcd. (found) for for 1·CH3OH: C: 64.74 (64.49), H: 6.65 (6.73), N: 10.53 (10.31).  (1,2-[N,N’-{p-Benzylamino}methyl]-N,N’-[6-{methoxycarbonyl}-pyridin-2-yl] methylamino)ethane (2). 1 (20 mg, 0.026 mmol) was dissolved in 2 mL CH2Cl2. TFA (0.5 mL) was added and the reaction mixture was stirred for 1 h. The acid was then quenched with saturated NaHCO3 (20 mL) and the aqueous solution was then extracted twice with EtOAc (20 mL) and twice with CH2Cl2 (20 mL). All organic fractions were collected and dried over MgSO4. The solvent was removed in vacuo to subsequently afford the free  121  amine 2 as a yellow oil which was immediately used in the follow-up reaction (3 mg, 0.005 mmol, 22 % Rf: 0.1). 1H NMR (CDCl3, 400 MHz, δ): 7.98 (d, 3J4, 5 = 6 Hz, H5, 2H), 7.71 (m, 3J4, 5 = 6 Hz, H3, H4, 4H), 7.06 (d, 3J11, 12 = 8 Hz, H11, 4H), 6.59 (d, 3J11, 12 = 8 Hz, H12, 4H), 4.02 (s, H14, 6H), 3.81 (s, H7, 4H), 3.47 (s, H9, 4H), 2.65 (s, H8, 4H). HR-ESI-MS: m/z calcd. for C32H37N6O4: 569.2869, found: 569.2869 [M + H]+.  (1,2-[N,N’-{p-Benzyl-isothiocyanato}-methyl]-N,N’-[6-{carboxylato}-pyridin-2-yl] methylamino)ethane (3, H2dp-N-NCS). Compound 1 (13 mg, 0.069 mmol) was dissolved in a 3:1 mixture of THF and H2O. LiOH (8 mg, 0.3 mmol) was added to the reaction mixture and the reaction was stirred for 1 h at room temperature to afford the free carboxylate intermediate. Reaction monitoring was performed via TLC and analytical HPLC (Rt(1) = 18.3 minutes, Rt(intermediate) = 15.8 minutes). The solvent was removed in vacuo and the intermediate was redissolved in 3 M HCl solution (2 mL) and stirred for 2 h to remove the t-Boc protecting groups. Again, HPLC was used to confirm full conversion of the intermediate into the fully deprotected second intermediate (Rt(sec. intermediate) = 9.4 minutes). CHCl3 (1 mL) and SCCl2 (106 μL, 20 eq) were added and the biphasic reaction was stirred vigorously for 18 h at room temperature to afford the final product 3 as a white precipitate; it was collected by vacuum filtration to yield 11 mg (0.017 mmol, 24 %, Rt(3) = 18.0 minutes). 1H NMR (400 MHz, CD3OD, δ): 8.11 (d, 3J3, 4= 7.5 Hz, H3, 2H), 8.02 (m, 3J3, 4= 7.5 Hz, 3J4, 5= 7.9 Hz, H4, 2H) 7.59 (d, 3J4, 5= 7.9 Hz, H5, 2H), 7.50 (d, 3J11, 12= 8.5 Hz, H12, 2H), 7.21 (d, 3J11, 12=  8.5 Hz, H11, 2H), 4.52 (s, H7, 4H), 4.32 (s, H9, 4H), 3.65 (s, H8, 4H). 13C NMR (100  MHz, CD3OD, δ): 165.6 (C1), 154.2 (C6), 147.5 (C2), 138.5 (C10), 136.2 (NCS), 133.9 (C4), 131.8 (C5), 130.1 (C13), 126.9 (C3), 125.9 (C12), 123.9 (C11), 57.0 (C7), 56.3 (C9), 49.0 (C8). IR (neat, cm-1): 2023 (s, br), 1731 (m), 1595 (w), 1505 (m), 1344 (m). HR-ESI-MS: m/z calcd. for C32H27N6O432S2: 623.1535, found: 623.1542 [M - H]-.  122  [N, N’-Bis{benzylamino}] 3-(4-Nitrophenyl)-propane-1,2-diamine (4). 3-(4-Nitrophenyl)-propane-1,2-diamine (204 mg, 1.046 mmol) was dissolved in 40 mL EtOH together with benzaldehyde (221 mg, 0.213 mL, 2.084 mmol) and the flask was cooled in an ice bath (Scheme 6.4). After two hours of reaction, the imine intermediate was confirmed through mass spectrometry. NaBH4 (80 mg, 2.5 mmol) was added to the reaction mixture at 0 °C and the reaction was stirred for another two hours. Subsequently the reaction was quenched with a saturated aqueous solution of NH4Cl (20 mL) and the product was extracted with 200 mL CH2Cl2. After removal of the solvent, 4 was afforded as an orange oil (378 mg, 1.01 mmol, 97% Rf: 0.1). 1H NMR (CDCl3, 400 MHz, δ): 8.13 (d, 3J12,  13  = 8.2 Hz, H13, 2H), 7.38 – 7.27 (m, 3J12,  13  = 8.2 Hz, H1-H3, H1’-H3’,H12,  2  12H), 3.79 - 3.70 (d, J7, 7 = 13.2 Hz, H7, H7’, 4H), 2.96 (m, 3J8, 9 = 8.8 Hz, 2J9, 9’ = 15.2 Hz, H8’, H9, 2H), 2.81 (dd, 3J8, 9’ = 8.8 Hz, 2J9, 9’ = 15.2 Hz, H9’, 1H), 2.63 (dd, 3J8, 10 = 3.6 Hz, 2J10, 10’ = 12.0 Hz, H10, 1H), 2.52 (dd, 3J8, 10’ = 6.9 Hz, 2J10, 10’ = 12.0 Hz, H10’, 1H). 13C NMR (CDCl3, 100 MHz, δ): 147.2 (C14), 146.4 (C11), 141.0, (C4) 139.9 (C12), 129.9 (C3), 128.4 (C3’), 128.3 (C2), 128.0 (C2’), 127.4 (C1), 127.0 (C1’), 123.5 (C13), 64.9 (C7), 57.5 (C7’), 53.6 (C8), 51.3 (C9), 38.9 (C10). HR-ESI-MS: m/z calcd. for C32H26N3O2: 376.2025, found: 376.2019 [M + H]+ .  [N,N’-Bis{benzylamino}]3-(4-nitrophenyl)-propane-1,2-diamino-N,N’-[6methoxycarbonyl}-pyridin-2-yl]methylamine (5). Compound 4 (181 mg, 0.48 mmol) and 6-bromomethylpyridine-2-carboxylic acid methyl ester (225 mg, 0.97 mmol, 2 eq) were dissolved in acetonitrile (35 mL). Na2CO3 (0.5 g) was added and stirred overnight at 70 °C. The milky yellow solution was filtered and the solvent was removed from the filtrate. The crude product was purified with column chromatography (5 % CH3OH in CH2Cl2) to afford 5 as a yellow solid (78 mg, 0.12 mmol, 25 % Rf: 0.6). 1H NMR (CDCl3, 400 MHz, δ): 8.02 (d, 3  J12, 13 = 8.5 Hz, H13, 2H), 8.00 – 7.08 (m, H18-20, H3/3’-H5/5’,16H), 7.05 (d, 3J12, 13 =  123  8.5 Hz, H12, 2H), 4.01 (s, H15/H15’, 6H), 3.94 (s, H16/H16’, 6H), 3.90 – 3.48 (d, 2J7, 7 = 10.8 Hz, 2J7’, 7’ = 13.7 Hz, H7/H7’, 4H), 3.18 (m, 3J8, 9 = 8.7 Hz, 3J8, 9’ = 8.2 Hz, 3J8, 10’ = 5.3 Hz , H8’, 1H), 2.97 (dd, 2J10, 10’ = 13.2 Hz, 3J8, 10’ = 5.3 Hz, H10/10’, 2H), 2.58 (dd, 2  J9, 9’ = 13.4 Hz, 3J8, 9 = 8.7 Hz, H9, 1H), 2.53 (dd, 2J9, 9’ = 13.4 Hz, 3J8, 9’ = 8.2 Hz, H9’,  1H).  13  C NMR (CDCl3, 100 MHz, δ): 165.6 (C1/1’), 160.5 (C6), 160.4 (C6’), 148.9  (C2/2’), 146.9 (C14), 146.1 (C11), 138.8 (C4/4’), 138.6 (C17), 130.0 (C12), 128.8 (C18), 128.4 (C19), 128.2 (C20), 127.4 (C5), 126.8 (C5’), 125.6 (C13), 123.5 (C3), 123.1 (C3’), 65.2 (C7’), 61.0 (C16), 59.6 (C8), 58.6 (C9), 53.9 (C7), 53.7 (C16), 53.4 (C15), 52.8 (C15’), 35.8 (C10). IR (neat, cm-1): 1719 (s), 1514 (m), 1342 (s), 1315 (m). HR-ESIMS: m/z calcd. for C39H40N5O6: 674.2979, found: 674.2960 [M + H]+. Elemental Analysis calcd. (found) for 6·2CH2Cl2: C: 58.37 (58.36), H: 5.10 (5.05), N: (8.30)8.72.  3-(4-Aminophenyl)-1,2-diamino-N,N’-bis[6-{methoxycarbonyl}-pyridin-2-yl] methylamino-propane (6, Me2dp-bb-NH2). Compound 5 (302 mg, 0.44 mmol) was dissolved in glacial acetic acid (20 mL). Pd/C (10% w/w, 80 mg) was added and the mixture was charged with H2 in a balloon and stirred overnight at room temperature. The reaction was found to be complete according to TLC (product stains bright purple with ninhydrin). Pd(OH)2 was filtered off and the solvent was removed in vacuo to afford the pure product 6 as the triacetate salt (149 mg, 0.32 mmol, 72 %, Rf: 0.05). 1H NMR (CDCl3, 400 MHz, δ): 8.00 – 7.62 (m, H5/5’, H4, 3H), 7.56 (m, 3J3, 4 = 7.6 Hz, H3, 1H), 7.46 (m, 3J3, 4 = 7.6 Hz, H3’, 1H), 7.32 (m, H4’, 1H), 6.91 (d, 3J12, 13= 7.9 Hz, H12, 2H), 6.56 (d, 3J12,  13=  7.9 Hz, H13, 2H), 4.76 – 4.09 (dd, 2J7, 7 = 16.5 Hz, 2J7’,  7’  = 17.1 Hz,  H7/H7’, 4H), 3.95 (s, NH2, 2H), 3.81 (s, H15, 3H), 3.79 (s, H15’, 3H), 3.63 (m, H8’, 1H), 3.02 (dd, 3J8, 10’ = 6.9 Hz, H10/10’, 2H), 3.04 (dd, 2J9, 9’ = 14.0 Hz, 3J8, 9’ = 6.4 Hz, H9’, 1H), 2.72 (dd, 2J9,  9’  = 14.0 Hz, 3J8,  9  = 8.2 Hz, H9, 1H), 2.61 (s, NH, 2H).  13  C NMR  (CDCl3, 100 MHz, δ): 164.9 (C1’), 164.7 (C1), 146.9 (C2’), 146.6 (C2), 144.7 (C14), 138.3 (C4), 137.2 (C4’), 129.9 (C11), 126.9 (C6), 126.2 (C6’), 126.0 (C12), 125.8 (C5), 124.3 (C5’), 124.0 (C3), 122.3 (C3’), 115.7 (C13), 57.1 (C7’), 53.5 (C9), 52.7 (C8), 52.6  124  (C7), 49.7 (C15), 48.7 (C15’), 35.9 (C10). HR-ESI-MS: m/z calcd. for C25H29N5O4Na: 486.2117, found: 486.2111 [M + Na]+.  3-(4-Isothiocyanatophenyl)-propane-1,2-diamino-N,N’-bis[6-{carboxylato}-pyridin2-yl] methylamine (7, H2dp-bb-NCS). Compound 6 (10 mg, 0.023 mmol) was dissolved in 3 M HCl (1 mL) to which CHCl3 (1 mL) and SCCl2 (19 μL, 10 eq) were added. The biphasic reaction was stirred vigorously for 18 h at room temperature. Subsequently, the solvent was removed and the crude was purified by preparative HPLC. The clean fractions were pooled and lyophilized to afford the final product as a yellow solid (3 mg, 0.006 mmol, 26 % Rt(7)= 13 minutes. 1H NMR (400 MHz, CD3OD, δ): 8.17 – 8.06 (m, H3/3’, H4/4’, 4H), 7.70 (m, H5/5’, 2H), 7.08 (d, 3J12, 13= 8.4 Hz, H13, 2H), 7.26 (d, 3J12, 13=  8.4 Hz, H12, 2H), 4.78 – 4.59 (dd, 2J7, 7 = 16 Hz, H7/H7’, 4H), 3.69 (m, H8’, 1H),  3.37 (dd, 3J8, 10’ = 3.2 Hz, H10/10’, 2H), 3.32 – 2.85 (dd, 2J9, 9’ = 13.7 Hz, 3J8, 9 = 9.7 Hz, H9, 1H).  13  C NMR (125 MHz, CD3OD, δ): 168.8 (C1’), 168.2 (C1), 162.7 (C6), 162.4  (C6’), 150.0 (C2), 149.6 (C2’), 139.6 (C4), 139.1 (C4’), 134.2 (NCS) , 133.1 (C11), 129.9 (C12), 129.8 (C5), 129.6 (C14), 125.9 (C5’), 125.8 (C13), 125.4 (C3), 124.3 (C3’), 56.7 (C8), 49.6 (C7’), 47.0 (C9), 46.3 (C7), 34.0 (C10). IR (neat, cm-1): 2076 (s, br), 1634 (m), 1505 (w), 1394 (m), 1346 (w). HR-ESI-MS: m/z calcd. for C24H22N5O432S: 476.1393, found: 476.1405 [M - H]-.  125  Me2dp-N-biotin (8). 2 (48 mg, 0.084 mmol ) was dissolved in DMF (5 mL) together with biotin-TFP (76 mg, 0.19 mmol, 2 equ.) and NEt3 (73 μL) and stirred at 55 °C for 20 h. Subsequently, the solvent was removed and the resulting colourless solid was purified by column chromatography (aluminium oxide, 10% CH3OH in CH2Cl2) to afford the dialkylated product 8 as a white solid (9 mg, 0.009 mmol, 10 % Rf: 0.5 on aluminium oxide, 10% CH3OH in CH2Cl2). 1H NMR (d6-DMSO, 400 MHz, δ): 9.80 (s, CO-NH, 2H), 7.87 (m, 3J3, 4 = 5.7 Hz, 3J3, 5 = 3.2 Hz, H5, H4, 2H), 7.62 (d, 3J3, 4 = 5.7 Hz, 4J3, 5 = 3.2 Hz, H3, 2H), 7.47 (d, 3  J11, 12 = 8.5 Hz, H12, 4H), 7.16 (d, 3J11, 12 = 8.5 Hz, H11, 4H), 6.42 – 6.35 (s, CO-NH,  4H), 4.29 (m, 3Jδ,β = 5.0 Hz, Hβ, 2H), 4.06 (m, Hχ, 2H), 3.85 (s, H15, 6H), 3.65 (s, H7, 4H), 3.45 (s, H9, 4H), 3.16 (m, Hε, 2H), 2.83 – 2.79 (dd, 2Jδ,δ = 12.6 Hz, 3Jδ,β = 5.0 Hz, Hδ, 4H), 2.56 (m, H9, 4H), 2.28 (m, CH2, 4H), 1.63 – 1.23 (m, CH2, 10H). 13C NMR (d6DMSO, 100 MHz, δ): 171.0 (CO-NH), 162.7 (Cα), 160.3 (C1), 138.1 (C6), 137.7 (C13, C4), 133.3 (C10), 129.0 (C11), 126.0 (C5), 123.2 (C12), 118.8 (C3), 61.0 (C7), 59.2 (Cχ), 57.7 (Cβ), 55.4 (Cε), 54.9 (Cβ), 52.4 (C8), 50.8 (C15), 40.0 (Cδ), 36.2, 28.3, 28.1, 25.2. HR-ESI-MS: m/z calcd. for C52H65N10O832S: 1021.4428, found: 1021.4451 [M + H]+.  H2dp-N-biotin (9). 8 (7 mg, 0.007 mmol) was dissolved in a 1:3 mixture of CH3OH and H2O (2 mL total volume). LiOH (1 mg, 0.041 mmol, 6 eq) was added to the solution which was then stirred at room temperature for 2 h. The reaction was found to be complete after 2 h according to TLC. Subsequently, the solvent was removed to afford 9 as a white solid (yield: 6 mg, 0.006 mmol, 85 %, Rf: 0.15). 1  H NMR (d6-DMSO, 400 MHz, δ): 9.83 (s, 2H, CO-NH), 7.87 (m, 3J3, 4 = 7 Hz, 4J3, 5 = 1.2 126  Hz, H5, H4, 4H), 7.47 (d, 3J11, 12 = 8.2 Hz, H12, 4H), 7.33 (d, 3J3, 4 = 7 Hz, 4J3, 5 = 1.2 Hz, H3, 2H), 7.06 (d, 3J11, 12 = 8.2 Hz, H11, 4H), 6.43 – 6.34 (d, 4H, CO-NH), 4.30 (m, 3Jδ, β = 4.8 Hz, Hβ, 2H), 4.19 (m, Hχ, 2H), 3.59 (s, H7, 4H), 3.38 (s, H9, 4H), 3.24 (m, CH2, 4H), 2.83 – 2.81 (dd, 3Jδ, β = 4.8 Hz, 2Jδ, δ = 12.3 Hz, Hδ, 2H), 2.57 (d, 2Jδ, δ = 12.3 Hz, Hδ, 2H), 2.30 (d, 3JCH2= 7.7 Hz, CH2, 2H), 2.23 (s, H8, 4H), 1.62 – 1.29 (m, CH2, 12H).13C NMR (d6-DMSO, 100 MHz, δ): 171.0 (CO-NH), 167.6 (Cα), 162.8 (C1), 157.5 (C12), 155.8 (C13), 138.2 (C6), 137.9 (C4), 131.8 (C10), 129.5 (C11), 124.0 (C12), 121.6 (C5), 118.8 (C3), 61.0 (C7), 59.2 (Cχ), 58.7 (Cβ), 56.9 (Cε), 55.4 (C8), 40.0 (Cβ), 36.2 (Cδ), 29.5, 28.3, 28.1, 25.2. IR (neat, cm-1): 1667 (s), 1573 (m), 1512 (s), 1428 (s), 1382 (s). HR-ESI-MS: m/z calcd. for C50H587LiN10O832S: 997.4041 found: 997.4065 [M -2H + Li]+.  6.2.2  Thiourea Coupling with [c(RGDyK)]  General procedure for RGD coupling: the cyclic RGD peptide (3 mg, 0.0048 mmol) was dissolved in aqueous buffered solution (0.1 M NaHCO3, pH 9 - 9.5, 1.5 mL). The isothiocyanate (H2dp-bb-NCS: 1.9 mg, 0.004 mmol or H2dp-N-NCS: 1.3 mg, 0.0022 mmol) was dissolved in CH3OH (0.5 mL). The two solutions were mixed and stirred at room temperature for 18 h in the dark. The solvent was subsequently removed and the crude product was redissolved in a minimal amount of a MeCN/H2O mixture (1:2) and then purified via semi-preparative HPLC. A Phenomenex Jupiter 5u C18 300 A 4.6 × 100 mm HPLC column was used. The gradient consisted of A(100%) – B(100%) in 25 minutes at 1mL/ min flow rate (A: H2O, 0.1 % TFA; B: MeCN).The fractions containing the desired product were pooled and the solvent was removed to afford the RGD conjugates as white solids in approximately 45 % yield. The product was analyzed with ESI-MS. H2dp-bb-NCS-RGD: Rt: 10.5 min; [M - H]- = 1862.0 (calculated for C87H110N23O20S2: 1861.8). H2dp-N-NCS-RGD: Rt: 11.5 min; [M + H]+ = 1097.6 (calculated for C51H65N14O12S: 1097.5).  127  6.2.3  Coordination Chemistry with Ga  [Ga(dp-N-biotin)](NO3). The Ga complex of 4 was synthesized according to general procedure (see 6.2.1). 1H NMR (d6-DMSO, 400 MHz, δ): 10.13 (s, 2H, CO-NH), 8.67 (m, 3J3, 4 = 8.2 Hz, 3J4, 5 = 7.5 Hz, H4, 2H), 8.36 (m, 3J4, 5 = 7.5 Hz, H5, 2H), 8.23 (m, 3J3, 4 = 8.2 Hz, H3, 2H), 7.64 (d, 3J11, 12 = 8.6 Hz, H12, 4H), 7.36 (d, 3J11, 12 = 8.6 Hz, H11, 4H), 6.41 – 6.36 (d, 4H, CO-NH), 4.98 – 4.06 (dd, 2J7, 7 = 16.3 Hz, H7, 4H), 4.31 (m, 3Jχ, β = 7.8 Hz, 3Jδ, β = 4.4 Hz, Hβ, 2H), 4.21 (m, 3Jχ, β = 7.8 Hz, 3Jχ, ε = 4.1 Hz, Hχ, 2H), 3.78 – 3.75 (dd, 3J9. 9 = 12.9 Hz, H9, 4H), 3.16 (m, 3Jχ, ε = 4.1 Hz, Hδ, 2H) 3.04 – 2.85 (dd, 3J8. 8 = 11.2 Hz, H8, 4H), 2.83 – 2.81 (m, 6H), 2.81 – 2.55 (dd, 3Jδ, β = 4.4 Hz, 2Jδ, δ = 12.3 Hz, Hδ, 2H), 2.31 (d, 3JCH2= 7.5 Hz, CH2, 2H), 1.62 – 1.32 (m, CH2, 12H).  13  C NMR (d9-DMSO, 75 MHz, δ): 171.5 (CO-NH), 162.7 (Cα), 162.4  (C1), 151.0 (C12), 146.5 (C13), 143.6 (C6), 140.1 (C4), 132.7 (C10), 128.4 (C11), 124.4 (C12), 123.3 (C5), 118.7 (C3), 61.1 (C7), 59.2 (Cχ), 55.9 (Cβ), 55.4 (Cε), 53.7 (C8), 46.1 (Cβ), 40.0 (Cδ), 36.3, 29.8, 28.2, 28.1, 25.1. HR-ESI-MS: m/z calcd. for C50H5869GaN10O832S: 1059.3136, found: 1059.3145 [M]+.  6.2.4  Radiolabelling with 67/68Ga, and Transferrin Stability  General procedure for labelling with  67/68  Ga: 100 μL of  67  Ga3+ (1 mCi, in 0.01 N  HCl) or 68Ga3+ (0.1 M HCl) was added into 10-4 M or 10-6 M solution of ligand in 10 mM NaOAc solution (pH 4.5, 950 μL) and left to react for 10 minutes at room temperature. The reaction was monitored by analytical HPLC (gradient: A: NaOAc buffer, pH 4.5, B: CH3CN. 0-100% B linear gradient 20 min). Transferrin competition:  67  GaCl3 was added  into 10-4 M (H2dp-N-biotin) or 10-6 M (H2dp-bb-NCS-RGD, H2dp-N-NCS-RGD) solution of ligand in 10 mM NaOAc solution (pH 4.5, 950 μL). Complex formation was followed by HPLC. A 400 μL aliquot was added to 1 mg/ mL apo-transferrin in a  128  NaHCO3 solution (10 mM, 600 μL) and incubated at 37 °C (water bath). Complex stability was followed at time points 10 minutes, 1 h and 2 h via analytical HPLC (for biotin-conjugate). PD-10 size-exclusion columns were used to determine the stability of the RGD conjugates, since the decomposition product could not be observed with previously used HPLC methods. Reaction mixtures were loaded onto the pre-rinsed PD10 columns (total loading volume: 2.5 mL). Subsequently, the column was rinsed with 3.5 mL PBS. The resulting eluate was collected and measured for radioactivity. The activity eluted corresponds to transferrin-bound 67Ga. [67Ga(dp-N-Biotin)]+: Rt of radiolabelled product on HPLC (gradient: A: NaOAc buffer, pH 4.5, B: CH3CN. 0-100% B linear gradient 20 min): 9.1 minutes. Stability versus transferrin (10 min/1 h/ 2 h; in % complex intact): 76/ 55/ 43. [67/68Ga(dp-bb-NCS-RGD)]+: Rt of  67  Ga radiolabelled product on HPLC (gradient: A:  NaOAc buffer, pH 4.5, B: CH3CN. 0-100% B linear gradient 20 min): 6.4 minutes. Stability versus transferrin (10 min/1 h/ 2 h; in % complex intact): 96/ 93/ 92. [67/68Ga(dp-N-NCS-RGD)]+: Rt of  67  Ga radiolabelled product on HPLC (gradient: A:  NaOAc buffer, pH 4.5, B: CH3CN. 0-100% B linear gradient 20 min): 7.6 minutes. Stability versus transferrin (10 min/1 h/ 2 h; in % complex intact): 92/ 73/ 73.  6.2.5  Radiolabelling with 64Cu, and Serum Stability  100 μL of 64Cu2+ (0.7 mCi, in dilute HCl) was added to 900 μL of a 10-5 or 10-6 M solution of ligand in 10 mM NaOAc solution (pH 5.5, 950 μL) and left to react for 10 minutes at RT. The reaction progress was monitored by analytical HPLC (gradient: A: H2O, 0.1 % TFA, B: CH3CN. 5-100% B linear gradient 30 min). Ligand concentrations of 10-6 M are capable of coordinating the radionuclide with an 82 % (H2dp-bb-NCS–RGD) to 97 % (H2dp-N-NCS–RGD) yield. Serum challenge: to evaluate complex stability, an aliquot (500 μL) of the reaction was added to a solution of mouse serum (500 μL). The mixture was incubated at 37 °C and analyzed by PD-10 column filtration after 15 min, 2 h and 24 h. PD-10 size-exclusion columns were used to determine the stability of the RGD conjugates because the decomposition product could not be observed with  129  previously used HPLC methods. Reaction mixtures were loaded onto the pre-rinsed PD10 columns (total loading volume: 2.5 mL). Subsequently, the column was rinsed with 3.5 mL PBS. The resulting eluate was collected and measured for radioactivity. The activity eluted corresponds to serum-bound 64Cu. [64Cu(dp-bb-NCS-RGD)]: Rt of  64  Cu radiolabelled product on HPLC (gradient: A: H2O,  0.1 % TFA, B: CH3CN. 5-100% B linear gradient 30 min): 8.4 minutes. Stability versus serum (15 min/ 2 h/ 24 h; in % complex intact): 94/ 90/ 72. [64Cu(dp-N-NCS-RGD)]: Rt of  64  Cu radiolabelled product on HPLC (gradient: A: H2O,  0.1 % TFA, B: CH3CN. 5-100% B linear gradient 30 min): 9.2 minutes. Stability versus serum (15 min/ 2 h/ 24 h; in % complex intact): 98/ 94/ 88.  6.2.6  Avidin binding  In order to evaluate target specific binding capabilities of conjugate [67Ga(dp-NBiotin)]+, an avidin blocking experiment was conducted in the following manner. The radiolabelled complex was incubated with a 10 fold excess of avidin for 30 minutes at 37 °C. As the corresponding control experiment, avidin was also pre-incubated with the corresponding 10 fold excess of native biotin for 30 minutes at 37 °C and subsequently incubated with the radiolabeled complex for 30 minutes at 37 °C. Subsequently, the reaction mixture was loaded onto a 30 kD MW cutoff separation filter and separated by centrifugation and washed three times with 1 mL of PBS pH 7.2. Avidin binding was determined as the percent radioactivity retained on the filter as compared to the total radioactivity.  130  6.3  Results and Discussion  6.3.1  Synthesis of H2dp-bb-NH2, H2dp-N-NH2, H2dp-bb-NCS and  H2dp-N-NCS In order to successfully furnish building blocks which connect H2dedpa to a biomolecule, different methodologies were taken into consideration. The synthesis of building blocks consistent with conjugation methods a, b, d and e (Scheme 6.2) were all attempted, but some proved unsuccessful. In the case of method a, orthogonal protection of the two different carboxylates is required. A hypothetical molecule A is shown in Figure 6.2.  Figure 6.2 Hypothetical, postulated building blocks for functionalization of the H2dedpa scaffold.  Synthesis of a tert-butyl protected pyridine carboxylate fragment was attempted (also as a convenient building block for compounds described in Chapter 3) but the corresponding molecule turned out to be extraordinarly acid-labile. Hence, synthesis of molecule B (compatible with method b) was also dismissed. Compound C was synthesized as a building block compatible with method b; however, attempted clickcoupling with both the free carboxylate, as well as the methyl-protected precursor, led to incorporation of the catalytically active Cu(I) with oxidation to Cu(II). This result prompted investigations of the H2dedpa scaffold for the purpose of coordination of both these Cu oxidation states on the macroscopic scale, and with  64  Cu (Chapter 5), but also  led to dismissal of this type of conjugation method. After careful evaluations of possible syntheses and remaining possible coupling  131  methodologies, the benzylamino- as well as the benzylisothiocyanato- derivatives were chosen. While their syntheses differs only by one step, they allow for conjugation to different functional groups.  Scheme 6.3 Synthesis scheme for compounds 1, 2 and 3. (i) Na2CO3, CH3CN, 70 °C, 18 h (ii) CH2Cl2/TFA, 30 min (iii) 1. LiOH (4 eq), THF/H2O (3:1), 2. HCl (3 M), 2 h, 3. SCCl2 , 18 h.  For the N-derivative, which has a somewhat simpler chemical makeup due to a lack of secondary amines and an inherent C2 symmetry, initial reduction of compound Me2dp-N-NO2 was attempted but (similar to reactivity of compounds reported in Chapter 3) led to complete debenzylation. Instead, the amine was introduced via alkylation of Me2dedpa with Boc-protected p-anilino fragment to form 1. 1 can be subsequently deprotected to afford 2, which can be used immediately for conjugation following method b. In order to furnish the corresponding isothiocyanato derivative, however, t-Bocdeprotection and extraction work-up was avoided since a large amount of the diamine 2 would be lost to the aqueous phase. Instead, an alternative sequence of deprotection conditions was sought to yield clean intermediate products. The synthesis was optimized to avoid tedious, low yielding purification steps of these highly polar compounds. Best results were obtained following a three-step one-pot reaction. First, the methyl esters  132  were removed under the usual basic conditions. Then, the intermediate was redissolved in 3 M HCl, leading to cleavage of the t-Boc protection groups. The reaction was monitored by analytical HPLC and was found to be complete after approximately 2 h. Subsequently, without isolation of the diamino-intermediate, SCCl2 was added to the reaction mixture and left to react overnight to form the di-SCN fragment 3 (H2dp-N-NCS) which, when the reaction is carried out on a larger scale, precipitates from the aqueous solution.  Scheme 6.4 Synthesis scheme for compounds 4, 5, 6 and 7. (iv) 1. EtOH, 0 °C, 2 h, 2. NaBH4, 0°C, 2 h (i) Na2CO3, MeCN, 70 °C, 18 h (v) Pd/C, AcOOH, 18 h (vi) 1. LiOH (4 eq), THF/H2O (3:1), 2. HCl (3 M), SCCl2, 18 h.  For the backbone derivatized fragments 6 and 7, the main difficulty was to overcome purification issues and the extremely low yielding reductive amination encoutered with compound Me2dp-bb-NO2 (see Chapter 4). To circumvent this reaction, the reductive amination was carried out with benzaldehyde first in order to generate 4, which can be easily alkylated using 6-bromomethyl-pyridine-2-carboxylic acid methyl ester forming 5, which can be purified in large quantities using column chromatography 133  (Scheme 6.4). One-pot debenzylation and transformation of the nitro functional group with Pd/C in one step yielded 6 (Me2dp-bb-NH2) as the triacetate salt. Subsequent onepot sequential deprotection and functional group transformation was monitored by analytical HPLC. In contrast to 3, 7 (H2dp-bb-NCS) did not precipitate out of the reaction solution and required purification by HPLC, which has been previously reported for this type of chelate building block.158 All products were thoroughly characterized by 1  H and  13  C NMR spectroscopy, as well as mass spectrometry. Notable spectroscopic  features of compounds H2dp-bb-NCS and H2dp-N-NCS include the characteristic  13  C  NMR resonances for the NCS carbon atoms, which are typically found around 135 ppm. 1  H NMR spectra of both isothiocyanato compounds are shown in Figure 6.3.  Figure 6.3 1H NMR spectra (300 MHz, CD3OD, 298 K) of H2dp-bb-NCS and H2dp-N-NCS, * denotes residual solvent peaks.  The NCS functional group can also be identified by its characteristic stretching frequency. For both compounds H2dp-bb-NCS and H2dp-N-NCS, the corresponding broad peaks were found between 2020 – 2100 cm-1, a typical value for the NCS functionality.  134  6.3.2  Amide Coupling with Biotin  In order to show the viability of coupling method b, biotin-TFP was synthesized and  mixed with either Me2dp-bb-NH2 or Me2dp-N-NH2 under standard coupling  conditions. Initial reaction control with mass spectrometry seemed promising. Me2dp-NBiotin was easily purified by column chromatography using aluminium oxide, then characterized and identified as the desired product. Deprotection yielded the desired compound H2dp-N-Biotin, which was also characterized and identified by 1H and  13  C  NMR spectroscopy, IR spectroscopy and mass spectrometry. In contrast, the identification of the product from the conjugation with the backbone derivatized ligand system proved to be rather problematic. After purification via preparative TLC and HPLC, the product was identified and was subsequently deprotected. Structural characterization using the 2D NMR techniques HMBC and HSQC suggested that the amido-carbon of the biotin fragment and the chelate backbone were covalently associated (Figure 6.4). This assumption was further confirmed when the corresponding coordination reactions with both non-radioactive Ga and  67  Ga remained  unsuccessful. In retrospect, the molecular connectivity observed is not surprising, because aliphatic, secondary amines are much stronger nucleophiles than aromatic amines under the chosen, basic reaction conditions. Hence, this coupling methodology, while efficient for the N-derivative, was abandoned for the backbone derivatized fragment.  Figure 6.4 Desired and observed products from coupling of biotin-TFP and Me2dp-bb-NH2.  135  6.3.3  Coordination Chemistry of Ga with H2dp-N-Biotin  The biotin conjugated fragment Me2dp-N-Biotin was successfully synthesized and characterized. Base catalyzed saponification of the methyl esters provides the readyto chelate fragment H2dp-N-Biotin. The coordination reaction proceeds smoothly, as with all H2dedpa derivatives. Since biotin is a reasonably small molecule, NMR spectroscopic methods were employed for complete identification of all hydrogens and carbon atoms of the complex structure. The observed coordination mode mirrors what was predicted by observations made on the model compound H2dp-N-NO2 and its corresponding Ga(III) coordination complex. The ligand adopts a C2 symmetric coordination mode upon chelation of the metal. Characteristic geminal splitting patterns are observed for the previously homotopic methylene hydrogens, that are rendered diastereotopic upon coordination (Figure 6.5).  Figure 6.5 1H NMR spectra (300 MHz, d6-DMSO, 298 K) of H2dp-N-Biotin and [Ga(dp-N-Biotin)]+, * denotes residual solvent peaks.  136  6.3.4  Radiolabelling of H2dp-N-Biotin with 67Ga and Avidin Binding  Radiolabelling of H2dp-N-Biotin with  67  Ga proceeds within 10 minutes at room  temperature in 98 % radiochemical yield. The subsequent apo-transferrin challenge experiment (Figure 6.6) revealed that 43 % of the radiolabelled complex remained intact after 2 h in the presence of excess apo-transferrin, a stability inferior to that of [Ga(dedpa)]+, but similar to the model derivative H2dp-N-NO2. To evaluate the affinity of the radiolabelled biotin conjugate for the target protein avidin, the radiochemical complex was incubated with the target protein for 30 minutes at 37 °C and the reaction mixture was subsequently filtered through a 30 kD cutoff filter. Any radiochemical complex associated with avidin is retained in the filter. Of the full amount of radiochemical complex added to avidin, 58 % was found to be bound to avidin. The corresponding blocking experiment, where the same amount of avidin was pre-incubated with a 10 fold excess of native biotin before incubation with the radiolabelled complex for 30 minutes at 37 °C, resulted in only 21 % of the biotin conjugate binding to avidin. Despite derivatization of the original biotin structure upon conjugation, the targeting moiety retains its affinity for the target molecule.42 100 % 76 % 55 % 43 %  67  Ga-Tf  0h 10 min 1h 2h 0  2  4  6  8  10  12  14  Figure 6.6 Stacked HPLC monitoring traces of transferrin challenge experiment for [67Ga(dp-N-Biotin)]+ .  137  6.3.5  Thiourea Coupling with [c(RGDyK)]  The great advantage of using an isothiocyanate functional group for the coupling of chelate to biomolecule, especially for the H2dedpa scaffold, is that under the chosen reaction conditions only primary amines are transformed into the NCS functional group. Also, the NCS functional group will only react with primary amines to form the thiourea functional group. This solves the problem encountered with the secondary amine of Me2dp-bb-NH2, which interfered with the coupling reactions using amines as nucleophiles. H2dp-bb-NCS or H2dp-N-NCS, dissolved in a minimal amount of CH3OH, was successfully coupled by addition of a slight excess of RGD peptide in pH 9 -9.5 aqueous solution (0.1 M NaHCO3), a condition key for efficient thiourea coupling. The coupled products H2dp-bb-NCS-RGD and H2dp-N-NCS-RGD were purified via preparative HPLC. It was noted that the reaction only proceeds if the NCS derivative is reasonably solubilized with CH3OH. The use of other solvents such as DMSO yielded no peptide-chelate coupling product.  Scheme 6.5 Synthesis scheme for compounds H2dp-bb-RGD and H2dp-N-RGD. (vii). 0.1 M NaHCO3 (pH 9 9.5)/ CH3OH (3:1), room temperature, under light exclusion, 18 h.  138  Radiolabelling and Stability of RGD Conjugates with 67/68Ga  6.3.6  Coordination of H2dp-bb-RGD to  67  Ga or  68  Ga affords the complex within 10  minutes at room temperature in 97 % radiochemical yield (Figure 6.7). As an added difficulty, HPLC turned out to be uninformative for the RGD transchelation experiments with apo-transferrin. Instead, PD-10 columns were used to filter off any transferrinbound  67  Ga. The subsequent apo-transferrin challenge experiment revealed that 92 % of  the radiolabeled complex remained intact after 2 h in the presence of excess apotransferrin. In the case of H2dp-N-RGD, the  67  Ga and  68  Ga complexes are formed within  10 minutes at room temperature in 99% radiochemical yield (trace Figure 6.7). The subsequent apo-transferrin challenge experiment yields 73 % of the complex remaining intact after 2 h. Both radiochemical coordination experiments are in accordance with what was observed with the model compounds H2dp-bb-NO2 and H2dp-N-NO2 (Chapter 4), including the high-yielding labelling at low ligand concentrations (1 nmol).  0  2  4  6  8  time (min)  10  12  14  16  0  2  4  6  8  10  12  time (min)  Figure 6.7 HPLC monitoring traces of labelling traces for [67Ga(dp-bb-RGD)]+ (left) and [67Ga(dp-N-RGD)]+ (right).  139  Radiolabelling and Stability of RGD Conjugates with 64Cu  6.3.7  Coordination of H2dp-bb-RGD to  64  Cu affords the complex within 10 minutes at room  temperature in 97 % radiochemical yield. The subsequent serum challenge experiment revealed 72 % of the radiolabeled complex remained intact after 24 h in the presence of excess serum. PD-10 columns were used to filter off any serum-bound 64  of H2dp-N-RGD, the  64  Cu. In the case  Cu complexes are formed within 10 minutes at room temperature  in 96 % radiochemical yield (Figure 6.8). The subsequent serum challenge experiment yields over 88 % of the complex remaining intact after 24 h. Both radiochemical coordination experiments are in accordance with what was observed with H2dedpa (Chapter 5), efficient labelling with radiochemical yields > 92 % can be observed with ligand concentrations as low as 10 nmol. Decrease of radiochemical yield at lower concentrations is due to the lower specific activity (compared to  67  Ga) of the  64  Cu stock  solution and also corresponds to the H2dedpa labelling experiment (Chapter 5).  0  2  4  6  time (min)  8  10  12  14  0  2  4  6  8  10  12  14  16  time (min)  Figure 6.8 Radioabelling traces for [64Cu(dp-bb-RGD)]+ (left) and [64Cu(dp-N-RGD)]+ (right). 64  Cu labelled bioconjugates show a non-negligible percentage of  64  Cu  transchelated with serum proteins after 24 hours, which might impose limitations for the use of slowly localizing biomolecules such as antibodies. For imaging with fast localizing  140  biomolecules such as RGD, the stability may be sufficient; in vivo experiments will provide further indication of the potential of these chelates for 64Cu labelling. The finding that the N-derivative is slightly more stable then the bb-derivative could be due to Cu(II) being a softer metal, which prefers softer, tertiary amines as donor ligands, compared to the harder Ga(III), which prefers harder, secondary amine ligand systems.  6.4  Conclusions In this chapter, the H2dedpa scaffold was successfully functionalized with  targeting biomolecules. While conjugation using a conventional amide coupling was only successful using the N-derivative, the cyclic peptide RGD was successfully conjugated with both derivatization modes using a thiourea forming coupling reaction. The novel isothiocyanato compounds necessary for this conjugation reaction were synthesized and characterized. Radiolabelling with radioisotopes  67/68  Ga and  64  Cu was successful under  the same mild conditions described previously for the model compounds H2dedpa, H2dpN-NO2)]+and H2dp-bb-NO2. In vitro stability evaluations also confirm findings from previous Chapters 4 and 5, establishing the benzyl nitro derivatives as good models for the derivatization with actual biomolecules. Successful bioconjugation of a novel chelate, as well as confirmation of preliminary labelling results with the latter is a great step towards the actual application of a novel chemical entity for clinical purposes. The ability of the H2dedpa derivatives, to coordinate the two PET isotopes  68  Ga and  64  Cu, both of  interest to the radiopharmaceutical community, in a fast and efficient manner renders this acyclic bifunctional chelate truly unique for its labelling properties. Due to decreased stability with  64  Cu at the 24 h in vitro competition timepoint, the complex might be more  suitable for targeted imaging with small, fast localizing biomolecules. In vivo evaluation, planned after submission of this thesis, will provide information on the potential of this chelate for the purpose of imaging with either radionuclide.  141  Chapter 7: Functionalization of the H2dedpa Scaffold (2): Small Lipophilic Cations for Potential Imaging of Myocardial Bloodflow§  7.1  Introduction: Cardiac Imaging Agents Evaluation of myocardial perfusion is done routinely with SPECT agents such as  201  Tl,  99m  Tc-sestamibi (trade name Cardiolite) or  99m  Tc-tetrofosmin (brand name  Myoview, Figure 7.1). Due to the great importance of early detection of coronary artery disease in the clinic, limitations such as low first-pass extraction and high liver uptake of such agents have been recognized.171 One way to improve upon the current  99m  Tc agents,  which are believed to be targeting mitochondrial uptake, is the development of new, lipophilic, cationic  99m  Tc complexes with improved in vivo properties.39 Another  approach focuses on using PET in order to increase spatial resolution. This can be done by  11  C-labelling of aliphatic carbon chains with terminal carboxylates, which  subsequently show increased retention in the myocardium since they function as metabolites for the heart muscle;172 however, the short 20 minute half-life and the need for a cyclotron to produce the radioisotope in this case imposes significant limitations.  Figure 7.1 Some commercially approved radiopharmaceuticals for heart imaging. §  Work presented in this chapter has accepted for publishing: "New Ga Derivatives of the H2dedpa Scaffold with Improved Clearance and Persistent Heart Uptake", E. Boros, C. L. Ferreira, B. O. Patrick, M. J. Adam, C. Orvig, Nucl. Med. Biol., in press (2011).  142  82  Rb, a generator produced PET isotope, has been in use in the clinic for perfusion  imaging; however, the emission properties of this radionuclide are not ideal and the liver uptake is high.173  68  Ga, also a generator produced PET isotope, has potential for  developing a 68Ga based cardiac imaging agent. Ga(III) forms stable complexes with many multidentate ligands (see Chapter 4), and a myriad of capable ligand systems has been reviewed recently;60,90 however, very few of the investigated ligands exhibit favourable properties such as high thermodynamic stability and kinetic inertness, as well as fast labelling under favourable conditions of room temperature and mild pH. Fulfillment of these criteria is crucial for a  68  Ga based  compound with potential use in clinical settings. From the findings described in Chapter 4 for the H2dedpa scaffold, it was concluded that through thoughtful functionalization of the chelate the physical properties, and biodistribution of the radiochemical complex should be modified accordingly. As [Ga(dedpa)]+ is a cation, investigations of more lipophilic derivatives could be of great interest and could potentially lead to compounds with increased and prolonged myocardial retention. Other ligand systems for  68  Ga have been investigated previously for this purpose,  ranging from tetra- to hexadentate, forming neutral or mono-cationic complexes upon coordination.51,52 Green and coworkers have investigated potential myocardial imaging agents based on acyclic bis(salicylaldimine) type chelates over the past decade (Figure 7.2).174  Figure 7.2 Previously investigated Ga-based compounds for heart imaging.  143  Despite recent, highly promising results, inferior heart-to-blood and heart-to-lung ratios compared to  99m  Tc-sestamibi, and a non-optimal, carrier-added labelling procedure  remain to be improved upon.173 In this chapter, seven new derivatives of the H2dedpa scaffold are described and investigated for their labelling properties, in vitro stability, log Po/w and biodistribution. All compounds bear two phenyl residues, which are derivatized with a varying number of methoxy or ethoxy groups. In the case of two ligands, an additional methoxy group was incorporated via derivatization of the pyridyl ring.  7.2  Experimental  7.2.1  Materials and Methods  All solvents and reagents were used as received. Refer to Chapter 2.2 for general information. The HPLC system used for analysis consisted of a Waters Alliance HT 2795 separation module equipped with a Raytest Gabbistar NaI detector and a Waters 996 photodiode array (PDA) detector. Analysis of radiolabeled complexes was done on a Waters XBridge BEH130 4.6 × 150 mm column (retention time of  67  Ga-Tf: 2.5 min). If  not stated otherwise, the Rf values are measured on standard TLC plates with 10% CH3OH in CH2Cl2 as the mobile phase.  7.2.2  Chemical Synthesis  Common starting materials for synthetic precursors such as 1, 3, 6, 9 and 10 were synthesized according to the literature references.108,170,175,176 Alkylation A (step 1, synthesis of 14 and 15, Scheme 7.1): Both secondary amine and 2.1 equivalents of the bromoaryl were dissolved in acetonitrile (100 mL). Na2CO3 (20 eq) was added into the reaction mixture and the reaction was stirred at 70 °C overnight. The resulting milky solution was filtered and the solvent was removed in vacuo to afford the crude product as an oil. This was then subsequently purified by column chromatography (10% CH3OH in CH2Cl2) to afford the product as a viscous oil that solidifies upon standing. 144  Alkylation B (step 1, synthesis of 13, 16, 17, 18 and 19): Both secondary amine and 2.1 equivalents of  6-bromomethyl-pyridine-2-carboxylic acid methyl ester or 9 were  dissolved in acetonitrile (100 mL). Na2CO3 (20 eq) was added to the reaction mixture and the reaction was stirred at 70 °C overnight. The resulting milky solution was filtered and the solvent was removed in vacuo to afford the crude product as an oil. This was then subsequently purified by column chromatography (10% CH3OH in CH2Cl2) to afford the product as a very viscous oil that solidifies upon standing. Deprotection procedure (step 2): The methylester-protected starting material was dissolved in 6 mL of a 3:1 mixture of THF and water (4 mL total volume). LiOH (4 eq) was added into the solution and the reaction mixture was stirred at room temperature until the reaction was determined to be complete by TLC (10% CH3OH in CH2Cl2). The solvent was removed in vacuo to afford the product as a white solid.  7.2.3  Synthesis of Lipophilic Ligand Systems  1,2-[N,N’-Bis(phenylmethyl)]-N,N’-bis[6-{methoxycarbonyl}-pyridin-2yl] methylamino)ethane (2). Precursors 1 and 6-bromomethyl-pyridine-2-carboxylic acid methyl ester were used according to alkylation method B to synthesize 2. Yield: 52 mg, 0.096 mmol, 46 %. Rf: 0.45. 1H NMR (CDCl3, 400 MHz, δ): 7.96 – 7.94 (dd, 3J3, 4  4  = 5.2 Hz,  J3, 5 = 2 Hz, H3, 2H), 7.72 – 7.68 (m, 3J4, 5 = 5.2 Hz, 4J3, 5 = 2  Hz, H4, H5, 4H), 7.26 – 7.19 (m, H11-H13, 10H), 3.97 (s, H14, 6H), 3.81 (s, H7, 4H), 3.57 (s, H9, 4H), 2.68 (s, H8, 4H).  13  C NMR (CDCl3, 75 MHz, δ): 166.0 (C1), 161.3  (C6), 147.2 (C2), 139.1 (C4), 137.4 (C10), 128.8 (C11), 127.2 (C12), 125.9 (C13), 123.6 (C3), 60.7 (C7), 59.3  (C9), 53.0 (C14), 52.2 (C8). HR-ESI-MS: m/z calcd. for  C32H35N4O4: 539.2658, found: 539.2645 [M + H]+.  145  1,2-[N,N’-Bis{3-methoxybenzyl}methyl]-N,N’-bis[6-{methoxycarbonyl}-pyridin-2-yl] methylamino)ethane (4). Precursors 3 and 1-(bromomethyl)-3-methoxybenzene were used according to alkylation method A to synthesize 4. Yield: 50 mg, 0.083 mmol, 59 %. Rf: 0.5. 1H NMR (CDCl3, 400 MHz, δ): 7.98 (d, 3J3, 4 = 4 Hz, H3, 2H), 7.73 – 7.72 (m, H14, H15, 4H) 7.18 (t, 3  J3, 4 = 4 Hz, , 3J4, 5 = 5.6 Hz, H4, 2H), 6.89 (m, H11, H12, 4H),  6.71 (d, 3J4,  5  = 5.6 Hz, H5, 2H), 4.0 (s, H17, 6H) , 3.85 (s, H7,  4H), 3.78 (s, H16, 6H), 3.58 (s, H9, 4H), 2.71 (s, H8, 4H). 13C NMR (CDCl3, 75 MHz, δ): 166.0 (C1), 161.3 (C12), 159.8 (C6), 147.3 (C2), 140.9 (C4), 137.5 (C10), 129.4 (C14), 126.0 (C5), 123.7 (C3), 121.2 (C15), 114.4 (C12), 112.4 (C13), 60.7 (C7), 59.3 (C9), 55.3 (C16), 53.0 (C8), 52.3 (C17). HR-ESI-MS: m/z calcd. for C34H39N4O6: 599.2870, found: 599.2882 [M + H]+.  (1,2-[N,N’-Bis{3,5-dimethoxybenzyl}methyl]-N,N’-bis[6-{methoxycarbonyl}-pyridin2-yl] methylamino)ethane (5). Precursors 3 and 3,5-dimethoxybenzyl bromide were used according to alkylation method A to synthesize 5. Yield: 51 mg, 0.077 mmol, 56 %. Rf: 0.45. 1H NMR (CDCl3, 400 MHz, δ): 7.96 (m, H4, 2H), 7.70 (m, H3, H5, 4H), 6.46 (s, H11, 4H), 6.30 (s, H13, 2H), 3.97 (s, H9, 6H) , 3.83 (s, H7, 4H), 3.73 (s, H14, 12H), 3.53 (s, H15, 4H), 2.69 (s, H8, 4H).  13  C NMR  (CDCl3, 75 MHz, δ): 165.9 (C1), 161.2 (C12), 160.9 (C6), 147.3 (C2), 141.8 (C4), 137.4 (C10), 126.0 (C5), 123.6 (C3), 106.6 (C11), 98.9 (C12), 60.7 (C3), 59.4 (C9), 55.4 (C14), 52.9 (C15), 52.4 (C8). HR-ESI-MS: m/z calcd. for C36H43N4O8: 659.3081, found: 659.3068 [M + H] +.  146  (1,2-[N,N’-Bis{2,4,6-trimethoxybenzyl}methyl]-N,N’-bis[6-{methoxycarbonyl}pyridin-2-yl] methylamino)ethane (7). Precursors 6 and 6-bromomethyl-pyridine-2-carboxylic acid methyl ester were used according to alkylation method B to synthesize 7. Yield: 79 mg, 0.11 mmol, 55 %. Rf: 0.4. 1H NMR (CDCl3, 300 MHz, δ): 8.27 (d, 3J3, 4 = 8.4 Hz, H3, 2H), 7.99 (d, 3J4, 5 = 7.5 Hz, H5, 2H), 7.86 (m, 3J3, 4 = 8.4 Hz, 3J4, 5 = 7.5 Hz, H4, 2H), 6.02 (s, H12, 4H), 4.31 (s, H7, 4H), 3.96 (s, H16, 6H), 3.94 (s, H9, 4H), 3.78 (s, H15, 6H), 3.68 (s, H14, 12H), 3.19 (s, H8, 4H) . 13C NMR (CDCl3, 75 MHz, δ): 165.2 (C1), 161.9 (C6), 159.9 (C11, C13), 146.8 (C2), 137.9 (C4, C10), 127.9 (C5), 124.2 (C3), 90.2 (C12), 58.5 (C7), 55.5 (C14), 55.3 (C15), 52.7 (C9), 50.4 (C8), 45.5 (C16). HR-ESI-MS: m/z calcd. for C38H47N4O10: 719.3292, found: 719.3303 [M + H]+.  (1,2-[N,N’-Bis{2,4,6-trimethoxybenzyl}methyl]-N,N’-bis[4-methoxy-6-{methoxycarbonyl}pyridin-2-yl]methylamino)ethane (8). Precursors 1 and 9 were used according to alkylation method B to synthesize 8. Yield: 311 mg, 0.4 mmol, 70 %. Rf: 0.4. 1H NMR (CDCl3, 400 MHz, δ): 7.32 (s, H3, H5, 4H), 5.89 (s, H12, 4H), 4.08 (s, H7, 4H), 3.83 (s, H9, 4H), 3.78 (s, H17, 6H), 3.64 (s, H16, 6H) 3.55 (s, H14, 12H), 3.25 (s, H15, 6H), 3.03 (s, H8, 4H). 13C NMR (CDCl3, 100 MHz, δ): 167.3 (C1), 165.3 (C4), 162.3 (C11), 160.1 (C13), 157.9 (C6), 148.4 (C2), 112.9 (C5), 111.3 (C3), 101.2 (C10), 90.5 (C12), 58.3 (C7), 56.3 (C15), 55.7 (C14’), 55.5 (C14), 52.9 (C16), 50.0 (C17), 49.2 (C8), 45.9 (C9). IR (neat, cm-1): 1721 (m), 1593 (m), 1455 (m), 1335 (m). HR-ESI-MS: m/z calcd. for C40H51N4O12: 779.3503, found: 779.3493 [M + H]+.  147  6-Bromomethyl-4-methoxy-pyridine-2-carboxylic acid methyl ester (9). The title compound was synthesized in 4 steps.170 The fist two steps include a two-step methylation of the two carboxylates and the alcohol, followed by a standard partial reduction as done for 1,2-[{6(methoxycarbonyl)pyridin-2-yl}methylamino]ethane,  followed  by  bromination of the alcohol to afford the product 9. Yield: 31 % (overall yield). 1H NMR (CDCl3, 400 MHz, δ): 7.46 (d, 4J3, 5 = 2.4 Hz, H3, 2H), 7.06 (d, 4J3, 5 = 2.4 Hz, H5, 2H), 4.73 (s, H7, 2H), 3.88 (s, H9, 3H), 3.82 (s, H8, 6H).  13  C NMR (CDCl3, 100 MHz, δ):  167.4 (C1), 165.4 (C4), 158.6 (C6), 149.3 (C2), 112.81 (C5), 110.9 (C3), 55.86 (C8), 53.15 (C9), 33.5 (C7). IR (neat, cm-1): 1731 (s), 1591 (m), 1439 (w), 1246 (m), 1043 (s). HR-ESI-MS: m/z calcd. for C9H10N79BrNaO3: 281.9742, found: 281.9739 [M + Na]+ . Elemental Analysis calcd. (found) for for C9H1079BrNO3: C: 41.56 (41.7), H: 3.88 (3.88), N: 5.31 (5.79).  2,4,6-Triethoxybenzaldehyde 2,4,6-Trihydroxybenzaldehyde (1.88 g, 9.9 mmol) was dissolved in DMF. NaH (0.960 g, 0.024 mol, in 60 % dispersion in mineral oil) was added, followed by EtBr (16.240 g, 11.05 mL, 0.149 mol). The mixture was stirred overnight at room temperature. After no tri-alkylated product was afforded, another batch of NaH (0.301 g, 0.0125 mol, in 60 % dispersion in mineral oil) was added, together with more EtBr (2.05 g, 1.397 mL, 0.0188 mol). The mixture was stirred overnight at 60 °C and quenched with H2O (50 mL). The crude product was extracted with CH2Cl2, and the organic phase was washed twice with saturated brine. Subsequently, the solvent was removed and the residual crude product was purified with column chromatography (SiO2, 3:2 mixture of hexanes/EtOAc) to afford the clean product as a colorless solid. Yield: 0.970 g, 3.4 mmol, 34 %. Rf = 0.21. 1  H NMR (CDCl3, 400 MHz, δ): 10.34 (s, H1, 1H), 5.99 (s, H4, 2H), 4.03 (m, H6, H8,  6H), 1.39 (m, H7, H9, 9H) .  13  C NMR (CDCl3, 100 MHz, δ): 187.9 (C1), 165.5 (C3),  163.5 (C5), 109.1 (C2), 91.6 (C4), 64.6 (C6), 63.9 (C8), 14.65 (C7, C9). IR (neat, cm-1): 1672 (m), 1599 (m), 1565 (s). HR-ESI-MS: m/z calcd. for C13H19O9: 239.1283, found:  148  239.1286 [M + H]+. Elemental Analysis calcd. (found) for for C13H18O9: C: 65.53 (65.37), H: 7.61 (7.48).  N,N'-Bis-(2,4,6-triethoxybenzyl)ethane1,2-diamine (10). This compound was synthesized according to a literature procedure.176 1H NMR (CDCl3, 400 MHz, δ): 6.01 (s, H5, 4H), 4.08 (s, H2, 4H), 3.91 (m, H7, H9, 12H), 3.74 (s, H1, 4H), 1.30 (m, H8, H10, 18H).  13  C NMR (CDCl3, 100 MHz, δ): 159.9 (C4),  158.8 (C6), 107.3 (C3), 92.04 (C5), 91.8 (C5’), 63.8 (C7, C9), 46.0 (C1), 40.3 (C2), 14.9 (C8, C10). IR (neat, cm-1): 1592 (m), 1438 (m). HR-ESI-MS: m/z calcd. for C28H45N2O6: 505.3278, found: 505.3269 [M + H]+.  (1,2-[N,N’-Bis{2,4,6-triethoxybenzyl}methyl]-N,N’-bis[6-{methoxycarbonyl}-pyridin 2-yl]methylamino)ethane (11). Precursors  10  and  6-bromomethyl-pyridine-2-  carboxylic acid methyl ester were used according to alkylation method B to synthesize 11. Yield: 112 mg, 0.13 mmol, 52 %. Rf: 0.2. 1H NMR (CDCl3, 400 MHz, δ): 8.01 (d, 3J3, 4 = 7.6 Hz, H3, 2H), 7.94 (d, 3J4, 5 = 7.6 Hz, H5, 2H), 7.82 (d, 3J3,  4  = 3J4,  5  = 7.6 Hz, H4, 2H),  5.94 (s, H12, 4H), 4.30 (s, H7, 4H), 3.94 (s, H9, 4H), 3.88 (m, 3J16, 17 = 3J14, 15 = 6.8 Hz, H14, H16, H18, 18H), 3.11 (s, H8, 4H), 1.35 (t, 3J16, 17 = 6.8 Hz, H17, 6H), 1.09 (m, 3J14, 15  = 6.8 Hz, H15, 12H). 13C NMR (CDCl3, 100 MHz, δ): 165.4 (C1), 161.5 (C13), 159.6  (C11), 156.2 (C6), 147.1 (C2), 138.4 (C4), 128.2 (C5), 124.7 (C3), 101.2 (C10), 91.7 (C12), 64.1 (C14), 63.7 (C16), 58.7 (C7), 52.9 (C18), 48.9 (C8), 45.6 (C9), 14.8 (C15, C17). HR-ESI-MS: m/z calcd. for C44H59N4O10: 803.4231, found: 803.4248 [M + H]+.  149  (1,2-[N, N’-Bis{2,4,6-triethoxybenzyl}methyl]-N,N’-bis[4-methoxy-6-{methoxycarbonyl}-pyridin-2-yl] methylamino)ethane (12). Precursors 9 and 10 were used according to alkylation method B to synthesize 12. Yield: 116 mg, 0.13 mmol, 52 %. Rf: 0.4. 1H NMR (CDCl3, 400 MHz, δ): 7.57 (s, H3, 2H), 7.47 (s, H5, 2H), 5.96 (s, H12, 4H), 4.24 (s, H7, 4H), 3.96 (s, H9, 4H), 3.91 (m, 3J16,  17  = 3J14,  15  =  6.8 Hz, H14, H16, H18, H19, 24H), 3.07 (s, H8, 4H), 1.35 (t, 3J16, 17 = 6.8 Hz, H17, 6H), 1.09 (t, 3J14, 15 = 6.8 Hz, H15, 12H). 13C NMR (CDCl3, 100 MHz, δ): 167.4 (C1), 165.4 (C4), 161.5 (C11), 159.6 (C13), 158.3 (C6), 148.5 (C2), 113.1 (C5), 111.9 (C3), 101.4 (C10), 91.8 (C12), 64.1 (C14), 63.8 (C16), 58.9 (C7), 56.5 (C15), 53.0 (C18), 48.9 (C8), 45.6 (C9), 14.8 (C15), 14.4 (C17). HR-ESI-MS: m/z calcd. for C46H63N4O12: 863.4405, found: 863.4424 [M + H]+.  (1,2-[N,N’-Bis{3-methoxybenzyl}methyl]-N,N’-bis[6-{carboxyato}-pyridin-2-yl] methyl amino)ethane (13, 0MeO). The general deprotection procedure was used with 2 as the starting material. Yield: 41 mg, 0.082 mmol, 85 %. Rf: 0.02. 1H NMR (CD3OD, 400 MHz, δ): 8.01 (d, 3J3,  4  = 7.6 Hz, H3, 2H),  7.89 (m, 3J3, 4 = 7.6 Hz, 3J4, 5 = 6.8 Hz, H4, 2H), 7.37 (d, 3J4, 5 = 6.8 Hz, H5, 2H), 7.25 – 7.21 (m, H11, H12, 6H), 7.04 (m, H13, 4H), 3.87 (s, H7, 4H), 3.41 (s, H9, 4H), 2.31 (s, H8, 4H).  13  C NMR (CD3OD, 100 MHz,  δ): 172.5 (C1), 159.5 (C6), 154.7 (C2), 139.8 (C4), 136.3 (C10), 131.4 (C11), 129.2 (C12), 128.5 (C13), 125.7 (C5), 123.2 (C3), 61.0 (C7), 57.6 (C9), 48.5 (C8). IR (neat, cm-1): 1616 (m), 1583 (m), 1433 (w), 1386 (m). HR-ESI-MS: m/z calcd. for C30H29N4O4: 509.2189, found: 509.2193 [M - H] -.  150  (1,2-[N,N’-Bis{3-methoxybenzyl}methyl]-N,N’-bis[6-{carboxyato}-pyridin-2-yl] methylamino)ethane (14, 2MeO). The general deprotection procedure was used with 4 as the starting material. Yield: 37 mg, 0.065 mmol, 78 %. Rf: 0.1. 1H NMR (CD3OD, 400 MHz, δ): 7.99 (d, 3J3, 4 = 7.6 Hz, H3, 2H), 7.89 (m, 3J3, 4 = 7.6 Hz, 3J4, 5 = 7.2 Hz, H4, 2H), 7.39 (d, 3J4, 5 = 7.2 Hz, H5, 2H), 7.15 (m, 3J14, 3  15  = 9.2 Hz, H14, 2H), 6.79 (d,  J14, 15 = 9.2 Hz, H15, 2H), 6.61 (m, H13,H11, 4H), 3.89 (s, H7, 4H), 3.73 (s, H16, 6H),  3.38 (s, H9, 4H), 2.38 (s, H8, 4H).  13  C NMR (CD3OD, 100 MHz, δ): 172.5 (C1), 160.8  (C12), 159.4 (C6), 154.6 (C2), 139.6 (C4), 137.7 (C10), 130.0 (C14), 125.5 (C3), 123.6 (C13), 122.9 (C5), 116.9 (C11), 113.5 (C15), 60.9 (C7), 57.2 (C9), 55.6 (C16), 47.5 (C8). IR (neat, cm-1): 1614 (m), 1584 (m), 1434 (w), 1387 (m). HR-ESI-MS: m/z calcd. for C32H33N4O6: 569.2400, found: 569.2396 [M - H]-.  (1,2-[N,N’-Bis{3,5-dimethoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2-yl] methylamino)ethane (15, 4MeO). The general deprotection procedure was used with 5 as the starting material. Yield: 42 mg, 0.063 mmol, 83 %. Rf: 0.15. 1  H NMR (CD3OD, 400 MHz, δ): 7.99 (d, 3J3, 4 = 8.0 Hz, H3,  2H), 7.88 (m, 3J3, 4 = 8.0 Hz, 3J4, 5 = 7.6 Hz, H4, 2H), 7.41 (d, 3  J4, 5 = 7.6 Hz, H5, 2H), 6.35 (s, H13, 2H), 6.32 (s, H11, 4H),  3.89 (s, H7, 4H), 3.69 (s, H14, 12H), 3.34 (s, H9, 4H), 2.36 (s, H8, 4H).  13  C NMR (CD3OD, 100 MHz, δ): 173.0 (C1), 162.2 (C12), 159.7 (C6), 154.8  (C2), 139.8 (C4), 138.8 (C10), 125.7 (C3), 123.1 (C5), 109.4 (C11), 100.1 (C13), 61.1 (C7), 56.8 (C9), 55.8 (C14), 47.8 (C8). IR (neat, cm-1): 1585 (m), 1428 (w), 1387 (m). HR-ESI-MS: m/z calcd. for C34H37N4O8: 629.2611. found: 629.2615 [M - H]-.  151  (1,2-[N,N’-Bis{2,4,6-trimethoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2yl] methylamino)ethane (16, 6MeO). The general deprotection procedure was used with 7 as the starting material. Yield: 61 mg, 0.088 mmol, 80 %. Rf: 0.2. 1  H NMR (CD3OD, 400 MHz, δ): 7.95 (d, 3J3,  4  = 7.6 Hz,  H3, 2H), 7.84 (m, 3J3, 4 = 7.6 Hz, 3J4, 5 = 7.2 Hz, H4, 2H), 7.30 (d, 3J4, 5 = 7.2 Hz, H5, 2H), 6.12 (s, H12, 4H), 3.93 (s, br, H7 4H), 3.62 (s, H15, 6H), 3.63 (s, H14, 12H), 3.41 (s, H8, 4H), 2.19 (s, H8, 4H).  13  C  NMR (CD3OD, 100 MHz, δ): 172.6 (C1), 162.4 (C13), 161.7 (C11), 160.9 (C6), 154.7 (C2), 139.5 (C4), 125.2 (C3), 122.6 (C5), 105.7 (C10), 91.5 (C12), 61.9 (C7), 55.9 (C14), 55.8 (C15), 48.7 (C8), 43.6 (C9). IR (neat, cm-1): 1585 (m), 1455 (m), 1434 (w), 1390 (m). HR-ESI-MS: m/z calcd. for C36H41N4O10: 689.2823, found: 689.2812 [M + H]+.  1,2-[N,N’-Bis{2,4,6-trimethoxybenzyl}methyl]-N,N’-bis[4-methoxy-6-{carboxylato} pyridin-2-yl]methylamino)ethane (17, 8MeO). The general deprotection procedure was used with 8 as the starting material. Yield: 61 mg, 0.088 mmol, 87 %. Rf: 0.35. 1H NMR (CD3OD, 300 MHz, δ): 7.50 (d, 4J3, 5 = 2.1 Hz, H3, 2H), 6.84 (d, 4J3, 5 = 2.1 Hz, H5, 2H), 6.13 (s, H12, 4H), 3.89 (s, H16, 6H), 3.77 (s, H15, 6H) 3.61 (s, H14, 12H), 3.41 (s, H7, 4H), 3.34 (s, H9, 4H), 2.10 (s, H8, 4H).  13  C NMR (CD3OD, 100 MHz,  δ): 172.4 (C1), 169.6 (C4), 162.7 (C13), 161.7 (C11), 156.7 (C6, C2), 110.9 (C5), 108.5 (C3), 106.0 (C10), 91.5 (C12), 65.1 (C7), 56.0 (C16), 55.8 (C14, C15), 48.7 (C8), 43.8 (C9). IR (neat, cm-1): 1585 (m), 1455 (m), 1433 (w), 1390 (m). HR-ESI-MS: m/z calcd. for C38H46N4O127Li: 757.3272, found: 757.3260 [M + Li]+.  152  (1,2-[N,N’-Bis{2,4,6-triethoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2-yl] methylamino)ethane (18, 6EtO). The general deprotection procedure was used with 11 as the starting material. Yield: 91 mg, 0.12 mmol, 92 %. Rf: 0.2. 1H NMR (CD3OD, 400 MHz, δ): 7.97 (d, 3J3, 4 = 7.6 Hz, H3, 2H), 7.87 (m, 3J3, 4 = 3J4, 5 = 7.6 Hz, H4, 2H), 7.29 (d, 3J4,  5  = 7.6 Hz, H5, 2H), 6.06 (s, H12, 4H), 3.97-3.81  (m, 3J16, 17 = 3J14, 15 = 6.8 Hz, H14, H16,H7, 16H), 3.43 (s, H9, 4H), 2.21 (s, H8, 4H), 1.38 (t, 3J16, 17 = 6.8 Hz, H17, 6H), 1.16 (t, 3J14, 15 = 6.8 Hz, H15, 12H).  13  C NMR (CD3OD, 100 MHz, δ): 171.2 (C1), 160.0 (C13, C11), 159.8 (C6),  153.4 (C2), 138.2 (C4), 123.9 (C3), 121.4 (C5), 104.7 (C10), 91.3 (C12), 63.5 (C15), 63.3 (C16), 61.5 (C7), 42.7 (C8), 28.7 (C9), 14.1 (C15, C17). IR (neat, cm-1): 1583 (m), 1434 (w), 1390 (m). HR-ESI-MS: m/z calcd. for C42H53N4O10: 773.3762, found: 773.3744 [M + H]+.  (1,2-[N,N’-Bis{2,4,6-triethoxy-benzyl}methyl]-N,N’-bis[4-methoxy-6-{carboxylato} pyri-din-2-yl]methylamino)ethane (19, 6EtO·2MeO). The general deprotection procedure was used with 12 as the starting material. Yield: 92 mg, 0.11 mmol, 84 %. Rf: 0.35. 1H NMR (CD3OD, 400 MHz, δ): 7.54 (d, 4J3, 5 = 2 Hz, H3, 2H), 6.82 (d, 4J3, 5 = 2.1 Hz, H5, 2H), 6.06 (s, H12, 4H), 3.97-3.84 (q, 3J16,  17  = 3J14,  15  = 6.8 Hz,  H14, H16,H7, 12H), 3.89 (s, H18, 6H), 3.41 (s, H9, 4H), 2.20 (s, H8, 4H), 1.33 (t, 3J16, 17 = 6.8 Hz, H17, 6H), 1.18 (t, 3J16, 17 = 6.8 Hz, H15, 12H).  13  C NMR (CD3OD, 100 MHz, δ): 171.0 (C1), 168.2 (C4), 161.3 (C13), 159.9  (C11), 159.8 (C6) , 155.3 (C2), 109.7 (C5), 107.0 (C3), 104.8 (C10), 91.3 (C12), 63.5 (C14), 63.2 (C16), 61.7 (C7), 42.8 (C8), 29.5 (C9), 14.1 (C5, C17). IR (neat, cm-1): 1587 (m), 1435 (w), 1390 (m). HR-ESI-MS: m/z calcd. for C44H57N4O12: 833.3973, found: 833.3979 [M - H]-.  153  7.2.4  Coordination Chemistry  General procedure for synthesis of non-radioactive Ga complexes: The ligand was dissolved in a CH3OH-water mixture (1:2, 6 mL total volume) with adjustment to pH 2 by addition of 0.1 M HCl. Ga(NO3)3·6H2O (1 eq) was added and the pH was adjusted by addition of 0.1 M NaOH to 4.5. The reaction mixture was stirred at 60 °C for 2 h. The solvent was removed in vacuo to afford the complex as a white solid in quantitative yield.  (1,2-[N,N’-Bis{3-methoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2yl]methyl- amino)ethane gallium(III) nitrate ([Ga(13)][NO3], [Ga(0MeO)][NO3]). The general complex synthesis procedure was used with 13 as the starting material. 1H NMR (CD3OD, 400 MHz, δ): 8.69 (m, 3J3, 4 = 7.6 Hz, 3  J4,  5  = 8 Hz, H4, 2H), 8.48 (d, 3J3,  4  = 7.6 Hz,  H3, 2H), 8.21 (d, 3J4, 5 = 8 Hz, H5, 2H), 7.45 (s, H11, H12, H13, 10H), 4.94 (d, 3J7, 7’ = 16.4 Hz, H7, 2H), 4.32 (d, 3J7, 7’ = 16.4 Hz, H7, 2H), 4.05 (d, 3J9, 9’ = 14 Hz, H9, 2H), 3.81 (d, 3J9, 9’ = 14 Hz, H9, 2H), 3.20 (dd, 3J8, 8’ = 10.8 Hz, H8, 4H). 13C NMR (CD3OD, 75 MHz, δ): 165.3 (C1), 152.3 (C6), 148.0 (C2), 145.5 (C4), 133.3 (C10), 130.9 (C11, C12), 130.3 (C13), 130.0 (C5), 125.3 (C3), 57.8 (C7), 56.6 (C9), 47.9. (C8) IR (neat, cm-1): 1686 (m), 1609 (m), 1345 (w), 1264 (m). HR-ESI-MS: m/z calcd. for C30H2869GaN4O4: 577.1366, found: 577.1368 [M]+.  (1,2-[N,N’-Bis{3-methoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2-yl] methyl-amino)ethane gallium(III) nitrate ([Ga(14)][NO3], [Ga(2MeO)][NO3]). The general complex synthesis procedure was used with 14 as the starting material. 1H NMR (CD3OD, 600 MHz, δ): 8.62 (m, 3J3, 3  J4,  5  = 7.2 Hz, H4, 2H), 8.41 (d, 3J3,  H3, 2H), 8.09 (d, 3J4,  5  4  = 9.6 Hz,  4  = 9.6 Hz,  = 7.2 Hz, H5, 2H), 7.36  (m, H14, 2H), 7.05 (m, H15, 2H), 6.86 (m, H11,  154  H13, 4H), 4.86 (d, 3J7, 7’ = 16.4 Hz, H7, 2H), 4.36 (d, 3J7, 7’ = 17.8 Hz, H7, 2H), 4.01 (d, 3  J9, 9’ = 15.2 Hz, H9, 2H), 3.78 (s, H16, 6H), 3.59 (d, 3J9, 9’ = 15.2 Hz, H9, 2H), 3.02 (dd,  3  J8, 8’ = 12 Hz, H8, 4H). 13C NMR (CD3OD, 125 MHz, δ): 164.7 (C1), 158.7 (C12), 147.8  (C6), 142.8 (C2), 130.0 (C4), 129.7 (C10), 128.3 (C14), 124.1 (C5), 124.0 (C3), 123.9 (C15), 117.1 (C11), 114.5 (C13), 55.8 (C7), 54.9 (C16), 54.7 (C9), 46.2 (C8). IR (neat, cm-1): 1681 (m), 1601 (m), 1346 (w). HR-ESI-MS: m/z calcd. for C32H3269GaN4O6: 637.1578, found: 637.1566 [M]+.  (1,2-[N,N’-Bis{3,5-dimethoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2-yl] methylamino)ethane gallium(III) nitrate ([Ga(15)][NO3], [Ga(4MeO)][NO3]). The general complex synthesis procedure was used with 15 as the starting material. 1H NMR (CD3OD, 400 MHz, δ): 8.69 (m, 3J3, 4 = 7.9 Hz, 3J4, 5 = 7.5 Hz, H4, 2H), 8.49 (d, 3J3, 4 = 7.9 Hz, H3, 2H), 8.20 (d, 3J4, 5 = 7.5 Hz, H5, 2H), 6.61 (br, s, H11, H13, 6H), 4.98 (d, 3  J7, 7’ = 16.7 Hz, H7, 2H), 4.37 (d, 3J7, 7’ = 16.7 Hz, H7,  2H), 3.98 (d, 3J9, 6H), 3.67 (d, 3J9,  9’  9’  = 14 Hz, H9, 2H), 3.18 (dd, 3J8,  = 14 Hz, H9, 2H), 3.81 (s, H14, 8’  = 11.6 Hz, H8, 4H).  13  C NMR  (CD3OD, 75 MHz, δ): 165.3 (C1), 162.9 (C12), 152.5 (C6), 147.9 (C2), 145.6 (C4), 132.8 (C10), 129.8 (C3), 125.3 (C5), 111.2 (C11), 102.3 (C13), 58.1 (C7), 56.8 (C9), 56.1 (C14), 47.9 (C8). IR (neat, cm-1): 1694 (m), 1596 (m), 1356 (w). HR-ESI-MS: m/z calcd. for C34H3669GaN4O8: 697.1789, found: 697.1803 [M]+.  (1,2-[N,N’-Bis{2,4,6-trimethoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2yl]methylamino)ethane gallium(III) nitrate ([Ga(16)][NO3], [Ga(6MeO)][NO3]). The general complex synthesis procedure was used with 16 as the starting material. 1H NMR (CD3OD, 400 MHz, δ): 8.68 (m, 3J3, 4 = 3J4, 5 = 7.6 Hz, H4, 2H), 8.48 (d, 3J3, 4 = 7.6 Hz, H3, 2H), 8.18 (d, 3J3,  4  = 7.6 Hz, H5,  2H), 6.31 (br, s, H12, 4H), 4.92 (d, 3J7, 7’ =  155  16.8 Hz, H7, 2H), 4.45 (d, 3J7, 7’ = 16.8 Hz, H7, 2H), 4.07 (d, 3J9, 9’ = 14 Hz, H9, 2H), 3.85 (s, H15, 6H), 3.79 (s, H14, 12H), 3.46 (d, 3J9, 9’ = 14 Hz, H9, 2H), 3.84 (dd, 3J8, 8’ = 11.4 Hz, H8, 4H). 13C NMR (CD3OD, 75 MHz, δ): 164.8 (C1), 163.2 (C13), 162.0 (C11), 153.2 (C2), 148.1 (C6), 145.9 (C4), 129.7 (C3), 125.2 (C5), 100.2 (C10), 92.2 (C12), 57.9 (C7), 56.5 (C14, C15), 56.2 (C8), 45.5 (C9). IR (neat, cm-1): 1689 (m), 1607 (m), 1352 (w). HR-ESI-MS: m/z calcd. for C36H40N4O1069Ga: 757.2000, found: 757.2005 [M]+. 1,2-[N,N’-Bis{2,4,6-trimethoxybenzyl}methyl]-N,N’-[4-methoxy-6-{carboxylato} pyri-din-2-yl]methylamino)ethane gallium(III) nitrate ([Ga(17)][NO3], [Ga(8MeO)] [NO3]). The general complex synthesis procedure was used with 17 as the starting material. 1H NMR (CD3OD, 400 MHz, δ): 7.85 (d, 4J3, 5 = 2.1 Hz, H3, 2H), 7.62 (d, 4J3, 5 = 2.1 Hz, H5, 2H), 6.27 (br, s, H12, 4H), 4.73 (d, 3J7, 7’ = 16.5 Hz, H7, 2H), 4.24 (d, 3J7, 7’ = 16.5 Hz, H7, 2H), 4.15 (s, H16, 6H), 4.00 (d, 3J9, 9’ = 13.8 Hz, H9, 2H), 3.43 (d, 3J9, 9’ = 13.8 Hz, H9, 2H), 3.82 (s, H15, 6H), 3.76 (s, H14, 12H), 2.99-2.77 (dd, 3J8,  8’  = 10.8 Hz, H8, 4H).  13  C NMR  (CD3OD, 75 MHz, δ): 175.6 (C4), 165.5 (C1), 164.9 (C13), 162.1 (C11), 154.6 (C6), 147.8 (C2), 113.9 (C5), 111.9 (C3), 100.4 (C10), 92.3 (C12), 58.8 (C7), 57.9 (C16), 56.6 (C14), 56.3 (C15), 49.9 (C8), 45.5 (C9). IR (neat, cm-1): 1667 (m), 1611 (m), 1372 (w). HR-ESI-MS: m/z calcd. for C38H4469GaN4O12: 817.2212, found: 817.2220 [M]+.  (1,2-[N,N’-Bis{2,4,6-triethoxybenzyl}methyl]-N,N’-bis[6-{carboxylato}-pyridin-2-yl] methylamino)ethane gallium(III)nitrate ([Ga(18)][NO3], [Ga(6EtO)][NO3]). The general complex synthesis procedure was used with 18 as the starting material. 1H NMR (CD3OD, 400 MHz, δ): 6.69 (m, 3J3, 4 = 7.2 Hz, 3J4, 5 = 7.8 Hz, H4, 2H), 8.45 (d, 3J3,  4  = 7.2 Hz, H3, 2H), 8.19 (d,  3  J3, 4 = 7.8 Hz, H5, 2H), 6.22 (s, H12, 4H), 4.97 (d,  3  J7, 7’ = 17.4 Hz, H7, 2H), 4.43 (d, 3J7, 7’ = 17.4 Hz,  156  H7, 2H), 4.09 (d, 3J9, 9’ = 13.8 Hz, H9, 2H), 3.43 (d, 3J9, 9’ = 13.8 Hz, H9, 2H), 4.01 (q, 3  J16, 17 = 3J14, 15 = 7.2 Hz, H14, H16, 12H), 3.29-2.84 (dd, 3J8, 8’ = 10.2 Hz, H8, 4H), 1.37  (t, 3J16, 17 = 7.2 Hz, H17, 6H), 1.17 (t, 3J14, 15 = 7.2 Hz, H15, 12H).  13  C NMR (CD3OD, 75  MHz, δ): 163.1 (C1), 161.9 (C13), 159.4 (C11), 150.9 (C6), 146.4 (C2), 144.1 (C4), 127.4 (C3), 123.3 (C5), 98.4 (C10), 91.4 (C12), 63.7 (C15), 62.9 (C16), 56.0 (C7), 48.4 (C8), 43.8 (C9), 13.3 (C15, C17). IR (neat, cm-1): 1693 (m), 1606 (m), 1586 (w), 1339 (w). HR-ESI-MS: m/z calcd. for C42H5269GaN4O10: 841.2939, found: 841.2953 [M]+.  (1,2-[N,N’-Bis{2,4,6-triethoxybenzyl}methyl]-N,N’-bis[4-methoxy-6-{carboxylato}pyri-din-2-yl]methylamino)ethane gallium(III) nitrate ([Ga(19)][NO3]), [Ga(6EtO·2MeO)][NO3]). The general complex synthesis procedure was used with 19 as the starting material. 1H NMR (CD3OD, 400 MHz, δ): 7.87 (s, H3, 2H), 7.68 (s, H5, 2H), 6.22 (s, H12, 4H), 5.12-4.80 (d, 3J7, 7’  = 16.8 Hz, H7, 2H), 4.15 (s, H18, 6H), 4.09  (d, 3J9,  9’  = 13.8 Hz, H9, 2H), 3.43 (d, 3J9,  9’  =  13.8 Hz, H9, 2H), 4.03 (q, 3J16, 17 = 3J14, 15 = 6.6 Hz, H14, H16, 12H), 3.10-2.80 (dd, 3J8, 8’ = 10.8 Hz, H8, 4H), 1.37 (t, 3J16, 17 = 6.6 Hz, H17, 6H), 1.25 (t, 3J14, 15 = 6.6 Hz, H15, 12H). 13C NMR (CD3OD, 75 MHz, δ): 175.5 (C4), 165.2 (C1), 165.1(C11), 163.6 (C13), 154.1 (C2), 147.6 (C3), 134.1 (C6), 113.6 (C5), 111.7 (C10), 93.2 (C12), 65.6 (C18), 63.2 (C16), 58.7 (C14), 57.5 (C7), 49.4 (C8), 45.4 (C9), 15.2 (C15), 15.0 (C17). IR (neat, cm-1): 1687 (m), 1607 (m), 1583 (w), 1357 (w). HR-ESI-MS: m/z calcd. for C44H5669GaN4O12: 901.3151, found: 901.3138 [M]+.  7.2.5  X-Ray Diffraction Structural Characterization of  [Ga(6EtO·2MeO)][ClO4] A colourless tablet crystal of C44H56N4O16GaCl.[1.28MeOH][1.18H2O] having approximate dimensions of 0.10 x 0.29 x 0.29 mm was mounted on a glass fiber. All measurements were made on a Bruker APEX DUO diffractometer with graphite  157  monochromated Mo-Kα radiation. The data were collected at a temperature of -183.0 + 0.1° C to a maximum 2θ value of 52.1 °. Data were collected in a series of φ and ω scans in 0.5 ° oscillations using 20.0-second exposures. The crystal-to-detector distance was 40.11 mm. Of the 57970 reflections that were collected, 9817 were unique (Rint = 0.043); equivalent reflections were merged. Data were collected and integrated using the Bruker SAINT78 software package. The linear absorption coefficient, μ, for Mo-Kα radiation is -1  6.82 cm . Data were corrected for absorption effects using the multi-scan technique (SADABS),79 with minimum and maximum transmission coefficients of 0.815 and 0.934, respectively. The data were corrected for Lorentz and polarization effects. The collection and analysis of the data was done by Dr. Brian O. Patrick.  7.2.6  Radiolabelling with 67/68Ga, Log Po/w Measurements and  Transferrin Stability General procedure for labelling with  67/68  Ga:  67  GaCl3 (100 μL, 1 mCi) in 0.01 M  HCl or 68Ga3+ in a 0.1 M HCl solution was added into 10-4 M solution of ligand in 10 mM or 200 mM NaOAc solution (pH 4.5, 950 μL) respectively and left to react for 10 minutes at room temperature. The reaction was monitored by analytical HPLC (gradient: A: NaOAc buffer, pH 4.5, B: CH3OH. 0-100% B linear gradient 20 min). Radioactivity for 68  Ga labelling decay corrected to elution time. Transferrin competition was done by addition of  67  GaCl3 into a 10-4 M solution of  ligand in 10 mM NaOAc solution (pH 4.5, 950 μL). Complex formation was confirmed by  HPLC. A 400 μL aliquot was added to 1 mg/mL apo-transferrin in a NaHCO3  solution (10 mM, 600 μL) and incubated at 37° C (water bath). Complex stability was checked at time points 10 minutes, 1 h and 2 h via analytical HPLC. Log Po/w measurements entail addition of a 20 μL aliquot of labelling solution to a mixture of 500 μL octanol and 480 μL aqueous PBS solution. The biphasic mixture was agitated for 30 seconds and subsequently centrifuged to separate the two layers. Aliquots (100 μL) of each phase were isolated and radioactivity was measured. Log Po/w measurements were executed in triplicate.  158  [67Ga(0MeO)]+: Rt of radiolabeled product on HPLC:  9.9 minutes. Stability versus  transferrin (10 min/1 h/ 2 h; % complex intact): 96/ 74/ 66.  68  Ga labelling: 10-6 M ligand,  97 %, 0.45 mCi/ nmol. Log Po/w: -1.25. [67Ga(2MeO)]+: Rt of radiolabeled product on HPLC: 10.8 minutes. Stability versus transferrin (10 min/1 h/ 2 h; % complex intact): 95/ 66/ 55.  68  Ga labelling: 10-6 M ligand,  96.5 %, 0.45 mCi/ nmol. Log Po/w: -0.83. [67Ga(4MeO)]+: Rt of radiolabeled product on HPLC: 11.6 minutes. Stability versus transferrin (10 min/1 h/ 2 h; % complex intact): 76/ 59/ 52.  68  Ga labelling: 10-6 M ligand,  95 %, 0.45 mCi/ nmol. Log Po/w: -0.32. [67Ga(6MeO)]+: Rt of radiolabelled product on HPLC: 11.8 minutes. Stability versus transferrin (10 min/1 h/ 2 h; % complex intact): 86/ 85/ 82. 68Ga labelling: 10-6 M ligand, 94 %, 0.45 mCi/ nmol. Log Po/w: -0.03. [67Ga(8MeO)]+: Rt of radiolabelled product on HPLC: 12.4 minutes. Stability versus transferrin (10 min/1 h/ 2 h; % complex intact): 97/ 96/ 96.  68  Ga labelling: 10-5 M ligand,  98.5 %, 0.45 mCi/ nmol. Log Po/w: 0.66. [67Ga(6EtO)]+: Rt of radiolabelled product on HPLC: 14.6 minutes. Stability versus transferrin (10 min/1 h/ 2 h; % complex intact): 95/ 92/ 91.  68  Ga labelling: 10-5 M ligand,  97.9 %. Log Po/w: 1.10. [67Ga(6EtO·2MeO)]+: Rt of radiolabeled product on HPLC:  15.4 minutes. Stability  versus transferrin (10 min/1 h/ 2 h; % complex intact): 94/ 93/ 93.  68  Ga labelling: 10-5 M  ligand, 98.3 %. Log Po/w: 1.16.  7.2.7  In Vivo Evaluation  The protocol used in the animal studies was approved by the Institutional Animal Care Committee of the University of British Columbia and was performed in accordance with the Canadian Council on Animal Care Guidelines. A total of 16 female ICR (20-30 g) mice were used for the animal study of each of the seven compounds.  67  Ga  monocationic complexes of 0MeO, 2MeO, 4MeO, 6MeO, 6EtO and 6EtO·2MeO were prepared as described above and then diluted in phosphate buffered saline to a concentration of 100 μCi/mL. Each mouse was i.v. injected with ~10 μCi (100 μL) of the  159  67  Ga complex and then sacrificed by CO2 inhalation at 2 min, 30 min, 1 h or 2 h after  injection (n = 4 at each time point). Blood was collected by cardiac puncture. Tissues collected included kidney, liver, spleen, femur, muscle, heart, lung, intestine and brain. Tissues were weighed and counted on a gamma counter and the counts were converted to % injected dose/gram (% ID/ g). The collection of the data was done by Dr. Dawn Waterhouse.  7.3  Results and Discussion  7.3.1  Ligand Synthesis  The seven ligand systems are synthesized using analogous synthetic pathways (Scheme 7.1), derived from methodology used for compounds reported in Chapter 4 and Chapter 6. For 0MeO, 6MeO and 6EtO, the corresponding benzaldehyde reacted with the ethylenediamine by reductive amination.  Scheme 7.1 General synthetic scheme for all compounds synthesized and reported in this chapter.  160  Then, 6-bromomethylpyridin-2-carboxylic acid methyl ester was used to alkylate the secondary amine to afford the desired scaffolds with methyl-protected carboxylates. 8MeO and 6EtO·2MeO were synthesized using the same methodology, using 9 as the alkylating agent. For the preparation of 2MeO and 4MeO, 3 was synthesized according to the literature.131 The secondary amines of 3 were alkylated with the corresponding bromo-methyl-phenyl derivatives to give rise to the corresponding methyl-protected carboxylate-bearing ligand scaffolds. Deprotection of all methyl esters was achieved using base-catalyzed saponification (Scheme 7.1). All chelates were characterized using 1  H and 13C NMR spectroscopic methods, mass spectrometry and IR spectroscopy.  7.3.2  Non-radioactive Complexes with Ga  The corresponding non-radioactive Ga complexes were formed by mixing Ga(NO3)3·6H2O with the ligand of interest. The pH was adjusted to 4.5 and the reaction mixture was stirred at 60 °C for 2 h in order to afford the desired complex quantitatively. 1  H NMR spectroscopy is used to determine complete chelation; a low field shift and  simultaneous splitting of the methylene protons can be observed. Two examples of 1H NMR spectra of ligand and corresponding Ga complex are shown (Figures 7.3 and 7.4). Geminal protons are easily identified by the characteristic, large geminal 2J coupling constants, which are found to be > 11 Hz for dedpa-type ligand systems. All complexes were characterized using 1H and  13  C NMR spectroscopic methods,  mass spectrometry and IR spectroscopy. One interesting observation from the spectroscopic data is the strong broadening of the methylene peaks, when the ligand is not complexed. This could be due to the fast rotation of the molecule in an attempt to decrease repulsion of hydrophilic and hydrophobic functional groups. This is supported by the observation that this effect increases with enhanced lipophilicity of the corresponding chelate.  161  Figure 7.3 1H NMR (300 MHz, CD3OD, 298 K) spectra of 6EtO and [Ga(6EtO)][NO3]. * denotes residual solvent peaks.  Figure 7.4 1H NMR spectra (300 MHz, CD3OD, 298 K) of 6EtO·2MeO and [Ga(6EtO·2MeO)][NO3]. * denotes residual solvent peaks.  162  7.3.3  X-Ray Diffraction Structural Characterization of  [Ga(6EtO·2MeO)][ClO4] To afford crystals suitable for solid state structure determination, Ga(ClO4)3·6H2O was used as starting material in place of the nitrate salt. The complex was dissolved in a H2O-CH3OH mixture and was left for slow solvent evaporation. After 5 days, crystals suitable for X-ray diffraction had formed. The solid state structure of complex [Ga(6EtO·2MeO)][ClO4] was determined by X-ray diffraction and is shown in Figure 7.5. When compared to the structural information for [Ga(dedpa)]+ (Chapter 4), a complex of similar structural composition with no overall increase in metal to ligand bond lengths was observed, despite the greatly increased steric bulk and additional functionality on the pyridyl moiety (Table 7.1).  Table 7.1 Selected bond lengths in [Ga(6EtO·2MeO)][ClO4] (bold), compared to selected bond lengths in [Ga(dedpa)][ClO4], ([Å]). bond  length [Å]  length [Å]  angle  degree [°]  N1-Ga  1.969(3)  1.992(5)  N(1)- Ga(1)- O(1)  80.25(12)  N2- Ga  2.163(3)  2.159(5)  O(1)- Ga(1)- O(3)  100.76(12)  N3- Ga  2.171(3)  2.188(5)  O(3)- Ga(1)- N(2)  92.12(12)  N4-Ga  1.970(3)  1.981(5)  N(1)- Ga(1)- N(3)  104.78(13)  O1- Ga  1.980(3)  1.967(4)  N(4)- Ga(1)- N(3)  78.98(13)  O2- Ga  1.998(3)  1.976(4)  O(1)- Ga(1)- N(3)  90.65(12)  163  Figure 7.5 ORTEP representation of [Ga(6EtO·2MeO)][ClO4], with corresponding molecular drawing. Shown is the atom numbering scheme (50% thermal ellipsoids). Hydrogen atoms are omitted for clarity.  7.3.4  Radiolabelling with 67/68Ga and In Vitro Stability  To investigate the viability of these new ligand systems for the purpose of labelling with  68  Ga, the ligands were subjected to extensive labelling studies with  67  Ga.  All chelates labelled in close to quantitative radiolabelling yields at concentrations as low as 10-6 M under the mild labelling conditions of only 10 minutes at room temperature. The labelling proceeds non-carrier added, which is advantagous compared to previously reported small cationic  67  Ga complexes.173,177 When the radiochemical complexes were  investigated for their stability to transferrin challenge, an unexpected trend was observed. Stability decreased from [67Ga(0MeO)]+ to [67Ga(2MeO)]+ to [67Ga(4MeO)]+, which was the complex with the lowest in vitro stability. [67Ga(6MeO)]+ however, showed increased stability, but was still inferior to the high stability observed for the more sterically crowded complexes [67Ga(6EtO)]+ and [67Ga(6EtO·2MeO)]+. This trend could  164  be explained by the increased lipophilicity of complexes [67Ga(6MeO)]+, [67Ga(6EtO)]+ and [67Ga(6EtO·2MeO)]+ compared to the other four. The binding site for Fe(III) and Ga(III) on transferrin lies in a shallow, polar pocket, which would experience more repulsion from an apolar, ball-like complex than from a more polar one.  7.3.5  Log Po/w Measurements  The increase in polarity was demonstrated by log Po/w measurements. While the octanol water partition coefficients for compounds [67Ga(0MeO)]+, [67Ga(2MeO)]+ [67Ga(4MeO)]+ and [67Ga(6MeO)]+ remain in the more polar, negative region, [67Ga(8MeO)]+, [67Ga(6EtO)]+ and [67Ga(6EtO·2MeO)]+ exhibit positive log Po/w values from 0.66 to 1.16 (Table 7.2), corresponding to the retention times measured by HPLC. According to a comprehensive review,171 one desirable property for  99m  Tc complexes to  have optimal myocardial retention and low liver uptake is a log Po/w value within the range of 0.5 to 1.2. Since the latter 3 compounds fall within these margins, they were considered good candidates for the development of a potential cardiac imaging agent. Table 7.2 Log Po/w values and corresponding HPLC retention times of the seven new compounds. Complex  Rt [10 min]  Log Po/w  Stability versus Tf [10 min/1 h/ 2 h; % complex intact]  [67Ga(0MeO)]+  9.9  -1.25  96/ 74/ 66  [67Ga(2MeO)]+  10.8  -0.83  95/ 66/ 55  [67Ga(4MeO)]+  11.6  -0.32  76/ 59/ 52  [67Ga(6MeO)]+  11.8  -0.03  86/ 85/ 82  [67Ga(8MeO)]+  12.4  0.66  97/ 96/ 96  [67Ga(6EtO)]+  14.6  1.10  95/ 92/ 91  [67Ga(6EtO·2MeO)]+  15.4  1.16  94/ 93/ 93  165  7.3.6  Biodistribution  To evaluate the biodistribution profile of the synthesized H2dedpa derivatives, each of the radiochemical complexes was prepared and subsequently diluted in phosphate buffered saline to a concentration of 100 μCi/mL. For each compound, 16 mice were injected intravenously and 4 animals were sacrificed at each time point after 2 minutes, 30 minutes, 1 h and 2 h. Activity is represented in % ID/g (Figure 7.6). The most hydrophilic complexes [67Ga(0MeO)]+, [67Ga(2MeO)]+ and [67Ga(4MeO)]+ have biodistribution profiles similar to those previously investigated in benzyl-nitro derivatives (Chapter 4). Predominantly renal excretion was observed, with bone uptake between 3 – 4 % ID/g (for complete tables see appendix B). This confirms the slightly decreased stability observed in the in vitro transferrin challenge for these 3 complexes. In the case of [67Ga(6MeO)]+ a distinct change in biodistribution behaviour was observed. Clearance was observed via both renal and hepatobilary excretion. [67Ga(6MeO)]+ seems to clear rapidly from all organs except the liver within 2 h; this had not yet been observed for any H2dedpa derivative. The rapid blood clearance was an important improvement. The very low bone uptake indicates high in vivo stability of this complex; however, due to the low lipophilicity, the compound showed no persistent retention in the myocardium. [67Ga(8MeO)]+ showed very similar behaviour. The fast clearance profile was maintained, still, no retention in the myocardium was observed. An increased uptake in the liver and the intestines was seen. [67Ga(6EtO)]+ was the first compound of the series to show a somewhat persistent heart uptake, above 1 % ID/g; however, blood and lung clearance were inferior to those of [67Ga(8MeO)]+. Due to increased lipophilicity, the uptake in the liver increased.  166  Figure 7.6 Biodistribution of [67Ga(0MeO)]+, [67Ga(2MeO)]+, [67Ga(4MeO)]+, [67Ga(8MeO)]+, [67Ga(6EtO)]+and [67Ga(6EtO·2MeO)]+ in female ICR mice over 2h.  [67Ga(6MeO)]+,  167  As the most lipophilic of the complexes investigated, [67Ga(6EtO·2MeO)]+ showed the best results in terms of biodistribution. The complex cleared well from both blood and lung after 30 minutes. Bone uptake was low, confirming the high stability observed in the transferrin challenge. After the initial, high uptake (above 9 % ID/g) the heart uptake dropped at the 30 minute time point, but it remained persistently above 1.5 % ID/g, resulting in heart/blood and heart/lung values greater then 3 (Table 7.3).  Table 7.3 Heart/blood, heart/lung and heart/liver ratios for compounds [67Ga(8MeO)]+, [67Ga(6EtO)]+, [67Ga(6EtO·2MeO)]+ Complex  2 min ± SD  30 min ± SD  1 h ± SD  2 h ± SD  heart/blood  0.50 ± 0.07  0.73 ± 0.12  0.64 ± 0.07  0.53 ± 0.05  heart/liver  0.17 ± 0.03  0.03 ± 0.02  0.02 ± 0.00  0.04 ± 0.01  heart/lung  0.38 ± 0.09  0.41 ± 0.08  0.50 ± 0.32  0.45 ± 0.30  heart/blood  0.29 ± 0.03  0.55 ± 0.13  0.65 ± 0.17  0.91 ± 0.70  heart/liver  0.16 ± 0.02  0.11 ± 0.13  0.07 ± 0.01  0.13 ± 0.08  heart/lung  0.51 ± 0.17  0.89 ± 0.15  0.84 ± 0.44  1.33 ± 0.59  heart/blood  0.33 ± 0.15  1.62 ± 0.43  3.37 ± 0.61  3.80 ± 0.81  heart/liver  0.31 ± 0.06  0.06 ± 0.00  0.07 ± 0.02  0.14 ± 0.60  heart/lung  0.66 ± 0.10  4.59 ± 5.19  2.29 ± 0.51  3.28 ± 0.60  [67Ga(8MeO)]+  [67Ga(6EtO)]+  [67Ga(6EtO·2MeO)]+  The significant liver uptake (above 13 % ID/g) even after 2 hours renders this compound a poor candidate for cardiac imaging with PET. The cause for the high liver uptake may not lie solely with the increased lipophilicity. When compared with the compounds based on the bis(salycilaldimine) scaffold,173 it becomes clear that an  168  increased number of benzylic groups could specifically target the liver, because this functional group is preferentially metabolized in liver microsomes to afford more watersoluble species, which are then subsequently excreted.178  7.4  Conclusion The synthesis, labelling and biodistribution of seven new lipophilic derivatives of  H2dedpa for the purpose of cardiac perfusion imaging were described. The solid state structure obtained for [Ga(6EtO·2MeO)][ClO4] showed that derivatization at the 4 position on the pyridyl ring is feasible and provides additional possibilities for functionalization without impacting metal coordination. While high initial uptake and slow clearance from liver remain issues, four of these novel complexes have shown improved blood, lung and kidney clearance compared to derivatives reported previously. The two complexes [67Ga(6EtO)]+ and [67Ga(6EtO·2MeO)]+ with log Po/w values > 1.1 have shown persistent heart uptake over the course of 2 h above 1 % ID/g, one of which shows a larger heart uptake value than the currently commercially used 82Rb+.  169  Chapter 8: Conclusions and Future Work 8.1  Further Enhancing the H2dedpa Technology Significant intellectual property is associated with the work in this thesis and one full  patent application has been filed; therefore, suggestions for future work are intentionally vague in order to not jeopardize this coverage and future filings. As described within the experimental portion of this thesis, a variety of acyclic chelates were investigated for their capability to chelate the (radio-)metals 63/64/65  Cu and  99m  Tc,  185/187  Re,  67/68/69/71  Ga. The most efficient, multifunctional chelate identified was  H2dedpa, which was subsequently functionalized to furnish a bioconjugate with the cyclic peptide RGD (Chapter 6) or to form small lipophilic cations upon coordination of Ga (Chapter 7). The techniques described within this work include a wide array of methods such as organic and inorganic synthesis, physico-chemical methods, radiochemistry and biological studies; typical for multidisciplinary fields like medicinal inorganic chemistry. In addition to the first applications of the scaffold described within this thesis, there is a large field of opportunities arising from this discovery. In vivo evaluations of the RGD conjugates are underway and will furnish information on how well bifunctionalized H2dedpa derivatives localize in tumours and clear from non-target tissues. Some suggestions for further elaborations, as well as new ideas and perspectives are given in this chapter.  8.1.1  Extension of the Denticity for Other Metals of Interest  While the investigated metals Ga(III) and Cu(II) have a preference for hexadentate chelation scaffolds, a wide variety of metals of relevance in the field of imaging and therapy requires greater denticities and bite angles. The chelate library described in Chapter 4 includes two octadentate chelate systems and two hexadentate chelates with larger bite angle due to their propyl backbones. Investigation of these systems for coordination with metals In, Y, Lu, Gd is of great interest. A second screening involving radionuclides of In, Y and Lu could be used to find lead chelates for these metal ions. Among selection criteria, more emphasis could be put on in vitro stability and less on fast  170  labelling conditions because of the longer half-lives of these radiometals. Once a lead compound is established, the corresponding non-radioactive complex must be formed and investigated for its structure and thermodynamic stability. Functionalization through the backbone is already explored and could also be investigated with octadentate ligand systems. The corresponding model derivatives are shown in Figure 8.1.  Figure 8.1 Proposed octadentate model compounds based on the H2dedpa scaffold.  8.1.2  H2dedpa Peptide Derivatives of Relevance for Clinical  Applications After successful conjugation to RGD, and subsequent in vivo imaging, it will become apparent if the clearance profile of the dedpa derivatives is satisfactory enough to consider other targeting moieties for H2dedpa-linked targeting entities. After successful derivatization of the pyridine moiety (Chapter 7) this could be a point of attachment for prosthetic groups, which can facilitate clearance or enhance circulation. In terms of application to other targeting moieties, the synthesized isothiocyanato derivatives can be easily conjugated to antibodies or other targeting moieties carrying a lysine that is not essential for the biological recognition of the target molecule. The minimum concentration necessary for quantitative labelling, especially with  67/68  Ga, is so  low that even kit formulations using any type of targeting moiety could be prepared easily. This could alleviate the current need for complicated automation and purification systems such as the ones currently used for the production of 68Ga-DOTATOC.166  171  8.2  Small Molecule Imaging Agents 8.2.1  New Lipophilic Ligand Systems for Imaging of Myocardial  Bloodflow A first compound series for imaging of myocardiac bloodflow was synthesized and described in Chapter 7. It was found that aryl functionalization increased the liver uptake greatly with increased lipophilicity of the ligand system. In order to decrease uptake in the liver, but maintain the high persistent uptake in the myocardium, other ways of introducing lipophilicity must be investigated. From investigations by others and those described in this thesis, it can be concluded that the optimal log Po/w window for Ga-based complexes might only partially overlap with the optimal window given for  99m  Tc-based complexes for the purpose of  heart imaging (Po/w = 0.5 – 1.2). Ga-complexes with log Po/w < 1 were all found to clear the myocardium to a significant extent before 2 h. The optimal log Po/w window for persistent heart upake of Ga-based complexes was found to be 1- 2. This, together with a reduced number of aryl functionalities must be taken into consideration when a new series of lipophilic H2dedpa derivatives for this purpose is designed and synthesized.  8.2.2  Alternative H2dedpa Derivatives as Small Molecule Imaging  Agents Besides modulating the physical properties of H2dedpa complexes in order to achieve accumulation in the myocardium, there are other functionalities, which can be attached to a chelate to subsequently direct localization and excretion. One of the currently most pursued and investigated small prosthetic groups is the nitroimidazole fragment, which is known to accumulate in hypoxic cells.179 Imaging hypoxic tissues is difficult and desperately sought. It was found that among nitroimidazole derivatives 2nitroimidazole can be reduced most efficiently to form a reactive chemical species, which can bind to cell components in the absence of sufficient oxygen. The PET tracer  18  F-  172  fluoromisonidazole (FMISO) is in clinical use for the imaging of hypoxia180 however, its biodistribution profile is not optimal and the development of non-cyclotron but generatorbased radiopharmaceuticals is a persistent, strong driving force.181 Some NOTA nitroimidazole conjugates and their uptake for imaging hypoxic tissues have been reported. Low tumor and high kidney and liver uptake rendered these conjugates poor candidates for further development.182 Since the more polar H2dedpa derivatives exhibit low uptake in those organs, a nitroimidazole derivative of this chelate could be an interesting target compound. Below, examples of a previously reported conjugate and a hypothetical H2dedpa nitroimidazole derivative are shown (Figure 8.2).  Figure 8.2 Nitroimidazole derivatives of NOTA (left) and H2dedpa (right).  Finally, it can be concluded that while acyclic chelates are sometimes disregarded due to their perceived lack of thermodynamic stability for the purpose of radiometal chelation, fast coordination and kinetic inertness can be more valued qualities. In some cases, an acyclic ligand system can exhibit high thermodynamic stability along with the expected rapid kinetics, and can give rise to a chelate of surprisingly superior qualities compared to the often favoured macrocyclic systems.  173  References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30)  Lippard, S. J. J. Am. Chem. Soc. 2010, 132, 14689-14693. Ehrlich, P. Deutsch. Med. Woch. 1910, 1893-1896. Maxwell, G.; Hollanger, S. J. Med. 1993, 24, 4-5. Sutton, B. M.; Hempel, J. C.; Hill, D. T. Curr. Clin. Pract. 1983, 7, 6-16. Holman, B. L.; Jones, A. 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Chem. 2010, 53, 6378–6385.  181  Appendices Appendix A Crystallographic Data [Re(Me2dipin-NO2)(CO)3] [Re(Br3(CO)3]  Complex  [Re(L1)(CO)3]  [Cu(dipin-NO2)(OH2)]  Formula  C21 H27 N2 O7 Re  C21 H18 Cu N4 O7  Fw  605.65  501.93  C27.50 H22 Br1.50 N4 O10.50 Re1.50 975.66  Description  colorless plate  green plate  colorless blade  colorless prism  Space group  P-1  P-1  P 21/c  P–1  a [Å]  6.8109(11)  7.143(2)  12.3723(10)  7.8880(12)  b [Å]  10.4134(19)  8.335(2)  17.5236(14)  8.9681(12)  c [Å]  17.057(4)  18.901(5)  14.8402(12)  13.264(2)  α [°]  102.114(8)  77.781(4)  90.00  87.389(7)  β [°]  94.058(13)  79.755(5)  112.128(2)  77.692(8)  γ [°]  102.511(9)  65.266(4)  90.00  85.503(7)  1146.1(4)  993.9(5)  2980.5(4)  913.5(2)  2  2  4  2  ρcalc [g/cm ]  1.755  1.677  2.174  1.809  T [K]  173  90  90  103  μ [mm ]  5.344  1.155  8.173  1.710  F 000  596  514  1856  504  reflns collcd/ unique  9144/ 3421  6190/ 2560  50217/ 8033  15309/ 4391  (Rint = 0.053)  (Rint = 0.072)  (Rint = 0.048)  (Rint = 0.020)  residuals (F , all data)  wR2 = 0.142  wR2 = 0.254  wR2 = 0.074  wR2 = 0.087  residuals (F, I > 2σ(I))  R1 = 0.063  R1 = 0.130  R1 = 0.035  R1 = 0.026  goodness of fit indicator  1.013  1.162  1.017  1.300  3  V [Å ] Z 3  -1  2  [Ga(dedpa)][ClO4] C16 H16 Cl Ga N4 O8 497.50  182  Complex  [Ga(dp-N-NO2)][ClO4]  [Cu(dedpa)]  [Ga(6EtO2MeO)][ClO4]  Formula  C31 H30 Cl Ga N6 O13  C16 H22 Cu N4 O7  C45.28 H63.14 Cl Ga N4 O18.47  Fw  799.78  445.92  1064.25  Description  colorless plate  blue prism  colorless tablet  Space group  P 21/c  P nn2  P 21/c  a [Å]  20.317(2)  11.352(2)  15.8043(13)  b [Å]  14.0298(17)  15.584(3)  15.1916(13)  c [Å]  24.060(3)  10.6461  20.8101(17)  α [°]  90.00  90.00  90.00  β [°]  98.966(2)  90.00  92.183(2)  γ [°]  90.00  90.00  90.00  V [Å ]  6774.4(14)  1883.4(6)  4992.7(7)  Z  8  4  4  ρcalc [g/cm3]  1.568  1.573  1.416  T [K]  90  90.0  90  μ [mm ]  0.968  1.207  0.682  F 000  3280  924  2234  Reflns collcd/ unique  29750/ 9225  40349/ 5484  57970/ 9817  (Rint = 0.085)  (Rint = 0.033)  (Rint = 0.043)  Residuals (F , all data)  wR2 = 0.167  wR2 = 0.053  wR2 = 0.192  Residuals (F, I > 2σ(I))  R1 = 0.107  R1 = 0.023  R1 = 0.089  Goodness of fit indicator  0.958  1.068  1.067  3  -1  2  183  Appendix B Biodistribution Data Biodistribution of Compounds [67Ga(dedpa)]+, [67Ga(dp-N-NO2)]+ , [67Ga(dp-bb-  B.1 NO2)]  Biodistribution data of [67Ga(dedpa)]+ in female ICR mice (% ID/g , n = 4). Organ/Tissue  30 min ± SD  1 h ± SD  2 h ± SD  4 h ± SD  Brain  0.26 ± 0.13  0.24 ± 0.12  0.21 ± 0.10  0.13 ± 0.06  Femur  2.18 ± 1.09  2.13 ± 1.06  1.89 ± 0.94  2.21 ± 1.10  Muscle  1.21 ± 0.60  1.15 ± 0.57  1.09 ± 0.54  1.12 ± 0.56  Spleen  1.36 ± 0.68  1.35 ± 0.67  1.27 ± 0.63  0.98 ± 0.49  Kidney  8.08 ± 4.00  7.82 ± 3.91  9.14 ± 4.57  8.30 ± 4.15  Liver  3.70 ± 1.85  3.69 ± 1.84  3.17 ± 1.58  2.73 ± 1.36  Intestine  2.09 ± 1.05  2.12 ± 1.06  1.99 ± 0.99  1.99 ± 0.99  Heart  2.64 ± 1.32  2.49 ± 1.24  2.41 ± 1.20  2.00 ± 1.00  Lung  4.87 ± 2.43  4.89 ± 2.44  4.84 ± 2.42  3.26 ± 1.63  Serum  16.06 ± 8.03  9.04 ± 4.52  12.04 ± 6.02  12.23 ± 6.11  184  Biodistribution data of [67Ga(dp-N-NO2)]+ in female ICR mice (% ID/g , n = 4). Organ/Tissue  30 min ± SD  1 h ± SD  2 h ± SD  4 h ± SD  Brain  0.16 ± 0.02  0.15 ± 0.01  0.16 ± 0.01  0.12 ± 0.01  Femur  1.54 ± 0.20  1.66 ± 0.09  1.97 ± 0.06  2.71 ± 0.88  Muscle  0.80 ± 0.07  0.60 ± 0.02  0.61 ± 0.02  0.57 ± 0.05  Spleen  2.32 ± 0.15  3.25 ± 0.27  3.20 ± 0.28  4.23 ± 0.79  Kidney  5.29 ± 0.14  4.19 ± 0.04  3.59 ± 0.40  4.45 ± 0.36  Liver  3.35 ± 0.57  3.42 ± 0.60  4.43 ± 0.42  4.51 ± 0.67  Intestine  1.70 ± 0.05  1.45 ± 0.12  1.89 ± 0.14  1.54 ± 0.12  Heart  1.80 ± 0.35  2.06 ± 0.07  1.88 ± 0.19  1.65 ± 0.22  Lung  11.18 ± 0.79  10.65 ± 0.56  8.52 ± 0.33  5.65 ± 0.54  Blood  5.77 ± 1.21  6.54 ± 0.05  5.90 ± 0.42  4.52 ± 0.46  Urine  411.14 ± 45.93  166.83 ± 48.91  47.17 ± 14.64  16.98 ± 3.51  Biodistribution data of [67Ga(dp-bb-NO2)]+ in female ICR mice (% ID/g , n = 4). Organ/Tissue  30 min ± SD  1 h ± SD  2 h ± SD  4 h ± SD  Brain  0.16 ± 0.02  0.18 ± 0.02  0.19 ± 0.01  0.13 ± 0.01  Femur  1.56 ± 0.12  2.29 ± 0.28  2.61 ± 0.21  3.44 ± 0.08  Muscle  0.91 ± 0.05  1.01 ± 0.24  0.72 ± 0.07  0.70 ± 0.05  Spleen  1.22 ± 0.12  1.13 ± 0.10  1.45 ± 0.92  1.17 ± 0.08  Kidney  12.98 ± 0.80  11.04 ± 0.92  9.40 ± 0.13  10.10 ± 0.23  Liver  4.40 ± 0.25  3.89 ± 0.50  3.24 ± 0.59  3.63 ± 0.15  Intestine  2.04 ± 0.08  2.07 ± 0.27  1.78 ± 0.26  1.87 ± 0.12  Heart  2.22 ± 0.20  2.19 ± 0.23  1.91 ± 0.16  1.74 ± 0.15  Lung  4.30 ± 0.45  3.79 ± 0.29  3.76 ± 0.43  3.09 ± 0.23  Blood  7.40 ± 0.65  7.36 ± 0.42  7.43 ± 0.61  5.40 ± 0.41  Urine  94.71 ± 22.67  55.80 ± 25.63  33.64 ± 18.18  13.60 ± 3.06  185  B.2 Biodistribution of Compounds [67Ga(0MeO)]+, [67Ga(2MeO)]+ , [67Ga(4MeO)]+ , 67 [ Ga(6MeO)]+ , [67Ga(8MeO)]+ , [67Ga(6EtO)]+ , [67Ga(6EtO·2MeO)]+. Biodistribution data of [67Ga(0MeO)]+, (% ID/g , n = 4). Organ  2 min ± SD  30 min ± SD  1h ± SD  2h ± SD  Brain  0.22 ± 0.13  0.16 ± 0.03  0.10 ± 0.04  0.13 ± 0.02  Muscle  1.25 ± 0.67  0.93 ± 0.14  0.81 ± 0.04  0.60 ± 0.24  Femur  2.24 ± 0.26  2.33 ± 0.50  2.55 ± 0.28  3.65 ± 0.57  Spleen  2.21 ± 0.53  1.08 ± 0.34  0.67 ± 0.12  1.02 ± 0.45  Kidney  19.40 ± 7.38  9.86 ± 1.11  7.87 ± 0.36  5.78 ± 2.34  Liver  5.31 ± 2.46  3.41 ± 0.27  2.01 ± 1.14  2.26 ± 1.56  Heart  4.39 ± 0.65  1.88 ± 0.42  1.14 ± 0.33  1.69 ± 0.12  Lung  5.90 ± 2.95  2.95 ± 0.48  1.87 ± 0.38  2.60 ± 0.33  Intestine  2.32 ± 0.49  2.48 ± 0.51  1.77 ± 0.22  1.63 ± 0.81  Blood  14.16 ± 5.22  6.70 ± 1.89  4.05 ± 1.36  6.33 ± 0.96  Heart/blood  0.34 ± 0.12  0.28 ± 0.02  0.28 ± 0.03  0.27 ± 0.04  Heart/liver  1.16 ± 1.01  0.55 ± 0.12  0.76 ± 0.56  1.86 ± 2.48  Heart/lung  1.06 ± 0.94  0.63 ± 0.04  0.60 ± 0.06  0.65 ± 0.09  186  Biodistribution data of [67Ga(2MeO)]+, (% ID/g , n = 4). Organ  2 min ± SD  30 min ± SD  1h ± SD  2h ± SD  Brain  0.24 ± 0.05  0.11 ± 0.01  0.11 ± 0.04  0.09 ± 0.06  Muscle  1.18 ± 0.21  1.00 ± 0.11  0.58 ± 0.05  0.56 ± 0.06  Femur  1.93 ± 0.51  2.47 ± 0.17  2.52 ± 0.15  3.77 ± 0.79  Spleen  1.61 ± 0.27  0.71 ± 0.20  0.75 ± 0.30  0.64 ± 0.40  Kidney  31.68 ± 17.82  10.90 ± 2.68  7.71 ± 1.27  5.34 ± 0.82  Liver  4.00 ± 1.03  3.24 ± 0.80  1.92 ± 1.25  2.37 ± 0.66  Heart  3.46 ± 0.71  1.20 ± 0.14  1.33 ± 0.40  0.97 ± 0.57  Lung  6.27 ± 1.39  2.13 ± 0.27  2.65 ± 1.01  1.55 ± 0.70  Intestine  1.79 ± 0.10  2.01 ± 0.25  1.72 ± 0.27  1.52 ± 0.13  Blood  12.11 ± 2.79  4.05 ± 1.34  4.85 ± 2.28  3.07 ± 2.36  Heart/blood  0.28 ± 0.01  0.31 ± 0.07  0.32 ± 0.12  0.34 ± 0.05  Heart/liver  0.88 ± 0.13  0.38 ± 0.05  1.28 ± 1.51  0.39 ± 0.14  Heart/lung  0.55 ± 0.05  0.56 ± 0.04  0.51 ± 0.09  0.60 ± 0.08  187  Biodistribution data of [67Ga(4MeO)]+, (% ID/g , n = 4). Organ  2 min ± SD  30 min ± SD  1h ± SD  2h ± SD  Brain  0.26 ± 0.14  0.14 ± 0.05  0.11 ± 0.05  0.11 ± 0.04  Muscle  1.55 ± 0.06  1.04 ± 0.31  0.83 ± 0.49  0.80 ± 0.26  Femur  2.51 ± 0.40  2.26 ± 0.56  3.21 ± 0.52  3.27 ± 2.11  Spleen  1.60 ± 0.29  0.73 ± 0.19  0.52 ± 0.41  0.81 ± 0.29  Kidney  23.71 ± 10.31  7.19 ± 1.43  4.17 ± 2.55  5.30 ± 0.80  Liver  4.15 ± 0.83  3.07 ± 1.34  2.20 ± 1.19  3.02 ± 0.43  Heart  3.16 ± 0.28  1.05 ± 0.63  1.34 ± 0.62  1.31 ± 0.47  Lung  6.87 ± 0.63  2.94 ± 0.70  2.01 ± 1.47  2.35 ± 0.68  Intestine  2.21 ± 0.25  2.01 ± 0.49  1.63 ± 0.15  1.48 ± 0.40  Blood  9.30 ± 1.34  4.44 ± 1.78  4.21 ± 2.63  3.97 ± 1.54  Heart/blood  0.34 ± 0.05  0.27 ± 0.17  0.35 ± 0.09  0.33 ± 0.05  Heart/liver  0.78 ± 0.14  0.49 ± 0.49  1.15 ± 1.52  0.43 ± 0.14  Heart/lung  0.46 ± 0.08  0.38 ± 0.23  1.29 ± 1.64  0.55 ± 0.09  188  Biodistribution data of [67Ga(6MeO)]+, (% ID/g , n = 4). Organ  2 min ± SD  30 min ± SD  1h ± SD  2h ± SD  Brain  0.20 ± 0.04  0.04 ± 0.01  0.02 ± 0.01  0.02 ± 0.01  Muscle  1.33 ± 0.11  0.23 ± 0.05  0.17 ± 0.04  0.16 ± 0.04  Femur  2.40 ± 0.43  0.52 ± 0.27  0.46 ± 0.11  0.75 ± 0.14  Spleen  2.42 ± 0.31  0.53 ± 0.11  0.27 ± 0.02  0.16 ± 0.04  Kidney  35.85 ± 8.19  3.52 ± 0.54  1.32 ± 0.15  0.81 ± 0.09  Liver  17.64 ± 2.84  7.07 ± 0.82  4.86 ± 1.14  4.32 ± 0.80  Heart  3.21 ± 0.52  0.59 ± 0.06  0.37 ± 0.05  0.29 ± 0.04  Lung  8.26 ± 0.53  1.17 ± 0.14  0.65 ± 0.07  0.41 ± 0.01  Intestine  2.44 ± 0.32  3.46 ± 2.80  1.78 ± 1.28  1.48 ± 0.44  Blood  7.16 ± 1.28  1.21 ± 0.32  0.80 ± 0.20  0.74 ± 0.11  Heart/blood  0.45 ± 0.05  0.50 ± 0.09  0.48 ± 0.10  0.40 ± 0.10  Heart/liver  0.18 ± 0.02  0.08 ± 0.01  0.08 ± 0.03  0.07 ± 0.02  Heart/lung  0.38 ± 0.06  0.50 ± 0.04  0.57 ± 0.09  0.71 ± 0.12  189  Biodistribution data of [67Ga(8MeO)]+, (% ID/g , n = 4). Organ  2 min ± SD  30 min ± SD  1h ± SD  2h ± SD  Brain  0.19 ± 0.06  0.04 ± 0.01  0.02 ± 0.01  0.02 ± 0.02  Muscle  1.34 ± 0.23  0.21 ± 0.07  0.12 ± 0.06  0.07 ± 0.02  Femur  2.15 ± 0.67  0.52 ± 0.09  0.29 ± 0.12  0.28 ± 0.04  Spleen  2.62 ± 0.86  0.49 ± 0.18  0.23 ± 0.03  0.10 ± 0.03  Kidney  33.12 ± 9.34  2.63 ± 0.66  0.92 ± 0.19  0.44 ± 0.05  Liver  21.91 ± 6.96  23.45 ± 12.82  15.09 ± 3.57  5.04 ± 2.24  Heart  3.82 ± 1.55  0.62 ± 0.14  0.33 ± 0.02  0.18 ± 0.04  Lung  9.85 ± 1.91  1.59 ± 0.65  0.43 ± 0.17  0.30 ± 0.10  Intestine  2.65 ± 0.51  2.58 ± 1.21  2.88 ± 0.54  1.83 ± 1.36  Blood  7.54 ± 2.47  0.84 ± 0.15  0.52 ± 0.06  0.35 ± 0.07  Heart/blood  0.50 ± 0.07  0.73 ± 0.12  0.63 ± 0.07  0.52 ± 0.05  Heart/liver  0.17 ± 0.03  0.03 ± 0.02  0.02 ± 0.00  0.04 ± 0.01  Heart/lung  0.37 ± 0.08  0.41 ± 0.08  0.50 ± 0.32  0.45 ± 0.30  190  Biodistribution data of [67Ga(6EtO)]+, (% ID/g , n = 4). Organ  2 min ± SD  30 min ± SD  1h ± SD  2h ± SD  Brain  0.38 ± 0.07  0.07 ± 0.02  0.06 ± 0.01  0.06 ± 0.03  Muscle  0.99 ± 0.24  0.49 ± 0.06  0.37 ± 0.06  0.43 ± 0.21  Femur  1.66 ± 0.42  0.90 ± 0.12  0.66 ± 0.14  0.65 ± 0.36  Spleen  2.43 ± 0.59  0.55 ± 0.04  0.42 ± 0.10  0.35 ± 0.03  Kidney  14.94 ± 4.75  3.26 ± 0.74  2.00 ± 0.47  2.30 ± 1.19  Liver  29.54 ± 8.83  23.36 ± 14.98  15.78 ± 4.88  10.52 ± 3.72  Heart  4.60 ± 1.10  1.19 ± 0.12  1.06 ± 0.30  1.17 ± 0.46  Lung  9.60 ± 2.40  1.36 ± 0.25  1.46 ± 0.74  0.89 ± 0.06  Intestine  2.24 ± 0.72  3.04 ± 1.80  2.36 ± 0.58  2.03 ± 0.58  Blood  15.71 ± 3.51  2.28 ± 0.55  1.64 ± 0.15  1.50 ± 0.40  Heart/blood  0.29 ± 0.03  0.55 ± 0.13  0.65 ± 0.17  0.91 ± 0.70  Heart/liver  0.16 ± 0.02  0.11 ± 0.13  0.07 ± 0.01  0.13 ± 0.08  Heart/lung  0.51 ± 0.16  0.89 ± 0.15  0.84 ± 0.44  1.33 ± 0.59  191  Biodistribution data of [67Ga(6EtO·2MeO)]+, (% ID/g , n = 4). Organ  2 min ± SD  30 min ± SD  1h ± SD  2h ± SD  Brain  0.66 ± 0.17  0.05 ± 0.01  0.04 ± 0.01  0.03 ± 0.01  Muscle  1.34 ± 0.39  0.66 ± 0.18  0.61 ± 0.21  0.56 ± 0.16  Femur  2.25 ± 0.66  0.77 ± 0.16  0.65 ± 0.17  0.59 ± 0.09  Spleen  4.56 ± 1.23  0.56 ±0.15  0.43 ± 0.18  0.24 ± 0.07  Kidney  13.66 ± 3.60  5.31 ± 1.32  4.40 ± 3.93  1.82 ± 0.50  Liver  30.16 ± 3.95  32.28 ± 7.98  28.86 ± 9.24  13.94 ± 6.10  Heart  9.31 ± 2.87  1.94 ± 0.42  1.91 ± 0.41  1.63 ± 0.36  Lung  14.34 ± 4.83  0.74* ± 0.45  0.85 ± 0.18  0.50 ± 0.06  Intestine  3.71 ± 0.51  5.35 ± 2.15  6.83 ± 3.48  4.39 ± 1.89  Blood  32.50 ± 14.98  1.29 ± 0.53  0.57 ± 0.11  0.44 ± 0.13  Heart/blood  0.33 ± 0.15  1.62 ± 0.43  3.37 ± 0.61  3.80 ± 0.81  Heart/liver  0.31 ± 0.06  0.06 ± 0.00  0.07 ± 0.02  0.14 ± 0.60  Heart/lung  0.66 ± 0.10  4.59 ± 5.19  2.29 ± 0.51  3.28 ± 0.60  *Animal 5 of 16 showed abnormal lung uptake not in accordance with the other 3 sacrificed animals at this time point. Q-test confirmed this data to be an outlier with 99% confidence.  192  

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