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Designed ligands for diagnosis and therapy Storr, Timothy J. 2005

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DESIGNED LIGANDS FOR DIAGNOSIS A N D T H E R A P Y  by  Timothy J. Storr B.Sc, University of Victoria, 1999  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH C O L U M B I A April 2005 © Timothy J. Storr  ABSTRACT This thesis discusses three distinct medicinal inorganic chemistry projects involving the design of well-tailored ligands for diagnostic and therapeutic applications. In one study, combination agents for diabetes therapy were developed utilizing vanadium and the thiazolidinediones. A hybrid approach was used whereby the two aforementioned active agents were combined into one molecule. A series of bifunctional thiazolidinedione-containing pro-ligands were synthesized and complexed to vanadium. One vanadium complex, and associated ligand precursor, were found to be quite effective in lowering plasma glucose levels in an acute animal model of diabetes. In another study numerous tetrahydrosalen compounds were synthesized and evaluated for potential use in Alzheimer's disease chelation therapy. Glycosylated pro-drug forms were developed to minimize systemic metal chelation and potentially enhance brain uptake. Solution studies of two tetrahydrosalen ligands exhibiting remote glycosylation indicated that these compounds form neutral complexes with C u  2+  and Z n  2+  at physiological pH (7.4). The  final study investigated the chemistry of carbohydrate-appended metal (Re and  99m  Tc)  complexes for use as target-specific radiopharmaceuticals. A series of bidentate and tridentate carbohydrate-containing ligands were attached to the/ac-{M(CO)3} (M = Re +  or  99m  T c ) core and in all cases the carbohydrate moiety was determined to remain  pendant. The bidentate analogs were found to be more susceptible towards ligand exchange than were the tridentate compounds. Clearly matching the tridentate binding capability of carbohydrate-appended ligands to the fac-{M(CO)3}  +  the resulting complexes to ligand substitution processes in vitro.  n  core greatly stabilized  T A B L E OF CONTENTS  Abstract  ii  Table of Contents  iii  List of Figures  vii  List of Schemes  x  List of Tables  xii  List of Equations  xiv  List of Abbreviations  xv  Acknowledgements  Chapter 1  Chapter 2  xxiii  Introduction 1.1  Medicinal Inorganic Chemistry  1  1.2  Vanadium and Diabetes  2  1.3  Carbohydrate-containing Molecules for Increased Drug Selectivity  8  1.4  Neurodegenerative Disease  11  1.5  Metal-sequestration as an A D Therapeutic Strategy  20  1.6  Target-specific Radiopharmaceuticals in Nuclear Medicine  26  1.7  Thesis Overview  28  1.8  References  29  Vanadyl-thiazolidinedione Combination Agents for Potential Use in Diabetes Therapy 2.1  Introduction  43  iii  Chapter 3  2.2  Experimental  48  2.2.1  Materials  48  2.2.2  Instrumentation  48  2.2.3  In vivo STZ-diabetic Rat Studies  49  2.2.4  Ligand Synthesis  51  2.2.5  Synthesis of Vanadyl-thiazolidinedione Complexes  64  2.3  Results and Discussion  66  2.3.1  Bifunctional Ligand Synthesis  66  2.3.2  Vanadyl-thiazolidinedione Complexes  71  2.3.3  Biological Studies  74  2.4  Concluding Remarks  77  2.5  References  78  ,  New Chelating Agents for Intervention in Neurodegenerative Disease 3.1  Introduction  82  3.2  Experimental  88  3.2.1  Materials  88  3.2.2  Instrumentation  89  3.2.3  Potentiometric Equilibrium Measurements  89  3.2.4  UV-vis Determination of Acidity Constants  90  3.2.5  ' H N M R Protonation Experiments  91  3.2.6  X-ray Crystallographic Analysis of H G L , C u G L , 7  and N i G L  3.2.7  7  2  Stability Constants for the Zn and Cu H GL  92  7  6 7  2  iv  Complexes of  93  3.2.8  Trolox Equivalent Antioxidant Capacity (TEAC) Assay  94  3.2.9  Enyzymatic Glycoside Cleavage  95  3.2.10  Toxicity Cell Studies and M T T Assay  96  3.2.11  Synthesis of Tetrahydrosalen Compounds (H2L ")  97  3.2.12  Synthesis of the Glycosylated Tetrahydrosalen Compounds (GL " and H G L " ) 1  5  6  102  7  2  Cu  3.3  Results and Discussion  122  3.3.1  Synthesis of the Tetrahydrosalen Compounds  122  3.3.2  Synthesis and Characterization of the Carbohydrate-derivatives of the Tetrahydrosalen Ligands  124  3.3.3  Synthesis and Characterization of the N i  141  2 +  and N i  Complexes of H G L  3.2.13  2 +  119  6 7  2  and Cu  67  Metal Complexes of Deprotonated H2GL "  Chapter 4  3.3.4  T E A C Values of the Tetrahydrosalen Compounds  159  3.3.5  Enyzymatic Cleavage of the Tetrahydrosalen Glycosides  161  3.3.6  Cell Toxicity Studies  164  3.4  Concluding Remarks  165  3.5  References  166  Carbohydrate-appended Metal Complexes ( Nuclear Medicine  99m  T c / Re) for Potential Use in  4.1  Introduction  172  4.2  Experimental  179  4.2.1  Materials  179  v  4.2.2  Instrumentation  180  4.2.3  X-ray Crystallography  180  4.2.4  [ Tc(H 0)3(CO) ] Labelling Studies  182  4.2.5  Cysteine and Histidine Challenge Experiments  183  4.2.6  Syntheses of the Re Complexes with the 1,3-Diaminocarbohydrates (L - L )  183  4.2.7  Syntheses of the Re Complexes with the Carbohydrate-appended 2,2'-Dipicolylamine ligands (L -L )  190  4.3  Results and Discussion  198  4.3.1  Synthesis and Characterization of the Re Complexes with L ' - L  198  Synthesis and Characterization of the with L - L  210  99m  +  2  3  8  12  7  4.3.2  ]  4.3.3  216  Synthesis and Characterization of the with L - L  228  8  Chapter 5  T c Complexes  7  Synthesis and Characterization of the Re Complexes with L - L 8  4.3.4  9 9 m  1 2  9 9 m  T c Complexes  1 2  4.4  Concluding Remarks  232  4.5  References  234  Future Work  5.1  Vanadyl-thiazolidinedione Combination Agents  239  5.2  Novel Chelating Agents for Alzheimer's Disease Therapy  240  5.3  Carbohydrate-appended Metal Complexes for Use in Nuclear Medicine  243  5.4  References  246  vi  LIST OF FIGURES  Figure 1.1  Examples of metal-based drugs.  1  Figure 1.2  Examples of different vanadium coordination geometries.  3  Figure 1.3  Bis(maltolato)oxovanadium(IV) (BMOV, R = C H ) , bis(ethylmaltolato)oxovanadium(IV) (BEOV, R = C H ) .  7  Figure 1.4  Pyranose (D-glucopyranose) and furanose (D-ribofuranose) sugar structures.  8  Figure 1.5  A drawing of A D pathological markers.  13  Figure 1.6  Cleavage pathways for the APP enzyme.  14  Figure 1.7  Proposed metal binding regions of the metal-Ap complexes.  19  Figure 1.8  Chemical structures of A D therapeutics.  21  Figure 1.9  Structures of metal chelators used in studies to disrupt metal-Ap interactions.  23  Figure 1.10  Promising molecules for A D chelation therapy.  25  Figure 2.1  Structures of the different drug classes discussed in Chapter 2.  44  Figure 2.2  Examples from the thiazolidinedione (TZD) drug class.  45  Figure 2.3  The structures of maltol and kojic acid 1.  47  Figure 2.4  Structure of streptozotocin (STZ).  50  Figure 2.5  Solution EPR spectrum of VO(L ) .  73  Figure 2.6  Hypoglycemic activity (% plasma glucose lowering) for four thiazolidinedione ligand precursors and three of their corresponding complexes, compared with B M O V and rosiglitazone.  76  Figure 3.1  Compounds of interest for Chapter 3.  83  Figure 3.2  Tetrahydrosalen pro-ligands and carbohydrate-protected tetrahydrosalen ligand precursors synthesized in Chapter 3.  84  3  2  5  4  2  vii  Figure 3.3  Schematic of the glycoside pro-drug strategy.  85  Figure 3.4  Reagents used in Chapter 3.  94  Figure 3.5  ORTEP diagram of H G L showing 50% thermal probability ellipsoids. 7  130  Figure 3.6  Compounds relevant to the potentiometric discussion.  133  Figure 3.7  *H N M R shifts vs. pD for H G L .  134  Figure 3.8  Variable pH (pH 9.7-13.5) U V spectra of H G L ([H GL ].  135  Figure 3.9  Solution speciation diagram for H G L .  136  Figure 3.10  *H N M R shifts vs. pD for H G L .  137  Figure 3.11  Variable p H (pH 8.5-14) U V spectra of H G L .  138  Figure 3.12  Solution speciation diagram for H G L .  140  Figure 3.13  UV-vis spectra (MeOH, 0.075M) of: A) H G L (black) and H G L (red). B) CuGL (black) and C u G L (red).  142  Figure 3.14  Frozen solution EPR spectrum CuGL .  144  Figure 3.15  Frozen solution EPR spectrum CuGL .  145  Figure 3.16  ORTEP diagram of CuGL showing 50% thermal probability ellipsoids.  146  Figure 3.17  Crystal packing in CuGL .  148  Figure 3.18  ORTEP diagram of NiGL showing 50% thermal probability ellipsoids.  148  Figure 3.19  (A) Solution speciation diagram for H G L and Z n ; (B) solution speciation diagram for H G L and C u .  152  (A) Solution speciation diagram for H G L and Z n ; (B) solution speciation diagram for H G L and C u .  153  p M diagrams for Z n  157  2  6  2  6  6  2  2  6  2  7  2  7  2  7  2  6  2  7  6  7  2  6  7  7  7  7  6  2+  2  6  2+  2  Figure 3.20  7  2+  2  7  2+  2  Figure 3.21  2+  and C u  viii  2 +  with relevant chelators.  Figure 3.22  T E A C Values at 1, 3, and 6 minutes for the tetrahydrosalen ligands, (±)-a-tocopherol, and BHT.  159  Figure 3.23  Silica-gel monitoring of enzymatic (Abg) deglycosylation reactions of selected tetrahydrosalen glycosides.  162  Figure 3.24  M T T plots for G L , H G L , and cisplatin.  164  Figure 4.1  Compounds discussed in the introduction.  174  Figure 4.2  1,3-Diaminocarbohydrates.  176  Figure 4.3  Carbohydrate-appended 2,2'-dipicolylamine ligands.  177  Figure 4.4  Compounds relevant to the experimental procedures.  Figure 4.5  *H N M R spectra of L and [Re(L )Br(CO) ].  200  Figure 4.6  ' H - N HSQC spectra of [Re(L )Br(CO) ].  201  Figure 4.7  ORTEP diagram of [Re(L )Br(CO) ] showing 50 % probability ellipsoids.  206  Figure 4.8  ORTEP diagram of [Re(L )Br(CO) ] showing 50 % probability ellipsoids.  208  Figure 4.9  ORTEP diagram of the two discrete molecules (about the one water) of [Re(L )Br(CO) ] in the unit cell showing 50 % probability ellipsoids.  210  H P L C radiation traces for the histidine challenge experiment with [ Tc(L )(H 0)(CO) ] .  215  Figure 4.11  ' H N M R spectra of A) L B) [Re(L )(CO) ]Br.  220  Figure 4.12  ' H N M R spectra of [Re(L " )(CO) ]Br in the region 4.8-5.6 ppm showing the pyridyl methylene hydrogen signals.  222  Figure 4.13  ORTEP diagram of [Re(L )(CO) ] showing 50 % probability ellipsoids.  226  Figure 4.14  H P L C comparison of a) [Re(L )(CO) ] (UV/254 nm) and b) [ Tc(L )(CO) ] (radiometric).  230  2  6  2  1  l  3  1 5  1  3  2  3  3  3  179  3  3  Figure 4.10  99m  1  +  2  3  8  8  3  8  12  3  8  +  3  10  +  3  99m  10  +  3  ix  LIST O F S C H E M E S  Scheme 2.1  The synthesis of V O C L ^ (83%), V O ( L ) (93%), and V O ( L ) 1  3  4  2  2  64  (88%). Scheme 2.2  The synthesis of H L .  67  Scheme 2.3  The synthesis of H L .  68  Scheme 2.4  The synthesis of H L .  69  Scheme 2.5  The synthesis of H L .  70  Scheme 3.1  General reaction of (±)-oc-tocopherol with a radical ( R ) .  86  Scheme 3.2  Synthesis of H L and H L .  122  Scheme 3.3  Synthesis of H L .  123  Scheme 3.4  Synthesis of H L . 5  123  Scheme 3.5  Synthesis of G L .  125  Scheme 3.6  Synthesis of G L .  126  Scheme 3.7  Synthesis of G L .  127  Scheme 3.8  Synthesis of G L .  127  Scheme 3.9  Synthesis of G L .  128  Scheme 3.10  Synthesis of H G L .  128  Scheme 3.11  Synthesis of H G L .  129  Scheme 3.12  Synthesis of the metal complexes ( M =Cu or Ni) of H G L " .  141  Scheme 4.1  Reaction scheme for the preparation of the Re complexes of the 1,3-diaminocarbohydrate ligands L ^ L .  199  Reaction scheme for the preparation of the Re complex of the bis-sugar analog L .  199  1  2  3  4  2  3  2  2  4  2  2  1  2  3  4  5  6  2  7  2  6  7  2  6  Scheme 4.2  7  x  Scheme 4.3  Reaction scheme for the synthesis of the 1,3-diaminocarbohydrate ligands Li -!-. .  9 9 m  T c complexes of the  211  Reaction scheme for the synthesis of the bis-sugar analog L .  9 9 m  T c complex of the  211  and L .  217  1  Scheme 4.4  6  7  Scheme 4.5  Reaction scheme for the preparation of L  Scheme 4.6  Reaction scheme for the preparation of the Re complexes of the carbohydrate-appended DPA ligands L - L .  218  Reaction scheme for the preparation of the Re complexes of the carbohydrate-appended DPA ligands L - L .  218  Reaction scheme for the synthesis of the  228  1 1  8  Scheme 4.7  1 0  M  Scheme 4.8  1 2  1 2  9 9 m  Q  T c complexes of the in  carbohydrate-appended DPA ligands L - L . Scheme 4.9  Reaction scheme for the synthesis of the  9 9 m  11  T c complexes of the  228  12  carbohydrate-appended D P A ligands L - L . Scheme 5.1  A potential route to a C-radiolabelled derivative of H 2 G L .  241  Scheme 5.2  The use of different amine starting materials to produce tetrahydrosalen analogs; structural diversity could be obtained by altering R and R' as well as the length (x) of the alkyl tether.  242  Scheme 5.3  Reaction scheme for the synthesis of the dioxorhenium(V) complex of (S)-2,3-diaminopropyl p-D-glucopyranoside.  244  Scheme 5.4  General reaction scheme for the synthesis of the dioxorhenium(V) complexes of the tridentate carbohydrate 2,2'dipicolylamine ligands.  245  6  14  xi  LIST OF T A B L E S  Table 2.1  Spin Hamiltonian Parameters for VO(L)2; L = L , L , L , ma (maltol), ka (kojic acid 1).  73  Table 2.2  Glucose lowering of four thiazolidinedione ligand precursors and three of their corresponding complexes compared with B M O V , and rosiglitazone.  75  Table 3.1  Selected bond lengths (A) in H G L .  130  Table 3.2  Deprotonation constants (p^ 's) of various tetrahydrosalen derivatives.  132  Table 3.3  Spin Hamiltonian parameters of the Cu complexes in MeOH, T = 130 K .  144  Table 3.4  Selected bond lengths (A) and angles (deg) in C u G L . 7  147  Table 3.5  Selected bond lengths (A) and angles (deg) in N i G L .  149  Table 3.6  Negative logarithms (pQ) of hydrolysis equilibrium constants for : aqueous solution. Z n and C u.2+ in  150  Stability constants of the C u H GL .  151  1  4  7  2  a  7  2 +  Table 3.7  3  z +  2 +  and Z n  2 +  complexes with  6 7  2  Table 3.8  Zn  Table 3.9  T E A C values ± SD at 1, 3, and 6 minutes.  160  Table 4.1  V c o IR bands, M S patterns, and conductivity measurements for [Re(L -L )(CO) Br].  203  Table 4.2  Selected bond lengths (A) and angles (deg) in [Re(L )(CO) Br].  207  Table 4.3  Selected bond lengths (A) and angles (deg) in [Re(L )(CO) Br].  209  Table 4.4  H P L C retention times (RT), and labelling yields (%) for the [ M ( L - L ) ( H 0 ) ( C O ) ] ( M = Re, T c ) complexes.  212  [ Tc(L -L )(H 0)(C0) ] complex stability (%) at 1, 4, and 24 hr towards ligand exchange in solutions of either 1 m M cysteine or 1 m M histidine in PBS.  214  2 +  and C u  1  2 +  stability constants of relevant chelators.  155  7  3  2  3  3  3  1  7  +  2  Table 4.5  99m  1  99m  3  7  +  2  3  xii  Table 4.6  vco IR bands, M S patterns, and conductivity measurements for [Re(L -L )(CO) ]Br.  225  Table 4.7  Selected bond lengths (A) and angles (deg) in [Re(L )(CO) ]Cl.  227  Table 4.8  H P L C retention times (RT), and labelling yields (%) for the [M(L -L )(CO) ] ( M = Re, T c ) complexes.  229  Table 4.9  [ T c ( L - L ) ( C O ) ] complex stability (%) at 1, 4, and 24 h towards ligand exchange in solutions of either 1 m M cysteine or 1 m M histidine in PBS (100-fold excess).  232  Table A l  Crystallographic data for H G L , CuGL and NiGL .  248  Table A2  Crystallographic data for [Re(L )(CO) Br] and [Re(L )(CO) Br], and [Re(L )(CO) ]Cl.  249  8  12  3  8  3  8  12  +  99m  3  99m  8  n  +  3  7  7  2  2  3  3  8  3  3  xiii  7  LIST OF EQUATIONS Eq. 2.1  50  Eq. 3.1  92  Eq.3.2  131  Eq.3.3  131  Eq.3.4  131  Eq. 3.5  131  Eq. 3.6  139  Eq.3.7  139  Eq.3.8  150  Eq. 3.9  150  Eq. 3.10  150  Eq.3.11  150  xiv  LIST OF ABBREVIATIONS  ~  approximate  a  alpha  A  angstrom, 1 x 10" metre 10  P  +  8  positron chemical shift in parts per million (ppm) from a standard (NMR) extinction coefficient (UV-vis) in L mol" cm" 1  y  gamma rays  X  wavelength  /Imax  wavelength of maximum absorption (UV-vis)  u.  micro (10" )  |a ff  effective magnetic moment in B M  6  e  v A  1  frequency m  molar conductivity  Q  ohms  Aiso  isotropic hyperfine coupling constant  An  parallel hyperfine coupling constant  A_i  perpendicular hyperfine coupling constant  A  hyperfine coupling constant in the x direction (rhombic)  A  y  hyperfine coupling constant in the y direction (rhombic)  A  z  hyperfine coupling constant in the z direction (rhombic)  A  absorbance  AP  P-amyloid  x  xv  Abg  agrobacterium sp. P-glucosidase enzyme  ABTS  2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)  AD  Alzheimer's disease  ALS  amyotrophic lateral sclerosis  Anal  analytical  APOE  apolipoprotein E  APP  amyloid precursor protein  Ar  aromatic  atm  atmosphere  ATP  adenosine triphosphate  BACE  P-secretase  BBB  blood-brain barrier  BC  bathocuproine  BEOV  bis(ethylmaltolato)vanadium(IV)  BHA  butylatedhydroxyanisole  BHT  butylatedhydroxytoluene  BM  Bohr magneton  BMOV  bis(maltolato)vanadium(IV)  Bn  benzyl  BP  bathophenanthroline  °C  degrees Celsius  Calcd  calculated  CH C1 2  2  methylene chloride  CHCI3  chloroform  CIS  coordination induced shift  cm"  1  wavenumber(s) (reciprocal centimeter)  CMC  carboxymethylcellulose  CNS  central nervous system  COSY  correlation spectroscopy (NMR)  2D  two-dimensional  d  doublet (NMR), day(s)  dd  doublet of doublets (NMR)  ddd  doublet of doublet of doublets (NMR)  Da  Dalton  DCC  dicyclohexylcarbodiimide  DCI-MS  desorption chemical ionization mass spectrometry  DFO  desferrioxamine  DGTA  1,3-N,N'-di-P-D-glucopyranosyldiethyltriamine  DMF  dimethylformamide  DPA  dipicolylamine  D-pen  D-penicillamine  DTPA  diethylenetriaminepentaacetic acid  EA  elemental analysis  EC  enzyme classification  ECDG  ethylenedicysteine-deoxyglucose  EDTA  ethylenediaminetetraacetic acid  xvii  t  EGTA  glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid  EI-MS  electron impact mass spectrometry  EPR  electron paramagnetic resonance  ES-MS  electrospray mass spectrometry  EtOAc  ethyl acetate  EtOH  ethanol  eV  electron volt  fac  facial arrangement  FAD  familial Alzheimer's disease  FDA  Food and Drug Administration (USA)  FDG  2-deoxy-2- [ F] fluoro-D-glucose  FFA  free fatty acids  g  gram  giso  isotropic Lande splitting factor, g-factor  gn  parallel g-factor  g  x  perpendicular g-factor  g  x  g-factor in the x-direction (rhombic system)  g  y  g-factor in the y-direction (rhombic system)  g  z  g-factor in the z-direction (rhombic system)  18  Gl  gastrointestinal tract  GLUT  glucose transporter  h  hour(s)  H bbpen 2  N,N'-bis(2-hydroxybenzyl)-N,N'-(2-methylpyridyl)-ethylenediamine  xviii  H salen  N,N'-bis(salicylidene)ethylenediamine  HBED  N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid  Hcq  clioquinol  His  histidine  HIV  human immunodeficiency virus  HMBC  heteronuclear multiple bond correlation (NMR)  HMIT  H / myo-inositol co-transporter  HMQC  heteronuclear multiple quantum coherence (NMR)  HPLC  high-performance liquid chromatography  HSQC  heteronuclear single quantum coherence (NMR)  HYBEDA  N,N'-bis(2-hydroxybenzyl)-ethylenediamine  Hz  hertz (s" )  /  ionic strength or nuclear spin  IC50  drug concentration at which 50% of cells are viable relative to the control  i.e.  that is  i.p.  intraperitoneal  IR  infrared radiation  J  coupling constant (NMR)  K  dissociation constant  2  +  1  K  a  acid dissociation constant  K  w  ionization constant for water (10"  ka  deprotonated kojic acid  L  litre or ligand  xix  13 7 4  )  LC  liquid chromatography  LMCT  ligand to metal charge transfer band  LSIMS  liquid secondary ion mass spectometry  m  metre or milli  M  molarity or metal  ma  deprotonated maltol  CH3CN  acetonitrile  MeOH  methanol  Met  methionine  metf  metformin  MHz  megahertz  min  minutes  mol  mole (6.02 x 10 molecules)  m.p.  melting point  MP AC  metal-protein attenuating compound  MRI  magnetic resonance imaging  MS  mass spectrometry  MTT  (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide  m/z  mass per unit charge  NFT  neurofibrillary tangles  NHS  N-hydroxysuccinimide  NMDA  N-methyl-D-aspartate receptor  NMR  nuclear magnetic resonance  23  xx  NSAID  non-steroidal anti-inflammatory drug  ORTEP  Oak Ridge Thermal Ellipsoid Plot  PBS  phosphate buffered saline  pD  -log [ D 0 ]  PD  Parkinson's disease  Pd/C  palladium on carbon  PET  positron emission tomography  PGc  plasma glucose in control mice  PGT  plasma glucose in treated mice  pH  -log [ H 0 ]  pH*  p H meter reading of a deuterium solution  pK  -log K  +  3  +  3  a  a  pM  - log [M], where [M] is the free metal concentration  pQ  -log Q (Q is the metal hydrolysis constant)  PPARoc  peroxisome proliferator-activated receptor alpha  PPARy  peroxisome proliferator-activated receptor gamma  ppm  parts per million  PSI, PS2  presenelin-1, presenelin-2  q  quartet (NMR)  ROS  reactive oxygen species  r.t.  room temperature  RT  retention time  s  second, singlet (NMR)  xxi  SD  standard deviation  SEM  standard error of the mean  SGLT  sodium-dependant glucose transporters  SOD  superoxide dismutase  SPECT  single photon emission computed tomography  STZ  streptozotocin  t  triplet (NMR)  t.  half-life  TEAC  trolox equivalent antioxidant capacity  TETA  triethylenetetraamine  Tg  transgenic  THF  tetrahydrofuran  TLC  thin layer chromatography  TPEN  N , N , N ' , N ' -tetrakis(2-pyridylmethyl)ethylenediamine  Tyr  tyrosine  TZD  thiazolidinedione  UBC  University of British Columbia  UV  ultraviolet  vis  visible  /2  VO(metf)2  N',N'-(dimethylbiguanidato)oxovanadium(IV)  VT  variable temperature  X-band  9 Ghz (EPR frequency)  ZDF  zucker diabetic fatty  xxii  ACKNOWLEDGEMENTS  I would like to thank my parents and sister for all of the encouragement over the past five years. Thanks to Anne for her support, companionship, and chemical songwriting. The band (Aaron and Pete) deserves acknowledgement for all the melodies, when are we going on tour? Thanks to the ski crew (Jonas, Guy, and the rest), the ultimate crowd, and the chemistry folks for the distraction. To Dr. Chris Orvig: I will get you out to another late night music show! Thanks for allowing me the freedom to pursue my interests, even if they were in far off places. I really enjoyed the independence you gave me to explore a diverse set of projects during my time at U B C . The optimism and kindness you convey as a research advisor promotes a great group atmosphere. I would like to thank Dr. Kathie Thompson for all of her interest and help with the biological side of my chemistry research. Dr. Harvey Schugar was instrumental in initiating the A D project and I would like to thank him for his interest and continued involvement. One can never have too many uncles! I would also like to thank Dr. Natia Frank for hosting me at the University of Washington as a visiting scientist and Dr. Shigenobu Yano for hosting me as a JSPS fellow in Japan. I would like to further acknowledge Dr. Yuji Mikata, and Team Yano (Dr. Makoto Obata, Yuko, Iza, Misaki, etc.) for their hospitality and continued collaboration. Thanks to Dr. Mike Adam for his interest and support at TRIUMF. None of this work would have gotten very far without the past and present members of the Orvig group: Dave, Cara, Barry, Cheri, Chuck, Meryn, Song Bin, Simon, Alex, Michael, Neil, Lauren, George, Vishaka, Kathy, Jess, Karin, Mike, Fabio, Khosro the list goes on. There is nothing like your peers to keep you honest and on your toes"Are you done yet?" Finally I would like to thank the support staff; Liane Darge (NMR),  xxiii  Marietta Austria (NMR), Dr. Nick Burlinson (NMR), Dr. Brian Patrick (X-ray), Dr. Yun Ling (MS), and the front office for all of their help.  xxiv  CHAPTER 1 Introduction  1.1 Medicinal Inorganic Chemistry  Medicinal inorganic chemistry can be divided into two main categories, (1) drugs that target metal ions in some form, and (2) metal-based drugs where the central metal ion is essential for the clinical application. Although the field of medicinal inorganic chemistry is not new, our continued increase in understanding of how metals interact with the body has enabled the development of many effective disease treatment strategies utilizing metal ions. Such treatments include the use of gold complexes (auranofm, Figure 1.1) for the treatment of rheumatoid arthritis, and platinum anti-cancer agents (cisplatin, Figure 1.1) for cancer therapy. Specific properties of metal ions have been exploited in the development of metal-based diagnostic agents, an example being the unique magnetic properties of gadolinium (Magnevist™, Figure 1.1) for magnetic resonance imaging (MRI).  OAc CI/,,  Auranofm  , NH V  3  Cisplatin Magnevist-TM  Figure 1 . 1 : Examples of metal-based drugs.  1  References start on p.29  As with any substance, there is a need to balance the potential toxicity with the positive impact of the diagnostic or therapeutic application of the metal-based drug. The 1  biodistribution and pharmacokinetics of metal ions can be significantly altered by the use of well-tailored ligands. Properties such as lipophilicity, charge, size, and solubility can be altered by careful ligand selection thus potentially reducing metal toxicity. In addition, ligands can be further elaborated to include biologically active molecules for improved targeting, or drug molecules to augment the activity of the metal ion. This thesis explores the development of novel ligands in an effort to enhance the therapeutic and/or targeting potential of vanadium in diabetes therapy (Chapter 2), and technetium in nuclear medicine (Chapter 4). As well, the design and testing of novel molecules for tissuespecific metal chelation (Chapter 3) are investigated.  1.2 Vanadium and Diabetes  Vanadium, element number 23, was discovered and characterized in 1831 and named after Vanadis the Norse goddess of beauty, in homage to the varied colours of the element in solution. Vanadium, a Group V first row transition metal, exists in a wide range of oxidation states from -3 to +5. However, due to conditions inherent to 3  biological systems, vanadium is most commonly found in the oxidation states +3 to +5 in this medium, with the vanadate ( [ H V 0 ] " and [H V 0 ]"), and vanadyl ( [ V O ] ) , v  2  v  4  2  lv  being the most common species. Interconversion between V and V 4  2+  4  v  1 V  is mediated by  oxygen levels, pH, and the presence of reducing agents such as ascorbate, glutathione, and catecholamines. ' Examples of vanadium as V 5 6  2  111  have been found in certain marine  References start on p.29  organisms such as ascidians. The diverse chemistry of vanadium can be attributed to the wide range of accessible oxidation states, as well as to coordinative and stereochemical flexibility, leading to vanadium compounds existing in numerous coordination geometries. Such geometries include tetrahedral, square pyramidal, octahedral, and pentagonal bipyramidal (Figure 1.2). " The non-oxo-containing V 8  10  I V  natural product  amavadin (Figure 1.2), found in Amanita muscaria, a species of mushroom, is an example of a stable 8-coordinate V  I V  compound.  11  2-  Figure 1.2: Examples of different vanadium coordination geometries: (a) vanadate as [H2VO4]",  tetrahedral geometry; (b) vanadyl ion in acid solution, octahedral geometry; (c)  diperoxovanadate, pentagonal bipyramidal geometry; (d) a vanadyl coordination complex with two bidentate ligands, square pyramidal geometry; (e) the natural product amavadin (eight coordinate non-oxo).  3  References start on p.29  Vanadium is found in less developed organisms (e.g. algae, bacteria, lichens, fungi), as well as in mammals, including humans. There has been considerable debate 12  about the physiological role(s)  and essentiality of vanadium in these biological  systems. " Vanadium is considered essential for many organisms; it is found in the 14  16  active site of vanadate-dependent haloperoxidases, an important metalloenzyme in sea algae and lichens, " and in vanadium nitrogenases found in the nitrogen fixing bacteria 17  21  22 24  Azobacter '  25  and Anabaena variabilis.  Even though vanadium has been determined to  elicit multiple effects in more complex mammals, no clearly essential role for vanadium has emerged. ' ' For example, vanadium as vanadate (VO4 ") is an inhibitor of the N a 14 15 24  +  3  K -ATPase pump in vitro? +  6  Crystal structure studies have shown that vanadate can substitute for phosphate in transition state analogs of phosphotyrosine phosphates (PTPases). " Vanadate is similar 27  29  to the phosphate anion (PO4 ") in size, charge, and geometry, and thus has the potential to interact with the phosphate binding site on kinases and phosphatases, as well as to 1  ^ 94 9Q  participate in a variety of phosphate metabolic processes. ' ' Interestingly, the discovery of the potential of vanadium compounds for diabetes treatment, among other diseases, was first discovered by French physicians in 1899. Vanadium was o  1  demonstrated more definitively to have insulin-like properties in vitro in 1979 , in skeletal muscle and adipose tissue, and further in vivo in 1985. The in vivo study 32  demonstrated that orally administered vanadium could reverse many of the symptoms of diabetes in a diabetic animal model. Since these initial findings there has been a great deal of interest in determining the biological role of vanadium, including its mechanism  4  References start on p. 29  of action, and in the development of new vanadium compounds as alternatives to conventional diabetes therapy. Diabetes mellitus is a major health problem, with an estimated worldwide prevalence of 150 million people in 2000; this is expected to increase to 220 million people by 2010.  Diabetes mellitus includes a heterogeneous group of disorders  characterized by hyperglycaemia arising as a consequence of a relative or absolute deficiency of insulin secretion, resistance to insulin action, or both. Insulin, a peptide and therefore not orally active, is produced by the P-cells of the pancreas to regulate blood glucose levels by stimulating glucose transport and uptake, as well as lipogenesis, and by inhibiting the breakdown of glycogen. A lack of treatment, or poor control of blood glucose levels, can lead to serious long term complications including blindness, kidney failure, heart disease, stroke, and pathophysiological conditions requiring amputation. The disease can be roughly divided into two classes: type 1, insulin dependent diabetes mellitus (IDDM) and type 2, non-insulin dependent diabetes mellitus (NIDDM). Type 2 diabetes mellitus is much more prevalent than is type 1, and has been associated with increased age and obesity. ' Given the dual problem of increasing rates of obesity and 34 35  the trend toward more sedentary lifestyles, diabetes is now considered an epidemic.  36  Treatment of type 1 diabetes requires regular intramuscular injection of insulin due to a lack of insulin-producing p cells in the pancreas. Insulin is a peptide and thus cannot be administered orally; direct intramuscular injection avoids breakdown in the gastrointestinal (Gl) tract. Treatment of type 2 diabetes is dependent on the progression of the disease and may involve daily subcutaneous injections of insulin, a change in diet, increased exercise, administration of one or more of the currently available oral drug  5  References start on p.29  therapies, or combination therapy. The development of new orally-active drug therapies to replace or augment the effect of insulin is however still much needed. A certain number of vanadium compounds are orally active and have been found to enhance the effects of insulin. More specifically, vanadium has been determined to 32  increase glucose uptake, inhibit lipolysis, and stimulate glycogen synthesis and lipogenesis. ' " Importantly, these effects occur without a concomitant increase in circulating insulin levels. ' Current evidence points to a site (or sites) of action 40 41  downstream from the insulin receptor in the insulin signaling cascade. ' The 42 43  mechanism of vanadium's in vivo effects however is still subject to debate, " due to the 44  multiplicity of its effects. '  13 46  47  The therapeutic value of inorganic vanadium, in the form of  vanadate ([V0 ] ") or vanadyl ([VCT]), as an orally active agent against diabetes has J  4  been well documented.  Poor absorption from the G l tract into the bloodstream, as well  as the toxicity associated with a therapeutically relevant dose, limit the utility of administering vanadium to diabetic patients in these forms. Vanadium complexes with suitable ligands have thus been developed to improve gastrointestinal absorption and bioavailability of the metal  4 9  Two general classes of compounds have been developed:  (1) coordination complexes, mostly of the general type V O L 2 , and (2) peroxovanadium complexes ([VO(0 )(H 0) (L-L')] ", n = 0 or 1, and [VO(0 ) (L-L')] ", n - 1,2, or 3). n  2  2  n  2  2  50  2  The peroxovanadates exhibit limited stability in aqueous solution and promote intracellular oxidative stress, reducing their potential use as therapeutic agents.  50  Complexes of the general formula V O L have shown a much greater utility for diabetes 2  treatment in vivo. Neutral, lipophilic, and water soluble vanadium compounds such as bis(maltolato)oxovanadium(IV), commonly known by its acronym B M O V ' 5  6  5 1  (Figure  References start on p.29  1.3), have shown increased efficacy over inorganic vanadium in a diabetic animal 59  model.  B E O V (the ethyl analog) completed phase 1 clinical trials in 2000.  Complexation of vanadyl ( V 0 ) with the approved food additive maltol improved G l 2+  absorption thereby decreasing the vanadium dose required for effective glucose lowering. '  52 53  R O  o R  Figure 1.3: Bis(maltolato)oxovanadium(IV) ( B M O V , R = C H ) , bis(ethylmaltolato)3  oxovanadium(IV) (BEOV, R = C H ) . 2  5  In Chapter 2 of this thesis the coupling of vanadium with ligands that contain an insulin-action-enhancing functionality is reported. The combination of complementary treatments has been proposed as a more effective means of achieving glycemic control by concurrently targeting different mechanisms in the body. In order to form stable neutral 33  complexes with the vanadyl ion, bifunctional ligands were developed, tethering the insulin-action-enhancing portion of the molecule to a suitable chelator. Chapter 2 presents the synthesis and characterization of vanadium-thiazolidinedione combination agents as well as preliminary in vivo biological investigations of these novel compounds.  7  References start on p.29  1.3 Carbohydrate-containing Molecules for Increased Drug Selectivity  Carbohydrates are of primary importance as energy sources for living organisms. Carbohydrates are the most abundant class of organic molecules, exhibiting an 54  empirical formula of C H 0 , with furanose (C5H10O5) and pyranose (C6H12O6) ring 2  structures being the most common (Figure 1.4). Glucose is the principal carbohydrate 55  metabolized by the body.  OH  D-Glucopyranose (D-Glucose)  D-Ribofuranose  Figure 1.4: Pyranose (D-glucopyranose) and furanose (D-ribofuranose) sugar structures.  The original definition of a carbohydrate has been broadened however to include polyhydroxy carbon-based compounds that include heteroatoms such as nitrogen, silicon, and sulphur. Due to the dependence of the human body on carbohydrates for energy, transport and utilization pathways for this class of polar water-soluble molecules are highly developed. The hydrophilicity of glucose prevents passage across lipid bilayers and thus the movement of this molecule across membranes relies on carrier-mediated transport systems. The transportation of glucose across membranes occurs via facilitative glucose transporters (GLUT) or sodium-dependent glucose transporters (SGLT). There  8  References start on p. 29  are six known sodium-dependent glucose transporters (SGLT1-6) which are primarily involved in the intestinal absorption of glucose. ' Thirteen facilitative glucose 56 57  transporters are known, including GLUT1-12 and HMIT-1 (HMIT-1 has been shown to be a H / myo-inositol co-transporter). ' +  58 59  The brain requires a significant amount of glucose to maintain normal bodily functions (up to 30% of total body glucose consumption ), and this demand is met by the 60  high density of GLUT1 transporters at the blood brain barrier (BBB). The B B B is defined as the junction at which the bloodstream meets the neuronal environment and consists of brain micro vessel endothelial cells that ensure that a vast majority of neurotoxic metabolites are excluded from the brain, while allowing for essential molecules to pass across. One method of gaining access to the brain is via passive diffusion whereby a 61  molecule of modest molecular weight (< 500 Da), and appreciable hydrophobicity (logP > 1.5, where P is the octanol/water partition coefficient) is able to passively diffuse across 62  the B B B .  Many essential nutrients however require the presence of transporters to gain  access to the brain. GLUT-1 represents essentially all of the facilitative glucose transporters at the B B B .  6 3  Other nutrients that are transported across the B B B include  amino acids which utilize either monocarboxylic acid transporters or the large neutral • i  64  amino acid transporter. The protection afforded by the B B B towards potential toxins prevents many drug molecules from gaining access to the brain. To enter the brain by passive diffusion, drug molecules need to be of modest molecular weight, and be neutral and lipophilic; unfortunately, many drugs do not fit these criteria. Treatment strategies for the human immunodeficiency virus (HIV) as well as for cerebral tumours would be much more 9  References start on p. 29  effective i f currently available therapies exhibited significant brain drug uptake.  In  order to address this, the coupling of drug molecules with nutrients such as glucose and amino acids that undergo transport across the B B B is being explored. The linking of carbohydrates to drug molecules, forming new derivatives and/or pro-drugs offers the potential to increase water solubility, minimize toxicity, and improve targeting. A prodrug is an inactive form of a drug that exerts its effects after metabolic processes within the body convert it to an active form. The high density of GLUT-1 transporters at the B B B , as compared to other tissues, make this transporter an attractive target for increasing the brain access of drug molecules. This glyconjugate approach has been used in an effort to enhance the central nervous system (CNS) targeting of anticonvulsants, analgesics, dopamine derivatives, 67  68,69  66  anti-cancer agents, " and HIV therapies ' 70  72  73 74  with some success. Cancer treatment, regardless of tissue location, may also benefit from the glycoconjugate approach due to the enhanced glucose metabolism of malignant tissues as compared to that in normal tissues. This difference in glucose metabolism is 75  currently utilized to determine the size and location of cancerous tissue by the administration of radiolabeled sugar derivatives. ' If pro-drug administration of a 76 77  carbohydrate-drug conjugate is successful at gaining access to the brain, enzymatic no nr\  cleavage by glucosidase enzymes ' offers the potential to release the drug molecule in the neuronal environment, thereby increasing selectivity and minimizing toxicity.  10  References start on p. 29  1.4 Neurodegenerative Disease  Oxidative Stress in the CNS The production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), nitric oxide (NO), superoxide (02~), and hydroxyl radicals (OH), is an intrinsic part of cellular metabolism. Cells regulate these potentially toxic species through a variety of defense mechanisms including antioxidants such as vitamin C , vitamin E , 8 0  81  catalase, thionein, superoxide dismutase, glutathione peroxidase, and glutathione reductase. '  82 83  Oxidative stress is the result of unregulated production of ROS and can  result in impaired cellular functions (such as perturbed C a  2+  signalling ' ), and coupled 84 85  with the formation of toxic species, can eventually lead to cell death. The brain is particularly susceptible to oxidative damage due to the high rate of metabolic activity coupled with relatively low antioxidant levels and low tissue regenerative capacity.  83  Increased oxidative stress, through impaired cellular energy metabolism and/or Fentontype processes involving redox active metal ions " , is a major feature of age-related 86  89  neurodegenerative diseases. The age-dependent rise in redox-active transition metals '  90 91  and oxidative stress in the brain, is consistent with the correlation between increased age and susceptibility to neurodegenerative disease. Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), are defined by the progressive loss of neuronal cell populations, protein aggregation, and extensive evidence of oxidative stress.  11  References start on p.29  Alzheimer's disease (AD) Alzheimer's disease (AD) was first described by Alois Alzheimer in 1907, who documented the behavioural changes of the disease along with the associated pathophysiological changes to the brain. A D currently affects 2% of the population in 92  industrialized countries with a predicted 3-fold increase in incidence over the next 50 years. In Canada, approximately 5% of the population over the age of 65 have A D . 93  9 4  A D is a progressive neurodegenerative disorder initially affecting brain regions involved in learning and memory, eventually resulting in death. A D can be classified as either early-(before 65 years) or late-(after 65 years) onset with more than 95% of the cases falling into the latter category. Genetic analysis of familial (inherited as an autosomal 95  dominant trait) early-onset cases of the A D has yielded many interesting findings. Point mutations in the amyloid precursor protein (APP) in regions involved in proteolytic processing, as well as mutations to the presenilin (PS1, PS2) genes, have been shown to lead to familial A D . ' 9 6  9 7  The cellular function of APP in the brain is unknown but 98  increasing evidence suggests it has an important role in regulating neuronal survival, as 99  well as in copper and zinc ion homeostasis. " 100  of membrane-bound proteins such as A P P .  106  105  The presenilins influence the cleavage  The vast majority of cases of A D are  however sporadic, and have not been linked genetically. Epidemiological findings also suggest that a low educational level, history of head trauma, consumption of high-calorie, high-fat diets and a sedentary lifestyle each may increase the risk of developing A D .  1 0 7  '  1 0 8  Diagnosis of A D , as opposed to other forms of dementia, requires post-mortem examination of the brain to determine the size and number of the hallmarks of the  12  References start on p. 29  disease; amyloid-(3 (Ap) plaques and neurofibrillary tangles (Figure 1.5). Neurofibrillary tangles are intracellular fibrillar aggregates of oxidatively-modified and hyperphosphorylated microtubule-associated protein tau. Amyloid-p plaques are extracellular deposits of fibrils and amorphous aggregates of the A p pepide (vide infra). It is still unclear as to whether Ap plaques, neurofibrillary tangles, or both, are a cause or an effect of the neurodegeneration in A D . However, neurofibrillary tangles are found in a wide variety of neurodegenerative conditions which suggests that they are a non-specific neuronal response to a variety of toxic insults, one of which may be Ap accumulation.  109  Furthermore, transgenic mouse studies support the theory that APP and AP influence the formation of neurofibrillary tangles. '  110 111  Normal  Alzheimer's  Figure 1.5: A drawing of A D pathological markers.  112  The figure shows a cartoon of the  normal brain (left) and the extracellular amyloid-p (Ap) plaques and intracellular neurofibrillary tangles (right) found in A D brains.  13  References start on p.29  The Ap peptide is a proteolytic product of APP, an integral membrane glycoprotein." Cleavage of APP occurs via a number of pathways, highlighted in 3  Figure 1.6. The enzymes responsible for APP cleavage are termed the a-, P-, and y96  secretases." The production of Ap (amyloidogenic processing) requires sequential 4  cleavage by p-secretase (BACE), followed by y-secretase. APP cleavage by B A C E releases sAPPp, and subsequent cleavage by y-secretase affords the A p peptide as predominantly AP1.40 or APM2 (a 40- or 42-residue peptide). Genetic analysis of familial cases of A D have offered strong evidence for altered homeostasis of Ap being a leading contributor to the disease.  Af340/42  Plasma membrane  Figure 1.6: Cleavage pathways for the APP enzyme. APP cleavage by the secretases to produce soluble fragments sAPPa and sAPPp as well as A P i ^ o and AP1.42. Adapted from reference [96].  14  References start on p.29  Presenilin mutations responsible for familial A D have been determined to alter ysecretase activity.  106  As well, point mutations of APP leading to early onset cases of A D  are situated in regions where proteolytic processing occurs. Finally, in all identified cases of early-onset familial A D where the genetic abnormality has been identified, the defective gene causes an increase in the production of the longer and more toxic form. ' 115  AP1.42  116  The A p peptide has been found in three general forms in the brain; membrane associated, aggregated, and soluble. In healthy individuals, most of the A P is membraneassociated, but in individuals with A D the aggregated and soluble fractions increase considerably. '  117 118  Aggregated A p exists as diffuse, amorphous deposits, as well as more  ordered fibrillar plaque deposits.  119  The initially deposited diffuse plaques are thought to  re-organize over time to form fibrillar neuritic plaques. Soluble forms of the peptide include monomers, dimers, oligomers, and protofibrils- metastable intermediates in 190  amyloid fibril formation.  AP1.42  is considered to be the main component of plaques, 191  followed by the shorter  AP1.40  form.  199  '  This correlates well with in vitro peptide  aggregation studies demonstrating that AP1.42 is much more prone to aggregation than is Ap!.  4 0  .  1 2 3  The amyloid hypothesis has long been the dominant theory to explain the cause of AD.  1 2 4  The amyloid hypothesis postulates that plaque A p depositions, or partially 125  aggregated soluble A p , trigger a neurotoxic cascade causing A D pathology.  Supporting  evidence for this hypothesis includes: (1) all familial forms of the disease where a genetic abnormality has been identified point towards altered proteolytic processing of APP and an increase in the production of  AP1.42;  96  (2) A p (in particular the longer form 15  AP1-42)  is  References start on p. 29  toxic to neurons;  "  and (3) there is a correlation between elevated brain levels of Ap 129  and cognitive decline.  Work with A D transgenic mouse models has shown a  correlation between memory deficits and A p elevation. ' '  124 130 131  The toxicity of the  various forms of the A p peptide have also been studied in great detail to determine the relative contribution of amyloid plaques, and soluble forms to A D pathology. Numerous studies have found that the soluble A p content of the human brain is better correlated with the severity of the disease as compared to plaques.  '  '  APP and A p are present  in normal, healthy brains, and indeed elderly non-demented individuals can exhibit extensive plaque development. '  133 134  APi.42  As well, the presence of excess soluble forms of  in the early stages of A D , and their ability to perturb memory-relevant signal  processing, point towards soluble forms of Ap being the active molecular pathogen. '  135 136  Evidence suggests that A p can be oxidatively modified in A D , particularly the sulphur atom of methionine-35 (Met-35). Api-42  137-139  The rate of aggregation of APi_ o and 4  is significantly impeded by the oxidation of Met-35,  140  and it is proposed that  oxidative modification results in soluble oxidized forms of the peptide that resist clearance and are toxic. ' 83  141  Oxidation of Met-35 can occur under a variety of conditions,  of which the interaction of Ap with redox-competent metal ions such as iron and copper is of particular importance. Metal ions and AD Evidence of oxidative stress is widespread in A D , with early neuronal and pathological changes showing indications of oxidative damage. '  142 143  The cause of  oxidative stress in A D has been attributed to a number of factors including the increased  16  References start on p. 29  production and aggregation of neurotoxic forms of A p , activation,  144  1 2 7  '  1 4 2  subsequent microglial  and the presence of high levels of redox-competent metal ions (Cu and Fe).  Increasing evidence points to the interaction of A p with transition metals as a major source of oxidative stress and free-radical production in A D .  1 4 5  "  1 4 7  As previously  discussed, there is an age-dependent rise in transition metals (Cu and Fe) in the brain, '  90 91  which is consistent with the correlation between increased age and susceptibility to neurodegenerative disease. Studies have shown that the concentrations of Cu, Fe, and Zn increase from 3- to 5-fold in A D brains as compared to age-matched controls.  148  Amyloid  plaques have been described as metallic sinks because remarkably high concentrations of Cu, Fe, and Zn have been found within these deposits in A D brains. '  148 149  In vitro studies  have shown that the interaction of Ap with metal ions leads to aggregation, with Z n  2+  rapidly accelerating the deposition process as compared to C u  150  2 +  and F e at pH 7.4. 3+  Cu -induced A p aggregation is however exaggerated under slightly acidic conditions (pH 6.8), and this metal ion has been shown to displace Z n  2+  under these conditions.  Mildly acidic conditions are believed to occur in the A D brain.  152  151  In addition, metal-Ap  interactions have recently been shown to initiate the formation of soluble oligomers in vitro, potentially implicating metal ions in the formation of these toxic forms of A p .  153  Soluble metal-Ap oligomers would be more mobile and could possibly generate oxidative damage over a widespread area. Further in vitro studies have demonstrated that Cu  2 + 1 5 4  and F e  3 + 1 4 5  potentiate the neurotoxicity of Ap via redox-cycling and the  production of H2O2 in the presence of dioxygen. The interaction of copper and iron with AP1.42 AP1.40,  produced a larger amount of ROS in vitro as compared to the interaction with supporting the enhanced toxicity of the longer  17  Api.42  form. ' 145  154  Peroxide  References start on p.29  generation in the presence of Cu or Fe  creates conditions whereby Fenton chemistry  can take place, resulting in the generation of the hydroxyl radical (OH). Evidence of O H radical formation has been inferred both in vitro  145  and in vivo '  on the basis of  155 156  characteristic oxidation products. Interestingly, it was found that the shorter length peptide A(3i_28 did not reduce Cu in vitro, potentially implicating the carboxy terminus and/or Met-35 as being a critical residue in the observed toxicity.  154  Evidence of  oxidative stress immediately surrounding plaque deposits is probably due to the presence of redox-active transition metal ions in these deposits, as well as microglial activation. Zn and Cu have been found to co-purify with A p from A D brain tissue, unlike F e .  158  157  Fe  in plaques is most likely involved in neuritic processes (for example complexed to ferritin ), and thus may not directly interact with A p . Fe in human amyloid deposits has 159  however been shown to be redox-active.  149  Interestingly, a recent in vitro study  highlighted the potential role of Fe in blocking Cu-mediated neurotoxicity.  160  The  extensive evidence of disrupted iron metabolism in A D still leaves many unanswered questions about the role of this metal ion in the disease.  161  The interaction of synthetic A p with Cu, Zn, and Fe, has been probed by a number of methods, including N M R ,  1 6 2  '  1 6 4  EPR,  1 4 1  '  1 5 4  '  1 6 3  '  1 6 5  '  1 6 6  and potentiometry. '  167 168  From these studies a high affinity metal binding site has been determined to involve three histidine (His) residues (His-6, His-13, His-14) as well as an oxygen ligand, most likely tyrosine-10 (Tyr-10) (Figure 1.7). Dimerization (leading to aggregation) may be mediated by a bridging histidine  169  (Figure 1.7) or di-tyrosine cross-linking.  170  In addition,  computational studies have implicated Tyr-10 as the pivotal residue in the catalytic production of H2O2 by the A p - C u complex.  171  18  The binding affinities of the A p peptide for  References start on p. 2 9  Cu and Zn are difficult to measure due to the propensity of the peptide to aggregate; a property that is significantly enhanced in the presence of metal ions. Binding affinity values have however been evaluated using a variety of methods. affinity C u  2 +  13  1,16X  1 7 2 1 7 3  A high-  -binding site on A p was determined, with the longer length AP1.42 peptide  exhibiting a much higher affinity for this metal ion as compared to that of binding constant reported for  A p  M  2  Aj$i-40.  The  and Cu is the second highest for any biomolecule,  exceeded only by Cu/Zn-superoxide dismutase. '  173 174  Figure 1.7: Proposed metal binding regions of the metal-AP complexes: (a) a monomeric C u - A p complex employing His-6, His-13, His-14, and Tyr-10 of A P for coordination; (b) dimerization (leading to aggregation) pathway of two Cu-AP complexes mediated by a bridging histidine. Adapted from ref [163].  The two additional amino acids (isoleucine-41, alanine-42) of the hydrophobic carboxyl-terminus of Api_4 (compared to A p M o ) are proposed to accelerate self2  aggregation,  173173  and enhance metal binding by increasing the P-sheet content  19  176  thereby  References start on p.29  thereby mediating other high-affinity Cu binding sites. In addition to the high-affinity metal binding site described, A(3 is able to bind another 2.5 equivalents of metal ions per 173  subunit.  Although these lower affinity metal binding sites have not been fully  characterized, they may play an important role in ROS generation, aggregation, and subsequent neurodegeneration. ' '  89 154 158  The high binding affinity of A(3 for metal ions has  led some researchers to postulate that one physiological role of the A p peptide is to interact with and sequester metal ions in the brain. Based on observations that A p binds metal ions, as well as proteins and macromolecules, a neuroprotective role for A p was postulated, termed the bioflocculant hypothesis.  177  The premise behind this hypothesis is  that A p is not toxic junk, but specifically designed to sequester contaminants,  178  the end  result being the deposition of amyloid plaques. The presence of excess contaminants such as metal ions may overload this defense system, leading to uncontrolled ROS production and A p aggregation observed in A D . 1.5 Metal-sequestration as an AD Therapeutic Strategy  Without basic knowledge of the biochemical origins of A D , the development of effective treatments is very difficult. Numerous therapeutic strategies have however been developed based on the current understanding of the disease. The therapeutic potential of antioxidant molecules and other therapies designed to minimize oxidative stress have garnered significant attention. For example, administration of vitamin E (Figure 1.8) has been shown to increase the median survival time in an A D trial.  179  Non-steroid anti-  inflammatory drugs (NSAIDS), such as ibuprofen (Figure 1.8), have also shown  20  References start on p.29  therapeutic potential, most likely by reducing inflammation associated with A D .  1 8 0  A  downstream effect of ROS generation is the overstimulation of glutamate receptors which can result in neuronal damage.  181  Memantine (Figure 1.8), a N-methyl-D-aspartate  (NMDA) receptor antagonist, has been approved by the Federal Drug Administration (FDA) for A D treatment as it has shown therapeutic benefit in patients with advanced 182  dementia.  The cholinesterase inhibitors, a class of compounds which inhibit the  breakdown of the neurotransmitter acetylcholine (deficient in AD), are the only other approved treatment strategy.  Cholinesterase inhibitors such as tacrine (Figure 1.8) have  been shown to slow the progression of the disease for a year or more.  184  While the  symptomatic treatments mentioned so far have shown modest effects, their therapeutic window is limited as they address the consequences of oxidative stress and not the underlying cause of ROS generation.  COOH  Vitamin E ((±)-a-tocopherol)  NH  (±)-Ibuprofen  2  Tacrine (acetylcholinesterase inhibitor)  Memantine  Figure 1.8: Chemical structures of A D therapeutics.  21  References start on p.29  Numerous strategies have been developed, based on the amyloid hypothesis, in order to lower the A(3 content in the brain in all of its forms. Inhibitors of the APP processing enzymes (3- and y-secretase 185  Immunization with  AP1.42  186  have been developed to limit A p generation.  in a transgenic mouse model of A D reduced amyloid plaque  formation as well as other AD-like neuropathologies.  131  These important findings led to  clinical trials of an Alzheimer's vaccine (AP1.42); unfortunately these trials were discontinued when 6% of the patients developed an inflammatory reaction in the C N S .  187  The exact cause of this inflammation is unknown but seems to be unrelated to the presence or absence of A p antibodies.  188  Antibodies that do not elicit an inflammatory  response (based on fragments of the AP) are now being investigated as potential therapies.  189  A treatment strategy that targets the underlying cause of oxidative stress as well as disrupting AP pathology in the form of metal ion chelators has shown considerable promise. The first attempt to target metal ions in neurodegenerative disease was the evaluation of desferoxamine (DFO) (Figure 1.9) administration to A D patients over a 24 month period. This study showed a significant decrease in the rate of decline of the treated subjects compared with the control group.  190  However, DFO is not able to cross  the B B B and thus must have exerted its effects systemically. While the results were promising, the toxicity of DFO and administration method (intramuscular) limit the longterm utility of this compound. The copper chelator D-penicillamine (Figure 1.9) has also exhibited therapeutic potential by reducing systemic copper levels as well as oxidative stress in A D patients. '  191 192  Unfortunately D-penicillamine exhibits toxic side-effects  limiting its long-term use. ' 191  193  Finally, tetrathiomolybdate (Figure 1.9) was shown to  22  References start on p.29  reduce neurological decline in Wilson's disease (characterized by excess Cu levels) most probably by systemic chelation of C u .  194  The above studies highlight the possibility that  systemic chelation and removal of metal ions may result in a lowering of brain metal levels and concomitant neurological decline. The use of chelators to sequester excess metal ions directly in the brain thereby limiting redox activity and facilitating redistribution and/or clearance may offer increased benefits over systemic metal depletion.  2HN. HOOC'. H  SH  2 1  s  D-Penicillamine  N I OH  Tetrathiomolybdate  DFO  HOOC N  / N  / N  s  HOOC  \ J  COOH  HOOC HN 9  COOH  N H  N H  NH?  C 0 0 H  TETA  DTPA  COOH L / N  k  Figure 1.9:  /  N  HOOC^  \ /—COOH  N  ^COOH EDTA  \/ \/ \ ^COOH O O N k.COOH  COOH  TPEN  V  BC  EGTA  Structures of metal chelators used in studies to disrupt metal-Ap interactions.  23  References start on p. 29  Studies have shown that the production of H2O2 via the interaction of Cu Fe  3+  with  AP1-42  and  is significantly attenuated in vitro by the presence of the chelators  diethylenetriaminetetraacetic acid (DTPA), triethylenetetraamine (TETA), and DFO (Figure 1.9).  145  In addition, metal chelators such as ethylenediaminetetraacetic acid  (EDTA) (Figure 1.9) are able to reverse Zn- and Cu-induced A(3 precipitation in vitro. '  151 195  Analogously, the chelators bathocuproine (BC), tetrakis(2-  pyridylmethyl)ethylenediamine (TPEN), and ethylene glycol-bis(2-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA) (Figure 1.9) re-solublize A P from post-mortem brain 118  tissue.  These studies further highlight the potential therapeutic benefit of metal ion  chelators in reducing plaque pathology and ROS generation, particularly i f the chelator can gain access to the brain. The most promising chelator for A D treatment is 5-chloro-7iodo-8-hydroxyquinoline (clioquinol, Hcq) (Figure 1.10), an antibiotic that was withdrawn from the market in the 1970's due to neurological side-effects that are now believed to be preventable with vitamin B12 supplementation.  196  Clioquinol is known to  have a moderate affinity for metal ions and to cross the B B B in humans. structures of Zn(cq)2 and Cu(cq)2 have also been recently reported.  197  The X-ray  In a similar fashion  to the chelators discussed above, clioquinol was demonstrated to reverse Zn/Cu-induced aggregation of synthetic A p , as well as to re-solublize post-mortem plaque deposits.  A  study in an A D mouse model demonstrated that the administration of clioquinol decreased brain A p deposition while stabilizing health and body-weight parameters. Clioquinol was also found to inhibit A p toxicity in neuronal cell cultures.  141  The potential  of clioquinol was further evaluated in a clinical trial where it showed indications of slowing neurological decline.  199  24  References start on p. 29  Due to the potential toxic effects of clioquinol, new chelators are being developed that are better tolerated and incorporate additional design elements to increase therapeutic potential. For example, a series of compounds based on the combination of a hindered phenol (antioxidant) and a pyridinone (chelator) (Figure 1.10) have been investigated as multifunctional drugs for AD treatment.  200  OH  Pyridinone-antioxidant conjugates  Clioquinol  OH Q  Carbohydrate-protected 3 -hydroxy-4-pyridinones  Figure 1.10: Promising molecules for AD chelation therapy.  As well, a pyridinone-carbohydrate conjugate, Feralex-G (Figure 1.10), has been evaluated as a potential treatment for AD.  The Orvig group has recently synthesized a  series of pyridinone-carbohydrate pro-drugs for potential use in treating AD (Figure 909  1.10).  These compounds carry carbohydrate functions to minimize systemic metal  25  References start on p.29  binding and improve targeting potential (via carbohydrate transport pathways), as well as to enhance compound solubility. Chapter 3 is a logical companion to this work, and describes the synthesis and evaluation of tetrahydrosalen-carbohydrate pro-drugs for A D therapy.  1.6 Target-specific Radiopharmaceuticals in Nuclear Medicine  Nuclear medicine relies on the use of radiopharmaceuticals (compounds containing radionuclides) for the diagnosis, and more recently the treatment, of disease. Diagnostic radiopharmaceuticals provide a non-invasive means of assessing the physiology and morphology of organs and tissues and have become very important in the diagnosis of disease and assessing the progress of treatment strategies. Radiopharmaceuticals are administered almost exclusively by intravenous injection in very low concentrations (10~ -10" M ) 6  8  2 0 3  as these compounds are not  intended to elicit a pharmacologic effect. There are two general techniques used to produce diagnostic images, single photon emission computed tomography (SPECT), and positron emission tomography (PET). In SPECT imaging, a radionuclide is used which decays by the emission of a y-ray, and the detected emissions are then re-constructed to afford a three dimensional image of the area of interest. PET imaging relies on much the same technology to produce useful images but differs in its use of radionuclides that decay by positron emission; a further annihilation reaction affords two 180° opposed yrays with an energy of 511 keV which are detected. While PET imaging affords better spatial resolution, the high cost of PET and the relatively short half-lives of PET emitters  26  References start on p. 2 9  ( F, ti/2 = 1.8 h; C , ti/2 = 20 min; N , t 18  n  1 3  V2  = 10 min) have limited the utility of this  imaging method. SPECT imaging is much more prevalent and allows for the use o f  99m  Tc  which has ideal nuclear properties (ti/2 = 6.01 h, y = 142.7 keV) and is easily isolated in the form of N a  99m  T c 0 4 from a M o generator. Indeed, 9 9  9 9 m  T c is the most widely used  isotope in nuclear medicine, with some 85% of diagnostic scans employing this radionuclide.  204  Radiopharmaceuticals containing  9 9 m  T c have many applications including  imaging myocardial perfusion, cerebral blood flow, and tissues and organs such as the kidney, liver, and bone.  205  Other less commonly used SPECT radionuclides include G a 67  citrate for imaging tumours, inflammation and infection, as well as complexes of I n 1H  oxyquinoline for imaging platelets.  206 1 1  ' i n and  9 9 m  T c have also been attached to peptides 907  in an effort to develop target-specific radiopharmaceuticals for tumour imaging.  The  development of target-specific radiopharmaceuticals is attracting significant attention and is based on the attachment of radionuclides to biologically active molecules for selective localization in diseased tissue. The therapeutic potential of nuclear medicine lies in the possibility of designing target-specific radiopharmaceuticals to deliver therapeutic doses of ionizing radiation to disease sites. Specificity is very important so as not to introduce widespread radiation damage. Two therapeutic radiopharmaceuticals have been approved; a Sm-phosphonate complex for bone cancer pain palliation, l53  208  and a Y -  labelled monoclonal antibody for the treatment of non-Hodgkin's lymphoma.  9 0  209  Chapter 4 of this thesis explores the possibility of attaching radionuclides to carbohydrates to develop target-specific radiopharmaceuticals.  9 9 m  T c was chosen as the  radionuclide due to its ideal characteristics and ease of isolation. Rhenium, the third row  27  References start on p. 29  transition metal analog of technetium^ exhibits similar chemistry to that of technetium, a fact which can be exploited in developmental work. In addition, Re itself has particle emitting radioisotopes ( Re t 186  m  = 3.68 days, p = 1.07 MeV, y = 137 keV;  188  R e t,/ = 2  16.98 h, p = 2.12 MeV, y = 155 keV) with physical properties useful in therapeutic applications if suitable target specificity in disease tissue can be obtained.  1.7 Thesis Overview  This introductory chapter provides a background for the experimental work described in this thesis. Chapter 2 furthers the discussion of vanadium and diabetes (Section 1.2) and reports a conjugate approach towards diabetes therapy utilizing vanadium and the thiazolidinediones. A hybrid approach is used whereby the two aforementioned drugs are combined into one molecule. Animal studies in a diabetic model were used to determine the efficacy of the synthesized compounds. Current A D research is reviewed in Section 1.5 with potential therapeutic strategies discussed in Section 1.6. 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Cancer Res. 1996, 2, 457.  42  References start on p. 29  CHAPTER 2  Vanadyl-thiazolidinedione Combination Agents for Potential Use in Diabetes Therapy  2.1 Introduction  In an effort to increase the efficacy of vanadium-containing drug candidates for diabetes treatment, the coupling of vanadium with a ligand that contains an insulinaction-enhancing drug was explored. Combining orally effective glucose-lowering drug molecules with different mechanisms of action may lead to synergistic and/or additive effects. Additive effects occur when two drug entities administered concurrently elicit an effect that is equal to the sum of the effects of the two drugs when given individually. Drug synergism occurs when the total effect of the two components is greater than the sum of the constituent parts. Combination agents containing the vanadyl ion with a series of biguanides (see Figure 2.1 for metformin and VO(metf)2) were prepared to test the possibility of additive or synergistic effects. The biguanides were introduced in the 1  1950s as synthetic oral glucose-lowering agents to treat type 2 diabetes, and belong to a class of compounds that enhance insulin action without altering insulin secretion. '  2 3  Metformin is in current pharmaceutical use. This series of vanadium combination agents failed to produce additive or synergistic effects. However, VO(metf)2 was no less effective than B M O V (Chapter 1, Figure 1.3) as a glucose lowering agent based on shortterm trials. The lack of an additive effect may have been due to the large dosage 1  43  References begin on p. 78  differences required for these two classes of drugs. In an advance of this combination approach we chelated the vanadium center with a series of thiazolidinedionecontaining ligands; dosage differences between oxovanadium(IV) compounds and 4  representative thiazolidinediones (TZDs) are potentially smaller. " 5  NH  N  n—^  H  „»Vr  °  -o-y^  2  Metformin (biguanide)  Clofibrate (fibrate)  VO(metf)  7  / = \  i?  N  -Oh ' ' B  C,H  Tolbutamide (sulfonylurea)  (alpha-glucosidase inhibitor)  2  Figure 2.1: Structures ofthe different drug classes discussed in Chapter 2.  The TZDs are named for the five-membered heteronuclear ring common to all compounds of this class (Figure 2.2). Of the TZD compounds, ciglitazone, troglitazone, englitazone, pioglitazone, and rosiglitazone have been clinically examined as potential Q  anti-diabetic compounds. Both pioglitazone (Actos™) and rosiglitazone (Avandia™) are available on the market for the treatment of type 2 diabetes. Troglitazone proved to be hepatotoxic and was withdrawn from the market after several cases of liver failure were reported.  9  44  References begin on p. 78  Ciglitazone  Troglitazone  Figure 2.2: Examples from the thiazolidinedione (TZD) drug class.  TZDs act by indirectly enhancing peripheral insulin sensitivity, thereby lowering the levels of both glucose and insulin. TZDs are known to activate the peroxisome 10  proliferator-activated receptor gamma (PPARY) which is predominantly expressed in adipose tissue. " Another class of antidiabetic agents, the lipid lowering fibrates (Figure 11  13  2.1), target a closely related nuclear receptor PPARct. PPARs are orphan receptors 11  belonging to the steroid/thyroid/retinoid receptor superfamily of ligand-activated transcription factors and play a central role in regulating the storage and catabolism of lipids in both animals and humans. PPARy regulates numerous metabolic pathways 12  involving lipoprotein lipase, glucose transporters and insulin signalling pathways. The 13  45  References begin on p. 78  crystal structure of PP ARy with rosiglitazone has been solved and provides for a starting point in the molecular design of drug candidates capable of increased affinity for the binding pocket of this receptor. However, binding affinity of the TZDs for PPARy does 14  not always correlate with glucose-lowering potential. A major benefit of the TZDs is 15  that, unlike sulphonylurea derivatives (Figure 2.1), alpha-glucosidase inhibitors (Figure 2.1), or insulin, they influence insulin resistance. Compounds which improve insulin resistance enable the continued treatment of type 2 diabetic patients without inducing hypoglycemia. This class of compounds has been shown to enhance the sensitivity of target tissues to insulin and to reduce plasma glucose, lipid, and insulin levels in humans. While the TZDs have many beneficial effects, weight gain (3-5 kg in most 16  *  *  *  17  patients) is a perceived negative consequence.  It is proposed, however, that the increase  in adipose tissue decreases available fatty acids and reduces fatty acid uptake by the muscle, which in turn leads to an improvement in insulin sensitivity. '  18 19  There have been numerous studies examining the combined effect of TZD agents along with other classes of antidiabetic drugs. TZD drugs have been used in combination with sulphonylureas, metformin, fibric acid derivatives (Figure 2.1), and insulin to test for increased efficacy. " The combination therapies have been shown to improve glycemic control without an increased risk in the rate of hypoglycemic incidents. A recent study using rosiglitazone (2.8 pmol/kg) and B E O V (0.1 mmol/kg) (Chapter 1, Figure 1.2) in Zucker diabetic fatty (ZDF) rats showed at least an additive effect with combination treatment. As a companion to this aforementioned study, combination 24  agents containing the vanadyl ion as well as thiazolidinedione moieties as a single entity were prepared and tested.  4  46  References begin on p. 78  In order to form stable neutral complexes with the vanadyl ion, bifunctional ligands were developed, tethering the active portion of the thiazolidinedione molecule to a suitable chelator. Kojic acid 1 (Figure 2.3) was chosen for this purpose because, like maltol (Figure 2.3), it forms, in its deprotonated form, water soluble, neutrally charged complexes with appropriate metal ions. Simple kojic acid complexes with V 0 25  2 +  have  also shown significant anti-diabetic activity. The planar ring system of kojic acid was 26  also well suited for interacting with the binding domain of the PPARy receptor. It was 14  hypothesized that deprotonated kojic acid would be the most versatile binding moiety as the planar ring system is also capable of chelating vanadium.  OH  Maltol  Kojic acid 1  Figure 2.3: The structures of maltol and kojic acid 1.  The concept of combining vanadium and a thiazolidinedione-containing ligand to form a compound that exhibits additive or synergistic effects is quite appealing. The preparation and characterization of a series of such bifunctional ligand precursors and their associated oxovanadium(IV) complexes are presented herein. Results from preliminary acute STZ-diabetic rat studies of four ligand precursors and three of the corresponding vanadium complexes are also reported and compared to rosiglitazone and B M O V , respectively.  47  References begin on p. 78  2.2 Experimental  2.2.1 Materials  Water was deionized, purified (Barnstead D9802 and D9804 cartridges) and distilled with a Corning MP-1 Mega-Pure Still. A l l solvents were purchased from Fisher except for dry D M F (Aldrich, "anhydrous", <0.003% H 0 ) . Other anhydrous solvents 2  that were required were dried according to standard procedures. Chemicals were from 27  Aldrich (Madison, WI) unless otherwise stated. A l l N M R solvents were purchased from Cambridge Isotope Laboratories. 3-Hydroxy-2-methyl-4-pyranone (maltol, Figure 2.3) and 5-hydroxy-2-(hydroxymethyl)-4-//-pyran-4-one (kojic acid 1, Figure 2.3) were obtained from Pfizer (Kirkland, PQ). Thin layer chromatography (TLC) was performed using silica T L C plates with aluminium backing (Merck). Silica for column chromatography was from Silicycle (Quebec City, PQ).  2.2.2 Instrumentation  Infrared spectra were recorded as K B r disks in the range 4000-500 cm" on a 1  Galaxy Series 5000 FTIR spectrometer and were standardized to polystyrene. Mass spectra were obtained on either a Kratos M S 50 (electron-impact ionization, EI-MS) spectrometer, a Kratos Concept II H32Q instrument (Cs , LSIMS; or N H , CI-MS), or a +  3  Macromass L C T (electrospray, ES-MS) instrument. ' H and ^Cf/H} N M R spectra were recorded on a Bruker AC-200E, AV-300 or an AV-400 instrument at 200 (50.2 for C 1 3  48  References begin on p. 78  N M R ) , 300.13 (75.48 for C N M R ) or 400.13 (100.62 for C N M R ) M H z , respectively. 1 J  1 J  The N M R spectra were calibrated with the deuterated solvent used in each case. Melting points were measured using a Gallenkamp melting apparatus and are uncorrected. Microanalyses for C, H , and N were done in this department (Mr. P. Borda or Mr. M . Lakha) or by Delta Microanalytical Services (Delta, BC). Room-temperature (293 K ) magnetic susceptibilities were measured on a Johnson Matthey balance, using Hg[Co(NCS)4] as the susceptibility standard; diamagnetic corrections were estimated using Pascal's constants.  Room temperature X-band EPR spectra were recorded on a  Bruker ECS-106 EPR spectrometer in 20pL quartz capillaries. The microwave frequency and magnetic field were calibrated with an EIP 625A microwave frequency counter and a Varian E500 gaussmeter, respectively. Computer simulations of the isotropic EPR spectra were performed using the Bruker WINEPR/SIMFONIA package.  2.2.3 In Vivo STZ-diabetic Rat Studies  These experiments were conducted by the research group of Prof. J. H . McNeill in the U B C Faculty of Pharmaceutical Sciences. Male Wistar rats weighing 190-210 g (Animal Care Unit, U B C ) were housed two per cage (polycarbonate) on a 12 h light:dark cycle. Animals were allowed ad libitum access to food (Purina Rat Chow #5001) and water, and were cared for in accordance with the principles and guidelines of the Canadian Council for Animal Care. Animals were allowed to acclimatize for a period of 7 days, and then diabetes was induced by a single tail vein injection of streptozotocin (STZ, Figure 2.4) (60 mg kg" in 0.9% NaCI) under light halothane anesthesia. Diabetes 1  49  References begin on p. 78  was confirmed three days after STZ-injection by tail-vein blood glucose determination (Ames Glucometer II and Glucostix), with blood levels over 13 m M being taken as diabetic. OH  Figure 2.4: Structure o f streptozotocin (STZ)  One week post-STZ, animals were divided into treatment groups: carboxymethylcellulose ( C M C ) alone, thiazolidinedione compound, congeneric vanadium complex, or B M O V . A l l drugs were administered by i.p. injection as suspensions i n 1% C M C . Animals were not fasted prior to drug administration. Blood, 50 p L , was collected for glucose analysis immediately prior to drug administration and at selected times up to 72 h after drug administration. B l o o d was collected from the tail vein into heparinized capillary tubes and centrifuged (10,000 g x 15 minutes) and plasma was collected for immediate determination o f glucose using Boehringer Mannheim kits (glucose oxidase method). The hypoglycemic activity o f the test compounds was calculated as shown i n equation 2.1 ' : 8  11  Hypoglycemic activity (%) =  50  PGc - PGr PGc  x!00%  (Eq. 2.1)  J  References begin on p. 78  PGc is plasma glucose in control mice and PGT is plasma glucose in the mice treated with test compounds. Values are presented as means ± S E M at 12, 24, and 48 hours. At all time points, the animals were observed for signs of toxicity (e.g. diarrhea). Studies were done on four separate occasions; in each study four groups were examined C M C , B M O V , ligand precursor, and vanadyl complex. For V O ( L ) , the animals were !  2  divided into C M C (n = 5), B M O V (n = 5), H L (n = 10), VO(L ) (n = 10); for H L , 1  1  2  2  C M C (n = 5), B M O V (n = 6), H L (n = 10), rosiglitazone (n = 10); for VO(L ) , C M C 2  3  2  (n = 6), B M O V (n = 10), H L (n = 10), V O ( L ) (n = 10); and for VO(L ) , C M C (n = 3  3  4  2  2  5), B M O V (n = 5), H L (n = 10), V O ( L ) (n = 10). A l l drug candidates were 4  4  2  administered by i.p. injection in a volume of 2.5 mL kg" at a dose of 0.1 mmol kg" . The 1  1  control groups (CMC) received an equivalent volume of 1% C M C alone. There was no significant effect of drug administration on body weight over the treatment period in any of the trials. Results are shown in Table 2.2 (p.75) and Figure 2.6 (p.76).  2.2.4 Ligand Synthesis  5-Benzyloxy-2-hydroxymethyl-pyran-4-one (2)  Q X n B n II  r  J  This compound was prepared in a manner similar to that described in the literature, with minor modifications. Sodium (2.30 g, 100 mmol) was 29  0  OH  added to dry MeOH (200 mL) and then kojic acid 1 (14.20 g, 100 mmol) 2 was added in portions. To this solution benzyl chloride (14 mL, 100  mmol) was added dropwise and the mixture was heated to reflux for 5 h, cooled to room 51  References begin on p. 78  temperature (r.t.) and then poured into H2O (1 L). The resulting precipitate was collected and recrystallized from a minimum of hot EtOH (80 mL) to afford the title compound 2 as colourless crystals (12.7 g, 55 %); m.p. 120-122 °C. H N M R (MeOH-<4 300.13 J  MHz): 5 8.00 (s, 1H; kojic ring//), 7.38 (m, 5H; Ar//), 6.50 (s, 1H; kojic ring//), 5.01 (s, 2H; AxCH ), 4.39 (s, 2H; kojicC// ). EI-MS m/z (relative intensity) = 232 ([M] , 60), 91 +  2  2  (100). Anal. Calcd. (found) for C i H i 0 : C, 67.23 (67.42); H , 5.21 (5.26). 3  2  4  Methanesulfonic acid 5-benzyloxy-4-oxo-4H-pyran-2-ylmethyl ester (3)  To a suspension of 5-benzyloxy-2-hydroxymethyl-pyran-4-one 2 (0.303  0  A / O B n  II  J  g, 1.30 mmol) in dry CH C1 (10 mL) was added triethylamine (0.36 2  OMs  2  mL, 2.60 mmol). The suspension was then cooled in an ice bath and 3 methanesulfonyl chloride (0.12 mL, 1.60 mmol) was added dropwise.  The mixture was stirred for 0.5 h, quenched with saturated N a H C 0 (15 mL), and then 3  extracted with CH C1 ( 2 x 1 5 mL); the combined organic extracts were dried over 2  2  anhydrous Na SC»4, and the solvent was removed under reduced pressure. The slightly 2  coloured oil 3 was used without further purification. H N M R ( C H C l - ^ i , 300 MHz): 8 !  3  7.57 (s, 1H; kojic ring//), 7.38 (m, 5H; Ar//), 6.55 (s, 1H; kojic ring//), 5.08 (s, 2H; A r C / / ) , 4.95 (s, 2H; kojicC// ), 3.08 (s, 3H; - S 0 C / / ) . 2  2  2  52  5  References begin on p. 78  5-(4-Nitro-benzyIidene)thiazolidine-2,4-dione (5)  N°2 [I  To a solution of 4-nitrobenzaldehyde 4 (5.00 g, 33.0 mmol) and  I  thiazolidine-2,4-dione (3.87 g, 33.0 mmol) in toluene (200 mL) was added c  l\ O  C = =  ^ H  X N  benzoic acid (0.68 g, 5.5 mmol) and piperidine (0.55 mL, 5.5 mmol). The resulting mixture was refluxed for 2 h with the continuous removal of  ^O  water using a Dean-Stark water separator. The reaction mixture was then cooled to r.t. and the resulting precipitate was collected and washed with CH2CI2 and EtiO to yield the product as a pale yellow solid (4.50 g, 55 %); m.p. 250 °C. H N M R (DMSO-d , 300 !  6  MHz): 5 8.35 (d, J = 8.6 Hz, 2H; AxH), 7.91 (s, 1H; alkene/7), 7.87 (d, J = 8.6 Hz, 2H; 3  3  AxH). EI-MS m/z (relative intensity) = 250 ([M] , 35), 179 (100). Anal. Calcd. (found) +  for C i H N O 4 S : C, 48.00 (48.00); H , 2.42 (2.46); N , 11.20 (10.99). 0  6  2  (±)-5-(4-Amino-benzyl)thiazolidine-2,4-dione (6)  f ^  This compound was prepared by a more direct method than that reported 30  in the literature. C0CI26H2O (0.30 g, 1.3 mmol) and dimethylglyoxime (0.56 g, 4.9 mmol) were added to H 0 (300 mL). After addition of NaOH  6  2  S  N ' ^  0  (3 mL, 1 M), the resulting brown solution was cooled to 0 °C in an ice  H  bath and N a B H (7.90 g, 209 mmol) was added in portions. 5-(4-Nitro4  benzylidene)thiazolidine-2,4-dione 5 (5.22 g, 20.9 mmol) was then added slowly as a slurry in THF (50 mL). The reaction mixture was stirred at r.t. for 16 h. The pH was then adjusted to 9 with C H 3 C O O H and the mixture was extracted with THF (3 x 200 mL). The  53  References begin on p. 78  combined organic extracts were dried over Na2S04, and the solvent removed under reduced pressure. The residue was purified by silica-gel chromatography (95:5 CHiCliMeOHeluent) to afford the title compound 6 as a pale yellow solid (3.31 g, 71 %); m.p. 152-153 °C. ' H N M R (DMSO-c/ , 300 MHz): 5 6.89 (d, J = 8.3 Hz, 2H; AxH), 3  6  6.49 (d, J = 8.3 Hz, 2H; ArH), 4.65 (dd, J= 9.3 Hz, V = 4.4 Hz, 1H; TZD/7), 3.20 (dd, 3  3  J= 14.4 Hz, J= 4.4 Hz, 1H; T Z D C / / ) , 2.91 (dd, J= 14.4 Hz, J = 9.3 Hz, 1H;  2  3  2  3  2  TZDC/7 ). EI-MS m/z (relative intensity) = 222 ([M] , 60), 106 (100). Anal. Calcd. +  2  (found) for CioH N 02S: C, 54.04 (54.26); H, 4.53 (4.53); N , 12.60 (12.45). 10  2  (±)-5-{4-[(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethyl)amino]benzyl}thiazolidine-2,4dione (7)  Methanesulfonic acid 5-benzyloxy-4-oxo-4H-pyran-2-ylmethyl ester 3 (2.67 g, 9.05 mmol) and (±)-5-(4-aminobenzyl)thiazolidine-2,4-dione 6 (2.00 g, 9.0 mmol) were dissolved in dry C H C N (50 mL). K C 0 (2.50 g, 18 mmol) was then added 3  2  3  as a solid and the resulting mixture was heated at 50 °C for 24 h with stirring. The solvent was then removed under reduced pressure and the residue partitioned between C H C 1 (50 mL) and saturated N a H C 0 (30 mL). The mixture was 2  2  3  extracted with C H C 1 (2 x 50 mL); the organic extracts were dried over Na SC>4 and 2  2  2  concentrated in vacuo. The crude product was purified by silica-gel chromatography (97:3 CH Cl2:MeOH eluent) to afford the title compound 7 as a pale yellow solid (2.73 g, 2  69 %); m.p. 57-59 °C. ' H N M R (acetone-</ , 300 MHz): 5 7.86 (s, 1H; kojic ring//), 7.37 6  54  References begin on p. 78  (m, 5H; AiH), 6.95 (d, J = 8.3 Hz, 2H; AiH), 6.59 (d, J = 8.3 Hz, 2H; AiH), 6.21 (s, 3  3  1H; kojic ring//), 5.02 (s, 2H; ArC/fc), 4.82 (dd, V = 8.4 Hz, J= 4.1 Hz, 1H; TZDH), 3  4.57 (s, 2H; kojicC// ), 3.33 (dd, J = 14.1 Hz, J = 4.1 Hz, 1H; TZDC/Y ), 3.09 (dd, J = 2  3  2  2  2  14.1, J= 8.4 Hz, 1H; T Z D C / / ) . EI-MS m/z (relative intensity) = 436 ([M] ,10), 196 3  +  2  (20), 106 (100), 91 (55). Anal. Calcd. (found) for C23H20N2O5S : C, 63.29 (63.39); H , 4.62 (4.59); N , 6.42 (6.43).  (±)-5- {4- [(5-Hy droxy-4-oxo-4H-pyran-2-y lmethyl)amino] benzyl} thiazolidine-2,4dione (HL ) 1  5 - {4- [(5 -Benzyloxy-4-oxo-4H-pyran-2-  0  ylmethyl)amino]benzyl}thiazolidine-2,4-dione 7 (3.75 g, 8.6 mmol) was dissolved in a mixture of concentrated HCI (20 mL) n  and C H 3 C O O H (40 mL) and heated at 70 °C with stirring for 48  h  O  h. The yellow solution was rotary evaporated to dryness and then H2O (3 mL) was added and the pH adjusted to 5. The resulting  precipitate was isolated and washed with a minimum of cold H2O to afford, after drying, the title compound as a pale yellow solid (2.71 g, 70 %); m.p. 146 °C. *H N M R ( D 0 , 2  300 MHz): 8 7.85 (s, 1H; kojic ring/7), 7.29 (d, V = 8.5 Hz, 2H; Ar//), 7.22 (d, V = 8.5 Hz, 2H; Ar//), 5.76 (s, 1H; kojic ring//), 4.58 (dd, J = 6.4 Hz, V = 4.8 Hz, 1H; TZDH), 3  4.51 (s, 2H; kojicC// ), 3.76 (dd, J= 15.5 Hz, V = 6.4 Hz, 1H; T Z D C / f c ) , 3.63 (dd, 2  2  J=  2  15.5 Hz, J = 4.8 Hz, 1H; T Z D C / / ) . EI-MS m/z (relative intensity) = 346 ([M] , 4), 106 3  +  2  55  References begin on p. 78  (100). Anal. Calcd. (found) for C H i N O S - 0 . 5 H O : C, 54.08 (54.21); H , 4.25 (4.06); 16  4  2  5  2  N , 7.88 (7.74).  5-Benzyloxy-2-hydroxymethyl-l-methyl-lH-pyridin-4-one (8)  O  This compound was synthesized in a higher yield than that reported in OBn  the literature.  5-Benzyloxy-2-hydroxymethyl-pyran-4-one 2 (0.99 g,  4.27 mmol) was suspended in a mixture of EtOH and H2O (10 mL/10  OH  8 mL) and methylamine (0.56 mL, 40% w/w, 6.42 mmol) was added. The mixture was heated to reflux for 6 h and then cooled to r.t. at which time the product crystallized from the solution. The solid was washed with Et20 and dried in vacuo to afford the title compound 8 as an off-white solid (0.81 g, 77 %); m.p. 218-220 °C. *H N M R (MeOH-^4, 300 MHz): 5 7.60 (s, 1H; kojic ring//), 7.48 (m, 2H; Ar//), 7.37 (m, 3H; Ar//), 6.61 (s, 1H; kojic ring//), 5.10 (s, 2H; A r C / / ) , 4.55 (s, 2H; kojicC// ), 3.77 (s, 2  2  3H; NC//?). DCI-MS m/z = 245 ([M] , 100), 168 (15), 139 (85), 91 (74). Anal. Calcd. +  (found) for C i H N 0 : C, 68.56 (68.42); H , 6.16 (6.22); N , 5.71 (5.81). 4  1 5  3  5-Benzyloxy-2-chloromethyl-l-methyl-lH-pyridin-4-one hydrochloride salt (9)  O  5-Benzyloxy-2-hydroxymethyl-l-methyl-lH-pyridin-4-one 8 (2.10 g, OBn  8.57 mmol) was added with stirring to form a suspension in dry CH C1 2  2  (20 mL). Thionyl chloride (5.0 mL, 68.9 mmol) was then added 9  dropwise. The resulting light yellow solution was stirred for 16 h under  56  References begin on p. 78  Ar and then the solvent was removed under reduced pressure. EtOAc (15 mL) was added and the triturated solid was collected and washed with hexanes to afford the title compound 9 as a white solid (2.45 g, 96 %); m.p. 170-172 °C. ' H N M R (DMSO-J , 300 6  MHz): 8 8.62 (s, 1H; kojic ring/*), 7.40 (m, 6H; A r / / , kojic ring//), 5.17 (s, 2H; ArC/7 , 2  or kojicC/fc), 5.04 (s, 2H; ArC/fc, or kojicC/6). DCI-MS m/z = 263 ([M] , 16), 228 (20), +  157 (30), 91 (100). Anal. Calcd. (found) for C i H i N 0 - H C l : C, 56.00 (56.00); H , 5.00 4  3  2  (5.04); N , 4.67 (4.72).  ( ± ) - 5 - {4- [(5-Benzy loxy- l-methyl-4-oxo-l ,4-dihydro-py ridin-2-  ylmcthyl)amino]benzyl}thiazolidine-2,4-dionc (10)  (±)-5-(4-Amino-benzyl)thiazolidine-2,4-dione 6 (1.55 g, 6.98 mmol) and 5-benzyloxy-2-chloromethyl-l-methyl-lH-pyridin-4one hydrochloride salt 9 (2.08 g, 6.98 mmol) were dissolved in D M F (50 mL). Triethylamine (3 mL, 21 mmol) was added 10  O  dropwise and the resulting mixture was stirred for 22 h. Water (60  mL) was added and the resulting mixture extracted with C H C 1 (2 x 70 mL). The 2  2  combined organic extracts were dried over N a S 0 , filtered, and then concentrated. The 2  4  crude product was purified by silica-gel chromatography (95:5 CH Cl :MeOHeluent) to 2  2  afford the title compound 10 as a pale yellow solid (2.65 g, 88 %); m.p. 85-87 °C. *H N M R (CHCl -rfi, 300 MHz): 8 7.32 (m, 5H; Ar//), 6.85 (s, 1H; kojic ring/*), 6.84 (d, J = 3  3  8.3 Hz, 2H; Ar//), 6.43 (d, J = 8.3 Hz, 2H; Ar//), 6.12 (s, 1H; kojic ring//), 5.08 (s, 2H; 3  ArC/6), 4.44 (dd, J = 8.0 Hz, J = 3.9 Hz, 1H; TZD//), 4.42 (s, 2H; kojicC/Y ), 3.35 (s, 3  3  2  57  References begin on p. 78  3H; NCH ), 3.20 (dd, J= 14.2 Hz, J = 3.9 Hz, 1H; T Z D C / / ) , 3.06 (dd, J= 14.2 Hz, J 2  3  2  3  3  2  = 8.0 Hz, 1H; T Z D C / / ) ; EI-MS m/z = 449 ([M] , 45), 344(10), 238(35), 106(80), +  2  91(100). Anal. Calcd. (found) for C  2  4H N3 2 3  0 S H C 1 : C, 59.32 (59.52); H , 4.98 (4.90); N , 4  8.65 (8.49).  (±)-5-{4-[(5-Hydroxy-l-methyl-4-oxo-l,4-dihydro-pyridin-2yImethyl)amino]benzyl}thiazolidine-2,4-dione (HL ) 2  The title compound H L (1.50 g, 73 %) was prepared as a light  0  2  OH  yellow solid from (±)-5-{4-[(5-benzyloxy-l-methyl-4-oxo-l,4dihydro-pyridin-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione \z^L  i 2  X N H  O  10 (2.60 g, 5.80 mmol) by a procedure analogous to that described for H L ; m.p. 108-110 °C. ' H N M R (DMSO-<4, 300 MHz): 5 1  HL 7.58 (s, 1H; kojic ring//), 6.94 (d, J= 8.1 Hz, 2H; AiH), 6.58 (d, J = 8.1 Hz, 2H; ArH), 3  3  5.99 (s, 1H; kojic ring/7), 5.06 (dd, J = 8.9 Hz, J= 4.2 Hz, 1H; TZDH), 4.69 (s, 2H; 3  3  kojicC// ), 3.69 (s, 3H; N C / / ) , 3.34 (dd, J= 14.1 Hz, V=4.2 Hz, 1H; T Z D C / / ) , 3.04 2  2  5  2  (dd, J= UA Hz, J=8.9 Hz, 1H; T Z D C / / ) . EI-MS m/z = 359 ([M] , 30), 254 (60), 181 2  3  +  2  (30), 106 (100). Anal. Calcd. (found) for C Hi7N O4S-0.5H O: C, 55.42 (55.19); H , 4.92 17  3  2  (4.75); N , 11.41 (11.28).  58  References begin on p. 78  4-(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzaldehyde (11) O OBn  Methanesulfonic acid 5-benzyloxy-4-oxo-4H-pyran-2-ylmethyl ester 3 (5.37 g, 17.3 mmol) was dissolved in D M F (150 mL) and 4-  O,  hydroxybenzaldehyde 14 (2.11 g, 17.3 mmol) was added. To this stirred CHO  solution K2CO3 (7.18 g, 52 mmol) was added and the mixture was  11  heated at 60 °C for 16 h. Water (250 mL) was then added and the mixture extracted with CH2CI2 (3 x 200 mL). The combined organic extracts were washed with a saturated aqueous NaCI solution (50 mL), dried over Na2S04, and the solvent removed under reduced pressure. The crude material was washed with acetone to afford 11 as a white solid (3.72 g, 64 %); m.p. 155 °C. ' H N M R (acetone-</ , 300 MHz): 5 9.93 (s, 1H; 6  CHO), 8.04 (s, 1H; kojic ring//), 7.92 (d, V = 8.5 Hz, 2H; Ar//), 7.40 (m, 5 H ; Ar//), 7.25 (d, J = 8.5 Hz, 2H; Ar//), 6.54 (s, 1H; kojic ring//), 5.15, (s, 2H; A r C / / , or kojicC// ), 3  2  2  5.07 (s, 2H; A r C / / , or kojicC/Y ). EI-MS m/z (relative intensity) = 336 ([M] , 4), 214 +  2  2  (10), 91 (100). Anal. Calcd. (found) for C o H , 0 : C, 71.42 (71.59); H , 4.79 (4.72). 2  6  5  5-[4-(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzylidene]thiazolidine-2,4-dione (12)  O  The title compound 12 (1.50 g, 57 %) was prepared as a light orange solid from 4-(5-benzyloxy-4-oxo-4H-pyran-2O,  ylmethoxy)benzaldehyde 11 (2.11 g, 6.28 mmol), thiazolidine-2,4dione (0.74 g, 6.28 mmol), benzoic acid (0.15 g, 1.25 mmol), and . 12  O  59  References begin on p. 78  piperidine (0.12 mL, 1.25 mmol) by a procedure analogous to that described for 5; m.p. 253-254 °C. ' H N M R (DMSO-d , 300 MHz): 5 12.52 (s, 1H; N / / ) , 8.27 (s, 1H; kojic 6  ring/7), 7.75 (s, 1H; alkene//), 7.58 (d, J = 8.6Hz, 2H; Ar//), 7.39 (m, 5H; Ar//), 7.20 (d, 3  3  J = 8.6Hz, 2H; Ar//), 6.56 (s, 1H; kojic ring//), 5.10 (s, 2H; ArC/fc, or kojic-C/fc), 4.94  (s, 2H; A r C / / , or kojicC// ). EI-MS m/z (relative intensity) = 435 ([M] , 7), 91 (100). +  2  2  Anal. Calcd. (found) for C H N 0 S : C, 63.44 (63.16); H , 3.93 (4.06); N , 3.22 (3.21). 2 3  1 7  6  5-[4-(5-Hydroxy-4-oxo-4H-pyran-2-ylmethoxy)benzyIidene]thiazoIidine-2,4-dione (HL ) 3  The title compound H L (1.60 g, 67 %) was prepared as a light beige solid from 5-[4-(5-benzyloxy-4-oxo-4H-pyran-2ylmethoxy)benzylidene]thiazolidine-2,4-dione 12 (3.00 g, 6.90 mmol) by a procedure analogous to that described for H L ; m.p. 1  O HL  275-276 °C. H N M R (DMSO-c/ , 300 MHz): 5 12.54 (s, 1H; N//), !  6  3  9.26 (s, 1H; OH), 8.12 (s, 1H; kojic ring//), 7.76 (s, 1H; alkene//),  7.59 (d, J = 8.8 Hz, 2H; AiH), 7.20 (d, J = 8.8 Hz, 2H; AiH), 6.57 (s, 1H; kojic ring//), 3  3  5.10 (s, 2H; kojicC/6). DCI-MS m/z (relative intensity) = 345 ([M] , 100), 221 (40), 203 +  (35), 150 (60), 125 (100). Anal. Calcd. (found) for C i H , i N 0 S : C, 55.65 (55.23); H , 6  6  3.21 (3.19); N , 4.06 (4.03).  60  References begin on p. 78  5-Benzyloxy-2-chloromethyl-pyran-4-one (13)  °  The title compound 13 (2.01 g, 62 %) was prepared as a white solid OBn  from 5-benzyloxy-2-hydroxymethyl-pyran-4-one 2 (3.00 g, 12.9 mmol) by a procedure analogous to that described for 9; m.p. 109-110 °C. ' H N M R (CHCl -^i, 300 MHz): 5 7.58 (s, 1H; kojic ring//), 7.38 (m, 5H; 3  Ar//), 6.56 (s, 1H; kojic ring//), 5.09 (s, 2H; A r C / / ) , 4.29 (s, 2H; kojicC// ). EI-MS m/z 2  2  (relative intensity) = 250 ([M] , 7), 126 (10), 108 (28), 91 (100). Anal. Calcd. (found) for +  C13H11CIO3:  C, 62.29 (62.41); H, 4.42 (4.41).  5-(4-Hydroxy-benzylidene)thiazolidine-2,4-dione (15)  The title compound (17.69 g, 98 %) was prepared as a bright yellow solid from 4-hydroxybenzaldehyde 14 (10.00 g, 81.9 mmol), thiazolidine-2,4-dione (9.58 g, 81.9 mmol), benzoic acid (1.51 g, 12.3 15  S  ^ ^0 ==  f  mmol), and piperidine (1.2 mL, 12.1 mmol) by a procedure analogous to  H  that described for 5; m.p. 291-292 °C. 'H N M R (MeOH-</ , 300 MHz): 5 7.72 (s, 1H; 4  alkene//), 7.43 (d, J = 8.6Hz, 2H; Ar//), 6.90 (d, J= 8.6Hz, 2H; Ar//). EI-MS m/z 3  3  (relative intensity) = 221 ([M] , 40), 151 (10), 150 (100), 121 (17), 75 (11). Anal. Calcd. +  (found) for C10H7NO3S: C, 54.29 (54.69); H, 3.19 (3.30); N , 6.33 (6.44).  61  References begin on p. 78  (+)-5-(4-Hydroxy-benzyl)thiazolidine-2,4-dione  (16)  C o C l - 6 H 0 (0.02 g, 0.08 mmol) and dimethylglyoxime (0.04 g, 0.3  O H  2  2  mmol) were added to H 0 (80 mL). After the addition of NaOH (1 M , 1 2  16 mL), the brown solution was cooled to 0 °C in an ice bath and N a B H 0 ^  -s ^ 0  N  4  (1.20 g, 31.7 mmol) was added in portions. 5-(4-Hydroxy-  H  benzylidene)thiazolidine-2,4-dione 15 (1.00 g, 4.5 mmol) was then added slowly as a solid over 15 min. and then the reaction mixture was stirred at r.t. for 16 h. The pH was adjusted to 6 with C H 3 C O O H and then the mixture was extracted with CHCI3 (3 x 60 mL). The combined organic extracts were dried over N a S 0 , filtered, and decolourized 2  4  with charcoal (refluxed for 5 minutes and filtered). Evaporation of the solvents afforded the title compound 16 as a white solid (0.64 g, 64 %); m.p. 156-157 °C. *H N M R (MeOH-c/ , 300 MHz): 5 7.06 (d, J = 8.6 Hz, 2H; Ar/7), 6.71 (d, J = 8.6 Hz, 2H; Ar//), 3  3  4  4.65 (dd, J = 9.2 Hz, J = 3.9 Hz, 1H; TZD//), 3.34 (dd, J = 14.2 Hz, J=3.9 Hz, 1H; 3  3  2  3  TZDC/Y ), 3.03 (dd, J= 14.2 Hz, J = 9.2 Hz, 1H; TZDC/Y ). EI-MS m/z (relative 2  3  2  2  intensity) = 223 ([M] , 35), 107 (100), 91 (4), 77, (13). Anal. Calcd. (found) for +  C0H9NO3S: C, 53.80 (53.76); H , 4.06 (3.95); N , 6.27 (6.15).  (+)-5-[4-(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzyl]thiazolidine-2,4-dione (17)  0  O B n  Sodium hydride (0.40 g, 16.7 mmol) was added to D M F (45 mL) cooled in an ice bath. To this stirring mixture (±)-5-(4-hydroxy-  62  References begin on p. 78  benzyl)thiazolidine-2,4-dione 16 (1.70 g, 7.6 mmol) was added as a solid in portions causing considerable gas evolution. After 0.5 h 5-benzyloxy-2-chloromethyl-pyran-4-one 13 (2.00 g, 8.0 mmol) was added dropwise as a solution in D M F (30 mL). The resulting mixture was warmed to room temperature and stirred for 18 h. Saturated NaHCCb (125 mL) was then added and the mixture was extracted with CH2CI2 (3 x 125 mL). The combined organic portions were washed with brine (100 mL), dried over Na SC>4, and 2  then concentrated. The crude product was purified by silica-gel chromatography (99:1 C H C l : M e O H eluent) to afford 17 as a pale yellow solid (1.20 g, 35 %); m.p. 165-166 2  2  °C. ' H N M R (CHCVrfi, 400 MHz): 5 7.57 (s, 1H; kojic ring/*), 7.40 (m, 5H; Ar/7), 7.16 (d, J= 8.6 Hz, 2H; Ar/7), 6.86 (d, V = 8.6 Hz, 2H; Ar/7), 6.56 (s, 1H; kojic ring//), 5.07 3  (s, 2H; ArC/7 ), 4.80 (s, 2H; kojicC/7 ), 4.47 (dd, J= 9.2 Hz, J = 3.9 Hz, 1H; TZD//), 3  2  3  2  3.42 (dd, J= 14.2 Hz, J = 3.9 Hz, 1H; TZDC/7 ), 3.11 (dd, J= 14.2 Hz, J = 9.2 Hz, 2  3  2  3  2  1H; TZDC/7 ). EI-MS m/z (relative intensity) = 437 ([M] , 3), 214 (10), 107 (53), 91 +  2  (100). Anal. Calcd. (found) for C H i N 0 S : C, 63.15 (63.09); H, 4.38 (4.40); N , 3.20 2 3  9  6  (3.32).  (+)-5-[4-(5-Hydroxy-4-oxo-4H-pyran-2-ylmethoxy)benzyI]thiazolidine-2,4-dione (HL ) 4  O  The title compound H L (0.50 g, 93 %) was prepared as a white 4  solid from (±)-5-[4-(5-benzyloxy-4-oxo-4H-pyran-2-  O.  ylmethoxy)benzyl]thiazolidine-2,4-dione 17 (0.68 g, 1.6 mmol) by O  a procedure analogous to that described for H L ; m.p. 174 °C. H  63  References begin on p. 78  N M R (MeOH-c/4, 300MHz): 5 8.04 (s, 1H; kojic ring//), 7.24 (d, J= 8.6 Hz, 2H; Ar//), 3  6.98 (d, V = 8.6 Hz, 2H; Ar//), 6.60 (s, 1H; kojic ring//), 4.99 (s, 2H; kojicC// ), 4.73 2  (dd, J = 8.9 H z , J = 4.2 Hz, 1H; TZDH), 3.42 (dd, J= 14.2 Hz, J = 4.2 Hz, 1H; 3  3  2  3  T Z D C / / ) , 3.11 (dd, V = 14.2 Hz, J=8.9 Hz, 1H; TZDCH ). EI-MS m/z (relative 3  2  2  intensity) - 347 ([M] , 10), 231 (70), 125 (100), 107 (63). Anal. Calcd. (found) for +  C i H N 0 S : C, 55.33 (54.87); H , 3.77 (3.75); N , 4.03 (4.03). 6  1 3  6  2.2.5 Synthesis of vanadyl-thiazolidinedione complexes  R=  Scheme 2.1: The synthesis of VO(L ) l  (83%), VO(L ) (93%), and VO(L ) (88%); 3  2  4  2  2  a) V O S C V 5 H 0 , H 0 . 2  2  64  References begin on p. 78  ((±)-5-{4-[(5-alkoxy-4-oxo-4H-pyran-2-ylmethyl)amino]benzyl}thiazolidine-2,4dionato)oxovanadium(IV) hydrate (VOfL^'HkO)  H L (0.91 g, 2.64 mmol) was dissolved in H 0 (15 mL) and V O S 0 5 H 0 (0.28 g, 1.12 1  2  4  2  mmol) was added. The p H was adjusted to 5 with 1 M NaOH and the mixture was refluxed under A r for 22 h. The reaction mixture was filtered hot and the precipitate was washed with H 0 to afford, after drying in vacuo, 0.71 g (83 % based on V ) of the title 2  compound as a brown solid; m.p. 175-176 °C. IR (cm" , K B r disk): 3500-3000 (v . , v . 1  N  H  c  H ) ; 1752, 1681 (vc=o thiazolidinedione); 1610, 1563, 1516, 1475 (pyrone vc=o, ringvc=c); 982 (v =o). ES-MS m/z (relative intensity) = 758 ([HVO(L ) ] ,(M+H) , 70), 444 1  v  +  +  2  ([VO(0) (L')] , 30), 412 ([VO(L )] , 60), 347 ([L+H] , 100). The room temperature solid +  1  +  +  2  state magnetic moment  u. ff e  = 1-75 B M . Anal. Calcd. (found) for C 3 H 6 N O n S V - H 0 : 2  2  4  2  2  C, 49.55 (49.25); H , 3.64 (3.54); N , 7.22 (6.94).  Bis((±)-5-[4-(5-alkoxy-4-oxo-4H-pyran-2-yImethoxy)benzylidene]thiazolidine-2,4dionato)oxovanadium(IV) hydrate (VO(L ) H 0) 3  2  2  The title compound V 0 ( L ) H 0 (1.00 g, 88 %) was prepared as a grey solid from H L 3  2  2  (1.03 g, 2.98 mmol) and V 0 S 0 4 5 H 0 (0.38 g, 1.60 mmol) by a procedure analogous to -  2  that described for VO(I/) ; m.p. 189-192 °C. IR (cm" , K B r disk): 3500-3000 (v . , v . 1  2  N  H  c  H); 1745, 1679 (vc=o thiazolidinedione); 1596, 1562, 1510, 1472 (pyrone vc=o, ring vc=c); 985 (w=o). ES-MS m/z = 756 ([HVO(L ) ] ,(M+H) , 20), 458 (10), 443 ([VO(0) (L )] , 3  +  +  3  2  +  2  56), 368 ([L+Na] , 20). The room temperature solid state magnetic moment +  65  u. ff e  =  1-72  References begin on p. 78  B M . Anal. Calcd. (found) for C 2H2oN Oi3S V-H20: C, 49.68 (50.01); H , 2.87 (2.67); N , 3  2  2  3.62 (3.71).  Bis((±)-5-[4-(5-alkoxy-4-oxo-4H-pyran-2-ylmethoxy)benzyl]thiazolidine-2,4dionato)oxovanadium(IV) hydrate (V 0(L ) *H 0) 4  2  2  The title compound V O ( L ) H 0 (0.51 g, 93 %) was prepared as a brown solid from 4  2  2  H L (0.71 g, 2.05 mmol) and V O S C V 5 H 0 (0.29 g, 1.21 mmol) by a procedure 4  2  analogous to that described for V O ( L ) ; m.p. 174 °C. IR (cm" , K B r disk): 3500-3000 1  1  2  1750, 1692 (vc=o thiazolidinedione); 1610, 1563, 1510, 1476 (pyrone  (VN-H, VC-H);  ring v =c); 975 c  (v =o)v  vc=o,  ES-MS m/z = 760 ([HVO(L ) ] , (M+H) , 55), 445 4  +  +  2  ([VO(0) (L )] , 100), 413 ([VO(L )] , 49), 370 ([L+Na] , 10). The room temperature 4  +  4  +  +  2  solid state magnetic moment  p ff e  = 1.69 B M . Anal. Calcd. (found) for  C 3 H N 0 S V H 0 : C, 49.43 (48.98); H , 3.37 (3.25); N , 3.60 (3.52). 2  2 4  2  1 3  2  2  2.3 Results and Discussion  2.3.1 Bifunctional Ligand Synthesis  A l l four ligand precursors H L , H L , H L , and H L were prepared by various 1  2  3  4  routes from kojic acid 1. Kojic acid 1 was first protected at the ring hydroxyl using the 90  method of Thomas and Marxer to afford the benzyl protected compound 2. This compound was further elaborated to synthesize the four distinct ligand precursors. A n  66  References begin on p. 78  effort was made to preserve the active portion of the thiazolidinediones (the 5-(4substituted-benzyl)thiazolidine-2,4-dione moiety) in these bifunctional ligands (Figure 2.2). For the amino-tethered ligand (Scheme 2.2), 5-benzyloxy-2-hydroxymethylpyran-4-one 2 was reacted with methanesulfonyl chloride to afford the mesylate 3. Compound 3 was then coupled with (±)-5-(4-amino-benzyl)thiazolidine-2,4-dione 6, which was synthesized by a more direct route than that available in the literature, to 30  afford 7. To synthesize 6, 4-nitrobenzaldehyde 4 was coupled with thiazolidine-2,4-dione using reported conditions " to afford 5, which was then reduced using N a B H i in the presence of a cobalt/dimethylglyoxime catalyst formed in situ?  3  OH  OH  1  OMs 2  3  Scheme 2.2: The synthesis of H L : a) thiazolidine-2,4-dione, piperidine, benzoic acid, 1  toluene, 55%. b) C0CI2, dimethylglyoxime, NaBFL;, H 0 , 71%. c) Na, benzyl chloride, 2  MeOH, 55%. d) triethylamine, methanesulfonyl chloride, CH C1 . e) K C 0 , C H C N , 2  2  2  3  3  69%. f) HCI, C H C O O H , 70%. 3  67  References begin on p. 78  Using this method, both the nitro group and the double bond were reduced simultaneously. Benzyl group removal from the coupled product 7 in strong acid afforded the amino-tethered thiazolidinedione ligand precursor H L . Hydrogenation using 10% 1  palladium on carbon as the catalyst was unsuccessful most probably due to binding of the thiazolidinedione sulfur to the catalyst thereby inhibiting the reduction reaction.  32  Synthesis of the pyridinone-type ligand precursor H L (Scheme 2.3) proceeded in 2  much the same manner as with H L . A pyridinone-type chelator was examined because 1  N-substitution into a pyrone ring has been shown to increase metal binding affinity. '  34 35  Benzyl-protected kojic acid 2 was reacted with methylamine to form the pyridinone 8 in an improved yield over that reported. Compound 8 was then converted to the chloride 9 31  with thionyl chloride in good yield. The coupling step proceeded to give the protected compound 10 from which the benzyl group was removed to afford the pyridinone-type ligand precursor H L . 2  2  8  9  Scheme 2.3: The synthesis of H L : a) methylamine, EtOH / H 0 , 77%. b) SOCl , 2  2  2  CH C1 , 96%. c) triethylamine, D M F , 88%. d) HCI, C H C O O H , 73%. 2  2  3  68  References begin on p. 78  The unsaturated ether-tethered ligand precursor (Scheme 2.4) H L was 3  synthesized using the mesylate 3 as the starting material.  Scheme 2.4: The synthesis of H L : a) 4-hydroxybenzaldehyde, K C 0 , D M F , 64%. b) 3  2  3  thiazolidine-2,4-dione, piperidine, benzoic acid, toluene, 57%. c) HCI, C H C O O H , 67%. 3  Compound 3 was coupled with 4-hydroxybenzaldehyde to afford the aldehyde 11 which then underwent a Knoevenagel condensation with thiazolidine-2,4-dione to yield compound 12. The original intent at this point was to reduce the C-C double bond connecting the thiazolidinedione ring. Unfortunately this step could not be completed selectively, despite the variety of conditions attempted (Mg/MeOH ; C0CI2, 36  dimethylglyoxime, N a B H ; H , Pd/C), due to the many other unsaturation points in the 4  2  molecule. Nonetheless, as previous work ' had shown that the activity of certain TZDs with an unsaturation point in an identical location exhibited equal or more impressive activity than their saturated analogs, we decided to continue by deprotecting 12 to form the unsaturated ether-tethered ligand precursor H L . 3  69  References begin on p. 78  The ether-tethered ligand precursor (Scheme 2.5) H L was synthesized in a manner similar to that employed for H L . 1  OH  OH  OH  OH  CI 2  13  Scheme 2.5: The synthesis of H L : a) thiazolidine-2,4-dione, piperidine, benzoic acid, toluene, 98%. b) C o C l , dimethylglyoxime, N a B H , H 0 , 64%. c) S O C l , CH C1 , 60%. 2  4  2  2  2  2  d) NaH, D M F , 35%. e) HCI, C H C O O H , 93%. 3  In this case 5-benzyloxy-2-chloromethyl-pyran-4-one 13 was used in the coupling step because the mesylate 3 did not afford any coupled product. The slower reactivity of the chloride derivative presumably minimized side-reactions allowing the coupling reaction to take place. Compound 13 was coupled with (±)-5-(4-hydroxy-benzyl)thiazolidine-2,4-dione 16 which was synthesized by a Knoevenagel condensation of 430  hydroxybenzaldehyde 14 with thiazolidine-2,4-dione to form 15, and then subsequent reduction. The coupled product 17 was then deprotected to afford the ether-tethered ligand precursor H L . 4  The saturated ligand precursors H L , H L , and H L each exist as a mixture of 1  2  4  enantiomers with the stereogenic center located at C-5 of the thiazolidinedione ring. No 70  References begin on p. 78  attempt was made to synthesize or isolate selectively one enantiomer because this center is prone to rapid racemization at physiological pH.  Interestingly, it has been reported  that the (S)-(-)-enantiomer of rosiglitazone was responsible for the binding affinity to PPARy with a t\a for racemization of 3 h at pH 7.2.  2.3.2 Vanadyl-thiazolidinedione complexes  The neutral oxovanadium(IV)-thiazolidinedione complexes were prepared (Scheme 2.1, p.22) by refluxing vanadyl sulfate and two equivalents of the appropriate ligand precursor in mildly acidic aqueous medium (pH ~5). The precipitated products were isolated and washed with water and diethyl ether to afford, after drying in vacuo light brown, grey, and brown solids, characterized as VOfJL'^'H^O, VO(L ) -H20, and 3  2  VO(L )2*H20, respectively. Unfortunately, the extremely low solubility of the 4  pyridinone-type ligand precursor H L precluded complexation to vanadium under a 2  variety of conditions. Characteristic stretching frequencies of the V = 0 bond in oxovanadium(IV) complexes generally occur in the region 930-1030 cm" . The complexes VO(L ) , 1  40  1  2  VO(L ) , and VO(L ) exhibit V = 0 stretching frequencies of 982, 985, and 975 cm" , 3  4  2  1  2  respectively. The corresponding bis(kojato)oxovanadium(IV) (VO(ka)2) complex exhibits a similar V = 0 stretch at 980 cm" . Elemental analyses of the three complexes were consistent with the calculated values. A l l exhibited one molecule of residual water which could not be eliminated with prolonged drying. The mass spectra (+ ES-MS)  71  References begin on p. 78  support the V O L 2 formulation showing parent ion M + l peaks ([HVOL2] ), as well as +  expected fragmentation patterns of the complexes. A l l complexes prepared were paramagnetic in the solid state. Room-temperature paramagnetic susceptibilities were obtained. By correcting for the diamagnetic susceptibilities of the ligands and vanadium using Pascal's constants, the effective 28  magnetic moments of the complexes were calculated. With an electronic configuration of [Ar]3d', vanadium(IV) has one unpaired electron for which the spin-only formula predicts a magnetic moment of 1.73 B M . The experimental values are in the range 1.691.75 B M for the three vanadyl complexes. Electron paramagnetic resonance spectroscopic data for the three complexes showed the characteristic 8-line pattern (7= 7/2) expected for V (IV). A representative spectrum for VO(L )2 is shown in Figure 2.5. The three vanadium-thiazolidinedione 4  complexes exhibited identical g-values and very similar isotropic vanadium nuclear hyperfine coupling constants (Table 2.1). In addition, the experimental values compare very well with those reported for B M O V  4 1  and VO(ka)  26 2  (Table 2.1). Dissolution of  each vanadium-thiazolidinedione complex in MeOFECFkCk (1:1) most probably results in solvated MeOH species. Possible sites for solvent coordination are trans or cis to the oxo-O, yielding six possible species in solution. X-ray crystal structure analysis would 42  give a much more detailed understanding of the coordination environment; however, many crystallization attempts did not produce the required single crystals.  72  References begin on p. 78  Table 2.1: Spin Hamiltonian Parameters for VO(L) ; L = L , L , L , ma (maltolate), ka 1  3  4  2  (kojate).  O  a giso  complex 1:1)  1.967  104.0  VO(L ) (MeOH:CH Cl2 1:1)  1.967  108.0  V O ( L ) ( M e O H : C H C l 1:1)  1.967  105.0  VO(ma) (BMOV) (CHC1 )  1.963  103.7  1.963  104.5  VO(L') (MeOH:CH Cl 2  2  2  3  2  2  4  2  2  2  2  C  3  VO(ka) ( C H C l ) 2  d  3  error ± 0.001. ± 0.1 x 10^ cm" . ref.[41]. ref.[26]. b  1  c  d  73  References begin on p. 78  2.3.3 Biological studies  Animal studies were completed in four sets of trials the first three of which used, in each trial, a diabetic control, a ligand precursor, the associated vanadyl complex of the ligand precursor, and B M O V as a representative standard. The fourth trial used a diabetic control, a ligand precursor, rosiglitazone, and B M O V . The percent decrease in blood glucose was calculated for each compound utilizing a common method ' and the results 8 11  are displayed in Table 2.2. A visual representation of the data is presented in Figure 2.6. The B M O V results are a combination of the four trials. The results for the amine tethered ligand precursor H L and the associated complex VO(L )2 suggest that these analogs 1  !  exhibit slow uptake as there was no observed glucose lowering at 12 hours. Positive effects were evident however at the 24 and 48 hour time points, with both compounds exhibiting half of the in vivo activity of B M O V in acute testing. The pyridinone-type ligand precursor H L showed minimal glucose lowering at 12 hours but 2  the effect was not sustained. The very low solubility of this compound leading to a lack of absorption in vivo was most probably responsible for the observed response. This low solubility precluded complexation with vanadium as well. The unsaturated ether-tethered ligand precursor H L and complex V O ( L ) showed the most efficacious hypoglycemic 2  effects in this study. Both compounds exhibited enhanced glucose lowering compared to B M O V at the 12 hour time point.  74  References begin on p. 78  Table 2.2: Glucose lowering activity of four thiazolidinedione ligand precursors and three of their corresponding complexes compared with B M O V , and rosiglitazone.  % decrease in blood glucose '  a b  Compound  12 h  24 h  48 h  HL  6.4 ± 2 . 0  19.9 ± 1.9  27.2 ± 5 . 3  v o r i A  3.9 ± 11.6  25.9 ± 11.0  24.5 ± 10.6  HL  2  10.9 ± 5 . 7  5.6 ± 3 . 3  2.0 ± 2 . 3  HL  3  54.5 ± 2 . 4  49.6 ± 3 . 5  27.5 ± 4 . 0  59.2 ± 3 . 3  57.5 ± 4 . 6  47.6 ± 6.0  3.6 ± 2 . 9  7.3 ± 1.8  0.9 ± 2 . 8  21.5 ± 6 . 7  28.8 ± 10.3  3.3 ± 2 . 5  6.4 ± 4 . 7  9.4 ± 5 . 1  10.2 ± 6 . 3  41.9 ± 6 . 0  57.1 ± 6 . 2  52.3 ± 5 . 3  1  VO(L ) 3  HL  2  4  VO(L ) 4  2  Rosiglitazone B M O V (average)  0  Glucose lowering of test compounds in STZ-diabetic rats was calculated as in eq. 2.1. Each value represents the mean ± S E M (n=10). Dose: 0.1 mmol/kg. combination of 4 trials (n=26). a  b  0  Whereas the glucose lowering effect of the ligand was lessened from 12 to 48 hours, the effect of V O ( L ) on blood glucose levels was sustained, similar to the 2  benchmark compound B M O V . Based on these promising results we tested the saturated analogs H  L  4  and  VO(L ) . 4  2  75  References begin on p. 78  70  n  l O) C  50  12 Hours I 24 Hours 48 Hours  I  I  IT  60  T  0)  o  40 H  -J  V I/) o 30 H o  T  III  O 20  H  10  H  It  I  CM  _l  1 CO  2  -I  o >  o >  o >  >  °  S  jS  CQ  O) 00  O  Figure 2.6: Hypoglycemic activity (% plasma glucose lowering) for four thiazolidinedione ligand precursors and three of their corresponding complexes, compared with B M O V and rosiglitazone.  The ligand precursor did not show any measurable glucose lowering, while the complex exhibited glucose-lowering at 12 and 24 hours but the effect was not sustained to 48 hours. The results for VO(L )2 contrast that for VO(L')2 in that the former complex 4  76  References begin on p. 78  was faster acting but the positive effects were not sustained. These results could be explained by quicker uptake of VO(L )2 followed by faster metabolism and/or excretion. 4  Both the ligand precursors H L and H L were more proficient in lowering plasma 1  3  glucose levels in this testing protocol when compared with rosiglitazone, a marketed PPARy agonist. It should be noted that rosiglitazone (2.8 pmol/kg) was effective in lowering plasma glucose levels in longer term (18 day) animal studies. H L showed 24  2  minimal hypoglycemic activity at 12 hours but the effect was not sustained. The very low solubility of this compound most probably hampered its activity. H L lowered plasma 3  glucose much more effectively than did rosiglitazone, a significant finding that warrants further attention. It has been reported that unsaturated thiazolidinediones show lesser fold transactivation of P P A R a and PPARy than do their saturated analogs. '  32 43  This suggests  that H L and other unsaturated thiazolidinediones exhibit their anti-diabetic effects 3  through other, as yet undetermined, mechanisms. Interestingly the saturated analog H L  4  did not show any activity in this testing protocol. This lack of activity could have been due to poor pharmacokinetics as the solubility of this ligand precursor was satisfactory.  2.4 Concluding Remarks  Designed vanadyl-thiazolidinedione complexes were appealing because of the potential for additive or synergistic effects via complexation of vanadium to thiazolidinedione-containing compounds. There is considerable evidence for the orally effective glucose-lowering properties of V(V) and V(IV) compounds, and a number of thiazolidinediones are either in late-stage clinical trials or are available for the treatment  77  References begin on p. 78  of type 2 diabetes. Neither additive nor synergistic effects were observed in this study; however, this could be due to the limited amount of information available from this shortterm testing protocol. From the in vivo study however, it is clear that VO(L )2 is 3  comparable to B M O V in lowering plasma glucose levels in STZ diabetic rats. As well, the TZD H L displays a significant increase in potency as compared to rosiglitazone. 3  Further testing would be prudent in a more specific model of type 2 diabetes whereby effects on other important parameters such as plasma triglycerides, and insulin could be determined. In this manner, the full potential of these combination agents could be 11  better determined.  2.5 References  (1) Woo, L. C. Y.; Yuen, V . G.; Thompson, K . H.; McNeill, J. FL; Orvig, C. J. Inorg. Biochem. 1999, 76, 251. (2) Sirtori, C. R.; Pasik, C. Pharm. Res. 1994, 30, 187. (3) Klip, A.; Leiter, L . A . Diabetes Care 1990,13, 696. (4) Storr, T.; Mitchell, D.; Buglyo, P.; Thompson, K . FL; Yuen, V . G.; McNeill, J. FL; Orvig, C. Bioconjugate Chem. 2003,14, 212. (5) Cohen, N . , Halberstam, M . , Shlimovich, P., Chang, C. J., Shamoon, FL, Rossetti, L. J. Clin. Invest. 1995, 95, 2501. (6) Chen, C. Am. J. Health-Syst. Pharm. 1998, 55, 905. (7) Perfetti, R.; Chamie, K. J. Endocrin. Invest. 2001, 24, 274. (8) Oguchi, M . , Wada, K., Honma, FL, Tanaka, A., Kaneko, T., Sakakibara, S., Ohsumi, J., Serizawa, N . , Fujiwara, T., Horikoshi, FL, Fujita, T. J. Med. Chem. 2000, 43, 3052. (9) Scheen, A. J. Diabetes Metab. 2001, 27, 305.  78  References begin on p. 78  (10) Wagenaar, L. G., Kuck, E. M . , Hoekstra, J. B. L. Neth. J. Med. 1999, 55, 4. (1 l)Sauerberg, P., Pettersson, I., Jeppesen, L., Bury, P. S., Mogenson, J. P., Wassermann, K., Brand, C. L., Sturis, J., Woldike, H . F., Fleckner, J., Anderson, A . T., Mortensen, S. B., Svensson, L. A . , Rasmussen, H . B., Lehmann, S. V., Polivka, Z., Sindelar, K., Panajotova, V . , Ynddal, L., Wulff, E. M . J. Med. Chem. 2002, 45, 789. (12) Henke, B. R. J. Med. Chem. 2004, 47, 4118. (13) Santiago, J. V . Compr. Ther. 1997, 23, 560. (14) Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M . PL, Kurokawa, R., Rosenfeld, M . G., Wilson, T. M . , Glass, C. K., Milbum, M . K. Nature 1998, 395, 137. (15) Raginato, M . J., Bailey, S. T., Krakow, S. L., Minami, C , Ishii, S., Tanaka, PL, Lazar, M . A . J. Biol. Chem. 1998, 273, 32679. (16) Mudaliar, S.; Henry, R. R. Annu. Rev. Med. 2001, 52, 239. (17) Mudaliar, S.; Henry, R. R. Curr. Opinion in Endocrin. Diabetes 2002, 9, 285. (18) Oakes, N . D.; Camilleri, S.; Furler, S. M . ; Chisholm, D. J.; Kraegen, E. W. Metabolism 1997, 46, 935. (19) Oakes, N . D.; Thalen, P. G.; Jacinto, S. M . ; Ljung, B. Diabetes 2001, 50, 1158. (20) Hanefeld, M . ; Brunetti, P.; Schemthaner, G. H.; Matthews, D. R.; Charbonnel, B. H . Diabetes Care 2004, 27, 141. (21) Wolffenbuttel, B. H . R.; Gomis, R.; Squatrito, S.; Jones, N . P.; Patwardhan, R. N . Diabetic Med. 2000,17, 40. (22) Chaput, E.; Saladin, R.; Silvestre, M . ; Edgar, A . D. Biochem. Biophys. Res. Commun. 2000, 271, 445. (23) Fonseca, V . ; Rosenstock, J.; Patwardhan, R.; Salzman, A . J. Amer. Med. Assoc. 2000, 283, 1695. (24) Yuen, V . G.; Bhanot, S.; Battell, M . L.; Orvig, C ; McNeill, J. H . Can. J. Physiol. Pharm. 2003, 81, 1049. (25) Finnegan, M . M . , Lutz, T. G., Nelson, W. O., Smith, A., Orvig, C. Inorg. Chem. 1987, 26, 2X1X. (26) Yuen, V . G., Caravan, P., Gelmini, L., Glover, N . , McNeill, J. H . , Setyawati, I. A., Zhou, Y., Orvig, C. J. Inorg. Biochem. 1997, 68, 109.  79  References begin on p. 78  (27) Perrin, D. D.; Armarego, W. L . F. Purification of Laboratory Reagents; Permagon Press: Oxford, 1980. (28) Mabbs, F. E., Machin, D. J. Magnetism and Transition Metal Complexes; Chapman and Hall: London, 1961. (29) Thomas, A . F., Marxer A . Helv. Chim. Acta. 1960, 61, 469. (30) Sohda, T., Mizuno, K., Imamiya, E., Sugiyama, Y . , Fujita, T., Kawamatsu, Y . Chem. Pharm. Bull. 1982, 30, 3580. (31) Teitei, T. Aust. J. Chem. 1983, 36, 2307. (32) Cantello, B. C. C , Cawthorne, M . A., Cottam, G. P., Duff, P. T., Haigh, D., Hindley, R. M . , Lister, C. A., Smith, S. A., Thurlby, P. L. J. Med. Chem. 1994, 37, 3977. (33) Tanis, S. P., Parker, T. T., Colca, J. R., Fisher, R. M . , Kletzein, R. F. J. Med. Chem. 1996, 39, 5053. (34) Finnegan, M . M . , Rettig, S. J., Orvig, C. J. Am. Chem. Soc. 1986,108, 5034. (35) Nelson, W. O., Rettig, S. J., Orvig, C. J. Am. Chem. Soc. 1987,109, 4121. (36) Profitt, J. A . ; Watt, D. S.; Corey, E. J. J. Org. Chem. 1975, 40, 127. (37) Reddy, K . A., Lohray, B. B., Bhushan, V., Reddy, A . S., Mamidi, N . V . S. R., Reddy, P. P., Saibaba, V . , Reddy, N . J., Suryaprakash, A., Misra, P., Vikramadithyan, R. K., Rajagopalan, R. J. Med. Chem. 1999, 42, 3265. (38) Prabhakar, C , Madhusudhan, G., Sahadev, K., Maheedara, C , Reddy, R., Sarma, M . R., Reddy, G. O., Chakrabarti, R., Rao, C. S., Kumar, T. D., Rajagopalan, R. Bioorg. Med. Chem. Lett. 1998, 8, 2725. (39) Parks, D. J., Tomkinson, N . C. O., Villeneuve, M . S., Blanchard, S. G., Willson, T. M . Bioorg. Med. Chem. Lett. 1998, 8, 3657. (40) Boas, L . V., Pessoa, J. C. Comprehensive Coordination Chemistry; Pergamon: New York, 1987. (41) Caravan, P., Gelmini, L., Glover, N . , Herring, F. G., L i , H., McNeill, J. H., Rettig, S. J., Setyawati, I. A . , Shuter, E., Sun, Y . , Tracey, A . S., Yuen, V . G., Orvig, C. J. Am. Chem. Soc. 1995,117, 12759. (42) Hanson, G. R., Sun, Y . , Orvig, C. Inorg. Chem. 1996, 35, 6507.  80  References begin on p. 78  (43) Cantello, B . C. C , Cawthorne, M . A., Haigh, D., Hindley, R. M . , Smith, S. A . , Thurlby, P. L., Bioorg. Med. Chem. Lett. 1994, 4, 1181.  81  References begin on p. 78  CHAPTER 3  New Chelating Agents for Intervention in Neurodegenerative Disease  3.1 Introduction  Molecules designed to sequester excess metals in the brain and aid in the removal of such metals are attractive therapeutic targets in neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob disease, and amyotrophic 1  3  lateral sclerosis. " In particular, dual function chelating antioxidant molecules designed to remove and/or re-distribute excess metals in the brain and reduce the concomitant oxidative stress are being investigated here as potential Alzheimer's disease (AD) therapies. While systemic levels of copper and iron are elevated in A D , the main target 4  5  organ of A D chelation therapy is the brain. High levels of the metals Cu, Zn, and Fe are thought to be responsible for amyloid-beta (AP) plaque development, at least in part due 6  to the resultant oxidative stress through metal (Cu / Fe) redox processes. " Whereas Fe 7  9  found within A p deposits is redox-active, ' it does not co-purify with A p from post10 11  mortem brain tissue, unlike Cu and Zn. Iron is found predominantly in neuritic 7  2 12  processes, probably complexed with ferritin in A p plaques. ' For these and other reasons discussed in detail in Section 1.3, the sequestration of excess Cu and Zn, and to a lesser extent Fe, is considered to be a promising A D therapeutic strategy. Specific, rather than systemic, chelation of excess metals in the brain of A D patients could lead to a disruption of abnormal metal-protein interactions associated with A D , leading to a 82  References start on p. 166  normalization of metal distribution, and a reduction of the associated oxidative stress. Metal chelators with associated antioxidant properties could also minimize the widespread oxidative stress in A D . Targeting drug molecules to the brain is a difficult undertaking because of the blood brain barrier (BBB), the physiological junction at which the bloodstream meets the neuronal environment. As explained in Section 1.3 the B B B consists of brain microvessel endothelial cells that ensure neurotoxic metabolites are excluded from the brain, while allowing for essential molecules to pass across. Clioquinol (Figure 3.1), a potential metal-protein attenuating compound ( M P A C ) ,  14  most  likely enters the brain via passive diffusion; however, this chelator also has the potential to systemically sequester metal ions and thus does not have target specificity for the brain.  Desferrioxamine  Figure 3.1: Compounds of interest for Chapter 3.  83  References start on p. 166  Long-term use of strong chelators that are not tissue-specific, such as desferrioxamine (Figure 3.1), likely affects the homeostasis of numerous biometals and the normal physiological functions of essential metal-requiring biomolecules such as metalloenzymes. ' Therefore, to prevent untimely systemic metal chelation prior to 15 16  entry to the B B B , a pro-drug that is de-protected after gaining access to the brain is desirable. Tetrahydrosalen compounds (Figure 3.2) were investigated here as potential metal sequestration agents for A D therapy.  HN  N H RO  RO  H L  R=H  GL  R = glucose  2  HjL GL  1  R= glucose, R' = H, R" = H  1  H L  2  2  GL  3  2  GL  2  GL  4  4  OH,OH  R = H, R' = benzyl, R" = H R= glucose, R'= benzyl, R" = H  3  H L  R = H, R = methyl, R" = H R= glucose, R'= methyl, R" = H  2  H L  R = H, R = H, R" = H  5  OH OH  R = H, R' = H, R" = t-butyl  R  H GL H GL 2  OH  R=H  2  R= glucose, R'= H, R" = t-butyl  Figure 3.2:  R'  7  R = t-butyl  Tetrahydrosalen pro-ligands and carbohydrate-protected tetrahydrosalen  ligand precursors synthesized in Chapter 3.  84  References start on p. 166  A pro-drug strategy was developed utilizing glucose as the masking group to protect the chelator, impart water solubility, and finally act as a targeting function. The carbohydrates were attached to the phenol moieties via p-glycoside bonds and a schematic of the glycoside pro-drug strategy is shown in Figure 3.3.  Brain  masked chelator  Pro-drug OH  Bioactivation (P-glucosidase)  HO^V^HO-A—*  G L U T - l  ()  OH  Chelator Glucose  (J-glycoside link  protecting group  Figure 3.3: Schematic of the glycoside pro-drug strategy; adapted from ref. [17].  In the first step, the glucose conjugates may gain access to the brain via the high density of G L U T - l transporters that are present at the B B B .  1 8  Once in the brain, pro-drug  bioactivation can occur as the glycoside link has the potential to be cleaved in vivo by a number of fJ-glucosidases, some of which are found naturally in the brain.  19  Tetrahydrosalen compounds (Figure 3.2) were investigated as chelators as they are known to have a high affinity for metal ions, " ' '  and were a complement to the  investigation of 3-hydroxy-4-pyridinones (Figure 3.1) for the same purpose. In addition 28  to the metal chelating ability of the tetrahydrosalen compounds, the antioxidant potential  85  References start on p. 166  of the phenol moieties could be beneficial; phenols can act as hydrogen donors, forming phenoxy radicals of varying stability depending on the ring substitution pattern.  Phenols are known to react with lipid radicals,  30,31  ultimately acting as chain  T9  breaking antioxidants. Examples of common phenolic antioxidants are butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), synthetic compounds used as a food preservatives, and (±)-a-tocopherol (Vitamin E), found naturally in foods (Figure 3.1). A generalized scheme for the reaction of a-tocopherol with a radical (-R), is shown in Scheme 3.1, although the exact nature of the oxidized products depends on the 90  conditions.  A series of tetrahydrosalen compounds, and their glycosylated analogs  (Figure 3.2), were developed to afford a diverse set of compounds for initial testing. As expected, subtle structural changes were found to have effects on the characteristics of the compounds tested (vide infra). Metal complexes of tetrahydrosalen compounds are not as common as are those of the corresponding salen (Figure 3.1) analogs. This can be attributed to the widespread interest in metal-salen complexes for catalytic applications, 86  References start on p. 166  33  as well as the increased difficulty in synthesizing the saturated analogs. However, unlike the imine CH=N linkages in salen compounds, the CH2-NH functionality in tetrahydrosalen compounds is very stable with respect to hydrolytic decomposition. In addition, the increased flexibility of the tetrahydrosalen compounds should allow for greater structural diversity of the resultant metal complexes. Of the tetrahydrosalen compounds E^L " , only H2L does not have precedent in the literature. Deprotonated 1  5  4  H2L has been extensively studied as a ligand for Fe(III), Sn(IV), Cr(III), Zn(II), 1  20  Cu(II), Ni(II), 23  23,24  21  22  23  and Co(II) ' . Deprotonated H L has been investigated as a ligand 23 24  2  2  for model M o (VIA'') centers of Mo hydroxylases and related enzymes, as well as for 25  potential catalytic applications complexed to Al(III) and Ti(IV) . Deprotonated H2L 26  27  has also been investigated for catalytic purposes as a ligand for Al(III)  2 6  3  Deprotonated  H2L has been studied as a ligand for Cr(III) as well as for Sn(IV) . The 5  22  21  61  tetrahydrosalen compounds H2GL " (Figure 3.2), exhibiting remote glycosylation at the para position of the phenol rings, were also developed here and do not have precedent in the literature. The use of glycosylated hydroquinone functions afforded compounds capable of chelating metals while exhibiting remote carbohydrate groups to enhance water solubility, and B B B uptake. The antioxidant properties of H2GL " may be enhanced as compared to H2L " due to the presence of the hydroquinone function. It is 1  5  ft 7  possible that each 'arm' of H2GL " could participate in a 2e" oxidation process (analogous to other hydroquinones and a-tocopherol (Scheme 3.1)) to the corresponding 67  quinone. In a relevant study, phenolic glycosides similar to the 'arms' of H2GL " were o shown to have significant antioxidant capability. 1  87  References start on p. 166  The tetrahydrosalen compounds were designed to gain access to the brain, bind metal ions associated with A D pathophysiology, and inhibit the associated oxidative stress, while allowing for endogenous clearance processes to cope more effectively with the accumulated Ap-peptide in the brain. The goal of this project was to synthesize a series of tetrahydrosalen compounds, and their glycosylated pro-drug forms, capable of removing excess metal ions from the brain. In addition, preliminary testing of the suitability of these compounds for A D therapy is reported.  3.2 Experimental  3.2.1 Materials  Most information related to this section is contained in Section 2.2.1. The reagents used for the synthesis of the tetrahydrosalen compounds as well as those for the Trolox Equivalent Antioxidant Capacity (TEAC) assays (vide infra) were obtained from Aldrich or Lancaster. The Agrobacterium sp. P-glucosidase (Abg) enzyme was a gift from Prof. S. G. Withers. Cisplatin was a gift from Prof. B. R. James. DC1, and NaOD were purchased from Cambridge Isotope Laboratories. Atomic absorption standards Cu(N03)2 and Z n C l were purchased from Aldrich and used directly in the potentiometry 2  experiments. N,N'-Bis(2-hydroxybenzyl)-ethane-l,2-diamine ( H 2 L ) (Figure 3.2) was 1  prepared by a literature method.  34  88  References start on p. 166  3.2.2 Instrumentation  Most information related to this section is contained in section 2.2.2. 2D N M R techniques such as ' H - ' H C O S Y and ' H - C H M Q C were used to aid in the 13  characterization of the compounds. Frozen solution X-band EPR spectra were recorded on a Bruker ECS-106 EPR spectrometer in 4 mm diameter quartz tubes. The temperature (-130 K ) was maintained by liquid nitrogen flowing through a cryostat in conjunction with a Eurotherm B-VT-2000 variable-temperature controller. Computer simulations of the EPR spectra were performed using the Bruker XSophe package. UV-vis spectra were recorded using a Hewlett-Packard 8543 diode array spectrometer and 96-well plates were analyzed by a Spectra Max 190 plate reader from Molecular Devices.  3.2.3 Potentiometric Equilibrium Measurements  Potentiometric equilibrium measurements were carried out using an automatic titration system consisting of a Metrohm 713 pH meter equipped with a Metrohm 6.0222.100 pH glass electrode, a model 665 Metrohm Dosimat autoburet, and waterjacketed 50 mL titration vessels maintained at 25.0 ±0.1 °C with a Julabo U C circulating bath. Both the pH meter and the autoburet were controlled by an IBM-compatible PC running a locally written Qbasic program. Ar, passed through water and 2 M NaOH to remove CO2, was passed through the solutions during the titrations. A l l solutions were prepared with deionized distilled water that was purged with Ar while boiling to remove CO2. The electrode was calibrated at the beginning of each  89  References start on p. 166  day by titrating a known amount of aqueous HCI with a known amount of NaOH. A plot of mV (calculated) vs. p H gave a working slope and intercept so that pH could be read as -log [H ] directly. The ionic strength (I) of the titration solution was kept constant with +  0.16 M NaCI. The value of pK used at / = 0.16 M NaCI and T= 25 °C was 13.76. The 35  w  NaOH solution was standardized potentiometrically with potassium biphthalate. Acidity constants for the compounds H 2 G L " were determined by titrating 50 mL 6  7  of aqueous 2.5 m M HCI (1= 0.16 M NaCI, T= 25 °C) in the presence of 0.6 m M H G L  6  2  or H G L , with 0.1M NaOH. The constants were calculated using the computer program 7  2  ~Kf\ T 7  H Y P E R Q U A D . ' The program operates by formulating separate mass balance equations and calculating the instantaneous concentration of all species for each point in the titration, based on the system model (i.e. pK values) and the total concentration of a  each component. The pK values of H G L were calculated with data in the range 4.5 < 6  2  a  pH < 10.5 while the pK values of H G L were calculated with data in the range 7  a  2  3.7 < pH < 10.0. Typically 90-100 data points were acquired per titration. The final acidity constant values for the two compounds were obtained from an average of 8 independent titrations and are listed in Table 3.2. Speciation diagrams for H G L and 6  2  H G L were calculated using the program H Y S S 7  37  2  (Figures 3.9 (p. 136) and 3.12 (p. 140),  respectively).  3.2.4 U V - v i s Determination of Acidity Constants  UV-vis measurements were used to corroborate and calculate the acidity constants for the two ligands above pH = 10. Solutions of either H G L or H G L 2  90  2  References start on p. 166  (0.125 m M , / = 0.16 M NaCI, 7 = 25 °C) were prepared, the pH of each solution adjusted, and the UV-vis spectrum then recorded using a Metrohm 713 p H meter equipped with a Metrohm 6.0222.100 pH glass electrode and an HP 8453 UV-vis spectrometer connected to a PC computer and an Isotemp 1016D water bath. A n aliquot from the solution was transferred into a cuvette (path length = 1 cm) and the absorption spectrum measured. After measuring the absorbance, the aliquot was carefully returned to the original solution. At least 20 readings were taken in the p H range 8-13.5. Four separate experiments were carried out for each ligand, and at two different wavelengths (237 nm and 308 nm for H G L ; 248 nm and 310 nm for H G L ) , the absorption and the 6  7  2  2  corresponding pH values were fitted by a locally written Basic program using a NewtonGauss nonlinear-least-squares curve-fit procedure. The experimental UV-vis experimental spectra for H G L and H G L are shown in Figures 3.8 (p. 135) and 3.11 6  7  2  2  (p. 138), respectively.  3.2.5 *H NMR Protonation Experiments  A solution of either H G L or H G L (8.2 mM) was prepared in D 0 and the pH* 6  2  7  2  2  adjusted to -13.4 with NaOD. pH* is the pH meter reading of a deuterium solution. A n aliquot was removed and the pH* of the resulting bulk solution was adjusted with DC1. The procedure was continued until a series of solutions with different pH* values for each ligand were obtained. The measured pH* values were converted to pD values based on the following relationship:  91  References start on p. 166  pD = pH* + 0.4  (Eq. 3.1)  The H N M R spectra were then measured and the shift of the hydrogen resonances correlated to the change in pD (Figures 3.7 (p. 134) H2GL , and 3.10 (p. 137) 6  H GL ). 7  2  3.2.6 X-ray Crystallographic Analysis of H G L , CuGL , and NiGL 7  7  7  2  The structures were solved by either Dr. B. O. Patrick or Dr. M . Merkel in the Department of Chemistry, U B C . Crystals of H 2 G L and CuGL were grown from 7  7  concentrated MeOFI/FIiO solutions of the respective compounds, whereas crystals of NiGL were obtained via the slow evaporation of a concentrated M e O H solution. The crystals were mounted on a glass fibre and the measurements made on a Bruker X8 A P E X diffractometer for H G L , and a Rigaku / A D S C C C D area detector for CuGL 7  7  2  and NiGL , with graphite monochromated Mo-Koc radiation. A l l data were processed and 7  corrected for Lorentz and polarization effects, and absorption using the S A D A B S 7  7  40  program for H GL' and the d*TREK 2  39  7  program for CuGL and NiGL . The structures  were solved by direct methods and expanded using Fourier techniques. A large 41  42  number of disordered water and MeOH molecules were removed from the unit cell of CuGL and the program P L A T O N / S Q U E E Z E 7  was used to account for the residual  electron density. Final refinements were carried out using S H E L X L - 9 7 for H G L and 44  7  2  teXsan for CuGL and NiGL . ORTEP diagrams of H G L , CuGL and NiGL are 45  7  7  7  7  7  2  shown in Figures 3.5 (p. 130), 3.16 (p. 146), and 3.18 (p. 148), respectively. Tables of  92  References start on p. 166  relevant bond lengths and angles are also shown for H G L (Table 3.1, p. 130), C u G L 7  7  2  (Table 3.4, p.147), and NiGL (Table 3.5, p.149). For details ofthe X-ray 7  crystallographic analyses for H2GL , CuGL and NiGL , please refer to Table A l 7  7  7  (p.248) in the Appendix.  3.2.7 Stability Constants for the Z n  The stability constants of C u  2 +  2+  and C u  and Z n  2 +  2+  Complexes of H G L  6  7  2  with H GL " were determined under the 6  7  2  same conditions as for the acidity constants (Section 3.2.3). The ligand-to-metal ratio for the titrations was approximately 1:1 with a slight excess of ligand present. Briefly, stability constants were determined by titrating a solution of 0.6 m M ( C u or Zn ) and 2+  2+  0.6 m M ( H G L or H G L ) (/= 0.16 M NaCI, T= 25 °C) with 0.1M NaOH. The stability 6  2  7  2  constants were calculated with data in the range 4.5 < pH < 10.0 using the program HYPERQUAD. '  Typically >100 data points were acquired per titration. The acidity 6 7  constants for H G L " (Table 3.2, p. 132) as well as the respective metal hydrolysis 2  constants (Table 3.6, p. 150) were used as constants in the calculations. The final values 46  for the stability constants for all the complexes were an average of at least 5 independent titrations. Speciation diagrams for the M :H GL " (M= Cu , Zn ) systems were 2  calculated using the program H Y S S  37  (Figures 3.19 (p. 152) and 3.20 (p. 153)). p M 24*  2+  diagrams were produced with a locally written Basic program for C u and Zn (1 mM) and selected ligands for a ratio of 1:1 M :ligand (Figure 3.21, p.157). The p M curves for 2+  EDTA  47  and A(3(l-28) with Cu were calculated based on data available in the literature. 48  93  References start on p. 166  3.2.8 Trolox Equivalent Antioxidant Capacity (TEAC) Assay  The tetrahydrosalen compounds (H2L " and H2GL " ) were tested for their ability 1  5  6  7  to scavenge free radicals using the T E A C assay. The results were compared to 49  antioxidant standards such as 6-hydroxy-2,3,7,8-tetramethylchroman-2-carboxylic acid (Trolox), (±)-a-tocopherol, and butylated hydroxytoluene (BHT) (Figure 3.4). A n 2,2'azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS* ) radical cation decolourization +  assay was used to determine relative T E A C values (Figure 3.4). 49  MTT (yellow)  Formazin (purple)  ABTS, diammonium salt  1,3 -D ibro mo - 5,5 -dimethylhydanto in  Figure 3.4: Reagents used in Chapter 3.  94  References start on p. 166  ABTS was dissolved in water (7 mM) and reacted with potassium persulfate (2.45 mM) in the dark for 16 h. During this time A B T S oxidizes to the coloured A B T S  , +  radical cation. The A B T S * solution was diluted with MeOH to an absorbance of +  0.70 (± 0.02) at 740 nm after equilibrating to 30°C (Fisher Isotemp circulating water bath). Stock solutions of the tetrahydrosalen compounds in M e O H were diluted so that addition of 20 pL to 2 mL of ABTS* solution caused a reduction of 20-80% in A o +  74  n m  as a result of the reduction to ABTS. The final concentrations of the aminophenols ranged from 2.5-15 p M to obtain the necessary absorbance change. After initial mixing, the  A740  nm was measured at 30 °C after 1, 3 and 6 minutes with each experiment  performed in triplicate. The percentage change of the absorbance at 740 nm was calculated and plotted as a function of tetrahydrosalen concentration. The slopes were then compared to the standard Trolox, with its T E A C value normalized to 1 (Table 3.9 (p. 160) and Figure 3.22 (p. 159)).  3.2.9 Enyzymatic Glycoside Cleavage  Preliminary reactivities of the glycosylated aminophenols with Agrobacterium sp. P-glucosidase (Abg) were tested and monitored by T L C and +ES-MS. To 1 mL Eppendorf tubes at 25 °C, glycosylated tetrahydrosalen compound (20 pL, 20 mM), phosphate buffer (50 pL, 75 mM, pH=7.4), and Abg (5 pL, 178 pg/mL) were added and the resulting solution mixed. After 2 h the reactions were monitored by T L C (1:1 MeOH:EtOAc eluent) using a U V lamp and charring ofthe T L C plates (5% H S 0 in 2  4  EtOH) to follow the cleavage process (Figure 3.23, p.162). The reactions were also  95  References start on p. 166  followed by mass spectrometry (+ES-MS) to correlate spectral changes with deglycosylation.  3.2.10 Toxicity Cell Studies and M T T Assay  Two representative glycosylated tetrahydrosalen compounds (GL , and H2GL ) 6  2  were used in this study to ascertain preliminary toxicity values. Human breast cancer cells were used (MDA-MB-435S) that had previously been cultured and prepared with 50  experimental details described elsewhere. Cells were transferred to a 96-well plate by 51  addition of 100 uL of the cell solution (1 x 10 cells) to 54 of the wells. Growth medium 4  (100 uL) was used as blanks in another 6 wells. The plate was incubated at 37 °C for 24 h and then solutions (100 uL) of G L at 8 different concentrations (10-5000 uM) in 2  phosphate buffered saline (PBS) were added to 48 wells. The remaining 6 wells containing cells served as the control; PBS was added (100 uL) and the plate was incubated at 37 °C for 3 days. A n identical procedure was used for H 2 G L and cisplatin 6  in this study. Cell toxicity was quantified by the M T T assay (MTT = 3-(4,5-dimethylthiazol52  2-yl)-2,5-diphenyltetrazolium bromide) (Figure 3.4). A PBS solution of M T T (50 uL, 2.5 mg/mL) was added to each well and the plate was then incubated for 3 h, by which time a purple precipitate of formazin (Figure 3.4) formed at the bottom of those wells that contained live cells. The contents of each well were then carefully decanted leaving the solid formazin which was dissolved in DMSO (150 u.L). The plate was then agitated and analyzed by a plate reader to determine the absorbance of each well at 570 nm. The  96  References start on p. 166  percentage cell viability was calculated by dividing the average absorbance of the cells 2  6  treated with G L , H2GL or cisplatin by that of the control. The percent cell viability vs. drug concentration (logarithmic scale) was plotted to determine the IC50 (drug concentration at which 50% of the cells are viable relative to the control), with the estimated error derived from the average of 6 trials (Figure 3.24, p. 164). Synthesis of Tetrahydrosalen Compounds (H2L " )  3.2.11  (H L )  N,N'-Bis(2-hydroxybenzyl)-N,N'-dimethyl-ethane-l,2-diamine  2  2  The title compound was synthesized by a method different from that in the literature. ' N,N'-dimethyl-ethane-1,2-diamine 1 (0.266 g, 3.02 25 26  mmol) and salicylaldehyde 3 (0.737 g, 6.04 mmol) were dissolved in 1,2-dichloroethane (9 mL) and sodium triacetoxyborohydride (1.790 g, h  2  l 2  8.45 mmol) was added in portions. After stirring the mixture for 24 h, a  saturated NaHC03 (20 mL) solution was added and the resulting mixture extracted with CH2CI2 (3  x 25 mL). The organic extracts were combined, dried with Na2S04, filtered,  and the solvent was removed in vacuo. The residue was purified by silica-gel chromatography (1:1 hexanes:EtOAc eluent) to afford the product H L as a white solid 2  2  (0.489 g, 54%). 'H NMR (CHCl -rfi, 300 MHz): 5 7.17 (ddd, V 3  Hz, \ (dd, J  b  4  = 8.1 Hz, 2H; Ar/Yb), 6.95 (d, V  M  = 1.5 Hz, %  d  = 1.0 Hz, V , = 8.1 Hz, 2H; Ar/7 a), 6.77 (ddd, J  = 7.4 Hz, 2H; Ar/Yd), 6.83  4  a;C  a  b  = 1-5 Hz, J b j C = 7.2 3  M  = 1.0 Hz, J , = 7.4 Hz, % 3  a>c  c  d  = 7.2 Hz, 2H; AiHe), 3.70 (s, 4H; ArC/7 ), 2.67 (s, 4H; NC/7 C/7 N), 2.29 (s, 6H; 2  2  97  2  References start on p. 166  c  NCH ).  13  3  C { ' H } N M R (MeOH-d , 75.48 MHz): 5 157.72 (Cf), 128.86, 128.46 (aromatic 4  CH), 121.53 (Ce), 119.10, 116.15 (aromatic CH), 61.71 (ArCH ), 54.02 ( N C H C H N ) , 2  2  2  41.64 (NCH ). M S (+ES-MS) m/z (relative intensity) = 323 ([L+Na] , 100), 217 ([L+  3  C H 0+Na] , 80). Anal. Calcd. (found) for C i H N 0 : C, 71.97 (71.66); H , 8.05 (8.10); +  7  6  8  2 4  2  2  N , 9.33 (9.35).  N,N'-Dibenzyl-ethane-l,2-diamine (2)  (  rj^^i  \  The  —1_ H  /  compound was prepared according to a reported method with  some modifications. Benzaldehyde (3.19 g, 30.06 mmol) and 53  ethylenediamine 4 (0.899 g, 15.00 mmol) were added to EtOH (75 mL) 2  and the reaction mixture heated to reflux with stirring. The reaction  2  mixture was evacuated to dryness after 4 h. The imine intermediate was dissolved in EtOH (75 mL) and N a B H (0.860 g, 22.72 mmol) was added in portions. The mixture 4  was heated to 50 °C for 2 h, cooled, and then HCI (6 M) was added to adjust the pH to -1.5. The precipitate was collected and dissolved in H 0 (60 mL) and the resulting 2  solution was adjusted to pH 11 by the addition of NaOH (5 % w/w in H 0 ) . The resulting 2  mixture was extracted into CH C1 (3x 60 mL), and the combined organics were dried 2  2  with N a S 0 , filtered, and evacuated to afford the product 2 as a colourless oil (3.020 g, 2  4  84% based on two steps). *H N M R (CHCl -di, 400 MHz): 5 7.27 (m, 6H; AiH), 7.21 (m, 3  4H; AiH), 3.73 (s, 4H; ArC/fc), 2.72 (s, 4H; N C / / C / / N ) . M S (EI-MS) m/z (relative 2  2  intensity) = 240 ([L] , 25), 120 ( [ L - C H i N ] , 100). Anal. Calcd. (found) for C i H N : +  +  8  0  6  2 0  2  C, 79.96 (79.47); H , 8.39 (8.44); N , 11.66 (11.33).  98  References start on p. 166  N,N'- Bis(2-hydroxybenzyl)-N,N'-dibenzyl-ethane-l,2-diamine (H2L ) 3  b H L 2  3  ) ' 2  The title compound (0.505 g, 63 %) was prepared from N,N'-dibenzylethane-l,2-diamine 2 (0.426 g, 1.78 mmol), salicylaldehyde 3 (0.433 g, 3.55 mmol), and sodium triacetoxyborohydride (1.051 g, 4.95 mmol) by a procedure analogous to that described for H 2 L and similar to a 2  recently reported method. ' H N M R (CHCl -t/i, 300 MHz): 5 7.28 (m, 26  3  12H; Ar/7), 6.91 (m, 6H; Ar/7), 3.73 (s, 4H; N C # C H O H ) , 3.52 (s, 4H; NCf7 C H ), 2  6  4  2  6  5  2.71 (s, 4H; N C / / C 7 / N ) . C{'H} N M R (CHCl -d,, 75.48 MHz): 6 157.42 (Cf), 136.12 13  2  2  3  (Cg), 129.44, 128.89, 128.72, 128.60, 127.72 (aromatic CH), 121.56 (Ce), 119.31, 116.13 (aromatic CH), 58.36, 58.04 (ArCH ), 49.77 ( N C H C H N ) . M S (EI-MS) m/z (relative 2  2  2  intensity) = 475 ([L+Na] , 100), 453 ([L+H] , 80). Anal. Calcd. (found) for C H N O : +  +  3 0  3 2  2  2  C, 79.61 (79.21); H , 7.13 (7.16); N , 6.19 (6.22).  N,N'-Bis(2-(4-tert-butyl)hydroxysalicylidene)-ethane-l,2-diamine (6)  The imine 6 was prepared by refluxing ethylenediamine 4 (0.203 g, 3.38 mmol) and 5-tert-butyl-2-hydrozybenzaldehyde 5 (1.203 g, 6.76 mmol) in EtOH (30 mL) for 4 h followed by solvent removal 2 6  under reduced pressure. ' H N M R (DMSO-d , 400 MHz): 5 8.62 (s, 6  2H; imine H), 7.60 (d, V  99  = 2.5 Hz, 2H; Ar7/d), 7.49 (dd, J , = 3  M  a  b  References start on p. 166  8.6 Hz,  V  = 2.5 Hz, 2H; Ar/Yb), 7.08 (d, J , = 8.6 Hz, 2H; Ar/Ya), 3.88 (s, 4 H ; 3  bjd  a h  NC/Y C/Y N), 1.25 (s, 18H; C(C/Y ) ). ES-MS m/z (relative intensity) = 403 ([M+Na] , +  2  2  5  3  100), 381 ([M+H] , 10). This compound was reduced in the next step without further +  purification.  N,N'-Bis(2-(4-tert-butyl)hydroxybenzyl)-ethane-l,2-diamine ( H L ) 4  2  The imine 6 was dissolved in MeOH (30 mL) and the solution cooled in an ice-bath. N a B H i (0.256 g, 6.77 mmol) was added in portions and then the reaction mixture was warmed to r.t. with stirring. The solvent was removed after 5 h and the residue 2  h  partitioned between a saturated NaHC03 (50 mL) solution and  l 4  CH2CI2 (30 mL) and the aqueous layer extracted with CH2CI2 (2x 20 mL). The combined organic fractions were dried with Na2S04, filtered, the solvent was removed in vacuo, and the residue purified by silica-gel chromatography (95:5 CH2Cl2:MeOH eluent) to afford the product H L as a light yellow solid (0.919 g, 71 %). *H N M R (CHCI3-J1, 300 4  2  MHz): 5 7.18 (dd, J , = 8.5 Hz, V ,d = 2.5 Hz, 2H; Ar/Yb), 6.97 (d, V 3  a  b  b  M  = 2.5 Hz, 1H;  Ar/Yd), 6.76 (d, V a > b = 8.5 Hz, 1H; Ar/Ya), 3.99 (s, 4 H ; ArC/Y ), 2.86 (s, 4 H ; 2  NC/Y C/Y N), 1.26 (s, 18H; C(C/Y ) ). C { ' H } N M R (CHCl -d,, 75.48 MHz): 5 155.41 13  2  2  3  3  3  (Cf), 141.84 (Cc), 125.55, 125.19 (aromatic CH), 121.35 (Ce), 115.76 (aromatic CH), 50.76 ( N C H C H O H ) , 48.79 ( N C H C H N ) , 34.21 (C(CH ) ), 31.52 (C(CH )3). M S 2  6  4  2  2  3  3  3  (+ES-MS) m/z (relative intensity) = 407 ([L+Na] , 100), 385 ([L+H] , 40). Anal. Calcd. +  +  (found) for C24H N 02: C, 73.60 (74.00); H , 9.47 (9.40); N , 7.15 (7.09). 36  2  100  References start on p. 166  N,N'-Bis(2-hydroxysalicylidene)-(-)-l,2-cyclohexane-(l/f,2/?)-diamine (8)  (li?,2£)-(-)-l,2-Diaminocyclohexane 7 (0.225 g, 1.96 mmol) and salicylaldehyde 3 (0.478 g, 3.91 mmol) were dissolved in  H O  \\  \ b EtOH (20 mL) and stirred for 4 h, and then the solvent was  HO  removed under reduced pressure to afford the imine 8. *H 8  N M R (DMSO-d , 400 MHz): 5 8.49 (s, 2H; imine H), 7.33 6  (dd, V \  = 1.5 Hz, J 3  M  Cjd  = 7.6 Hz, 2H; AiHA), 7.24 (ddd, V  = 8.2 Hz, 2H; AiHb), 6.85 (dd, J 3  b  = 1.5 Hz, J ,c = 7.3 Hz, 3  M  b  = 7.3 Hz, Jc = 7.6 Hz, 2H; AiH c), 6.81 (d, 3  bjC  >d  J ,b = 8.2 Hz, 2H; AiH a), 3.41 (m, 2H; ring CHN), 1.88 (m, 2H; ring H), 1.81 (m, 2H;  3  a  ring H), 1.62 (m, 2H; ring H), 1.05 (m, 2H; ring H). M S (+ES-MS) m/z (relative intensity) = 413 ([L+Na] , 100). This compound was reduced in the next step without +  further purification.  N,N'-Bis(2-hydroxybenzyl)-(-)-l,2-cyclohexane-(lit,2/()-diamine (H L ) 5  2  The imine 8 was dissolved in MeOH (20 mL) and cooled in an ice bath. NaBH4 (0.148 g, 3.91 mmol) was added in portions A a  \b  and the mixture was stirred for 24 h at room temperature and then the solvent was removed in vacuo. The residue was  b H L 2  5  partitioned between a saturated NaHC03 (20 mL) solution and  101  References start on p. 166  CH2CI2  (30 mL) and the aqueous layer extracted with  CH2CI2  (2 x 20 mL). The combined  organic fractions were dried with Na2S04, filtered, evacuated, and then purified by silicagel chromatography (95:5 C H C l : M e O H eluent) to afford the product H L as a light S  2  2  2  yellow solid (0.458 g, 73 %). ' H N M R (CHCI3-J1, 300 MHz): 5 7.17 (ddd, V 3  J  = 7.3 Hz, Ja, = 7.9 Hz, 2H; Ar/Yb), 6.98 (overlapping dd, J 3  b j C  3  b  d), 6.82 (d, J ,b = 7.9 Hz, 2H; Ar/Ya), 6.76 (dd, J 3  3  a  4.05 (d, J 2  =13.9 Hz, 2H; ArC/Y ), 3.93 (d, J  b j C  2  g e m  2  g e m  = 7.3 Hz, V  c > d  M  = 1-6 Hz,  = 7.0 Hz, 2H; Ar/Y  = 7.0 Hz, 2H; Ar/Yc),  C;d  =13.9 Hz, 2H; ArC/Y ), 2.45 (m, 2H; 2  ring C/YN), 2.15 (m, 2H; ring H), 1.71 (m, 2H; ring /Y), 1.23 (m, 4 H ; ring H). C { ' H } 13  N M R (CHC1 -^I, 75.48 MHz): 8 157.93 (Cf), 128.75, 128.31 (aromatic CH), 122.99 3  (Ce), 119.18, 116.36 (aromatic CH), 59.64 (CHN ring),  49.54,  30.36, 24.14 ( C H ring). 2  MS (+ES-MS) m/z (relative intensity) = 349 ([L+Na] , 40), 327 ([L+H] , 100). Anal. +  +  Calcd. (found) for C 2 H N O 2 : C, 7 3 . 5 9 (72.99); H , 8.03 (7.95); N , 8.58 (8.18). 0  2 6  2  3.2.12 Synthesis of the Glycosylated Tetrahydrosalen Compounds (GL " and 1  H GL 2  5  6-7^  )  N,N'-Bis(2-(phenyl P-D~glucopyranoside)salicylidene)-ethane-l,2-diamine (10)  Salicylaldehyde-P-D-glucopyranoside (helicin) 9 (0.461 g, 1.62 mmol) and ethylenediamine 4 (0.048 g, 0.81 mmol) were added to EtOH (12 mL) and the 2 1 0  suspension was heated to reflux for 24 h. The reaction  mixture was filtered and the precipitate washed with EtOH and E t 0 to afford the product 2  102  References start on p. 166  1 0 as a light yellow solid (0.438 g, 91%). ' H N M R (MeOH-d , 300 MHz): 5 8.85 (s, 2H; 4  imine H), 7.86 (d, \  d  Ar/Yb), 7.23 (d, Y  = 8.4 Hz, 2H; Ar/Ya), 7.08 (dd, Y , = 7.2 Hz, Y  3  = 7.5 Hz, 2H; Ar/Yd), 7.43 (dd, J  = 7.2 Hz, J , = 8.4 Hz, 2H;  3  3  b j C  a  3  a;b  = 7.5 Hz, 2H;  3  b  c  b  Cjd  Ar/Yc), 4.89 (d, J , = 6.9 Hz, 2H; /Y-l), 3.86 (m, 4H; N C / / C / Y N ) , 3.73 (dd, J 3  3  X 2  2  2  5M  = 5.4  Hz, V ,6b = 11.7 Hz 2H; //-6a), 3.45 (m, 2H; /Y-5), 3.44 (m, 8H; H-2, /Y-3, /Y-4, /Y-6b). 6a  MS (+LSIMS) m/z (relative intensity) = 593 ([L+H] , 100). Anal. Calcd. (found) for +  C28H36N2O12:  C, 56.75 (56.55); H , 6.12 (6.20); N , 4.73 (4.77).  N,N'-Bis(2-(phenyl P-D-gIucopyranoside)benzyl)-ethane-l,2-diamine (GL ) 1  Compound 1 0 (0.438 g, 0.74 mmol) was suspended in MeOH (7 mL) and N a B H (0.030 g, 0.79 mmol) was 4  added. The reaction mixture was heated to reflux and another equivalent of N a B H (0.028 g, 0.79 mmol) 4  was added after 3 h. The reaction mixture was cooled after 6 h, H2O (6 mL) was added, and the solvent removed in vacuo. The residue was then purified by size-exclusion chromatography on Sephadex G-10 (MeOH eluent) to afford the product G L as a white 1  solid (0.234 g, 53 %). ' H N M R (MeOH-</ , 400 MHz): 5 7.52 (m, 4H; Ar/Yb and d), 7.41 4  (d, \  = 8.5 Hz, 2H; Ar/Ya), 7.21 (dd, J 3  b  V,, = 7.7 Hz, 2H; /Y-l), 4.47 (d, J 2  2  6a  6b  = 7.6 Hz, 2H; Ar/Yc), 5.01 (d,  = 12.8 Hz, 2H; ArC/Y ), 4.39 (d, Y  2  V ,  C ; d  2  g e m  2H; ArC/Y ), 3.94 (overlapping dd, J , 2  = 7.2 Hz, J 3  b j C  6a 6b  2  g e m  = 12.8 Hz,  = 11.8 Hz, 2H; //-6b), 3.75 (dd, J ,6a = 5.4 Hz, 3  5  =11-8 Hz, 2H; //-6a), 3.54 (m, 12H; H-2, /Y-3, /Y-4, /Y-5, NC/Y C/Y N). 2  2  C{ rl}  ]3  l  N M R (MeOH-<4 100.62 MHz): 5 156.39 (Cf), 129.90 (aromatic CH), 129.30 (Ce),  103  References start on p. 166  128.27, 121.91, 115.66 (aromatic CH), 102.71 (CI), 77.26, 76.45 (C3/C5), 73.62 (CI), 69.79 (C4), 60.87 (C6), 48.52, 47.96 ( A r C H , N C H C H N ) . M S (+ES-MS) m/z (relative 2  2  2  intensity) = 597 ([L+H] , 100), 435 ( [ L - C H n 0 ] , 20). Anal. Calcd. (found) for +  +  6  5  i  C H o N O i - H 0 : C, 54.72 (54.47); H , 6.89 (6.91); N , 4.56 (4.54). 28  4  2  2  2  2-(Methylphenyl)-23A6-tetra-0-acetyl-P-D-glucopyranoside (12)  The title compound was prepared by a different method  0 A c 6  AcoH^-*-<\ I A c O - ^ - - * * ^ * * * ^ - - - ^ /--W^d 0  3  OAc'  ||  from that previously reported.  2-Methylphenol 11 (2.380  ^  g> 22.03 mmol) andpentaacetylglucose (7.171 g, 18.37 12  mmol) were dissolved in dry C H C 1 (150 mL) and 2  2  BF3-Et 0 (10.081 g, 70.99 mmol) was added dropwise whilst stirring. The reaction was 2  quenched after 4 h with a saturated NaHC03 (80 mL) solution, and the organic layer was washed with H 0 (80 mL) and a saturated NaCI (50 mL) solution, and then the solvent 2  was removed in vacuo. Recrystallization from hot EtOH (40 mL) afforded the product 12 as a white crystalline solid (5.95 g, 74 %). H N M R (CHCl -di, 400 MHz): 5 7.14 (m, [  3  2H; Ar/7), 6.98 (m, 2H; Ar/7), 5.33 (m, 2H; H-2, H-3), 5.18 (dd, J 3  Hz, 1H; Z7-4), 5.03 (d, V i , = 7.4 Hz, 1H; H-\), 4.29 (dd, 2  3  J 6a 5;  2 ; 3  = 9.5 Hz, V , = 9.1 4  = 5.4 Hz,  2  6a  2  5  Hz,  3  J ,6a 5  = 5.4 Hz,  3  J ,6b 5  6  = 12.3 Hz,  7 ,6b  1H; /7-6a), 4.15 (dd, V , b = 1.7 Hz, J , b = 12.3 Hz, 1H; H-6b), 3.84 (ddd, J 3  6a  6  5  4 ] 5  = 9.1  = 1.7 Hz, 1H; H-5), 2.17 (s, 3H; ArC/7 ), 2.08 (s, 3H; COC/7 ), 3  3  2.06 (s, 3H; COC/7 ), 2.05 (s, 3H; COC/7 ), 2.04 (s, 3H; COC/7 ). M S (+CI-MS) m/z 5  5  5  (relative intensity) = 456 ([L+NH ] , 100). Anal. Calcd. (found) for C i H O , : C, 57.53 +  4  2  2 6  0  (57.43); H , 5.98 (5.97). 104  References start on p. J 66  2-(Bromomethyl)phenyl-2,3,4,6-tetra-0-acetyl-P-D-gIucopyranoside (13)  OAc  The title compound was prepared using a reported  AcO  method.  dissolved in dry benzene (10 mL) and to this solution 1,3-  b  13  Compound 12 (3.501 g, 7.99 mmol) was  dibromo-5,5-dimethylhydantoin (1.170 g, 4.09 mmol) (Figure 3.4) was added followed by 2,2'-azobisisobutyronitrile (AIBN) (0.025 g, 0.15 mmol) (Figure 3.4) and the mixture stirred and heated to reflux while irradiating with a tungsten lamp. A further 0.025 g A I B N was added after 30 min and the reaction was filtered after a total of 1.5 h. The filtrate was evacuated and then recrystallized from CHCl3/hexanes to afford the product 13 (~4.3 g) contaminated with a small amount of the sugar starting material. ' H N M R 300 MHz): 8 7.36 (d, J 3  (CHCl -rfi, 3  C j d  = 7.3 Hz, 1H; A r / / d ) , 7.26 (m, 1H; A r / / b ) , 7.05  (m, 2H; A r / / a and c), 5.36 (m, 2H; H-2, H-3), 5.19 (m, 1H; H-4), 5.16 (d, \ 1H; H-l), 4.64 (d, J  = 9.8 Hz, 1H; A r C / / ) , 4.35 (d, J  2  (dd, V 5 , 6 a = 5.4 Hz,  2  J  2  6a;6b  = 12.2 Hz, 1H; //-6a), 4.15 (dd,  Hz, 1H; //-6b), 3.90 (ddd,  3  J ,5 4  = 9.7 Hz J 3  5M  = 5.4 Hz,  3H; C O C / / ) , 2.07 (s, 3H; COC//3), 2.05 (s, 3H; 5  3  g e m  3  2  J, 5  J ,6b 5  COC//3),  = 7.4 Hz,  = 9.8 Hz, 1H; A r C / / ) , 4.29  2  gem  2  = 2.5 Hz, J , 2  6b  6a 6b  = 12.2  = 2.5 Hz, 1H; H-5), 2.11 (s,  2.04 (s, 3H; COCZ/j). M S  (+ES-MS) m/z (relative intensity) = 541, 539 ([L+Na] , 60), 461 ([starting material +  +Na] , 10), 331 ([L-C H OBr] , 100). +  +  7  6  105  References start on p. 166  N,N'-Bis(2-(phenyl  2,3,4,6-tetra-0-acetyl-P-D-glucopyranoside)benzyl)-N,N'-  dimethyl-ethane-l,2-dianiine (14)  Compound 13 (2.04 g, 3.95 mmol) and N , N ' dimethyl-ethane-l,2-diamine 1 (0.158 g, 1.79 mmol) were dissolved in THF (30 mL); N E t (0.399 g, 3.95 3  mmol) was then added. The reaction mixture was stirred for 6 h and then the solvent was removed in vacuo. The residue was partitioned between a saturated NaHCC»3 (15 mL) solution and portion was extracted with  CH2CI2  CH2CI2  (15 mL) and the aqueous  (2 x 20 mL). The organic extracts were combined,  dried over Na2S04, filtered, and evacuated. The crude material was purified by silica-gel chromatography (95:5 C H C i 2 : M e O H eluent) to afford the product 1 4 as a white solid 2  (0.890 g, 52 %). ' H N M R (CHCl -t/i, 300 MHz): 5 7.45 (d, J 3  3  (dd, J 3  = 7.2 Hz, J 3  b ; C  = 8.1 Hz, 2H; A r / / b ) , 7.05 (dd, J 3  3 ; b  c > d  = 7.3, 2H; A r / / d ) , 7.19  = 7.3 Hz, J , = 7.2 Hz, 2H; 3  C ; d  b  c  A r / / c ) , 6.99 (d, J , = 8.1 Hz, 2H; Ar//a), 5.30 (m, 4H; H-2, H-3), 5.17 (dd, 3  a  Hz,  3  J ,5 4  b  = 9.1 Hz, 2H; H-4), 5.06 (d, J , 3  = 12.2 Hz, 2H; H-6a), 4.15 (dd, J , 3  J ,5 4  = 9.5 Hz J 3  5 > 6 a  = 5.5 Hz,  3  / 6b 5 j  = 7.6 Hz; H-\), 4.26 (dd,  = 2.3 Hz, J ^  3  5  j 2  2  6 b  6H;  2  5  2>  = 5.5 Hz,  = 9.5 2  J ,6 6 a  COC//3),  2.05 (s, 6H; COCH ), 2.04 (s, 3  2.03 (s, 6H; COCH ). M S (+LSIMS) m/z (relative intensity) = 961 3  ([L+H] , 80), 494 ([L-C H28NO, )] , 80), 481 ([L-C 3H oNOio)] , 100). Anal. Calcd. +  +  22  +  0  2  3  (found) for C 6H oN202o-H 0: C, 55.42 (55.45); H , 6.47 (6.11); N , 2.81 (2.94). 4  6  b  = 12.2 Hz, 2H; //-6b), 3.84 (ddd, 2  5  COC//5),  J ,6a  J 3  = 2.3 Hz, 2H; H-5), 3.51 (s, 4H; A r C / / ) , 2.57 (s, 4H;  N C / / C / / N ) , 2.21 (s, 6H; N C / / ) , 2.06 (s, 6H; 2  3  3  2  106  References start on p. 166  N,N'-Bis(2-(phenyl P-D-gIucopyranoside)benzyl)-N,N'-dimethyl-ethane-l,2-diamine (GL ) 2  9  \  HO'  b  Compound 14 (0.610 g, 0.63 mmol) was dissolved in dry MeOH (12 mL) and whilst stirring NaOMe (0.137 g, 2.54 mmol) was added. Rexyn (H form) was added +  after 2 h, and then the mixture was filtered and the  ' 2  solvent was removed in vacuo. The crude material was purified by silica gel chromatography (1:1 EtOAc:MeOH eluent) followed by size-exclusion chromatography on Sephadex G-10 (MeOH eluent) to afford the product G L as a white solid (0.181 g, 46 2  %). H NMR (MeOH-^4, 300 MHz): 5 7.41 (m, 6H; Ar/7 a, b, and d), 7.14 (dd, % !  Hz, J b > c = 7.2 Hz, 2H; Ar/7c), 4.94 (d, J 3  l>2  2  2  = 12.7 Hz, 2H; ArC/7 ), 3.92 (dd, J ,6b = 1.8 Hz, J , 2  3  g e m  = 7.3  = 7.4 Hz, 2H; /7-1), 4.12 (d, V g e m = 12.7 Hz,  3  2H; ArC/7 ), 4.01 (d, J  d  2  6 a  5  6 b  = 12.0  Hz, 2H; //-6b), 3.73 (dd, V ,6a = 5.4 Hz, J , = 12.0 Hz, 2H; //-6a), 3.50 (m, 8H; H-2, 2  6 a  5  6 b  Z7-3, /7-4, /7-5), 3.09 (s, 4H; NC/7 C/7 N), 3.09 (s, 6H; NC//j). C{'H} NMR (MeOH13  2  d , 75.48 MHz):  2  8 158.52 (Cf), 133.85, 132.47 (aromatic CH), 124.89 (Ce), 124.73,  4  118.31 (aromatic CH), 104.20 (CI), 78.94, 78.42 (C3/C5), 75.48 (C2), 71.74 (C4), 62.84 (C6), 57.52 (ArCH ), 53.44 (NCH CH N), 42.26 (NCH ). MS (+ES-MS) m/z (relative 2  2  2  3  intensity) = 625 ([L+H] , 100), 326 ([L-Ci H NO )] , 30). Anal. Calcd. (found) for +  +  4  20  6  C oH44N Oi -H 0: C, 56.07 (55.97); H, 7.21 (7.45); N, 4.36 (4.09). 3  2  2  2  107  References start on p. 166  2-Formylphenyl-2,3,4,6-tetra-0-acetyl-P-D-glucopyranoside (15)  OAc  Helicin 9 (1.006 g, 3.54 mmol) was dissolved in pyridine  AcO' d  (2 mL) and the mixture cooled in an ice bath. To this solution, acetic anhydride (2.913 g, 28.54 mmol) was  a  added dropwise. The solvent was removed under reduced  1 5  pressure after 2 h and the residue dissolved in EtOAc (30 mL) and extracted with H 0 2  (3x 50 mL). The organic layer was dried with N a S 0 , filtered, and the solvent removed 2  4  in vacuo. The crude material was re-crystallized from hot EtOH (10 mL) to afford the product 15 as colourless crystals (0.717 g, 83 %). H N M R ( C H C l - ^ i , 300 MHz): 5 !  3  10.35 (s, 1H; CHO), 7.86 (dd, V 1.6 Hz,  3  J ,c b  2H; Ar/Yc), 7.11 (d, 1, /Y-4), 4.30 (dd, 2  J a,6b 6  V  = 7.2 Hz,  3  3  J ,b a  J ,6a 5  M  = 1.6 Hz, %  d  = 7.5 Hz, 1H; Ar/Yd), 7.56 ( d d d , V = M  = 8.1 Hz, 2H; Ar/Yb), 7.19 (dd, J 3  a>b  = 7.2 Hz, J 3  b ; C  C j d  = 7.5 Hz,  = 8.1 Hz, 2H; Ar/Ya), 5.36 (m, 2H; /Y-3, /Y-4), 5.18 (m, 2H; H-  = 5.1 Hz,  2  Y ,6b 6a  = 123 Hz, 1H; /Y-6a), 4.18 (dd; V ,6b = 2.7 Hz,  = 12.3 Hz, 1H; H-6b), 3.90 (ddd,  5  3  J ,5 4  = 10.1 Hz Y 3  5;  6a  = 5.1 Hz,  3  J ,6b 5  = 2.7 Hz, 1H;  /Y-5), 2.07 (s, 3H; COC/Y ), 2.06 (s, 3H; COC/Yj), 2.06 (s, 3H; COC/Y3), 2.05 (s, 3H; 5  COCH3). M S (+CI-MS) m/z (relative intensity) = 470 ([L+NH ] , 100). Anal. Calcd. +  4  (found) for C i H 0 n : C, 55.75 (55.83); H, 5.35 (5.39). 2  2 4  108  References start on p. 166  N,N'-Bis(2-(phenyI 2,3,4,6-tetra-0-acetyl-P-D-glucopyranoside)benzyl)-N,N'dibenzyl-ethane-l,2-diamine (16)  Compound 15 (0.482 g, 1.07 mmol) and N , N ' dibenzyl-ethane-l,2-diamine 2 (0.128 g, 0.53 mmol) were dissolved in 1,2-dichloroethane (3 mL) and sodium triacetoxyborohydride (0.316 g, 1.49 mmol) 2  was added in portions. The reaction was stirred for 24  16  h and then quenched with a saturated NaHCO"3 (10 mL) solution. The reaction solution was then extracted with EtOAc (3x10 mL), the organic fractions dried with Na2SO"4, filtered, and the solvent removed in vacuo. The crude material was purfied by silica-gel chromatography (97.5:2.5 CH Cl :lV[eOH eluent) to afford the product 16 as a white solid 2  (0.316  g,  2  53 %). ' H N M R (CHCl -rfi, 300 MHz): 5 7.54 (dd, V 3  M  = 1.5 Hz,  3  J , c  d  = 7.8 Hz,  2H; AiHd), 7.20 (m, 12H; Ar//), 6.95 (m, 4H; Ar//), 5.25 (m, 4H; H-2, H-3), 5.13 (m, 2H; H-4), 4.95 (d, J i , = 7.8 Hz, 1H; HA), 4.24 (dd, J 3  3  2  //-6a), 4.15 (dd, J ,6b = 2.1 Hz, 3  5  V , 5  V  = 5.4 Hz, J ,6b = 2.1 Hz, 2H; H-5), 3.56 (d, J  gem  2  5  3  J , 4  = 9.5 Hz  5  2  3.51 (d, Vgem =15.4 Hz, 2H; N C / / C H ) , 3.44 (d, J 6  = 12.3 Hz, 2H;  6 b  = 13.5 Hz, 2H; N C / / C H O H ) ,  2  5  2  6 a  = 12.3 Hz, 2H; //-6b), 3.90 (ddd,  6 a > 6 b  3  6 a  = 5.4 Hz, J , 2  5M  gem  6  4  =13.5 Hz, 2H; N C / ^ C e ^ O H ) ,  3.41 (d, Vgem = 15.4 Hz, 2H; N C / / C H ) , 2.55 (m, 4H; N C / / C / / N ) , 2.03 (s, 6H; 2  6  5  2  2  COCH ), 2.02 (s, 6H; COC//?), 2.01 (s, 6H; COCH ), 1.89 (s, 6H; C O C / / ) . M S 3  3  5  (+LSIMS) m/z (relative intensity) =1113 ([L+H] , 100), 675 ( [ L - C i H O ] , 30), 570 +  +  2  24  10  ([L-C H NOio] , 50), 556 ([L- C aH3 NO ] , 70). Anal. Calcd. (found) for +  28  +  32  2  4  10  C 8 H N 0 o - 3 H 0 : C, 59.63 (59.28); H , 6.47 (5.90); N , 2.40 (2.36). 5  69  2  2  2  109  References start on p. 166  N,N'-Bis(2-(phenyl P-D-glucopyranoside)benzyI)-N,N'-dibenzyl-ethane-l,2-diamine (GL ) 3  Compound 16 (1.18 g, 1.06 mmol) was dissolved in dry MeOH (20 mL) and NaOMe (0.231 g, 4.28 OH  mmol) was added with stirring. Rexyn (H form) was  HO' H  °  2  3  OH  O  added after 2 h, and then the mixture was filtered and  1  •  '  the solvent removed under reduced pressure. The  2  crude material was purified by silica gel chromatography (4:1 MeOH:CH3CN eluent) and the isolated material was then triturated with acetone (15 mL) to afford the product GL  3  as a white solid (0.378 g, 46 %). 'H NMR (MeOH-rf,, 300 MHz): 8 7.40 (m, 12H; Ar/7), 7.28 (m, 4H; Ar/7), 7.09 ((ddd, % = 1.2 Hz % = 7.2 Hz, % = 7.5 Hz, 2H; Ar/7c), c  4.85 (d, J 3  1 > 2  c  d  = 7.8 Hz, 1H; /7-1), 3.92 (m, 10H; NC// C H OH, NC/7 C H , /7-6b), 3.73 2  6  4  2  6  5  (dd, J ,6a = 5.4 Hz, J ,6b = 12.0 Hz, 2H; /7-6a), 3.46 (m, 8H; H-2, H-3, H-4, H-5), 3.01 3  2  5  6a  (s, 4H; NC/7 C/7 N). C{ R} l3  2  2  l  NMR (MeOH-^, 75.48 MHz): 8 158.43 (Cf), 137.06  (Cg), 133.35, 131.76, 131.59, 130.27, 129.80 (aromatic CH), 126.65 (Ce), 124.52, 117.88 (aromatic CH), 104.17 (CI), 78.92, 78.36 (C3/C5), 75.41 (C2), 71.73 (C4), 62.92 (C6), 60.50 (NCH C H OH), 54.71 ( N C H ^ H s ) 50.79 (NCH CH N). MS (+ES-MS) m/z 2  6  4  2  2  (relative intensity) = 778 ([L+H] , 100), 402 ([L-C H NO ] , 10). Anal. Calcd. (found) +  +  20  24  6  for C H N 0, -2H 0: C, 62.06 (62.20); H, 6.94 (6.71); N, 3.45 (3.33). 42  52  2  2  2  110  References start on p. 166  4-Tert-butyl-2-formylphenyl-2,3»4,6-tetra-0-acetyI-P-D-gIucopyranoside (18)  OAc  5-Tert-butyl-2-hydrozybenzaldehyde 5 (2.061 g, 11.56  AcO'  mmol) and a-D-glucopyranosyl bromide tetraacetate 17 (2.382 g, 5.79 mmol) were dissolved in acetone (20 18  mL) and NaOH (4 mL, 5% w/w) was added dropwise.  The reaction was stirred for 24 h and then H 0 (10 mL) was added. The reaction mixture 2  was extracted with CH2CI2 (3x 20 mL), the combined organic extracts were dried over Na S04, filtered, and then the solvent was removed in vacuo. The residue was purified by 2  silica-gel chromatography (1:1 Et20:pet. ether eluent) to afford the product 18 as a white solid (0.971 g, 33%). K N M R (CHCl -rfi, 300 MHz): 5 10.34 (s, 1H; CHO), 7.87 (d, V l  3  = 2.6 Hz, 1H; A r i / d ) , 7.59 (dd, \  = 8.7 Hz, V  b  M  = 2.6 Hz, 1H; AxHb), 7.04 (d, \  M  =  b  8.7 Hz, 1H; Ar//a), 5.34 (m, 2H; H-2, H-3), 5.19 (m, 1H; H-4), 5.15 (d, V i , = 7.5 Hz, 2  1H; H-\), 4.30 (dd, 2  J ,6b 6a  = 5.2 Hz,  V ,6a 5  2  J a,6b 6  = 12.4 Hz, 2H; //-6b), 3.89 (ddd,  3  H-5), 2.08 (s, 3H; COC/f?), 2.06 (s, 3H; COC//3),  = 12.4 Hz, 2H; //-6a), 4.18 (dd,  J ,5 4  = 9.6 Hz J  = 5.2 Hz,  3  5M  COC//3),  2.05 (s, 3H;  3  J ,6b 5  3  J 6b 5 j  = 2.4 Hz,  = 2.4 Hz, 2H; 2.02 (s, 3H;  COC//3),  1.31 (s, 18H; C(CH ) ). M S (+CI-MS) m/z (relative intensity) = 526 ([L+NH ] , +  3  3  4  100), 331 ( [ L - C H i 0 2 ] , 50). Anal. Calcd. (found) for C 5H 2Oir0.5H O: C, 58.02 +  n  3  2  3  2  (57.92); H , 6.43 (6.42).  111  References start on p. 166  N,N'-Bis(2-(4-tert-butyIphenyl-2,3,4,6-tetra-0-acetyl-P-Dglucopyranoside)salicyIidene)ethane-l,2-diamine (19)  The title compound 19 (0.708 g, 72%) was prepared from 4-tert-butyl-2-formylphenyl-2,3,4,6-tetra-0acetyl-P-D-glucopyranoside 18 (0.971 g, 1.91 2  mmol) and ethylenediamine 4 (0.057 g, 0.95 mmol)  19  by a procedure analogous to that described for compound 10. H N M R (DMSO-ck, !  400.13 MHz): 5 8.45 (s, 2H; imine H), 7.80 (d, V = 8.7 Hz, V  M  = 2.5 Hz, 2H; Ar//d), 7.49 (dd, V , A  = 2.5 Hz, 2H; Ar//b), 7.08 (d, J , = 8.7 Hz, 2H; AiH a), 5.46 (d, J , , = 3  B>D  3  a  b  2  7.9 Hz, 2H; H-l), 5.42 (dd, V , = 9.0 Hz, J ,4 = 9.1 Hz, 2H; H-3), 5.14 (dd, J 3  2  3  Hz, JI = 7.9 Hz, 2H; H-2), 5.00 (dd, J 3  3  j2  2H; H-5), 4.19 (dd, J ,6a =5.2 Hz, 3  5  3  3  = 9.1 Hz, J 3  3>4  4;5  2 ; 3  = 9.0  = 9.5 Hz„ 2H; HA), 4.22 (m,  = 12.6 Hz, 2H; //-6a), 4.08 (broad d, J , = 2  J ,b  2  6a 6b  6a 6  12.6 Hz, 2H; //-6b), 3.79 (m, 4H; N C / / C / / N ) , 2.01 (s, 6H; C O C / / ) , 2.00 (s, 6H; 2  2  3  COC//3), 1.99 (s, 6H; COCH ), 1.98 (s, 6H; COC//?), 1.24 (s, 18H; (C(CH ) )). M S 3  3  3  (+LSIMS) m/z (relative intensity) =1041 ([L+H] , 100), 551 ([L-C H Oio] , 30). Anal. +  +  25  30  Calcd. (found) for C H N O : C, 59.99 (59.94); H, 6.58 (6.65); N , 2.69 (2.72). 5 2  6 8  2  2 0  N,N'-Bis(2-(4-tert-butylphenyl-P-D-glucopyranoside)benzyl)ethane-l,2-diamine (GL ) 4  Compound 19 (0.791 g, 0.67 mmol) was added to MeOH (15 mL). To this suspension N a B H (0.0251 g, 0.67 mmol) was added and the reaction mixture heated to reflux. The 4  112  References start on p. 166  B  reaction mixture was cooled to room temperature after 3 h and stirred for a further 16 h before adding H2O (2 mL) to quench the reaction. The solvent was removed and the crude material was purified by silica-gel chromatography (1:1 EtOAc:MeOH eluent) to afford the product G L as a white solid 4  (0.186 g, 39%). ' H N M R (MeOH-J , 300 MHz): 5 7.26 (m, 4H; Ar/Yb and d), 7.13 (d, 4  3  J , = 8.7 Hz, 2H; Ar/Ya), 4.83 (d, J 3  a  b  = 7.4 Hz, 2H; /Y-l), 3.88 (d, J 2  1 ; 2  ArC/Y ), 3.86 (broad d, J a,6b = 12.0 Hz, 2H; /Y-6b), 3.65 (dd, J 2  3  2  6  12.0 Hz, 2H; /Y-6a), 3.60 (d, J 2  g e m  = 12.5 Hz, 2H;  = 5.0 Hz, J , 2  5 ! 6 a  6a 6b  =  = 12.5 Hz, 2H; ArC/Y ), 3.41 (m, 8H; H-2, /Y-3, /Y-4, 2  /Y-5), 2.70 (s, 4H; NC/Y C/Y N), 1.27 (s, 18H; (C(C#j) )). C{ H} u  2  g e m  2  l  3  N M R (MeOH-J , ¥  75.48 MHz): 5 155.80 (Cf), 146.64 (Cc), 129.13 (Ce), 128.95, 126.86, 117.00 (aromatic CH), 103.97 (CI), 78.33, 78.17 (C3/C5), 75.02 (C2), 71.45 (C4), 62.66 (C6), 50.76 ( N C / Y C H O H ), 48.79 ( N C H C H N ) , 35.13 (C(CH ) ), 32.08 (C(CH ) ). M S (+ES-MS) 2  6  4  2  2  3  3  3  3  m/z (relative intensity) = 709 ([L+H] , 100), 598 (5). Anal. Calcd. (found) for +  C H56N Oi : C, 61.00 (61.17); H , 7.96 (8.19); N , 3.95 (4.22). 36  2  2  N,N'-Bis(2-(P-D-glucopyranoside)saIicylidene)-(-)-l,2-cycIohexane-(l/?,2i?)-diamine (20)  The title compound 20 (0.371 g, 93%) was prepared from (IR, 2R)-(-)-\,2diaminocyclohexane 7 (0.071 g, 0.62 mmol) and helicin 9 (0.351 g, 1.23 mmol) by a procedure analogous to that described for compound 10. *H N M R (MeOH-d , 300 MHz): 4  5 8.81 (s, 2H, imine/Y), 7.66 (dd, V d = 1-6 Hz, Y 3  b>  113  Cjd  = 7.8 Hz, 2H; Ar/Yd), 7.37 (ddd,  References start on p. 166  V  1.6 Hz,  =  M  3  J ,c b  = 7.2 Hz,  3  Ja  ; b  = 8.1 Hz, 2H; Ar//b),  7.23 (d, J , = 8.1 Hz, 2H; Ar//a), 7.03 (dd, V 3  a  Hz, J  b  = 7.8 Hz, 2H; AiH c), 4.90 (d, J  3  3  C j d  U2  b;C  = 7.2  = 7.5 Hz, 2H;  H-1), 3.91 (dd, V ,6b = 1.5 Hz, Ve^b = 12.0 Hz, 2H; H5  6b), 3.72 (dd, V , = 5.1 Hz, J 2  5  6a  6 a j 6 b  = 12.0 Hz, 2H; #-6a),  3.55 (m, 8H; H-2, H-3, H-4, H-5, Hg, Hg'), 1.88 (m, 6H; ring H), 1.58 (m, 2H; ring H). MS (+LSIMS) m/z (relative intensity) = 647 ([L+H] , 70), +  381 ([L- C H i 0 ] , 50), 323 ([L-Ci H N0 ] , 100). Anal. Calcd. (found) for +  13  3  +  6  4  18  6  C H 2N Oi2: C, 59.43 (59.05); H, 6.55 (6.85); N, 4.33 (4.27). 32  4  2  N,N'-Bis(2-(phenyl P-D-glucopyranoside)benzyl)-(-)-l,2-cyclohexane-(l/?,2/f)diamine (GL ) 5  The title compound G L (0.274 g, 76%) was prepared 5  from compound 20 (0.350 g, 0.54 mmol) and NaBH e  GL  OH  HN  wo\^^\^o-K\ 3  2  OH  1  i  d  (0.031 g, 0.82 mmol) by a procedure analogous to that  p >  described for G L . *H NMR (MeOH-J , 300 MHz): 8 2  4  7.28 (m, 6H;Ar//a, b, and d), 7.02 (ddd,V a,c  a  £  1.2 Hz, J 3  >c \L_j/ a b 3  J,,  12.0 Hz, 2H; //-6b), 3.84 (d, J 2  2  3  J 6a 5>  = 7.9 Hz, J , - 7.2 Hz, 2H; ArHc), 4.95 (d, 3  C j d  b  = 7.5 Hz, 2H; H-l),  2  c  3.90 (dd, J ,6b = 2.1 Hz, J a,6b 3  2  5  = 5.7 Hz,  = 12.6 Hz, 2H; ArC/fc), 3.78 (d, J  gem  2  J a.6b 6  :  6  2  gem  ArC// ), 3.72 (dd,  4  = 12.6 Hz, 2H;  = 12.0 Hz, 2H; //-6a), 3.46 (m, 8H; H-2, H-3, H-  4, H-5), 2.36 (m, 2H; ring Hg, g'), 2.02 (m, 2H; ring H), 1.69 (m, 2H; ring H), 1.19 (m,  114  References start on p. 166  N M R ( M e O H - < 4 75.48 MHz): 8 155.45 (Cf), 130.67, 129.73  4H; ring//). C{ H) l3  l  (aromatic CH), 128.09 (Ce), 123.02, 115.52 (aromatic CH), 100.74 (CI), 76.03, 75.61 (C3/C5), 72.77 (C2), 69.34 (C4), 60.63 (C6), 58.43 (Cg/Cg'), 44.62 (ArCH ) 29.47, 2  24.16 (ring CH ). M S (+ES-MS) m/z (relative intensity) = 673 ([L+Na] , 5), 651 +  2  ([L+H] , 100), 489 ( [ L - C H 0 ] , 5). Anal. Calcd. (found) for C H N 0 , - H 0 : C, +  +  6  9  5  3 2  4 6  2  2  2  57.47 (57.10); H , 7.23 (7.03); N , 4.19 (4.21).  N,N'-bis-(3-(4-Hydroxyphenyl-P-D-glucopyranoside)benzyl)-N,N'-dimethyl-ethane1,2-diamine ( H G L ) 6  2  4-Hydroxyphenyl-p-D-glucopyranoside (arbutin) 21 (10.01 g, 36.8 mmol), paraformaldehyde (1.20 g, 40.00 mmol), and N,N'-dimethyl-l,2-ethaneH GL  diamine 1 (1.470 g, 16.68 mmol) were added to  6  2  EtOH (150 mL) and the reaction was heated to reflux for 24 h and then evacuated. The residue was purified by silica-gel chromatography (1:1 MeOH:EtOAc eluent) to afford the product H G L as a white solid (4.122 g, 39 %). ' H N M R (MeOH-</ , 300 MHz): 5 6  2  6.96 (dd, V  M  4  = 2.8 Hz, %  b  =8.7 Hz, 2H; A r / / b ) , 6.90 (d, V  M  = 2.8 Hz, 2H; Ar/Yd),  6.70 (d, ./ ,b =8.7 Hz, 2H; Ar/Ya), 4.77 (d, J = 7.5 Hz, 2H; /Y-l), 3.89 (dd, 3  3  U  a  Hz,  2  Y  6 a j  6b  = 12.0 Hz, 2H; /Y-6b), 3.73 (dd,  3  Y 6a= 5 ;  5.4 Hz,  2  J ,6b= 6 a  3  1.7  Y 6b= 5 ;  12.0 Hz, 2H; /Y-6a),  3.68 (s, 2H; ArC/Y ), 3.30-3.45 (m, 8H; H-2, /Y-3, /Y-4, /Y-5), 2.69 (s, 4H, NC/Y C/Y N ), 2  2.28 (s, 6H, N C / / ) . 3  2  1 3  2  C N M R (MeOH-d , 75.48 MHz): 8 153.91, 152.26 (Cf, Cc), 4  124.50 (aromatic Ce), 119.47, 118,70, 117.26 (aromatic CH), 103.68 (CI), 78.12 (C3/C5), 75.14 (C2), 71.62 (C4), 62.76, 61.36 (C6/ArCH ), 55.03 ( N C H C H N ) , 42.06 2  115  2  2  References start on p. 166  (NCH ). M S (+ES-MS) m/z (relative intensity) = 657  ([L  C i H i 0 ] , 40). UV-visible spectrum (MeOH): ^  ( w ) = 226 nm (1.1 x 10 L mol"  3  +  3  5  7  + H] , +  100), 373.6 ( [ L 4  1  cm" ), 289 nm (5.4 x 1 0 L mol" cm" ). Anal. Calcd (found) for C oH4 N Oi4-H 0: C, 1  3  1  1  3  4  2  2  53.41 (53.65); H , 6.87 (7.12); N , 4.15 (4.03).  3-Tert-butyl-4-hydroxyphenyl-2,3,4,6-tetra-0-acetyl-P-D-glucopyranoside (23P)  The title compound was synthesized by a method different from that available in the literature. Tert31  "OH  butyl-hydroquinone 22 (0.535 g, 3.22 mmol) and pentaacetylglucose (1.05 g, 2.69 mmol) were  dissolved in dry C H C 1 (30 mL) and with stirring B F OEt (1.90 g, 13.41 mmol) was 2  2  3  2  added dropwise. A saturated N a H C 0 (30 mL) solution was added after 4 h and the 3  resulting mixture extracted with CH C1 (2 x 30 mL). The combined organic extracts 2  2  were dried over Na S04, filtered, and then the solvent was removed in vacuo. The crude 2  product was purified by silica-gel chromatography (1:1 then 2:1 Et 0:pet. ether eluent) to 2  afford the product 23p as a white solid (0.931 g, 70 %). *H N M R ( C H C 1 M , 300 MHz): 5 6.94 (d,  4  J ,d= b  2.9 Hz, 1H; Ar/Yd), 6.71 (dd, V b > d = 2.9 Hz,  3  J, a  b  = 8.5 Hz, 1H; AiHb),  6.57 (d, J , = 8.5 Hz, 1H; Ar/Ya), 5.19 (m, 3H; H-2, /Y-3, /Y-4), 4.96 (d, J i , = 7.5 Hz, 3  3  a  b  2  1H; /Y-l), 4.73 (br. s, 1H; OH), 4.22 (dd, (dd,  3  J 6b= 5 ;  2.4 Hz,  2  Y a,6b= 6  1 2  3  J ,6a= 5  5.2 Hz,  - Hz, 1H; H-6b), (ddd, J 6  2  Y ,6b  3  6a  = 12.6 Hz, 1H; /Y-6a), 4.15  = 9.5 Hz, Y 3  4 ; 5  5 j 6 a  = 5.2 Hz, %  6b  = 2.4 Hz, 2H; /Y-5), 2.08 (s, 6H; 2x COC/Yj), 2.04 (s, 3H; COC/Y ), 2.03 (s, 3H; COC/Y5), 5  1.38 (s, 18H; C(C/Y ) ). M S (+CI-MS) m/z (relative intensity) = 514 ([L+NH ] , 90), 331 +  3  3  4  116  References start on p. 166  ( [ L - C i H i O ] , 100). Anal. Calcd (found) for C 2 4 H 3 2 O 1 1 : C, 58.06 (57.95); H , 6.50 +  0  3  2  (6.47).  3-Tert-butyl-4-hydroxyphenyl-2,3 4,6-tetra-0-acetyI-a-D-glucopyranoside (23a) ?  ? i - A . 5^0  The title compound 23a (2% yield) was isolated from the  A c  6  AcO-^^^A  1  previous coupling reaction between tert-butylj| V  \  hydroquinone 22 and pentaacetylglucose. ' H N M R  (CHCl -^i, 300 MHz): 5 6.98 (d, V = 3.0 Hz, 1H; ArH  3 2 3 a  3  d), 6.78 (dd, J = 3.0 Hz, % 4  M  M  = 8.6 Hz, 1H, AvHb), 6.58 (d, J , = 8.6 Hz, 1H; ArH a), 3  b  a  5.69 (dd, J , = 10.2 Hz, J , = 10.0 Hz, H-3), 5.60 (d, J 3  3  2  3  3  3  4  = V , = 10.0 Hz, 1H; HA), 5.01 (dd, J = 3.6 Hz, J 3  4  5  1H; OH), 4.27 (dd, V 3  J ,6a= 5  3  1;2  5;  4.4 Hz,  6a=  2  J ,6b= 6 a  = 3.6 Hz, H-l), 5.15 (dd, J , 3  1 ; 2  3  3  5  3  J ,6b= 5  4  = 10.2 Hz, 1H; H-2), 4.65 (br. s,  12.1 Hz, 1H; 77-6a), 4.18 (ddd,  4.3 Hz, J , = 10.0 Hz, 1H; H-5), 4.06 (dd, 4  2 ; 3  b  2.0 Hz,  V ,6b= 6a  3  J ,6b= 5  2.0 Hz,  12.1 Hz, 1H;  H-6b), 2.07 (s, 3H; COCH ), 2.06 (s, 3H; COCH ), 2.05 (s, 3H; COCH ), 2.04 (s, 3H; 3  3  3  COCH3), 1.39 (s, 18H; C(CH ) ). M S (+ES-MS) m/z (relative intensity) = 519 ([L+Na] , +  3 3  100). Anal. Calcd (found) for C H 0 u : C, 58.06 (58.56); H , 6.50 (6.55). 2 4  3 2  N,N'-Bis-(3-(5-tert-butyll-4-hydroxyphenyl-2,3,4,6-tetra-0-acetyl-p-Dglucopyranoside)benzyI)-N,N'-dimethyl-ethane-l,2-diamine (24)  Compound 23 (3.90 g, 7.86 mmol), paraformaldehyde (0.26 g, 8.57 mmol), and N , N ' dimethyl-ethane-l,2-diamine 1 (0.315 g, 3.57 mmol) were added to dry benzene (40 mL).  117  References start on p. 166  With stirring, the reaction mixture was heated to \  OAc  4 1 * ^  / \  d / ~  6  A c O ^ ^ ^ AcO-V^<T->^0—(\  \  reflux for 22 h. The solvent was then removed in  Q  3  z  OAc  ^  1  b  /)—OH .  U  V  / 2  , ,  .,  ._  vacuo and the residue purified by silica-gel chromatography (3:1 Et20:pet. ether eluent) to afford the product 24 as a white solid (2.49 g, 63  %). ' H N M R (CHCl -rfi, 300 MHz): 8 10.60 (s, 2H; OH), 6.87 (d, V = 2.7 Hz, 2H; Ar/7 3  M  b or d), 6.50 (d, % = 2.7 Hz, 2H; Ar/Yb or d), 5.25 (m, 6H; H-2, /Y-3, /Y-4), 4.94 (d, d  3  Ji, = 2  3  7.5 Hz, 2H; /Y-l), 4.27 (dd, J ,6a= 5.2 Hz, J , = 12.3 Hz, 2H; //-6a), 4.15 (dd, 3  2  5  6 a  6 b  J ,6b= 2.2 Hz, V ,6b= 12.3 Hz, 1H; //-6b), 3.81 (ddd, y a= 5.2 Hz, Y 3  5  6a  3  5;6  5>6b  = 2.2 Hz,  V,  4 5  = 9.6 Hz, 2H; /Y-5), 3.61 (s, 4H; ArC/Y ), 2.59 (s, 4H, NC/Y2CYY2N ), 2.25 (s, 6H; NC7/j), 2  2.08 (s, 6H; COC/Y5), 2.07 (s, 6H; COC/Y5), 2.04 (s, 6H; COC/Y3), 2.0 (s, 6H; COC/Y3),  1.36 (s, 18H; C(C/Y ) ). M S (+LSIMS) m/z (relative intensity) = 1105 ([L+H] , 30), 552 +  5  3  ([L-C 7H NOn] , 100). Anal. Calcd (found) for C 4H N 022: C, 58.69 (59.11); H, 6.93 +  2  38  5  76  2  (J'.OT); N , 2.53 (2.69).  N,N'-bis-(3-(5-tert-Butyl-4-hydroxyphenyl-P-D-gIucopyranoside)benzyI)-N,N'dimethyl-ethane-l,2-diamine (H2GL ) 7  Compound 24 (5.90 g, 7.68 mmol) was dissolved in dry MeOH (100 mL) and NaOMe (1.15 g, 21.3 mmol) was added with stirring. Rexyn ( H form) +  was added after 2 h, and then the mixture was filtered and the solvent removed under reduced pressure. The crude material was purified by silica gel chromatography (4:1  118  References start on p. 166  EtOAc:MeOH eluent) to afford the product H G L as a white solid (3.81 g, 93 %). U 7  l  2  N M R ( M e O H - ^ , 300 MHz): 5 7.02 (d, V b > d = 2.8 Hz, 2H; ArH, b or d), 6.73 (d,  4  J = M  2.8 Hz, 2H; ArH, b or d), 4.86 (d, J i , = 7.5 Hz, 2H; HA), 3.83 (dd, J ,6b= 1.7 Hz, J , 3  3  = 12.1 Hz, 2H; H-6b), 3.74 (dd, J 3  2  5  2  6 a  6 b  = 3.1 Hz, J a,6b= 12.1 Hz, 2H; H-6a), 3.68 (s, 4H; 2  5>6a  6  AiCH ), 3.44 (m, 8H; H-2, H-3, HA, H-5), 2.66 (s, 4H, N C / 7 C / / N ), 2.27 (s, 6H, 2  2  N C / / ) , 1.39 (s, 18H; C(CH ) ). 3  1 3  3 3  2  C N M R (MeOH-^ , 75.48 MHz): 5 153.56, 151.84 (Cf, 4  Cc), 139.02 (Ca), 124.52 (Ce), 117.11, 116.93 (aromatic CH), 104.2 (CI), 78.46 (C3/C5), 75.45 (C2), 71.96 (C4), 63.57, 63.08 (C6/ArCH ), 54.80 ( N C H C H N ) , 41.95 (NCH ), 2  2  2  3  36.16 (C(CH ) ), 30.52 (C(CH ) ). M S (+ES-MS) m/z (relative intensity) = 791 3  3  3  3  ([L+Na] , 10), 769 ([L+H] , 100). UV-visible spectrum (MeOH): ^ +  +  (s ) = 231 nm max  (1.2 x 10 L mol" cm" ), 288 nm (6.2 x 10 L mol" cm" ). Anal. Calcd (found) for 4  1  1  3  1  1  C H oN20,4-H 0: C, 58.00 (58.40); H, 7.94 (7.92); N , 3.56 (3.57). 38  6  2  3.2.13 C u  2 +  and N i  2 +  Complexes of Deprotonated H G L  6  7  2  Synthesis of C u G L - 2 H 0 6  2  H G L (0.096 g, 0.15 mmol) and Cu(C10 ) -6H 0 (0.054 g, 0.15 mmol) were dissolved 6  2  4  2  2  in MeOH (5 mL) and aqueous NaOH (0.5 mL, IM) was added with stirring. The resulting green suspension was stirred for 2 h and then the solvent was removed in vacuo. The residue was dissolved in a minimum of H 0 and purified by size-exclusion 2  chromatography on Sephadex G-10 ( H 0 eluent) to afford the product C u G L - 2 H 0 as a 6  2  2  dark green solid (0.047 g, 47 %). M S (+ES-MS) m/z (relative intensity) = 720/718  119  References start on p. 166  ([M+H] , 100), 657 ([L+H] , 20), 557/555 ([M-C Hn0 ] , 50). EPR (130 K, MeOH): A +  +  +  6  5  L  = 20 x 10" cm" , g = 2.037, A , , = 179 x 10" cm" , g„ = 2.215. UV-visible spectrum 4  1  4  1  x  (MeOH): / I ™ * ( w ) = 243 nm (2.8 x 10 L mol" cm" ), 290 nm (1.5 x 10 L mol" cm" ), 4  1  1  4  1  1  420 nm (2.0 x 10 L mol" cm" ), 601 nm (7.0 x 10 L mol" cm" ). The room temperature 3  1  1  2  1  1  solid state magnetic moment p ff = 1.74 BM. Anal. Calcd (found) for e  C oH CuN 0, -2H 0: C, 47.77 (47.82); H, 6.15 (5.90); N, 3.71 (3.63). 3  42  2  4  2  Synthesis of N i G L - 3 H 0 6  2  H G L (0.101 g, 0.15 mmol) and Ni(C10 ) 6H 0 (0.055 g, 0.15 mmol) were dissolved 6  2  4  2  2  in MeOH (4 mL) and NEt (0.031 g, 0.31 mmol) was added with stirring. The reaction 3  mixture turned a dark red colour upon base addition. CH CN (2 mL) was added to the 3  reaction mixture after 12 h and the product formed an oil. The reaction solution was decanted and the oily residue was re-dissolved in H 0 and precipitated using CH CN (2 2  3  mL) to afford the product NiGL -3H 0 as a red-brown solid upon drying (0.027 g, 25%). 6  2  MS (+ES-MS) m/z (relative intensity) = 713 ([M+H] , 100). Anal. Calcd (found) for +  C H N NiOi4-3H O: C, 46.95 (47.03); H, 6.30 (6.21); N, 3.65 (3.42). 30  42  2  2  Synthesis of C u G L - 2 M e O H 7  H G L (0.082 g, 0.11 mmol) and Cu(C10 ) -6H 0 (0.040 g, 0.11 mmol) were dissolved 7  2  4  2  2  in MeOH (4 mL) and aqueous NaOH (0.5 mL, IM) was added with stirring. The green solution was stirred for 2 h and then the solvent was removed in vacuo. The residue was  120  References start on p. 166  dissolved in a minimum of M e O H and purified by size-exclusion chromatography on Sephadex G-10 (MeOH eluent) to afford the product C u G L - 2 M e O H as a dark green 7  solid (0.050 g, 56 %). M S (+ES-MS) m/z (relative intensity) = 854/852 ([M+Na] , 100), +  830 ([M+H] , 10). EPR (130 K , MeOH): A = 24 x 10" cm" , g = 2.040, A = 30 x IO" +  4  1  x  4  x  y  cm" , g = 2.020, A = 178 x 10" cm" , g = 2.215. UV-visible spectrum (MeOH): 1  4  y  ^  1  z  z  (£max) = 247 nm (1.7 x 10 L mol" cm" ), 297 nm (1.2 x 10 L mol" cm" ), 445 nm (2.7 x 4  1  1  4  1  1  10 L mol" cm" ), 620 nm (1.4 x 10 L mol" cm" ). The room temperature solid state 3  1  1  3  1  1  magnetic moment p ff = 1.79 B M . Anal. Calcd (found) for C 8H CuN 0i4-2Me0H: C, e  3  58  2  53.71 (53.38); H , 7.44 (7.30); N , 3.13 (3.23).  Synthesis o f N i G L H 0 7  2  H G L (0.051 g, 0.07 mmol) and Ni(C10 ) -6H 0 (0.024 g, 0.07 mmol) were dissolved 7  2  4  2  2  in MeOH (2 mL) and N E t (0.036 g, 0.36 mmol) was added with stirring. The colour of 3  the reaction mixture changed from light pink to dark red over a period of 24 h. The reaction mixture was filtered and the filtrate left to stand for 3 days in which time red crystals of the product N i G L - H 0 formed. The X-ray quality crystals were isolated by 7  2  decanting the supernatant liquid and washing with a minimum amount of cold MeOH to afford the crystalline product N i G L - H 0 (0.012 g, 20%). M S (+ES-MS) m/z (relative 7  2  intensity) = 847 ([M+Na] , 60), 826 ([M+H] , 90), 792 ([L+Na] , 10), 769 ([L+H] , 100). +  +  +  +  Anal. Calcd (found) for C H 8 N N i O i H 0 : C, 54.10 (53.86); H , 7.17 (7.18); N , 3.32 38  5  2  4  2  (3.42).  121  References start on p. 166  3.3 Results and Discussion  3.3.1 Synthesis of the Tetrahydrosalen Compounds  The tetrahydrosalen pro-ligands were synthesized using literature procedures or adapted versions thereof. In each case the appropriate salicylaldehyde was coupled with a diamine and the resulting salen derivative was either isolated or immediately reduced in situ to afford the corresponding tetrahydrosalen compounds H L . 1 5  2  CHO R  / — \ ,R N N H H  H L  OH +  R = Methyl  2  2  H L 2  3  R = Benzyl  1 R = Methyl 2 R = Benzyl  Scheme 3.2: Synthesis of H2L and H L : a) sodium tris(acetoxy)borohydride, 1,23  2  dichloroethane, 54 % for H L , 63 % for H L . 2  2  3  2  H2L (Figure 3.2) was synthesized by a literature method. 1  34  H 2 L and H 2 L were 2  3  synthesized using the same protocol as shown in Scheme 3.2. In both cases reductive amination of salicyladehyde 3 with the corresponding diamine (1 or 2) using sodium triacetoxyborohydride as the reducing agent afforded the tetrahydrosalen compounds 56  H2L and H L . N,N'-Dibenzyl-ethane-l,2-diamine 2 was prepared via the method of 2  Frost and Freedman with minor modifications.  122  53  References start on p. 166  Scheme 3.3: Synthesis of H L : a) EtOH. b) N a B H , MeOH, 71 % for two steps. 4  2  4  H L (Scheme 3.3) was synthesized in two steps via isolation of the diimine, 4  2  followed by reduction. Initially ethylenediamine 4 and 5-tert-butyl-2hydroxybenzaldehyde 5 were combined to form the salen derivative 6. Compound 6 was then reduced using N a B H in MeOH to afford H L in 71 % yield over the two steps. It 4  4  2  was postulated that the presence of the t-butyl group para to the phenol moiety would enhance the antioxidant potential of this compound. H L (Scheme 3.4) was synthesized 5  2  in much the same manner as was H L utilizing (l/?,2^)-(-)-l,2-diaminocyclohexane 7 4  2  and salicylaldehyde 3 as the starting materials to afford this tetrahydrosalen derivative in 73 % yield over two steps.  8  H L  5  2  Scheme 3.4: Synthesis of H L : a) EtOH. b) N a B H , MeOH, 73 % for two steps. 5  2  4  123  References start on p. 166  3.3.2 Synthesis and Characterization of the Carbohydrate-derivatives of the Tetrahydrosalen Ligands  The synthesis of the carbohydrate-protected tetrahydrosalen compounds (GL ) 1-5  proceeded by a variety of routes, either by reductive amination, or N-alkylation of the diamines with the appropriate carbohydrate-protected phenols. A l l of the carbohydrateprotected tetrahydrosalen compounds were isolated as their p-anomers to afford potential substrates for the p-glucosidase enzymes. Initial attempts to directly glycosylate the tetrahydrosalen compounds H L'" using the Koenigs-Knorr reaction with either silver 5  57  2  carbonate or silver triflate and a-D-glucopyranosylbromide tetraacetate 17 were unsuccessful. Steric interference may inhibit the formation of the bis-glycosylated tetrahydrosalen compounds by this route. Glycoside bond formation utilizing B F O E t 2  58  3  and pentaacetylglucose was also attempted, however this route was not successful. Boron complexation by the tetrahydrosalen compounds, analogous to the stable boronpyridinone complexes synthesized in the Orvig group, ' could explain why BF OEt2 59 60  3  may not be an ideal coupling reagent. Phenols were thus glycosylated prior to the formation of the tetrahydrosalen framework to circumvent the lack of reactivity discussed above. Using this general route, further structural diversity could easily be obtained through the use of a series of secondary diamines. G L (Scheme 3.5) was formed by the reaction of salicylaldehyde-P-D1  glucopyranoside (helicin) 9 with ethylenediamine 4 to form the imine 10 which was isolated and characterized. The imine was then reduced using NaBFL; in MeOH to afford G L in 48% yield over two steps. G L was found to be associated with one molecule of 1  1  124  References start on p. 166  water in the solid state. This result was not unexpected as the glycosylated tetrahydrosalen compounds discussed here were found to be quite hygroscopic.  Scheme 3.5: Synthesis of G L : a) ethylenediamine 4, EtOH, 91%. b) N a B H , MeOH, 53%. 1  4  G L was synthesized as shown in Scheme 3.6. 2-Methylphenol 11 was first glycosylated using BF OEt2 and pentaacetylglucose to afford compound 12. 3  Bromination of compound 12 with l,3-dibromo-5,5-dimethylhydantoin in the presence of A I B N afforded compound 13.  55  Compound 13 was seen as a useful building block for the  attachment of glycoside-protected phenols to suitable compounds such as amines and other nucleophiles. Unfortunately, compound 13 was difficult to purify and was found to contain a residual amount of the starting material 12. In any case, N-alkylation of two equivalents of compound 13 withN,N'-dimethylethylenediamine 1 in the presence of NEt3 and subsequent acetyl group removal using NaOMe in M e O H afforded G L in 2  moderate yield.  125  References start on p. 166  Scheme 3.6: Synthesis of G L : a) pentaacetylglucose, B F E t 0 , CH C1 , 74%. b) 1,32  3  2  2  2  dibromo-5,5-dimethylhydantoin, A I B N , benzene, c) NEt , THF, 52%. d) NaOMe, 3  MeOH, 46%.  Initial attempts to synthesize G L (Scheme 3.7) via the route used for G L were only partially successful. The coupling of N,N'-dibenzylethylenediamine 2 with compound 13 resulted in the isolation of 16 in only 9% yield. However, a reductive amination reaction between aldehyde 15 and N,N'-dibenzylethylenediamine 2, using sodium tris(acetoxy)borohydride afforded 16 in increased yield (53%>). Acetyl group removal afforded G L in 46% yield.  126  References start on p. 166  GlucO.  Scheme 3.7: Synthesis of G L : a) acetic anhydride, pyridine, 83%. b) N , N ' 3  dibenzylethylenediamine 2, sodium triacetoxyborohydride, 1,2-dichloroethane, 53%. c) NaOMe, MeOH, 46%.  The synthesis of G L i s shown in Scheme 3.8. Coupling of 5-tert-butyl-24  hydroxybenzaldehyde 5 and oc-D-glucopyranosylbromide tetraacetate 17 using a published protocol, afforded compound 18 in 33% yield. Coupling of compound 18 61  with ethylenediamine afforded the salen derivative 19 which was then reduced with NaBH4 in MeOH. Under these conditions the sugar acetyl groups were also removed to afford G L in one step. Interestingly, use of the mild reducing agent sodium 4  tris(acetoxy)borohydride, as opposed to NaBHj, for the synthesis of compound 16 did not lead to acetyl group removal as with G L . 4  Scheme 3.8: Synthesis of G L : a) NaOH, acetone, 33%. b) ethylenediamine 1, EtOH, 4  72%. c) N a B H , MeOH, 39%. 4  127  References start on p. 166  G L is a more rigid version of G L exhibiting a 1,2-diaminocyclohexane 5  1  backbone. G L was synthesized (Scheme 3.9) by coupling (li?,2i?)-(-)-l,25  diaminocyclohexane 7 with helicin 9 to afford the diimine 20. Reduction with NaBH  4  afforded G L in 71% yield over two steps. 5  GL  20  5  Scheme 3.9: Synthesis of G L : a) EtOH, 93%. b) NaBH , MeOH, 76%. 5  4  1  6  H2GL and H G L were designed to chelate metal ions in their deprotonated 2  forms while carrying carbohydrates to enhance solubility and improve targeting ability. For H2GL , commercially available arbutin 21 was coupled with N,N'6  dimethylethylenediamine via a Mannich condensation to afford H 2 G L in 39% yield 6  62  (Scheme 3.10). \ / — \  / OGluc  Scheme 3.10: Synthesis of H G L : a) paraformaldehyde, EtOH, 39%. 6  2  128  References start on p. 166  The synthesis of H 2 G L is shown in Scheme 3.11. In the first step 7  t-butylhydroquinone 22 was coupled with pentaacetylglucose to afford the compound 23.  OGluc(OAc)  4  23  H GL  7  2  Scheme 3.11: Synthesis of H G L : a) pentaacetylglucose, B F O E t , CH C1 , 70%. b) 7  2  3  2  2  2  paraformaldehyde, benzene, 63%. c) NaOMe, MeOH, 93%.  B F O E t was used as the Lewis acid promoter and the yield of 23 was almost 3  2  identical to a literature report of the same coupling using /7-toluenesulfonic acid under Helferich conditions.  Glycosylation occurred exclusively at the less hindered alcohol in  72% overall yield (97% f3: 3% a). The two anomers were separated by silica-gel chromatography and compound 23P was then coupled with N , N ' dimethylethylenediamine via a Mannich condensation to afford 24 in 63% yield. Compound 24 was then deprotected under standard conditions to afford H G L in 93% 7  2  yield. The use of a series of secondary diamines, replacing the methyls of N , N ' dimethylethylenediamine, could easily lead to analogs of H G L and H G L . 6  2  129  7  2  References start on p. 166  X-ray quality crystals of H G L were grown via the slow evaporation of a 7  2  concentrated solution of the compound in MeOH/HiO. A n ORTEP diagram of H G L is 7  2  shown in Figure 3.5 with selected bond lengths and H-bonding interactions presented in Table 3.1. The triclinic unit cell contained four molecules of H G L as well as numerous 7  2  solvent molecules. As the four molecules of H G L are crystallographically independent, 7  2  yet chemically equivalent, only one of them is shown in Figure 3.5.  Figure 3.5: ORTEP diagram of H G L showing 50% thermal probability ellipsoids. 7  2  Table 3.1: Selected bond lengths (A) in H G L . 7  2  C(19)-C-(38) N(l)-C(17) N(l)-C(19) N(2)-C(36) N(2)-C(38) C(10)-O(7) C(29)-0(14)  1.53(1) 1.471(8) 1.469(9) 1.468(8) 1.462(9) 1.387(8) 1.374(7)  C(7)-0(l) C(l)-0(1) C(20)-O(8) C(26)-0(8)  1.407(8) 1.380(9) 1.398(7) 1.404(7)  H-bonding 0(7)-(H7) •-N(l) 0(14)-(H14) N(2)  2.64(1) 2.68(1)  130  References start on p. 166  The carbohydrate moieties participate in a network of hydrogen bonding interactions in the solid state, accounting for the observed packing arrangement in the crystal. As well, intramolecular H-bonding between the amine nitrogen atoms and the H atoms of the hydroxyl groups are evident (shown by dashed lines in Figure 3.5). These H bonds were determined to have an important stabilizing effect in solution for the neutral H GL 2  6  and H 2 G L species (vide infra). 7  6  7  Both H2GL and  H G L 2  were investigated by potentiometry to determine the  ability of these compounds to compete with biological ligands (notably A(3) for excess Cu and Zn. Potentiometric titrations of these two ligands were first completed to determine the acidity constants (K \-K 4) of these compounds (Table 3.2, and Equations a  a  3.2-3.5); the results were then used as constants in the metal binding studies discussed in Section 3.3.3. The acidity constants (Table 3.2) were also compared to other tetrahydrosalen derivatives shown in Figure 3.6.  _[[H GL - ] ][H ] 6  [H GL " ] 6  7  2+  4  ± 5 [H GL " ] + H 6  7  +  +  K  7  +  +  3  3  [[H GL " ] ] 6  7  2+  (Eq. 3.2)  4  [H GL " ][H ] 6  [ H G L - ] t" H G L " + H 6  7  3  +  6  7  K>2  2  7  +  2  +  =  [[H GL - ] ] 6  7  +  (Eq. 3.3)  3  _ [[HGL - ]-][FT] 6  H G L " ± 5 [HGL " ]" + FT 6  7  6  7  2  7  [H GL  ]  2  [[GL - ] "][H ] 6  [HGL " ]" ± 5 [GL " ] " + H 6  7  6  7  2  + K  a4  131  -  7  2  +  [[HGL ]] 67  (Eq. 3.5)  References start on p. 166  Due to the limitations of potentiometry (inaccurate because of ionic strength changes when pH < 2.5 or pH>l 1), UV-vis spectrophotometry was used to determine pK  a  values outside the usable p H range of potentiometry. In addition, as potentiometry does not provide microscopic information involving the identification of protonation sites on the ligands, spectroscopic methods (UV-vis / ' H N M R ) were therefore used to assign pK  a  values to ionizable sites on the ligands.  Table 3.2: Deprotonation constants (pK s) of various tetrahydrosalen derivatives (Figure a  3.6) (25 °C, 7= 0.16 M NaCI unless otherwise noted; errors are for the last digit).  equil. quotient  HBED  HYBEDA"  H2bbpen  H GL  [L][H]/[LH]  12.60  10.50  11.33(4)  11.3(3)  [HL][H]/[H L]  11.00  9.80  10.64(7)  10.30(5)  A  c  H GL  6  2  7  2  d  13.7(5) (pK ) 13.1(5)  d  a4  2  [H L][H]/[H L]  8.44  8.37  9.09(7)  [H L][H]/[H L]  4.72  6.17  6.87(3)  [H L][H]/[H L]  2.53  -  4.70(3)  [H L][H]/[H L]  1.74  -  2.17(8)  2  3  e  (P^a3)  (pKa3)  8.03(6)  6.47(4)  d  (P^al) 3  4  5  a  4  5  6  5.01(4)  4.02(9)  (P^al)  (P*al)  -  -  -  -  ref.[63], / = 0.1 M K N 0 ; ref.[23], 1= 0.1 M KCI; °ref[64]; determined b  3  spectrophotometrically; determined by both spectrophotometric (10.2(2)) and e  potentiometric (10.30(5)) methods.  132  References start on p. 166  There are four ionizable groups o f interest on H 2 G L or H 2 G L , two tertiary 6  7  amines and two phenol moieties. It is unlikely that deprotonation ofthe O H groups o f glucose occurs in the p H range examined here. While the pK value o f glucose at C-1 a  (p/v =12.3. / = 0.2 M N a C I , 25 ° C )  65  a  is within the p H range studied here, the glycoside  bond at C-1 for H 2 G L and H 2 G L precludes this de-protonation/protonation from 6  7  occurring. The ionization values o f the other four O H groups o f glucose are expected to be higher (pK> 14.2) , similar to ethylene glycol (pK = 14.77) and glycerol (pK = 65  66  x  {  14.40) , and beyond the range o f interest for this study. 66  HOOC  COOH  NH  H N ^  OH H O - ^ ^ ~ \ \  R  -OH HO  A^Af'-Bis(2-hydroxybenzyl)-ethylenediamine-A'iA^'-diacetic acid (HBED)  MA^'-Bis(2-hydroxybenzyl)-ethylenediamine (HYBEDA)  OH HO'  ^ O H H O - ^ J  OH  R H GL  /  ^  R= H  6  2  N,A^'-Bis(2-hydroxybenzyl)-yV,7V'-(2-methylpyridyl)-ethylenediamine  ^  H GL 2  7  R = t-butyl  (H bbpen) 2  /—COOH HOOC^ / HOOC-^  N /  V _  C  0  0  H  N  Ethylenediaminetetraacetic acid ( E D T A )  Figure 3.6: Compounds relevant to the potentiometric discussion.  133  References start on p. 166  O  H  For H2GL , three acidity constants (pK \, pK^, and pK^) could be accurately 6  a  determined by potentiometry, while two acidity constants (pK^ and pK 4) could be a  evaluated spectrophotometrically. The values for pK^ determined by the two methods (pATa3 =  10.2(2) via UV-vis; pK^ = 10.30(5) via potentiometry) were found to agree very  well. The higher error associated with the p i ^ value determined spectrophotometrically is due to the lower number of data points (-20) used in the fitting procedure and thus the number determined by potentiometry is quoted in Table 3.2. The first two acidity constants (pK , pK^) for H 2 G L were assigned to the tertiary amine moieties on the 6  ai  basis of variable pD ' H N M R (Figure 3.7) and pH UV-vis (Figure 3.8) studies.  134  References start on p. 166  Shifts in the ethylenediamine backbone as well as the N-methyl hydrogen signals in the pD range 4-8 signified that deprotonation of the two ammonium moieties was occurring. The increased shift of the hydrogen signal a to the phenol hydroxyl (H ) in the a  basic pH region leads to the tentative assignment of p/Ca3 and pK 4 to deprotonation of the a  two phenols. This assignment is further substantiated from the variable pH UV-vis experiment (Figure 3.8), in which a bathochromic shift of the aromatic 7T.—»7t* transition (and an increase in s  max  ) due to deprotonation of the two phenol moieties occurs in the  basic pH region.  ° C , / = 0.16 M NaCI).  135  References start on p. 166  The UV-vis trace remains unchanged until pH~9.5. The order of deprotonation for the ammonium and phenol moieties in H 2 G L parallels that for the related 6  compounds H Y B E D A , H bbpen , and H B E D 2 3  64  63  2  (Figure 3.6, Table 3.2).  The weak basicity of the ammonium moieties is most probably due to the stability afforded by the formation of intramolecular hydrogen bonds (6-membered rings) between the amine nitrogen atoms and the H-atoms of the hydroxyl groups for the neutral species H2GL . H-bonding thus weakens the proton affinity of the amino groups, increasing the 6  stability ofthe phenol O-H bond. ' ' 23  63  68  pH Figure 3.9: Solution speciation diagram for H 2 G L (0.6 M). 6  This same H-bonding pattern is evident in the X-ray structure (Figure 3.5) of the related compound  H GL . 7  2  Using the acidity constants determined and described above, a  solution speciation diagram for  H GL 2  6  was calculated (Figure 3.9).  136  References start on p. 166  For H2GL , two acidity constants (pK \, and pKso) could be accurately determined a  by potentiometry, while both pK^ and pK had to be evaluated spectrophotometrically a4  (Table 3.2). Variable pD H N M R (Figure 3.10) and pH UV-vis (Figure 3.11) !  experiments confirmed that the order of deprotonation for the four ionizable sites corresponded to that for H 2 G L . 6  pD  Figure 3.10: *H N M R shifts vs. pD for H G L (8.2 m M , D 0 ) . 7  2  2  Shifts in the ethylenediamine backbone as well as the N - methyl hydrogen signals in the pD range 4-8 suggested that deprotonation of the two ammonium moieties was occurring. Interestingly, the sensitivity of the hydrogen signal (Hb d) on the phenol o r  137  References start on p. 166  moiety (evidenced by a slight change in chemical shift) in this same pD range may be due to the formation of intramolecular H-bonds for the H 2 G L species. The assignment of 7  64  the latter two deprotonation events (p/Ca3 and p/C 4) to the phenol moieties was based on a  variable pH UV-vis experiments (Figure 3.11), wherein the shift of the aromatic 71—»7i* transition occurs in the basic pH region.  220  240  260  280  300  320  340  360  380  400  Wavelength (nm)  Figure 3.11: Variable pH (pH 8.5-14) U V spectra of H G L ( [ H G L ] = 0.13 m M , 25 7  7  2  2  ° C , / = 0.16 M NaCI).  However, the UV-vis trace remains unchanged until p H ~ l 1.5, signifying that the phenol moieties of H G L are much more basic than those of H G L . The increased 7  6  2  2  138  References start on p. 166  basicity of the phenols of H 2 G L could be due to the greater strength of the 7  intramolecular H-bonds, discussed previously for H G L , and/or the added electron 6  2  density on the phenol due to the inductive effect of the t-butyl group in the ortho position. The X-ray structure of H G L (Figure 3.5) exhibits H-bonding in the solid-state between 7  2  the amine nitrogen atoms and the H-atoms of the hydroxyl groups; the same H-bonding arrangement that is hypothesized to stabilize the neutral H G L species in solution. The 7  2  first two deprotonation events ($K \ and p/^2) are lower for the t-butyl analog, signifying a  the increased stability of the neutral species H G L compared with H G L . In addition, 7  6  2  2  the latter two deprotonation events (p^a3 and pK ) are much higher, at the limit of the a4  measurable range in an aqueous system. Simulation of the spectrophotometric titration data for H G L afforded only one pK with a value of 13.4(3). The calculated value most 7  2  a  likely corresponds to an average of pK^ and pK 4 as the two phenols are equivalent and a  thus the acidity constants should be similar. In addition, the increased uncertainty in the measured pH in this region (pH 12-14) may lead to the calculation of one pK value. a  Statistical effects can be used to separate p i ^ and pK from the single measured value. a4  For the first phenol deprotonation there are two positions that can be deprotonated, but only one position that can be protonated. This requires the measured acidity constant to be twice as acidic. For the second deprotonation step, the reverse situation exists. Considering this statistical effect, the two acidity constants should differ by a factor of four, and the values for pK^ and pK can be calculated according to equations 3.6 and a4  3.7. ptfa3=(p*a3 + P*a4)/2-0.3  (Eq. 3.6)  p^ =(p^a3+P^a4)/2 + 0.3  (Eq. 3.7)  a4  139  References start on p. 166  69  Based on the experimentally determined value of 13.42, the values for pK 2 and a  pK can be calculated to be 13.1(5) and 13.7(5) respectively. These results could be a4  evaluated differently, whereby pK^ is equal to 13.4(5) and the value for the final pK  a  estimated (pK 4 is >14) based on the experimental results. However, as the effect of the ta  butyl group decreased the difference between pK and pK^ (3 log units for H G L , 2.5 6  a{  2  log units for H G L ) it seems unlikely that the final two pK values would differ by more 7  2  a  than one log unit. The increased uncertainty in the calculated values for pK^ and pK  a4  should account for either scenario. Using the acidity constants determined and described above, a solution speciation diagram for H G L was produced and is shown in Figure 7  2  3.12.  Figure 3.12: Solution speciation diagram for H G L (0.6 M). 7  2  140  References start on p. 166  3.3.3 Synthesis and Characterization of the Ni and Cu metal complexes of deprotonated H G L  6  7  2  The neutral N i  2 +  and C u  2 +  complexes of H2GL " were synthesized in MeOH with 6  7  the addition of base (Scheme 3.12). The Cu complexes were characterized by M S , EA, UV-vis, EPR, and  while the N i complexes were characterized by M S and E A . The  p ff, e  Cu and N i complexes of H 2 G L were further characterized by X-ray crystallography. 7  a or b GlucO—<\  />— OH  HO—(f  H GL " 6  x  >—OGluc  *"  GlucO—<\  />— O'  O—('  MGL "  7  6  2  x  >—OGluc  7  Scheme 3.12: Synthesis of the metal complexes ( M =Cu or Ni) of H GL " : a) 6  7  2  Cu(C10 ) -6H 0, MeOH, NaOH; CuGL = 47%, C u G L = 56%. b) Ni(C10 ) -6H 0, 6  4  2  7  2  4  2  2  MeOH, NaOH; NiGL = 25%, NiGL = 20%. 6  7  The green Cu complexes exhibited characteristic [Cu+Na] and/or [Cu+H] ion +  +  peaks, with the correct isotope patterns by +ES-MS. Cu exists as a mixture of isotopes ( Cu = 69.2%, Cu = 30.8%) affording diagnostic peak patterns. Elemental analysis of the bulk samples correlated well for CuGL -2H 0 and CuGL -2MeOH respectively. The 6  7  2  different associated solvents ( H 0 vs. MeOH) are a result of the purification procedure 2  used and these could not be removed upon prolonged drying. It is assumed that due to Jahn-Teller distortion, there is a weak axial interaction ^between the residual solvents and the square-planar Cu centers. The red/brown N i complexes exhibited characteristic  141  References start on p. 166  [Ni+Na] and/or [Ni+H] ion peaks by +ES-MS. Elemental analysis of the bulk samples +  +  correlated well for NiGL H 0 and NiGL 3 H 0 , respectively. 6  7  2  2  The UV-vis spectra at room temperature of H GL " and the corresponding Cu 6  7  2  complexes are shown in Figure 3.13.  3.0 - _  30 ,  Wavelength (nm)  Wavelength (nm)  Figure 3.13: UV-vis spectra (MeOH, 0.075M) of: (A) H G L (black) and H G L (red). 6  7  2  2  (B) C u G L (black) and CuGL (red). 6  7  The pro-ligands H GL " display two similar bands below 300 nm attributable to 6  7  2  phenol to  TT—>7r*  transitions. Two new absorption bands appear in the visible region for  the complexes C u G L and CuGL . The absorption at 420 nm (2.0 x 10 L mol" cm" ) for 6  7  3  1  1  CuGL and 445 nm (2.7 x 10 L mol" cm" ) for CuGL can be assigned to a phenolate6  3  1  1  7  to-Cu(H) charge transfer transition (LMCT), analogously to other characterized  142  References start on p. 166  70  Cu(II)-phenolate systems.  7  The red-shift of the band for CuGL in comparison to that  for C u G L could be due to the inductive effect of the t-butyl group stabilizing the excited 6  state. The low energy bands at 601 nm (7.0 x 10 L mol" cm" ) and 2  1  1  620 nm (1.4 x 10 L mol" cm" ) for CuGL and CuGL , respectively, can be assigned to 3  1  1  6  7  a ligand field (d—>d) transition on the basis of other work on similar systems. '  71 72  Cu(II) with an electronic configuration of [Ar]d has a single unpaired electron. 9  The spin-only formula predicts a magnetic moment of 1.73 B M for one unpaired electron. The experimental values for C u G L (1.74 B M ) and C u G L (1.79 B M ) , were 73  6  7  found to very close to the spin only value at room temperature. Frozen solution (T = 130 K ) X-band EPR spectra for the two Cu complexes were recorded. The experimental and simulated EPR spectra for C u G L are shown in 6  Figure 3.14 with the appropriate spin Hamiltonian parameters reported in Table 3.3. This compound was determined to be axially symmetric (g = g = gj_ = 2.037; A = A = A± = x  y  x  y  20 x 10" cm" ), exhibiting a typical 4-line ( ' Cu, 1= 3/2) pattern for the peaks in the 4  1  63  65  parallel direction (g = g//= 2.215; A = A//= 179x 10" cm"'). These values are within the 4  z  z  typical range for a tetragonal Cu(lT)-N 02 center. In contrast, computer simulation of 74  2  the EPR pattern for C u G L suggests that the structure is rhombic (g *g * g \ 7  x  y  z  A *A * x  y  ^ ( F i g u r e 3.15; Table 3.3).  143  References start on p. 166  —I—i—.—'—[—'  2100  2BO0  '—I—'—'  2800  1  1  3CCC  3250  j—•—•—'—1—'—'—'—1—'—'—'—1—'—'—'—I—  rwu p i  3*00  3600  3600  '1000  1200  Figure 3.14: Frozen solution EPR spectrum (T = 130 K, v = 9.5713 GHz) of C u G L in 6  MeOH, experimental (red) and simulated (green).  Table 3.3: Spin Hamiltonian parameters of the Cu complexes in MeOH, T = 130 K.  Complex  A  b 2  v (GHz)  CuGL  6  2.037  2.037  2.215  20  20  179  9.5713  CuGL  7  2.040  2.020  2.215  24  30  178  9.5710  Error ± 0.001; Units ± 0.1 x 10" cm" b  4  1  The distortion of C u G L from ideal square planar geometry (vide infra) may be 7  enhanced by the bulky t-butyl groups, and could be the reason for the observed rhombic pattern in comparison to C u G L . A similar rhombic EPR pattern has been determined for 6  144  References start on p. 166  a distorted square planar bis(phenolate-imidazole)Cu(H) complex.  The spin-  Hamiltonian parameters for C u G L are also within the typical range for a CuOO-ASO? 7  center. Superhyperfine features, resulting from the interaction of the unpaired electron 74  with the nuclear spin of the coordinated N-atoms ( N / = 1), were not clearly evident in 14  the EPR spectra of C u G L or C u G L . 6  -i  1  2403  , 1.  1 .  2600  ,  .  1  .  7  •—.  2800  1  .  »  300©  .  ,  3200  .  .  .  j  >  3400  •  »  \  •  «  3800  FMJ P J  >  1  3800  >  •  i  1  •  4000  i  i  1  •  >-  4200  Figure 3.15: Frozen solution EPR spectrum (T = 130 K, v = 9.5710 GHz) of C u G L in 7  MeOH, experimental (red) and simulated (green).  X-ray quality crystals of C u G L and N i G L were grown via the slow evaporation 7  7  of concentrated MeOH/H20 and MeOH solutions of the respective compounds. There are a significant number of Cu and Ni complexes containing carbohydrate-derived ligands reported in the literature; "* in almost all cases the carbohydrate moiety is bound to the 75  1  metal center. Examples include complexes utilizing N-glycosylamine ligands containing amines such as l^-diaminopropane, ^ tris(2-aminoethyl)amine, 7  145  76  References start on p. 166  and 1,4,7-triazacyclononane (Ni, Cu), in order to enhance the stability of the resulting complexes. In addition, di- and tri-metallic complexes (Cu, N i ) ' 77  78  have been prepared  and structurally characterized with N-glycosylamine ligands. A Cu complex of a carbohydrate-appended dipicolylamine ligand has also been recently structurally 79  characterized. In contrast to the complexes mentioned above, only two X-ray structures of N i and Cu complexes containing pendant carbohydrate ligands exist; a N i complex of a 1,3-diaminosugar, and a Cu complex with xylose-appended serine ligands. 80  81  Similarly, the carbohydrate moieties remain pendant in both the N i and Cu complexes of 7  7  H2GL . The structure of C u G L is shown in Figure 3.16 with selected bond lengths and angles presented in Table 3.4. C u G L possesses a distorted square planar geometry; the 7  angle between the Cu(l)N(l)0(7) and Cu(l)N(2)0(14) planes is 22.1°.  146  References start on p. 166  C u G L crystallized with two molecules in the asymmetric unit, one of each 7  diastereomer. The packing in the crystal is displayed in Figure 3.17. The carbohydrate moieties participate in a network of hydrogen bonding interactions, both between and along the sheets, accounting for the observed packing arrangement. Removal of the solvent molecules while solving the structure precluded the observation of H-bonding interactions mediated by the solvent molecules.  Table 3.4: Selected bond lengths (A) and angles (deg) in CuGL . 7  Cu(l)-N(l) Cu(l)-N(2) Cu(l)-0(7) Cu(l)-0(14) C(7)-0(6) C(l)-0(6) C(20)-O(13) C(l)-0(1) C(10)-O(7) C(29)-0(14) C(17)-N(l) C(36)-N(2) C(19)-C(38)  2.002(6) 2.042(7) 1.893(5) 1.906(5) 1.430(8) 1.415(8) 1.381(8) 1.449(8) 1.307(9) 1.343(9) 1.470(9) 1.499(9) 1.53(1)  N(l)-Cu(l)-N(2) N(l)-Cu(l)-0(7) N(2)-Cu(l)-0(14) 0(7)-Cu(l)-0(14) Cu(l)-N(l)-C(17) Cu(l)-N(2)-C(36) 0(6)-C(l)-0(l) O(13)-C(20)-O(8)  86.4(3) 92.7(2) 93.2(2) 92.2(2) 112.1(4) 109.4(5) 106.7(6) 106.5(5)  Similarly to CuGL , NiGL crystallized with two molecules in the asymmetric 7  7  unit. The structure of NiGL is shown in Figure 3.18 with selected bond lengths and 7  angles presented in Table 3.5. NiGL possesses a slightly distorted square planar 7  geometry as the angle between the Ni(l)N(l)0(7) and Ni(l)N(2)0(14) planes is 13.8°; the distortion is less than that exhibited in the Cu complex. Hydrogen bonding  147  References start on p. 166  interactions are mediated by MeOH solvent molecules in N i G L , not directly between 7  carbohydrate moieties as with C u G L . 7  Figure 3.17: Crystal packing in C u G L . 7  Figure 3.18: ORTEP diagram of N i G L showing 50% thermal probability ellipsoids.  148  References start on p. 166  Table 3.5: Selected bond lengths (A) and angles (deg) in N i G L . 7  Ni(l)-N(l) Ni(l)-N(2) Ni(l)-0(7) Ni(l)-0(14) C(7)-0(6) C(l)-0(6) C(20)-O(13) C(l)-0(1) C(10)-O(7) C(29)-0(14) C(17)-N(l) C(36)-N(2) C(19)-C(38)  1.90(1) 1.942(9) 1.907(8) 1.838(8) 1.42(1) 1.40(1) 1.42(1) 1.42(1) 1.38(1) 1.32(1) 1.51(1) 1.47(2) 1.51(2)  N(l)-Ni(l)-N(2) N(l)-Ni(l)-0(7) N(2)-Ni(l)-0(14) 0(7)-Ni(l)-0(14) Ni(l)-N(l)-C(17) Ni(l)-N(2)-C(36) 0(6)-C(l)-0(l) O(13)-C(20)-O(8)  87.8(4) 94.2(4) 92.6(4) 87.0(3) 113.9(8) 111.2(8) 105(1) 105.9(9)  Unfortunately the complexation of the pro-ligands H2GL " with Zn was only 6  7  partially successful on the macroscopic scale. Evidence for complexation via +ES-MS ([Zn+Na] and/or [Zn+H] ) was encouraging, yet the complexes could not be purified +  +  satisfactorily; however, the stability constants of H2GL " with both Zn and Cu were 6  7  determined by potentiometry in aqueous solution. The solution speciation of C u  2 +  and Z n  2+  with H2GL " was studied by 6  7  potentiometric titrations. The pK values for the ligands, described in section 3.3.2, were a  used as constants for the stability constant calculations. As well, hydrolysis reactions of free C u  2+  and Z n  2 +  were also included in the calculations (Table 3.6). The calculated 46  values are summarized in Table 3.7 and defined by Equations 3.8-3.11.  149  References start on p. 166  M»  +  [HGL ]" ft [MHGL'Y  V,r  M»  +  [ G L « f ft M G L "  ^ „= ^g.!  67  MGL"  +  H i  0L  O ft [MGL«(OH)]- H  =[  M  [M  ^  S  ]  <«* » >  ( E , . 3.10)  f]  - ^"^gS'j  +  +  11  "'  1  ( « * 3-")  Table 3.6: Negative logarithms (p0 of hydrolysis equilibrium constants for Z n Cu  2+  in aqueous solution (/= 0.16, 25 °C).  Reaction M  2 +  M  2 +  M  2 +  M  2 +  2 +  vQ Cu  2 +  + H 0 ±5 M(OH) + F f  9.25  8.29  + 2H 0 ±? M(OH) + 2FT  17.2  17.6  + 3H 0 ±5 M(OH) - + 3FT  28.4  27.8  + 4H 0 ±5 M(OH) ~ + 4Ff  40.6  39.0  2 M + H 0 5 M (OH) + FT  8.71  -  2 M + 2H 0  -  10.65  57.5  -  +  2  2  2  3  2  2  4  2+  3+  2  2  2+  2  M (OH) 2  2+ 2  + 2FT  2 M + 6H 0 5 M (OH) " + 6PT 2+  2  2  2  6  and  46  Zn 2  2 +  150  References start on p. 166  The values in Table 3.7 show that H G L exhibits larger binding constants 7  2  (log AT  . ) with Zn and C u 2+  6  2 +  7  compared with the analagous values for H GL . 6  2  Table 3.7: Stability constants of the C u  2+  and Z n  2+  complexes with H GL " (1= 0.16 M 6  7  Cu  2 +  2  NaCI, 25 °C).  H GL  H GL  6  2  Constant  Zn  g^[MH GL«]^  l 0  Cu  2 +  7  2  Zn  2 +  2+  -  7.23(5)  -  5.75(8)  7.8(2)  14.05(6)  9.3(2)  14.83(5)  12.90(8)  20.46(4)  16.83(5)  24.18(2)  -11.0(6)  -11.1(4)  -11.1(4)  -10.9(3)  2  §^[MHGL - ]  l 0  6  l 0  l 0  7  +  g*MGL"  8^[MGL - (0H)]6  7  This result is expected as binding constants for a particular metal ion should increase with increasing ligand basicity for a series of similar ligands. " Based on the 82  84  calculated constants, solution speciation diagrams were prepared for Zn and Cu with 6  7  H GL (Figure 3.19) and H GL (Figure 3.20). Except for the presence of 2  2  [CuH GL " ] in the C u 6  7  2+  2  9+  2 +  systems, the species present in the p H range examined are 9+  *  identical for both Zn and Cu .  151  References start on p. 166  pH  Figure 3.19: (A) Solution speciation diagram for H2GL and Zn ; (B) solution speciation diagram for H G L and C u 6  2  2 +  ( [ H G L ] : [ M ] , 1.1:1; [ M ] = 0.6 mM). 6  2+  2+  2  152  References start on p. 166  Figure 3.20: (A) Solution speciation diagram for H 2 G L and Z n ; (B) solution 7  speciation diagram for H G L and C u 7  2  2+  2+  ([H GL ]:[M ], 1.1:1; [ M ] = 0.6 mM). 7  2+  2+  2  153  References start on p. 166  The [CuH GL ] 6 7  2  2 +  species was a necessary addition to the model for C u  in  2 +  order to fit the pH curve adequately in the acidic pH region. The Irving-Williams series ' of stability (Cu > Zn ) correctly accounts for the experimental results. In addition, the ligands studied here easily adopt a square-planar binding arrangement that is better suited to C u . From the speciation diagrams it is obvious that no free C u 2+  2 +  exists  above pH 5 with either ligand, while free Zn exists up to pH 7. Due to the higher 2+  stability of the CuGL " species as compared to ZnGL " , the former species are more 6  7  6  7  predominant at lower pH and exist over a larger pH range. Indeed, at pH 7, CuGL " is 6  7  the sole species present for C u , while ZnGL " , Z n , and [ZnHGL " ] exist for the 2+  Zn  2+  6  7  2+  6  7  +  systems. While hydrolysis reactions of C u and Z n (Table 3.6) were included in the 2+  2+  fitting procedure, it was found that metal hydrolysis did not influence the stability constants in the range studied. However, the hydrolysis species [MGL " (OH)]" does 6  become important above pH 9 for both C u hydrolysis of Z n  2+  is similar to that of C u  role in the solution speciation of Zn  2+  2 +  and Z n . Although the tendency towards 2+  (Table 3.6), hydrolysis usually plays a greater  due to its weaker coordination to ligands as  9+  hydrolysis does not play a larger role for Z n [MGL6  .  7(QH)]  f\ 7  9+  compared to Cu . Interestingly, despite the weaker coordination of Zn  log^  7  2+  to both H GL " , 2  compared with C u . The 2+  . values (Table 3.7) are identical (within experimental error) regardless of  system studied. The larger error associated with thelog^  [MGL6  .  7(OH)]  . values is due to the  increased difficulty of modelling systems at high pH. 2+  2+  6 7  Comparison of the calculated Cu and Zn stability constants of H GL " with 2  relevant chelators (Table 3.8) is of significant interest. The log K (Zn ) values for 2+  154  References start on p. 166  H GL " are higher than that reported for H Y B E D A , yet lower than the value for 6  7  2 3  2  H B E D (Figure 3.6).  The two additional acid functions of H B E D (and the possibility for  hexadentate coordination) evidently increases the stability of the resultant Z n  2 +  complex.  The potentially hexacoordinate E D T A exhibits log K (Zn ) intermediate between H 2 G L 2+  and H2GL . The acid moieties of H B E D or E D T A do not increase the binding to C u 7  comparison to H GL " ; the stability of the C u 6  7  2+  2  2+  in  complexes can be attributed to strong  coordination of the metal ion with the amino nitrogens and phenolate oxygens in an approximately co-planar arrangement.  Table 3.8: Z n  2+  Ligand  H GL  6  H GL  7  2  2  HYBEDA HBED EDTA  3  b  C  Clioquinol Apl-28  f  a  2 +  stability constants of relevant chelators.  log/C(Zn )  log£(Cu )  solvent  12.90(8)  20.46(4)  H 0  16.83(5)  24.18(2)  H 0  11.97  20.5  H 0  18.37  23.69  H 0  16.44  18.70  H 0-  15.8  1:1 E t O H : H 0  2+  12.47  h  2 +  6  11.03  Api-40 Api-42  d  and C u  s  -  2  2  2  2  2  e  2  15.5  H 0  10.3  H 0  2  h  2  17.2  H 0 2  ref.[23]; refs.[63,86]; ref.[47]; ref.[87]; Ligand forms 1:2 metakligand complexes, c  e  values converted from log J3 values; ref.[48]. ref.[88]. ref.[89]. f  8  155  h  References start on p. 166  6  As a result of Jahn-Teller distortion, the carboxylate oxygens of H B E D (or EDTA) are probably only weakly bound in the axial positions, and thus the resultant Cu complex stability is not increased significantly. Comparing stability constants of chelators can be difficult when different experimental and physical conditions (solvent systems, temperature, methods of determination, ligand:metal ratios) are used. Although stability studies of clioquinol with Z n  2+  and C u have been published, comparison of 2+  87  the data with that presented here for H GL " is difficult due to the different solvent 6  7  2  system used (1:1 EtOH:H 0) for clioquinol. 2  While stability constants are a measure of the overall stability of a metal-ligand species, they do not reflect the metal-binding affinity of ligands at a specific pH value, and thus a direct comparison can be misleading. For a given complex equilibrium system and pH, the concentration of free metal ion (commonly referred to as p M = -log[M]) represents a direct estimate of the ligand-metal affinity, taking into account all relevant • • • 82 91 92  equilibria. ' ' p M depends on ligand and metal concentration, temperature, ionic strength, and pH of the solution. Figure 3.21 shows the calculated pZn values for solutions containing 1 m M of Z n  2+  and 1 m M of either H G L , H G L , or E D T A , as 6  7  2  2  well as calculated pCu values for solutions containing 1 m M of C u  2 +  and 1 m M of either  H G L , H G L , Apl-28, or E D T A . Interestingly, although H G L has larger zinc and 6  2  7  7  2  2  7  fx  copper binding constants compared to H G L , the zinc and copper affinity of H G L 2  2  does not exceed that of H G L until pH>l 1.5 due to the greater proton affinity of 6  2  H GL . 7  2  156  References start on p. 166  157  References start on p. 166  At physiological pH (pH = 7.4) the pZn values for H G L (pZn = 4.2) and H G L 6  2  7  2  (pZn = 3.9) are quite similar, yet substantially lower than that for E D T A (pZn = 7.9). Again, the pZn values can be attributed to the difference in proton affinity of H GL " 6  7  2  compared with E D T A . Much the same trend exists for copper; the related pCu values (pH 7.4) are H G L (pCu = 8.0), H G L (pCu = 7.5), and E D T A (pCu = 9.4). In 6  7  2  2  addition, the pCu value (pH 7.4) for the amyloid peptide (Api-28, pCu = 6.3) could be calculated from published potentiometric data, and shows that both H G L and H G L 48  6  2  7  2  should be able to compete with this A p peptide fragment for C u . 2+  The p M values (Cu/Zn) for H GL " were found to be moderate at pH 7.4, when 6  7  2  compared to the strong chelator EDTA. H GL " may be ideally suited for the disruption 6  7  2  of the abnormal metal-protein interactions in the brain possibly responsible for the observed toxicity in A D . The moderate affinity of H GL " for metal ions at 6  7  2  physiological pH may obviate the toxicity commonly associated with chelation therapy, especially when coupled with the potential for increased tissue specificity due to the attached carbohydrate functions. Whether or not H GL " can compete for metal ions 6  7  2  with the longer length A p peptide fragments (Api-40, Api-42) remains to be determined. The binding constant reported for Api-42 and C u  2 +  is the second highest for  89 93  any biomolecule,  1  exceeded only by Cu/Zn-superoxide dismutase, and thus it may be  difficult to disrupt this metal-peptide interaction. It is possible however that the pathophysiology of A D is mediated by the low-affinity metal-binding sites on A p . These 2  low affinity sites (Ap can bind up to 3.5 moles of Cu or Zn per subunit) may be 89  responsible for the observed aggregation and associated toxicity. The tetrahydrosalen compounds have the potential to interact with the low-affinity metals bound to A p . 158  References start on p. 166  3.3.4 T E A C Values of the Tetrahydrosalen Compounds  The tetrahydrosalen compounds H 2 L  and H2GL " were monitored for  15  6  7  antioxidant activity by the T E A C assay. This assay has been used as a simple method to 49  quantify the antioxidant activity of biological fluids, extracts, and pure compounds by measuring the disappearance of the ABTS* radical cation via UV-visible spectroscopy. +  The ability of the tetrahydrosalen compounds to quench the ABTS* radical cation was +  compared to Trolox (Figure 3.4), a more water soluble analog of oc-tocopherol, and the results are shown in Figure 3.22 and Table 3.9. The H2L " series displayed a similar 1  5  ability to quench the ABTS* radical cation and exhibited T E A C values that were +  enhanced in comparison to (±)-oc-tocopherol and BHT.  3.0  -1  X  2.5 H CD  1 min 3 min. 6 min.  2.0 H  _3  CO >  O <  T  1.5 H  LU  1.0 0.5 0.0 H L' 2  H L2  H L  3  2  H L 2  5  H GL 2  6  H UL 2  7  1  a-Tocophcrol  dl  BHT  Figure 3.22: T E A C Values at 1, 3, and 6 minutes for the tetrahydrosalen ligands, (±)-octocopherol, and BHT. Error bars represent ± SD above and below the average T E A C value (determined in triplicate).  159  References start on p. 166  It should be noted however that each tetrahydrosalen compound contains two phenolic moieties capable of quenching the ABTS* radical cation. Introduction of a +  t-butyl group para to the ring hydroxyl moiety in H2L did not increase the antioxidant 4  properties of this analog in comparison to the other compounds in the series. The 67  compounds H2GL " were the most potent in this assay, displaying enhanced T E A C values compared to H2L " . 1  5  Table 3.9: T E A C values ± SD at 1, 3, and 6 minutes.  Compound  1 min  3 min  6 min  H2L  1  1.04 ± 0 . 0 4  1.28 ±0.07  1.46 ± 0 . 0 7  H L  2  1.13 ±0.04  1.45 ±0.06  1.64 ±0.07  H L  3  1.17 ±0.07  1.49 ±0.09  1.8 ± 0.1  H L  4  0.98 ± 0.07  1.25 ±0.09  1.39 ± 0 . 0 9  H L  5  0.93 ± 0.05  1.32 ±0.09  1.5 ± 0 . 1  2  2  2  2  H GL  6  1.93 ±0.05  2.21 ±0.07  2.34 ± 0 . 0 7  H GL  7  2.39 ±0.07  2.5 ± 0 . 1  2.7 ± 0 . 1  (±)-a-Tocopherol  0.72 ± 0.02  0.72 ± 0.03  0.72 ± 0.03  BHT  0.13 ± 0 . 0 1  0.24 ±0.01  0.34 ± 0 . 0 1  2  2  These two analogs are essentially protected hydroquinones, and oxidation to the associated quinones could be the reason for the enhanced antioxidant properties. The 7  6  increased activity of H2GL , as compared to the hydrogen analog H2GL , is most likely  160  References start on p. 166  due to the increased stabilization of the phenoxy radical through inductive and/or steric effects from the t-butyl group ortho to the ring hydroxyl. It is clear from this study that 32  the tetrahydrosalen compounds H L 2  1 5  and H GL " have the potential to act as 6  7  2  antioxidant compounds.  3.3.5 Enyzymatic Cleavage of the Tetrahydrosalen Glycosides  The tetrahydrosalen glycosides GL " require that the glucose protecting group be 1  5  removed before metal binding can take place. It was hypothesized that this could occur enzymatically in vivo as there are a number of enzymes that cleave (3-glycosides in humans. Examples of such enzymes include a cytostolic broad specificity (3-glucosidase 94  (EC 3.2.1.21), glycosylceramidase (EC 3.2.1.62), and the membrane-bound lactasephlorizin hydrolase (EC 3.2.1.62/108). ' As the human glucosidase enzymes are 19 94  difficult to obtain, and more than one enzyme could potentially act as a site for glycoside cleavage, rat brain homogenate has been used as a model system to determine overall (3glucosidase activity. ' Using this method, glucosidase activity of approximately 4-4.5 17 95  nmol/hr/mg brain homogenate has been determined in male Wistar rats. Additionally, 95  p-glucosidase activity was found to be very low in plasma, signifying that carbohydrate pro-drugs should be stable in this medium.  17  Initial screening of the glycosylated tetrahydrosalen compounds GL " was 1  5  undertaken to determine if the glycoside moieties could be enzymatically cleaved to afford the corresponding pro-ligands H L . Even though removal of the glucose 1 - 5  2  moieties of H GL " are not required for metal binding, these compounds were included 6  7  2  161  References start on p. 166  in this assay. A broad specificity 13-glucosidase, Agrobacterium (3-glucosidase (Abg), was used to test for glycoside cleavage and the reactions were monitored by T L C (Figure 3.23) and mass spectrometry after incubating for two hours. Of the seven glycosylated tetrahydrosalen compounds, only G L did not act as a 3  substrate for Abg. In all other cases significant reactivity was observed by both T L C and mass spectrometry. As an example, the T L C for G L is shown in Figure 3.23A. From this 2  TLC one can see the complete loss of GL (lane 1) and the appearance of H L 2  (lane 2 vs. 3) and free glucose (lane 2 vs. 4) in the enzyme reaction (lane 2).  A  B  C  4M  •  1 2 3 4  1 2 3 4  1 2 3 4 5  Figure 3.23: Silica-gel monitoring of enzymatic (Abg) deglycosylation reactions of selected tetrahydrosalen glycosides. Spots containing sugars are dark (exposed with H2SO4  / EtOH, heat), and U V active (254 nm lamp) spots are outlined. Reactions were  monitored by T L C after 2 hours. A : 1) G L . 2) G L and Abg. 3) H L . 4) Glucose. B : 2  2  2  2  G L . 2) G L and Abg. 3) H L . 4) Glucose. C: 1) H G L and Abg. 2) H G L . 3) H G L 3  3  3  7  2  2  7  2  2  and Abg. 4) H G L . 5) Glucose. 6  2  162  References start on p. 166  6  The T L C of GL (Figure 3.23B) indicates that no de-glycosylation has occurred as the only spot in the enzyme reaction (lane 2) corresponds exactly with G L (lane 1). 3  The additional steric bulk of the benzyl groups, compared to the other tetrahydrosalen compounds, could inhibit the interaction of G L with Abg. 3  The T L C (Figure 3.23C) of H2GL " indicates that the compounds act as 6  7  substrates for Abg. Both compounds exhibit the complete loss of the glycosylated material and the presence of free glucose in the enzyme reactions. Additional T L C spots for H2GL , accompanied by a colour change from colourless to black, indicates that 6  further reactions/decomposition of this compound may be occurring once the glucose moieties are removed. This did not occur with the t-butyl analog H 2 G L . 7  Mass spectrometric (+ES-MS) analysis of the diluted enzyme reactions correlated nicely with the results from the T L C study. In all cases, except G L , mass peaks 3  corresponding to the loss of either one or two glucose moieties were present in the enzyme reactions. The reactions of G L and G L with Abg were not complete at 2 hours 1  5  as small peaks corresponding to the glycosylated starting materials were evident in addition to the expected deglycosylated compounds. This preliminary study is quite encouraging and shows that the glycosylated pro-drugs (except GL ) undergo glycoside 3  cleavage in the presence of the Abg enzyme to afford the desired tetrahydrosalen proligands. The next step is to monitor whether the glycosylated pro-drugs act as substrates for the glucosidase enzymes found in rat brain homogenates.  163  95  References start on p. 166  3.3.6 Cell Toxicity Studies  Human breast cancer cells" were used in this study to determine the biological activity (toxicity) of two representative glycosylated tetrahydrosalen compounds ( G L and H2GL ) in comparison to the cytotoxic agent cisplatin (Figure 3.24). 6  180  n  Concentration (mM)  Figure 3.24: MTT plots for G L , H2GL , and cisplatin. The 2  6  IC50  value for cisplatin in  this study was determined to be 35 ± 5 u.M. The error bars indicate one standard deviation above and below the average cell percent viability. Each point is an average of six wells.  164  References start on p. 166  Ideally, neither compound would be cytotoxic in vitro over a large dosage range. A n MTT assay, which is an in vitro colourometric method of determining cell viability, was used here to assess for cell toxicity. ' The results of the M T T assay clearly show 51 96  that the glycosylated tetrahydrosalen compounds are not toxic in this cell line at the concentrations studied. Cisplatin was toxic, exhibiting an IC50 value of 35 ± 5 uM. Toxicity studies on the full series of tetrahydrosalen compounds, and their glycosylated analogs, is warranted in a more relevant cell model.  3.4 Concluding Remarks  A series of tetrahydrosalen compounds, and their glycosylated analogs, were synthesized and characterized by a variety of methods. The synthetic routes reported here are amenable to the future synthesis of derivatives with potentially improved pharmacokinetic properties. The glycosylated analogs, with the exception of G L , were 3  determined to be substrates for Abg, indicating the potential of the carbohydrate pro-drug strategy. Two analogs, G L and H2GL , were determined to be non-toxic in vitro. The 2  6  tetrahydrosalen compounds also displayed antioxidant ability in an A B T S " radical cation +  6 7  competition assay. H2GL " were determined to be the most potent antioxidants probably due to the presence of hydroquinone functions. The affinity of H G L " for Zn and Cu 6  7  2  were also determined via potentiometry. These pro-ligands were found to be moderate Zn and Cu chelators at physiological pH.  165  References start on p. 166  3.5 References  (1) Barnham, K . J.; Masters, C. L.; Bush, A . I. Nat. Rev. Drug Discov. 2004, 3, 205. (2) Bush, A . I. Trends Neurosci. 2003, 26, 207. (3) Doraiswamy, P. M . ; Finefrock, A . E. Lancet Neurol. 2004, J , 431. (4) Squitti, R.; Lupoi, D.; Pasqualetti, P.; Dal Fomo, G.; Vemieri, F.; Chiovenda, P.; Rossi, L.; Cortesi, M . ; Cassetta, E.; Rossini, P. M . Neurology 2002, 59, 1153. (5) Ozcankaya, R.; Delibas, N . Croat. Med. J. 2002, 43, 28. (6) Lovell, M . 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Cancer Res. 1988, 48, 589.  171  References start on p. 166  CHAPTER 4  Carbohydrate-appended Metal Complexes (  m  T c / Re) for Potential Use in Nuclear  Medicine  4.1 Introduction  The recent interest in the development of radiopharmaceuticals utilizing the Group VII metals is a direct result of the ideal properties of diagnosis and therapy, respectively. " 1  39 9 m  9 9 m  T c and  1 8 6 / 1 8 8  R for e  T c is the most commonly used isotope in single  photon emission computed tomography (SPECT) due to its ideal nuclear properties (ti/2 = 6.01 h, y = 142.7 keV) and easy isolation as N a  9 9 m  T c 0 4 from a M o generator. Rhenium, 9 9  the third row transition metal analog of technetium, exhibits chemistry similar to that of technetium and has particle emitting radioisotopes ( Re ti/2 = 3.68 days, P = 1.07 MeV, 186  y = 137 keV;  l88  Re t  1/2  = 16.98 h, p = 2.12 MeV, y = 155 keV) with physical properties  applicable to therapeutic nuclear medicine. The possibility of designing radiopharmaceuticals for diagnosis (  99m  Tc) and therapy (  186/188  R e ) by exploiting the  similar chemistries of these two Group VII metals is thus a highly desirable goal in areas such as tumour imaging and therapy. One compound that has proven very useful for the detection of tumours and metastatic tissue is 2-deoxy-2-[ F]fluoro-D-glucose (FDG) (Figure 4.1), imaged by positron emission tomography (PET). Because the production of 4  F requires a cyclotron and the isotope has a short (110 min) half-life, its utility is somewhat limited compared to that of single photon emitters in nuclear medicine. The  172  References start on p. 234  design of carbohydrate-appended  m  T c complexes for potential use in nuclear medicine  is thus of considerable interest. One approach is to attach a chelating ligand to a carbohydrate that, in a subsequent reaction, will bind to the metal center. This approach is necessary in order to minimize the potential interactions between the carbohydrate and the tracer group and provides a stable and easily characterized complex. Direct metal ioncarbohydrate interactions are difficult to study due to the inherent multifunctionality, complicated stereochemistry, and weak coordinating ability typical of carbohydrates. Carbohydrate ligands with well-tailored binding groups for metal ions have previously been developed in order to overcome these difficulties. Designed binding groups include iminodiacetic acid, ' tris(2-aminoethyl)amine, ' 1,4,7-triazacyclononane, imino- and 5 6  7 8  amino -phenols, ethylenediamine, 11  12-14  9  1,3-propanediamine, ' '  13 15 16  10  and  17  ethylenedicysteine.  Examples of this approach in medicinal inorganic chemistry include  carbohydrate-appended cis-platin analogs as potent antitumour agents, ' ' antifungal 16 18 19  Ni(II) complexes derived from amino sugars, as well as carbohydrate-appended metal 20  complexes of the radioisotopes  9 9 m  T c and  186  R e for potential use in nuclear imaging and  6 11 17 21 23  therapy. • • • '  In many cases however the resultant metal complexes exhibit binding  of the carbohydrate moiety to the metal center which limits the targeting potential of these compounds. Tc-carbohydrate conjugates have not been extensively studied in the literature, with early work, typical of metal-carbohydrate chemistry, plagued by poor characterization as well as potential interactions/chelation of the carbohydrate itself with the metal center. " Factors that most likely effect recognition in vivo include the size of 24  27  the metal chelate as well as the distance between the chelate and the pendant 173  References start on p. 234  carbohydrate moiety. One recent study utilizing ethylenedicysteine-deoxyglucose (ECDG) (Figure 4.1) as the chelate for  9 9 m  T c displayed hexokinase activity as well as  17  tumour uptake.  Further studies have shown that this compound is active in the  hexosamine biosynthetic pathway and has significant in vivo tumour imaging potential. Uptake in tumour cells has also been demonstrated for the  9 9 m  28  T c complex of l,3-^/,7Y'-di-  P-D-glucopyranosyldiethylenetriamine (DGTA) (Figure 4.1), even in the presence of excess glucose in the culture medium carbohydrate-labelled  9 9 m  2 9  Both studies highlight the potential of  T c compounds for tumour imaging. HO.  HO  Ethylenedicysteine-deoxyglucose (ECDG)  M'  H 0^ |  ^ C O CO  2  Organometallic precursor [M(H 0) (CO) ] (M= Re, +  2  3  3  99m  ljS-A'./V-di-p-D-Glucopyranosyldiethylenetriamine (DGTA)  Tc) OH  NH  N-(2'-Hydroxybenzyl)-2-amino-2-deoxy-D-glucose  Figure 4.1:  Compounds discussed in the introduction. 174  References start on p. 234  Stable core structures have found utility in the chemistry of Tc due to the wide range of accessible oxidation states (Tc(-I) to Tc(VII)). For example, the {M(CO)3} (M +  = Tc/Re) (Figure 4.1) core has garnered significant interest ever since its development by 30  Jaouen  31  and Alberto and co-workers. This organometallic core offers advantages in  terms of stability, kinetic inertness, and size. The low-spin d -electron configuration and 6  the stability of the C O ligands to substitution protect the metal center from further ligand substitution and/or oxidation. For the above reasons there has been widespread interest in the development of target-specific radiopharmaceuticals exploiting the {M(CO)3} (Tc/Re) core.  +  32-41  The labeling precursor [ Tc(H 0)3(CO)3] (Figure 4.1) is easily prepared from a 99m  +  2  kit formulation in aqueous conditions utilizing a boranocarbonate as a dual-function reducing agent and in situ CO source. The three labile water molecules are readily 42  exchanged for suitable chelating ligands. Previous work investigating the coordination chemistry of the {M(CO)3} (Tc / Re) core has established that heterocyclic and aliphatic +  amines as well as carboxylate donors are effective chelating agents. ' More recently it 43 44  has been found that aromatic or aliphatic amines are the best match for the {M(CO)3} (  99m  Tc) core.  +  45  Studies investigating the utility of 1,3-diaminocarbohydrates (Figure 4.2) as ligands for the {M(CO) } ( +  3  99m  T c / Re) core are presented in this Chapter. The 1,3-  diaminocarbohydrates have been developed by Yano and co-workers '  for potential use  in bioinorganic chemistry. ' ' ' In all cases the carbohydrate moiety is connected to 16 18 46 47  the chelating unit via the C - l position. As previously discussed the 1,3-diaminopropyl chelating group is very well-suited for the {M(CO)3} (Tc / Re) core +  175  4 3 - 4 5  References start on p. 234  OH  l,3-Diamino-2-propyl P-D-glucopyranoside (L )  l,3-Diamino-2-propyl P-D-xylopyranoside (L )  1,3-Diamino-2-propyl P-D-galactopyranoside (L )  ,3-Diamino-2-propyl P-(a-D-glucopyranosyl-( 1,4)-D -glucopyranoside) (L )  5  1  6  OH  Bis(aminomethyl)bis[(P-D-glucopyranosyloxy)methyl]methane) (L )  Figure 4.2: 1,3-Diaminocarbohydrates.  Utilizing the ligands shown in Figure 4.2, the solution and solid state properties of a number of different carbohydrates with the {M(CO)3} (Tc / Re) core were examined. +  176  References start on p. 234  In order to design robust carbohydrate-appended metal complexes of Tc and Re with better pharmacokinetic profiles, the tridentate dipicolylamine (DPA) function was investigated as the binding group (Figure 4.3).  OH  2-(Bis(2-pyridinylmethyl)amino)ethyl-p-D-glucopyranoside (L ) 8  2-(Bis(2-pyridinylmethyl)amino)ethyl-P-D-xylopyranoside (L ) 9  OH  2-(Bis(2-pyridinylmethyl)amino)methyl-glucosamide (L )  2-(Bis(2-pyridinylmethyl)amino)methyl-l,3,4,6-tetra-0-acetyl-P-D-  11  glucosamide (L ) 12  Figure 4.3: Carbohydrate-appended 2,2'-dipicolylamine ligands.  This tridentate chelator (containing two pyridines and one tertiary amine) avidly binds the {Re(CO)3} core; more recently, this chelate system has been used to attach +  44  {Re(CO)3} as a proof of principle for the preparation of peptide-targeted +  177  References start on p. 234  radiopharmaceuticals,  and to prepare molecules to monitor dopamine transporter sites,  and image the hepatobiliary system. A preliminary study by Mikata et al. concerning 50  51  the synthesis and characterization of a glucose-pendant D P A copper complex shows the usefulness of this approach for the carbohydrate labeling of metal ions. A series of carbohydrate-appended D P A conjugates (Figure 4.3) were investigated as ligands for the {M(CO) } ( +  3  99m  Tc/Re) core. For compounds L - L 8  1 0  the carbohydrate moiety is  connected to the chelating unit via the C-1 position, similar to the 1,3diaminocarbohydrates. Ligands L - L n  1 2  exhibit an amide linkage between the  carbohydrate (glucosamine) function and the DPA fragment. Glucosamine was chosen as the carbohydrate due to the recent success of  99m  T c - E C D G (Figure 4.1) as an imaging  17  agent.  As well, the Orvig group has recently investigated the utility of N-(2'-  hydroxybenzyl)-2-amino-2-deoxy-D-glucose (Figure 4.1) as a ligand for the {M(CO)3} ( Tc/Re) core. 99m  +  11  The goal of this project was to design carbohydrate-appended Re and Tc complexes for potential use in nuclear medicine. Structural investigations were initially carried out on the 'cold' Re derivatives with the carbohydrate-appended bidentate (L^-L ) 7  and tridentate (L -L ) ligands to ascertain solution and solid state configuration. 8  12  Labeling studies and preliminary in vitro stability measurements for the analogous  9 9 m  Tc  derivatives are also presented.  178  References start on p. 234  4.2 Experimental  4.2.1 Materials  Most information related to this section is contained in Section 2.2.1. The 1,3diaminocarbohydrates L Z - L , 7  1 3  (bis(2-pyridinylmethyl)amino)acetic acid 1 (Figure 4.4), 44  1,3,4,6-tetra-O-acetyl-p-D-glucosamine-HCl  52  (Figure 4.4), and [NEt4]2[Re(CO)3Br ]  53  3  were synthesized according to previously published procedures. 2-(Bis(2pyridinylmethyl)amino)ethyl-P-D-glucopyranoside L , 8  5 1  2-(bis(2-  pyridinylmethyl)amino)ethyl-p-D-xylopyranoside L , and 2-(bis(29  pyridinylmethyl)amino)ethyl-P-D-mannopyranoside L  1 0  were gifts from Prof. Yuji  Mikata and Prof. Shigenobu Yano, Nara Women's University, Japan. Isolink™ boranocarbonate kits for  9 9 m  T c labeling were a gift of Mallinkrodt Inc., and N a [  99m  Tc04]  was supplied by the U B C Hospital Department of Nuclear Medicine.  OAc  (Bis(2-pyridinylmethyl)amino)acetic acid  1,3,4,6-tetra-O-acetyl-p-D-glucosamineHCl  Figure 4.4: Compounds relevant to the experimental procedures.  179  References start on p. 234  4.2.2 Instrumentation  Most information related to this section is contained in Section 2.2.2. 2D N M R techniques such as ' H - ' r i COSY, H - C H M Q C and H - N HSQC were used to aid in !  1 3  ]  1 5  the characterization of the Re complexes. C, H , N analyses were performed either at U B C by Mr. M . Lakha (Carlo Erba analytical instrumentation) or by Prof. C. Ohtsuki (Perkin Elmer PE2400 Series II CHNS/O Analyzer) at the Nara Institute of Science and Technology, Nara, Japan. Conductivity measurements were performed using a Serfass conductance bridge model RCM151B (Arthur Thomas Co. Ltd.) connected to a 3403 cell (Yellow Springs Instrument Co.). The cell was calibrated using a 0.01000 M KCI solution with a molar conductance ( A ) of 141.3 fi" cm m or ' at 25 °C to determine the 1  2  m  cell constant to be 1.016 c m . ' -1  54  55  Solutions were prepared at 10' M . 3  4.2.3 X-ray Crystallography  The structures were solved by Dr. B. O. Patrick or Ms. C. A. Barta in the Department of Chemistry, U B C , or Dr. M . Obata at the Nara Women's University, Japan. Colourless crystals of [Re(L )(CO) Br]MeOH and [Re(L )(CO) Br]H 0 were obtained 2  3  3  3  2  from the slow evaporation of M e O H / H 0 solutions of the respective compounds. 2  Colourless crystals of [Re(L )(CO) ]ClEt OMeOH were obtained via slow vapor 8  3  2  diffusion of E t 0 into a concentrated MeOH solution of the Re compound containing 2  excess NaCI. The crystals were mounted on a glass fibre, cooled to -100.0 ± 0 . 1 °C, and the data collected on a Bruker X8 A P E X diffractometer using graphite-monochromated  180  References start on p. 234  Mo-Kct radiation. Data were collected and integrated using the Bruker SAINT  software  package. The data were corrected for Lorentz and polarization effects, as well as absorption ( S A D A B S ) . The structures were solved by direct methods (SIR92 ) with all 57  58  non-hydrogen atoms refined anisotropically. Hydrogen atoms were added but not refined. Residual electron density peaks consistent with one methanol molecule were found in the asymmetric unit of [Re(L )(CO)3Br], however, the molecule proved to be disordered and could not be modelled satisfactorily. As a result the S Q U E E Z E  59  function in P L A T O N  6 0  was used to measure the unassigned electron density, and correct the data to account for this residual electron density. [Re(L )(CO)3Br] crystallizes with two Re complexes and 3  one water molecule in the asymmetric unit. One mannose moiety was disordered and was refined in two orientations. The major disordered fragment was refined anisotropically, while the minor fragment was refined isotropically. A l l other non-hydrogen atoms were refined anisotropically. Final refinements of all three structures were carried out utilizing SHELXL-97.  ORTEP diagrams of [Re(L )(CO) Br], [Re(L )(CO) Br], and  61  2  3  3  3  [Re(L )(CO) ] are shown in Figures 4.8 (p.208), 4.9 (p.210), and 4.13 (p.226), 8  +  3  respectively. Tables of relevant bond lengths and angles are also shown for [Re(L )(CO) Br] (Table 4.2, p.208), [Re(L )(CO) Br] (Table 4.3, p.210), and 2  3  3  3  [Re(L )(CO)3]Cl (Table 4.7, p.227). For details of the X-ray crystallographic analyses for [Re(L )(CO) Br], [Re(L )(CO) Br] and [Re(L )(CO) ]Cl, please refer to Table A2 in the 2  3  3  8  3  3  Appendix (p.249).  181  References start on p. 234  4.2.4 [  99m  T c ( H 0 ) ( C O ) 3 ] Labeling Studies +  2  3  The organometallic precursor [ Tc(H 0)3(CO) ] was prepared from a saline 99m  +  2  solution of N a [  99m  3  T c 0 ] (1 mL, 200 MBq) using an Isolink™ kit which contains a 4  mixture of sodium tartrate, sodium tetraborate, sodium carbonate, and sodium boranocarbonate. Briefly, a 1 mL solution of Na[ Tc04] (200 MBq) was added to an 99m  IsoLink™ kit and the vial heated to reflux for 20 min. Upon cooling, 1 mL of a 0.1 M HCI solution was added to adjust the pH to 9-10. Labeling was achieved by mixing an aliquot (200 uL) of the [ Tc(H 0)3(CO) ] precursor with 1 mL of a 1 m M solution of 99m  +  2  1  3  1  one of L - L or histidine in PBS (pH 7.4, 1 mL) and incubating at 75 °C for 30 min. In the case of the tridentate ligands L - L , 1 mL of a 0.1 m M solution of one of the ligands 8  1 2  was used for labeling. H P L C analyses were performed on a Knauer Wellchrom K-1001 HPLC equipped with a K-2501 absorption detector and a Kapintek radiometric well counter. A Synergi 4 um C-12 Max-RP analytical column with dimensions of 250 x 4.6 mm was used for the bidentate ligands L ' - L , while a Synergi 4 (am C-18 Hydro-RP 7  analytical column with dimensions of 250 x 4.6 mm was used for the tridentate ligands L - L . HPLC solvents consisted of 0.1 % w/w 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). A n example H P L C trace of [  99m  T c ( L ) ( H 0 ) ( C O ) ] is shown in Figure 4.10 (p.215). Example H P L C traces of 1  +  2  99tii  3  8  8  ~r"  -f~  r T c ( L ) ( C O ) r , and the corresponding Re complex [Re(L )(CO) ] m  0  8  3  3  1  are shown in  Figure4.l4(p.230). 182  References start on p. 234  4.2.5 Cysteine and Histidine Challenge Experiments  To a 900 p L solution of either cysteine or histidine in PBS (10~ M , pH = 7.4) was 3  added a solution of the 10~ M for 5  L  8  - L  U  9 9 m  T c complex (final ligand concentration 10" M for L Z - L and 4  7  The samples were incubated at 37 °C and aliquots were removed at 1,  ) .  4, and 24 h for analysis by H P L C . The histidine challenge experiment with [  99m  T c ( L ) ( H 0 ) ( C O ) ] is shown in Figure 4.10 (p.215). 1  +  2  3  4.2.6 Syntheses of the Re Complexes with the 1,3-Diaminocarbohydrates  Bromotricarbonyl(l,3-diamino-2-propyl  (T-Z-L ) 7  P-D-glucopyranosyl)rhenium(I)  [Re(L )Br(CO) ] 1  3  9 H  0  H  H B  T^OH  °  2  A solution of rNEt4]2[ReBr (CO)3] (0.076 g, 0.099  B r  3  \ _ < \^CO H  C  C  M  M  O  1  )  A N D  L (0.025 g, 0.099 mmol) in M e O H (10  O  2  mL) was heated to reflux for 6 h. The solvent was then [Re(L )(CO) Br] 1  3  removed in vacuo and the residue purified by silica gel chromatography (9:1 EtOAc:MeOH eluent) to afford the product as a white solid (0.033 g, 55 %). ' H N M R (DMSO-c/ /D 0, 400.13 MHz): 5 5.38 (s, 1H; N/Y), 5.37 (s, 6  1H; "NH), 4.28 (d, J 3  2  J , 6 a  2  = 7.6 Hz, 1H; /Y-l), 4.07 (m, 1H; /Y-a), 3.66 (dd, J ,6a = 6.0 Hz, 3  U  5  = 11.6 Hz, 1H; 7Y-6a), 3.44 (m, 2H; H-b, H-c), 3.40 (dd, J ,6b = 2.5 Hz, J ,6b 3  6 b  2  5  6a  =  11.6 Hz, 1H; H-6b), 3.13 (m, 2H; /Y-3, /Y-5), 2.98 (m, 3H; H-2, H-4, N/Y), 2.70 (m, 3H;  183  References start on p. 234  N H , H-V, H-V).  13  C { ' H } N M R (DMSO-d /D 0, 100.62 MHz): 5 195.55, 195.45, 192.99 6  2  (fac-Rc(CO) ), 102.69 (CI), 77.06, 76.39 (C3/C5), 73.96 (Ca), 73.44 (C2), 69.98 (C4), 3  61.07 (C6), 48.07, 47.17 (Cb, Cc). IR (cm" , K B r disk): 3335 (br) (v(NH )), v(OH)); 1  2  2023 (vs), 1881 (vs, br) (v(/ac-Re(CO) )); 1581 (5(NH ). +ES-MS m/z (relative intensity) 3  2  = 625 ([M+Na] , 40), 523, 521 ([M-Br] , 95), 361, 359 ( [ M - B r - C H i O ] , 100). A = 98 +  +  +  6  0  5  M  Q" cm mol" (1:1 electrolyte). Anal. Calcd. (found) for C i H B r N O R e : C, 23.93 1  2  1  2  20  2  9  (23.53); H, 3.35 (3.40); N 4.65 (4.57).  BromotricarbonyI(l,3-diamino-2-propyl p-D-xylopyranosyl)rhenium(I) [Re(L )Br(CO) ] 2  3  "2 Br  b  3  2  OH  The title compound [Re(L )Br(CO) ] (0.075 g, 44 %) 2  3  L?  co  [Re(L )(CO) Br] 2  was prepared from [NEt ] [ReBr (CO) ] (0.226 g, 4  0  2  3  9  3  m  m  °  1  )m  d  L  '  (  0  '°  6  2  5  3  g  '  0  2  9  3  3  m  m  °  1  )b  y  a  procedure analogous to that described for [Re(L )Br(CO) ]. *H N M R (DMSO-c/ /D 0, 1  3  6  2  400.13 MHz): 5 5.49 (d, VNH.NH = 9.6 Hz, 1H; N/7), 5.41 (d, J H,NH = 11.2 Hz, 1H; N//), 2  N  4.28 (d, J i 3  = 7.5 Hz, 1H; H-l), 4.09 (m, 1H; //-a), 3.70 (dd, J 5a= 5.2 Hz, J ,5b = 11.2 3  >2  2  4)  5a  Hz, 1H; //-5a), 3.33 (m, 2H; H-b, H-c)), 3.24 (ddd, J , = 9.7 Hz, J , = 5.2 Hz, J , = 3  3  3  4  3  4  5 a  4  5 b  8.8 Hz, 1H; HA), 3.03 (m, 4H; H-2, H-3, //-5b, NH), 2.67 (m, 3H; N / / , H-V, H-c'). 13  C { H } N M R ( D M S O - J / D 0 , 100.62 MHz): 5 195.50, 195.35, 192.95 (fac-Re(CO) ), J  6  2  3  103.50 (CI), 76.30 (C3), 73.98 (Ca), 73.31 (CI), 69.38 (CA), 65.83 (C5), 47.99, 47.10 (Cb, Cc). IR (cm" , K B r disk): 3300 (br) (v(NH ), v(OH)); 2033 (vs), 1902 (vs), 1871 1  2  (vs) (v(/ac-Re(CO) )); 1585 (5(NH )). +ES-MS m/z (relative intensity) = 595 ([M+Na] , +  3  2  184  References start on p. 234  20), 492, 490 ([M-Br] , 50), 361, 359 ( [ M - B r - C H 0 ] , 100). A = 108 Q" cm mol" +  +  5  8  1  4  2  1  M  (1:1 electrolyte). Anal. Calcd. (found) for C n H i B r N 0 R e H 0 : C, 22.38 (22.19); H , 8  2  8  2  3.41 (3.74); N 4.74 (4.76).  Bromotricarbonyl(l,3-diamino-2-propyl a-D-mannopyranosyl)rhenium(I) [Re(L )Br(CO) ] 3  3  ? HO  The title compound [Re(L )Br(CO) ] (0.054 g, 50 %)  H  3  Q  H  3  \  4  .  H O - ^ ? ^ i  H  __2 gj-  /—N  / / / (  |•  .  \_ < H  Q  w  a  s  prepared from [NEt4]2[ReBr (CO)3] (0.144 g, 0.187 3  I^CO  N  C  C  c  mmol) and L (0.047 g, 0.187 mmol) by a procedure  o  2  analogous to that described for [Re(L )Br(CO) ]. ' H 1  [Re(L )(CO) Br] 3  3  3  N M R ( D M S O - d / D 0 , 400.13 MHz): 5 5.23 (m, 2H; 2N/Y), 4.77 (d, J 3  6  2  H-\), 4.08 (m, 1H; H-a), 3.69 (dd, J  - 1.5 Hz, V  3  1>2  2;3  1 > 2  = 1.5 Hz, 1H;  = 3.9 Hz, 1H; H-2), 3.60 (m, 2H;  H-3, //-6a), 3.40 (m, 2H; H-b, H-c)), 3.35 (m, 4H; /Y-4, /Y-5, H-6b, N/Y), 2.63 (m, 3H; N H , H-b\ /Y-c'). C { ' H } N M R ( D M S O - J / D 0 , 100.62 MHz): 5 195.50, 195.39, 192.91 I3  6  2  (/ac-Re(CO) ), 98.63 (CI), 74.84 (C4/C5), 70.73 (Ca), 70.29 (C3), 70.25 (C2), 67.00 3  (C4/C5), 61.21 (C6), 47.74, 45.40 (Cb, Cc). IR (cm , K B r disk): 3300 (br) (v(NH ), -1  2  v(OH)); 2029 (vs), 1924 (vs), 1863 (vs) (v(/ac-Re(CO) )); 1582 (8(NH )). +ES-MS m/z 3  2  (relative intensity) = 625 ([M+Na] , 20), 523, 521 ([M-Br] , 40), 361, 359 ([M-Br+  +  C H i O ] , 100). A = 94 Q" 'cm mol" (1:1 electrolyte). Anal. Calcd. (found) for +  6  0  I  5  2  1  M  C i H B r N O R e : C, 23.93 (23.95); H , 3.35 (3.59); N 4.65 (4.57). 2  20  2  9  185  References start on p. 234  Bromotricarbonyl(l,3-diamino-2-propyl  a-D-galactopyranosyl)rhenium(I)  [Re(L )Br(CO) ] 4  3  The title compound [Re(L )Br(CO) ] (0.037 g, 51 %) was  HO OH  4  3  b  HO  tsr  /~^">- I,^CO prepared from [NEt ] [ReBr (CO) ] (0.093 g, 0.121 4  H  c  analogous to that described for [Re(L')Br(CO) ]. H  3  3  N M R ( D M S O - J / D 0 , 400.13 MHz): 5 5.53 (d, V 2  = 11.4 Hz, 1H; NH), 4.81 (d, V 3  J ,6b 5  6  = 11.2, 1H; NH), 5.35 (d,  2  JNH,NH  5M  = 6.0 Hz, 1H; H-5), 3.68 (m, 1H; 77-4), 3.64 (dd, J 3  = 12.4 Hz, 1H; H-6a), 3.49 (dd,  2 ; 3  3  = 3.2 Hz, J  =  3  3 ; 4  2 j 3  = 11.2 Hz. 1H; H-2), 3.54 (dd, V ,6a=  3  2  J a,6b  ,NH  = 3.9 Hz, 1H; H-\), 4.00 (m, 1H; H-a), 3.78 (dd, J  3  2  N H  3  U  11.2 Hz, 1H; H-3), 3.51 (dd, J i , = 3.9 Hz, J 6.0 Hz,  3  4  4  = 6.0 Hz,  3  mmol) and L (0.030 g, 0.121 mmol) by a procedure  o  2  [Re(L )(CO) Br]  6  2  5  J 6b= 5>  6.0 Hz, J , 2  6a 6h  = 12.4 Hz, 1H; //-6b),  3.40 (m, 2H; H-b, H-c), 3.16 (m, 1H; NH), 2.70 (m, 2H; H-b\ H-V), 2.59 (m, 1H; NH). 13  C{'H} N M R ( D M S O - ^ / D 0 , 100.62 MHz): 5 195.50, 195.36, 193.03 (/ac-Re(CO) ), 6  2  3  99.69 (CI), 73.24 (Ca), 72.27 (C5), 69.49, 68.93 (C3/C4), 68.27 (C2), 60.80 (C6), 47.88, 46.43 (Cb, Cc). IR ( c m , K B r disk): 3350 (br) (v(NH ), v(OH)); 2023 (vs), 1879 (vs, br) 1  2  (v(/ac-Re(CO) )); 1581 (5(NH )). +ES-MS m/z (relative intensity) = 625 ([M+Na] , 50), +  3  2  523, 521 ([M-Br] , 60), 361, 359 ( [ M - B r - C H i O ] , 100). A = 100 Q cm mof (1:1 +  +  6  0  5  _1  2  1  M  electrolyte). Anal. Calcd. (found) for C i H B r N O R e : C, 23.93 (23.99); H , 3.35 (3.50); 2  20  2  9  N4.65 (4.71).  186  References start on p. 234  Bromotricarbonyl(l,3-diamino-2-propyl P-D-galactopyranosyl)rhenium(I) [Re(L )Br(CO) ] 5  3  HO OH  H  The title compound [Re(L )Br(CO) ] (0.033 g, 49 %) 5  2  B  r  3  < \^*CO was prepared from [NEt ]2[ReBr3(CO) ] (0.086 g, 4  3  0.112 mmol) and L (0.028 g, 0.112 mmol) by a [Re(L )(CO) Br] s  3  procedure analogous to that described for [Re(L )Br(CO) ]. ' H N M R ( D M S O - d / D 0 , 300.13 MHz): 5 5.49 (d, JNH,NH = 10.5 Hz, 1  2  3  6  2  1H; NH), 5.41 (d, VNH.NH = U . l Hz, 1H; NH), 4.24 (d, \  2  = 7.2 Hz, 1H; HA),  4.10 (m,  1H; H-a), 3.66 (d, J , = 3.3 Hz, 1H; H-4), 3.56 (m, 2H; //-6a, //-6b), 3.40 (m, 5H; H-2, 3  3  4  H-3, H-5, H-b, H-c), 3.11 (m, 1H; NH), 2.91 (m, 3H; NH, H-b\ H-V). C { ' H } N M R 13  (DMSO-flf /D 0, 75.48 MHz): 5 195.51, 195.39, 192.95 (/ac-Re(CO) ), 103.11 (CI), 6  2  3  75.40 (C5), 73.61 (C3), 73.19 (Ca), 70.53 (C2), 67.89 (C4), 60.35 (C6), 48.19, 46.97 (Cb, Cc). IR (cm , K B r disk): 3350 (br) (v(NH ), v(OH)); 2023 (vs), 1908 (vs), 1877 (sh) -1  2  (v(/ac-Re(CO) )); 1582 (5(NH )). ES-MS m/z (relative intensity) = 625 ([M+Na] , 10), +  3  2  523, 521 ([M-Br] , 20), 361, 359 ( [ M - B r - C H i O ] , 100). A = HO Q c m m o l (1:1 +  +  6  0  5  _1  2  -1  M  electrolyte). Anal. Calcd. (found) for C i H B r N O R e : C, 23.93 (24.39); H , 3.35 (3.59); 2  20  2  9  N 4.65 (4.39).  187  References start on p. 234  P-(a-D-glucopyranosyI-(l,4)-D-  BromotricarbonyI(l,3-diamino-2-propyl  glucopyranosyl)rhenium(I) [Re(L )Br(CO)3] 6  OH  A solution of [NEt ]2[ReBr (CO)3] 3  4  HO * HO-  (0.060 g, 0.078 mmol) and L (0.032 6  g, 0.078 mmol) in M e O H (10 mL) was heated to reflux for 6 h. The solvent was then removed in vacuo  [Re(L )(CO) Br] 6  3  and the residue purified by silica gel chromatography (4:1 EtOAc:MeOH eluent) to afford the product as a white solid (0.040 g, 68 %). *H N M R ( D M S O - d / D 0 , 400.13 6  MHz): 5 5.27 (s, 1H;N/Y), 5.25 (s, 1H;N/Y), 5.02 (d, *J  = 3.5 Hz, 1H; /Y-l'), 4.31 (d,  vy  J i , = 7.7 Hz, 1H; /Y-l), 4.12 (s, 1H; H-a), 3.68 (d, J ,  3  = 11.6 Hz, 1H; H-6a), 3.59 (d,  2  2  6 a  2  6 b  Je \6V = 9.6 Hz 1H; /Y-6a'), 3.41 (m, 6H; /Y-3, /Y-4, /Y-6b, /Y-6b', H-b, H-c), 3.33 (m, 1H;  2  a  /Y-3'), 3.25 (m, 2H; /Y-4, /Y-5), 3.22 (dd, J  = 3.5 Hz, J',3' = 9.7 Hz, 1H; H-2), 3.07  3  3  VX  2  (m, 2H; H-2, /Y-5'), 2.94 (m, 1H; N/Y), 2.65 (m, 3H; N/Y, H-b\ H-c'). C{ ll} u  l  NMR  (DMSO-c/ /D 0, 100.62 MHz): 8 195.96, 193.41 (/ac-Re(CO) ), 102.71 (CI), 101.09 6  2  3  (CV), 79.59 (C4), 76.51 (C3), 75.66 (C5), 74.60 (Ca), 73.80 (C4'), 73.55 (C3'), 73.40 (C2), 72.65 (C2'), 70.22 (C5'), 61.26, 61.08 (C6/C6') 48.61, 47.70 (Cb, Cc). IR (cm , -1  KBr disk): 3350 (br) (v(NH ), v(OH)); 2021 (vs), 1903 (vs), 1881 (vs) (v(/ac-Re(CO) )); 2  3  1582 (8(NH )). +ES-MS m/z (relative intensity) = 787 ([M+Na] , 70), 685, 683 ([M-Br] , +  +  2  70), 361, 359 ([M-Br-Ci H oOio] , 100). A = 150 Q" cm mol" (1:1 electrolyte). Anal. +  2  2  1  2  1  M  Calcd. (found) for Ci H BrN Oi4Re: C, 28.28 (28.38); H , 3.95 (4.17); N 3.66 (3.26). 8  30  2  188  References start on p. 234  (Bis(aminomethyl)bis[(P-D-glucopyranosyIoxy)methyl] methane) bromotricarbonylrhenium(I) [Re(L )Br(CO)3] 7  OH  The title compound [Re(L )Br(CO) ] (0.030 g, 7  3  HO HO4  H  Br  2  45 %) was prepared from [NEt4]2[ReBr (CO)3] 3  ' /*- !..,*\CO N  R  I^CO HO HO-  (0.064 g, 0.083 mmol) and L (0.038 g, 0.083 7  4  2  3  mmol) by a procedure analogous to that  OH i [Re(L )(CO) Br]  described for [Re(L )Br(CO) ]. H N M R  7  6  3  !  3  ( D M S O - J / D 0 , 400.13 MHz): 5 5.24 (m, 2H; N/7), 4.14 (d, V i , = 7.6 Hz; H-l), 4.11 (d, 6  3  2  2  J > = 7.6 Hz; H-V), 3.77 (d, J , ' = 10.0 Hz, 1H; H-d), 3.67 (m, 2H; //-6a, //-6a'), 3.58 2  r>2  d  d  (d, J ,e' = 9.8 Hz, 1H; H-e), 3.53 (d, J 2  2  e  d>d  ' = 10.0 Hz, 1H; H-d'), 3.45 (m, 2H; H-6b, H-  6b'), 3.31 (d, J ' = 9.8 Hz, 1H; H-e'), 3.13 (m, 11H; H-2, H-T, H-3, H-V, HA, HA', 2  e>e  H-5, H-5\N//,  H-b, H-c), 3.02 (m, 1H; N/7), 2.66 (m, 2H; H-V, H-V).  13  C{'H} N M R  (DMSO-t/ /D 0, 100.62 MHz): 5 195.91, 192.97 f/ac-Re(CO) ), 103.84 (CI), 103.61 6  2  3  (CV), 77.27, 77.26, 77.08, 76.94 (C3/C37C5/C5'), 73.69, 73.40 (C2/C2'), 72.42 (Ce), 71.34 (Cd), 70.36, 70.21 (C4/C4'), 61.39, 61.27 (C6/C6'), 47.33, 47.18 (Cb, Cc), 42.12 (Ca). IR (cm" , K B r disk): 3350 (br) (v(NH ), v(OH)); 2023 (vs), 1898 (vs, br) (v(fac1  2  Re(CO) )); 1582 (5(NH )). +ES-MS m/z (relative intensity) = 831 ([M+Na] , 10), 729, +  3  2  727 ([M-Br] , 60), 567, 565 ( [ M - B r - C H O ] , 50), 405, 403 ( [ M - B r - C i H O ] , 100). +  +  6  A  +  5  2  20  10  = 140fi" cm mol" (1:1 electrolyte). Anal. Calcd. (found) for 1  M  10  2  1  C H B r N O i R e - H O : C, 29.03 (28.83); H, 4.51 (4.13); N 3.38 (3.11). 20  35  2  3  2  189  References start on p. 234  4.2.7 Syntheses of the Re Complexes with the Carbohydrate-appended 2,2'Dipicolylamine ligands (L -L ) 8  12  (2-(Bis(2-pyridinylmethyl)amino)ethyl-(3-D-glucopyranosyl)tricarbonylrhenium(I) bromide [Re(L )(CO) ]Br 8  3  A solution of [NEt4] [ReBr (CO) ] (0.076 g, 2  OH  3  0.099 mmol) and L (0.040 g, 0.099 mmol) in 8  H O ^ ^ ^ 3  3  Br  OH 1  z  MeOH (10 mL) was heated to reflux for 6 h. The solvent was removed in vacuo and the residue purified by alumina (Brockman  [Re(L )(CO) ]Br 8  3  activity I, neutral) chromatography (8.5:1.5 C H C N : H 0 eluent) to afford the product as 3  2  a white solid (0.035 g, 46 %). ' H N M R (MeOH-</ , 400.13 MHz): 5 8.89 (d, J , =5.3 3  4  Hz, 2H; #-14), 7.96 (dd, J ,n =7.3 Hz, J , 3  3  u  1 2  Hz, 1H; if-11), 7.59 (d, Ju\\r 3  1 3  1 4  =8.0 Hz, 2H; H-12), 7.60 (d, Y n , i = 7.3 3  1 3  2  =7.3 Hz, 1H; H-IV) 7.39 (dd, J , 3  2 ; 1 3  =8.0 Hz, V i , i = 3  4  5.3 Hz, 2H; /Y-l3), 5.11 (overlapping d, J a,9b/9'a,9'b = 16.8 Hz, 2H; /Y-9a, /Y-9'a), 4.99 2  9  (overlapping d, J a,9b/9'a,9'b = 16.8 Hz, 2H; /Y-9b, /Y-9'b), 4.49 (d, J 3  2  h2  9  = 7.8 Hz, 1H; /Y-l),  4.45 (m, 1H; /Y-7a), 4.20 (m, 2H; /Y-8a, /Y-8b), 4.12 (m, 1H; /Y-7b), 3.87 (dd, J , 3  5  6 a  = 1.5  Hz, Y , = 11.7 Hz, 1H; /Y-6a), 3.76 (dd, J ,6b = 5.2 Hz, Y ,6b = 11.7 Hz, 1H; /Y-6b), 2  3  6a  6b  2  5  6a  3.30-3.49 (m, 4H; H-2, /Y-3, /Y-4, /Y-5). C{'H} N M R ( M e O H - ^ , 100.62 MHz): 5 13  197.22, 196.36 (/ac-Re(CO) ), 162.53 (C10), 162.51 (C10'), 153.02 (C14), 141.61 (C12), 3  126.84 (CI 1), 124.70 (C13), 124.66 (C13'), 104.59 (CI), 78.16, 78.08 (C3/C5), 75.03 (C2), 71.57 (C4), 70.77 (C8), 69.48, 69.45 (C9/C9'), 67.49 (C7), 62.61 (C6). IR (cm" , 1  190  References start on p. 234  thin film, AgBr plate): 3379 (br) (v(OH)); 2930 (w) (v(CH)); 2027 (vs), 1931 (vs) (vifacRe(CO) )); 1611 (w) (v(C=C or C=N)). +ES-MS m/z (relative intensity) = 676, 674 3  ([M] , 100). A = 165 Q ' W m o l " (1:1 electrolyte). Anal. Calcd. (found) for +  1  M  C H B r N 0 R e - 2 H 0 : C, 34.90 (35.17); H , 3.95 (3.76); N , 5.31 (5.20). 23  27  3  9  2  (2-(Bis(2-pyridinylmethyl)amino)ethyl-P-D-xylopyranosyl)tricarbonylrhenium(I) bromide [Re(L )(CO) ]Br 9  3  The title compound [Re(L )(CO) ]Br (0.087 g, 9  3  75 %) was prepared from 3  'OH 1  Br  f7-N//, | . > C O Re>^  [NEt ] [ReBr3(CO) ] (0.108 g, 0.141 mmol)  IQP  4  2  3  and L (0.053 g, 0.141 mmol) by a procedure 9  13  analogous to that described for  [Re(L )(CO) ]Br 9  3  [Re(L )(CO) ]Br. H N M R (MeOH-tf , 400.13 MHz): 5 8.89 (d, J , i = 5.6 Hz, 2H; H8  !  3  3  4  14), 7.98 (dd, J n , i =7.6 Hz, V , 3  2  11), 7.65 (d,  1 3  =8.0 Hz, 2H; #-12), 7.67 (d, J , 3  2 j l 3  J  W  ,  I  M 2  = 7.6 Hz, 1H; H-  =7.6 Hz, 1H; H-1V), 7.41 (dd, J i , i =8.0 Hz, J i , i =5.6 Hz, 2H; 3  3  4  T  3  2  3  3  4  #-13), 5.07 (overlapping d, V g ^ t W b = - 7 Hz, 2H; #-9a, #-9'a), 5.01 (d, y ,9b = 16.7 16  2  9a  Hz, 1H; #-9b) 4.99  (d, J 2  9%9  -  B  = 16.7 Hz, 1H; #-9'b), 4.45 ( d , J 3  l i 2  =7.6 Hz, 1H; #-1),  4.36 (m, 1H; #-7a), 4.19 (m, 2H; #-8a, #-8b), 4.13 (m, 1H; #-7b), 3.96 (dd, J 3  Hz, J a,5b = 11.4 Hz, 1H; #-5a), 3.56 (ddd, 5  1H; H-4), 3.42 (dd, J , = 8.7 Hz, J 3  3  2  13  3  3 j 4  = 10.2 Hz, J , 3  2  3  J  3  A  4  = 5.3 Hz, J 3  5 a  4 ; 5 b  4 j 5 a  = 5.3  = 8.8 Hz,  = 10.2 Hz, 1H; #-3), 3.33 (m, 2H; H-2, H-5b).  C{'H} N M R (MeOH-d , 100.62 MHz): 5 197.18, 196.33 (/ac-Re(CO) ), 162.46 (C10), 4  3  162.37 (C10'), 153.06 (C14), 153.03 (C14'), 141.63 (C12), 141.61 (C12'), 126.87 ( C l l ) ,  191  References start on p. 234  126.86 (Cll'),124.72 (CI 3), 124.69 (C13'), 105.34 (CI), 77.90 (C3), 74.87 (C2), 71.15 (C4), 70.62 (C8), 69.70, 69.34 (C9, C9'), 67.63 (C7), 67.15 (C5). IR (cm- , thin film, 1  AgBr plate): 3378 (br) (v(OH)); 2935 (v(CH)); 2029 (vs), 1920 (vs) (v(/ac-Re(CO) )); 3  1611 (w) (v(C=C or C=N)). +ES-MS m/z (relative intensity) = 646, 644 ([M] , 100). A +  M  - 141 Q-'cm moF (1:1 electrolyte). Anal. Calcd. (found) for C H25BrN 0 Re-3H20: C, 2  1  3  22  8  33.89 (33.83); H , 4.01 (3.97); N , 5.39 (5.78).  (2-(Bis(2-pyridinyImethyl)amino)ethyl-a-D-mannopyranosyl)tricarbonylrhenium(I) bromide [Re(L )(CO) ]Br 10  3  ,OH  The title compound [Re(L )(CO) ]Br (0.064 10  3  g, 63 %) was prepared from Br  [NEt ] [ReBr (CO) ] (0.105 g, 0.136 mmol) 4  and L l(k  2  1 0  3  3  (0.055 g, 0.136 mmol) by a procedure  analogous to that described for  [Re(L*")(CO) ]Br 3  [Re(L )(CO) ]Br. ' H N M R (MeOH-d , 400.13 MHz): 5 8.89 (d, J i 8  3  3  4  14), 7.98 (dd, J i 2 =7.6 Hz, V ,  =7.8 Hz, 2H; #-12), 7.62 (d, J ,n  3  U  3 ; 1 4  3  2 > 1 3  n  11), 7.60 (d, Vn',i2' =7.6 Hz, 1H; #-11'), 7.39 (dd, J ,u  = 5.5 Hz, 2H; #= 7.6 Hz, 1H; #-  =7.8 Hz, J , i =5.5 Hz, 2H;  3  3  n  1 3  4  #-13), 5.07 (overlapping d, J ,9b/9' ,rb = 16.8 Hz, 2H; #-9a, #-9'a), 4.99 (d, J 9b = 16.8 2  2  9a  Hz, 1H; #-9b), 4.98 (d,  3  Ji,  a  9a>  = 1.3 Hz, 1H; #-1), 4.97 (d, J > 2  2  9%9  b  = 16.8 Hz, 1H; #-9'b),  4.28 (m, 1H; #-7a), 4.17 (m, 2H; 2x #-8), 4.04 (m, 2H; #-2, #-7b), 3.96 (dd, V Hz, J 2  6 a j 6 b  5 > 6 a  = 1.6  = 11.0 Hz, 1H: #-6a), 3.80 (m, 2H; #-3, #-6b), 3.68 (m, 2H; #-4, #-5).  C{'H} N M R (MeOH-<4 100.62 MHz): 8 197.17, 196.29 (/~ac-Re(CO) ), 162.26 (C10), 3  192  References start on p. 234  162.23 (CIO'), 153.05 (C14), 141.61 (C12), 126.92 (CU), 126.90 (CIV), 124.83 (C13), 124.81 (C13'), 102.15 (CI), 75.45 (C4/C5), 72.57 (C3), 71.86 (C2), 70.41 (C8), 69.66, 69.30 (C9, C9'), 68.61 (C4/C5), 65.52 (CI), 62.98 (C6). IR (cm" , thin film, AgBr plate): 1  3389 (br) (v(OH)); 2935 (v(CH)); 2027 (vs), 1932 (vs) (v(/ac-Re(CO) )); 1611 (w) 3  (v(C=C or C=N)). +ES-MS m/z (relative intensity) = 676, 674 ([M] , 100). A = 160 fi" +  M  W m o l " (1:1 electrolyte). Anal. Calcd. (found) for C H27BrN 0 Re-2H20: C, 34.90 1  23  3  9  (34.77); H, 3.95 (3.65); N , 5.31 (5.59).  (2-(Bis(2-pyridinylmethyl)amino)methyl-glucosamido)tricarbonyIrhenium(I) bromide [Re(L )(CO) ]Br u  3  2-(Bis(2-pyridinylmethyl)amino)methyl-glucosamide (L ) 11  9 1 6  To a mixture of (bis(2-pyridylmethyl)amino)acetic  H  -  -Q  /=\'  |  acid (0.384 g, 1.49 mmol), N-hexylcarbodiimide  l  Ho-V-^^^rvOH  ?  N  io> N12  13  44  14-  (0.338 g, 1.64 mmol), N-hydroxysuccinimide (0.189 g i 64 m o l ) , and dimethylaminopyridine (0.020 g, ;  m  0.16 mmol) was added D M F (15 mL) at 0° C. The  flask was warmed to room temperature after 1 fir and then the reaction mixture was filtered after 6 h. To the resulting solution glucosamine-HCl (0.353 g, 1.64 mmol) and triethylamine (0.327 g, 3.23 mmol) were added as a solution in DMF/H2O (2 mL/1 mL). The solvent was removed in vacuo after a further 24 h and the residue purified by silica gel chromatography (7:3 EtOAc: MeOH eluent) to afford the product L as a light 1 1  193  References start on p. 234  yellow solid (0.542 g, 87 %). ' H N M R (MeOFW , 400.13 MHz): 8 8.54 (d,  J =4.0  3  4  Hz, 2H; 77-14), 7.80 (m, 2H; 7/-12), 7.53 (d, J ,n = 8.0 Hz, 1.4H; H-lla), 3  u  = 8.0 Hz, 0.6H; H-\ip),  7.32 (m, 2H; 7/-13), 5.13 (d, Jy 3  X  nM  7.49  (d, J ,n 3  n  =2.0 Hz, 0.7H; /7-la), 4.74  (d, J,, = 8.0 Hz, 0.3H; 7/1-P), 3.93 (d, J b/9'a,9'b = 15.0 Hz, 2H; 7/-9a, 7/-9'a), 3.90 (d, 3  2  2  V ,«>h/9'a,9'b  9a;9  =  1 5  9a  '°  H  z  '  2  H  ;  / / _ 9  ' ' ^-9'b), 3.85 (m, 2.1H, 7/-2a, 77-3a, 7/-4a), 3.71 (m, a  1.9H; 7/-2p, //-6aa, 77-6bp, 7/-3p or /7-4P), 3.45 (m, 0.7H; i7-5a), 3.32 (m, 2.9H; 7/-6ap, i/-6bp, 7/-8a, 7/-8b, 7/-5p, 7/-3P or 77-4p). ' ^ { ' H } N M R (MeOH-t/4, 100.62 MHz): 5 174.41 (C7P), 173.67 (C7a), 159.12 (ClOa), 159.07 (C10p), 150.04 (C14P), 150.01 (C14a), 138.68 (C12a), 138.68 (C12p), 125.28 ( C l i p ) , 125.11 ( C I l a ) , 124.00 (C13a and p), 97.01 (Clp), 92.56 ( C l a ) , 77.98, 75.82 (C3p/C4p), 73.13, 72.83 (C3a/C4a), 72.19 (C5a), 72.04 (C5p), 61.04 (C9), 60.79 (C6a), 59.13 (C6p), 58.92 (C8), 58.46 (C2p), 55.68 (C2a). IR (cm" , thin fdm, AgBr plate): 3311 (br) (v(OH)); 2965 (m) 1  (v(CH)); 1653 (m) (v(amide I)). +ES-MS m/z (relative intensity) = 441 ([M + Na] , 100). +  Anal. Calcd. (found) for C oH26N CV1.5H 0: C, 53.92 (53.82); H , 6.56 (6.30); N , 12.58 2  4  2  (12.61).  [Re(L )(CO) ]Br n  3  OH  The title compound [Re(L )(CO) ]Br (0.084 n  3  g, 58 %) was prepared from "N//,  I  , x\CO ilX  CO  Br  [NEt ] [ReBr (CO) ] (0.145 g, 0.189 mmol) 4  and L  1 1  2  3  3  (0.079 g, 0.189 mmol) by a procedure  13 [Re(L )(CO) ]Br n  3  194  References start on p. 234  analogous to that described for [Re(L )(CO) ]Br. H N M R (MeOH-d , 400.13 MHz): 8 8  !  3  4  8.90 (d, Vi3,i4 = 5.7 Hz, 2H; #-14), 7.98 (m, 2H; #-12), 7.59 (m, 2H; #-11), 7.42 (m, 2H; #-13), 5.38 (d,V a 9b = 15.6 Hz, 1H; #-9a), 5.28 (d, J ^ 2  9  9  ;  9  b  = 17A Hz, 1H; #-9'a) 5.26 (d,  J ,2' =3.3 Hz, 0.7H; #-la), 5.02 (m, 2H; #-9b, #-9'b), 4.73 (m,2H; #-8a, #-8b), 4.66  3  V  (d,Vi  = 7.6 Hz, 0.3H; #-lp), 3.85 ( d d , J  =3.3 Hz, J',3' = 10.5 Hz, 0.7H; #-2oc),  3  ) 2  3  vx  2  3.92 (dd, J ',6a' =2.1 Hz, ./ ., . = 12.0 Hz, 0.7H; #-6aa), 3.80 (m, 3H; #-6ba, #-6ap, 3  2  6a  5  6b  #-6bp, #-2p, #-3a, #-4a), 3.35 (m, 0.9H; #-3p, #-4p, #-5p). C { ' H } N M R (MeOH13  d , 100.62 MHz): 8 196.81, 196.73, 196.19 (/ac-Re(CO) ), 169.70 (C7P), 169.45 (C7a), 4  3  162.71, 162.35, 162.33, 162.07 (C10, C10'), 153.19, 153.08 (C14, C14'), 141.73 (C12, C12'), 126.97, 126.93, 126.79 (CI 1, C I T ) , 124.82, 124.75, 124.54 (C13, C13'), 96.89 (Clp), 92.58 ( C l a ) , 78.08, 75.94 (C3p/C4p), 73.12, 72.80, 72.43 (C3a/C4a/C5a), 72.08 (C5p), 70.44, 69.98, 69.60, 69.50 (C8a, C8p, C9, C9'), 62.74, 62.70 (C6a/C6p), 58.36 (C2P), 55.64 (C2a). IR (cm" , thin film, AgBr plate): 3390 (br) (v(OH)); 2960 (m) 1  (v(CH)); 2030 (vs), 1922 (vs) (v(/ac-Re(CO) )); 1665 (w) (v(amide I)). +ES-MS m/z 3  (relative intensity) = 689, 687 ([M] , 100). A = 145 Q" cm mol" (1:1 electrolyte). Anal. +  1  2  1  M  Calcd. (found) for C H B r N 0 R e H 0 : C, 35.12 (35.34); H , 3.59 (3.61); N , 7.12 2 3  2 6  4  9  2  (7.09).  195  References start on p. 234  (2-(Bis(2-pyridinylmethyl)amino)methyl-l,3,4,6-tetra-0-acetyl-P-Dglucosamido)tricarbonylrhenium(I) bromide [Re(L )(CO)3]Br 12  2-(Bis(2-pyridinylmethyl)amino)methyl-l,3,4,6-tetra-0-acetyl-P-D-glucosamide (L ) 12  TO a mixture of (bis(2-pyridylmethyl)amino)acetic  LC  A  c  0  \ — ^ ^  0  A  /==\ '  c  2  a c i d 4 4  (  L 1 6 0  g» 4  5 1  mmol), N-hexylcarbodiimide  N—( , (1.020 g, 4.94 mmol), N-hydroxysuccinimide  i  4  (0.571 g, 4.94 mmol), and dimethylaminopyridine i  (0.051 g, 0.42 mmol) was added D M F (40 mL) at L  1 2  0° C. The reaction flask was warmed to room temperature after 1 hr and then the reaction mixture was filtered after 6 h. To the resulting solution 1,3,4,6-tetra-O-acetyl-P-D-glucosamine-HCl (1.901 g, 4.96 mmol) 52  and triethylamine (0.944 g, 9.33 mmol) were added as a solution in D M F (10 mL). The solvent was removed in vacuo after a further 24 h and the residue purified by silica gel chromatography (19:1 EtOAc: MeOH eluent) to afford the product L a s a light yellow 1 2  solid (1.426 g, 60 %). H N M R (MeOH-d , 400.13 MHz): 5 8.55 (d, J [  3  4  77-14), 7.79 (dd, J ,n 3  n  =7.8 Hz, J i , 3  2  3  77-1), 5.50 (dd, V  =6.8 Hz, 2H; #-12), 7.46 ( d , J 3  1 3  77-11), 7.33 (dd, Vi2,i3 =6.8 Hz, J , 1 3  UM  =4.3 Hz, 2H; 77-13), 5.99 ( d , J 3  1 4  = 10.3 Hz, J , =9.6 Hz, 1H; H-3), 5.09 (dd, J 3  2 ; 3  3  3  Hz, 1H; H-4), 4.34 (dd, J 3  4  3  3  2  2  5  3  =9.8 Hz, V 5 j 6 a = 4.5 Hz, J 3  4 j 5  = 8.8 Hz, 1H;  =9.6 Hz, J 3  3 j 4  3  6a  Hz, J ,3 = 10.3 Hz, 1H; H-2), 4.16 (dd, J ,6b =2.2 Hz, J (ddd, J  1>2  = 7.8 Hz, 2H;  = 4.5 Hz, J ,6b = 12.5 Hz, 1H; H-6a), 4.21 (dd, J 2  5M  1 U 2  =4.3 Hz, 2H;  5 ; 6 b  =2.2 Hz, 1H; 196  6 a ; 6 b  H-5),  1 > 2  4 > 5  =9.8  = 8.8  = 12.5 Hz, 1H; 77-6b), 4.04 3.80 (s, 4H; H-9, 77-9'), 3.28 References start on p. 234  (d, J > = 16.8 Hz, 1H; #-8), 3.22 (d, %$> = 16.8 Hz, 1H; #-8'), 2.08 (s, 3H; COC# ), 2  ifi  3  2.05 (s, 3H; COC# ), 1.91 (s, 3H; COC/Y3), 1.88 (s, 3H; COC# ). C { ' H } N M R 13  3  3  ( M e O H - ^ , 100.62 MHz): 5 173.91 (C7), 172.18, 171.52, 171.18, 170.38 (4COCH ), 3  158.96 (CIO), 150.03 (C14), 138.67 (C12), 124.97 (CI 1), 124.01 (C13), 93.35 (CI), 73.85, 73.74 (C3/C5), 69.84 (C4), 62.94 (C6), 60.70 (C9), 58.65 (C8), 53.94 (C2), 20.61 (2COCH ), 20.57 (COCH3), 20.55 (COCH ). IR (cm" , thin film, AgBr plate): 2939, 2880 1  3  3  (m) (v(CH)); 1751 (vs) (v(C=0)CH )); 1676 (m) (vfamide I)). +ES-MS m/z (relative 3  intensity) = 609 ([M + N a f , 100). Anal. Calcd. (found) for C28H34N4O10: C, 57.33 (57.34); H , 5.84 (5.91); N , 9.55 (9.68).  [Re(L )(CO) ]Br ,2  3  OAc U A  C  A  0  The title compound [Re(L )(CO) ]Br (0.086 12  y—KS '  14'  ~'  9  o  3  13'  ; o \ ^ ^ O A c -.11 3 HN. 1^ N  g, 79 %) was prepared from Br  r^-N//, |.,^\\co  [NEt4]2[ReBr (CO) ] (0.094 g, 0.122 mmol) 3  and L  1 2  3  (0.068 g, 0.122 mmol) by a procedure  analogous to that described for  [Re(L )(CO) ]Br Ii  3  [Re(L )(CO)3]Br. Unfortunately the product could not be separated fully from the NEt Br 4  formed during the reaction. H N M R (MeOH-d , 400.13 MHz): 5 8.84 (d, V i J  4  2H; HA4), 7.94 (dd, J ,n  =7.8 Hz,  3  u  7.38 (dd,  3  Ji i3 2 j  5.43 (dd, J 3  2>3  =8.0 Hz,  3  Ji ,i 3  4  Ji ,i3 2  ,i4  = 5.4 Hz,  =8.0 Hz, 2H; #-12), 7.55 (m,2H; #-11),  = 5.4 Hz, 2H; #-13), 5.95 ( d , V i , = 8.8 Hz, 1H; #-1),  = 10.0 Hz, % =9.6 4  3  3  2  Hz, 1H; #-3), 5.27 (d, V a,9b = 17.0 Hz, 1H; #-9a), 9  197  References start on p. 234  5.25 (d, J >  = 17.0 Hz, 1H; //-9'a), 5.05 (dd, J  2  9%9  3  b  = 9.6 Hz, J 3  3 > 4  4 ; 5  =9.8 Hz, 1H; H-4),  4.90 (d, J ,9b = 17.0 Hz, 1H; H-9b), 4.86 (d, J 9'b = 17.0 Hz, 1H; //-9'b), 4.52 (s, 2H; 2  2  9a  9X  H-S, //-8'), 4.29 (dd, V 5 ; 6 a = 4.5 Hz, J a,6b = 12.3 Hz, 1H; //-6a), 4.10 (dd, V 6b =2.1 Hz, 2  6  V a,6b 6  5>  = 12.3 Hz, 1H; //-6b), 3.96 (m, 2H; H-2, H-5), 2.11 (s, 3H; C O C / / ) , 2.05 (s, 3H; 3  COC//3),  2.04 (s, 3H; C O C / / 3 ) , 2.02 (s, 3H; C O C / / 3 ) . C{*H} N M R (MeOD, 100.62 13  MHz): 5 196.72, 196.05 (/ac-Re(CO) ), 172.25 (C7), 172.18, 171.22, 170.72, 169.76 3  (4C=0), 162.28 (CIO), 162.08 (CIO'), 153.22 (C14), 152.99 (C14'), 141.78 (C12), 141.57 (C12'), 126.94 (C13), 124.73 ( C l l ) , 124.62 (CIV), 92.97 (CI), 73.65 (C3/C5), 70.05, 69.96, 69.68, 69.61, (C4/C8/C9/C9'), 62.93 (C6), 54.79 (C2), 20.98, 20.85, 20.69, 20.54 (4CH ). IR (cm , thin film, AgBr plate): 3360 (br) (v(OH)); 2031 (vs), 1920 (vs) -1  3  (v(/~ac-Re(CO) ); 1741 (m) (v(C=0)CH ); 1679 (w) (v(amide I)); 1611 (w) (v(C=C or 3  3  C=N)). +ES-MS m/z (relative intensity) = 857, 855 ([M] , 100). +  4.3 Results and Discussion  4.3.1 Synthesis and Characterization of the Re Complexes with L ' - L  7  The synthesis of the Re complexes proceeded in a straightforward manner from [NEt ] [ReBr (CO) ] and one of L / - L in refluxing methanol to afford neutral [RefL 53  4  2  3  7  1  3  7  1  L )(CO)3Br] in moderate yield after chromatography (Schemes 4.1 and 4.2). H N M R spectral analysis of the crude product was consistent in each case with the formation of the proposed structures as well as the presence of the by-product NEt Br. This salt was 4  198  References start on p. 234  then removed by silica-gel chromatography to afford the complexes as white powders. The compounds were found to be stable in the solid state, but to decompose slowly over a period of months in aqueous solution.  a  Scheme 4.1: Reaction scheme for the preparation of the Re complexes of the 1,3diaminocarbohydrate ligands L ' - L a ) [NEt ]2[ReBr (CO) ], MeOH, 6 h, 44-68 % yield. 6  4  OH  3  3  OH  OH  OH  Scheme 4.2: Reaction scheme for the preparation of the Re complex of the bis-sugar analog L a ) [NEt ] [ReBr (CO) ], MeOH, 6 h, 45 % yield. 7  4  2  3  3  N M R analysis of the Re compounds in DMSO-flk/E^O was indicative of the mode of ligand binding (N-atoms) and illustrated the lowered symmetry of the ligands once bound to the {Re(CO) } core. The largest changes in chemical shift induced by ligand +  3  binding were for the hydrogens within the 6-membered chelate ring formed between the 1,3-propanediamine moiety and the Re metal center. In all cases (except L ) the C H 7  199  References start on p. 234  propyl (H-a) hydrogen signal shifted downfield by -0.6 ppm (Figure 4.5). As a further consequence of metal binding the signals for the CH2-propyl hydrogens (H-b/b' and H c/c') were split into two separate resonances due to the asymmetric environment produced upon chelation to the metal. A certain amount of asymmetry is already present in the ligands due to the many chiral centers of the sugar ring. This asymmetry extends to the CH2-propyl carbon atoms (Cb, Cc) of the binding moiety as two separate C signals 1 3  are visible for these carbon atoms in the ligand and the associated Re complexes (vide 13  infra).  Figure 4.5: ' H N M R spectra of L (upper) and [Re(L )Br(CO) ] (lower) in D M S O d /D 0. 1  1  3  6  2  200  References start on p. 234  Interestingly, the N H protons of the Re complexes were also visible in the *H N M R experiments, assigned unequivocally by ' H - N HSQC (Figure 4.6). The N H I 5  protons of the free ligand are readily exchangeable in protic solvents and thus are not visible in the *H N M R spectra.  (ppm)  'HCppm)  Figure 4.6: ' H - N HSQC spectra of [Re(L )Br(CO) ] (DMSO-d ). 1 5  1  3  6  Evidently this is not the case with the Re complexes; as a result of N-binding to the Re center, the N H exchange process becomes slow compared to the N M R time-scale and these resonances are visible. ' The three or four separate N H resonances 62 63  201  References start on p. 234  (depending on signal overlap) are indicative of the asymmetry in the Re complexes [Re(L -L )(CO) Br]. 1  7  3  The hydrogen and carbon resonances of the sugar moieties in the complexes were unchanged as compared to those in the free ligands (values similar in DMSO-c4, MeOHd4,  and  D2O).  1 3  The lack of coordination induced shifts  64-66  (CIS), due to carbohydrate  ligation to the metal center, confirms the pendant nature of the carbohydrate functions in solution. Interestingly, binding of L , the bis-glucose derivative, to the {Re(CO) } core 7  +  3  resulted in two sets of similar yet distinct ' H and C N M R resonances for the 1 3  carbohydrate moieties. This is in contrast to the free ligand L which displays only one 7  set of resonances for the carbohydrate groups. Evidently metal coordination exerts a 13  far-reaching (yet minor) effect with this derivative. The identity of the Re complexes in solution was probed by a number of methods. Based on previous work, ' it has been concluded that the weakly coordinated Br" 53  67  ligands of [ReBr (CO) ] " (labilized by the strong trans influence of the carbonyl ligand) 2  3  3  undergo facile exchange in aqueous conditions. The extent of this exchange for [RefL 1  L )(CO) Br] was probed directly by conductivity measurements. The Re compounds 3  (1(T M) were dissolved in deionised water and the conductivity was measured and compared to a number of 1:1 electrolyte 10" M solutions (NaCI = 125 fi" cm mol" , 3  1  2  1  [NBti4]Br = 113 fi" cm mol" , [MePPh ]Br = 108 fi" cm mol" ). The conductivity 1  2  1  1  2  1  3  values for the Re compounds correspond to 1:1 electrolytes ' at the concentrations 54 55  measured (Table 4.1). The values offer clear evidence that facile H 2 O for Br~ exchange is occurring in solution with the complexes exclusively existing in the form [Re(L " )(H 0)(CO) ]Br. 1  7  2  3  202  References start on p. 234  Table 4.1: IR vco bands, M S patterns, and conductivity measurements for [RefX 1  L )(CO) Br]. 7  3  Complex  IR (KBr) v(/tfc-Re(CO) ) (cm" ) 3  [Re(L)(CO) Br] 3  M S Peak Assignment (+ES-MS)  Conductivity (10" M,H O) A (Q" cm mol' ) 3  2  1  1  2  m  1  L  1  2023, 1881  625 [M+Na] 523,521 [M-Br] 361,359 [M-Br-C Hio0 ]  98  +  +  6  2033, 1902, 1871  595 [M+Na] 492, 490 [M-Br] 361,359 [ M - B r - C H 0 4 ] +  +  8  625 [M+Na] 523,521 [ M - B r f 361,359 [ M - B r - C H O ]  93  +  6  2023, 1879  108  +  5  2029, 1924, 1863  +  5  10  +  5  625 [M+Na] 523,521 [M-Br] 361,359 [ M - B r - C H i O ]  100  +  +  6  2023, 1908, 1877  0  625 [M+Na] 523,521 [M-Br] 361,359 [ M - B r - C H i O ]  110  +  +  6  2021, 1903, 1881  +  5  0  787 [M+Na] 685, 683 [M-Br] 361, 359 [M-Br  +  5  150  +  +  C12H20O10]  L  7  2023, 1898  831 [M+Na] 729, 727 [M-Br] 567, 565 [M-Br-C Hio0 ] 405, 403 [M-Br-  140  +  +  6  +  5  C12H20O10]  203  References start on p. 234  The IR spectra of the Re compounds were consistent with the proposed structures as bands attributable to the {Re(CO)3} core were present between 2100 and 1800 cm +  -1  (Table 4.I). '" In most cases, three bands were present (indicative of a low symmetry 44  environment) however overlap of the two lower energy bands occurred with the Re complexes of L , L , and L . Bands attributable to the NH2 group were also present 1  4  5  -3300 cm- , and 1580 cm" . 1  1  14  The Re compounds were further examined by mass spectrometry. Re exists as a mixture of  185  R e / R e isotopes (37.4% and 62.6% abundance respectively) affording 187  diagnostic peak isotope patterns. The compounds were run as dilute solutions in MeOH and in all cases displayed molecular ion plus sodium peaks [M + Na] (Table 4.1). This +  shows indirectly that the Re compounds exist (at least partially) in the neutral form in this solvent. The limited interaction of the {Re(CO)3} core with nucleophiles such as MeOH, +  D M S O and D M F has been demonstrated. The addition of NaBr further increased the 67  intensity of the [M + Na] ion peaks. Peaks due to the loss of weakly coordinated Br~ +  ligand [M-Br] were always present and of a greater intensity compared with that of the +  molecular ion. Further fragmentation patterns were also consistent with the proposed structures. Peaks due to the loss of a sugar moiety (fragmentation at C-1), as well as B r , -  for Re compounds of L Z - L were present in each spectrum. These high intensity peaks 6  further corroborate the proposed structures and also indicate the strong binding of the 1,3propanediamine to the metal center. Fragments corresponding to the successive loss of two glucose moieties were present for the bis-glucose derivative [Re(L )(Br)(CO)3]. 7  Two ofthe Re compounds, [Re(L )(CO) Br], and [Re(L )(CO) Br], were 2  3  3  3  crystallized from MeOH/H20 solutions of the respective compounds and analyzed by X -  204  References start on p. 234  ray crystallography (see Appendix for relevant data). The data represent the first reported crystal structures of Re organometallic carbohydrate compounds. A manganese dicarbonyl(carbohydratocarbene) complex is the only other Group VII organometallic 68  carbohydrate X-ray structure that has been described. Structures of oxorhenium(V) complexes with carbohydrate moieties directly bound to the metal center have also been reported, using hydridotris(pyrazolyl)borato as the ancillary tridentate ligand. Very few 65  carbohydrate-containing transition metal complexes in which the carbohydrate moiety is not only unprotected, but also unbound to the metal center have been crystallized. ' - ' ' 14 16  21 23 69  A n ORTEP diagram of [Re(L )(CO)3Br] is displayed in Figure 4.7, and selected 2  bond lengths and angles are presented in Table 4.2. A salient feature of this structure is the pendant carbohydrate (xylose) group in accordance with the solution characterization data. In order to take advantage of hexose transport and metabolic pathways in vivo it is imperative that the effects of the tracer group on the properties of the carbohydrate molecule be minimized. Carbohydrate binding to the metal center is most likely incompatible with this hypothesis. The Re coordination sphere is approximately octahedral occupied by three facially arranged carbonyls, with the two amino groups from the l,3-diamino-2-propyl linker group and the bromine occupying the remaining three positions. A l l bond distances and angles involving Re and its ligating donor atoms are within typical values. ' ' 43  44  70-73  The amine functions form a 6-membered chelate ring  with the Re center in a chair conformation. Interestingly, the bulky xylose moiety is present in an axial position which is generally a higher energy conformation. This geometry is evidently stabilized by the presence of hydrogen bonding interactions  205  References start on p. 234  between the N ( l ) - H ( l b ) Crystal structures of P d  69  0(2), N(2)-H(2a)  0(1), and N(2)-H(2a) -0(2) atoms.  and Pt with 1,3-diaminocarbohydrate ligands also exhibit the 16  carbohydrate moiety in the same axial orientation. The presence of only one diastereomer is likely due to weak hydrogen bonding interactions between N(l)-H(lb)  Br(l) and  N(2)-H(2a)'Br(l).  Figure 4.7: ORTEP diagram of [Re(L )Br(CO) ] showing 50 % probability ellipsoids. 2  3  206  References start on p. 234  Table 4.2: Selected bond lengths (A) and angles (deg) in [Re(L )(CO) Br]. 2  3  Re(l)-Br(l) Re(l)-N(l) Re(l)-N(2) Re(l)-C(9) Re(l)-C(10) Re(l)-C(ll) N(l)-C(7) N(2)-C(8) C(6)-C(8) C(6)-C(7) C(6)-0(2) C(l)-0(2)  2.6382(6) 2.236(4) 2.230(4) 1.902(6) 1.917(5) 1.890(6) 1.489(7) 1.482(7) 1.536(7) 1.499(7) 1.433(5) 1.397(6)  N(l)-Re(l)-N(2) N(l)-Re(l)-Br(l) N(2)-Re(l)-Br(l) C(ll)-Re(l)-N(l) C(10)-Re(l)-Br(l) C(9)-Re(l)-C(10) C(9)-Re(l)-C(ll) C(10)-Re(l)-C(ll)  83.0(2) 85.5(1) 84.4(1) 95.2(2) 91.2(2) 86.5(2) 88.5(3) 91.9(2)  H-bonding N(l)-H(lb) N(2)-H(2a) N(2)-H(2a) N(l)-H(lb) N(2)-H(2a)  2.694(8) 3.139(9) 2.81(1) 3.323(9) 3.28(1)  0(2) 0(1) •0(2) Br(l) Br(l)  The bite angle between the coordinating amines and the Re center (N(l)-Re(l)N(2) 83.00(16)) is less than 90° and is governed by the Re-N bond lengths in conjunction with the geometrical constraints of the l,3-diamino-2-propyl binding unit. The distance between the coordinating N-atoms is fixed due to the rigid trimethylene bridge and thus the bite angle solely depends on the Re-N bond length. Further deviation from ideal geometry is evident by the angles (N(l)-Re(l)-Br(l) 85.54(11), N(2)-Re(l)-Br(l) 84.38(11)) formed between the bromine ligand, Re, and the chelating amines. Hydrogen bonding between the bromine ligand and hydrogen atoms attached to the chelating amines may account for the observed bond angles. Finally, the xylose moiety exists exclusively as the P-anomer in the solid state structure, correlating with solution N M R data.  207  References start on p. 234  A n ORTEP diagram of [Re(L )(CO) Br] is displayed in Figure 4.8, and selected bond 3  3  lengths and angles are presented in Table 4.3. The crystal structure of [Re(L )(CO) Br], 3  3  while similar to that of the xylose derivative, exhibited notable differences. The pendant nature of the carbohydrate (mannose) moiety in the solid state was in agreement with the solution characterization data. The asymmetric unit was determined to contain one water molecule and two symmetry-independent [Re(L )(CO) Br] molecules where the metal 3  3  ions exhibit similar coordination environments (Figure 4.9). The Re coordination sphere in [Re(L )(CO) Br] matched the [Re(L )(CO) Br] derivative as previously described. The 3  2  3  3  pendant mannose moiety occupies an axial position of the 6-membered chelate ring formed between the amine functions and Re. Hydrogen bonding arguments can again be invoked to explain the observed axial orientation of the mannose substituent attached to the chelate ring as well as the presence of one diastereomer (Figure 4.8, Table 4.3).  Figure 4.8: ORTEP diagram of [Re(L )Br(CO) ] showing 50 % probability ellipsoids. 3  3  208  References start on p. 234  Table 4.3: Selected bond lengths (A) and angles (deg) in [Re(L )(CO) Br]. 3  3  Re(2)-Br(2) Re(2)-N(3) Re(2)-N(4V Re(2)-C(22) Re(2)-C(23) Re(2)-C(24) N(3)-C(20) N(4)-C(21) C(19)-C(20) C(19)-C(21) C(19)-0(ll) C(13)-0(ll)  2.625(1) 2.247(7) 2.230(7) 1.931(9) 1.92(1) 1.911(9) 1.497(9) 1.49(1) 1.53(1) 1.52(1) 1.444(8) 1.408(8)  N(3)-Re(2)-N(4) N(3)-Re(2)-Br(2) N(4)-Re(2)-Br(2) C(22)-Re(2)-Br(2) C(23)-Re(2)-N(3) C(22)-Re(2)-C(23) C(22)-Re(2)-C(24) C(23)-Re(2)-C(24)  83.9(3) 82.7(2) 84.1(2) 93.5(3) 95.1(7) 89.8(4) 89.3(4) 91.3(4)  H-bonding N(3)-H(3d)' N(4)-H(4c)N(3)-H(3d) N(4)-H(4c)-  2.771(9) 2.874(9) 3.23(1) 3.27(1)  0(11) 0(11) Br(2) Br(2)  The difference between the two molecules of the asymmetric unit lies in the orientation of the sugar moiety with respect to the Re center. While the sugar moiety occupies an axial position of the 6-membered chelate ring in each molecule, rotation around the C(l)-0(2)/C(13)-0(l 1) bond (in the two separate molecules), attaching the sugar ring to the propane moiety, results in the two symmetry independent molecules. One of the molecules in the asymmetric unit closely resembles [Re(L )(CO)3Br] with the 2  sugar ring directed away from Re, while in the other case the sugar moiety is bent towards the Re center (Figure 4.9, right-hand side). The mannose unit in the latter molecule was disordered and refined in two orientations. The mannose moiety exists exclusively as the a-anomer in the solid state structure, correlating nicely with the solution N M R data.  209  References start on p. 234  Figure 4.9: ORTEP diagram of the two discrete molecules (about the one water) of [Re(L )Br(CO)3] in the unit cell showing 50 % probability ellipsoids. 3  4.3.2 Synthesis and Characterization of the  The  9 9 m  9 9 m  T c Complexes with L - L !  7  T c labelled carbohydrate compounds were synthesized utilizing a  previously established method. ' The formation ofthe [ 43 71  99m  T c ( H 0 ) ( C O ) ] precursor +  2  3  3  was verified by H P L C (RT =13.9 min, Table 4.4) before labeling with the carbohydrate ligands L ' - L (Schemes 4.3 and 4.4). The labelled compounds [ Tc(L " )(H 0)(CO) ] 7  99m  1  7  2  +  3  were then characterized by their associated radioactive H P L C traces and compared, via co-injection, with the corresponding Re complexes (monitored at 254 nm). In all cases the retention times (RT) of the Re and  9 9 m  T c complexes were identical within  experimental error (Table 4.4).  210  References start on p. 234  Scheme 4.3: Reaction scheme for the synthesis of the  Tc complexes of the 1,3-  diaminocarbohydrate ligands L -L a)  3  l  6  [ Tc(H 0)3(CO) ] , 70° C, 40 min, PBS buffer. 99m  +  2  Scheme 4.4: Reaction scheme for the synthesis of the  m  T c complex of the bis-sugar  analog L a) [ Tc(H 0)3(CO) ] , 70° C, 40 min, PBS buffer. 7  99m  +  2  3  211  References start on p. 234  Table 4.4:  H P L C retention times (RT), and labeling yields (%) for the [ M f L 1  L ) ( H 0 ) ( C O ) ] ( M = Re, 7  +  2  3  99m  T c ) complexes. Synergi 4 um C-12 Max-RP analytical  column (solvent: 0.1 % w/w C F C O O H in H 0 to 100 % C H C N over 30 min). 3  Complex [M(L)(H 0)(C0) ] 2  +  3  2  3  RT(min) M = Re (254 nm)  RT (min) M = Tc (radiometric) 9 9 m  Labeling Yield (%) ± SD (n =3)  L  1  10.7  10.6  99 ±  L  2  11.2  11.3  99 ±  L  3  10.4  10.5  97 ±  L  4  10.7  10.8  99 ±  L  5  10.9  11.2  99 ±  L  6  9.7  9.7  97 ±  L  7  9.1  9.1  99 ±  [M(His)(H 0)(CO) ]  -  13.2  99 ±  [M(H 0) (CO) ]  -  13.9  2  2  3  3  3  +  These results confirm that the  m  -  T c complexes produced on the tracer level are  identical to the Re complexes produced and characterized (vide supra) on the macroscopic scale. The labeling yields for the seven compounds were essentially quantitative under the conditions studied and the yields shown in Table 4.4 are the average of at least three separate experiments. As expected, the more polar complexes of 6  7  L (maltose) and L (bis-glucose) exhibited the shortest retention times, while the least  212  References start on p. 234  polar complex of L (xylose) exhibited the longest retention time on the C-18 reversephase column used for separation. The in vitro stability of the  9 9 m  T c complexes was assessed by incubation with  solutions of either cysteine or histidine. The susceptibility of the complexes to ligand 43  exchange by these amino acids was assessed over a 24 hr period (Table 4.5). While both cysteine and histidine are potentially tridentate mono-anionic ligands, it has been previously determined that histidine displays a much higher affinity for the {M(CO)3} 74  +  core due to the ideal binding geometry and better-suited binding atoms (N,N,0 vs. N,0,S). On the basis of our work with the Re complexes of L - L it is clear that these 1  7  ligands chelate in a bidentate fashion to the {M(CO)3} core via the primary amine +  functions. While it has been previously shown that primary amines are a good match for the {M(CO)3} core, ' the ligands studied herein leave a coordination site open for +  44 45  potential attack by adventitious ligands. Complexes of bidentate ligands with {  99m  Tc(CO) } , while exhibiting moderate stability towards ligand exchange, have +  3  previously been found to exhibit poor clearance due to plasma protein binding, most likely through the free coordination site on the metal center  213  4 3  References start on p. 234  Table 4.5: [ Tc(L -L )(H 0)(CO) ] complex stability (%) at 1, 4, and 24 h towards 1  99m  7  +  2  3  ligand exchange in solutions of either 1 m M cysteine or 1 m M histidine in PBS.  Complex [  99m  Cysteine ± SD (n=3)  Tc(L)(H 0)(CO) l  1h  4h  24 h  1h  4h  24 h  1  99 + 1  99 ± 1  98 ± 1  98 ± 1  97 ± 1  68 ± 5  L  2  99+1  99 ± 1  97+1  98 ±.1  98 + 1  70 ± 2  L  3  97+1  95 ± 2  90+1  97 ± 1  96 ± 1  66 + 2  L  4  99 + 1  99 ± 1  98 + 1  98 ± 1  98 + 1  69 ± 4  L  5  99 ± 1  99 + 1  98 ± 1  99 ± 1  98 ± 1  61 ± 2  L  6  99 ± 1  99 + 1  98 ± 1  99 ± 1  98 + 1  73 ± 3  L  7  99 ± 1  99 ± 1  98 ± 1  99 ± 1  98 ± 1  86 ± 4  +  3  2  L  Histidine ± SD (n=3)  Incubation of [ Tc(L " )(H 0)(CO) ] with solutions of either cysteine or 99m  1  7  +  2  3  histidine showed that the complexes were moderately stable over the test period. Cysteine was found to have a very minor effect on complex stability over the 24 h period as the complexes were > 90 % intact at 24 h. Histidine only exhibited a measurable effect at the 24 h time point as the appearance of a second peak in the H P L C trace ([ Tc(His)(CO) ], confirmed by the preparation of an authentic sample) established 99m  43  3  that ligand exchange was occurring (Figure 4.10).  214  References start on p. 234  [  1 hr  4hr  99m  Tc(L')(H 0)(CO) ] 2  [  99m  [  99m  +  3  Tc(L )(H 0)(CO) ] 1  2  3  IS  24 hr  Tc(L )(H 0)(CO) ] 2  [  10  1  99m  +  3  Tc(His)(CO) ]  15  3  20  25  30  Time (min)  Figure 4.10: H P L C radiation traces for the histidine challenge experiment with [  99m  Tc(L )(H 0)(CO) ] . Synergi 4 um C-12 Max-RP analytical column (solvent: 0.1 % 1  +  2  3  w/w C F 3 C O O H in H 0 to 100 % C H C N over 30 min). 2  3  215  References start on p. 234  The  9 9 m  T c complexes of L - L were determined to be from 61 % to 86 % intact 1  7  at 24 h with the stability of the complexes roughly paralleling the steric size of the carbohydrate ligands; the disaccharide L and the bis-glucose analog L were the most 6  7  stable towards histidine ligand substitution. The increased steric bulk of these ligands likely reduces the exchange of the coordinated water molecule for cysteine or histidine, thus inhibiting the ligand exchange process.  4.3.3 Synthesis and Characterization of the Re Complexes with L - L 8  Ligands L -L  12  were gifts from Prof. Yuji Mikata and Prof. Shigenobu Yano,  Nara Women's University, Japan. The synthesis of L has been reported. L 8  51  1 1  and L  1 2  were synthesized according to Scheme 4.5. The amide coupling of (bis(2pyridylmethyl)amino)acetic acid 1 with 1,3,4,6-tetra-O-acetyl-P-D-glucosamine-HCl 44  afforded L  1 2  in 60% yield. The original intent was to remove the acetyl groups of L  1 2  52  to  afford the glucosamine derivative L . However, acetyl group removal using catalytic n  NaOMe / MeOH, or NH3 / MeOH, led to a significant amount of uncharacterized byproducts. The direct coupling of (bis(2-pyridylmethyl)amino)acetic acid 1 with glucosamine-HCl was successful in affording L  1 1  in 87 % yield as a mixture of anomers  (70 % a: 30 %> P). Dissolving the glucosamine-HCl in a small amount of water before addition to the reaction mixture was found to be necessary for the coupling to take place.  216  References start on p. 234  OAc  HOOC  Scheme 4.5: Reaction scheme for the preparation of L  1 1  and L  1 2  a) 1,3-  dicyclohexylcarbodiimide, N-hydroxysuccinimide, dimethylaminopyridine, 1,3,4,6-tetraO-acetyl-P-D-glucosamine-HCl, D M F , 60%. b) NaOMe, MeOH. c) 1,3dicyclohexylcarbodiimide, N-hydroxysuccinimide, dimethylaminopyridine, glucosamine-HCl, D M F / H 0 , 87%. 2  The synthesis of the Re complexes proceeded in a straightforward manner from [NEt4]2[ReBr3(CO)3] and each of L - L in refluxing methanol to afford the compounds 8  12  1  [Re(L " )(CO) ]Br in moderate yield after chromatography (Schemes 4.6 and 4.7). H 3  N M R spectral analysis of the crude product was consistent in each case with the quantitative formation of the proposed structures as well as the presence of the by-  217  References start on p. 234  product NEt4Br. This salt was then removed by column chromatography to afford the complexes as hygroscopic white powders stable in the solid state and in aqueous solution. Unfortunately the elemental analysis of [Re(L )(CO)3]Br was not acceptable, most 12  probably due to the contamination of the bulk material by NEt4Br.  Scheme 4.6: Reaction scheme for the preparation of the Re complexes of the carbohydrate-appended D P A ligands L - L : a) [NEt ]2[ReBr (CO) ], MeOH, 6 h, 46-75 8  1 0  3  4  3  % yield.  Scheme 4.7: Reaction scherrie for the preparation of the Re complexes of the carbohydrate-appended D P A ligands L - L : a) [NEt ] [ReBr (CO) ], MeOH, 6 h, 58 % H  1 2  4  2  3  3  yield for L . 1 1  218  References start on p. 234  N M R analysis of the Re compounds [Re(L " )(CO) ]Br in M e O H - ^ was 8  10  3  indicative of the mode of ligand binding (N-atoms) and illustrated the lower symmetry of the ligands once bound to the {Re(CO)3} core (Figure 4.11). Resonances were assigned +  on the basis of ID ( W C) l  n  as well as by 2D ( ' H - ' H C O S Y , and H - C H M Q C ) N M R !  1 3  experiments. Due to the electronic influence of the Re(I) center, a significant downfield shift of the hydrogen resonances in close proximity to the ligating N-atoms was observed. The pyridine hydrogens H-14/H-14' (Figure 4.11) move downfield by approximately 0.5 ppm, while the hydrogens of the ethylene linker (H-8a/8b), adjacent to the tertiary amine, shift downfield by 1.5 ppm upon ligand binding. As well, the hydrogen signals of the methylene groups (H-9a/H-9b and H-9'a/H-9'b) adjacent to the pyridine rings shift downfield by 1.1 ppm and become diastereotopic exhibiting geminal coupling (~17 Hz). The hydrogen and carbon resonances of the sugar moieties were either unchanged or exhibited very minor shifts compared to those in the free ligands. Coordination induced shifts " (CIS) would suggest carbohydrate ligation to the metal center. The lack of these 64  66  shifts confirms the pendant nature of the carbohydrate functions in solution. The carbohydrate moieties do however exert a long range asymmetric effect on the compounds described herein. This asymmetric effect is present in the H N M R spectrum l  of the ligands as hydrogens H-7a/H-7b are diastereotopic (Figure 4.11). However, the effect of the carbohydrate is extended upon binding to the {Re(CO) } core, most likely +  3  due to the conformational restriction of the ligand upon chelation. The two methylene carbons (9/9') adjacent to the pyridines, exhibit separate C signals in all three Re , 3  complexes.  219  References start on p. 234  no^iM^° 8 3  2  O H  l  Y  / = \'  n  f  N-< . 4  11 f 12  N  \l4 13  H-9b/H-9'b H-14/H-14'  H-12/H-12'  H-13/H-13'  H-9a/H-9'a  H-l  H-ll/H-11'  H-8 H-7a  to O  H-7b  u H-9  H-ii/H-ir H-14/H-14'  >3  H-12/H-12  H-l  H-13/H-13'  1  Iuu H-7a  3  L  JL 8.8  8.4  —i 8.0  '  1  '  1 7.6  1  '  1 7.2  -/I • — i — 5.2  4.8  4.4  I H-7b  4.0  s is.)  Figure 4.11: *H N M R spectra ( M e O H - ^ , 400 MHz) of A) L B) [Re(L )(CO) ]Br. 8  8  3  3.6  As well, a complicated pattern arises for the diastereotopic methylene hydrogen signals (H-9a/H-9b and H-9'a/H-9'b). The observed pattern can be explained by the fact that the four hydrogens are non-identical. The effect is more pronounced (due to larger chemical shift differences) for the xylose and mannose derivatives in the ' H N M R spectrum (Figure 4.12). Long range ^ - ' H COSY measurements highlight weak 4-bond coupling (~l-2 Hz) between H - l 1/H-l 1' and the hydrogens of the methylene groups (H9a/H-9b and H-9'a/H-9'b), leading to further broadening of the latter hydrogen signals. The pyridine rings also display inequivalency by N M R . The pattern displayed by H 11/H-l 1' changes substantially upon metal binding (Figure 4.11) with two distinct doublets evident. Two sets of signals are apparent for the pyridine carbons in the  C  N M R spectra of the Re complexes. It is intriguing that the pendant carbohydrate moiety exerts an effect as distant as the pyridine rings. This is in contrast to the complex of (bis(2-pyridylmethyl)amino)acetic acid with the {Re(CO)3} core whereby a single set of +  resonances (indicative of C symmetry) was found in both the *H and C N M R spectra. 1 3  44  s  11  Interestingly, the  C N M R data of the complexes reveal that there are only two  Re-CO peaks in a 2:1 peak height ratio. This result can be explained by a) simple overlap, or b) that two of the C O groups are magnetically equivalent because of the mirror symmetry along the axis formed by the Re with one C O group. The latter argument is mostly obviated by the N M R results already discussed.  221  References start on p. 234  I  5.5  I 5.3  I  5.4  I 5.2  I 5.1  I 5.0  I • 4.9  Figure 4.12: U N M R spectra (MeOH-d ) of [Re(L " )(CO) ]Br in the region 4.8-5.6 l  8  12  4  3  ppm showing the pyridyl methylene hydrogen signals.  222  References start on p.  The solution structures of the glucosamine derivatives [Re(L )(CO)3]Br were n_,2  also analyzed by N M R spectroscopy. The acetylated ligand L  1 2  and associated complex  [Re(L )(CO)3]Br exhibited easily interpretable N M R patterns due to the presence of a 12  single anomer (P). The N M R spectra of the unprotected glucosamine derivatives were however more complicated due to the presence of the two anomers in addition to the asymmetry attributable to the chiral centers of the glucosamine moiety. The relative proportions of the two anomers (70% a: 30% P) was found to be identical for L [Re(L )(CO) ]Br, signifying that complexation of L n  3  1 1  1 1  and  to the {Re(CO) } core has little +  3  effect on the relative thermodynamic stabilities of the two anomers. This result is in contrast to similar work with the glucosamine ligand N-(2'-hydroxybenzyl)-2-amino-2deoxy-D-glucose (Figure 4.1), whereby a change in the anomeric ratio occurred upon ligand binding to the {Re(CO) } core. In this case a direct interaction of the +  11  3  carbohydrate moiety with the Re center is most likely responsible for the observed change in anomeric ratio upon complexation. Similar to the complexes [Re(L - )(CO) ]Br, the pyridine hydrogens (H-14/H-14') of [Re(L 8  10  3  1M2  )(CO) ]Br move 3  downfield by approximately 0.4 ppm, while the hydrogens of the methylene link (Ff8a/8b), adjacent to the tertiary amine, shift downfield by -1.4 ppm upon ligand binding. As well, the hydrogen signals of the methylene groups (H-9a/H-9b and H-9'a/H-9'b) adjacent to the pyridine rings shift downfield (-1.1 ppm) and become non-identical due to a combination of the conformational restriction upon chelation and differences in environment exerted by the glucosamine function. The presence of the two anomers (a/p) for [Re(L )(CO)3]Br leads to a more complicated pattern (Figure 4.12). The hydrogen n  and carbon resonances of the glucosamine moieties in [Re(L )(CO)3]Br were either 1M2  223  References start on p. 234  unchanged or exhibited very minor shifts compared to those in the respective free ligands. Similar to the discussion for [Re(L " )(CO) ]Br, the lack of coordination8  10  3  induced shifts (CIS) of the glucosamine functions suggests that the carbohydrate 64  moieties in [Re(L " )(CO)3]Br remain pendant in solution. n  12  Two complete sets of signals are evident in the C N M R spectrum of L 1 3  1 1  due to  the presence of the two anomers. The C N M R spectrum becomes more complicated 1 3  upon ligand binding as the asymmetric effect of the glucosamine moiety is extended due to the increased rigidity upon chelation to the {Re(CO) } core. In addition to the effect +  3  of the two anomers, the D P A function exhibits further asymmetry so that up to four 13  separate  C N M R signals are present for each pyridine carbon atom. Three sharp Re-CO  resonances are present in the C N M R spectrum indicating the low symmetry of the 1 3  complex. The identities of the pure compounds were probed in solution via conductivity measurements. The Re compounds with L - L 8  n  (1 mM) were dissolved in deionised water  and the conductivity was measured and compared to a number of 1:1 electrolyte solutions at IO" M (NaCI = 125 Q" cm mol" , [NBu4]Br = 113 Q" cm mol" , [MePPh ]Br = 108 3  1  2  1  1  2  1  3  1 2  1  Q" cm mol"). As expected, the conductivity values for the Re compounds correspond to 1:1 electrolytes ' at the concentrations measured (Table 4.6). The values are higher 54 55  than expected (yet still within the range of 1:1 electrolytes) which could be due to NEt4Br by-product which was not removed during column purification; however, no NEt4Br could be detected in the N M R spectra.  224  References start on p. 234  Table 4.6: IR vco bands, M S patterns, and conductivity measurements for [Re(L L )(CO) ]Br. 12  3  Complex  IR (AgBr plate) v(/ac-Re(CO) ) (cm" )  M S Peak Assignment (+ES-MS)  3  [Re(L)(CO) ]Br  1  3  Conductivity (10" M , H 0 ) A (Q" cm mol" ) 3  2  1  2  m  1  L  8  2027, 1931  676, 674 [ M ]  +  165  9  2029, 1920  646, 644 [ M ]  +  141  1 0  2027, 1932  676, 674 [ M ]  +  160  1 1  2030, 1922  689, 687 [ M ]  +  145  1 2  2031, 1920  857, 855 [ M ]  +  L L L L  -  The IR spectra of the Re compounds [Re(L " )(CO) ]Br were consistent with the 8  12  3  proposed structures; bands attributable to the {Re(CO) } core were present between +  3  2100 and 1800 cm" (Table 4.6). ' In each ofthe five metal complexes two CO bands 1  44 53  were present. The broader, lower band at -1930 cm" is most probably due to the overlap 1  of two CO absorptions based on symmetry considerations. The Re compounds were further examined by mass spectrometry. The compounds were run as dilute solutions in MeOH and displayed the molecular ion [M] peak in 100% abundance for the five +  complexes (Table 4.6). In all cases the theoretical isotope patterns matched the experimental spectra. The Re compound [Re(L )(CO) ]ClEt OMeOH, was crystallized as the chloride 8  3  2  salt and analyzed by X-ray crystallography. A n ORTEP diagram of the cation  225  References start on p. 234  [Re(L )(C0)3] is shown in Figure 4.13 with the relevant bond lengths and angles presented in Table 4.7.  C12  Figure 4.13: ORTEP diagram of the cation [Re(L )(CO) ] in its CI salt showing 50 % 8  +  3  probability ellipsoids.  The structure consists of discrete CI" anions, [Re(L )(CO)3] cations, and solvent 8  +  (Et20/MeOH) in the lattice. A salient feature of this structure is the pendant carbohydrate (glucose) in accordance with the solution characterization data. The distorted octahedral environment of Re(I) is occupied by three facially arranged carbonyls and the amine and pyridyl nitrogen donors of the ligand. The facial arrangement of the ligand, imposed by the {Re(CO)3} core, is in contrast to the meridional binding seen in a Cu(II) complex of +  226  References start on p. 234  the same ligand, highlighting the versatility of the DP A function for metal ion 51  chelation.  Table 4.7: Selected bond lengths (A) and angles (deg) in [Re(L )(CO) ]Cl. 8  3  Re(l)-N(l) Re(l)-N(2) Re(l)-N(3) Re(l)-C(21) Re(l)-C(22) Re(l)-C(23) C(22)-0(8)  2.227(3) 2.167(3) 2.177(3) 1.930(4) 1.926(4) 1.918(3) 1.154(8)  N(l)-Re(l)-N(2) N(l)-Re(l)-N(3) N(2)-Re(l)-N(3) N(l)-Re(l)-C(21) N(l)-Re(l)-C(22) N(l)-Re(l)-C(23) C(21)-Re(l)-C(22) C(21)-Re(l)-C(23)  78.4(1) 77.7(1) 80.6(1) 94.3(1) 97.7(1) 173.2(1) 90.1(1) 89.7(1)  The Re-CO distances (1.918-1.930A) are analogous to those found in similar complexes. ' The Re(l)-N(l) distance (2.227(3)A) is slightly longer than the Re44 72  pyridinefN) distances (Re(l)-N(2) 2.167(3); Re(l)-N(3) 2.177(3)) consistent with the hybridization of the N-donors (sp vs. sp ). This trend is further substantiated by the Re-N 3  2  distances in [Re(L )(CO) Br] and [Re(L )(CO) Br] discussed in Section 4.3.2. The most 2  3  3  3  significant distortion in the structure are the angles N(l)-Re(l)-N(2) = 78.4(1)° and N(l)-Re(l)-N(3) = 77.7(1)° due to the formation of strained 5-membered chelate rings. Finally, the glucose moiety exists exclusively as the p-anomer in the solid state structure, correlating well with solution N M R data.  227  References start on p. 234  4.3.4 Synthesis and Characterization of the  The  99m  9 9 m  T c Complexes with L - L 8  Tc-labelled carbohydrate compounds [  99m  1 2  Tc(L - )(CO) ] were 8  12  +  3  synthesized utilizing a previously established method. ' The formation of the 43 71  [ Tc(H 0)3(CO) ] precursor was verified by H P L C (RT =13.0 min, Table 4.8) before 99m  +  2  3  labeling with the carbohydrate ligands L - L 8  1 2  (Schemes 4.8 and 4.9).  Scheme 4.8: Reaction scheme for the synthesis of the carbohydrate-appended DPA ligands L - L 8  1 0  a) [  99m  Tc complexes of the  T c ( H 0 ) ( C O ) ] , 70° C, 40 min, +  2  3  3  PBS  buffer.  Scheme 4.9: Reaction scheme for the synthesis of the  Tc complexes of the  carbohydrate-appended DPA ligands L - L  2  u  1 2  a) [  99m  T c ( H 0 ) ( C O ) ] , 70° C, 40 min, +  3  3  PBS buffer.  228  References start on p. 234  The labelled derivatives were then characterized by their associated radioactive HPLC traces and compared, via co-injection, with the corresponding Re complexes (monitored at 254 nm). In all cases the retention times of the Re and  9 9 m  T c complexes  were identical within experimental error (Table 4.8 and Figure 4.14).  Table 4.8: H P L C retention times (RT), and labeling yields (%) for the [M(L -L )(CO) ] 8  12  ( M = Re,  +  3  99m  T c ) complexes. Synergi 4 (am C-18 Hydro-RP analytical  column (solvent: 0.1 % w/w C F C O O H in H 0 to 100 % C H C N over 30 min). 3  Complex [M(L)(CO) ]  +  3  3  RT (min) M = Re (254 nm)  RT (min) M= Tc (radiometric)  Labeling Yield (%) ± SD (n =3)  W  L  8  13.8  13.7  99+1  L  9  14.5  14.6  99 ± 1  L  1 0  13.7  13.9  99 ± 1  L  1 1  13.7  13.9  99 ± 1  L  1 2  16.2  16.0  -  -  12.2  -  -  13.0  -  [M(His)(CO) ] 3  [M(H 0) (CO) ] 2  2  3  3  +  These results confirm that the complexes produced on the tracer level are identical to the Re complexes produced and characterized (Section 4.3.3) on the macroscopic scale. The labeling yields for the four compounds [  229  99m  Tc(L  8_11  )(CO) ] were essentially +  3  References start onp.234  quantitative under the conditions studied and the yields shown in Table 4.8 are the average of at least three separate experiments. While [  99m  T c ( L ) ( C O ) 3 ] was of interest 12  +  from a synthetic perspective, it was not deemed to have as significant in vivo potential (due to the acetyl protecting groups) as do the other carbohydrate analogs.  a) [Re(L )(CO) ] 10  (13.7 min)  +  3  l b) [  99m  T c ( L ) ( C O ) f (13.9 min) 10  3  Time (min)  Figure 4.14: H P L C comparison of a) [Re(L )(CO) ] (UV/254 nm) and b) 10  +  3  [ Tc(L )(CO) ] (radiometric). Synergi 4 pm C-18 Hydro-RP analytical column 99m  10  +  3  (solvent: 0.1 % w/w C F C O O H in H 0 to 100 % C H C N over 30 min). 3  2  3  230  References start on p. 234  It is important to note that two different columns were used for the  Tc HPLC  studies; a Synergi 4 pm C-12 Max-RP analytical column with dimensions of 250 x 4.6 mm was used for the bidentate ligands L ' - L , while a Synergi 4 pm C-18 Hydro-RP 7  analytical column with dimensions of 250 x 4.6 mm was used for the tridentate ligands L -L 8  1 2  .  The retention time ofthe starting material [ Tc(H 0)3(CO) ] (RT =13.9 min, 99m  +  2  3  Table 4.4, Max-RP: RT = 13.0 min, Table 4.8, Hydro-RP), as well as that ofthe histidine complex [ Tc(His)(CO) ] (RT = 13.2 min, Table 4.4, Max-RP: RT = 12.2 min, Table 99m  3  4.8, Hydro-RP), differed depending on the column used. The in vitro stability ofthe  9 9 m  T c complexes [  99m  Tc(L  8_11  ) ( C O ) ] w a s assessed by +  3  incubation with solutions of either cysteine or histidine. The susceptibility of the 43  complexes to ligand exchange by these amino acids was assessed over a 24 h period. One recent study using the D P A framework for ligating to the {  99m  Tc(CO) } core 3  demonstrated the exceptional stability of the resulting complex towards ligand exchange in solutions of histidine or human serum. The tridentate DPA ligands were thus 52  expected to form stable complexes resistant to ligand exchange processes and this was indeed the case; no decomposition occurred upon incubation of [  99m  T c ( L ) ( C O ) ] with 8  +  3  an excess (10-fold) of either cysteine or histidine over a 24 h period. This is in contrast to the results in Section 4.3.2 where the  9 9 m  T c complexes of L - L exhibited decomposition !  7  by the 24 h time point in the presence of excess histidine. This decomposition is most 21  likely due to the substitution of the coordinated water molecule by histidine leading to subsequent displacement of the carbohydrate ligands. In order to better examine the stability of the  9 9 m  T c complexes of L - L , the stability studies were run with a 100-fold 8  N  excess of either cysteine or histidine. Even at higher amino acid concentrations the  231  9 9 m  Tc  References start on p. 234  complexes were > 94 % intact at the 24 h timepoint (Table 4.9). Clearly matching the tridentate binding capability of these ligands to the fac- [M(CO) ] core greatly stabilizes +  3  the resulting complexes to ligand substitution processes in vitro, and hopefully in vivo as well.  Table 4.9: [  99m  T c ( L - L ) ( C O ) ] c o m p l e x stability (%) at 1, 4, and 24 h towards ligand 8  n  +  3  exchange in solutions of either 1 m M cysteine or 1 m M histidine in PBS (100-fold excess).  Complex  [  99m  [  99m  Cysteine ± SD (n=3)  99m  [  99m  4h  24 h  1h  4h  24 h  Tc(L )(CO) ]  +  99 ± 1  99+1  98 + 1  99 ± 1  98 ± 1  95 ± 5  Tc(L )(CO) ]  +  99 ± 1  98 + 1  98 ± 1  99 ± 1  99 ± 1  95 ± 3  8  3  [  1h  Histidine ± S D ( n=3)  9  3  Tc(L )(CO) ]  +  99 ± 1  98 ± 1  97 ± 1  98 + 1  98 ± 1  95 ± 2  Tc(L )(CO) ]  +  99 ± 1  98 + 1  96 ± 1  98 + 1  97 ± 1  94 ± 2  10  3  n  3  4.4 Concluding Remarks  This chapter has described the synthesis, and resultant solution and solid state properties of a series of novel carbohydrate-appended metal complexes employing the {M(CO) } (M = +  3  9 9 m  T c / R e ) core. The pendant nature of the carbohydrate groups was  confirmed for the Re compounds in solution by N M R spectroscopies as well as in the solid state by X-ray crystallography. Three analogs [Re(L )Br(CO) ], [Re(L )Br(CO) ], 2  3  3  232  3  References start on p. 234  and [Re(L )(CO)3]Cl were analyzed by X-ray crystallography and represent the first 8  reported structures of Re organometallic carbohydrate compounds. The design of a drug molecule exhibiting a pendant carbohydrate offers considerable advantages as this moiety is freely available to interact with carbohydrate transport and metabolic pathways in the body. The potential effect of the tracer group on the biological properties of the radiopharmaceutical is most likely minimized by excluding the carbohydrate function from binding to the metal center. Conductivity measurements for the bidentate analogs showed that ligand exchange of the weakly coordinated Br" ligand for H 0 in aqueous 2  media was a facile process to afford complexes of the general formula [Re(L )(H 0)(CO) ]Br. The tridentate analogs [ R e ( L ' ) ( C O ) ] B r were also confirmed 1_7  8  2  n  3  3  to be 1:1 electrolytes in solution, as expected, on the basis of conductivity measurements. Radiolabeling of the 1,3-diaminocarbohydrates ( L ' - L ) , as well as the carbohydrate7  appended 2,2'-dipicolylamine ligands ( L - L ), using the labeling precursor [ Tc(H 0)3(CO)3] was essentially quantitative and afforded compounds identical to the 99m  +  2  Re analogs on the basis of H P L C comparison. The radiolabeled compounds [  99m  T c ( L - L ) ( H 0 ) ( C O ) ] were determined to exhibit moderate stability towards ligand ,  7  +  2  3  exchange in the presence of an excess of either cysteine or histidine over a 24 h period. In contrast, the radiolabeled compounds [  99m  T c ( L - L ) ( C O ) 3 ] were determined to be 8  n  +  exceptionally stable towards ligand exchange, even under increased concentrations of cysteine and histidine.  233  References start on p. 234  4.5 References  (1) Nicolini, M . ; Bandoli, G.; Mazzi, U . , Eds. Techentium, Rhenium and Other Metals in Chemistry and Nuclear Medicine; Raven Press: New York, 2000; Vol. 5. (2) Liu, S. Chem. Soc. Rev. 2004, 33, 445. (3) Jurisson, S. S.; Lydon, J. D. Chem. Rev. 1999, 99, 2205. (4) Fowler, J. S.; Wolf, A . P. Acc. Chem. Res. 1997, 30, 181. (5) Dumas, C.; Schibli, R.; Schubiger, P. A. J. Org. Chem. 2003, 68, 512. (6) Petrig, J.; Schibli, R.; Dumas, C.; Alberto, R.; Schubiger, P. A . Chem. Eur. J. 2001, 7, 1868. (7) Tanase, T.; Doi, M . ; Nouchi, R.; Kato, M . ; Sato, Y . ; Ishida, K.; Kobayashi, K.; Sakurai, T.; Yamamoto, Y . ; Yano, S. Inorg. Chem. 1996, 35, 4848. 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(71) Alberto, R.; Schibli, R.; Egli, A . ; Schubiger, A . P.; Abram, U ; Kaden, T. A . J. Am. Chem. Soc. 1998,120, 7987. (72) Reger, D. L.; Gardinier, J. R.; Pellechia, P. J.; Smith, M . D.; Brown, K . J. Inorg. Chem. 2003, 42, 7635. (73) Schibli, R.; Schwarzbach, R.; Alberto, R.; Ortner, K.; Schmalle, H.; Dumas, C.; Egli, A.; Schubiger, P. A . Bioconjugate Chem. 2002,13, 750. (74) Egli, A . ; Alberto, R.; Tannahill, L.; Schibli, R.; Abram, U . ; Schaffland, A . ; Waibel, R.; Tourwe, D.; Jeannin, L.; Iterbeke, K.; Schubiger, P. A . J. Nucl. Med. 1999, 40, 1913.  238  References start on p. 234  Chapter 5  Future Work  5.1 Vanadyl-thiazolidinedione Combination Agents  Chapter 2 details the synthesis, characterization, and in vivo studies of a series of vanadyl-thiazolidinedione combination agents for potential use in diabetes therapy. The compounds were tested for their glucose-lowering ability in an acute STZ-diabetic animal study over a period of 72 hours. Neither additive nor synergistic properties were observed in this study; however, this could be due to the limited amount of information available from this short-term testing protocol. The compound VO(L ) did however show glucose 2  lowering effects similar to those of B M O V in the acute trial. It was interesting that the •y  unsaturated thiazolidinedione H L and complex VO(L ) were considerably more active 2  than the saturated analogs H L and VO(L ) . H L was the most active thiazolidinedione 4  4  3  2  in the acute study, lowering plasma glucose levels much more effectively than did the commercially available rosiglitazone, a significant finding that warrants further attention. While glucose lowering is an effective screen for potential efficacy, the thiazolidinediones are known to elicit other beneficial effects such as reducing insulin resistance, triglyceride levels, and fatty acid uptake. ' A longer-term trial studying the 1 2  effect these combination agents have on parameters such as plasma triglycerides and insulin, as well as on plasma glucose would be worthwhile. Based on the results reported in Chapter 2 it would be prudent to test further the potential of H L and VO(L ) for 3  3  2  239  References start on p. 246  diabetes treatment in a more specific animal model of type 2 diabetes such as the db/db 3,4  mouse. '  5.2 Novel Chelating Agents for Alzheimer's Disease Therapy  The synthesis, characterization, and preliminary testing of novel chelating agents for Alzheimer's disease (AD) therapy were discussed in Chapter 3. Initial testing demonstrated that the tetrahydrosalen compounds can act as antioxidants, as well as metal ion chelators. The glycosylated pro-drugs were determined to be substrates for a glucosidase enzyme in vitro, highlighting the possibility that these compounds could be activated in vivo by brain glucosidases. Continued testing of these compounds will allow 5  the Orvig group to evaluate the importance of certain structural features in the design of B B B permeable chelators for use in A D therapy. The ability ofthe synthesized tetrahydrosalen compounds to interrupt Ap-peptide metal interactions should be assessed. Various methods exist, one of which involves monitoring the turbidity of solutions (by UV-vis) containing the Ap-peptide and metal ions (Cu , Z n , and Fe ) in the presence and absence of the tetrahydrosalen 2+  2+  3+  compounds. Turbidity experiments have been used previously to show that chelators such as E D T A can reverse metal-induced Ap-peptide aggregation. ' Another means of 6 7  assessing the ability of the tetrahydrosalen compounds to reverse metal-induced A p peptide aggregation is to monitor the solublization of Ap from post-mortem A D brain tissue in the presence of chelators.  240  References start on p. 246  Testing of the glycosylated tetrahydrosalen compounds for B B B permeability is an important goal. The extent of brain uptake can be assessed by determining the biodistribution of radiolabeled analogs and efforts in this area are already underway in the Orvig group. Using C as the radionuclide minimizes the potential pharmacokinetic 1 4  changes which may arise when introducing larger radionuclides such as  I. A method of  producing C-containing analogs of H2GL ' would be to employ C-formaldehyde in 6  14  7  14  the Mannich condensation as shown in Scheme 5.1. One drawback of this method is the need for two equivalents of C-formaldehyde in the Mannich condensation to form 14  H GL . 6  2  OGluc  H GL  6  2  Scheme 5.1: A potential route to a C-radiolabelled derivative of H 2 G L . 6  14  The trolox equivalent antioxidant capacity (TEAC) assay demonstrated the antioxidant capability of the tetrahydrosalen compounds. Further cell studies assessing the ability of these compounds to minimize oxidative stress are warranted. In collaboration with researchers at Rutgers University, New Jersey, the tetrahydrosalen compounds are being evaluated for neuroprotective efficacy in the PC-12 cell line. The PC-12 cell line has been used extensively for neuronal differentiation studies and mimics neuronal oxidative stress when exposed to suitable toxins. '  9 10  241  References start on p. 246  Metal-binding affinities of H G L  for C u  6 7  2  2+  and Z n  2+  were assessed by  potentiometry and, as expected, the stability constants were found to be much higher for the C u  2 +  complexes. Unfortunately the Z n  macroscopic scale; in contrast the C u  2+  2+  complexes could not be prepared on the  complexes of H G L  6 - 7  2  The potentiometric results do however suggest that the Z n  were well characterized.  complexes are stable at  2+  neutral pH and thus efforts should be continued to isolate these compounds. The next logical step would be to study the interaction of Fe with H G L 3+  2  Preparation of the F e  3+  complement to the C u  6 - 7  via potentiometry.  complexes on the macroscopic scale would also be a useful  2 +  complexes reported in Chapter 3.  Further evaluation of the compounds discussed in Chapter 3 could lead to the synthesis of new tetrahydrosalen derivatives with improved characteristics for A D chelation therapy. For example, using the developed synthetic routes reported in Chapter 3, changing the R-groups of the secondary amines and/or altering the alkyl tether between amines could lead to more active compounds, as shown in Scheme 5.2. CHO  R  H  H  '\ j ^ s  R  ^k/OGluc  A\  GlucO  Scheme 5.2: The use of different amine starting materials to produce tetrahydrosalen analogs; structural diversity could be obtained by altering R and R' as well as the length (x) of the alkyl tether.  242  References start on p. 246  Determining the A D therapeutic potential of the tetrahydrosalen compounds reported in Chapter 3 should be a priority before new chelators are synthesized.  5.3 Carbohydrate-appended Metal Complexes for Use in Nuclear Medicine  The synthesis and characterization of novel carbohydrate-appended Re and  9 9 m  Tc  complexes were reported in Chapter 4. The in vitro stability of the Tc-carbohydrate 99m  complexes was assessed by competition experiments in the presence of either excess cysteine or histidine. These studies highlighted the greater stability of tridentate carbohydrate-appended ligands for the {M(CO)3} (M = Re / +  99m  T c ) core as compared to  that of the bidentate analogs. ' While the bidentate analogs exhibited moderate stability 11 13  towards cysteine and histidine exchange, studies have shown that a free coordination site on the metal results in plasma protein binding and subsequent slow clearance, limiting the in vivo application of these derivatives. The dioxorhenium(V) {0=Re=0} core could 14  +  be used as an alternative platform for the bidentate ligands discussed in Chapter 4. Dioxorhenium(V) complexes with ethylenediamine and 1,3-propanediamine have 15  16  been reported previously and thus it seems feasible that the bidentate 1,3diaminocarbohydrates could also be attached to this core. The synthesis of a dioxorhenium(V) complex with the bidentate sugar ligand (5)-2,3-diaminopropyl p-D•  17  glucopyranoside  (Scheme 5.3; experimental details follow scheme) has been achieved.  It will be interesting to ascertain i f this strategy will furnish the corresponding 1,3diaminocarbohydrate analogs.  243  References start on p. 246  Scheme 5.3: Reaction scheme for the synthesis of the dioxorhenium(V) complex of (5)2,3-diaminopropyl P-D-glucopyranoside a) Re02l(PPh ) , MeOH, 5 hrs, 63 % yield. 18  3  The synthesis  of bis-((S)-2,3-diaminopropyl  2  f3-D-glucopyranosyl)-transdioxorhenium  iodide  (5)-2,3-Diaminopropyl p-D-glucopyranoside  17  and Re02l(PPH3)2 were made according to 18  literature procedures. (S)-2,3-Diaminopropyl PD-glucopyranoside (0.147 g, 0.58 mmol) was dissolved in M e O H (7 mL) and Re0 I(PPH ) (0.241 g, 0.28 mmol) was added as a solid 2  3  2  with stirring. A brown precipitate was isolated after 5 h, washed with M e O H and Et20, and dried in vacuo to afford the product as a beige solid (0.150 g, 64 %). ' H N M R (D 0, 2  300 MHz): 5 4.40 (d, J 3  = 7.8 Hz, 2H; 77-1), 4.19 (dd, J , > = 11.4 Hz, J , = 3.6 Hz, 2  1 > 2  3  C C  b  2H; H-c), 3.97 (dd, J - = 11.4 Hz, J < = 5.7 Hz, 2H; 77-c'), 3.80 (dd, J , 2  3  2  CjC  3  b>c  6 a  J ,6b = 1.5 Hz, 2H; 77-6b), 3.60 (dd, J , 2  5  c  6 a  = 12.3 Hz, J  5M  12H; 77-2, 77-3, 77-4, 77-5, H-b, 77-a), 2.67 (m, 1H; 77-a).  244  = 12.3 Hz,  = 6.0 Hz, 2H; 77-6a), 3.24 (m,  3  6 b  6 b  C{ H}  l3  l  N M R (D 0, DSS 2  References start on p. 246  standard, 75.48 MHz): 5 97.38 (CI), 70.56, 70.11 (C3/C5), 64.14 (CI), 63.18 (CA), 61.13 (C6), 55.98, 55.21 (Ca/Cb), 45.31 (Cc). IR (cm' , K B r disk): 3232 (br) (v(NH ), v(OH)); 1  2  814 (s) (v(0=Re=0)). M S (+ES-MS) m/z (relative intensity) = 723, 721 ( M , 100). Anal. +  Calcd. (found) for Cis^oIN^ieRe-IfcO: C, 24.03 (24.09); H , 4.71 (5.02); N , 6.23 (6.07).  Stability studies and  9 9 m  T c labeling would be the next logical step. Additionally,  reaction of the dioxorhenium(V) {0=Re=0} core with the tridentate carbohydrate 2,2'+  dipicolylamine ligands ( L - L ) discussed in Chapter 4 could be investigated (Scheme 8  N  5.4).  Scheme 5.4: General reaction scheme for the synthesis of the dioxorhenium(V) complexes of the tridentate carbohydrate 2,2'-dipicolylamine ligands a) Re02l(PPh )2.  18  3  The iodine could be subsequently replaced by any number of monodentate ligands, allowing for further tuning of the properties of the product. The targeting ability of the Tc-carbohydrate conjugates discussed in Chapter 4 99m  needs to be assessed in an animal model to determine in vivo applicability. Biodistribution studies could be used initially to determine tissue uptake as well as  245  References start on p. 246  clearance times in vivo. Evaluating the utility of the  Tc-carbohydrate conjugates for  imaging potential in tumour-bearing rodents would also be a worthwhile endeavour.  19  Assuming that suitable tumour target specificity can be obtained, the synthesis of the 186/188  corresponding  Re analogs could be investigated for radiotherapeutic applications.  Indeed, the synthesis of the  186  R e complex of 2-(Bis(2-pyridinylmethyl)amino)methyl-  glucosamide (Chapter 4, L ) has recently been reported by our group. n  12  5.4 References  (1) Mudaliar, S.; Henry, R. R. Ann. Rev. Med. 2001, 52, 239. (2) Oakes, N . D.; Thalen, P. G.; Jacinto, S. M . ; Ljung, B. Diabetes 2001, 50, 1158. (3) Sauerberg, P., Pettersson, I., Jeppesen, L., Bury, P. S., Mogenson, J. P., Wassermann, K., Brand, C. L., Sturis, J., Woldike, H. F., Fleckner, J., Anderson, A . T., Mortensen, S. B., Svensson, L . A., Rasmussen, H. B., Lehmann, S. V., Polivka, Z., Sindelar, K., Panajotova, V., Ynddal, L., Wulff, E. M . J. Med. Chem. 2002, 45, 789. 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Radiology 2003, 226, 465.  247  References start on p. 246  Table A l : Crystallographic data for H G L , CuGL and N i G L . 7  7  7  2  H GL  Crystal Data Formula Formula Weight Crystal Color, Habit Crystal System Crystal Size [mm] Space group Lattice parameters  C38H68N Oi8. 5 2  vrA'i z  [g/cm ] p(MoKa) [cm" ] Fooo Temperature (K) D aic  3  C  1  ©min-max  No. of reflections Measured  2  3  844.95 colourless, needle triclinic 0.50x0.10x0.08 Pl a = 10.498 (1) A b = 10.748 (1) A c = 39.414 (3) A a = 87.66(1)° [3 = 86.31 (1)° y = 89.89 (2)° 4434.3 (8) 4 1.266 1.00 1824 173 (2) 1.55,22.49° Total: 53561 Unique: 18652 (R =0.066) wR = 0.179 1.025 Ri = 0.063 0.87 e " / A int  Residuals (refined on F , all data) Goodness of Fit Indicator Residuals (refined on F, I>2a (I)) Max. peak in Final Diff. Map Min. peak in Final Diff. Map 2  CuGL C 8H58CuN 0 830.40 green, block triclinic 0.50x0.25x0.10 P l (#1) a = 10.691 (2) A b = 8.7559 (9) A c = 13.602 (2) A a = 95.700(1)° p = 90.080(1)° y = 92.050 (7)° 2349.4 (7) 2 1.174 52.3 882 173 (2) 2.16, 27.87° Total: 20637 Unique: 15770 (R =0.044) wR = 0.213 1.048 R i = 0.082 0.95 e " / A -0.96 e " / A 7  7  2  2  3  -0.38e"/A  j  2  14  int  2  3  j  NiGL C H 4N NiOi 953.74 pink, platelet monoclinic 0.40x0.10x0.02 P2i (#4) a = 14.993 (3) A b = 9.2197 (18) A c = 16.607 (3) A a = 90.00° P = 94.823 (7)° Y = 90.00 ° 2287 (7) 2 1.385 50.0 1024 173 (2) 2.46,21.96° Total: 12661 Unique: 5383 (R =0.089) wR = 0.189 0.974 R i = 0.084 0.73 e " / A -0.43 e " / A 7  42  7  2  8  int  2  J  J  Table A2: Crystallographic data for [Re(L )(CO) Br] and [Re(L )(CO) Br], and [Re(L )(CO) ]Cl. 2  3  8  3  Crystal Data Formula Formula Weight Crystal Color, Habit Crystal System Crystal Size [mm] Space group Lattice parameters  [Re(L )(CO) Br] 604.43 colourless, plate monoclinic 0.25x0.15x0.03 C2 (#5) a= 15.142(1) A b = 6.4896 (5) A c = 20.026 (2) A a = 90.0° P = 104.814(4)° 7 = 90.0° 1902.5 (3) 4 2.110 85.33 1160 173 (2) 2.71,27.80° Total: 28862 Unique: 4313 (R ,= 0.034) w R = 0.062 1.055 Ri = 0.025  [Re(L*)(CO) ]Cl C2 H 6ClN Oio. Re 780.23 colourless, needle monoclinic 0.30x0.12x0.10 P2]2 2 a = 36.523 (6) A b = 8.220(1) A c = 10.172 (2) A a = 90° p = 90° y = 90° 3053.8(8) 4 1.697 41.26 1024 173 (2) 2.00, 28.01° Total: 67291 Unique: 7305 (R =0.085) w R = 0.051 1.038 R i = 0.022  1.20 e 7 A -0.85 e7 A  1.27 e 7 A -0.76 e 7 A  1.47 e 7 A -0.58 e7 A  j  3  3  Ci H22BrN20 Re 2  Dcaic [g/cm ] 3  p(MoKa) [cm ] Fooo Temperature (K) -1  0min-max  No. of reflections Measured  9  in  Residuals (refined on F , all data) Goodness of Fit Indicator Residuals (refined on F, I>2CT (I)) Max. peak in Final Diff. Map Min. peak in Final Diff. Map 2  3  [Re(L )(CO) Br] Ci H2iBrN209. Re 611.41 colourless, flake triclinic 0.25 x 0.07 x 0.03 Pl (#1) a = 6.6037 (7) A b = 7.8941 (9) A c= 18.899 (2) A a = 93.946 (5)° p = 95.075 (5)° y= 111.779 (5)° 905.8 (2) 2 2.242 89.64 586 173 (2) 2.16, 27.87° Total: 22644 Unique: 7894 (R =0.028) wR = 0.076 1.10 Ri = 0.024  z  V[A'] Z  3  2  3  2  5  int  2  3  3  3  6  3  3  X  int  2  3  3  5  3  

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