Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Fluorinated nitroimidazoles and their ruthenium complexes : potential hypoxia-imaging agents Baird, Ian Robert 1999

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1999-463141.pdf [ 16.82MB ]
Metadata
JSON: 831-1.0228854.json
JSON-LD: 831-1.0228854-ld.json
RDF/XML (Pretty): 831-1.0228854-rdf.xml
RDF/JSON: 831-1.0228854-rdf.json
Turtle: 831-1.0228854-turtle.txt
N-Triples: 831-1.0228854-rdf-ntriples.txt
Original Record: 831-1.0228854-source.json
Full Text
831-1.0228854-fulltext.txt
Citation
831-1.0228854.ris

Full Text

FLUORINATED  A N D THEIR R U T H E N I U M C O M P L E X E S : POTENTIAL H Y P O X I A - I M A G I N G A G E N T S  NITROPAIIDAZOLES  By  I A N ROBERT BAIRD B . S c , University of British Columbia, 1994  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A March 1999 © Ian Robert Baird, 1999  In  presenting  degree freely  at  this  the  thesis  in  University  of  available for reference  copying  of  department publication  this or of  thesis by  this  for  his thesis  partial and her  of  The University of British C o l u m b i a Vancouver, Canada  DE-6 (2/88)  the  study.  requirements  I further agree that  purposes  may  representatives.  be  It  for financial gain shall not  permission.  Department  of  British Columbia, I agree that scholarly  or  fulfilment  is  the  an  advanced  Library shall make  permission for  granted  by  understood be  for  the that  allowed without  it  extensive  head  of  my  copying  or  my  written  Abstract  With the goal of synthesizing nitroimidazole-based hypoxia imaging agents for the detection of cancerous tumours, the chemistry of halogen incorporation, especially fluorine, into the N I side-chain of nitroimidazoles was investigated. The coordination of these nitroimidazoles and other methyl (Me)-substituted imidazoles to Ru(II) and Ru(III) centres was also investigated. The general objective was to combine the hypoxia-selective nature of the nitroimidazole moiety and the DNA-binding properties of Ru to deliver the active species selectively to its target. The nitroimidazole compounds and Ru complexes were characterized in general by a combination of N M R , IR and UV-Visible spectroscopies, as well as mass spectrometry, cyclic voltammetry, conductivity and elemental analysis; six nitroimidazoles and eleven Ru complexes were also characterized by X-ray crystallography. The halogenated nitroimidazoles were synthesized using a standard amide coupling reaction; this was used for the synthesis of EF5 [2-(2-nitro-l-H-imidazol-l-yl)-N(2,2,3,3,3-pentafluoropropyl) acetamide], a known, sensitive probe for quantifying the amount of hypoxia within cells. Treatment of a desired acid with N-methylmorpholine and zso-butylchloroformate, H2NCH2CX3  followed by  addition  of the  appropriate  amine  (either  or H N C H C X C X 3 , where X= H , F, CI and/or Br) led to formation of the 2  2  2  halogenated nitroimidazole; 2-nitro- (2N0 ), 2-methyl-5-nitro- (2Me5N0 ) and 2-methyl2  2  4-nitro- (2Me4N0 ) imidazole compounds were isolated in yields of 15 to 83 %. 2  The synthesis of EF5 (typical yield 45 %) was improved by coupling iodoacetic acid  with H N C H C F C F 2  2  2  to  3  give I C H C ( 0 ) N H C H C F C F 2  2  2  3  (TF5)  which  was  subsequently reacted with 2N0 Im and C s C 0 to yield the final product (78 %); this 2  2  3  2-step method circumvented the previously required 5-step synthesis. Other analogous compounds were less stable than IF5, especially those species containing a C-Br moiety, and they often decomposed in the presence of heat and/or light. Alteration of the associated side-chain on EF5 by 'reversal' of its amide linkage was performed; however, the biological activity of the derivative was significantly less than that of EF5. A nitroimidazole (N0 lm) containing side-chain with a highly reactive terminal 2  group, with the potential to exchange fluoride, was also sought. Attempts to isolate a  ii  tosylated  derivative  of SR2508 [2-(2-mtro-l-H-irnidazol-l-yl)-N-(2-hydroxyethyl)  acetamide] proved to be futile. The reaction with tosyl chloride led to the formation of a monochloro analogue of SR2508, presumably via the reactive tosylate intermediate, while reaction with triflic anhydride led to an intramolecular cyclization of the side-chain to give a compound whose X-ray structure was determined. The focus was then shifted to F-for-Br exchange; however, attempts to perform such a task revealed that the C - N 0  2  bond of the Im ring was more labile than the C-Br bond, and this resulted in the formation of a 2-fluoroimidazole species. Further efforts to incorporate  an F-atom into a  nitroimidazole side-chain were unsuccessful.  SR2508  EF5  [Ru(II)(L) ]  metro  complexes were synthesized from [Ru(DMF) ][CF S03]3; D M F =  2+  6  6  3  dimethylformamide, L = imidazole (Im), N-methylimidazole (NMelm) and 5-methylimidazole (5MeIm). The 2-methylimidazole complex fram-[Ru(CO)(DMF)(2MeIm) ] 4  [CF S03]2 was synthesized via a reaction involving abstraction of CO from D M F ; this 3  complex loses C O reversibly at ambient temperature to form [Ru(DMF)(2MeIm) ] 4  [ C F S 0 ] , and the D M F can be removed to generate [Ru(CF S0 )x(2MeIm) ][CF3S0 ] 3  3  2  3  3  4  3  y  (x = 2, y = 0, or x = 1 = y). The X-ray structures of [Ru(Im) ][CF S0 ] , 6  [Ru(NMeIm) ][CF S0 ] 6  [Ru(II)(L) ] x  2+  3  3  and [Ru(5MeIm) ][CF S0 ]  2  6  3  3  2  were  obtained.  3  3  2  Analogous  nitroimidazole complexes (L = 2N0 Im, x = 6; L = 4N0 Im, x = 6; L = 2  2  2Me5N0 Im, x = 5) were isolated from reaction with [Ru(DMF) ][CF S03] . The 2  6  3  reaction with EF5 and SR2508 in EtOH yielded the bis-substituted  3  complexes  [Ru(DMF) (EF5) (EtOH) ][CF S03] and [Ru(DMF) (SR2508) ][CF SO3] , respectively. 2  2  2  3  3  4  2  3  3  Ru(III) complexes of composition R u C l L (L = 2N0 Im, 4N0 Im, 2Me5N0 Im, 3  3  metro) and RuCl L (EtOH) (L= EF5, SR2508) were 3  2  2  2  synthesized  2  directly from  RuCl »3H 0. Their *H N M R spectra were typically broad and sometimes signals were not 3  2  observed; however, their paramagnetic, d low-spin composition was confirmed using the 5  Evans method.  iii  Some new Ru(II) and Ru(III) bis-P-diketonate (acac = acetylacetonate; hfac = 1,1,1,5,5,5-hexafluoroacetylacetonate) Reaction  of two  Im and N 0 l m 2  complexes were synthesized.  equiv. of an imidazole with c/s-[Ru(acac) (MeCN) ][CF S0 ] 2  2  3  3  (synthesized from Ru(acac) and C F S 0 H in MeCN) yielded Ru(III) complexes with 3  3  3  composition [Ru(acac) (L) ][CF S0 ] (L = Im, NMelm, 2MeIm, 5MeIm, 2N0 Im, metro, 2  2  3  3  2  E F 5 and SR2508), the first four being structurally characterized by X-ray crystallography. The  analogous  Ru(II)  hfac  complexes  were  isolated  from  a  reaction  of  cz'5-Ru(hfac) (MeCN) (71) (synthesized from either Ru(hfac) or Na[Ru(hfac) ] and 2  2  3  3  C F S 0 H in MeCN) with two equiv. of imidazole to yield Ru(hfac) (L) (L = Im, NMelm, 3  3  2  2  2MeIm, 4(5)MeIm, 2N0 Im, E F 5 and SR2508). X-ray structures of 71 and Ru(hfac) 2  3  were obtained. Reaction of 71 with neat NMelm gave [Ru(hfac)CNMeIm) ][hfac]. The 4  mixed ligand complex c/5-Ru(hfac)(acac)(MeCN) (X-ray) was synthesized from the new 2  species Ru(hfac) (acac), which was isolated from a reaction of 71 with Hacac. 2  In vitro  assays for toxicity and monoclonal antibody (MoAb) binding with SCCVLT  cells were used to evaluate the potential of selected nitroimidazoles as hypoxia-selective imaging agents. The toxicity, MoAb binding, cell accumulation and DNA-binding assays were used to test the utility of selected Ru complexes for transporting and/or localizing the coordinated nitroimidazoles within the cell. A preliminary radiosensitization study was also performed with two Ru complexes. In general, the nitroimidazoles (N0 lms) and Ru complexes were non-toxic under 2  both oxic and hypoxic conditions. The accumulation of the N 0 l m s within the cell 2  required hypoxic conditions, while the amount of N 0 l m bound within the hypoxic cells 2  correlated with its one-electron reduction potential, the N 0 l m s with the more positive 2  reduction potentials giving the higher concentrations. The interaction of the fluorescently labeled MoAbs (ELK3-51 and ELK5-A8) with the N 0 l m s depended on the length, size 2  and composition of the halogenated side-chains. The R u - N 0 l m complexes displayed 2  relatively high cell accumulation and DNA-binding levels when compared to literature data for other Ru complexes. The most interesting result came from the MoAb assay in which the cells treated with [Ru(acac) (EF5) ] [CF S0 ] afforded a fluorescence signal four 2  2  3  times greater than that seen for EF5 itself.  iv  3  Table of Contents Abstract  ii  Table of Contents  v  List of Figures  xv  List of Schemes  xx  List of Tables  xxi  List of Abbreviations  xxvi  Key to Chemical Compound Numbers and Abbreviations  xxix  Acknowledgements  xxxv  Chapter 1 - Introduction 1.1 Introduction  1  1.2 Hypoxia  2  1.2.1 What is it?  2  1.2.1.1 Resistance to Treatment: Radioresistance 1.2.2 Hypoxia: The Aggressor? 1.3 Role of Nitroimidazoles in Cancer Therapy  3 5 6  1.3.1 Hypoxic Radiosensitizers  6  1.3.2 Chemistry of Nitroimidazoles in Biological Systems  7  1.4 Imaging of Hypoxic Tumours  10  1.4.1 Radiolabelling of Hypoxic Cells  11  1.4.2 Nuclear Medicine Techniques  11  1.4.3 Fluorescent Probes  12  1.4.4 Magnetic Resonance Imaging (MRI)  14  1.4.5 Positron Emission Tomography (PET)  14  1.5 Role of Metals in Cancer Therapy  16  1.6 Ruthenium Complexes  18  1.6.1 Chemical Properties Relevant to Tumour Treatment  18  1.6.2 D N A Binding  20  v  1.6.3 Tumouricidal Effects of Ru Complexes  22  1.7 Thesis Overview  24  1.8 References  26  Chapter 2 - General Experimental 2.1 Materials  33  2.1.1 Chemicals  33  2.1.1.1 Imidazoles  33  2.1.1.2 Amines  33  2.1.1.3 Miscellaneous Reagents  33  2.1.2 Solvents  34  2.2 Analytical Techniques  34  2.2.1 Nuclear Magnetic Resonance Spectroscopy  34  2.2.2 Infrared Spectroscopy  37  2.2.3 UV-Visible Spectroscopy  37  2.2.4 Mass Spectrometry  37  2.2.5 Gas Chromatography  38  2.2.6 Cyclic Voltammetry  38  2.2.7 Conductivity  39  2.2.8 X-Ray Analysis  40  2.2.9 Elemental Analysis  40  2.3 Compound Purification Techniques  40  2.3.1 Column Chromatography  40  2.3.2 Preparative Thin Layer Chromatography  41  2.3.3 Chromatotron  41  2.4 General Methodologies  41  2.4.1 Amide Coupling Reaction 2.5 Ruthenium Precursors  42  2.5.1 R u C l ' 3 H 0 3  42  2  2.5.2 [Ru(DMF) ][CF S0 ]3 6  41  3  42  3  vi  2.5.3 [Ru(DMF) ][CF S0 ]2 6  3  43  3  '2.5.4 c/s-RuCl (DMS0)(DMSO)  43  2.5.5 trans-RuCl (pMSO)  44  2.5.6 cw-RuCl (TMSO)  44  2  3  2  4  2  4  2.5.7 /we/--RuCl (DMSO)  44  2.5.8 [RuCl (COD)]  45  2.5.9 [RuCl (dppb)] (u-dppb)  45  3  2  2  3  x  2  References  46  Chapter 3 - Synthesis a n d Characterization of New 2-, 4- a n d 5-Nitroimidazoles w i t h Halogenated Side-Chains  3.1 Introduction  48  3.2 Experimental Section  49  3.2.1 Synthesis of Side-Chains  49  3.2.1.1 2-Iodo-/V-(2,2,3;3,3-pentafluoropropyl)acetamide  [IF5]  3.2.1.2 2-Iodo-7V-(3-bromopropyl)acetamide [IBr]  49 49  3.2.1.3 2-Chloro-/V-(2,2,3,3,3-pentafluoropropyl)acetamide [C1F5] .... 51 3.2.1.4 Reduction of IF5 to ICH CH NHCH CF CF (3)  52  3.2.1.5 3-Fluoropropylamine Hydrochloride (5)  52  2  2  2  2  3  3.2.2 2-Nitroimidazole Compounds 3.2.2.1 2-(2-Nitro-l-H-imidazol-l-yl)acetic acid (7)  54 54  3.2.2.2 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide [EF5] 3.2.2.3  56  2-(Imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide  [ImF5]  57  3.2.2.4 N-(2-nitro-l-H-imidazol-1-ethyl) pentafluoropropionamide  [RevEF5]  58  3.2.2.5 2-(2-Nitro-l-H-imidazol-l-yl)-N-(3-bromo-2,2,3,3tetrafluoropropyl) acetamide [EF4Br]  vii  61  3.2.2.6 2-(2-Nitro-l-H-imidazol-l-yl)-N-(3,3,3trifluoropropyl)acetamide [EF3]  62  3.2.2.7 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(2,2,2-trifluoroethyl)acetamide  [EF3(-1>]  63  3.2.2.8 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(3 -bromo-3,3 -difluoropropyl) acetamide [EF2Br]  63  3.2.2.9 2-(2-Nitro-l-H-imidazol-l-yl)-N-(3,3difluoropropylene)acetamide [E=F2]  64  3.2.2.10 2-(2-Nitro-l-H-imidazol-l-yl)-N-(3-fluoropropyl)acetamide  [EF1]  65  3.2.2.11 2-(2-Nitro-l-H-irmdazol-l-yl)-N-(2-fluoroethyl)acetamide  [EFl(-l)]  67  3.2.2.12 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(3 -chloropropyl)acetamide  [ECU]  68  3.2.2.13 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(2-chloroethyl)acetamide  [ECU(-l)]  69  3.2.2.14 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(3 -bromopropyl)acetamide  [EBrl]  70  3.2.2.15 2-(2-Nitro-1 -H-imidazol-1 -yl)-N-(2-bromoethyl)acetamide  [EBrl(-l)]  71  3.2.2.16 2-(2-Nitro-l-H-imidazol-l-yl)-N-(propyl)acetamide [EPrA].. 72 3.2.2.17 2-(2-Nitro-l-H-imidazol-l-yl)-N-(/'50-amyl)acetamide  [EIAA]  73  3.2.3 2-Methyl-5-Nitroimidazole Compounds 3.2.3.1 2-(2-Methyl-5-nitro-IB-imidazol-l-yl)acetic acid (13)  74 74  3.2.3.2 2-(2-Methyl-5-nitro-l/f-imidazol-l-yl)-N-(2,2,3,3,3) pentafluoropropyl) acetamide [MF5]  74  3.2.3.3 2-(2-Methyl-5-nitro-l#-imidazol-1 -yl)-N-(2,2,2-trifluoroethyl) acetamide [MF3(-1)]  viii  76  3.2.3.4 2-(2-Methyl-5-mtro-l^-irnidazol-l-yl)-N-(2fluoroethyl)acetamide [MFl(-l)]  76  3.2.3.5 2-(2-Methyl-5 -nitro- 177-imidazol-1 -yl)-N-(2chloroethyl)acetamide [MCIl(-l)]  77  3.2.3.6 2-(2-Methyl-5-nitro- l#-imidazol- l-yl)-N-(2bromoethyl)acetamide [MBrl(-l)]  79  3.2.3.7 (2-Methyl-5-mtro-l/f-irmdazol-l-yl)-N-(2-chloroethane)  (16).. 80  3.2.4 2-Methyl-4-Nitroimidazole Compounds  81  3.2.4.1 2-(2-Methyl-4-nitro-l-H-imidazol-l-yl)propionic acid (17)  81  3.2.4.2 3-(2-Methyl-4-nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3pentafluoropropyl)propionamide  [2M4NF5]  81  3.2.4.3 3-(2-Methyl-4-mtro-l-H-imidazol-l-yl)-N-(2,2,2-trifluoroethyl) propionamide [2M4NF3(-1)]  82  3.2.4.4 3-(2-Methyl-4-nitro-l-H-imidazol-l-yl)-N-(2-fluoroethyl) propionamide [2M4NF1(-1>]  83  3.2.4.5 3-(2-methyl-4-nitro-1 -H-imidazol-1 -yl)-N-(2chloroethyl)propionamide  [2M4NC11(-1)]  84  3.2.4.6 2-(2-methyl-4-nitro-l-H-imidazol-l-yl)-N-(2bromoethyl)propionamide [2M4NBrl(-l)]  85  3.2.5 Reactions of SR2508 with Tf 0  85  2  3.2.5.1 2-(2-Nitro-l-H-imidazol-l-yl)-N-(ethylformate)acetamide 3.2.5.2 CycF3 (19)  (18) 85 86  3.2.6 Reactions with RSU1111  87  3.2.6.1 Synthesis of (20), a pentafluorinated derivative ofRSUllll.... 87 3.2.6.2 Synthesis of (21), a dibromo derivative ofRSUllll  88  3.2.6.3 Synthesis of (22), a terminal nitrile derivative ofRSUllll  88  3.2.7 Reactions of Nitroimidazoles with Bu NF»H 0 4  90  2  3.2.7.1 Reaction of E B r l with Bu NF«H 0 4  90  2  3.2.7.2 Reaction of E B r l with Bu NF«H 0 and C H C 0 H  91  3.2.7.3 Reaction of SR2508 with Bu NF«H 0  91  4  2  4  ix  3  2  2  3.2.7.4 Reaction of « N 0 I m (n = 2, 4, 5) with Bu NF«H 0 2  4  92  2  3.2.8 Cyclic Voltammetry of Nitroimidazoles  94  3.3 Results and Discussion  96  3.3.1 Synthesis of the Nitroimidazole Side-Chains 3.3.2 Nitroimidazoles  96 '.  101  3.3.3 Electrochemistry of Nitroimidazoles  117  3.3.4 Incorporation of Fluorine into an Existing Side-chain  122  3.4 References  129  Chapter 4 - Synthesis and Characterization of Ru(II) and Ru(III) Imidazole Complexes 4.1 Introduction  134  4.2 Experimental Section  135  4.2.1 Complexes Synthesized from [Ru(DMF) ][CF S03]3 6  4.2.1.1 [Ru(Im) ][CF S03] 6  3  135  3  (28)  2  135  4.2.1.2 [Ru(NMeIm) ][CF S0 ] (29)  135  4.2.1.3 [Ru(5MeIm) ][CF3S0 ]  136  6  3  3  6  4.2.1.4  3  2  (30)  2  fra»5-[Ru(CO)(DMF)(2MeIm)4][CF3S0 ]  3 2  (31),  [Ru(DMF)(2MeIm)4][CF S0 ] (32) and [Ru(CF S0 ) 3  3  2  3  (2MeIm) ][CF S03] (x=2, y=0; or x=l=y) (33) 4  3  y  3  x  137  4.2.1.5 [Ru(2N0 Im) ][CF S03] (34)  138  4.2.1.6 [Ru(5N0 Im) ][CF3S0 ]  138  2  6  2  3  2  6  3  (35)  2  4.2.1.7 [Ru(2Me5N0 Im) ][CF3S0 ] (36)  139  4.2.1.8 [Ru(DMF) (SR2508) ][CF SO ] (37)  140  4.2.1.9 [Ru(DMF) (EF5) (EtOH) ][CF S0 ] (38) 3  140  4.2.2 Complexes Synthesized from RuCl *3H 0  141  2  5  3  4  2  2  2  3  2  3  2  3  3  3  4.2.2.1 /wer-RuCl (2N0 Im) (39) 3  2  3  2  141  3  4.2.2.2 fac- and /wer-RuCl (5N0 Im) (40)  142  4.2.2.3 "RuCl (2Me5N0 Im) *3C0 " (41)  143  3  3  2  2  3  x  3  2  4.2.2.4 RuCl (metro) (42)  143  4.2.2.5 RuCl (SR2508) (EtOH) (43)  144  4.2.2.6 RuCl (EF5) (EtOH) (44)  145  4.2.2.7 RuCl (metro) (45)  146  3  3  3  2  3  2  2  4  4.2.3 Complexes Synthesized from RuCl (DMSO) 2  146  4  4.2.3.1 c/.v-RuCl (DMSO) (en) (46)  146  4.2.3.2 /ram-RuCl (DMSO) (en) (47)  147  4.2.3.3 RuCl (DMSO) (EF5)(acetone) (48)  148  2  2  2  2  2  2  4.2.4 Miscellaneous Complexes  148  4.2.4.1 [RuCl(dppb)(EF5)] (u-Cl) (49) 2  148  2  4.2.4.2 ds-RuCl (MeCN) (51) and roer-RuCl (MeCN) (52)  149  4.2.4.3 Other Attempted Reactions  150  2  4  3  3  4.3 Results and Discussion  153  4.3.1 Complexes Synthesized from [Ru(DMF) ][CF S0 ] 6  3  3  153  3  4.3.1.1 Hexakis(imidazole) Ru(II) Complexes  153  4.3.1.2 Reaction of [Ru(DMF) ][CF S0 ] with 2MeIm  158  6  3  3  3  4.3.1.3 Hexakis(nitroimidazole) Ru(II) Complexes, and some Ru(III) mixed DMF-SR2508 and -EF5 Complexes  160  4.3.2 Complexes Synthesized from RuCl »3H 0 3  165  2  4.3.3 Complexes Synthesized from c/V*ram-RuCl (DMSO) 2  4  4.3.4 Miscellaneous Ru Complexes 4.4 References Chapter 5 -  173 175 182  Synthesis and Characterization of Ru(II) and Ru(III) (3-Diketonate Imidazole Complexes  5.1 Introduction  187  5.2 Experimental  188  5.2.1 Ruthenium(III) Acetylacetonato Complexes 5.2.1.1 Ru(acac) (53)  188 188  3  xi  5.2.1.2 c/5-[Ru(acac)2(MeCN)2][CF S03] (54)  189  5.2.1.3 cw-[Ru(acac) (Im)2][CF S03] (55)  189  5.2.1.4 c/5-[Ru(acac) (NMeIm) ][CF3S0 ] (58)  190  5.2.1.5 c/5-[Ru(acac) (2MeIm) ][CF S0 ] (60)  191  5.2.1.6 cw-[Ru(acac) (5MeIm) ][CF S0 ] (62)  192  5.2.1.7 [Ru(acac) (2N0 Im)2][CF S03] (64)  193  5.2.1.8 [Ru(acac) (SR2508) ][CF SO ] (65)  193  5.2.1.9 [Ru(acac) (EF5) ][CF S0 ] (66)  194  5.2.1.10 [Ru(acac) (metro) ][CF S0 ]  195  3  2  3  2  2  2  3  2  2  3  2  2  3  2  3  3  2  2  2  3  3  2  2  3  3  3  2  3  3  (67)  5.2.1.11 Reaction of c/5-[Ru(acac) (MeCN)2][CF3S0 ] (54) with 2  3  EtOH  195  5.2.1.12 Reaction of c/5-[Ru(acac)2(MeCN)2][CF3S0 ] (54) with 3  H 0  196  2  5.2.2 Ruthenium(II) 1,1,1,5,5,5-Hexafluoroacetylacetonate  Complexes.. 197  5.2.2.1 [Na][Ru(hfac) ] (68) and [Ru(hfac)(EtOH) ][hfac] (69)  197  5.2.2.2 Ru(hfac) (70)  198  3  4  3  5.2.2.3 C75-Ru(hfac) (MeCN) (71)  198  5.2.2.4 c/5-Ru(hfac) (Im) (72)  199  5.2.2.5 c/5-Ru(hfac) (NMeIm) (74)  200  5.2.2.6 [Ru(hfac)(NMeIm) ][PF ] (76)  201  5.2.2.7 cz5-Ru(hfac) (2MeIm) (77)  202  5.2.2.8 c«-Ru(hfac) (4MeIm)(5MeIm) (80)  203  5.2.2.9 Ru(hfac) (2N0 Im)  203  2  2  2  2  2  2  4  6  2  2  2  2  2  2  (82)  5.2.2.10 Ru(hfac) (EF5) (83) 2  204  2  5.2.2.11 Attempted Synthesis of Ru(hfac) (SR2508) (84) 2  5.2.3 Ruthenium(II) and (III) acac/hfac Complexes 5.2.3.1 Ru(acac) (hfac) (85) 2  (86)  5.2.3.3 Ru(hfac) (acac) (87)  205  206 206  2  5.2.3.4 m-Ru(acac)(hfac)(MeCN) (88) 2  xii  205  205  2  5.2.3.2 [Na][Ru(hfac) (acac)]  2  207  5.2.3.5 c/.v-Ru(acac)(hfac)(Im) (89)  208  5.2.3.6 c/s-Ru(acac)(hfac)(NMeIm) (90)  208  2  2  5.2.4 Cyclic Voltammetry of Ru(II) and Ru(III) p-diketonato Complexes  209  5.3 Results and Discussion  211  5.3.1 Ruthenium tris(P-diketonato) complexes  211  5.3.2 Bis-MeCN Complexes  219  5.3.3 Bis-imidazole Complexes  227  5.3.3.1 Bis(acetylacetonato)bis(imidazole)ruthenium(III) complexes .. 227 5.3.3.2 Bis( 1,1,1,5,5,5 -hexafluoroacetylacetonato)bis(imidazole) njthenium(II) complexes  238  5.3.3.3 Cyclic Voltammetric Results for the Bis-imidazole Complexes 245 5.4 References  248  Chapter 6 - In Vitro Evaluation of Selected Nitroimidazoles a n d Ruthenium Nitroimidazole Complexes  6.1 Introduction  252  6.2 Experimental  253  6.2.1 Materials and Methods  253  6.2.1.1 Media  253  6.2.1.2 General Solutions  253  6.2.1.3 Cell Handling  255  6.2.1.4 Preparation of Compound Solutions  255  6.2.1.5 Cell Incubation Procedures  256  6.2.2 Instrumentation  257  6.2.2.1 Atomic Absorption Spectroscopy (AAS)  257  6.2.2.2 UV-Vis Spectroscopy  258  6.2.2.3 Flow Cytometry (FCM)  258  6.2.2.4 Image Cytometry (ICM)  258  xiii  6.2.3 Biological Assays  260  6.2.3.1 Toxicity Assays  260  6.2.3.2 Cell Accumulation (Uptake) Assay  260  6.2.3.3 DNA-binding Assay (Isolation of D N A from SCCVII Cells)... 261 6.2.3.4 Radiosensitizing Assays  261  6.2.3.5 Monoclonal Antibody Assay  263  6.3 Results and Discussion  265  6.3.1 Compound Solubility  265  6.3.2 Toxicity  266  6.3.2.1 Nitroimidazoles  266  6.3.2.2 Ruthenium Complexes  268  6.3.3 Cellular Accumulation of Ru Complexes  269  6.3.4 DNA-binding by Ru Complexes  272  6.3.5 Radio sensitization  274  6.3.6 Nitroimidazole Adduct Recognition using Monoclonal Antibodies. 276 6.3.6.1 Recognition of N 0 l m Adducts by ELK3-51  278  6.3.6.2 Recognition of N 0 l m Adducts by ELK5-A8  284  2  2  6.4 Refereneces  287  Chapter 7 - Conclusions a n d Recommendations f o r F u t u r e W o r k  7.1 General Remarks  291  7.2 Halogenated Nitroimidazoles (Chapter 3)  291  7.3 Ruthenium Imidazole Complexes (Chapter 4)  295  7.4 Ruthenium P-Diketonate Imidazole Complexes (Chapter 5)  297  7.5 In Vitro Evaluation of Nitroimidazoles and their Ru Complexes  298  7.6 References  301  xiv  Appendix I - X-ray data  Appendix 1-1 X-ray data for EF5  302  Appendix 1-2 X-ray data for EF2Br»0 5 H 0  304  Appendix 1-3 X-ray data for MF5  306  Appendix 1-4 X-ray data for 2M4NF5  308  Appendix 1-5 X-ray data for 2M4NF1(-1)  311  Appendix 1-6 X-ray data for cycF3 (19)  313  2  Appendix 1-7 X-ray data for [Ru(Im) ][CF S03]2 6  316  3  Appendix 1-8 X-ray data for [Ru(NMeIm)6][CF S0 ]2  318  Appendix 1-9 X-ray data for [Ru(5MeIm) ][CF S0 ]  320  3  3  6  3  3  2  Appendix 1-10 X-ray data for 7wer-RuCl (MeCN) »CHCl 3  Appendix 1-11 X-ray data for Ru(hfac)  3  322  3  324  3  Appendix 1-12 X-ray data for Ru(hfac) (MeCN) 2  327  2  Appendix 1-13 X-ray data for Ru(acac)(hfac)(MeCN)  330  2  Appendix 1-14 X-ray data for [Ru(acac) (Im) ][CF S0 ]»benzene  333  Appendix 1-15 X-ray data for [Ru(acac) (NMeIm) ][CF S0 ]  335  2  2  3  2  3  2  3  3  Appendix 1-16 X-ray data for [Ru(acac) (2MeIm) ][CF SO ]«0.5 hexane... 338 2  2  3  3  Appendix 1-17 X-ray data for [Ru(acac) (5MeIm) ][CF S0 ] 2  2  3  340  3  Appendix II - Miscellaneous Biological Data  343  List of Figures Figure i: Imidazole position numbering  xxviii  Figure 1-1: Oxygen gradient observed in a typical tumour (adapted from ref. 6)  2  Figure 1-2: Typical results for X-ray irradiation of Chinese Hamster Ovary cells under aerobic ( 0 ) or hypoxic (N ) conditions (adapted from ref. 6) 2  2  3  Figure 1-3: Structures of metronidazole, misonidazole and etanidazole  7  Figure 1-4: Nitroimidazole reduction scheme (adapted from ref. 30)  8  Figure 1-5: Ranges of reduction potentials for nitroimidazoles vs. N H E at p H 7  8  xv  Figure 1-6: Nucleophilic addition of water to l-methyl-2-nitrosoimidazole  9  Figure 1-7: Proposed reactions of 2-hydroxyaminoimidazole within the cell  10  Figure 1-8: Iodoazomycin arabinoside (IAZA)  12  Figure 1-9: Indolizine-linked 2-nitroimidazole, fluorescent hypoxia probe  12  Figure 1-10: Structures of the hypoxia-selective fluorinated 2-nitroimidazoles CCI-103F and EF5  13  Figure 1-11: y-ray production from positron emitting isotope [ F]  15  18  Figure 1-12: Chemical structures of antitumour agents cisplatin, carboplatin, JM216 and AMD473  17  Figure 1-13: Cisplatin/DNA interaction to form a G G intrastrand cross-link  21  Figure 2-1: Diagramatic representation of a capillary (containing a solution of the complex) within a N M R tube (containing solvent only) used for the Evans method  36  Figure 2-2: Cyclic voltammetry cell (A) containing a Pt reference electrode  39  Figure 2-3: Cyclic voltammetry cell (B) containing an A g reference electrode  39  Figure 3-1: T L C analysis of products obtained from 2-(2-Nitro-l-H-imidazol- l-yl)acetic acid (7) synthesis  55  Figure 3-2: T L C analysis of products obtained from EF1 synthesis  66  Figure 3-3: T L C analysis of MXl(-l) compounds (X =F, CI, Br)  79  Figure 3-4: T i N M R spectrum of EF1 in d -acetone  111  Figure 3-5: * H N M R spectrum of EFl(-l) in d -acetone  Ill  Figure 3-6: Typical IR spectrum of a 2-nitroimidazole, EF1  113  Figure 3-7: ORTEP view of EF5; 33 % probability thermal ellipsoids are shown  114  Figure 3-8: ORTEP view of MF5; 33 % probability thermal ellipsoids are shown  115  6  6  Figure 3-9: ORTEP view of 2M4NF5; 33 % probability thermal ellipsoids are shown.. 116 Figure 3-10: ORTEP view of 2M4NF1(-1); 33 % probability thermal ellipsoids are shown  117  Figure 3-11: A lowering of pH levels in hypoxic cells due to increased [C0 ]  118  Figure 3-12: Quasi-reversible C V plot for EF1 referenced to FeCp  120  2  Figure 3-13: C V plot for MCIl(-l) referenced to FeCp  xvi  2  2  121  Figure 3-14: ORTEP view of 19; 33 %'probability thermal ellipsoids are shown  123  Figure 3-15: ORTEP view of EF2Br; 33 % probability thermal ellipsoids are shown....128 Figure 4-1: T L C analysis of products obtained from synthesis of 52 150 Figure 4-2: ORTEP view of Ru(Im)  2+ 6  (28); 50% probability thermal ellipsoids are  shown  154  Figure 4-3: ORTEP view of Ru(NMeIm)  2+ 6  (29); 33% probability thermal ellipsoids are  shown  155  Figure 4-4: ORTEP view of Ru(5MeIm)  2+ 6  (30); 50% probability thermal ellipsoids are  shown  156  Figure 4-5: Proposed stepwise D M F displacement by 2N02lm for the synthesis of [Ru(2N0 Im) ][CF,S0 ]2 (34) 2  161  3  6  Figure 4-6: In situ T i N M R spectrum in C D O D during the synthesis of 34  162  Figure 4-7: T i N M R spectrum for 39 in d -dmso  167  3  6  Figure 4-8: Geometrical isomerization of complex 40 in d -dmso  168  6  Figure 4-9: Possible configurations of a dimer formed from 2Me5N0 Im  168  Figure 4-10: IR spectrum for the isolated, orange complex 41  169  Figure 4-11: *H N M R spectra for complex 41 in C D O D (initial and after 6 d)  170  Figure 4-12: Successive aquation of 43 (L = SR2508) and 44 (L = EF5)  172  2  3  Figure 4-13: TI N M R (200 MHz) spectrum (Evans Method) of 43 (0.0032 g / mL D 0 , 2  in a 0.1 mm capillary tube) referenced to the residual proton peak, HOD. 172 Figure 4-14: Isomerization of c/'s-RuCl (DMSO) (en) from 46a to 46b  174  Figure 4-15: P{ H} N M R spectrum for complex 49, with proposed structure  176  2  31  2  1  Figure 4-16: P{TI} N M R spectrum for the proposed 31  [RuCl (dppb)(EF5)] (u>dppb) 2  (50)  2  177  Figure 4-17: [RuCl (diop)] (u.-diop) and the proposed structure for 50 2  2  178  Figure 4-18: ORTEP view of /wer-RuCl (MeCN) (52); 50% probability thermal ellipsoids 3  3  are shown  180  Figure 5-1: T L C analysis for the synthesis of 88 from 85  207  Figure 5-2: Resonance structures for acetylacetonate (acac")  211  xvii  Figure 5-3: ORTEP view of Ru(hfac) (70); 50 % probability thermal ellipsoids are 3  shown  213  Figure 5-4: TT N M R spectrum of Ru(hfac) (acac) (87) in CDC1 at r.t 2  217  3  Figure 5-5: Relationship between E i and I o in 0.1 M TBAP at 25°C. — • —, this /2  m  work (CV data in MeCN); — • —, Patterson and Holm (polarographic data in D M F ) .  218  15  Figure 5-6: Proposed reaction mechanism for acid catalyzed displacement of acac (adapted from ref. 19)  220  Figure 5-7: ORTEP view of c/5-Ru(hfac) (MeCN) (71); 50 % probability thermal 2  2  ellipsoids are shown  222  Figure 5-8: ORTEP view of cw-Ru(acac)(hfac)(MeCN) (88); 50 % probability thermal 2  ellipsoids  227  Figure 5-9: H N M R spectrum of czs-[Ru(acac) (Im) ][CF S0 ] (55) in d -acetone l  2  2  3  3  6  230  Figure 5-10: *H N M R spectrum of cis-[Ru(acac) (NMeIm) ][CF S0 ] (58) in 2  2  3  3  d -acetone  232  6  Figure 5-11: ORTEP view of the cation of czs-[Ru(acac) (Im) ][Tf] (55); 50 % probability 2  2  thermal ellipsoids are shown  234  Figure 5-12: ORTEP view of cz5-[Ru(acac) (NMeIm) ][Tf] (58); 50 % probability thermal 2  2  ellipsoids  235  Figure 5-13: ORTEP view of the cation of czs-[Ru(acac) (2MeIm) ][Tf] (60); 50 % 2  2  probability thermal ellipsoids  236  Figure 5-14: ORTEP view of the cation of czs-[Ru(acac) (5MeIm) ][Tf] (62); 50 % 2  2  probability thermal ellipsoids  237  Figure 5-15: TT N M R spectra for complexes 72 and 73 in d6-acetone  241  Figure 5-16: Proton signals for the coordinated NMelm ligands of 74 in d -acetone.... 242 6  Figure 5-17: *H N M R spectrum of [Ru(hfac)(NMeIm) ][PF ] (76) in d -acetone 4  6  6  242  Figure 5-18: TT N M R spectra of cis- and zra«s-Ru(lrfac) (2MeIm) (77 and 79) in 2  2  d6-acetone  243  Figure 5-19: TT N M R of the mixed-ligand complex Ru(hfac) (4MeIm)(5MeIm) in 2  d6-acetone  244  xviii  Figure 5-20: Cyclic voltarnmograms for cis- and rra«s-Ru(hfac) (2MeIm) in 0.1 M 2  2  Bu NC10 in M e C N 4  247  4  Figure 6-1: Diagram of 'tox' vessel used for the accumulation and toxicity experiments  257  Figure 6-2: Different types of SCCVII cells observed on microscope slides  260  Figure 6-3: Setup used for irradiation of the "ducks" in the radiosensitzation assay  263  Figure 6-4: Pictorial summary of the in vitro MoAb assay  264  Figure 6-5: Comparison of toxicity for selected compounds (at 100 u M except for TF5) in oxic and hypoxic SCCVII cells (incubated for 3 h); average of 2 or 3 experiments. (The plating efficiencies for the other 2N0 Ims, which also 2  show little oxic/hypoxic variability, reported in Table II-1, Appendix II.)... 267 Figure 6-6: Accumulation data for RuCl (SR2508) (EtOH) in SCCVII cells after a 3 h 3  2  incubation; reported as ng Ru/10 cells (± 5 %)  272  6  Figure 6-7: DNA-binding data for RuCl (SR2508) (EtOH) in SCCVII cells after a 3 h 3  2  incubation; reported as ng Ru/mg D N A (± 5 %)  274  Figure 6-8: Radio sensitization by SR2508 (600 uM) and EF5 (150 uM) and lack thereof by their Ru complexes RuCl (SR2508) (EtOH) (308 uM) and RuCl (EF5) 3  2  3  2  (EtOH) (60.6 uM); after 3 h pre-incubation with hypoxic SCCVII cells. ... 275 Figure 6-9: The indocarbocyanine fluorescent dye Cy3  276  Figure 6-10: Direct comparison of relative median fluorescence intensity and radioactive drug uptake for 9L cells incubated with 100 u M EF5 for 3 h at indicated oxygen partial pressures (adapted from ref. 13)  277  Figure 6-11: Relative mean fluorescence intensity for SCCVII cells incubated with lOOuM drug for 3 h under N and then treated with ELK3-51 (Cy3); 2  determined using flow cytometry (average of 2 or 3 expts.; ± 10 %). (The oxic data are reported in Table II-2, Appendix II.)  279  Figure 6-12: Log of mean fluorescence signal determined by flow cytometry versus the compound's reduction potential (determined by CV; chapter 3, Section 3.2.8). See Table II-3, Appendix II  xix  280  Figure 6-13: Relative mean fluorescence intensity for SCCVTI cells incubated with lOOuM drug for 3 h under N and then treated with ELK3-51; determined 2  using image cytometry (data from 2 expts.; ± 1 0 %). (The oxic data are reported in Table II-4, Appendix II.)  281  Figure 6-14: Relative mean fluorescence intensity for SCCVTT cells incubated with drug for 3 h under N and treated with ELK3-51; determined using flow 2  cytometry (data for Ru complexes from 2 experiments; ± 1 0 %). All values are normalized for EF5 content (100 uM). The m-PtCl (NH )(EF5)(PtEF5) 2  3  results were obtained from ref. 13  283  Figure 6-15: Relative mean fluorescence intensity for SCCVII cells incubated with lOOuM drug for 3 h under N and treated with ELK5-A8; determined using 2  flow cytometry. (The oxic data are reported in Table II-5, Appendix II.) . 285 Figure 6-16: Proposed recognition sites for the ELK5-A8 MoAb  286  Figure 7-1: Proposed coordination geometry of [RuCl(PPh ) (2M4NF3(-l))]Cl  296  3  2  List of Schemes Scheme 2-1: Standard amide bond forming reaction  42  Scheme 3-1: Synthesis of petafluoropropylamine from perfluoropropionic acid.  35  96  Scheme 3-2: Synthesis of IF5, the precursor to E F 5  97  Scheme 3-3: Formation of aziridine ring upon exposure of 3 to U V light  98  Scheme 3-4: Cyclization of IBr in the presence of heat and/or UV-radiation  98  Scheme 3-5: Synthesis of 3-fluoropropylamine reported by Pattison et al.  99  45  Scheme 3-6: Reaction of 3-hydroxypropylamine with DAST  100  Scheme 3-7: High yield Gabriel synthesis of 3-fluoropropylarnine hydrochloride  101  Scheme 3-8: Reaction mechanism for addition of side-chain via an amide linkage  102  Scheme 3-9: Synthesis of 7, via 8 from SR2508  102  Scheme 3-10: Oxidation of metronidazole with Jone's Reagent to yield the carboxylic acid derivative, 13  103  Scheme 3-11: Acid hydrolysis of 2-methyl-4-nitro-l-imidazolepropionitrile (abbreviated as  xx  RCN)  104  Scheme 3-12: New synthetic route to EF5  104  Scheme 3-13: Formation of the isobutylester (11) in the presence of H 0  106  Scheme 3-14: The 3-step synthesis of RevF5 from 2-bromoethylphthalamide  107  Scheme 3-15: Stepwise reduction of 2-nitroimidazole to 2-aminoimidazole  118  Scheme 3-16: Synthesis of ECll(-l) via the tosylate intermediate  124  2  Scheme 3-17: Synthesis of fluoroetanidazole (EFl(-l)) via a tosylate intermediate. ... 125 77  Scheme 3-18: Mechanism for reaction of SR2508 with D M F to yield the formate ester  (18)  126  Scheme 4-1: Conversion of 31 -> 32 — > 33, with suggested formulations (L= 2MeIm). 158 Scheme 5-1: Synthesis ofNa[Ru(hfac) ] (68) from R u C l ' 3 H 0 3  3  212  2  Scheme 5-2: Synthesis of c/5-Ru(acac)(hfac)(MeCN) (88) from Ru(acac) (hfac) (85)..223 2  2  Scheme 5-3: Attempted synthesis of c/'s-Ru(acac)(hfac)(MeCN) (88) from 2  Ru(hfac) (acac) (87)  224  2  Scheme 7-1: Proposed reduction of bis-CN compound (24) with BH »THF  292  3  Scheme 7-2: Proposed synthesis of ligands containing two or three - C H C F C F units..294 2  2  3  Scheme 7-3: Proposed synthesis of [ F]-EF1 similar to that used by Tewson to 18  synthesize [ F]-fluoroetanidazole  295  18  Scheme 7-4: Possible F addition to fluoroalkenes to yield EF4 and EF5  296  2  List of Tables Table 3.1: Summary of the T i N M R , F{ H} N M R and UV-Vis data for « N 0 I m 19  l  2  compounds and for their reaction with Bu4NF»H 0 2  Table 3.2: Summary of reduction potentials for the 2-nitroimidazoles vs. SCE  93 94  Table 3.3: Summary of reduction potentials for the 2-methyl-5-nitroimidazoles vs. SCE.95 Table 3.4: Summary of reduction potentials for the 2-methyl-4-nitroimidazoles vs. SCE 95 Table 4.1: Conductivity measurements versus time for 44 in H 0 2  146  Table 4.2: Summary of attempted reactions for which no pure product was isolated or characterized  151  Table 5.1: Summary of Ru(III/II) reduction potentials vs. SCE  xxi  210  Table 5.2 Summary of UV-Vis data for complexes 53, 70, 85 and 87 in M e O H [U Table 5.3  !  215  (exlO' )] 3  216  H and F { H } N M R data for the Ru(III) tris(p-diketonato) complexes 19  J  Table 5.4 N M R data for the Im and Melm complexes [Ru(acac) (L) ][CF S03] in 2  2  3  231  d6-acetone at r.t  Table 5.5 N M R data for the free imidazole ligands and their complexes Ru(hfac) (L) 2  2  240  in d6-acetone at r.t  Table 6.1 Actual drug concentrations used for in vitro experiments  265  Table 6.2 Aerobic and hypoxic toxicity data (PE at 3 h) of Ru complexes in SCCVII 269  cells. (See Table 6.1 for complex concentrations.) Table 6.3 Accumulation of Ru complexes (normalized to 100 uM) in SCCVTI cells incubated for 3 h; expressed as ng Ru/10 cells (± 5 %) 6  270  Table 6.4 Amount of Ru bound to D N A extracted from SCCVII cells after 3 h exposure to complex (normalized to 100 uM; ng Ru/mg D N A (± 5 %))  273  Table 7-1 : Summary of most significant in vitro assay results obtained during this thesis 300 Al  Table 1-1 1 Experimental details for EF5 Table 1-1 2 Atomic coordinates and B i / B for EF5  A2  Table 1-1 3 Bond lengths (A) for EF5  A2  Table I-1 4 Bond angles (°) for EF5  A2  S0  eq  A3  Table 1-2 1 Experimental details for EF2Bi-0 5 H 0 2  Table 1-2 2 Atomic coordinates and B / B i80  eq  for EF2Br«0.5 H 0 2  A4  Table 1-2 3 Bond lengths (A) for EF2BH3.5 H 0 2  A4  Table 1-2 4 Bond angles (°) for EF2Br«0 5 H 0  A4  Table 1-3 1 Experimental details for MF5  A5  2  Table 1-3 2 Atomic coordinates and B i / B for MF5  A6  Table 1-3 3 Bond lengths (A) for MF5  A6  Table 1-3 4 Bond angles (°)for MF5  A6  Table 1-4 1 Experimental details for 2M4NF5  A7  S0  eq  Table 1-4.2 Atomic coordinates and B / B for 2M4NF5 eq  A8  Table 1-4.3 Bond lengths (A) for 2M4NF5  A8  Table 1-4.4 Bond angles (°)for 2M4NF5  A9  is0  Table 1-5.1 Experimental details for 2M4NF1(-1) Table 1-5.2 Atomic coordinates and B / B i s o  e q  A10  for 2M4NF1(-1)  Al1  Table 1-5.3 Bond lengths (A) for 2M4NF1(-1)  Al1  Table 1-5.4 Bond angles (°)for 2M4NF1(-1)  Al1  Table 1-6.1 Experimental details for cycF3  A12  Table 1-6.2 Atomic coordinates and B / B for cycF3  A13  Table 1-6.3 Bond lengths (A) for cycF3  A13  Table 1-6.4 Bond angles (°) for cycF3  A14  iso  eq  Table 1-7.1 Experimental details for [Ru(Im) ][CF S0 ]2 6  3  Al5  3  Table 1-7.2 Atomic coordinates and B / B for [Ru(Im) ][CF S0 ] is0  eq  6  Table 1-7.3 Bond lengths (A) for [Ru(Im) ][CF S0 ] 6  3  3  Table 1-7.4 Bond angles (°)for [Ru(Im) ][CF S0 ] 6  3  3  3  A16 A16  2  6  i s o  e q  A16  2  2  Table 1-8.1 Experimental details for [Ru(NMeIm) ][CF S0 ] Table 1-8.2 Atomic coordinates and B / B  3  3  3  A17  2  for [Ru(NMeIm) ][CF S0 ] 6  Table 1-8.3 Bond lengths (A) for [Ru(NMeIm) ][CF S0 ] 6  3  3  Table 1-8.4 Bond angles (°)for [Ru(NMeIm) ][CF S0 ] 6  3  3  3  A18 A18  2  3  3  A19  2  Table 1-9.2 Atomic coordinates and B / B for [Ru(5MeIm) ][CF S0 ] iso  eq  6  Table 1-9.3 Bond lengths (A) for [Ru(5MeIm) ][CF S0 ] 6  3  3  Table 1-9.4 Bond angles (°)for [Ru(5MeIm) ][CF S0 ] 6  3  3  Table 1-10.2 Atomic coordinates and B / B is0  eq  3  A20  2  3  A21  3  for /wer-RuCl (MeCN) 'CHCl 3  3  Table 1-10.4 Bond angles (°)for we/--RuCl (MeCN) »CHCl 3  3  Table 1-11.1 Experimental details for Ru(hfac)  Table 1-11.2 Atomic coordinates and B / B for Ru(hfac)  xxiii  3  3  3  3  A22 A22 A22 A23  3  eq  A20  2  A20  Table 1-10.3 Bond lengths (A) for / w ^ - R u C l ( M e C N y C H C l  iso  3  2  Table 1-10.1 Experimental details for /«e/--RuCl (MeCN) 'CHCl 3  A18  2  2  Table 1-9.1 Experimental details for [Ru(5MeIm) ][CF S0 ] 6  3  3  A24  Table 1-11.3 Bond lengths (A) for Ru(hfac) Table 1-11.4 Bond angles (°)for Ru(hfac)  A24  3  A25  3  Table 1-12.1 Experimental details for Ru(hfac) (MeCN) 2  Table 1-12.2 Atomic coordinates and B / B iso  for Ru(hfac) (MeCN)  eq  2  Table 1-12.3 Bond lengths (A) for Ru(hfac) (MeCN) 2  Table 1-13.2 Atomic coordinates and B / B iso  A26  2  A27  2  A29  2  for Ru(acac)(hfac)(MeCN)  eq  Table 1-13.3 Bond lengths (A) for Ru(acac)(hfac)(MeCN) Table 1-13.4 Bond angles (°) for Ru(acac)(hfac)(MeCN)  A30  2  A30  2  A31  2  Table 1-14.1 Experimental details for [Ru(acac) (Im) ][CF S0 ]»benzene 2  Table 1-14.2 Atomic coordinates and B / B is0  2  3  A32  3  for [Ru(acac) (Im) ][CF S0 ]'benzene..A33  eq  2  2  3  3  Table 1-14.3 Bond lengths (A) for [Ru(acac) (Im) ][CF S0 ]«benzene  A33  Table 1-14.4 Bond angles (°) for [Ru(acac) (Im) ][CF S0 ]»benzene  A33  Table 1-15.1 Experimental details for [Ru(acac) (NMeIm) ][CF S0 ]  A34  2  2  2  3  2  3  3  3  2  Table 1-15.2 Atomic coordinates and B i / B s o  2  3  3  for [Ru(acac) (NMeIm) ][CF S0 ]  e q  2  2  3  3  A35  Table 1-15.3 Bond lengths (A) for [Ru(acac) (NMeIm) ][CF S0 ]  A35  Table 1-15.4 Bond angles (°)for [Ru(acac) (NMeIm) ][CF S0 ]  A36  2  2  2  3  2  3  3  3  Table 1-16.1 Experimental details for [Ru(acac) (2MeIm) ][CF SO ]«0.5 hexane 2  Table 1-16.2 Atomic coordinates and B / B iso  2  3  3  A37  for [Ru(acac) (2MeIm) ][CF SO ]»0.5  eq  2  2  3  3  hexane  A3 8  Table 1-16.3 Bond lengths (A) for [Ru(acac) (2MeIm) ][CF SO ]»0.5 hexane  A38  Table 1-16.4 Bond angles (°) for [Ru(acac) (2MeIm) ][CF SO ]»0.5 hexane  A38  Table 1-17.1 Experimental details for [Ru(acac) (5MeIm) ][CF S0 ]  A39  2  2  2  3  2  3  2  Table 1-17.2 Atomic coordinates and B / B i s o  e q  3  3  2  3  3  for [Ru(acac) (5MeIm) ][CF S0 ] 2  2  3  3  A40  Table 1-17.3 Bond lengths (A) for [Ru(acac) (5MeIm) ][CF S0 ]  A41  Table 1-17.4 Bond angles (°) for [Ru(acac) (5MeIm) ][CF S0 ]  A41  2  2  2  2  3  3  3  3  Table II-1: Toxicity (PE ± 10 %) for selected compounds (at 100 uM) in oxic and hypoxic SCCVII cells (incubated for 3 h)  xxiv  A42  Table II-2: Relative mean fluorescence intensity for SCCVII cells incubated with l O O u M drug for 3 h under N and then treated with ELK3-51 (Cy3); determined using 2  flow cytometry (average of 2 or 3 expts.; ± 10 %)  A42  Table II-3: Log of mean fluorescence signal determined by flow cytometry and the compound reduction potential (determined by C V vs. SCE)  A42  Table II-4: Relative mean fluorescence intensity for SCCVII cells incubated with l O O u M drug for 3 h under N and then treated with ELK3-51; determined using image 2  cytometry (data from 2 expts.; ± 10 %)  A43  Table II-5: Relative mean fluorescence intensity for SCCVII cells incubated with l O O u M drug for 3 h under N and treated with ELK5-A8; determined using flow 2  cytometry  A43  XXV  List of Abbreviations Abbreviation Sr  2D AAS Ab acacH B.M. b.p. BCCRC BSA BSO CCD CHO CNS COSY CTRON CV d DAST DCC DCU dd DEM DMAD DMF dmso DMSO DMSO DNA dppb ELISA ELK en EPR equiv. FAB FCM FDG GC GI GSH Gy  Meaning relative permitivity 2-dimensional atomic absorption spectroscopy antibody acetylacetone Bohr magneton boiling point British Columbia Cancer Research Centre bovine serum albumin buthionine sulphoximine charge coupled device Chinese hamster ovary (cell line) central nervous system correlated spectroscopy chromatotron cyclic voltammetry day diethylaminosulfonytrifluoride dicylcohexylcarbodiimide dicyclohexylurea doubly distilled diethylmaleate dimethyladenine N,N-dimethylformamide dimethylsulfoxide (solvent) dimethylsulfoxide coordinated through O-atom dimethylsulfoxide coordinated through S-atom deoxyribonucleic acid 1,4 - bis(diphenylphosphino)butane enzyme-linked immunosorbent assay Edith Lord Koch 1,2-diaminoethane (ethylenediamine) electron paramagnetic resonance equivalent fast atom bombardment flow cytometry fluorodeoxyglucose gas chromatography gastrointestinal glutathione gray xxvi  List of Abbreviations (cont.) HEPES hfacH HR-MS HSC iBuClFrm ICM Im IOI K LAH LET lit. LMCT LR-MS LSJJVIS m.p. MALDI MLCT MO MoAb MRI MWCO NBS NEM NHE NHS NMM N0 N0 lm NVIm OD ORTEP P PBS PE PET pF r.t. RBF RF Rf RNA RS a  2  2  N-2-hydroxylethylpiperazine-N'-2-ethane sulfonic acid 1,1,1,6,6,6-hexafluoroacetylacetone high resolution mass spectrometry hypoxia selective cytotoxin z'so-butylchloroformate image cytometry imidazole integrated optical intensity acid dissociation constant lithium aluminum hydride linear energy transfer literature reference ligand-to-metal charge transfer low resolution mass spectrometry low energy secondary ionization mass spectrometry melting point matrix assisted laser desorption ionization metal-to-ligand charge transfer molecular orbital monoclonal antibody magnetic resonance imaging molecular weight cutoff N-bromo succinimide N-ethyl maleimide normal hydrogen electrode N-hydroxysuccinimide N-methylmorpholine nitro nitroimidazole N-vinylimidazole optical density Oakridge Thermal Ellipsoid Program partition coefficient phosphate buffered saline plating efficiency positron emission tomography paraformaldehyde room temperature round-bottom flask radio-frequency retention frequency ribonucleic acid radiosensitizing  xxvii  List of Abbreviations (cont.)  SCCVII SCE SDS SER SF SR2508 SR4233 STMU'BF T/C TBAP TE Tf Tf 0 TF5 2  TLC TMSO TNE TsCl V  4  squamous cell carcinoma VII (cell line) saturated calomel electrode sodium dodecylsulphate sensitivity enhancement ratio surviving fraction etanidazole tirapazamine (1,2,4-benzotriazin-3-amine-1,4-di-N-oxide) 0-(N-succinimidyl)-N, N , N ' , N'-tetramethyluronium tetrafluoroborate treated/control tetrabutylammonium perchlorate tris(hydroxymethyl)aminomethane + ethylenediaminetetraacetic acid triflate (CF3SO3") trifluoro sulfonic anhydride 3 -N-pentafluoropropylamido-1,2,4-benzotriazine-1,4-di-Noxide thin layer chromatography tetramethylenesulfoxide T E + 150 m M NaCl p-toluenesulfonyl chloride vinyl  xxviii  Key to Chemical Compound Numbers and Abbreviations The imidazole ring is numbered as shown in Figure i . The 1-position is typically referred to as the N-position at which various types of side-chains were added. For this work the 2-, 4- and 5-positions were usually substituted with either Me or N 0 groups to 2  give the corresponding imidazole compounds (Ri = R = R 4 - R 5 - H , Im; R = R 4 = R 5 = H 2  2  and R i = Me, NMelm; R = R 4 = R = H and R = Me, 2MeIm; R = R = R4/5 = H and R4/5 x  5  2  x  2  = Me, 4(5)MeIm; R i = R 4 = R = H and R = N 0 , 2N0 Im; Ri = R = R4/5 = H and R4/5 = 5  2  2  2  2  N 0 , 4(5)N0 Im; Rj = R 4 = H and R = Me and R = N 0 , 2Me5N0 Im; R = R = H and 2  2  2  5  2  2  x  5  R = Me and R 4 = N 0 , 2Me4N0 Im). 2  2  2  R4  R5  Figure i: Imidazole position numbering.  Numerous compounds were synthesized and used during the course of this thesis. The nitroimidazoles synthesized in chapter 3 were typically given names corresponding to the derivative from which they were synthesized (E = etanidazole (SR2508), M = metronidazole, 2M4N = 2-methyl-4-nitro-) followed by the type and number of halogen atoms on the terminal propyl group attached to the amide N-atom of the N I side-chain (e.g. EF5 has a terminal - C F - C F group, 5 F-atoms). A (-1) included at the end of the 2  3  compound's name (e.g. EFl(-l)) corresponds to a terminal ethyl group (i.e. one - C H 2  group shorter). Other starting materials, side-products and incompletely characterized compounds are numbered 1 thru 27 in chapter 3. The Ru complexes reported in chapters 4 and 5 are numbered in the sequence in which they appear. It is suggested that the Quick Reference sheets on the following pages are used when reading the specific chapters.  xxix  Chapter 3 Quick Reference Compound Library  2  Y  N  N0  CH CH CH CH CH CH CH CH CH CH CH CH CH CH  R  N  Compound  R  2N0 Im compounds  O 2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  CF CF CF CF Br CH CF CF CH CF Br CH=CF CH CH F CH F CH CH C1 CH C1 CH CH Br CH Br CH CH CH(CH )  EF5 EF4Br EF3 EF3(-1) EF2Br E=F2 EF1 EFl(-l) ECU ECll(-l) EBrl EBrl(-l) EPrA EIAA  CF CF CF CH F CH C1 CH Br  MF5 MF3(-1) MFl(-l) MCll(-l) MBrl(-l)  2  3  2  2  2  3  2  2  3  2  2  2  2  2  2  2  2  2  2  2  3  3  2  5N0 Im compounds 2  N0  CH CH CH CH CH  2  R O  Me  2  2  2  2  2  2  3  3  2  2  2  4N0 Im compounds 2  0 N  CH CH CH CH CH  0  2  N'  ,R  I  Me  2  2  2  2  2  CF CF CF CH F CH C1 CH Br 2  3  3  2  2  2  2M4NF5 2M4NF3(-1) 2M4NF1(-1) 2M4NC11(-1) 2M4NBrl(-l)  H  Misc. Compounds  R-  ^CF  o  R = I, IF5 = CI, C1F5  3  i  RevEF5  IBr  XXX  ImF5  H  -X  H  H  0  1  1  .~  N.  CF  —  2  ^CF  3  3  2  H a N ^ ^ ^ ^ F .HC1  4  5 0  N 0  T N 0  2  o 2  7 /  °  8  \  N  6  9  JSTH CI 3  N Y  N 0  T  10  2  F  F  11 N 0  N 0  2  f \  .OH  2  r K . 0 .  VT  Me  Me  13 N 0  12  oN  Me  V  OH  7  Y  N  2  0  °  O  O  N ^ N ^ V ^ ^ I j r ^ CBr H 2  20  O—l  21  19 N  Io  H  0 H  2  22  ^V  I  T  0.  2  ^  24  \ / ^ C \  NOj  H  23  T F  0  NO, 25  H  18  >  N  0  T  17  16  NO,  N  N0  Me  SO, N  H  0  N y N ^ Me  15  14  2  2  Js.  T F  26  xxxi  0  27  Chapter 4 Quick Reference Compound Library  Compound Number  Composition  28  [Ru(Im) ][CF S0 ]2  29  [Ru(NMeIm) ][CF S0 ]  30  [Ru(5MeIm) ][CF S0 ]  31  rra/75-[Ru(CO)(DMF)(2MeIm) ][CF S0 ]2  32  [Ru(DMF)(2MeIm) ] [CF S 0 ]  33  [Ru(CF S0 ) (2MeIm) ] [CF S 0 ]  6  3  3  6  3  6  3  3  3  2  2  4  4  3  3  3  x  3  4  3  2  3  3  y  (x=2, y=0, or x=l=y)  34  [Ru(2N0 Im) ][CF S0 ]  2  35  [Ru(5N0 Im) ][CF S0 ]  2  36  [Ru(2Me5N0 Im) ][CF S0 ]  37  [Ru(DMF) (SR2508) ] [CF S 0 ]  38  [Ru(DMF) (EF5) (EtOH) ][CF S0 ]  39  /wer-RuCl (2N0 Im)  40  fac- and /wer-RuCl (5N0 Im)  41  "RuCl (2Me5N0 Im) -3 C 0 "  42  RuCl (metro)  43  RuCl (SR2508) (EtOH)  44  RuCl (EF5) (EtOH)  45  RuCl (metro)  46  cw-RuCl (DMSO) (en)  47  *ra«s-RuCl (DMSO) (en)  48  RuCl (DMSO) (EF5)(acetone)  49  [RuCl (dppb)(EF5)] (u-Cl)  50  [RuCl (dppb)EF5] (u-dppb)  51  cw-RuCl (MeCN)4  52  /wer-RuCl (MeCN)  2  6  2  3  6  3  3  2  3  5  3  4  3  2  2  3  2  3  3  3  3  2  2  3  3  2  2  3  2  3  3  2  3  3  2  3  2  2  4  2  2  2  2  2  2  2  2  2  2  2  3  xxxii  3  2  3  3  3  3  Chapter 5 Quick Reference Compound Library  Compound Number  Composition  53  Ru(acac)  54  c/'5-[Ru(acac)2(MeCN)2][CF S03]  55  czs-[Ru(acac) (Im) ][CF S0 ]  56  toms-[Ru(acac) (Im) ][CF S0 ]  57  c/5-[Ru(acac) (MeCN)(Im)][CF S0 ]  58  C75-[Ru(acac)2(NMeIm) ][CF S0 ]  59  cz5-[Ru(acac) (MeCN)(NMeIm)][CF S0 ]  60  cw-[Ru(acac) (2MeIm) ][CF S0 ]  61  fraws-[Ru(acac) (2MeIm)][CF S0 ]  62  c/5-[Ru(acac) (5MeIm) ][CF S0 ]  63  c/5-[Ru(acac) (4MeIm)(5MeIm)] [CF S0 ]  64  [Ru(acac)2(2N0 Im) ] [CF S0 ]  65  [Ru(acac) (SR2508) ][CF SO ]  66  [Ru(acac) (EF5) ][CF S0 ]  67  [Ru(acac) (metro) ][CF S0 ]  68  [Na][Ru(hfac) ]  69  [Ru(hfac)(EtOH) ] [hfac]  70  Ru(hfac)  71  c/5-Ru(hfac) (MeCN)  72  cz'5-Ru(hfac) (Im)  73  cw-Ru(hfac) (Im)(MeCN)  74  c/'5-Ru(hfac) (NMeIm)  75  [Ru(hfac)(NMeIm) ] [hfac]  76  [Ru(hfac)(NMeIm) ] [PF ]  77  c/s-Ru(hfac) (2MeIm)  78  ds-Ru(hfac) (2MeIm)(MeCN)  3  3  2  2  2  3  2  3  3  3  2  3  2  3  3  3  2  3  2  2  3  2  3  3  2  2  3  3  3  2  3  2  2  2  3  2  2  2  3  2  3  3  2  3  3  3  3  3  4  3  2  2  2  2  2  2  2  4  4  2  2  xxxiii  3  6  2  3  Compound Number  Composition  79  fr-a«5-Ru(hfac) (2MeIm)2  80  c/5-Ru(rrfac)2(4MeIm)(5MeIm)  2  81  fra«5-Ru(rrfac)2(4MeIm)(5MeIm)  82  Ru(hfac) (2N0 Im)  83  Ru(hfac) (EF5)  84  Ru(hfac) (SR2508)  85  Ru(acac) (hfac)  86  [Na][Ru(hfac) (acac)]  87  Ru(hfac) (acac)  88  c/5-Ru(acac)(hfac)(MeCN)  89  cz'5-Ru(acac)(hfac)(Im)2  90  cz5-Ru(acac)(hfac)(NMeIm)  91  cw-Ru(acac)(hfac)(NMe!m)(MeCN)  2  2  2  2  2  2  2  2  2  2  EF5  SR2508  xxxiv  2  2  Acknowledgements  I would like to thank my supervisors Brian James and Kirsten Skov for their guidance, encouragement, and support throughout the duration of this work. The low-key role they played was essential in helping me become a better scientist. I would also like to thank the departmental services, especially Lianne Diarge, Marietta Austria, Peter Borda, Steve Rak, the late Steve Rettig, and the people at mass spectrometry for their support. From the B C Cancer Research Centre I am indebted to Hans Adomat, "the allknowing-one," for help with essentially everything associated with the in vitro experiments. The help provided by Haibo Zhou and Jennifer Decker (thanks for staying late with me so many times) was also greatly appreciated. I must also give special thanks to Cameron Koch and Alex Kachur at the University of Pennsylvania and Mike Adam at TRTUMF for their collaboration on this project. From BRJ's lab a special thanks must go to Jeff Posakony (Mr. Late Night) for being a great lab partner, running buddy, workout guru and diving mate. Never did I think that my second year T A would become my lab companion; thanks for waiting around Jeffy. Matt LePage (PoochBoy) must be commended for being the weirdest person in the lab B Y FAR! Thanks for making sure things did not get too serious Matt. Other members of the James group past and present: Ken MacFarlane, Terrance Wong, Patrie Meessen, Paul Cyr, Nathan Jones, Erin Ma, Elizabeth Cheu, Dave Kennedy (and all the rest) deserve honorable mention. Thanks to all for being my friend and also helping with the proofreading of parts of this thesis. I must also thank Jeff and Katja for the fishing trips to rainbow country and Paul (my hunting buddy) and Helen for the many fantastic moose hunting trips up north. I am sure that it was these trips away from the city which helped me keep my sanity in an otherwise chaotic world. Most importantly I must thank my beautiful wife, Kerry, for being the most caring, understanding and giving person I know. Without her encouragement throughout these past five years I surely would not have achieved this goal. I would also like to thank my parents and the rest of my family for all their love and support over the years. Deluxe! Asta-lavista baby!  xxxv  Chapter 1 Introduction  1.1  Introduction Cancer is a complex family of diseases that is characterized by abnormal gene  expression, resulting in uncontrolled growth and spread of abnormal cells. If the spread (metastases) of the cancer cells is not controlled it can ultimately lead to death. Approximately one in three people in North America will develop some form of cancer in their lifetime, and of these people approximately two-thirds will die of the disease. The most common cancers in the western world are lung, breast and colorectal cancer, which account for approximately 50 % of all cancer related deaths.  1  Although it is now possible to successfully treat ("cure") testicular cancer with cisplatin, most other cancers have limited response to chemotherapy and have only prolonged survival at best. Hence, a major goal is to develop a system of cancer 2  treatment which cures patients of their tumours while avoiding harmful side-effects to normal tissues. Our increased understanding of tumour biology and the cellular processes determining the behavior of cancer cells has allowed treatments to be directed specifically towards the tumour.  3  The differences in tumour versus normal tissue stem from the rapid growth of cells within tumours which leads to formation of a tumour microenvironment. Typically, solid tumours contain areas of low extracellular p H and regions of low oxygen concentration (hypoxia). Exploitation of these properties, especially hypoxia, has been advantageous  for the treatment of cancer using both organic compounds (e.g.  radiosensitizers, hypoxic-selective cytotoxins) and metal complexes (e.g. antineoplastic and antimetastatic agents, see Section 1.6.3). However, the early detection of cancer using known imaging procedures (i.e. MRI, PET), prior to metastasis, is essential to ensuring patient survival. With early detection there would be approximately a 30 % increase in patient survival post treatment.  1  1  References on page 26  Chapter 1  This chapter introduces the concept of hypoxia, how it develops in tumours, how it is measured/imaged and the postulation that hypoxia may make tumours more aggressive. The use of metal complexes in cancer therapy, focussing on Ru, is also briefly reviewed. These two topics are the major focus of this thesis and are reflected in the design and synthesis of the new compounds reported.  1.2  Hypoxia  1.2.1 What is it? Hypoxic cells are those that are deficient in oxygen, yet remain viable, and are often found in tumours. Such cells may also exist in other living tissue; for example, both heart tissue damaged by cardiac arrest or a stroke and retinal tissue in the eye contain hypoxic cells. The tissue in malignant tumours often outgrows its supply of oxygen and nutrients as the tumour enlarges. This is due to an inadequate production of a network of functioning blood vessels and capillaries. This poorly organized vasculature accounts for many of the environmental features unique to tumours. Although cells deprived of oxygen 4  and nutrients ultimately die, at any given time a tumour may possess viable hypoxic cells. There are currently two accepted mechanisms by which hypoxia may develop in tumours. First is the classical "chronic" diffusion-limited model of hypoxia. With chronic 5  hypoxia, as oxygen diffuses from the blood, a gradient is formed where oxygen concentration decreases with increased distance from the vasculature (Figure 1-1).  Hypoxic Necrotic Figure  1-1: Oxygen gradient observed in a typical tumour (adapted from ref. 6).  2  References  on page 26  Chapter 1  Studies have determined the mean distance from the vasculature to the necrotic regions of a tumour to be 150 urn, consistent with the estimated diffusion range of oxygen. The cells 5  just inside this distance, a layer of one to two cells thick, were proposed to be hypoxic. The second mechanism is illustrated by the "acute" form of hypoxia which results from a localized pinching-off of blood vessels (i.e. when blood flow within the tumour 7  vasculature fluctuates, resulting in a temporary cessation due to the transient opening and closing of vessels ' ). The cells affected from acute hypoxia will, however, only be 8 9  resistant to radiotherapy if the hypoxic environment lasts throughout the treatment (Section 1.2.1.1). The presence of viable hypoxic cells (both chronic and acute) has been observed in both animal ' and human tumours. 9 10  11  1.2.1.1 Resistance to Treatment: Radioresistance Ionizing radiation is commonly used to kill tumour cells, however, it is not equally effective on each cell within a tumour. The fate of cells after radiation can be attributed to the biological effect of ionizing radiation which is greatly affected by the oxygen partial pressure within the cells (the "oxygen effect"). The ratio of hypoxic to oxic doses needed to achieve the same biological effect is referred to as the oxygen enhancement ratio (OER). For sparsely ionizing radiations, such as X - or y-rays, the O E R at high doses has a value between 2.5 and 3 (Figure 1-2). For densely ionizing radiation T  I O  i_i S  1  1  1  L.  i  «S  i> •  15  20  Dose (Gy)  1  r  25  30  Figure 1-2: Typical results for X-ray irradiation of Chinese Hamster Ovary cells under aerobic (O2) or hypoxic (N2) conditions (adapted from ref. 6).  3  References  on page 26  Chapter 1  there is no oxygen effect. For the oxygen effect to be observed, oxygen must be present during the irradiation because the lifetime of the radicals produced by radiation via the indirect effect of low L E T radiation is very short. The free radicals formed (typically from the hydrolysis of water) result in cleavage of chemical bonds leading to lesions which initiate the chain of events that results in the final expression of biological damage. The chemical damage produced by free radicals in D N A can be sometimes repaired under hypoxia, but the damage is "fixed", that is, made permanent and not infrequently irrepairable if molecular oxygen is present. The sensitivity of living cells to damage induced by radiation is due to a competition between oxygen and endogenous thiols (e.g. glutathione) for radiation induced radicals on the D N A .  12  The electron affinic oxygen reacts with the unpaired  electrons produced on the D N A resulting in lesions which the cell has difficulty repairing, particularly the double-strand break. At the same time, hydrogen atom donation to the 12  unpaired electron from molecules such as glutathione yield lesions which are more readily repaired and it is this process which predominates in the absence of oxygen. To combat this effect various chemicals (DEM, N E M , diamide) which oxidize thiols have been tested against D N A repair with limited success. However, buthionine sulphoximine (BSO), a compound which selectively inhibits y-glutamyl cysteine synthetase (the  enzyme  responsible for glutathione synthesis), exhibits some radiosensitization and has been shown to increase the effectiveness of other radiosensitizers (e.g. nitroimidazoles). '  13 14  As a result of the radioresistance of hypoxic cells, a number of different methods have been employed to treat many tumours which might contain hypoxic regions. 1) Fractionated radiation treatment (2-2.5 Gy administered 5 days a week for 6 weeks) ensures that the treated cells could not accumulate and repair sublethal damage and the effect of increased cellular proliferation (repopulation) would be nullified. Moreover, the variation in the radio sensitivity of cells in different phases of the cell cycle and the ability of hypoxic cells to undergo reoxygenation would both be accounted for; 2) hyperbaric oxygen could be used to increase oxygen concentration within the blood in hopes of increasing the oxygen concentration in the tumour; 3) hypoxic-selective radiosensitizers (Section 1.3.1), many of which are oxygen mimetic, are useful. Like oxygen, these species  4  References on page 26  Chapter 1  are highly electron affinic, but unlike oxygen they are not metabolized as they diffuse through the tissue and so actually reach and hence accumulate within the hypoxic cells; and 4) the use of bioreductive cytotoxins, which selectively target and directly kill hypoxic cells, can be beneficial. Methods 2-4 (all containing selectivity for hypoxia) provide a therapeutic advantage over other treatments because hypoxic cells are rare in normal tissue while they occur frequently in tumour tissue.  1.2.2 Hypoxia: The Aggressor? Over the past 60 years there has been increasing evidence that hypoxia is prevalent in certain tumours and it poses a major problem in the treatment of cancer with either radiotherapy or chemotherapy. ' ' A meta analysis reporting all hypoxic sensitizer 5 15 16  studies (>10,000 patients), including those agents now known to be ineffective, showed only a 14 % improvement in outcome. ' There is clearly an urgent need to find better 17 18  methods of detection, and to select those patients who would benefit from adjuvant therapy such as a sensitizer or hypoxic cytotoxin. There is compelling evidence that hypoxia in the tumour has far greater implications than the response to treatment of the primary tumour itself. Direct evidence for this comes from the assessment of hypoxia in patients containing carcinomas of the uterine cervix, using an Eppendorf electrode to determine oxygen concentration prior to treatment. ' The treatment of patients with radiation during this study showed a direct 19 20  correlation between tumour inhibition and low hypoxic fractions. More importantly though, is that hypoxia was prognostic for outcome (curability) after surgery. For patients containing either cervix carcinomas or soft tissue sarcomas, the result was significantly 21  22  better in those whose tumours were relatively well oxygenated and were treated with surgery, implying that tumours with hypoxia are more aggressive. Indirect evidence for hypoxia as an aggressor includes reports that hypoxia promotes genomic instability, a potential factor in tumour heterogeneity and progression. The report that the expression 23  of the p53 gene (which encodes a tumour supressor protein important in chemical and viral carcinogenesis, apoptosis and cell cycle drug resistance) is induced by hypoxia and that cells exposed to hypoxia die by p53-mediated apoptosis is essential to our  5  References on page 26  Chapter 1  understanding of hypoxia. Hypoxic regions were shown to correlate spatially with highly apoptotic regions of tumours. However, cells lacking intact p53 were resistant to low 24  oxygen concentrations; hence hypoxia selects for mutant p53, and these cells survive. Hypoxia was also shown to be present in metastases which affected the cell's response to radiation; this may potentially increase the metastatic potential of a tumour. The above findings which suggest that hypoxia makes a tumour more "aggressive" (or vice-versa), greatly increase the importance of determining which patients have hypoxia-containing tumours (not all tumours contain hypoxia). Finding treatments which will not only eradicate the hypoxic cells in the primary tumour, but also be directed towards the metastatic disease accompanying the aggressiveness caused by hypoxia, is also imperative.  1.3  Role of Nitroimidazoles in Cancer Therapy  1.3.1  Hypoxic Radiosensitizers In the early 1970s nitroimidazoles were first proposed as radiosensitizers because  of the high electron affinity of the nitro group. The 5-nitroimidazole, metronidazole (Figure 1-3), demonstrated radiosensitization in a variety of murine tumour systems and 25  its low toxicity and long biological half-life made it a good candidate for clinical testing. The drug at high doses, however, resulted in severe gastrointestinal (GI) intolerance which led to the search for a more effective, less toxic radiosensitizer. A number of 2-nitroimidazoles (2NC«2lrn) were investigated due to their higher electron affinity (more positive E1/2 value) and were shown to be more active than their 5-nitroimidazole analogues in vitro. The leading compound of this series was misonidazole (Figure 1-3) which showed marked activity in many different tumour models in vivo using several different assays.  25  Initial clinical trials with misonidazole using single doses appeared  promising, with a decrease in GI intolerance from that of metronidazole. Notwithstanding, when multiple doses were administered patients developed neurological complications, 27  limiting the doses required to produce significant hypoxic cell sensitization.  6  References  on page 26  26  Chapter 1  K  N.  H  l=\  OH  OCH  OH  3  Me  Metronidazole (flagyl)  Misonidazole (Ro 07-0582)  Etanidazole  (SR2508)  Figure 1-3: Structures of metronidazole, misonidazole and etanidazole. Investigations of the mechanism of neural toxicity observed for misonidazole and other related compounds have revealed that toxicity is related to lipophilicity of the compounds. This study revealed that lowering the partition coefficient (lipid:water) of a 28  2-nitroimidazole, via alteration of side-chain substituents on N l of the imidazole ring, decreased the penetration of drug across the blood-brain barrier. The reduction of lipophilicity also led to poorer absorption of the drug into the blood from the peritoneal cavity. This problem was overcome by using intravenous injection. These studies also revealed that the partition coefficient of a  2N02lm  could be lower than that of  misonidazole by a factor of up to 20 without compromising tumour penetration or the radio sensitizing effectiveness. The  2N02lm, etanidazole  (Figure 1-3), exhibited a greatly  reduced lipophilicity compared to misonidazole (0.046 compared to 0.43) resulting in more rapid plasma clearance and less penetration into neural tissue, while maintaining the potency of misonidazole in vivo  29  Clinical trials of etanidazole demonstrated that patients  could tolerate a total dose of three times that of misonidazole without significant side-effects; however, etanidazole did result in nausea, vomiting and peripheral neuropathies which limited its use.  29  1.3.2 Chemistry of Nitroimidazoles in Biological Systems The importance of using nitro-containing compounds (e.g. nitroimidazoles) for treatment and/or detection of hypoxia stems from the reducibility of the nitro group at physiological pH. A mechanism involving the reduction of the nitro group has been proposed (Figure 1-4). Under hypoxic conditions the nitro group is reduced (it has been 30  suggested that cytochrome P450 reductase is the dominant enzyme for the reduction of 2N0 Ims ) through a number of reactive intermediates, one or more of which is 31  2  7  References on page 26  Chapter 1  presumed to be toxic to the cells. The selectivity towards hypoxic cells is because the 32  reverse oxidation to the parent nitroimidazole is effectively reduced in the presence of low oxygen concentrations. In aerobic cells the reverse oxidation results in futile cycling of the nitro group with concomitant production of the superoxide radical and hydrogen peroxide. A direct correlation between the sensitizing efficiency and electron affinity has 33  been supported by various pulse radiolysis studies on one-electron transfer reactions between known radiosensitizers.  r=\  N^  Nitroreductase  .NR NO,  n W  2  •  Futfle  Redox Cycling  n W  34  NR  r\ -  e',2H  e  -H,0  N0 '  M  "  - r\  e  »  Hydroxylamine  Nitroso  Radical Anion  y  NHOH  NO  2  2  N  N.  .NR NH, Amine  Figure 1-4: Nitroimidazole reduction scheme (adapted from ref. 30). The position of the nitro group on the imidazole ring is also of marked importance to the effectiveness of the nitroimidazole as a sensitizer. The electron affinity of the nitro group is measured by the one-electron  reduction potential  of the  nitroimidazole; the more positive the Em value the easier the reduction to the radical nitro anion. The reduction potentials for 2-, 4-, and 5-nitroimidazoles fall in typical ranges (Figure 1-5), with those of the  2NC«2lms  being the most positive.  32  Ei/ (mV) 2  2-nitroimidazole  -360 to -400  5-rritroimidazole  -510 to -545  4-nitroimidazole  -540 to -685  NO,  NO,  0,N N ^ N - R  Figure 1-5: Ranges of reduction potentials for nitroimidazoles vs. N H E at pH 7.  8  References on page 26  Chapter 1  In the nitroimidazole reduction scheme, focussing on 2-nitroimidazoles, three products are expected from reduction in two-electron jumps. 2-Aminoimidazoles can be prepared as stable compounds by chemical methods and have been detected as metabolites of 2-nitroimidazoles. However, the amines lack significant biological activity, leading to 35  the consensus that one of the intermediate reaction products, either the 2-hydroxylaminoimidazole or the 2-nitrosoimidazole, is involved in adduct formation within the cell.  36  The nitrosoimidazole, l-methyl-2-nitrosoimidazole, has been isolated in the solid state, but was found to be unstable in aqueous solution with a maximum half-life of 10 h at neutral pH. The product in acidic conditions, typical of hypoxic regions, has been identified, and results from the nucleophilic addition of water to the imidazole ring (Figure 1-6). Products at neutral pH are more complex, but appear to arise from ring 37  opening reactions of the hydrolyzed species. Reactions have also been observed with phosphines and aromatic amines, but the products have not yet been identified. 2-Nitrosoimidazole is more electron affinic than oxygen, can cause strand breaks in D N A and its half-life in cells with [GSH] > 0.1 m M is estimated to be less than one second. The rapid redox reaction with G S H is thought to be the major mode of activity for the nitrosoimidazole, leading to the formation of hydroxylaminoimidazole.  37  H H HO. I I ^OH /  Ns^  y  \  .N—Me  2 H 0 , H+ 2  **  H - N o . .N—Me  © y  NO  NHOH  Figure 1-6: Nucleophilic addition of water to l-methyl-2-nitrosoimidazole. The 2-hydroxylaminoimidazoles have a longer half-life (-3-5 min) than the 2-nitrosoimidazoles and form stable species in acid. Products of further reaction have 37  been identified as the dihydrodihydroxyimidazolium ions resulting from the N-0 bond heterolysis of the hydroxylamine that yields the nitrenium ion, which subsequently reacts with two equivalents of water. In the presence of other nucleophiles, the chemistry is modified such that the nucleophile competes with water for the nitrenium ion intermediate, and nucleophile-incorporated products are formed (Figure 1-7). These products include  9  References  on page 26  Chapter 1  covalent binding with G S H and other protein thiols,  guanine (DNA), phosphates (DNA)  and other biological amines. '  37 39  r\  H-N(T~}NR  ©y  f=\ N.  N^NR  NHOH 2 -hydroxylarninoirnidazolium ion  NH  ©  W  .NR  Y® NH  nitreniurn ion  ^OH^  H0 2  00 3 v jH_ LH^ O H HPO,  ^0 N  ©  H  1  PO  e  y NH,  H H  I  j \ / ^NR N  R  n  i  ». H-N*. I V .NR ro  ©y'  NH,  NH RSH  H H PhNH^I ^OH  H H  H-N^ .NR © y NH,  H-N^ -NR © y NH,  Figure 1-7: Proposed reactions of 2-hydroxyaminoimidazole within the cell.  1.4  Imaging of Hypoxic Tumours The ideal marker of tissue oxygenation should: 1) have a large ratio of absolute  binding between hypoxic tissue and aerobic tissue (large dynamic range); 2 ) exhibit an oxygen dependency that is similar to that of cellular radiosensitivity; 3) have a partition coefficient which facilitates its rapid diffusion into both well-perfused and poorly-perfused tissues; 4) contain a probe which can be accurately measured by invasive and/or noninvasive procedures; and 5) have no toxicity at its optimum concentration for this diagnostic use.  30  Many techniques have been used in an attempt to meet these  requirements, a few of which are summarized in the following subsections.  10  References  on page 26  Chapter 1  AAA  Radiolabelling of Hypoxic Cells By taking advantage of the selective bioreduction and subsequent binding of  nitroimidazoles under conditions of low oxygen concentration, it was proposed that incorporation of H or C into the imidazole would allow for detection of hypoxia by 3  1 4  radioactivity. For C-misonidazole, for example, it was determined using this technique 14  that the binding to cellular molecules within hypoxia was proportional to the square root of the drug concentration. immunochemical techniques  40  Detection methods are liquid scintillation techniques, or through autoradiography  of histological sections  depending on the label attached to the nitroimidazole. This last mentioned technique was used for patients with different metastatic lesions and, although there was a large tumour to plasma ratio of radiolabeled nitroimidazole, the high dose of radiolabels required made this approach unsuitable as a predictive assay of tumour hypoxia.  41  1.4.2 Nuclear Medicine Techniques Similar to the technique described above, this approach uses high energy y-emitting radionuclides as labels on nitroimidazoles. The  82  Br-labelled 4-bromo-  misonidazole compound was one of the first compounds developed in an attempt to quantify the amount of hypoxia in a tumour. Biodistribution and blood clearance studies 42  revealed that the label was retained on the compound in vitro and in vivo and that the tumour and normal tissue distribution kinetics were similar or superior, from the perspective of imaging, to those of the parent compound, misonidazole. There was also higher uptake in the tumour for the bromo derivative, likely resulting from the significantly higher partition coefficient when compared to that of misonidazole (2.9 vs. 0.43). Moreover, this property also led to increased toxicity, presumably due to CNS toxicity. Hence, the usefulness of this compound as a hypoxia imaging agent was abandoned. More recently, a series of 2-nitroimidazoles linked to iodinated sugars has been developed. These I-radioiodinated azomycin (2N02im) nucleosides show promise for 123  use in the detection of tumour hypoxia while avoiding the toxicity of previously developed compounds. The leading compound in this series is iodoazomycin arabinoside (IAZA, Figure 1-8) and was shown to undergo hypoxia-dependent binding in murine tumour  11  References  on page 26  Chapter 1  models in vitro."' ' Tumour to blood ratios of 5-10 at 8 h post injection were observed 1  indicating areas of hypoxia in vivo for EMT-6 tumours in B A L B / c mice. These promising results led to the clinical study investigating  123  I - I A Z A as a potential non-invasive marker  of hypoxia which is currently under consideration.  44  CM  Figure 1-8: Iodoazomycin arabinoside (IAZA).  1.4.3  Fluorescent Probes Analogous to the methods used to develop radiolabeled compounds for the  detection of hypoxia, the addition of a fluorophore chemically linked to the side-chain of the 2-nitroimidazole moiety was pursued in hopes of utilizing the selective binding of the nitro species in hypoxia. Provided that the chain linking the 2-nitroimidazole moiety does not suffer metabolic cleavage from the fluorophore, the fluorescence should be "fixed" within the hypoxic cells. The best compound developed using this technique was an indolizine-linked 2-nitroimidazole species (Figure 1-9) which yielded a maximum fluorescence differential between oxic and hypoxic cells of 12.5:1 when tested in vitro. Unfortunately, when this probe was tested in vivo there was no observable difference between oxic and hypoxic cells, presumably due to an insufficient concentration of compound remaining in the cells.  \  45  ,CH CH(OH)CH —N 2  2  CONH(CH ) N 2  2  \  •(CH ) OH 2 2  Figure 1-9: Indolizine-linked 2-nitroimidazole, fluorescent hypoxia probe. 12  References on page 26  Chapter 1  Fluorescent markers for the detection of hypoxia have also been used. The first 46  study of this kind involved raising polyclonal antibodies against the protein adduct of reductively-activated CCI-103F. The antibodies were coupled with a fluorescent dye and when introduced to hypoxic cells pretreated with CCI-103F, the antibodies remained intact within the cells, as determined by fluorescence. Studies with murine tumour models in vivo have shown that the antibody binds to the drug adduct in tumour sections with patterns similar to those revealed by autoradiographic studies of Ff-labelled CO-1COF. 3  46  H  CCI-103F  EF5  Figure 1 - 1 0 : Structures of the hypoxia-selective fluorinated 2-nitroimidazoles CCI-103F and E F 5 . More recently, a pentafluorinated  derivative of etanidazole ( E F 5 )  was  synthesized and a monoclonal antibody (ELK3-51), significantly more specific than the previously used polyclonal antibodies for E F 5 and EF5-protein adducts, was isolated. The dynamic range (between nitrogen and air) of binding by E F 5 , as measured by radioactive drug uptake, was a factor of at least 50 in human cells and 100 in rodent cells. '  47 48  Consequently, it is important that the drug adduct-detecting antibodies have very high sensitivity and specificity. This was the case for ELK3-51, which revealed, by flow cytometric analysis, the same dynamic range and oxygen dependence as the radioactivity assay. The advantage of the immunological method is that the signal difference between oxic and hypoxic cells can be detected on an individual cell basis which allows for measurement of inter- and intra-tumour heterogeneity. The development of solid state microscopy techniques now enables quantitative assessment of the hypoxia distribution within a tumour.  49  This technique is currently the most powerful method available for  hypoxia imaging and quantification.  13  References on page 26  Chapter 1  1.4.4  Magnetic Resonance Imaging (MRI) M R I is a biological multi-slice imaging technique utilizing the effect of atoms on  an applied magnetic field, and is able to access specific areas of the body. The application of a magnetic field results in the orientation of nuclear spins, usually parallel to the magnetic field. For MRI, the patient is subjected to energy in the form of radiowaves via an RF (radio-frequency) pulse. This energy causes some of the low energy atoms (P, H , or F) to flip to their high energy (anti-parallel) state. A two-dimensional Fourier transform then manipulates the signal into an output which allows a computer to transform it into a signal strength versus intensity picture. M R I has been used as a non-invasive diagnostic method for detecting tumour hypoxia for molecules containing P , *H and F . P - M R I makes use of the fact that 31  19  31  compounds associated with cellular energetics contain phosphorus atoms (especially ATP). In animal tumours these spectra were shown to correlate with changes in oxygen concentration as measured by microelectrodes, but there was too much scatter in the data to be clinically useful. ^ - M R I makes use of the high rate of glycolytic metabolism found 50  in tumours resulting from lactate production. However, interference from other cellular signals make conclusive measurement difficult. The incorporation of  19  51  F atoms into known hypoxia-selective agents like  2-nitroimidazoles has also been investigated. '  52 53  The hexafluorinated 2-nitroimidazole  CCI-103F was shown to lose the hexafluoropropyl substituent from its side-chain in vivo, but the trifluorinated hypoxia probe SR4554 was shown to remain intact upon enzymatic reduction in vivo, displaying selective uptake into the tumour in preliminary M R I studies. M R I studies with EF5, which revealed a dynamic range of at least 50 using immunohistochemical techniques (as above), are currently underway in hopes of producing a clinically useful M R I hypoxia probe.  1.4.5  54  Positron Emission Tomography (PET) PET is a biological imaging technique that allows one to measure in microscopic  detail the function and/or metabolism of a labelled compound in specific areas throughout the body. This technique uses short-lived, neutron-poor, positron-emitting isotopes  14  References on page 26  53  Chapter 1  produced by a cyclotron such as 0 (half-life of 2 min), 1 5  18  1 3  N (10 min), C (20 min) and n  F (110 min) which are incorporated into biological substrates, substrate analogues or  drugs. PET, like M R I , allows for multi-slice imaging. The most attractive candidate radionuclide is  18  F due to its sufficiently long half-life which allows for complex or  multistep organic synthesis. The decay of F is predominantly by positron emission (97 18  %) and the positron is of relatively low energy (0.635 M e V maximum) and thus has a short mean range (2.39 mm in water).  55  When the positron meets an electron they  annihilate each other. A l l that remains is energy in the form of two y-rays, of equal intensity, going in opposite directions (Figure 1-11). The y-rays are then registered by detectors within the PET camera to generate a useful image.  Figure 1-11: y-ray production from positron-emitting isotope [ F]. Fluorine is an interesting substituent in medicinal chemistry with a long and successful history of F-incorporation into organic pharmaceuticals. Fluorine is small (atomic radius, 0.57 A) and exhibits high C-F bond energies (~ 106 kcal/mol). Fluorine is also extremely electronegative (x = 3.98) and its substitution into a molecule can often produce large changes in physiochemical and biological properties. A good example of this is the differences observed between SR2508 (no F-atoms) and E F 5 (5 F-atoms). The most widely used PET agent to date, fluorodeoxyglucose (FDG), incorporates an F atom 18  into the ring of D-glucose and is used for imaging in both cancerous and non-cancerous tissues.  56  15  References  on page 26  Chapter 1  In order to provide a non-invasive detection method for hypoxia,  F was  incorporated into a proven, hypoxia-selective 2-nitroimidazole. The first and most investigated compound of this type was [ F]-fluoromisonidazole; 18  compound is not ideal due to its hypoxia-independent tissue retention. syntheses of [ F]-fluoroetanidazole 18  59  and [ F]-EF1 18  60  57  however, this  58  More recently,  were reported, both showing  promising hypoxia imaging results in cells and small animals. These findings indicate that the introduction of a non-invasive hypoxia imaging agent into the clinic is not far off.  1.5  Role of Metals in Cancer Therapy Initially the use of metals and metal complexes as antitumour agents was not  considered by most scientists due to the fact that numerous cases of poisoning, especially with heavy metals, had been documented. Researchers did not assume that such metals could be used to synthesize effective therapeutic agents when introduced to different ligand environments. Systematic studies into the relationship between chemical structure 61  and efficacy or toxicity of metal compounds began only about sixty-five years ago. Collier and Krauss in 1931 descibed the first metal complex tumour-inhibiting study and developed a theory, which remains valid: "the effect of a heavy metal on experimental murine cancer is not only due to the metal alone, but also the structure of the compounds and the type of compound." Nevertheless, it was not until 34 years later (1965) when the 62  first metal-containing anticancer drug was discovered, somewhat serendipitously by Rosenberg et al., that the field of metal complexes in cancer therapy escalated. 63  Rosenberg's  new platinum "wonder-drug"  was initially generated as an  electrolysis product formed by the association of dissolved platinum (from a Pt electrode) with the ammonium cation from a culture medium containing E. coli bacteria. '  The  63 64  resultant product was identified as (NFL^MPtCle] which converted to c/s-[PtCl4CNH ) ] via 3  2  a photochemical reaction. Similar neutral, cz's-amine Pt species were synthesized and the series was tested for possible antitumour activity against the mouse tumour, Sarcoma 180. All of the compounds reduced tumour growth, but the most promising complex was cis-[PtCl2(NH ) ] (now known as cisplatin, Figure 1-12), which entered clinical trials in 65  3  2  1971 and was approved by the US Food and Drug Administration in 1978 for treatment of  16  References on page 26  Chapter 1  testicular and ovarian cancer. Due to the severe toxic side-effects associated with 2  cisplatin therapy, in particular nausea and vomiting, neuropathy, ototoxicity and most significantly, nephrotoxicity, the search for metal complexes having similar activity but with decreased side-effects was initiated.  cisplatin  carboplatin  JM216  AMD473  Figure 1-12: Chemical structures of antitumour agents cisplatin, carboplatin, JM216 and AMD473. A less toxic analogue of cisplatin, carboplatin (Figure 1-12), was introduced into clinical practice in 1981, but disappointingly the two drugs were effective against the same population of tumours and thus shared cross-resistance with one another. '  66 67  A novel  class of platinum (IV) dicarboxylates which demonstrated promising oral activity against a number of murine tumour models was discovered within the past ten years. In preclinical 68  studies, one such compound (bis-acetatoammine(dichloro)cyclohexylamineplatinum (IV)) (JM216, Figure 1-12) provided a structural lead to platinum complexes which may circumvent transport-determined resistance to cisplatin; '  69 70  this complex contains  toxicological properties reminiscent of carboplatin rather than cisplatin. More recently, 71  the sterically hindered Pt(II) complex AMD473 has entered clinical trials because of its circumvention of cisplatin resistance.  72  Since the discovery of cisplatin, a myriad of different metal complexes (Ti, V , Mo, Tc, Fe, Ru, Co, Rh, Ni, Pd, Cu, Au, Ga, Ge, Sn and Bi) have been synthesized and tested for their antitumour activity both in vitro and in vivo in hopes of discovering drugs which have activity against Pt resistant tumours. A few examples are Au(I) auranofin which displays irreversible inhibition of D N A synthesis in HeLa cells ; Rh(II) carboxylates 73  which act to inhibit murine ascitic tumours ; and Ti(IV) titanocene dichloride which 74  17  References  on page 26  Chapter 1  demonstrates a reduced growth of solid animal tumour systems and in some cases this effect was more pronounced than that of cisplatin. For the most part these complexes do 75  not have the chemical properties required for an antitumour agent (see below). Most metal complexes have dose-limiting side-effects and are not tumour specific, restricting their possible use in the clinic. For example, Ga(III) nitrate was shown to inhibit tumour growth by greater than 90 % in 6 of 8 solid tumours transplanted subcutaneously,  76  but the  gallium concentration was 130 times higher in the kidney than in the tumour. The lack of 77  selectivity likely explains this drug's poor clinical effect. It is important, when considering the utility of a potential metal-containing antitumour agent, to take into account its kinetic characterisitics. If too labile, the complex is likely to interact with physiological nucleophiles before reaching its site of action in the tumour and, if too inert, the complex may not interact with its biomolecular target as required to produce the antitumour effect. In this regard, many metals form complexes 78  that would be deemed too labile. However, the Pt group metals (Ru, Os, Rh, Ir, Pd, Pt) are capable of forming many relatively inert complexes whose kinetic properties can be tailored by variation of the ligands about the metal centre. Although the main focus over the past 30 years has been on Pt antitumour complexes, the biological effects of Ru complexes, with the possibility of producing Rubased drugs active in the treatment of human malignancies, have also been widely investigated. " 79  85  The Ru complexes differ from cisplatin, particularly when tumour  sensitivity and the effects of tumour reduction on host survival time are concerned. This suggests that Ru complexes (the focus of this thesis work) have a select place in cancer therapy, opening further directions in the development of a new generation of anticancer agents.  79  1.6  Ruthenium Complexes  1.6.1  Chemical Properties Relevant to Tumour Treatment As antitumour drugs are generally cytotoxic, the development of drugs which  selectively target the tumour to ensure a selective cell kill is essential. The use of Ru over Pt is one approach to avoid the problems associated with Pt drug therapy. 18  References on page 26  Chapter 1  There are two significant differences between Ru- and most of the Pt-based antitumour drugs which make their prospective mechanism of action advantageous: 1) the usually six-coordinate, octahedral geometry of Ru complexes (versus square planar Pt (II)) may allow for new modes of binding to nucleic acids, and 2) the ease with which electron-transfer takes place for the Ru ™ couple because of a small activation barrier (see 1  below).  83  The most stable complexes in aqueous solution are generally Ru and R u and 11  m  these are usually readily interconverted via oxidation/reduction within biological systems. The Ru  111  ion (d , low-spin) usually accepts 7i-electron density into its partially filled 5  dn-orbital and thus has a strong attraction for halides and anionic oxygen ligands; a low-spin, d Ru ion has little affinity for 7i-donors, but readily binds 7i-acceptor ligands. 6  11  This is due to further extended rL. orbitals which lead to better 7r-orbital overlap. The 86  R u reduction potentials can be tuned by changing the type of coordinated ligand. For m  87  example, the reduction potential for [L(NH3) Ru ] varies from -0.08 V (L = OH) to 1.1 V m  5  (L = N 2 ) . In general, anionic a-donor ligands lower the reduction potential, while neutral or cationic, 7c-acceptor ligands raise it. As the Franck-Condon barrier to electron transfer is low for Ru ™ couples, owing to small bond distortions between these two ions, redox 1  reactions are often rapid.  88  The reduction of R u to Ru allows for the use of Ru m  11  111  complexes as "prodrugs"  for Ru species which have an elevated propensity to coordinate to biomolecules. In 11  particular, the binding of R u with the imine N of imidazole rings on histidine and n  purines '  89 90  predominates where reducing power is relatively high and oxygen, which can  reoxidize Ru back to R u , is virtually non-existent. Such an environment occurs in many 11  tumours  91,92  m  which are hypoxic due to their higher metabolism and diffusion limited  oxygen supply. ' Tumours also typically have a lower pH which favours pH-dependent 93 94  reduction of some complexes. Consequently, the R u / R u ratio should be higher in most 95  n  m  types of tumours than in other tissues leading to increased binding and hence localized cytotoxicity. And so, the treatment of tumours with Ru complexes (potentially toxic for 11  all tissues) should be avoided in favour of the more inert R u prodrugs. m  19  References on page 26  Chapter 1  The accumulation of Ru ions within tumour tissues is not only due to reduction of R u , but is also significantly increased by coupling with transferrin. ' m  96 97  phenomenon is due to the similarities between Ru affinity for phenolate ligands '  98 99  ffl  This  and F e , which both have a high m  which are integral in complexing F e in transferrin for m  transport through the blood to the tissues. Release of R u from transferrin (like Fe ) may m  be facilitated by lower p H or reduction to Ru , 11  100  m  which has little affinity for phenolate.  1.6.2 DNA Binding Antitumour agents are expected to block the D N A activity of tumour cells in order to be effective, and accordingly D N A is thought to be the major target for most antitumour drugs. Ru complexes are no exception. The bulk of evidence suggests that D N A binding is possible, and in some instances is as frequent and effective as with cisplatin, for which a D N A interaction has been well documented using X-ray crystallography.  101  Cisplatin (czs-DDP) is believed to result mainly from the 1,2 (GG) and 1,3 (GXG) intrastrand cross-links within D N A which trans-DDP cannot form. The 1,2 cross-link involves binding of Pt to N7 of adjacent deoxyguanosines along the same strand of D N A to form a 17-membered chelate ring (Figure 1-13).  102  This intrastrand adduct can  be removed by nucleotide excision repair (an entire oligomer containing the adduct is excised ), but some results suggest that the repair of a G G intrastrand cross-link is 103  significantly impaired when compared with that for monofunctional adducts.  104  The  intrastrand cross-link also results in a bending of the D N A double helix (a 33° kink) which promotes binding of a specific class of damage-recognition proteins.  It has been  105  postulated that these proteins may be important in cisplatin toxicity by blocking access of repair enzymes to damaged D N A .  1 0 6  The trans isomer of cisplatin (transplatin) was  determined to be much less active likely due to its steric inability to form the major intrastrand cross-links. Ru complexes, and more importantly Ru complexes, were shown to be capable of 11  strong interaction with oligonucleotides (e.g. with calf thymus D N A )  89  for which  preferential N - N adduct formation between two adjacent guanines, identical to that of  20  References on page 26  Chapter 1  cisplatin, was observed.  However, Clarke reports that the G G cross-link formation with  cr  H  Figure 1-13: Cisplatin/DNA interaction to form a G G intrastrand cross-link. D N A is not essential for antitumour activity, with, for example, pentaammine-Ru complexes as it is for the platinum drugs. The N7 of guanine is relatively exposed in the 83  major groove of B - D N A and the metal complexes, typically positively charged species (e.g. [Ru(NH ) (H 0)] ), undergo a fairly strong electrostatic attraction for the 2+  3  5  2  polyanionic D N A . Once bound to the D N A the Ru complex is also thought to induce localized denaturation of the D N A which makes the interior sites (N6 of adenine and N4 of cytosine) available for Ru interaction. 11  107  Interestingly, a comparison of D N A binding  for cis- and zraws-RuCl2(DMSO)4 reveals that the trans isomer covalently binds at a markedly higher reaction rate (behaviour opposite to that of cis- and trans-DDP) and protects G-rich sequences from cutting by specific restriction endonucleases, indicating a preferential interaction with adjacent guanines.  90  DNA-bound Ru was also found after in vivo treatment with antitumour doses of either Ru or Ru 11  111  complexes; this was measured by atomic absorption spectroscopy, in  samples of D N A extracted from tumour cells and from cells of lung, internal mucous, liver  21  References on page 26  Chapter 1  and spleen tissue. These data displayed unequal distribution of DNA-bound Ru, which was more concentrated (selective localization) in the lung and tumour tissue than in the spleen or liver.  108  1.6.3 Tumouricidal Effects of Ru Complexes The selectivity of Ru complexes for tumours makes them highly appropriate for investigation in antitumour research. As none of the existing antitumour drugs are devoid of severe and dose-limiting side-effects, the search for novel non-toxic compounds continues. Preliminary studies have revealed that "Ru complexes may help to overcome the dose-limiting complication, and it would be hypothesized that new derivatives could find a place in therapy as new-generation target-specific anticancer drugs."  79  There is a myriad of newly synthesized Ru complexes reported in the literature each year, but those which exhibit antitumour activity can be grouped into three categories. The first is the Ru-ammine complexes, the first series of Ru complexes to undergo screening for antitumour activity. The compound, /ac-RuCl3(NH3)3 exhibited excellent activity in several tumour screens (up to T/C of 190 %); however, its poor solubility impedes it from adequate formulation as a drug.  80  Keppler and co-workers  synthesized a number of similar complexes, yielding species which were more soluble in aqueous media due to their anionic charge. For example, cw-[RuCl2(NH ) ]Cl (with T/C 3  4  values up to 160 %) is active against P388 leukemia, a highly sensitive platinum tumour model used to compare Ru drug performance to cisplatin. '  109 110  The second group of complexes is that characterized by the presence of D M S O , which, as a ligand, facilitates transport through, and penetration into membranes, increases complex water solubility and is significantly labile to undergo displacement by stronger binding nitrogenous D N A bases (especially N7 of guanine). Both cis- and transRuCl (DMSO)4 exhibit interesting antitumour properties, with the trans isomer exhibiting 2  significantly higher toxicity than the cis isomer. More importantly, both exhibit antimetastatic activity greater than cisplatin.  82  The third group (the focus of this thesis) is comprised of Ru complexes containing one or two heterocyclic ligands such as imidazole, pyrazole, indazole and their  22  References on page 26  Chapter 1  methyl substituted derivatives. Generally, these complexes exhibit significant antitumour activity.  81  For example, the water soluble, bis-imidazole complex [ImH][RuCl4(Im) ] 2  showed a T/C value of about 200 % against the P388 leukemia (cf. cisplatin, T/C 175 %).  1 U  The complex also possessed potent and selective antitumour activity in a  carcinogen-induced colorectal tumour of the rat. Assessment of cytotoxicity with all three classes of Ru antitumour complexes generally revealed a low in vitro effectiveness. This is not the case for all Ru complexes, 79  but it is clearly evident that most of them, despite being capable of reducing in vivo tumours and/or increasing the lifespan of tumour-bearing animals, are essentially void of any capacity to reduce cell viability when tested in vitro This implies that if the new Ru 19  complexes are initially screened using in vitro assays, the result might be misleading and hence fail to unmask the real effectiveness of these complexes against cancer growth. The development of Ru complexes by examining the general effect of tumour growth, or by examining tumour size variation with solid tumours, may also limit the scope of these complexes as potential antitumour agents. It is clear that each Ru complex must be used at a maximum tolerated dose to significantly reduce primary tumours, while the reduction of solid tumour metastases is achievable at doses with undetectable cytotoxic side-effects for the hosts.  112  The development of antimetastatic drugs for the treatment of solid tumours is essential in combating cancer which can readily disseminate throughout the host. Tumour metastases behave quite differently from their primary neoplasms due to differences in drug sensitivity, antigenicity and clonogenic capacity.  113  The drugs available for the  management of human tumours are usually only active on primary lesions. Therefore, i f the tumour has metastasized, the antimetastatic drug becomes equally as important in prevention of tumour growth. The first compound capable of selective activity against metastatic tumours (frara-Na[RuCl (DMSO)(Im)]) was developed by Sava et al. and 4  showed a significant advantage for the postsurgical prognosis when associated with ablation of primary tumours.  114  The mechanism of action of this complex is, however,  poorly understood and so obtaining a full understanding of this process is essential to the  23  References on page 26  Chapter 1  preparation  of complexes  which  will  circumvent  the  actual  limits  of  trans-  Na[RuCl (DMSO)(Im)]. 4  1.7  Thesis Overview At the onset of this thesis work the major goal was to synthesize, characterize  and assess in mamalian cells in vitro new Ru nitroimidazole complexes for use as potential non-toxic  radiosensitizers.  pentafluorinated (ELK3-51), ' 47  49  However, because  of the  development  of E F 5 (a  2N02lm) and a highly selective monoclonal antibody to E F 5 the focus shifted to the synthesis and characterization of new 2-, 4- and  5-nitroimidazoles with halogenated side-chains (Chapter 3). Alteration of the side-chain and position of the nitro group on the imidazole ring provides valuable information on the interaction of the monoclonal antibody with the drug (Chapter 6). This thesis consists of seven chapters plus appendices of 17 X-ray structures. Chapter 2 describes the general experimental procedures used in the characterization of the newly synthesized complexes, including the synthetic procedures for all the Ru starting complexes used in Chapter 4. Each of Chapters 3, 4 and 5 contains its own experimental and results and discussion sections. Chapter 3 contains the synthesis and complete characterization of nearly 30 new 2-, 4-, and 5-nitroimidazoles, containing N l halogenated side-chains, including their reduction potentials as determined by cyclic voltammetry (CV). Chapter 4 describes the synthesis and characterization of many Ru complexes obtained from the reaction of imidazoles and nitroimidazoles with different Ru precursors (e.g. [Ru(DMF) ][CF S03]3, RuCl *3H 0, cis/trans-RuC\ (DMSO) ). 6  3  3  2  2  4  Many of  these complexes were paramagnetic Ru(IIT) species which made their characterization by N M R more difficult. Chapter 5 is comprised of the synthesis and characterization of Ru(II) and Ru(III) bis-acetylacetonate (acac), bis-l,l,l,6,6,6-hexafluoroacetylacetonate (hfac) and mixed acac/hfac complexes with imidazole and nitroimidazole ligands. A comprehensive study of the reduction potentials of these complexes, as determined by C V , suggests the capability of synthesizing a complex with a reduction potential in the desired biological range by altering the substituents on the chelating P-diketonato ligands. Chapter 6  focuses  on  the  in  vitro  testing  24  of  selected  nitroimidazoles  and  Ru  References on page 26  Chapter 1  imidazole/nitroimidazole complexes. The toxicity and monoclonal antibody recognition (using both M o Abs ELK3-51 and ELK5-A8) of some of the nitroimidazoles from Chapter 3 are evaluated in SCCVII cells. The Ru complexes are tested for their toxicities and abilities to accumulate within the cell and to coordinate to D N A , and a preliminary radiosensitization study of two of these complexes is included. Conclusions and suggestions for future experiments, including a report on the studies that are currently underway to answer some of the chemical and biological questions generated from this thesis work, are reported in Chapter 7.  25  References on page 26  Chapter 1  1.8 1  References Cook, A . R. The New Cancer Source Book, Health Reference Series, Vol. 12; Omnigraphics, Inc., Detroit, 1996, p. 3.  2  Flicker, S. P. In Metal Compounds in Cancer Therapy; S. P. Flicker, Ed.; Chapman and Hall, London, 1994, p.3.  3  Ruddon, R. W. Cancer Biology; Oxford University Press, New York, 1995, p. 3.  4  Jain, R. K . J. Natl. Cancer Inst. 1989, 81, 570.  5  Thomlinson, R. H.; Gray, L . H . Br. J. Cancer 1955, 9, 539. "  6  Farrell, N . In Transition Metal Complexes as Drugs and Chemotherapeutic Agents; R. Ugo and B. R. James, Eds.; Kluwer Academic, Dordrecht, 1989, p. 183.  7  Brown, J. M . Br. J. Radiol. 1979, 52, 650.  8  Dudar, T. E.; Jain, R. K . Cancer Res. 1984, 44, 605.  9  Chaplin, D . J.; Durand, R. E . ; Olive, P. L . Int. J. Radial Oncol. Biol. Phys. 1986, 12,  1279. 10 Moulder, J. E.; Rockwell, S. Int. J. Radiat. Oncol. Biol. Phys. 1984,10, 695. 11 Vaupel, P.; Kallinowski, F.; Okunieff, P. Cancer Res. 1989, 49, 6449. 12 Ward, J. F. In Radioprotectors and Anticarcinogens; O. F. Nygaard and M . G. Simic, Eds.; Academic Press, New York, 1983, p. 73. 13 Bump, E. A ; Yu, N . Y.; Brown, J. M . Pharmac. Ther. 1990, 47, 117. 14 Koch, C. J.; Skov, K . A . Int. J. Radiat. Oncol. Biol. Phys. 1994, 29, 345.  15 Bush, R. S.; Jenkin, R. D. T.; Allt, W. E. C ; Beale, F. A ; Bean, H . A ; Dembo, A . J.; Pringle, J. F. Br. J. Cancer 1978, 37, 255. 16 Teicher, B . A ; Laxo, J. S.; Sartorelli, A. C. Cancer Res. 1981, 41, 73. 17 Coleman, C. N . Br. J. Cancer 1996, 74, S297. 18 Overgaard, J. Conference Summary - clinical. 9 Int. Conf. on Chem. Modifiers of th  Cancer Treatment, Oxford, U . K., Aug. 22-26, 1995. 19 Vaupel, P.; Schlenger, K.; Knoop, C ; Hdckel, M . Cancer Res. 1991, 57, 3316. 20 Hockel, M . ; Schlenger, K.; Knoop, C ; Vaupel, P. Cancer Res. 1991, 51, 6098.  26  Chapter 1  21 Hockel, M . ; Schlenger, K.; Aral, B.; Mitze, M . ; Schaffer, U . ; Vaupel, P. Cancer Res. 1996, 56, 4509. 22  Brizel, D. M . ; Scully, S. P.; Harrelson, J. M . ; Layfield, L . J.; Bean, J. M . ; Prosnitz, L . R.; Dewhirst, M . W. Cancer Res. 1996, 56, 941.  23  Paquette, B.; Little, J. B . Cancer Res. 1994, 54, 3173.  24 Graeber, T. G.; Osmanian, C ; Jacks, T.; Housman, D. E.; Koch, C. J.; Lowe, S. W.; Giaccia, A. J. Nature 1996, 379, 88. 25  Fowler, J. F.; Denekamp, J. Pharmacol. Therapeut. 1979, 7, 413.  26 Dische, S. Int. J. Radiat. Oncol. Biol. Phys. 1991, 20, 147. 27  Brown, J. M . Int. J. Radiat. Oncol. Biol. Phys. 1984, 52, 650.  28 Brown, J. M . ; Workman, P. Radiat. Res. 1980, 82, 171. 29 Brown, J. M . ; Y u , N . Y . ; Brown, D. M . ; Lee, W. W. Int. J. Radiat. Oncol. Biol. Phys. 1981, 7, 695. 30 Chapman, J. D.; Lee, J.; Meeker, B. E. In Selective Activation of Drugs by Redox Processes; G. E. Adams, A. Breccia, E. M . Fielden, P. Wardman, Eds.; Plenum Press, New York, 1990, p. 316. 31 Joseph, P.; Jaiswal, A. K.; Stobbe, C. C ; Chapman, J. D. Int. J. Radiat. Oncol. Biol. Phys. 1994, 29, 351. 32  Adams, G. E . ; Flockhart, I. R.; Smither, C. E.; Stratford, I. J.; Wardman, P.; Watts, M . E. Radiat. Res. 1976, 67, 9.  33  Biaglow, J.; Varaes, M ; Roizen-Towle, L . ; Clarke, E.; Epp, E.; Astor, M . ; Hall, E. Biochem. Pharmacol. 1986, 35, 77.  34 Adams, G. E.; Cooke, M . S. Int. J. Radiat. Biol. 1969, 75, 457. 35 Walton, M . I.; Workman, P. Biochem. Pharmacol. 1987, 36, 887. 36 Palcic, B . ; Josephy, D.; Skov, K ; Skarsgard, L . D. Proceedings of a Conference Sponsored by the Radiosensitizer/Radioprotector Working Group DCT/NCI, 1979, p. 16.  27  Chapter 1  37 McClelland, R. A . In Selective Activation of Drugs by Redox Processes; G. E . Adams, A. Breccia, E. M . Fielden, P. Wardman, Eds.; Plenum Press, New York, 1990, p. 125. 38 Raleigh, J. A.; Koch, C. J. Biochem. Pharmacol. 1990, 40, 2457. 39 McClelland, R. A.; Panicucci, R ; Rauth, A . M . J. Am. Chem. Soc. 1987,109, 4308. 40  Koch, C. J.; Stobbe, C. C ; Baer, K . A. Int. J. Radiat. Oncol. Biol. Phys. 1984, 10,  mi. 41 Urtasun, R. C ; Chapman, J. D.; Raleigh, J. A ; Franko, A . J.; Koch, C. J. Int. J. Radiat. Oncol. Biol. Phys. 1986,12, 1263.  42 Rasey, J. S.; Krohn, K . A.; FreaufF, S. Radiat. Res. 1982, 91, 555. 43 Mannan, R. H . ; Somayaji, V . V.; Lee, L ; Mercer, J. R.; Chapman, J. D.; Wiebe, L . I. J. Nucl. Med. 1991, 32, 1764.  44 Parliament, M . B . ; Chapman, J. D.; Urtasun, R. C ; McEwan, A . J.; Goldberg, L . ; Mercer, J. R ; Mannan, R. H.; Wiebe, L . I. Br. J. Cancer 1992, 65, 90. 45 Parrick, J.; Hodgkiss, R. J.; Jones, G. W.; Middleton, R. W.; Rami, H . K.; Wardman, P. In Selective Activation of Drugs by Redox Processes; G. E. Adams, A . Breccia, E. M . Fielden, P. Wardman, Eds.; Plenum Press, New York, 1990, p. 249. 46 Raleigh, J. A ; Miller, G. G.; Franko, A. J.; Koch, C. I ; Fuciarelli, A . F.; Kelly, D. A . Br. J. Cancer 1987, 56, 395.  47 Koch, C. J.; Evans, S. M . ; Lord, E. M . Br. J. Cancer 1995, 72, 869. 48 Matthews, J.; Adomat, H . ; Farrell, N . ; King, P.; Koch, C ; Lord, E.; Palcic, B . ; Poulin, N . ; Sangulin, I ; Skov, K. Br. J. Cancer 1996, 74, S200. 49 Evans, S. M . ; Joiner, B . ; Jenkins, W. T.; Laughlin, K . M . ; Lord, E. M . ; Koch, C. J. Br. J. Cancer 1995, 72, 875.  50 Fu, K . K.; Wendland, M . F.; Iyer, S. B.; Lam, K . N . ; Eugeseth, H . ; James, T. L . Int. J. Radiat. Oncol. Biol. Phys. 1990,18, 1341.  51  Stone, H . B . ; Brown, J. M . ; Philips, T. L . ; Sutherland, R. M . Radiat. Res. 1993, 136, 422.  28  Chapter 1  52 Raleigh, J. A.; Franko, A. J.; Treiber, E. O.; Lunt, J. A . ; Allen, P. S. Int. J. Radiat. Oncol. Biol. Phys. 1986, 72, 1243. 53 Aboagye, E. O.; Kelson, A. B . ; Workman, P. Anti-cancer Drug Design 1998,  13,  703. 54 Siemann, D. W.; Inglis, B . A . ; Cranston, B . A.; Lord, E. M . ; Koch, C. J. 10th International Conference on Chemical Modifiers of Cancer Treatment, Jan. 28-31, 1998, Clearwater, Florida, Abstract p. 43. 55 Kilbourn, M . R. Nucl. Med. (Nuclear Science Series); National Academy Press, Washington, D. C , 1990, p. 1. 56 Brennan, M . B . Chemical and Engineering News 1996, 2, 26. 57 Grierson, J. R.; Link, J. M . ; Mathis, C. A.; Rasey, J. S.; Krohn, K . A . J. Nucl. Med.  1989, 30, 343. 58 Rasey, J. S.; Nelson, N . J.; Chin, L.; Evans, M . L . ; Grunbaum, Z. Radiat. Res.  1990,  122, 301. 59 Tewson, T. J. Nucl. Med. Biol. 1997, 24, 755. 60 Kachur, A . V.; Evans, S. M . ; Shiue, C.-Y.; Dolbier, Jr., W. R.; L i , A.-R.; Roche, A . ; Skov, K . A.; Baird, I. R.; James, B. R.; Koch, C. J. Appl. Rod. hot. (submitted Oct., 1998). 61 Keppler, B . K . In Metal Complexes In Cancer Chemotherapy; B . K . Keppler, Ed.; V C H , Weinheim, 1993, p.3. 62 Source of information, ref. 61. 63 Rosenberg, B.; Van Camp, L.; Krigas, T. Nature 1965, 205, 698. 64 Rosenberg, B . ; Van Camp, L . ; Grimely, E. B . ; Thomson, A. J. J. Biol. Chem. 1967, 242, 1347. 65 Rosenberg, B.; Van Camp, L.; Trosko, J. E.; Mansour, V . H . Nature 1969, 222, 385. 66 Gore, M . ; Fryatt, I.; Wiltshaw, E.; Dawson, T.; Robinson, B . A.; Calvert, A . H . Br. J. Cancer 1989, 60, 767.  29  Chapter 1  67 Eisenhauer, E.; Swerton, K . ; Sturgeon, J. et al. In Carboplatin: Current Perspectives and Future Directions; P. Burn, R. Caretta, R. Ozols, M . Rozencweig, Eds.; W. B . Saunders Company, Philadelphia, 1990, p. 133. 68 Harrap, K . R.; Murrer, B . A.; Giandomenico, C. In Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy; S B . Howell, Ed.; Plenum Press, New York, 1991, p. 391. 69 Kelland, L . R ; Mistry, P.; Abel, G.; Freidlos, F.; Loh, S. Y.; Roberts, J. J.; Harrap, K . R. Cancer Res. 1992,  52, 1710.  70 Kelland, L . R.; Jones, M . ; Abel, G.; Harrap, K . R. Cancer Chemother. Pharmacol.  1992, 30, 43. 71 McKeage, K . J.; Morgan, S. E.; Boxall, F. E.; Murrer, B. A.; Hard, G. C ; Harrap, K . R. Proc. Am. Assoc. Cancer Res. 1992,  33, A3197.  72 Holford, J.; Sharp, S. Y . ; Murrer, B . A ; Abrams, M . ; Kelland, L . R. Br. J. Cancer  1998, 77, 366. 73  Simon, T. M . ; Kunishima, D. H.; Vilbert, G. J.; Lorber, A. Cancer 1979, 44, 1965.  74 Howard, R. A ; Sherwood, E.; Erck, A ; Kimball, A. P.; Bear, J. L . J. Med. Chem. 1911, 20, 943. 75 Kbpf-Maier, P.; Kopf, H . Arzneim.-Forsch./DrugRes.  1987, 70, 103.  76 Adamson, R. H . ; Canellos, G. P.; Sieber, S. M . Cancer Chemother. Rep. 1975,  59,  599. 77 Hall, S. W.; Yeung, K.; Benjamin, R. S. Clin. Pharmacol. Ther. 1979, 25, 82. 78 Buckley, R. G. In Metal Compounds in Cancer Therapy; S. P. Flicker, Ed.; Chapman and Hall, London, 1994, p. 92. 79 Sava, G. In Metal Compounds in Cancer Therapy; S. P. Flicker, Ed.; Chapman and Hall, London, 1994, p. 65. 80 Clarke, M . J. In Progress in Clinical Biochemistry and Medicine; Springer-Verlag, Berlin, 1989, Vol. 10, p. 26.  30  Chapter 1  81 Keppler, B . K . ; Hern, M . ; Juhl, U . M . ; Berger, M . R ; Niebl, R ; Wagner, F. E . In Progress in Clinical Biochemistry and Medicine; Springer-Verlag, Berlin, 1989, Vol. 10, p. 41. 82 Mestroni, G.; Alessio, E.; Calligaris, M . ; Attia, W. M . ; Quadrifoglio, F.; Cauci, S.; Sava, G.; Zorzet, S.; Pacor, S.; Monti-Bragadin, C ; Tamaro, M . ; Dolzani, L . In Progress in Clinical Biochemistry and Medicine; Springer-Verlag, Berlin, 1989, V o l . 10, p. 71. 83 Clarke, M . J. In Metal Complexes in Cancer Chemotherapy; B . K . Keppler, Ed.; V C H , Weinheim, 1993, p. 129. 84 Mestroni, G.; Alessio, E.; Sava, G.; Pacor, S.; Coluccia, M . In Metal Complexes in Cancer Chemotherapy; B . K. Keppler, Ed.; V C H , Weinheim, 1993, p. 157. 85 Keppler, B . K.; Lipponer, K.-G.; Stenzel, B.; Kratz, F. In Metal Complexes in Cancer Chemotherapy; B . K . Keppler, Ed.; V C H , Weinheim, 1993, p. 188. 86 Wishart, J. F.; Bino, A ; Taube, H . Inorg. Chem. 1986, 25, 3318. 87 See Chapter 5, Table 5.1. 88 Chou, M . ; Creutz, C ; Sutin, N . J. J. Am. Chem. Soc. 1977, 99, 5615. 89 Kelly, J. M . ; Feeney, M . M . ; Tosi, A . B.; Lecomte, J. P.; Kirsch-De Mesmaeker, A . Anticancer Drug Des. 1990, 5, 69. 90 Loseto, F.; Mestroni, G.; Lacidogna, G.; Nassi, A ; Giordano, D.; Coluccia, M . Anticancer Res. 1991,  77,1549.  91 Palmer, B . D.; Wilson, W. R ; Pullen, S. M . J. Med. Chem. 1990, 33, 112. 92 Luk, C. K.; Veinot-Drebot, L.; Tjan, E. J. Natl. Cane. Inst. 1990, 82, 684. 93 Vaupel, P.; Kallinowski, F.; Okunleff, P. Cancer Res. 1989, 49, 6449. 94 Sasai, K.; Ono, K.; Hiraoko, M . Int. J. Radiat. Oncol. Biol. Phys. 1989,16, 1477. 95 Herman, T. S.; Teicher, B . A ; Holden, S. A. Cancer Res. 1989, 49, 3338. 96 Som, P.; Oster, Z. H . ; Matsui, K . ; Gugliemi, G.; Persson, B . R ; Pellettieri, M . L . ; Srivastava, S. C ; Richards, P.; Atkins, H . L.; Brill, A. B . Eur. J. Med. 1983, 8, 491.  31  Chapter 1  97 Srivastava, S. C ; Mausner, L . F.; Clarke, M . J. In Ruthenium and other Non-Platinum Metal Complexes in Cancer Chemotherapy; M . J. Clarke, Ed.; Springer-Verlag, Heidelberg, 1989, Vol.10, p. 11. 98  Snetsinger, P. A . ; Chasteen, N . D.; van Willigen, H . J. J. Am. Chem. Soc. 1990, 112, 8155.  99 Pell, S. J.; Salmousen, R.; Abelliera, A.; Clarke, M . J. Inorg. Chem. 1984, 23, 385. 100 Bomford, A.; Young, S. P.; Williams, R. Biochem. 1985, 24, 3472. 101 Takahara, P. M . ; Frederick, C. A.; Lippard, S. J. J. Am. Chem. Soc. 1996,  118,  12309. 102 Fichtinger-Schepman, A. M . J.; van der Veer, J. L . ; den Hartog, J. H . J.; Lohman, P. H. M . ; Reedijk, J. Biochem. 1985, 24, 707. 103 Beck, D. J.; Popoff, S.; Sancar, A.; Rupp, W. D. Nucleic Acid Res. 1985, 13, 7395. 104 Page, J. D.; Husain, I.; Sancar, A.; Chaney, S. G. Biochem. 1990, 29, 1016. 105 Hughes, E. N . ; Engelsberg, B. N . ; Billings, P. C. J. Biol. Chem. 1992, 267, 13520. 106 Donahue, B. A.; Augot, M . ; Bellon, S. F.; Treiber, D. K.; Toney, J. H.; Lippard, S. J.; Essigmann, J. M . Biochem. 1990, 29, 5872. 107 Clarke, M . J.; Buchbinder, M . ; Kelman, A. D. Inorg. Chim. Acta 1978, 27, L87. 108 Sava, G. Unpublished results, from ref. 18. 109 Keppler, B . K.; Wehe, D.; Endres, H.; Rupp, W. Inorg. Chem. 1987, 26, 844. 110 Keppler, B . K.; Wehe, D.; Endres, H.; Rupp, W. Inorg. Chem. 1987, 26, 4366. 111 Keppler, B . K . ; Rupp, W.; Juhl, U . M . ; Endres, H . ; Niebl, R.; Balzer, W. Inorg. Chem. 1987, 26, 4366. 112 Sava, G ; Pacor, S.; Mestroni, G ; Alessio, E. Anti-Cancer Drugs 1992, 3, 25. 113 Talmadge, J. E. Cancer Metast. Rev. 1983, 2, 25. 114 Sava, G.; Pacor, S.; Coluccia, M . ; Mariggio, M . ; Cocchietto, M . ; Alessio, E . ; Mestroni, G Drug Invest. 1994, 8, 150.  32  Chapter 2 General Experimental 2.1  Materials  2.1.1  Chemicals  2.1.1.1 Imidazoles Im, 2MeIm and 4MeIm (from Aldrich) and NMelm (from Merck) were used as received. 2N02lm was synthesized according to literature procedures or purchased from 1  Aldrich.  Other  nitroimidazoles  purchased  from  Aldrich  included:  4(5)N02lm,  2Me5N0 Im, 2-methyl-4-nitro-l-imidazolepropionitrile and 2-methyl-4-nitro-l-imidazole2  propionic acid. SR2508 was kindly donated by Dr. C. Koch (Univ. of Perm.), while metronidazole and RSU1111 were donated from the laboratory of Dr. M . Naylor (MRC Chilton).  2.1.1.2 Amines The following amines were synthesized and kindly donated from Prof. W. Dolbier's group  (Univ.  of Florida): H NCH CF CF «HC1, 2  2  2  H NCH CF CF Br'HCl,  3  2  2  2  2  H N C H C H C F ' H C 1 , H N C H C H C F B P H C 1 , H NCH CH CH F«HF. A l l other amines 2  2  2  3  2  2  2  2  2  2  2  2  used throughout this work were purchased from Aldrich and generally used without further purification. E t N was distilled prior to use. 3  2.1.1.3 Miscellaneous Reagents RuCl *3H 0 was supplied on loan from both Johnson Matthey Ltd. and Colonial 3  2  Metals, Inc. with a Ru composition of 39 to 41 %. N-methylmorpholine (NMM) and isobutylchloroformate (iBuClFrm) were purchased from Aldrich and were vacuum distilled prior to use. A l l other commercial chemicals were used as purchased, unless otherwise indicated, from Aldrich Chemical Co., Fisher Scientific Co., Strem, Sigma Chemical Co., B D H Chemicals, Merck and Co., Inc. or Mallinckrodt Chemical Works. N B S and TsCl  33  References on page 46  Chapter 2  were recrystallized before use. All gases (N , H and CO; minimum purity 99.99 %) were 2  2  purchased from Linde (Union Carbide, Inc.) and used as received.  2.1.2 Solvents Reagent grade D M F (Fisher) and dmso (Fisher) were distilled and dried over molecular sieves, and M e C N and EtOAc (anhydrous, Aldrich) were used as received. Other solvents were dried using the appropriate drying agent and distilled prior to use: M e O H (Mg), E t O H (Mg), THF (Na-K alloy), E t 0 (Na), hexanes (Na), pentane (CaH ), 2  2  acetone (K2CO3) and CH C1 (CaH ). Deuterated solvents (d -acetone, d -dmso, C D O D , 2  CDCI3,  2  2  6  6  3  C D C N , D 0 ) were supplied by M S D Isotopes or Isotech Inc. and used 3  2  immediately upon opening of an ampule (except for D 0 ) to ensure dryness. Please note 2  that throughout this thesis dmso is used to indicate solvent while D M S O is used to indicate a coordinated ligand.  2.2  Analytical Techniques  2.2.1  Nuclear Magnetic Resonance Spectroscopy TI (200 MHz), H (46.1 MHz), C{TI} (50.3 MHz) and F{TI} (188.2 MHz) 2  13  19  N M R spectra were recorded on a Bruker AC200F spectrometer.  19  F N M R (282.2 MHz)  spectra requiring larger sweep widths (SW up to 100,000) were recorded on a Varian XL300 spectrometer, while large sweep width *H N M R spectra were recorded on either the Varian XL300 (300.0 M H z ) or the Bruker WH400 (400.0 M H z ) spectrometers. 31  P { H } (121.5 MHz) and C{TI} (75.4 MHz) N M R spectra were recorded on a Varian 1  13  XL300 spectrometer. The TI N M R chemical shifts were referenced to the residual proton signal of the internal deuterated solvent (5 7.24 for CDCI3, 4.78 for D 0 , 3.30 for 2  CD3OD,  2.50 for (CD ) SO, 2.20 for ( C D ) C 0 ; all are reported relative to the external 3  2  3  2  standard of tetramethylsilane (8 0.00)) while the C { H } N M R chemical shifts were also 13  J  referenced to the shift from the solvent (5 29.8 for Me of acetone). The F { T i } N M R 19  chemical shifts were referenced to trifluoroacetic acid in D 0 (external reference) and the 2  31  P { H } N M R chemical shifts were reported relative to 85 % H P 0 (external reference) 1  3  4  with the downfield shifts observed as positive. All spectra were recorded at r.t.  34  References on page 46  Chapter 2  The peak multiplicities are reported as observed in a given spectrum (s = singlet; d = doublet; t = triplet; q = quartet; p = pentet; sex = sextet; spt = septet; br = broad, vbr = very broad, including a combination of these symbols, for example, qt = quartet of triplets). For those signals where peak coincidences have altered the peak multiplicity for a specific set of protons, the expected multiplicity is reported in brackets (e.g. for - N H CH2-CH2- one would expect to observe a doublet of triplets; however, a quartet is observed and so is recorded as q (dt)). The J values are reported for those nitroimidazole compounds containing F atom(s) on their side-chain and those Ru complexes containing more than two P-bound ligands. The computer program MestRe-C (Magnetic Resonance Companion, Version 1.5.0) was used to expand various spectra obtained on the AC200F N M R spectrometer. This program was especially useful for observing the F-F coupling in the ^ { T T } N M R spectra which could not be observed with the resolution of the standard HP plotter. Determination of the p, fr and number of unpaired electrons for selected e  paramagnetic Ru(III) complexes (in chapter 4) was performed at room temperature (r.t.) using the Evans method. Two resonance lines were obtained from the residual solvent 2  protons in two solutions owing to the difference in their volume susceptibilities, with the line from the more paramagnetic solution lying at higher frequencies. Typically the solution of the complex (of known concentration) is outside and the solvent only is placed within the capillary tube; however, because of problems with overlap of peaks of the complex with those of the residual solvent this was reversed (Figure 2-1). The A8 values were determined using both the 200 and 300 M H z N M R machines and the u. ff calculated e  for each case. The \x & values agreed within ± 0.1. Of note, the Evans method e  measurements were made for a compound dissolved in the same deuterated solvent as reported for the *H N M R unless otherwise stated.  35  References on page 46  Chapter 2  • , complex dissolved in deuterated solvent (~ 2-3 mgin I O O J I L )  ,deuterated solvent (-0.5 mL)  Figure 2-1: Diagramatic representation of a capillary (containing a solution of the complex) within a N M R tube (containing solvent only) used for the Evans method. The following equations used to calculate p ff were obtained from various e  literature sources. " The equations are listed in the order in which they were used. 2  4  m = concentration of sample (g/mL)  3A/  r  = —-—i-  Af = frequency separation between two lines (Hz)  y  f = frequency at which proton resonances are studied (Hz) Xo = mass susceptibility of solvent (cm /g) 3  M = compound molecular weight (g/mol) X M  XM  7'XL  XM ' = molar susceptibility of metal ion (cm /mol) 3  Xn  XL = diamagnetic correction for ligand (cm /mol) 3  X m = diamagnetic correction for metal (cm /mol) 3  ju  eff  -  2&3ylx ,T = yjn(n + 2) M  T= absolute temperature (K)  n = number of unpaired electrons on metal ion  36  References on page 46  Chapter 2  The diamagnetic correction values for the ligands (XL) were calculated from a table of Pascal's constants (XL= 34, 34, 46, 74, 91 and 115 (x 10" cmVmol) for 2N0 Im, 5  6  2  4N0 Im, 2Me5N0 Im, metronidazole, SR2508 and EF5, respectively). 2  2  2.2.2 Infrared Spectroscopy IR spectra were recorded on an ATI Mattson Genesis FTIR spectrophotometer using either K B r (by compression to 17,000 psi of a mixture of finely ground K B r and the compound, ~ 100:3 ratio) or a thin film on a K B r disc (from slow evaporation of a solution of a compound on the surface of a 0.5 mm K B r disc). Only the major bands in the spectra (transmittance <70%) are reported. Only select bands are assigned (stretching frequencies unless otherwise indicated) according to references from standard IR texts. " 6  8  Values are given in cm" (± 4 cm" ). 1  1  2.2.3 UV-Visible Spectroscopy U V - V i s absorption spectra were recorded on an spectrophotometer and are given as Ama (± 2 nm), X  [Smax  HP 8452A diode array  x lO^fJvf^cm" )], sh=shoulder. 1  2.2.4 Mass Spectrometry The mass spectra for selected compounds were obtained from the Mass Spectrometry (MS) lab at U B C (under the supervision of Dr. G. Eigendorf). The following spectrometers were used: KRATOS MS80 (DCI), K R A T O S Concept IIHQ (LSEVIS) and K R A T O S MS50 (EI). For the most part, the most valuable M S technique used for determination of the composition of the nitroimidazoles was DCI+ M S . For those nitroimidazoles which did not yield acceptable elemental analyses, a high resolution determination of the exact elemental composition of the parent peak was performed. The Ru complexes were all run using +LSLMS, but unfortunately only a small number of the Ru-nitroimidazole complexes displayed acceptable M S spectra using this technique. For the nitroimidazole complexes other M S techniques were used in addition to LSEVIS; these  37  References  on page 46  Chapter 2  included M A L D I , Electrospray and EI mass spectrometries; however, none of these techniques yielded acceptable/useful M S data.  2.2.5 Gas Chromatography GC Analysis was done on an HP 5890A instrument using a 30 kPa He flow at 75 °C with an HP-17 50% phenyl, 50% methyl, polysiloxane intermediate polarity column, using a flame ionization detector and an HP 3 3 92A Integrator.  2.2.6 Cyclic Voltammetry Electrochemistry was performed on 10" M solutions of selected compounds with 3  0.1 M T B A P in dry, degassed M e C N under Ar or N in the cells illustrated in Figures 2.2 2  and 2.3; the potential was scanned down at 100 mV/s. The potentiometer was a Pine Biopotentiostat Model AFCBP1 attached to a 80MHz computer using Pine Chem (Version 2.00) cyclic voltammetry software. The working and counter electrodes for cell A (Figure 2-2) were Pt plates (6 mm ) and the reference electrode was a Pt wire, while the 2  working and counter electrodes for cell B (Figure 2-3) were Pt wire, and the reference electrode was A g wire. For each sample, an initial run was performed to determine the potential range required to observe all of the expected reductions; a fresh solution of the compound and electrolyte was then prepared and the scan performed again. If necessary, further solutions were prepared and analyzed until the values were in good agreement (AEi/2 < ± 5 mV). The reduction potentials (Ei/ ) were obtained from the average of the 2  potentials of the reduction and oxidation peaks [(Ep. .+E . .)/2] and the reported E1/2 value c  p a  (+ 3 mV) is the average of the combined runs. Because of the irreproducibility of the reduction potential of the ferrocene/ferrocenium couple for both of the cells, ferrocene was added as an internal standard to the solution of each compound measured. The ferrocene/ferrocenium couple in M e C N vs. the SCE is reported as 424 m V ; hence the 9  conversion factor used to report the selected compound's E i / vs. the S C E was 2  424-Ei/2(FeCp ). 2  38  References  on page 46  Chapter 2  -*  >•  15 cm  Figure 2-2: Cyclic voltammetry cell (A) containing a Pt reference electrode.  Pt Working Electrode  15 cm  Figure 2-3: Cyclic voltammetry cell (B) containing an A g reference electrode.  2.2.7 Conductivity Conductivity measurements were made on a 10" M solution of the selected 3  complex at room temperature (r.t.) using a Serfass conductance bridge model RCM15B1 (Arthur H . Thomas Co. Ltd.) connected to a Yellow Springs Instrument Co. 3404 cell (cell constant =1.016), and are reported as A (+ 0.5 ohm^mol^cm ). 2  M  39  References  on page 46  Chapter 2  2.2.8 X-Ray Analysis All single crystal X-ray diffraction studies were performed by the late Dr. S. J. Rettig of the U B C chemistry department on a Rigaku/ADSC C C D area detector or a Rigaku AFC6S diffractometer, both of which use graphite monochromated C u - K a radiation.  2.2.9 Elemental Analysis Elemental analyses were performed in the U B C chemistry department by Mr. P. Borda using either a Carlo Erba (Model 1106) or a Fisons Instruments (Model E A 1108) CFfN-0 elemental analyzer. The results reported have an absolute accuracy of ± 0.3%.  2.3  Compound Purification Techniques In general, the visualization of nitroimidazoles on TLC plates was achieved with  the aid of a U V lamp (Mineralight® Lamp Model UVG-54 short wave UV-254 nm), but imidazoles and other non-UV absorbing compounds were visualized by exposing the developed TLCs to I or B r . For each chromatographic technique used, the solvent 2  2  combination, including the ratio, follows in brackets (e.g. solvent A : solvent B , 10:1) and in specific cases where the eluent strength was altered an arrow is included (e.g. 10:1 —» 5:1).  2.3.1 Column Chromatography Column chromatography was performed using Merck silica gel (230-400 mesh). The eluent was added to the silica gel in a large Erlenmeyer flask; the contents were stirred with a glass rod for 1 min and then poured into the column on a 45° angle to ensure that no air-bubbles were introduced. (If air-bubbles were present the column was tapped gently to agitate them to the surface of the silica gel.) The silica gel was packed using a pressurized air-flow adapter and then eluted to the edge of the silica gel. The compound was introduced onto the silica gel two ways: 1) by dissolving the compound in a minimum volume of the eluent and then loading onto the silica gel, making sure not to  40  References  on page 46  Chapter  2  disturb the top of the gel layer; 2) by adding silica gel to a solution of the mixture to be purified, followed by removal of the solvent under vacuum and then addition of the silica gel (with the compound) to the top of the column. For this technique the solvent must be slightly above the gel layer. The solvent (containing a mixture of compounds) was eluted to the gel front, sea sand (~2 cm) was then added, and the column eluted dropwise, unless otherwise stated. The Rf values of the pure compounds were measured on Merck silica gel 60F  254  T L C A l sheets.  2.3.2 Preparative Thin Layer Chromatography Preparative TLCs of the Ru complexes were performed with Merck silica gel 60, 0.5 mm plates (without indicator) and for nitroimidazoles and other organic compounds, preparative TLCs were performed with silica gel 60  F254,  1.0 mm plates. All plates used  for preparative T L C had dimensions of 20cm x 20cm.  2.3.3 Chromatotron Some products  were  purified on a Harrison Research  model 7924T  Chromatotron (a rotating disc TLC) using silica as the stationary phase and an eluent flow rate of ~3 mL/min. The thickness of the plate used (1.0 mm or 2.0 mm) was dependent on the amount of compound used for purification (up to 250 mg or 750 mg, respectively). This technique was extremely useful for compounds which had similar Rf values as the band separation could be observed during the elution using a U V lamp, and hence the amount of compound overlap in the collected fractions could be reduced to a minimum.  2.4  General Methodologies  2.4.1 Amide Coupling Reaction The synthesis of most of the nitroimidazole compounds was performed using a standard amide coupling technique (Scheme 2-1) '  10 11  in which a carboxylic acid and an  amine are coupled to form an amide linkage. The N-methylmorpholine (TSMM) deprotonates the acid group, making it a better nucleophile, which attacks the acyl carbonyl group of /'so-butylchloroformate (iBuClFrm); this displaces the chlorine as CI"  41  References  on page 46  Chapter 2  and gives formation of the mixed anhydride species. Addition of R-NH «IIX and another 2  equivalent of N M M (to mop up H X ) leads to attack at the activated carbonyl nearest the nitroimidazole ring by the amine N-atom to yield an amide bond. In the presence of any H 0 , the iso-butylcarbonate hydrolyzes to form CO2 and wo-butanol, which can then react 2  with the activated anhydride; hence extremely anhydrous conditions are pertinent to the success of this reaction.  u  1 Me  +  R—C—N—R'  I  HO  R'-NH *HX  O  2  H  Scheme 2-1:  2.5  Standard amide bond forming reaction.  Ruthenium Precursors  2.5.1 RuCI «3H 0 3  2  This starting material, for which the oxidation state of the metal (possibly a mixture of R u and R u oxidation states) and degree of hydration of the compound are m  w  ill-defined, ' was kindly donated from both Johnson Matthey Ltd. and Colonial Metals, 12 13  Inc. The formulation of the chloride for the purpose of estimating a known number of millimoles was taken as RuCl3 3H 0. ,  2  [Ru(DMF)6][CF S0 ]3  2.5.2  14  3  3  In a 2-neck flask, RuCl «3H 0 (2.00 g, 8.13 mmol) was dissolved in D M F (120 3  2  mL) and then Sn metal (7.00 g, 58.9 mmol) was added. The mixture was stirred at r.t. for 1 h, the colour changing from orange-brown to green to blue. To the blue solution, P b ( C F S 0 ) (6.51 g, 12.9 mmol; synthesized from Pb(C0 ) and reagent grade C F S 0 H , 3  3  2  3  42  3  References  3  on page 46  Chapter 2  1:2) were added and the mixture was heated to 50 °C for 2 h (the colour changed to red-brown). The mixture was gravity filtered and then the volume was reduced to -25 mL before the addition of CH C1 (300 mL). The mixture instantly formed a dark brown slurry 2  2  which was stirred for 12 h at r.t. The brown solid was filtered off and the filtrate's volume was reduced to -30 mL. The addition of 1,2-dichloroethane (100 mL) to the solution at 0 °C led to the formation of a yellow precipitate. After 1 h at 0 °C the yellow solid was collected via suction filtration and washed with 1,2-dichloroethane (3 x 10 mL). This crude solid was recrystallized from CH Cl :pentane to yield a yellow microcrystalline solid 2  2  (4.85 g, 56%). Anal, calc for C^H^NsO.sSjFgRu: C, 25.56; H , 4.29; N , 8.52; found: C, 25.43; H, 4.30; 8.54. U V - V i s (DMF): 254 (1.72), 273 (6.95), 342 (5.80). IR (KBr): 1642 (C=Cw  DMF)  (cf. 1673 for  C=O  FREED  MF).  X  H N M R (300 MHz, d -dmso): 6 22.50 (br s, 6  -Mej), 19.80 (br s, -Me ); the lower-field Me resonance has been assigned to the M e 2  group cis to the carbonyl oxygen atom. These data agree well with those reported in the literature.  2.5.3  15  [Ru(DMF) ][CF S0 ]2  14  6  3  3  In a Schlenk tube, [Ru(DMF) ][CF S0 ]3 (0.237 g, 0.240 mmol) was dissolved in 6  3  3  5 mL D M F (purged with N for 10 min) to give a yellow solution. A trace amount (<1 2  mg) of Pt black (Adam's catalyst) was added and the mixture was stirred at 50 °C for 1.5 h under H bubbling. The Pt black was filtered off and the red-orange filtrate's volume 2  was reduced to -1 mL prior to the addition of n-pentanol (5 mL). Vigorous stirring for 20 min at 0 °C yielded an orange solid that was washed with E t 0 ( 3 x 5 mL) and dried in 2  vacuo (0.207 g, 87 %). UV-Vis (DMF): 494 (0.182). IR (KBr): 1635 (C=O  . DMF). H X  coord  N M R (200 MHz, d -dmso): 5 7.50 (s, - C M ) ) , 3.10 (s, 3H, -Me,), 2.89 (s, 3H, -Me ). 6  2  These data agree well with those reported in the literature.  2.5.4 c/s-RuCI(DMSO)(DMSO) 2  15  16  3  In a 50 mL flask RuCl »3H 0 (1.00 g, 4.06 mmol) was dissolved in 10 mL dmso, 3  2  and the mixture was refluxed for 30 min in air. The reaction mixture was cooled to r.t. and addition of acetone (20 mL) led to the accumulation of a yellow precipitate. The yellow  43  References  on page 46  Chapter 2  solid was collected, washed with acetone ( 3 x 1 0 mL) and dried in vacuo at 78 °C for 24 h (1.32 g, 84 %). Anal, calc for C H Cl 0 S4Ru: C, 19.38; H , 4.99; found: C, 19.23; H , 8  24  2  4  4.81. U V - V i s (CHCI3): 305 (0.74), 355 (1.05). IR (KBr): 1116 (S=0), 963 (S=<9). H X  N M R (200 M H z , CDC1 ): 8 3.55, 3.51, 3.45, 3.44 (S-bonded DMSO); 2.76 (O-bonded 3  DMSO); 2.59 (free DMSO). The spectroscopic data agree well with those previously reported. '  16 17  2.5.5 fra/?s-RuCI(DMSO) 2  18  4  In a 25 mL flask, R u C l ' 3 H 0 (0.500 g, 2.03 mmol) was dissolved in 3 mL 3  2  dmso, and the mixture was heated to 70 °C and stirred for 15 min in air. The resulting red solution was cooled to room temperature and acetone (15 mL) was added slowly with continuous stirring. A red solid precipitated which was collected, washed with acetone (3 x 10 mL) and dried in vacuo at 78 °C for 24 h (0.525 g, 67 %). Anal, calc for C H 4Cl20 S4Ru: C, 19.38; H , 4.99; found: C, 19.53; H , 5.16. U V - V i s (CHC1 ): 440 8  2  4  3  (0.22). IR (KBr): 1086 (S=0). H N M R (200 MHz, CDC1 ): 8 3.41 (s, S-bonded C H ) , l  3  3  2.59 (free DMSO). The spectroscopic data agree well with those previously reported. '  17 18  2.5.6 cis  (TMSO)  2  19  4  In a 100 mL RBF, RuCl *3H 0 (2.00 g, 8.13 mmol) was refluxed in M e O H (40 3  2  mL) under H for 4 h when the colour of the solution turned from brown-orange through 2  a green intermediate to finally become dark blue. TMSO (8 mL, 89.1 mmol) was added and refluxing under H was continued for an additional 4 h, generating a yellow-green 2  precipitate. The solid was collected, washed with E t 0 ( 3 x 1 0 mL) and dried in vacuo at 2  78 °C for 24 h (4.02 g, 84 %). Anal, calc for Ci6H Cl 0 S4Ru: C, 32.60; H , 5.44; found: 32  2  4  C, 32.29; H , 5.19. U V - V i s (CHC1 ): 355 (1.07), 300 (0.58). IR (KBr): 1121, 1086 ( S O ) . 3  !  H N M R (200 M H z , CDC1 ): 8 4.15, 3.47 (m, 2 H each, -CH -S(0)-CH -), 3  -CH -CH -). 2  2  2  2  2.26 (m, 4H,  The spectroscopic data agree well with those previously reported.  2.5.7 /ner-RuCI(DMSO) 3  19  20  3  In a large R B F , [(DMSO) H][Ru(DMSO) Cl ] (1.12 g, 2.13 mmol; kindly 2  2  4  donated by E. Cheu of this group) was suspended in acetone (80 mL), and then dmso (0.5  44  References  on page 46  Chapter 2  mL, 7 mmol) was added. The mixture was refluxed with vigorous stirring. To the refluxing mixture, a A g B F solution (0.400 g, 2.05 mmol, dissolved in 20 mL acetone) 4  was added dropwise to give a bright orange solution which becomes turbid over time (due to formation of AgCl). The AgCl was filtered off (whilst the mixture was hot) and then the volume of the filtrate was reduced to ~ 10 mL before the addition of 5 mL E t 0 . Storage 2  at 4 °C for 12 h yielded a fine, microcrystalline red solid that was collected, washed with E t 0 and dried in vacuo at 78 °C for 24 h (0.297 g, 34 %). U V - V i s (CH C1 ): 446 (1.28), 2  2  2  382 (3.54), 268 (sh) (6.43). IR (KBr): 1123, 1105 (SK)); 910 (S=0). *H N M R (300 M H z , CDC1 ): 6 9.45 (br s, DMSO), 2.62 (free DMSO), -15.35 (br s, DMSO). The 3  spectroscopic data agree well with those previously reported.  2.5.8  20  [RuCI(COD)]  21  2  x  In a Schlenk tube RuCl «3H 0 (2.0 g, 8.13 mmol) was dissolved in E t O H (80 3  2  mL), and 1,5-cyclooctadiene (7.5 mL, 61.0 mmol) was added under N to the resulting 2  solution. The orange mixture was heated at reflux for 3 d, at which point the solution was pale yellow after a brown product had precipitated. The brown solid was collected, washed with EtOH (3 x 10 mL) and dried in vacuo (2.01 g, 86 %). Anal, calc for [C Hi Cl Ru] : C, 34.30; H , 4.32; found: C, 34.49; H , 4.58. 8  2  2  x  2.5.9 [RuCI(dppb)](n-dppb) ' 22  2  23  2  This complex was kindly donated by Dr. K . S. MacFarlane, formerly of this group. The physical and spectroscopic data of this precursor agree with those previously reported.  22,23  45  References  on page 46  Chapter 2  2.6  References  1  Davis, D. P.; Kirk, K . L.; Cohen, L . A. J. Heterocyclic Chem. 1982,19, 253.  2  Evans, D. F. J. Chem. Soc. 1959, 2003.  3  Live, D. H.; Chan, S. I. Anal. Chem. 1970, 42, 791.  4  Crawford, T. H . ; Swanson, J. J. Chem. Educ. 1971, 48, 382.  5  Figgis, B . N . ; Lewis, J. In Modern Coordination Chemistry; J. Lewis and R. G. Wilkins, Eds.; Interscience Publishers, Inc., London, 1960, p. 402.  6  Silverstein, R. M . ; Bassler, G. C ; Morrill, T. C. Spectrometric Identification of Organic Compounds; John Wiley and Sons, Inc., Toronto, 1991.  7  Nakamoto, K . ; McCarthy, P. J. Spectroscopy and Structure of Metal Chelate Compounds; John Wiley and Sons, Inc., New York, 1968.  8  Nakamoto, K . Infrared Spectra of Inorganic and Coordination Compounds, 2nd Ed.; Wiley-Interscience, Toronto, 1970.  9  Connelly, N . G ; Geiger, W. E. Chem. Rev. 1996, 96, 877.  10 Bock, M . G.; DiPardo, R. M . ; Evans, B. E.; Rittle, K . E.; Veber, D. F.; Freidinger, R.  M . Tetrahedron Lett. 1987, 28, 939. 11 Holzapfel, C. W.; Pettit, G. R. J. Org. Chem. 1985, 50, 2323. 12 Seddon, E. A . ; Seddon, K . R. The Chemistry of Ruthenium; Elsevier, Amsterdam,  1984 13 Hui, B . C ; James, B . R. Can. J. Chem. 1974, 52, 348. 14 Cao, R. M.Sc. Thesis, University of Bern, 1992. 15 Judd, R. J.; Cao, R.; Biner, M . ; Armbruster, T.; Burgi, H.-B.; Merbach, A. E.; Ludi, A. Inorg. Chem. 1995, 34, 5080. 16 Evans, I. P.; Spencer, A.; Wilkinson, G. J. Chem. Soc, Dalton Trans. 1973, 204. 17 Alessio, E.; Milani, B . ; Mestroni, G ; Calligaris, M . ; Faleschini, P.; Attia, W. M .  Inorg, Chim. Acta 1990, 177, 255. 18 Jaswal, J.; Rettig, S. J.; James, B. R. Can. J. Chem. 1990, 68, 1808. 19 Yapp, D. T. T. Ph. D. Thesis, University of British Columbia, 1993.  46  Chapter 2  20 Alessio, E.; Balducci, G.; Calligaris, M . ; Costa, G.; Attia, W. M . Inorg. Chem.  1991,  30, 609.  21  Genet, J. P.; Pinel, C ; Ratovelomanana-Vidal, V . ; Mallart, S.; Pfister, X . ; Cafio De Andrade, M . C ; Laffitte, J. A . Tetrahedron: Asymmetry 1994, 5, 665.  22 Joshi, A. M . Ph. D. Thesis, University of British Columbia, 1990. 23 Bressan, M . ; Rigo, P. Inorg. Chem. 1975,14, 2286.  47  Chapter 3 Synthesis a n d C h a r a c t e r i z a t i o n of N e w 2-, 4- a n d 5-Nitroimidazoles w i t h Halogenated Side-Chains  3.1  Introduction The use of nitroimidazoles provides a sensitive technique for the detection of  hypoxia in human cancers; as a result of hypoxia-dependent bioreduction of nitroimidazoles by cellular nitroreductases, they can be used to determine the variation of oxygen concentration within a tumour based on the amount of reduction product that forms adducts with cellular macromolecules. Recently, a monoclonal antibody (ELK3-51) 1  was raised against the bioreduction adduct of a fluorine-containing analogue of etanidazole, named EF5 [2-(2-nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide], ' and the amount of binding in tumour cells was determined based on the 2 3  fluorescence of a fluorophore called Cy3 that was chemically coupled to the antibody. 4  5  This technique has been used to assess hypoxia in vitro and in various animal tumour models. ' The detection and quantification of hypoxia in tumours should provide better 6 7  insight into which patients would most likely benefit from hypoxia-targeted therapies. The National Cancer Institute (US) has approved clinical trials on EF5 through support of large scale synthesis, formulation and toxicology. In this chapter we report the sythesis and characterization of various halogenated nitroimidazole compounds, including their one-electron reduction potentials. The major goal was to expand this area of chemistry in hopes of developing a nitroimidazole compound that was more biologically active than EF5. The preliminary studies for the development  of a new PET imaging agent are also  48  reported.  References on page 129  Chapter 3  3.2  Experimental Section  3.2.1  Synthesis of Side-Chains  3.2.1.1  2-Iodo-7V-(2,2,3,3,3-pentafluoropropyl)acetamide [EF5]  Iodoacetic acid (0.224 g, 1.20 mmol) was dissolved in THF (15 mL) at 0°C (ice-bath) under a constant N -flow. N M M (140 p.L, 1.28 mmol) was added, and the 2  mixture was stirred for 10 min after which iBuClFrm (195 uL, 1.50 mmol) was added. The mixture was further stirred for 30 min during which a slurry was formed. The ice-bath was removed, and H NCH CF CF «HC1 (0.249 g, 1.45 mmol) and N M M (165 uL, 1.51 2  2  2  3  mmol) were added. Stirring was continued for 20 h at r.t. after which the resulting yelloworange precipitate was filtered off and washed with dry THF. The dark orange washings were combined and eluted through a silica gel column (CH Cl :MeOH, 10:1); a purple 2  2  band (I ) appeared, followed by a yellow band that was analyzed using T L C and found to 2  contain two products. The yellow band was eluted off and, after removal of the solvents, the residue was placed in a sublimation apparatus and heated to 110 °C under vacuum, when pure I F 5 sublimed as a pale yellow solid (0.202 g, 53%); mp 90-93 °C. Anal. calc. for C H N O F I : C, 18.94; H , 1.59; N , 4.42; found: C, 18.76; H , 1.67; N , 4.61. IR (v, 5  5  5  cm" ): 3305 (N-H); 3088 (C-Hm); 2963, 2858 (C-H); 1658 (C=0); 1204, 1185, 1141, 1  1067, 1036. H N M R (300 MHz, d -acetone): 5 8.06 (s, IH, N-//), 4.05 (td, 2H, JHF = 3  X  6  6.5 Hz, JHH = 0.9 Hz, -Cr7 -CF -), 3.81 (s, 2H, l-CH -). 3  2  2  2  VCH}  19  N M R (188 M H z ,  de-acetone): 8 -8.18 (t, -CF ), -45.34 (q, -CF -). 3  2  H  O  IF5  49  References  on page 129  Chapter 3  2-Iodo-A -(3-bromopropyl)acetamide [IBr]  3.2.1.2  /  A similar procedure to that for the synthesis of EF5 was used (see above). Iodoacetic acid (0.979 g, 5.27 mmol) and N M M (576 uL, 5.27 mmol) were dissolved in THF (20 mL), and iBuClFrm (752 uL, 5.78 mmol) was added. After 40 min, H N C H C H C H B r ' H B r (1.26 g, 5.79 mmol) and N M M (639 uL, 5.85 mmol) were then 2  2  2  2  added, the mixture giving a yellow slurry. Stirring was continued for 4 h at r.t. during which the colour changed to a red/brown and a yellow precipitate was formed. This was filtered off and washed with THF (3 x 15 mL). The washings were combined and the solvent was removed by rotoevaporation yielding a dark red oil; subsequent T L C analysis revealed several products. Purification of the desired product was achieved via column chromatography (CH Cl :MeOH, 20:1). The first band (yellow, low concentration) was 2  2  followed by a minor red band after which the product band (bright orange) emerged; T L C analysis of the orange band revealed a single species. Subsequent removal of solvent afforded an orange oil (0.978 g, 61 %). L R - M S [DCI(+)]: 306 (TVf), 255 (I ), 227 ( M " 2  - Br), 180 Qs/T - I), 169 (TVf - N H C H C H C H B r ) , 128 (I). Tf N M R (300 M H z , 2  2  2  de-acetone): 5 7.90 (s, I H , N-//), 3.77 (s, 2H, I-CrY -), 3.52 (t, 2H, -Gr7 -Br), 3.36 (dt, 2  2  2H, -NH-C# -), 2.07 (m, 2H, -NH-CH -Cr7 -, partially overlapped with residual acetone 2  2  2  peak). (These chemical shifts are comparable to those seen for compounds of similar composition, e.g. IF5 and E B r l (Section 3.2.2.14)). Unlike with IF5, purification of I B r by sublimation (at 70°C) was unsuccessful due to thermal instability as evidenced by N M R spectroscopy (see Discussion, p. 98). Of note, mass spectrometric analysis of I B r after such a sublimation procedure revealed the presence of species attributable to compounds containing cyclic rings, such as 1 and 2 of the same molecular weight (see Scheme 3-4, section 3.3.1, p. 98). L R - M S [DCI(+)]: 226 fJVf), 128 (I), 99 (TVf - I). T i N M R , including 2D-cosy (200 MHz, d -acetone): (1) 5 4.81 (t, 2H, 0-CH -), 4.12 (s, I H , 6  2  l-CH=), 3.74 (t, 2H, -NH-CfY -), 2.51 (s, IH, -N#-), 2.37 (p, 2H, -CH -Cr7 -CH -); (2) 5 2  2  2  2  4.82 (s, 2H, I-C# -), 3.55 (t, 2H, -N-CH -), 3.32 (t, 2H, -N-Cr7 -), 2.03 (m, 2H, - C H 2  2  2  2  C// -CH -). Of note, partial formation of 1 and 2 was observed when neat IBr was 2  2  exposed to U V radiation (TLC lamp) at r.t. for 2 d.  50  References  on page 129  Chapter 3  IBr  3.2.1.3  1  2  2-Chloro-iV-(2,2,3,3,3-pentafluoropropyl)acetamide [C1F5] Chloroacetic acid (0.101 g, 1.08 mmol) was dissolved in THF (10 mL) under 1  atm N . N M M (120 uL, 1.08 mmol) was then added, and the reaction mixture was stirred 2  at 0 °C for 10 min. Afterwards, iBuClFrm (151 pL, 1.16 mmol) was added and a white precipitate was formed instantaneously. Stirring was continued for 30 min after which the ice-bath was removed, and H NCH CF CF »HC1 (0.189 g, 1.10 mmol) and N M M 2  2  2  3  (128  pL, 1.17 mmol) were added. The final mixture was stirred at r.t. for 8 h before the white solid was collected and washed with THF. The solvent was subsequently removed giving a pale yellow oil whose TLC analysis revealed two products, one visible under U V light and the other appearing after development using LVhexanes. The mixture was sublimed at 60 °C under vacuum, and a crystalline white solid was obtained (the remaining non-sublimed product was a dark yellow oil). TLC analysis revealed a single species, not visible under U V light (0.148 g, 61 %); mp 44-45 °C. L R - M S [DCI(+)]: 225 (M+), 190 (M+ -CI), 176 (TVf - CH C1). H R - M S [DCI(+)] calc. for C H N 0 C 1 F : 224.99799; found 224.99782. 35  2  5  5  5  JR (v, cm" ): 3324 (N-H); 3106 ( C - H M ) ; 2966, 2872 (C-H); 1653 (C=0); 1206, 1180, 1  1146, 1067, 1037.  :  H NMR  Cl-Gr7 -), 4.17 (td, 2H, 2  MHz,  3  JHF  (300 MHz, = 6.7 Hz,  d -acetone): 8 8.12 (s, I H , N-H), 4.24 (s, 2H, 6  3  = 2.2 Hz, -C/f -CF -).  JHH  2  2  19  F { H } N M R (188 J  d -acetone): S -8.21 (t, -CF ), -45.26 (q, -CF -). 6  3  2  H  C1F5  51  References  on page  129  Chapter  3.2.1.4  3  Reduction of IF5 to ICH CH NHCH CF CF (3) 2  2  2  2  3  IF5 (0.040 g, 0.126 mmol) was placed in a 25 mL two-neck flask and THF (5 mL) was added. To the resulting pale yellow solution was added BH *THF (1.0 M , 290 3  uL, 0.290 mmol), when the reaction mixture turned bright yellow within 1 min. After 15 min the colour reverted to pale yellow; additional BH «THF (50 uL) was added and the 3  solution became colourless. A condenser was attached and the solution was refluxed at 80 °C for 1 h to ensure completion of the reaction. The reaction mixture was then cooled to r.t. and M e O H (10 mL) was added to decompose unreacted B H .  The solvent was then  3  removed to give a yellow oil; TLC revealed the presence of one major and several minor products. The major product was purified via preparative TLC (CH Cl :MeOH, 50:1) as a 2  2  yellow oil, 3 (0.028 g, 73 %). Note: the product spot on the TLC plate, on exposure to UV  light, turned yellow, a phenomenon not seen for 1F5 T i N M R (300 M H z ,  dg-acetone): 5 7.75 (br s, IH, -NH-), 4.02 (td, 2H,  3  JHF  = 10 Hz,  3.59 (t, 2H, l-CHr-), 3.31 (dt, 2H, -C77 -NH-). ^ { T T } N M R 2  3  JHH  = 2 H Z , -Cr7 -CF ), 2  (188 MHz,  2  d -acetone): 5 6  -8.13 (t, -CF ), -45.27 (q, -CF -). After exposure to U V light the solution became dark 3  2  red/brown and a new species (4) formed. *H N M R 3  = 10 Hz,  JHF  ¥{ H}  19  l  3  NMR  JHH  d6-acetone): 5 4.02 (t, 2H,  (300 MHz,  = 1.6 Hz, -Cr7 -CF ), 3.62 (m, 2H, -C7f -), 2.05 (m, 2H, -C/7 -). 2  (188 MHz,  2  eq  ax  d -acetone): 5 -8.16 (t, -CF ), -45.19 (q, -CF -). Two of the 6  3  2  minor bands, one yellow and one red, were determined by U V - V i s spectophotometric analysis to be I " and I , respectively. UV-Vis (CH C1 ): [band 2] 292, 362; [band 3] 502. 8  3  2  2  2  4 3.2.1.5  3-Fluoropropylamine Hydrochloride (5) The parent amine for this compound was previously synthesized by Pattison et  al. from 3-fluoropropionic acid (see Scheme 3-5, p. 99), but the yield was low. Here 9  other possible routes to the HC1 salt of 3-fluoropropylamine were investigated.  52  References  10  on page  129  Chapter 3  Synthesis 1 (Scheme 3-6, p. 100) The use of dialkylaminosulfur fluorides as effective fluorinating agents has been reported previously; the hydroxyl group in alcohols can be replaced with fluorine atoms, and high yields are obtained for the non-rearranged fluoro compound. ' In a 25 mL two11 12  neck flask at 0°C under a constant flow of N was added DAST (1 mL) to 5 mL CH C1 2  2  2  to give a pale yellow solution. 3-Hydroxypropylamine (500 uL) was then added dropwise, when the mixture turned orange and then quickly brown. The mixture was stirred for 1 h at 0 °C, and then frozen using liquid N . The volatile components were then vacuum 2  transferred to another flask as a pale yellow solution through which HCl( ) was bubbled for g  5 min. A white solid was formed, and E t 0 (10 mL) was added to complete the 2  precipitation before the solid was isolated via suction filtration. ( N B . This compound is extremely hygroscopic and is best handled in an inert atmosphere.) H N M R X  spectroscopic  analysis of the solid revealed that 5 was formed, but only as a minor product. The major product was Et NH«HCl and according to spectral integration was present in a six-fold 2  excess over 5. H N M R (300 MHz, d -dmso): (5) 8 8.09 (br s, 2H, -NH-), 4.52 (dt, 2H, X  6  2  JHF  = 44 Hz,  3  2.00 (dp (dtt), 2H, IH,  = 4.8 Hz, -C# F), 2.91 (peak overlaps with H 0 peak, -NH-Cr7 -),  JHH  2  3  JHF  = 20 Hz,  3  2  JHH  = 4.7 Hz, -C# -CH F); ( E t N H - H C l ) 8 8.80 (br s, 2  2  2  -NH), 2.91 (m, 2H, NH-C# ), 1.20 (t, 3H, C/f -CH -). 2  d -dmso): (5) 8 -144.94 (spt (tt), 6  2  JHF  2  3  = 47.4 Hz,  3  JHF  2  19  F N M R (282 M H z ,  = 24.0 Hz, -CH F). Separation of 2  these two products was extremely difficult and was not further pursued.  Synthesis 2 (Scheme 3-7, p. 101) This procedure was adapted from that reported by Gibson and Bradshaw on the Gabriel synthesis of primary amines. In a Schlenk tube under 1 atm N was added 10 mL 13  2  D M F to potassium phthalamide (0.500 g, 2.70 mmol) to produce a white slurry. 1-Bromo3-fluoropropane (225 | i L , 2.45 mmol) was then added, and the reaction mixture was stirred at 70 °C for 24 h. Afterwards, the mixture was filtered and the filtrate was chromatographed using preparative T L C (CH C1 :acetone, 20:1); the major band (Rf = 2  2  0.85) was isolated as a colourless oil which when cooled yielded a white crystalline solid  53  References on page 129  Chapter 3  (0.334 g, 66 %), N-3-fluoropropylphthalamide (6). L R - M S [DCI(+)]: 207 (M*), 187 (TVT - F), 160 (IVF - C H C H F ) . HR-MS [DCI(+)]: calc. for C n H N Q F 207.06955; found 2  2  10  2  207.06938. *H N M R (300 M H z , de-acetone): 5 7.85 (s, 4H, -CH -), 4.56 (dt, 2H, Bz  51 Hz, 3  JHF  (tt),  3  JHH  = 6.2 Hz, -C# F), 3.80 (t, 2H,  = 27 Hz, 2  JHF  2  3  JHH  = 5.6 Hz, -Gr7 -CH F).  = 49.4 Hz,  2  3  JHF  19  2  3  JHH  = 6.8 Hz, -N-CH -\ 2  2  JHF  =  2.10 (dp (dtt), 2H,  F N M R (282 M H z , de-acetone): -142.08 (spt  = 24.0 Hz, -CH F). 2  The hydrazinolysis of 6 was adapted from a procedure described by Sheehan and Ryan. 6 (0.120 g, 0.579 mmol) was suspended in 10 mL EtOH and the mixture was 14  heated to 50 °C to facilitate dissolution. Hydrazine monohydrate (40 uL) was then added and the mixture was refluxed for 2.5 h and then was stirred for 16 h at r.t.; the initially colourless solution became a white slurry. The volume was reduced to ~5 mL, 6 M HC1 (10 mL) added, and the mixture stirred at 80 °C for 1 h. The resulting white precipitate was filtered and extracted with warm H 0 (3 x 10 mL). The solvent volume was then 2  reduced resulting in the re-formation of the white precipitate which was filtered off. The rotoevaporation/filtration procedure was then repeated to obtain additional product. The final filtrate was taken to dryness to recover the remaining product (0.060 g, 91 %). The *H N M R spectrum revealed only the presence of 5. Analysis calc. for C H NC1F: C, 3  9  31.72; H , 7.93; N , 12.33; found C, 31.56; H , 8.20; N , 11.83. L R - M S [DCI(+)] 78 (M+), 15  :  59  -F), 30 (H NCH -). HR-MS [DCI(+)]: calc. for C3H9NF 78.07190; found 2  2  78.07158.  3.2.2  2-Nitroimidazole Compounds  3.2.2.1  2-(2-Nitro-l-H-imidazol-l-yl)acetic acid (7) (see Scheme 3-9, p. 102) In a 250 mL RBF, SR2508 (3.70 g, 0.0173 moi) was dissolved in M e O H (100  mL) to give a clear, colourless solution. Dowex 50-X8(Fr) (40.3g, previously washed with equiv. amounts (50 mL) of 0.1M HC1, dd H 0 and MeOH) was then added followed 2  by an additional 50 mL MeOH. The mixture was refluxed for 24 h when amide bond cleavage occurs to form the intermediate methyl ester, 8, as shown by T L C analysis; the reaction mixture was then filtered and the filtrate was rotary evaporated to dryness  54  References on page 129  Chapter 3  yielding an orange oil (the accompanying orange colour comes from the cation exchange resin and, if the methyl ester is purified via column chromatography at this stage, 8 can be isolated as a white solid). The oil was dissolved in 100 mL 0.1M N a O H and the mixture stirred at 100 °C for 45 min to hydrolyze the ester (monitored by TLC). The mixture was cooled to r.t. and 2 M HC1 added dropwise until the pH was ~ 4. Saturated NaCl solution (50 mL) was added and the product was extracted with EtOAc (10 x 100 mL). The organic extracts were combined and dried with M g S 0 ; the mixture was filtered and the 4  solvent removed to give a pale yellow solid (0.805 g, 27%). UV-Vis (MeOH): (7) 316 (7.7); (8) 316 (7.7). H N M R (200 MHz, d -dmso): (7) 8 13.42 (s, I H , -OH), 7.63 (s, I H , X  6  lm-H ), 7.22 (s, IH, lm-H ), 5.20 (s, 2H, -CH -); (8) 8 7.64 (s, I H , lm-H ), 7.22 (s, I H , 5  4  2  5  Im-H ), 5.33 (s, 2H, -CH -), 3.72 (s, 3H, -CH ). Data are in agreement with those 4  2  previously reported.  3  16  o OH  I  N 0  H  2  OMe  OH  NO,  NO,  7  SR2508  8  The aqueous layers were also combined and rotovapped to dryness. The resulting yellow solid was then dissolved in M e O H (40 mL) and the solution was filtered; removal of the solvent from the filtrate gave a bright yellow solid (1.35 g). T L C analysis (shown below) showed the presence of large amounts of 7 that was not extracted with EtOAc.  Eluent (20: L CH Cl :MeOH) 2  2  (E = SR2508)  Figure 3-1:  T L C analysis of products obtained from 2-(2-Nitro-1 -H-imidazol-1 • yl)acetic acid (7) synthesis.  55  References  on page 129  Chapter 3  5.2.2.2  2-(2-Nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide  [EF5] Synthesis 1 (cf. Scheme 3-8, p. 102) 7 (0.200 g, 1.16 mmol) was dissolved in THF (25 mL) at 0°C under N . N M M 2  (130 uL, 1.18 mmol) was then added and the colourless mixture was stirred for 10 min. iBuClFrm (167 pL, 1.28 mmol) was added and the mixture was stirred for 30 min during which a white slurry was formed. The ice-bath was removed, and H NCH CF CF «HC1 2  2  2  3  (0.241 g, 1.41 mmol) and N M M (142 uL, 1.30 mmol) were added. The reaction slurry was stirred at r.t. for 4.5 h and the resulting white precipitate was filtered off and washed with THF ( 3 x 5 mL). The yellow filtrate was rotovapped to dryness yielding a pale yellow oil that was purified via column chromatography (CH C1 :acetone, 10:1 —» 5:2). The 2  2  major band (colourless) followed a minor yellow band and the combined fractions were rotovapped to dryness to yield EF5. Recrystallization from EtOAc/hexanes yielded a white microcrystalline solid (0.116 g, 33%).  Synthesis 2  17  2N0 Im (0.0257 g, 0.227 mmol) was dissolved in D M F (10 mL) at r.t. under 2  N , and powdered C s C 0 (0.0720 g, 0.221 mmol) was added to give a white slurry. The 2  2  3  mixture was heated to 50 °C when a pale yellow solution resulted; after 30 min a white precipitate was formed. The slurry was stirred for 2 h and IF5 (0.0724 g, 0.229 mmol) was then added, and the mixture stirred for 3 h at 50 °C. The resulting pale yellow precipitate was filtered off and washed with D M F ( 3 x 5 mL); the filtrate was then worked up as described in Synthesis 1 (0.0535 g, 78%); mp 136-137 °C. X-ray quality crystals were obtained by slow evaporation of a concentrated M e O H solution of EF5. Anal. calc. for C H N 4 0 F : C, 31.80; H , 2.34; N , 18.54; found: C, 31.96; H , 2.24; N , 18.37. L R - M S 8  7  3  5  [DCI(+)]: 303 O v T ) , 256 (M+ -N0 ). IR ( v , cm" ): 3317 (N-H); 3086 (C-H^); 2921, 2850 1  2  (C-H); 1689 (C=0); 1490 (N-O (obtained in  riVdmso  asym  ); 1367 (N-O ). UV-Vis (H 0): 323 (6.8). T I N M R sym  2  and in C D O D to show the effect that H/D exchange of the N - H 3  proton has on the -Gr7 -CF - signal): (300 MHz, CD OD) 5 7.47 (s, IH, lm-H ), 7.20 (s, 2  2  3  56  5  References on page 129  Chapter  1H, Im-H \ 5.25 (s, 2H, -Gr7 -CO-), 4.01 (t, 2H, 4  2  3  JHF  = 15 Hz, -Gr7 -CF -); (200 2  2  d -dmso) 6 9.05 (t, I H , N-#), 7.66 (s, I H , Im-H ), 7.21 (s, I H , lm-H ), 5  6  CH2-CO-),  MHz,  4.06 (td, 2H,  CD3OD):  3  JHF  = 15 Hz,  3  JHH  4  19  5 -10.66 (t, -CF ), -47.24 (q, -CF -); 2  3  2  (188 MHz,  MHz,  5.24 (s, 2H, -  = 1.5 Hz, -C# -CF -). F{TI} N M R 2  3  (188  d -dmso) 6 -4.34 (t, 6  -CF ), -41.31 ( q , - C i v ) . 3  H  '2  EF5 3.2.2.3  2-(Imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide [ImF5] Sodium imidazolate (0.0312 g, 0.344 mmol) was added in DMF  (5 mL) at r.t.  under N to give a yellow slurry. TF5 (0.117 g, 0.366 mmol) was then added and the 2  mixture was stirred for 4 h at 70 °C. The volume was reduced under vacuum to yield a yellow oil that was subsequently loaded onto a preparative TLC  plate (CH Cl :MeOH, 2  2  20:1). (Two major bands with similar Rf values were visible only after development using I .) The isolated brown solid from the two bands was heated to 120 °C under vacuum in a 2  sublimation apparatus when a yellow oil deposited onto a cold finger maintained at 5 °C (0.0365 g, 41%). Of note, exposure of the oil to air caused a colour change to dark purple while re-dissolution and exposure to air in acetone generated a bright yellow colour; removal of the acetone regenerated the dark purple colour. L R - M S [EI]: 257 OvT), 81 OVf  - C O N H C H C F C F ) . HR-MS [EI] calc. for C H N O F : 257.05875; found 2  257.05903. T I N M R  2  3  8  JHF  5  = 17 Hz,  3  JHH  5  6  Im-H ), 7.16 (s, I H , Im-H ), 6.97 (s, I H , lm-H ), 3  3  d -acetone): 5 7.96 (br. s, IH, N-#), 7.65 (s, I H ,  (300 MHz,  2  8  4  4.92 (s, 2H, -Cr7 -CO-), 4.10 (td, 2H,  = 6.4 Hz, -Gr7 -CF -). F{TI} N M R 19  2  2  2  (188 MHz,  d -acetone): 8 -8.16 6  (t,-CF ), -44.87 (q,-CF -). 3  2  57  References  on page  129  Chapter 3  H  O  ImF5  3.2.2.4  N-(2-nitro-l-H-imidazol-l-ethyl) pentafluoropropionamide [RevEF5]  Synthesis 1 (see Scheme 3-14, p. 107)  Step 1 In a 250 mL 2-neck flask, 2N0 Im (0.870 g, 7.70 mmol) was dissolved in 50 2  mL D M F under a constant flow of N . C s C 0 (2.75 g, 8.47 mmol) was then added and 2  2  3  the resulting yellow slurry was stirred at 90 °C for 3 h when dissolution was completed. Bromoethylphthalamide (3.96 g, 15.6 mmol) was added and the resulting yellow slurry was stirred for 15 h during which a white solid appeared. The solid was filtered off and the filtrate was reduced in volume to 2 mL before M e O H (20 mL) was added to precipitate a white solid (9). The solid was filtered, washed with hot M e O H (3 x 25 mL) and dried in vacuo at 80 °C for 3 d (1.95 g, 99 %). L R - M S [DCI(+)]: 287 fJVT + H), 240 (TVf - N 0 ) , 2  160  - 2N0 ImCH ). HR-MS [DCI(+)] calc. for  (Wt  2  2  C13H11N4O4:  287.07803; found  287.07810. TI N M R (300 MHz, d -acetone): 5 7.78 & 7.73 (overlapping multiplets, 4H, 6  Bz-H),  7.01 (s, IH, Im-H ), 6.89 (s, IH, 5  Im-H \ 4  4.70 (t, 2H, -C# -Phth), 4.18 (t, 2H, 2  -C# -CH -Phth). 2  2  Step 2 In a 25 mL RBF, 9 (0.0705 g, 0.273 mmol) was suspended in 10 mL EtOH. H N N H » H 0 (35 uL, 0.721 mmol) was then added via syringe and the mixture was 2  2  2  stirred at 80 °C for 18 h during which a clear, colourless solution was formed; T L C analysis revealed that 9 had completely reacted. 6 M HC1 (10 mL) was added to the reaction mixture and the EtOH was then removed to yield a white slurry. The solid [LR-MS DCI(+): 162 (M*), 132 (IVf - N H ) , 104 (TVT - C O - N H ) , 76 (M+ - 2CO 2  2  2  2  - N H ).] was isolated and washed with copious amounts of H 0 . The solvent was 2  2  2  removed from the pale yellow filtrate to yield an extremely hygroscopic, cream coloured  58  References  on page 129  Chapter 3  solid (10) (0.0565 g, 90 %). L R - M S [DCI(+)]: 157 ( M + H), 110 (IVf - N 0 ) . [Only the +  2  parent cation is observed with DCI (+), so it is unclear whether 1 or 2 moi of HC1 is associated with the compound.] H N M R (300 MHz, D 0 ) : 6 7.48 (d, I H , lm-H ), 7.18 !  2  5  (d, IH, lm-H ), 4.75 (t, 2H, Im-Cr7 -), 3.51 (t, 2H, -C# -NH ). 4  2  2  2  Step 3a In a 3-neck 50 mL flask, D C C (0.182 g, 0.880 mmol) and NHS (0.101 g, 0.880 mmol) were dissolved in 4 mL D M F under N to give a clear, colourless solution. 2  C F C F C 0 H (92 pL, 0.870 mmol) was added and a white microcrystalline precipitate 3  2  2  was immediately formed. The mixture was stirred for 1 h at r.t. before a D M F solution (3 mL) of 10 (0.206 g, 0.900 mmol) and N M M (198 uL, 1.80 mmol), was added. Stirring was continued for 4 h after which TLC analysis revealed that 10 had completely reacted. The white precipitate [DCU according to L R - M S [DCI(+)]: 225 (TVf), 143 (Tyf - C H ) , 6  5  99 (CeH -NH-)] was isolated and washed with D M F ; the filtrate was reduced in volume to 5  ~ 1 mL before being loaded onto a preparative T L C plate (CH Cl :MeOH, 18:1). The 2  2  product band (Rf = 0.45) was isolated to yield RevEF5 as an off-white solid (0.0757 g, 29 %). Step 3b , This reaction was continued from Step 2 prior to the addition of HC1 (same amount of 9 used for this synthesis). The colourless EtOH solution was taken to dryness to yield an oily, white film which was subsequently taken up in C H C I 3 (10 mL) to give a white slurry. N M M (1 equiv.) was added and the mixture stirred for 30 min at r.t. Upon addition of ( C F C F C O ) 0 (1 equiv.) the mixture became a clear solution which slowly 3  2  2  deposited a white residue on the walls of the flask over 5 h. Addition of E t O H led to the formation of a white precipitate which was filtered off and washed with E t O H ( 3 x 5 mL). T L C analysis of the filtrate revealed two major products which were separated using preparative T L C (CH Cl :MeOH, 20:1). The band corresponding to RevEF5 was 2  2  identified by comparison of the Rf value with that of an authentic sample of the compound. The isolated RevEF5 was reprecipitated from an EtOAc/hexanes mixture to yield a white solid (0.0265 g, 32%).  59  References  on page 129  Chapter 3  H N,  .N.  TO  N,  Y  N0  Y  N0  2  CF3  2  O RevEF5  10  9  Synthesis 2 Reaction conditions similar to those used for synthesis 1 of E F 5 (section 3.2.2.2) were used: C F C F C 0 H (25 uL, 0.238 mmol) was dissolved in THF (10 mL) 3  2  2  after which N M M (25 uL, 0.238 mmol) and iBuClFrm (36 uL, 0.276 mmol) were added giving a white slurry. 10 (0.0605 g, 0.262 mmol) and N M M (60 pL, 0.571 mmol) were then added and the mixture was stirred at r.t. for 4 h. The white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL); the crude product (filtrate) was then purified using preparative T L C (CH Cl :MeOH, 20:1). Four bands were observed and the 2  2  minor band with R = 0.37 was isolated to yield RevEF5 (0.0115 g, 16 %). Anal. calc. for f  C8H7N4O3F5:  C, 31.80; H , 2.34; N , 18.54; found: C, 31.85; H , 2.33; N , 18.59. L R - M S  [DCI(+)]: 320 (Tvf + N H / ) , 303 (M* + H), 287 ( V f - O), 273 (Jvf - NO), 256 (JVf - N 0 ) . H R - M S [DCI(+)] calc. for C H N 0 F : 303.05164; found 303.05137. IR (v, 2  8  8  4  3  5  cm- ): 3327 (N-H); 2965, 2932, 2866 (C-H); 1613 (C=0); 1457 (N-O 1  asym  ); 1383 (N-O ). sym  U V - V i s (MeOH): 316 (6.2). H N M R (300 MHz, d -acetone): 5 8.89 (br s, I H , -NH-), J  6  7.41 (s, I H , 1m-H ), 7.05 (s, IH, Im-H ), 4.68 (t, 2H, Im-Cr7 -), 3.86 (q (dt), 2H, s  4  2  -C// ,-NH-). F{ H} N M R (188 MHz, d -acetone): 5 -6.79 (t, -CF ), -46.43 (q, -CF -). 19  J  2  6  3  2  Synthesis 3 Based on published peptide-coupling techniques, '  18 19  C F C F C 0 H (30 pX, 3  2  2  0.286 mmol) and E t N (1 equiv.) were dissolved in D M F (2 mL) and STMT>BF (0.0941 3  4  g, 0.313 mmol), a reagent used to produce the -NHS esters was then added. The clear mixture was then stirred for 18 h at r.t. after which T L C analysis (CH Cl :MeOH, 20:1) 2  2  indicated the presence of a new product in high yield, presumed to be the activated succinimidyl ester. 7 (0.0652 g, 0.288 mmol) and E t N (2 equiv.) were subsequently 3  added, and the mixture, now cloudy, was stirred for 24 h when T L C analysis indicated the  60  References  on page 129  Chapter 3  presence of two major products. The solvent was removed under reduced pressure, and the residue was chromatographed using preparative T L C (CH Cl :MeOH, 25:1) to give 2  2  two pale yellow oils; comparison of T L C data with those of an authentic sample of RevEF5 revealed that neither oil contained the desired product. A minor band with the same Rf as RevEF5 was observed but the product was not isolated due to low concentrations (<1%).  3.2.2.5  2-(2-Nitro-l-H-imidazoI-l-yI)-N-(3-bromo-2,2,3,3-tetrafluoropropyI) acetamide [EF4Br] Reaction conditions similar to those used for the synthesis of EF5 (synthesis 1)  were employed: 7 (0.0760 g, 0.442 mmol) was dissolved in THF (15 mL) and N M M (50 pL, 0.458 mmol) and iBuClFrm (63 pL, 0.483 mmol) were added. To the resulting pale yellow slurry, H NCH CF CF Br«HCl (0.120 g, 0.487 mmol) and N M M (53 pL, 0.485 2  2  2  2  mmol) were added, and the mixture was then stirred at r.t. for 5 h. The white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The filtrate was taken to dryness to give a yellow oil that was purified via preparative T L C (CH Cl :MeOH, 20:1). 2  2  The isolated product band appeared to have two species present (with almost identical Rf values) and so a second preparative T L C was performed (CH Cl :MeOH, 12:1). The 2  2  major band separated from the minor band; however, when isolated and eluted with acetone the eluate was taken to dryness to give back the original oil. The oil was dissolved in 1 mL EtOAc followed by addition of hexanes (15 mL) which resulted in formation of a white precipitate. The fine white solid was isolated via suction filtration, washed with E t 0 2  ( 3 x 5 mL) and dried in vacuo (0.0425 g, 27%). Anal. calc. for CgH7N O F4Br»0.15 E t 0 : 4  3  2  C, 27.61; H , 2.29; N , 14.97; found C, 27.76; H , 2.13; N , 14.94. L R - M S [DCI(+)]: 365, 363 (Wt+ H), 335, 333 (Wt-NO), 318, 316 (M^ - N 0 ) , 283 (Wt- Br). H R - M S [DCI(+)] 2  calc. for CgHgOsNdVTir (CgH 0 N4F4 Br): 364.96954 (362.97163); found 364.96834 79  g  3  (362.97100). IR (v, cm ): 3310 (N-H); 3076 (C-H^); 2960, 2851 (C-H); 1697 (C=0); -1  1493 (N-O  asym  ); 1370 (N-O ). UV-Vis (MeOH): 316 (6.9), 232 (3.5). TI N M R (300 sym  M H z , de-acetone): 8 8.18 (br s, IH, -NH-), 7.49 (s, I H , lm-H ), 7.20 (s, I H , lm-H ), 5.31 s  61  4  References  on page 129  Chapter 3  (s, 2H, -C#2-C0-), 4.10 (td, 2H, J - = 15.6 Hz, -NH-C/^,-CF -). ^Ff/H} N M R (188 3  H  2  F  MHz, de-acetone): 5 10.04 (t, -CF Br), -38.75 (t, 2  -CU -CF -). 2  2  H  3.2.2.6  2-(2-Nitro-l-H-imidazol-l-yl)-N-(3,3,3-trinuoropropyI)acetamide [EF3] Compound 7 (0.150 g, 0.873 mmol) was dissolved in THF (20 mL) at 0°C  under N . N M M (96 pL, 0.873 mmol) was then added and the clear, colourless mixture 2  was stirred for 10 min. iBuClFrm (125 uL, 0.961 mmol) was added and the solution was stirred for 30 min during which the mixture became a white slurry. The ice-bath was removed and H NCH CH CF *HC1 (0.199 g, 0.961 mmol) and N M M (105 uL, 0.961 2  2  2  3  mmol) were added. Stirring was continued for 5.5 h at r.t. before the white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL); the yellow filtrate was taken to dryness to yield a yellow solid that was subsequently recrystallized from EtOAc/hexanes to give white microcrystalline needles (0.193 g, 83 %). Anal. calc. for C8H9N4O3F3: C, 36.10; H , 3.41; N , 21.05; found: C, 36.35; H , 3.35; N , 21.05. IR (v, cm" ): 3312 (N-H), 1  3026, 2923, 2851 (C-H), 1669 (C=0), 1493 (N-O  a9yra  ), 1370 (N-O ). U V - V i s (dmso): sym  328 (9.4); (MeCN) 322 (4940). *H N M R (300 MHz, d -acetone): 5 7.81 (br s, IH, -NH-), 6  7.50 (s, IH, Im-H ), 7.15 (s, IH, Im-H ), 5.25 (s, 2H, -CH -CO-), 5  4  -NH-Cr7 -), 2.50 (qt, 2H, J - F = 11 3  2  H  2  HZ,  -CH CF ), 2  3  '^{'H)  3.50 (q, 2H,  N M R (188 M H z ,  de-acetone): 5 10.84 (s, -CF ). 3  H  •N, NO,  62  References on page 129  Chapter 3  3.2.2.7  2-(2-Nitro-l-H-imidazol-l-yl)-N-(2,2,2-trifluoroethyl)acetamide [EF3(-1)] Reaction conditions similar to those used for the synthesis of EF3 were  employed: 7 (0.172 g, 0.998 mmol) was dissolved in THF (17 mL) and N M M (109 uL, 0.996 mmol) and iBuClFrm (142 pL, 1.09 mmol) were added. To the resulting pale yellow slurry, H NCH CF «HC1 (0.149 g, 1.10 mmol) and N M M (120 uL, 1.10 mmol) 2  2  3  were added, and the mixture was then stirred at r.t. for 4 h. The white precipitate that formed was filtered off and washed with Et 0 ( 3 x 5 mL); the filtrate was taken to dryness 2  to give a yellow oil that was purified using a C T R O N (THF:acetone, 10:1). The fourth, most concentrated band was isolated to yield a white solid (0.208 g, 83%). Anal. calc. for C H N O F ' 0 . 1 5 acetone: C, 34.40; H , 3.08; N , 21.39; found C, 34.25; H , 2.86; N , 7  7  4  3  3  21.31. IR (v, cm" ): 3299 (N-H); 3117 (C-H^); 2923, 2851 (C-H); 1685 (C=0); 1489 1  (N-O  asym  6 8.21  ); 1374 (N-O ). UV-Vis (MeOH): 316 (8.5). TI N M R (300 M H z , d -acetone): sym  6  (br s, I H , -NH-), 7.53  -CH2-CO-),  4.06  (qd,  2H,  3  J -F H  (s,  I H , Im-H \ 7.15 5  (s,  = 9.4 Hz, -CH -CF ). 2  3  I H , lm-H ), 5.36 4  F{ H}  19  l  (s, 2H,  N M R (188 M H z ,  de-acetone): 5 4.30 (s, -CF ). 3  H  3.2.2.8  2-(2-Nitro-l-H-imidazol-l-yl)-N-(3-bromo-3,3-difluoropropyl) acetamide [EF2Br] Reaction conditions similar to those in the synthesis of EF3 were used: 7 (0.206  g, 1.20 mmol) was dissolved in THF (20 mL) and N M M (131 uL, 1.20 mmol) and iBuClFrm (172 uL, 1.32 mmol) were added. To the resulting pale orange slurry were added H N C H C H C F B r ' H C l (0.273 g, 1.30 mmol) and N M M (142 uL, 1.30 mmol). 2  2  2  2  The mixture was then stirred at r.t. for 6 h. The white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The filtrate was taken to dryness to give an orange oil that was purified via C T R O N (Et 0:acetone, 10:0 - » 10:3). A n orange band eluted 2  63  References  on page 129  Chapter 3  from the plate first using Et20; the product band was then eluted using acetone which when taken to dryness yielded an off-white solid (0.242 g, 62%). Crystals suitable for X-ray analysis were obtained from slow evaporation of a concentrated M e O H solution. Anal. calc. for C HoN 0 F Br: C, 29.38; H , 2.77; N , 17.13; found C, 29.35; H , 2.87; N , 8  4  3  2  17.22. L R - M S [DCI(+)]: 346, 344 (tvf + N H / ) , 329, 327 (TVT+ H), 299, 297 (Ivf - NO), 282, 280 OVT - N0 ), 247 ( M + - Br). HR-MS [DCI(+)] calc. for C H i O N 2  8  0  3  81 4  Br  (C Hio0 N4 Br): 328.98838 (326.99047); found 328.98733 (326.99038). IR (v, cm" ): 79  1  3  8  3287 (N-H); 3120 (C-Hm,); 3062, 2963 (C-H); 1671 (C=0); 1494 (N-O  asym  ); 1371  (N-O ). U V - V i s (MeCN): 328 (6.6). TT N M R (300 MHz, d -acetone): 5 7.86 (br s, I H , sym  6  -NH-), 7.50 (s, I H , Im-H ), 7.14 (s, I H , Im-H \ 5.25 (s, 2H, -CH -CO-), 5  4  -NH-Cr7 -), 2.73 (tt, 2H,  3  2  J -F H  3.55 (q, 2H,  2  = 17 Hz, -C# -CF Br). 2  2  19  F{TI} N M R (188 M H z ,  de-acetone): 5 32.71 (s, -GF Br). 2  H  3.2.2.9  2-(2-Nitro-l-H-imidazol-l-yl)-N-(3,3-difluoropropylene)acetamide [E=F2] An attempt to synthesize EF3 from EF2Br using a standard halogen exchange  procedure (Br for F) led mainly to the "-HBr elimination product" E=F2; no EF3 was observed according to ^ { / H } N M R . Addition of EF2Br (0.0401 g, 0.122 mmol) to Bu NF»H 0 (0.0684 g, 0.245 mmol) in 2 mL M e C N led to formation of a bright yellow 4  2  solution. Stirring was continued at r.t. for 10 h before the solvent was removed and the major product isolated as a yellow oil via preparative T L C ( C H C l : M e 0 H , 25:1). The oil 2  2  was dissolved in EtOAc, and hexanes were added to precipitate a white solid, which was filtered off and dried in vacuo (0.0132 g, 44 %). Anal. calc. for C H N 0 F : C, 39.03; H , 8  8  4  3  2  3.28; N , 22.76; found C, 39.12; H , 3.20; N , 22.55. IR (v, cm" ): 3311 (N-H); 1  3 1 0 4 ( 0 - ^ ) ; 2923, 2851 (C-H); 1668 (C=0); 1487 (N-O  asym  ); 1375 (N-O ). U V - V i s sym  (MeCN): 320 (3.9). H N M R (300 MHz, d -acetone): 5 7.75 (br s, I H , -NH-), 7.50 (s, X  6  IH, Im-Hi), 7.12 (s, I H , Im-H ), 5.22 (s, 2H, -CH -CO-), 4  2  64  4.55 (dtd, I H ,  ^(trans)  References  = 25  on page 129  Chapter 3  Hz, Hz,  3  = 7.7 Hz,  JHH  Vctrans) =  3  J H F (  C  I  S  = 2.2 Hz, -CH=CF ),  )  3.85 (dddd, 2H,  2  JHH  2  = 9.2 Hz,  3  JHH  = 7.1  2.1 Hz, Vcds) = 1.4 Hz, -Cft-CH=). ^ { T I } N M R (188 M H z ,  ds-acetone): 5 -12.78 (d,  2  JFF  = 26.3 Hz, =C-F ), -13.82 (d, cis  2  JFF  = 26.3 Hz, ^ - i w ) .  3.2.2.10 2-(2-Nitro-l-H-imidazol-l-yl)-N-(3-fluoropropyl)acetamide [EF1] Reaction conditions similar to those in the synthesis of EF3 were used: 7 (0.124 g, 0.722 mmol) was dissolved in THF (15 mL) and N M M (79 uL, 0.717 mmol) and iBuClFrm (103 pL, 0.789 mmol) were added. To the resulting white slurry were added H N C H C H C H F ' H F (0.241 g, 1.41 mmol) and N M M (142 pL, 1.30 mmol). The 2  2  2  2  mixture was stirred at r.t. for 4.5 h before the white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The filtrate was taken to dryness to give a yellow oil that was purified using preparative TLC (CH Cl :MeOH, 20:1); the major band isolated 2  2  yielded a creamy white solid (0.0146 g, 9 %). The hydrochloride salt of 3-fluoropropylamine (5) was used in order to eliminate problems caused by the presence of HF in the hydrofluoride salt. Identical reaction conditions were used as described above, but using 0.264 g (1.41 mmol) of the hydrochloride, and a white solid was isolated (0.0249 g, 15 %). Anal. calc. for C8H11N4O3F:  C, 41.74; H , 4.82; N , 24.34; found C, 40.69; H , 4.62; N , 21.34. L R - M S  [DCI(+)]: 231 (M++H), 184 (M+-N02). HR-MS [DCI(+)J calc. for CgHjzOsN^: 231.08933; found 231.08881. IR (v, o n ) : 3284 (N-H); 3124 (C-Hfa); 2926, 2851 (C-H); 1  1660 (C=0); 1492 (N-O  a8ym  ); 1369 (N-O ). UV-Vis (MeOH): 316 (6.7), 210 (5.2). T I sym  N M R (300 MHz, d -acetone): 8 7.64 (br s, IH, -NH-), 7.52 (s, IH, lm-H ), 7.18 (s, I H , 6  5  lm-H ), 5.22 (s, 2H, -CH -CO-\ 4.51 (dt, 2H, J . = 66 Hz, J . = 9.4 Hz, -CH F), 3.38 2  4  3  2  H  F  H  H  2  (q (dt), 2H, -NH-Cr7 ,-), 1.91 (dp (dtt), 2H, J . = 42 Hz, J . = 9.7 Hz, -C# -CH F). 3  2  3  H  65  F  H  H  2  2  References on page 129  Chapter 3  19  F N M R (282 MHz, d -acetone): 5 -142.50 (spt (tt), 6  2  J  H  .  F  = 49.1 Hz,  3  J -F H  = 25.1 Hz,  -CH F). 2  T  o  N02  Comparison of the TLCs obtained from the above reactions shows that both gave two other major products having higher Rf values than that of EF1; a small amount of unreacted 7 (R = 0) was also observed (Figure 3-2). The difference in yield of EF1 f  (9% vs. 15%) between the two reactions probably indicates that the presence of HF does not limit the reaction yield.  Band 1 = 2N0 Im-methylester (8) 2  Band 2 = 2N0 Im-isobutylester (11) 2  Band 3 = EF1 Band 4 = 2N0 Im-acetic acid (7) 2  N  Y  N  N0  Figure 3-2:  R= OH (7) OMe (8) 0"Bu (11) 2  T L C analysis of products obtained from EF1 synthesis.  Bands 1 and 2 were also isolated and characterized in order to determine the identities of the side-products responsible for the low yields of EF1. The pale yellow solid isolated from band 1 was identified as 8 as evidenced by H N M R spectroscopy (p. 55). X  This species is likely formed from reaction of the acyl carbonate with M e O H during preparative T L C separation. The species isolated from band 2 was more difficult to identify; the *H N M R spectrum shows signals corresponding to the isobutyl protons and  66  References  on page 129  Chapter 3  JK revealed the presence of a Vco band. M S analysis definitely showed that the species is the isobutylester (11) (see Scheme 3-13, p. 106). L R - M S [DCI(+)]: 228 (Tvf), 182 (JVT - N 0 ) . IR (v, cm ): 3125 (C-H^); 2964, 2876 (C-H); 1751 (C=0); 1490 (N-O -1  2  asym  ); 1369  (N-O ). *H N M R (200 M H z , d -acetone): 5 7.59 (s, IH, Im-H ), 7.19 (s, IH, lm-H ), sym  6  5  4  5.40 (s, 2H, -C#2-CO-), 4.01 (d, 2H, O-C/^-CH-), 1.92 (m, IH, -Ci7-(CH ) ), 0.87 (d, 3  2  6H, -CH-(C# ) ). 3  2  3.2.2.11 2-(2-Nitro-l-H-imidazoI-l-yl)-N-(2-nuoroethyl)acetamide [EFl(-l)]  Synthesis 1 Reaction conditions similar to those in the synthesis of EF3 were used: 7 (0.154 g, 0.891 mmol) was dissolved in THF (10 mL), and N M M (105 pL, 0.953 mmol) and iBuClFrm (140 uL, 1.07 mmol) were added. To the resulting pale yellow slurry were added H NCH CH F-HC1 (0.132 g, 1.33 mmol) and N M M (120 pL, 1.10 mmol). The 2  2  2  mixture was stirred at r.t. for 6 h before the white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The filtrate and washings were taken to dryness to give a yellow oil that was chromatographed using C T R O N (CH C1 :THF, 10:3). A white solid 2  2  was isolated from the fourth band and was recrystallized from acetone/Et 0 (1:1) (0.124 2  g, 64%). Anal. calc. for C H N 0 F : C, 38.89; H , 4.20; N , 25.92; found C, 38.88; 7  9  4  3  H,  4.25; N , 25.77. IR (v, cm" ): 3296 (N-H); 3120 (C-HmO; 2926, 2851 (C-H); 1669 (C=0); 1  1488 (N-O  asym  ); 1372 (N-O ). UV-Vis (MeOH): 316 (6.2), 212 (4.7). *H N M R (300 sym  M H z , de-acetone): 5 7.87 (br s, IH, -N/7-), 7.50 (s, IH, Jm-H ), 7.14 (s, IH, Jm-H ), 5.26 5  (s, 2H, -CH2-CO-), 4.48 (dt, 2H,  2  J . H  F  = 48 Hz,  3  J . H  H  4  = 6.5 Hz, -C# F), 3.55 (dq (dtd), 2  2H, J . F = 30 Hz, JHH = 6.2 Hz, -NH-Cf^-). F N M R (282 M H z , de-acetone): 6 -144.94 3  3  19  H  (spt (tt), J - F = 49.1 Hz, 2  H  3  J H  F  = 26.2 Hz, -CH F). 2  1 3  C N M R (50 MHz, d -acetone): 5  166.89 (-CO-), 130.92 and 129.92 (Jm-C  ), 84.70 (d,  (-CH2-CO-), 42.89 (d,  2  4and5  2  J  C F  6  = 166.5 Hz, -CH F), 54.33 2  = 21 Hz, -NH-CH ,-). H  67  References on page 129  Chapter 3  Synthesis 2 In a N -flushed 50 mL, 2-neck flask at -78 °C, CH C1 (10 mL) was added to 2  2  2  SR2508 (0.100 g, 0.466 mmol) to give a white slurry; D A S T (80 uL, 0.606 mmol) was then added dropwise and the mixture was stirred for 3 h at r.t. The resulting yellow/orange solution was chromatographed using C T R O N (Et 0 —> THF), but neither of the two 2  products was the desired EFl(-l). A wafer-like, crystalline, white solid was isolated from the second band (R = 0.40 with THF) (~ 10 mg). *H N M R (300 M H z , d -acetone): 8 f  6  7.59 (s, I H , lm-H ), 7.38 (dd, IH, -CF -N#-), 7.13 (s, I H , Im-H ), 5.34 (q, 2H, 5  2  4  -C/f -CF -), 4.38 (dt, 2H, -NH-C# ), 3.71 (t, 2H, -C# OH). ^ F l / H } N M R (188 M H z , 2  2  2  2  d6-acetone): 8 -6.22 (s, -GF -NH-). N M R data indicate fluorine substitution at the 2  carbonyl group (12) rather than at the O H group, and so this reaction procedure was pursued no further. H  12  3.2.2.12 2-(2-Nitro-l-H-imidazol-l-yl)-N-(3-chloropropyl)acetamide  [ECU]  Reaction conditions similar to those for the synthesis of EF3 were used: 7 (0.103 g, 0.596 mmol) was dissolved in THF (15 mL) and N M M (64 uL, 0.581 mmol) and iBuClFrm (83 pL, 0.636 mmol) were added. To the resulting pale yellow slurry were added H NCH CH CH C1«HC1 (0.0871 g, 0.700 mmol) and N M M (71 pX, 0.650 mmol). 2  2  2  2  The mixture was then stirred at r.t. for 3.5 h after which the yellow precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The filtrate and washings were taken to dryness and the resulting yellow oil was chromatographed using preparative T L C (CH Cl :MeOH, 20:1). The major band yielded a white solid (0.0945 g, 64%). Anal. calc. 2  2  for C g H n N ^ s C l : C, 38.96; H , 4.50; N , 22.71; found: C, 39.36; H , 4.64; N , 22.70. IR (v, cm ): 3287 (N-H); 3099 (C-HfoO; 2946, 2868 (C-H); 1657 (C=0); 1484 (N-O -1  asym  ); 1368  (N-O ). U V - V I S (MeOH): 316 (6.1), 216 (3.4). *H N M R (300 M H z , d -acetone): 8 sym  6  68  References  on page 129  Chapter 3  7.70 (br s, 1H, -NH-),  7  4  9  (> > Im-#j), 7.14 (s, IH, Im-/^), 5.23 (s, 2H, -CH -CO-\ s  1H  2  3.66 (t, 2H, -CftCl), 3.39 (q (dt), 2H, -NH-Cft-),  1  9  8  (P ( )> > -CH -C# -). tt  2H  2  2  H  a  3.2.2.13 2-(2-Nitro-l-H-imidazol-l-yl)-N-(2-chloroethyl)acetamide [ECU(-l)] Synthesis 1 Reaction conditions similar to those for the synthesis of EF3 were used: 7 (0.100 g, 0.585 mmol) dissolved in THF (15 mL) and N M M (65 uL, 0.590 mmol) and iBuClFrm (84 pL, 0.644 mmol) were added. To the resulting yellow slurry were added H NCH CH C1»HC1 (0.0746 g, 0.644 mmol) and N M M (72 uL, 0.659 mmol). The 2  2  2  mixture was then stirred at r.t. for 4.5 h before the white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The filtrate and washings were taken to dryness to give a yellow oil which was chromatographed using preparative T L C (CH Cl :MeOH, 20:1); the major band yielded a white solid (0.0939 g, 69%). Anal. calc. 2  for  2  C7H9N4O3CI:  C, 36.14; H , 3.90; N , 24.08; found: C, 36.17; H , 3.92; N , 24.04. L R - M S  [EI(+)]: 233 (M ), 196 (M* - CI), 186 (TS/t - N 0 ) . IR (v, cm" ): 3292 (N-H); 3117 4  1  2  (C-Hfa); 2926, 2851 (C-H); 1665 (C=0); 1489 (N-O  asym  ); 1373 (N-O ). U V - V i s sym  (MeOH): 316 (5.8), 218 (3.7). *H N M R (300 MHz, d -dmso) 8 8.64 (br t, I H , -NH-), 6  7.53 (s, I H , lm-H X 7.20 (s, I H , lm-H ), 5.15 (s, 2H, -CH -CO-), 3.61 (t, 2H, -CH C\), 5  4  3.44 (q (dt), 2H, -NH-C# -). 2  and 127.58 (Im-C  1 3  2  2  C N M R (50 MHz, d -acetone): 8 166.17 (-CO-), 128.90 6  ), 51.63 (-CH -CO-), 43.46 (-OLC1), 40.90 (-NH-CH -).  4and5  2  2)  H  69  References  on page 129  Chapter 3  Synthesis 2 (see Scheme 3-16, p. 124) In a flask equipped with a reflux condenser, CHC1 (10 mL) was added to 3  SR2508 (0.403 g, 1.88 mmol) at 0 °C to yield a white slurry. D M F (3 mL) was then added, resulting in a clear, colourless solution. TsCl (0.553 g, 2.90 mmol) and py (350 pL, 4.34 mmol) were added (together), and the reaction mixture was stirred for 0.5 h. The icebath was then removed and the reaction mixture was allowed to warm to r.t. E t 0 was 2  then added dropwise ( - 3 0 mL) and the white precipitate that formed was isolated via suction filtration. (From *H N M R , this solid was found to be a mixture of pyridinium hydrochloride and pyridinium tosylate.) The filtrate was taken to dryness and the resulting residue was dissolved in minimal acetone before being loaded onto a column. The column was then eluted with 5% acetone in CH2CI2 and two major bands were isolated. The first band yielded unreacted TsCl, while the second band gave ECll(-l) (0.207 g, 47 %).  3.2.2.14 2-(2-Nitro-l-H-imidazol-l-yl)-N-(3-bromopropyl)acetamide [EBrl] Reaction conditions similar to those for the synthesis of EF3 were used: 7 (0.100 g, 0.585 mmol) was dissolved in THF (15 mL), and N M M (65 pL, 0.590 mmol) and iBuClFrm (84 pL, 0.644 mmol) were added. To the resulting pale yellow slurry were added H NCH CH CH Br»HBr (0.141 g, 0.644 mmol) and N M M (72 pL, 0.659 mmol). 2  2  2  2  The mixture was then stirred at r.t. for 4 h before the white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The filtrate and washings were reduced in volume and subsequently loaded onto a column and eluted dropwise (CH C1 :THF, 5:1). 2  2  The first 200 mL of eluate was discarded and the first band was collected in 3 x 50 mL fractions. The eluent strength was increased (using 50% THF) and after 150 mL of eluate, the second band emerged containing the desired product. Eight 50 mL tractions were collected (the purity of the product being confirmed by T L C analysis) and combined before being taken to dryness to give a pale yellow solid (0.113 g, 67%). Anal. calc. for C H n N 0 B r : C, 33.04 ; H , 3.83; N , 19.22; found: C, 32.19; H , 3.68; N , 18.57. L R - M S 8  4  3  [DCI(+)]: 293, 291 (Wt + H), 246, 244 (M+ - N 0 ) . H R - M S [DCI(+)] calc. for 2  C Hi 0 N 8  2  3  8 1 4  Br (C H 0 N 8  12  3  79 4  B r ) : 293.00723 (291.00932); found 293.00732 (291.00932).  70  References  on page 129  Chapter 3  IR (v, cm- ): 3286 (N-H); 3100 (C-Hfa); 2929 (C-H); 1656 ( C O ) ; 1484 (N-0 1  asym  ); 1366  (N-0 ). UV-Vis (MeOH): 316 (6.2), 212 (3.7). H N M R (300 MHz, d -acetone): 6 7.70 X  sym  6  (br s, I H , -NH-), 7.51 (s, IH, lm-H ), 7.08 (s, IH, Jm-H ), 5.21 (s, 2H, -CH -C0-), 3.50 5  4  2  (t, 2H, -CH Br), 3.39 (q (dt), 2H, - N H - C ^ - ) , 2.01 (m (tt), 2H, -C# -CH Br). 2  2  H  2  .Br  3.2.2.15 2-(2-Nitro-l-H-imidazol-l-yl)-N-(2-bromoethyl)acetamide [EBrl(-l)] Synthesis 1 Reaction conditions similar to those for the synthesis of EF3 were used: 7 (0.237 g, 1.37 mmol) was dissolved in THF (25 mL), and N M M (154 uL, 1.40 mmol) and iBuClFrm (196 uL, 1.50 mmol) were added. To the resulting pale yellow slurry were added H NCH CH Br«HBr (0.312 g, 1.53 mmol) and N M M (167 uL, 1.53 mmol). The 2  2  2  mixture was then stirred at r.t. for 4 h before the white precipitate was filtered off and washed with Et 0 ( 3 x 5 mL). The pale yellow filtrate and washings were taken to dryness 2  and the residue was chromatographed on a silica gel column. The desired product was collected in the first 3 fractions as its R was higher than those of the side-products and f  unreacted starting material. The purity of these fractions was confirmed by T L C analysis; the combined fractions were taken to dryness to give a pale yellow solid (0.269 g, 71%). Anal. calc. for C v H o N ^ B r ' f O . S THF): C, 35.90 ; H , 3.63 ; N , 17.53; found: C, 36.04 ; H , 3.92 ; N , 17.60. IR (v, cm' ): 3293 (N-H); 3108 (C-H^); 2923, 2854 (C-H); 1668 ( C O ) ; 1  1488 (N-Oa ); 1370 (N-O ). UV-Vis (MeOH): 316 (6.4), 214 (3.5). *H N M R (300 Sym  sym  M H z , CD OD): 8 7.41 (s, I H , lm-H ), 7.15 (s, IH, lm-H ), 5.13 (s, 2H, -C# -CO-), 3.58 3  5  4  2  (t, 2H, -NH-CH -), 3.43 (t, 2H, -C# Br); (300 M H z , d -acetone) 7.99 (br s, I H , 2  2  6  -N#-CH -), 7.53 (s, I H , Im-H ), 7.17 (s, IH, lm-H ), 5.28 (s, 2H, -CH -CO-\ 3.64 (q 2  s  4  2  (dt), 2H, -NH-Cr7 -), 3.52 (t, 2H, -CH Br). [The analysis in d -acetone permitted 2  2  6  identification of the triplet -NH-CH - (methylene) signals found in the C D O D spectrum.] 2  3  71  References  on page 129  Chapter 3  13  C N M R (50 M H z , d -acetone): 5 172.75 (-CO-), 134.84 and 134.26 (Im-C ), 6  58.60  4and5  (-CH2-CO-), 48.24 (-NH-CH2,-), 37.88 (-CH Br). 2  H  r=\  •N.  N02  Synthesis 2 In a Schlenk tube under a flow of N , SR2508 (0.114 g, 0.533 mmol) was 2  dissolved in D M F (6 mL). The solution was then acidified using HBr( (pH acidity tests g)  were accomplished using pH paper, the D M F sample first being added to H 0; the HBr ) 2  (g  line was flushed with N prior to use as this reduces fuming that results from the presence 2  of H 0.) The N -flow was resumed and PBr (62.5 uL, 0.659 mmol) was added via 2  2  3  syringe. The reaction was monitored by T L C and, after 3 h M e O H (10 mL) was added to quench unreacted PBr . The mixture was then stirred for 5 min after which E t N was 3  3  added until the mixture was strongly basic. Excess E t N and D M F were removed under 3  vacuum and the remaining residue was chromatographed (CH Cl :MeOH, 10:1). The 2  2  eluate was collected in fractions, each analyzed using T L C ; the fractions containing the product were combined and taken to dryness to yield a pale yellow solid (0.0177 g, 12 %).  3.2.2.16 2-(2-Nitro-l-H-imidazol-l-yl)-N-(propyl)acetamide  [EPrA]  Reaction conditions similar to those for the synthesis of EF3 were used: 7 (0.0808 g, 0.469 mmol) was dissolved in THF (10 mL), and N M M (50 uL, 0.467 mmol) and iBuClFrm (70 pL, 0.536 mmol) were added. To the resulting yellow slurry were added H NCH CH CH »HC1 (0.0466 g, 0.488 mmol) and N M M (56 uL, 0.513 mmol). 2  2  2  3  The mixture was then stirred at r.t. for 4.5 h before the white precipitate was filtered off and washed with THF ( 3 x 5 mL). The filtrate and washings were taken to dryness and the desired product was isolated using preparative T L C (CH Cl :MeOH, 10:1) to give a white 2  2  solid (0.0301 g, 30 %). Anal. calc. for C H i N 0 : C, 45.28 ; H , 5.70 ; N , 26.40; found: g  2  4  3  C, 45.21 ; H , 5.58 ; N , 26.27. IR (v, cm ): 3290 (N-H); 3113 (C-Hm,); 2969, 2878 (C-H) -1  1662 ( C O ) ; 1490 (N-O  asym  ); 1361 (N-O ). UV-Vis (MeOH): 316 (5.6), 228 (3.1). TI sym  72  References  on page 129  Chapter 3  N M R (300 M H z , d -acetone): 5 7.52 (br s, IH, -Nr7-CH -, overlapping with peak at 6  2  7.50), 7.50 (s, IH, 1m-H ), 7.13 (s, IH, Im-H ), 5.20 (s, 2H, -C# -CO-), 3 20 (q (dt), 2H, 5  4  2  -NH-Cr7 -), 1.52 (sex (qt), I H , -NHCH C# -), 0.89 (t, 3H, -CH -C# ). 2  2  2  2  3  H  •N.  r=\  3.2.2.17  2-(2-Nitro-l-H-imidazol-l-yl)-N-(wo-amyl)acetamide [EIAA] Reaction conditions similar to those for the synthesis of E F 3 were used: 7  (0.0812 g, 0.472 mmol) dissolved in THF (10 mL), and N M M (50 pL, 0.467 mmol) and iBuClFrm (70 pL, 0.536 mmol) were added. To the resulting yellow slurry was added H N C H C H C H ( C H ) (59 pL, 0.508 mmol). The mixture was then stirred at 2  2  2  3  2  r.t. for 4 h before the white precipitate was filtered off and washed with THF ( 3 x 5 mL). The filtrate and washings were taken to dryness and the desired product was isolated using preparative TLC (CH Cl :MeOH, 20:1) to give a white solid (0.0773 g, 2  2  68%). Anal. calc. for C i H N O : C, 49.99 ; H , 6.71 ; N , 23.32; found: C, 49.64 ; H , 0  16  4  3  6.56 ; N , 22.98. IR (v, cm ): 3267 (N-H); 3127 (C-H^); 2958, 2875 (C-H); 1656 -1  ( C O ) ; 1490 (N-O  ); 1374 (N-O ). UV-Vis (MeOH): 316 (7.1), 228 (4.0). H X  asym  sym  N M R (300 M H z , d -acetone): 5 7.52 (br s, I H , -Nr7-CH -, overlapping with peak at 6  2  7.50), 7.50 (s, I H , lm-H ), 7.11 (s, IH, Im-H ), 5.20 (s, 2H, -C# -CO-), 3.27 (q (dt), s  4  2  2H, -NH-C# -), 1.65 (m, I H , -C#-(CH ) ), 1.38 (q (dt), 2H, -NHCH C# -), 0.88 (d, 2  3  2  2  2  6H, -CH(C# ) ). 3  2  H  73  References on page 129  Chapter 3  3.2.3  2-Methyl-5-Nitroimidazole Compounds  3.2.3.1  2-(2-Methyl-5-nitro-lfl-imidazol-l-yl)acetic acid (13) A slightly modified literature synthetic procedure was used. In a 3-neck flask, 20  metronidazole (3.33 g, 19.5 mmol) was dissolved in acetone (120 mL) under N . To the 2  resulting white slurry was added Jones' Reagent (20 mL of (7g C r 0 + 50 mL H 0 + 6.1 3  2  mL H2SO4}) dropwise via a pressure-equalized addition funnel, when a clear, dark orange solution resulted; this was then stirred at r.t. for 24 h to ensure complete oxidation of the alcohol to the corresponding acid. [After 3 h, T L C analysis revealed the presence of three compounds, the unreacted metronidazole, trace aldehyde and the acid > Rf(acid)).]  (Rflaidehyde) > Rf^icohoi)  The final mixture was now dark green and a green, oily deposit was observed.  The mixture was filtered twice through a plug of Celite to remove the green oil, and the clear, pale yellow filtrate was rotovapped to dryness. The resulting residue was then dissolved in H 0 (50 mL) and the solution was transferred to a separatory funnel and 2  washed with EtOAc (5 x 80 mL). The organic fractions were combined and dried over MgS04 (10 min) before filtration and rotary evaporation yielded a pale yellow solid (3.61g, 55 %); mp 176-178 °C (lit. mp 179-180 °C). TI N M R (200 M H z , D 0 ) : 5 8.00 21  2  (s, I H , Im-H ), 4.85 (s, 2H, -CH -\ 4  2  2.40 (s, 3H, Im-Gr7 ). The N M R data are in  agreement with previously reported values.  3  20  Metronidazole  3.2.3.2  13  2-(2-Methyl-5-nitro-l#-imidazol-l-yl)-N-(2,2,3,3,3) pentafluoropropyl) acetamide [MF5] Compound 13 (0.130 g, 0.70 mmol) was dissolved in a 5:1 mixture of THF  (25 mL) and D M F (5 mL) at 0°C under N [13 is sparingly soluble in neat THF]. 2  74  References on page 129  Chapter 3  N M M (80 u.L, 0.73 mmol) was then added and the clear, colourless mixture was stirred for 10 min before iBuClFrm (130 uL, 1.00 mmol) was added. Stirring was continued for 30 min during which the reaction mixture became a pale yellow slurry. The ice-bath was removed, and H NCH CF CF «HC1 (0.149 g, 0.87 mmol) and N M M 2  2  2  3  (95 u,L, 0.87 mmol) were added. The reaction slurry was then stirred at r.t. for 3 h when T L C analysis confirmed the presence of a single product. The creamy white precipitate that formed was filtered off and washed with THF ( 3 x 5 mL). The pale yellow filtrate was taken to dryness to give a yellow oil that was purified via column chromatography using THF as the eluent. Two bands were observed: the first, a pale yellow band and the second, a colourless one containing M F 5 . The second band was collected in several fractions and these were combined and taken to dryness to yield a white solid (0.117 g, 53 %). X-ray quality crystals were grown by slow evaporation of a concentrated M e O H solution of M F 5 . Anal. calc. for  C9H9N4O3F5:  C, 34.19; H ,  2.87; N , 17.53; found: C, 34.25; H , 2.99; N , 17.53. IR (v, cm" ): 3212 (N-H); 3028 1  (C-HM);  2921, 2851 (C-H); 1670 (C=0); 1473 (N-O  asym  ); 1368 (N-O ). UV-Vis sym  (MeOH): 310 (8.1), 230 (3.7), 212 (3.9). H N M R (300 MHz, CD OD): 8 8.07 (s, J  3  IH, lm-H ), 5.24 (s, 2H, -CH -CO-), 4.08 (t, 2H, -CH CF -), 2.54 (s, 3H, Im-Cflj); 4  2  2  2  (300 M H z , CDCI3) 8 7.93 (s, IH, lm-H ), 6.28 (br s, IH, -NH-), 4.93 (s, 2H, 4  -CH -CO-), 3.97 (dt, 2H, -CH CF ), 2.47 (s, 3H, \m-CH ). ^ { T J . } N M R (188 M H z , 2  2  2  3  CD3OD): 8 -7.56 (t, -CF ), -44.63 (q, -CF -); (188 MHz, CDCh) 8 -8.17 (t, -CF ), 3  2  3  -45.79 (q, -CF -). 2  75  References  on page 129  Chapter 3  3.2.3.3  2-(2-Methyl-5-nitro-l/?-imidazol-l-yl)-N-(2,2,2-trifluoroethyl) acetamide [MF3(-1)] Reaction conditions similar to those for the synthesis of MF5 were employed:  13 (0.252 g, 1.359 mmol) was added to THF (15 mL) and N M M (150 uL, 1.361 mmol) and iBuClFrm (192 pL, 1.472 mmol) were added (13 is not completely soluble in THF but the subsequent addition of base increases solubility). The resulting off-white slurry was then stirred at 0°C before H NCH CF «HC1 (0.202 g, 1.490 mmol) and N M M (164 pX, 2  2  3  1.489 mmol) were added. The mixture was then warmed to r.t. and stirred for 4.5 h. The precipitate that formed was filtered off and the filtrate taken to dryness. Purification of the desired product from the residue was achieved via column chromatography (THF). It was imperative to elute dropwise (slowly) to ensure the separation of the desired product from a number of side-products (as seen from TLC analysis). MF3(-1) was the third and most concentrated band to elute from the column. The fractions from this band were combined and taken to dryness to give an off-white solid (0.291 g, 80 %). Anal. calc. for C g H o N ^ F s : C, 36.10; H , 3.41; N , 21.05; found: C, 36.24; H , 3.25; N , 20.01. IR (v, cm ): 3304 (N-H); 3117 (C-H^); 2960, 2871 (C-H); 1678 (C=0); 1471 (N-O m); 1373 -1  asy  (N-O ). UV-Vis (MeOH): 310 (8.6), 230 (3.5), 210 (4.0). sym  X  H N M R (300 M H z ,  de-acetone): 5 8.26 (br s, IH, -NH-), 7.92 (s, IH, lm-H ), 5.25 (s, 2H, -C/^-CO-), 4.05 4  (qd, 2H, -Gr7 CF ), 2.43 (s, 3H, lm-CH ). E{ U) N M R (188 M H z , d -acetone): 6 4.76 l9  2  3  3  l  6  (s, -CF,).  Me  3.2.3.4  2-(2-Methyl-5-nitro-lfl-imidazol-l-yl)-N-(2-fluoroethyl)acetamide [MFl(-l)]  Reaction conditions similar to those for the synthesis of MF3(-1) were used: 13 (0.251 g, 1.359 mmol) was dissolved in THF (15 mL), and N M M (150 pX, 1.361 mmol)  76  References on page 129  Chapter  3  and iBuClFrm (192 pL, 1.472 mmol) were added. To the resulting white slurry at 0°C were added H NCH CH F«HC1 (0.155 g, 1.560 mmol) and N M M 2  2  2  (164 pL, 1.489 mmol).  The mixture was warmed to r.t. and stirred for 4.5 h before the precipitate formed was filtered off. The filtrate was taken to dryness and the residue purified via column chromatography (THF); dropwise (slow) elution yielded MFl(-l) (fourth and most concentrated band) as several fractions from the column. These fractions were combined and  taken to dryness to yield a pale yellow solid (0.207 g, 66 %). Anal. calc. for  CgHnNtOsF: C, 41.74; H , 4.82; N , 24.34; found: C, 41.85; H , 4.80; N , 24.11. IR (v, cm" ): 3288 (N-H); 3110 1  (C-Hjm);  2956, 2874 (C-H); 1668 (C=0); 1463  (N-O  asy  m);  1367  (N-O ). U V - V i s (MeOH): 310 (9.2), 226 (4.7), 210 (5.4). TI N M R (300 M H z , sym  dg-acetone) 8 7.80 (br s, 2H, -NH- + lm-H ), 5.16 (s, 2H, -Gr7 -CO-), 4.50 (dt, 2H, 4  -CH2F,  2  JHF  2  = 42 Hz), 3.55 (dq (ddt), 2H, -NH-C# -, 2  \m-CH ). F N M R  (282 MHz,  19  3  3  = 26 Hz), 2.45 (s, 3H,  JHF  d -acetone) 8 -144.91 (spt (tt), 6  2  JHF  = 50.5 Hz,  3  JHF  = 25.7  Hz, -CH2F).  3.2.3.5  2-(2-Methyl-5-nitro-lfl-imidazol-l-yl)-N-(2-chloroethyl)acetamide [MCU(-l)] Reaction conditions similar to those for the synthesis of MFl(-l) were used: 13  (0.301 g, 1.62 mmol) was dissolved in THF (20 mL), and N M M (165 uL, 1.62 mmol) and iBuClFrm (220 uL, 1.73 mmol) were added. To the resulting white slurry at 0°C were added H N C H C H C K H C 1 (0.200 g, 1.73 mmol) and N M M 2  2  2  (184 uL, 1.73 mmol). The  mixture was warmed to r.t. and stirred for 5.5 h. The precipitate formed was removed via filtration and the pink filtrate was taken to dryness to yield an orange-pink oil that was purified using preparative TLC (CH Cl :MeOH, 10:1). The major band (lowest Rf) was 2  2  isolated to give a white, microcrystalline solid (0.190 g, 47 %). Anal. calc. for  77  References  on page  129  Chapter 3  C8H11N4O3CI:  C, 38.96; H , 4.50; N , 22.71; found: C, 37.28; H , 4.68; N , 20.89. L R - M S  [DCI(+)]: 249, 247 (Tvf + H), 202, 200 (TVf - N 0 ) . HR-MS [DCI(+)] calc. for 2  C H i i N 0 3 C l (C HiiN 03 Cl): 249.05684 (247.05980); found 249.05725 (247.05896). 37  8  35  4  8  4  IR (v, cm ): 3312 (N-H); 3028 (C-H ); 2921, 2851 (C-H); 1670 (C=0); 1473 (N-O -1  ta  asym  );  1368 (N-O ). U V - V i s (MeOH): 310 (6.9), 232 (2.9), 210 (3.7). *H N M R (300 M H z , sym  de-acetone) 5 7.94 (br s, 2H, -NH- + lm-H ), 5.16 (s, 2H, -CH -CO-\ 4  -CH2CI), 3.61 (q (dt), 2H, -NH-CH -), 2  2  3.69 (t, 2H,  2.46 (s, 3H, lm-CH ). 3  Of note, this reaction was not as clean when compared to those for the preparation of MF3(-1) and MFl(-l). T L C analysis reveals the presence of two other major products having Rf values higher than that of MCll(-l); a small amount of unreacted 13 (R = 0) was also observed. Comparison of the T L C for authentic samples of f  MFl(-l) and MBrl(-l) with the reaction mixture facilitated identification of the desired product (Figure 3-3). *H N M R spectroscopic analysis of band 1 revealed the presence of the methyl ester (14),  probably formed from the reaction of acyl carbonate with M e O H on the  preparative T L C plate [TI N M R (300 M H z , de-acetone): 5 7.99 (s, I H , Jm-H ), 5.11 (s, 4  2H, -CH -X 3.72 (s, 3H, -C# ), 2.42 (s, 3H, Im-C# )]. For band 2, the isolated white 2  3  3  crystalline solid (in relatively high concentration) was determined to be the isobutyl ester side-product (15) (analogous to 11, Scheme 3-13, p. 106). This finding suggests that H 0 2  was present during the reaction, likely coming from the extremely hygroscopic amine hydrochloride. H N M R (300 MHz, de-acetone): 8 7.96 (s, I H , lm-H ), 5.26 (s, 2H, X  4  -CH2-CO-), 3.97 (d, 2H, O-CH2-), 2.49 (s, 3H, lm-CH \ 3  1.93 (m, IH, -C#-(CH ) ), 0.91 3  2  (d, 6H, -CH-(C# ) ). 3  2  78  References  on page 129  Chapter 3  NO  2  Band 1 = 2Me5N0 Im-methyl ester (14) 2  Band 2 = 2Me5N0 Im-isobutyl ester (15) 2  Band 3 = MCIl(-l)  o  J.  Band 4 = 13  A = MFl(-l); B = MCll(-l) rxn. mixture; C = MBrl(-l)  Figure 3-3: 3.2.3.6  T L C analysis of MXl(-l) compounds (X =F, CI, Br).  2-(2-Methyl-5-nitro-lfl-imidazol-l-yl)-N-(2-bromoethyl)acetamide [MBrl(-l)] Reaction conditions similar to those for the synthesis of MFl(-l) were used: 13  (0.108 g, 0.586 mmol) was dissolved in THF (10 mL) and N M M (65 uL, 0.590 mmol) and iBuClFrm (83 pL, 0.636 mmol) were added. To the resulting white slurry at 0°C were added H NCH CH Br«HBr (0.135 g, 0.660 mmol) and N M M (73 uL, 0.663 mmol). The 2  2  2  mixture was warmed to r.t. and stirred for 5 h. The precipitate was filtered off and the filtrate was taken to dryness. The resulting residue was chromatographed  using  preparative T L C (CH Cl :MeOH, 20:1), and the major band was isolated to yield an off2  2  white solid (0.137 g, 80 %). Anal. calc. for C H n N 0 B r : C, 33.01; H , 3.81; N , 19.25 8  4  3  [with 0.1 moi acetone C, 33.58; H , 3.94; N 18.87]; found: C, 33.69; H , 3.75; N , 18.66. I R ( v , cm ): 3295 (N-H); 3100 (C-H^); 2926, 2854 (C-H); 1661 ( C O ) ; 1458 (N-O -1  asym  );  1372 (N-O ). UV-Vis (MeOH): 310 (8.9), 228 (3.7), 210 (4.8). TT N M R (300 M H z , sym  de-acetone) 5 7.91 (br s, 2H, -NH- + lm-H ), 5.16 (s, 2H, -C# -CO-), 3.65 (q (dt), 2H, 4  2  -NH-C# -), 3.53 (t, 2H, -C# Br), 2.45 (s, 3H, lm-CH ). 2  2  3  79  References  on page 129  Chapter 3  N.  3.2.3.7  Br  (2-Methyl-5-nitro-lfl-imidazol-l-yl)-N-(2-chloroethane)(16) Reaction conditions similar to those described for the synthesis of ECU(-l)  were used (see synthesis 2, p. 70). In a flask equipped with a reflux condenser, CHCI3 (10 mL) was added to metronidazole (0.434 g, 2.54 mmol) at 0 °C to give a white slurry. TsCl (0.672 g, 3.53 mmol) and py (380 pL, 4.71 mmol) were then added simultaneously, and the reaction mixture was stirred for 0.5 h. The ice-bath was removed and the mixture was permitted to warm to r.t. over ~2 h. In attempts to isolate both the tosylated intermediate and the chlorinated metronidazole at this stage, the mixture was taken to dryness and the residue was column chromatographed. CH C1 was first used to elute unreacted TsCl. 2  2  Afterwards, the eluent strength was then increased to 5% M e O H and a pale yellow band emerged followed by a colourless band. These were identified to contain the desired product (16) and the unreacted metronidazole, respectively. No evidence for the presence of the tosylate was seen. The yellow fractions were combined and taken to dryness. A small amount of py present, as evidenced by T L C analysis, was removed under vacuum. The final product was isolated as a yellow, microcrystalline solid (0.125 g, 26%). Anal. calc. for C H N 0 C 1 : C, 38.01; H , 4.25; N , 22.16; found: C, 37.79; H , 3.96; N , 22.56. 6  8  3  2  L R - M S [DCI(+)]: 190 O f ) . HR-MS [DCI(+)] calc. for C H N 0 C 1 ( C H N 0 C 1 ) : 37  6  8  3  35  2  6  3  8  2  192.03539 (190.03833); found 192.03610 (190.03792). *H N M R (300 M H z , d -acetone) 6  8 7.96 (s, I H , Im-H \ 4.80 (t, 2H, -CH -C\), 4.09 (t, 2H, lm-CH -), 2.60 (s, 3H, 4  2  2  Im-CH ). 3  •CI Me 16  80  References on page 129  Chapter 3  3.2.4  2-Methyl-4-Nitroimidazole Compounds  3.2.4.1  2-(2-Methyl-4-nitro-l-H-imidazol-l-yl)propionic acid (17) This synthesis was adapted from the nitrile hydrolysis procedure reported by  Mann and Porter.  22  2-Methyl-4-nitro-l-imidazolepropionitrile (2.709 g, 15.0 mmol) and  N a N 0 (1.384 g, 20.1 mmol) were refluxed in aqueous 40% H S 0 solution (25 mL). 2  2  4  T L C analysis revealed that the reaction was complete after 2.5 d. The solution was neutralized with 6 M NaOH and then the pH adjusted to 3.23 using 0.1 M HC1 and 0.1 M NaOH. The resulting 17 precipitate was collected, washed with H 0 (2 x 25 mL) and 2  dried in vacuo to yield a white powder (2.287g, 76 %). The purity of the compound was confirmed by melting point (mp: found 223-225 °C, reported 221-225°C (Aldrich)) and X  H N M R (200 MHz, d -dmso) 5 8.32 (s, IH, lm-H ), 4.18 (t, 2H, -C# -CO-), 2.84 (t, 2H, 6  s  2  Im-C# -), 2.39 (s, 3H, 1m-CH ). 2  3  Me 17  3.2.4.2  3-(2-Methyl-4-nitro-l-H-imidazol-l-yl)-N-(2,2,3,3,3-pentafluoropropyl)propionamide [2M4NF5] 17 (0.305 g, 1.53 mmol) was placed in a 25 mL two-neck flask under N , and 2  THF (10 mL) was added. To the resulting white slurry was added D M F dropwise (~3 mL) until a clear, colourless solution was obtained. N M M (169 uL, 1.53 mmol) was then added and the mixture stirred for 10 min at 0°C before iBuClFrm (215 uL, 1.65 mmol) was next added. Stirring was continued for 30 min during which a pale yellow slurry formed. The ice-bath was removed, H NCH CF CF «HC1 (0.311 g, 1.82 mmol) and N M M 2  2  2  3  (200 pL, 1.83 mmol) were added and the slurry was stirred at r.t. for 4 h. The white precipitate that formed was filtered off and washed with dry THF ( 3 x 5 mL). The filtrate was reduced in volume to ~5 mL before E t 0 was added slowly. A resulting pale yellow 2  81  References  on page 129  Chapter 3  precipitate was isolated via filtration, but TLC analysis revealed later that this precipitate was not 2M4NF5. The filtrate was stored at 0°C overnight, resulting in the formation of clear, pale yellow crystals which were isolated via filtration (0.403 g, 83%). X-ray quality crystals were obtained from slow evaporation of a concentrated M e O H solution of 2M4NF5. Anal. calc. for C i o H n N ^ F s : C, 36.37; H , 3.36; N , 16.97; found: C, 36.67; H , 3.45; N , 16.69. JR (v, cm ): 3260 (N-H); 3128 -1  1506 (N-O  asym  (C-HnO;  3079, 2921 (C-H); 1674 (C=0);  ); 1400 (N-O ). UV-Vis (MeOH): 300 (6.2), 224 (3.6), 210 (4.2). *H sym  N M R (300 M H z , d -dmso) 5 8.69 (t, I H , -NH-), 8.22 (s, I H , Jm-H ), 5  6  3.96 (td, 2H,  -CH2-CO-),  Jm-CH ). 3  FCH}  19  3  JHF  = 16.5 Hz, -CH -CF -), 2  2  4.23 (t, 2H,  2.78 (t, 2H, Im-C# -), 2.38 (s, 3H, 2  N M R (188 MHz, d -dmso) 5 -7.06 (t, -GF ), -43.99 (q, -CF -). 6  2  3  Me  3.2.4.3  3-(2-Methyl-4-nitro-l-H-imidazol-l-yl)-N-(2,2,2-trifluoroethyl) propionamide [2M4NF3(-1)] Reaction conditions similar to those for the synthesis of 2M4NF5 were used:  17 (0.308 g, 1.55 mmol) was dissolved in THF (10 mL) and D M F (~3 mL), and N M M (170 pL, 1.55 mmol) and iBuClFrm (220 pL, 1.68 mmol) were added. To the resulting yellow/pink slurry were added H NCH CF «HC1 (0.234 g, 1.73 mmol) and N M M (190 2  2  3  pX, 1.74 mmol). The mixture was stirred at r.t. for 4 h before the white solid was filtered off and washed with THF ( 3 x 5 mL). The THF volume was reduced to ~ 5 mL under vacuum and then Et 0 was added until a cloudy white mixture persisted. This mixture was 2  stored at 0°C for 16 h to yield a white, microcrystalline solid (0.377 g, 87%). Anal. calc. for  C9HHN4O3F3:  C, 38.58; H , 3.96; N , 19.99; found: C, 38.79; H , 3.93; N , 19.78. IR (v,  cm ): 3263 (N-H); 3122 (C-Hm,); 3076, 2946 (C-H); 1675 (C=0); 1506 (N-O -1  asym  ); 1400  (N-O ). UV-Vis (MeOH): 300 (6.3), 224 (3.8), 212 (3.9). *H N M R (300 M H z , sym  82  References on page 129  Chapter 3  ds-dmso) 8 8.70 (t, I H , -NH-), 8.21 (s, IH, Im-H ), 4.20 (t, 2H, -C# -CO-), 3.90 (sex s  2  (qd), 2H, -C# -CF ), 2.73 (t, 2H, Im-C# -), 2.37 (s, 3H, lm-CH ). F{ B.} N M R 19  2  3  2  (188  l  3  MHz, de-dmso) 8 5.63 (s, -CF ). 3  H  Me  3.2.4.4  3-(2-MethyI-4-nitro-l-H-imidazol-l-yl)-N-(2-fluoroethyl) propionamide [2M4NF1(-1)] Reaction conditions similar to those for the synthesis of 2M4NF5 were used:  17 (0.308 g, 1.55 mmol) was dissolved in THF (10 mL) and DMF  (~3 mL), and  NMM  (170 uL, 1.55 mmol) and iBuClFrm (220 pL, 1.68 mmol) were added. To the resulting yellow/pink slurry was added H NCH CH F-HC1 (0.174 g, 1.74 mmol) and N M M 2  2  (190  2  uL, 1.74 mmol). The mixture was stirred at r.t. for 5 h before the white solid was filtered off and washed with THF ( 3 x 5 mL). The volume of the pink filtrate was reduced to ~3 mL  under vacuum, followed by the addition of E t 0 until a cloudy white mixture 2  persisted. Sonication of the mixture for 5 min at 5 °C and then filtration yielded a white microcrystalline solid (0.290 g, 77%). X-ray quality crystals were grown from slow evaporation of a concentrated M e O H solution of 2M4NF1(-1). Anal. calc. for C H i N 0 F : C, 44.25; H , 5.37; N , 22.94; found: C, 44.38; H , 5.34; N , 22.75. IR (v, 9  3  4  3  cm" ): 3298 (N-H); 3110 (C-H^); 3073, 2947 (C-H); 1668 ( C O ) ; 1504 (N-O 1  a8ym  ); 1396  (N-Osym). U V - V i s (MeOH): 300 (9.1), 210 (7.0). *H N M R (300 MHz, d -dmso) 8 8.28 (t, 6  IH,  -N#-), 8.23 (s, IH, lm-H ), 4.37 (dt, 2H, -C# F, 5  2  -Cr7 -CO-), 3.31 (dq (ddt), 2H, -C# -CH F, 2  2  3H, Im-CHs). F N M R 19  (282 MHz,  2  3  JHF=  2  JHF  = 56 Hz), 4.19 (t, 2H,  28 Hz), 2.69 (t, 2H, Im-Cr7 -), 2.38 (s,  d -dmso) 8 -142.64 (spt (tt), 6  2  2  JHF  = 49.2 Hz,  3  JHF  =  24.2 Hz).  83  References on page 129  Chapter 3  3.2.4.5  3-(2-methyl-4-nitro-l-H-imidazol-l-yl)-N-(2-chIoroethyI)propionamide [2M4NC11(-1)] Reaction conditions similar to those for the synthesis of 2M4NF5 were used:  17 (0.306 g, 1.54 mmol) was dissolved in THF (10 mL) and D M F (~3 mL), and N M M (169 pL, 1.54 mmol) and iBuClFrm (220 uL, 1.68 mmol) were added. To the resulting yellow/pink slurry were added H NCH CH C1»HC1 (0.195 g, 1.69 mmol) and N M M (188 2  2  2  pL, 1.69 mmol). The mixture was stirred at r.t. for 4 h before the white solid was filtered off and washed with THF ( 3 x 5 mL). The colourless filtrate was taken to dryness to give an oily white residue; T L C analysis indicated several side-products. The residue was chromatographed using preparative TLC (CH Cl :MeOH, 20:1); the major band yielded a 2  2  colourless oil. Addition of a small volume (~3 mL) of M e O H to the oil, followed by addition of E t 0 (15 mL) led to a cloudy white mixture. The white slurry was stored at r.t. 2  for 2 h, before sonication (5 min at 5 °C) and then filtration gave a white, microcrystalline solid (0.315 g, 79%). Anal. calc. for C9H13N4O3CI: C, 41.47; H , 5.03; N , 21.49; found: C, 41.83; H , 4.81; N , 20.64. IR (v, cm ): 3258 (N-H); 3119 ( C - H ^ ; 3079, 2923 (C-H); -1  1666 (C=0); 1499 (N-O  asym  ); 1384 (N-O ). UV-Vis (MeOH): 302 (6.7), 226 (3.9), 212 sym  (4.3). *H N M R (300 MHz, d -acetone) 8 8.03 (s, IH, lm-H ), 7.60 (br s, I H , -NH-), 4.38 6  5  (t, 2H, -CH2-CO-) 3.60 (t, 2H, -C# C1), 3.51(q (dt), 2H, - C / / - C H C l ) , 2.84 (t, 2H, 2  2  2  Im-C// -), 2.44 (s, 3H, Im-CH ). 2  3  84  References on page 129  Chapter 3  3.2.4.6  2-(2-methyl-4-nitro-l-H-imidazol-l-yl)-N-(2-bromoethyl)propionamide [2M4NBrl(-l)] Reaction conditions similar to those for the synthesis of 2M4NF5 were used:  17 (0.307 g, 1.54 mmol) was dissolved in THF (10 mL) and D M F (~3 mL), and N M M (169 pL, 1.54 mmol) and iBuClFrm (220 pX, 1.68 mmol) were added. To the resulting pale yellow slurry were added H NCH CH Br«HBr (0.347 g, 1.70 mmol) and N M M (189 2  2  2  pL, 1.70 mmol). The mixture was stirred at r.t. for 6 h. A workup identical to that used for 2M4NC11(-1) yielded clear, colourless crystals (0.098 g, 21%). Anal. calc. for C H N403Br: C, 35.43; H , 4.29; N , 18.36; found: C, 35.62; H , 4.28; N , 18.65. IR (v, 9  13  cm' ): 3259 (N-H); 3115 (C-Hm,); 3072, 2918 (C-H); 1660 (C=0); 1499 (N-O 1  asym  ); 1399  (N-O ). UV-Vis (MeOH): 300 (7.4), 226 (4.4), 210 (5.6). *H N M R (300 M H z , sym  ds-acetone) 5 8.03 (s, IH, lm-Hs), 7.67 (br s, I H , -N/7-), 4.36 (t, 2H, -C# -CO-), 3.56 (q 2  (dt), 2H, -C# -CH Br), 3.44 (t, 2H, -C# Br), 2.83 (t, 2H, Im-C# -), 2.42 (s, 3H, 2  2  2  2  Im-CHs).  3.2.5  Reactions of SR2508 with Tf 0  3.2.5.1  2-(2-Nitro-l-H-imidazol-l-yl)-N-(ethylformate)acetamide (18)  2  This reaction was carried out in an attempt to synthesize the triflate analogue of SR2508; however, the presumed triflate was too reactive as demonstrated by the results from the attempted synthesis. In a Schlenk tube under 1 atm N , CH C1 (15 mL) and 2  2  2  D M F (3 mL) were added to SR2508 (0.303 g, 1.14 mmol) to give a clear, colourless solution; this was then stirred at 0 °C for 30 min before py (320 pL, 3.95 mmol) and subsequently ( C F S 0 ) 0 (342 pL, 3.95 mmol) were added dropwise. The ice-bath was 3  2  2  removed and the dark orange reaction mixture was stirred for 3 h at r.t. T L C analysis  85  References  on page 129  Chapter 3  (using THF) revealed the presence of three products characterized by a bright yellow band (R = 0.95, 19), a colourless band (R = 0.65, 18) and another colourless band (R = 0.10, f  f  f  unidentified). The solvent was removed (under high vacuum) and 18 was isolated using column chromatography (THF); further purification via sublimation (at 130 °C under vacuum) yielded a white solid (0.703 g, 67 %). Anal. calc. for  C, 39.67; H ,  C8H10N4O5:  4.16; N , 23.13; found: C, 39.90; H , 4.15; N , 23.03. L R - M S [EI(+)]: 243 OVT), 196 OVf - N 0 ) . IR (v, cm" ): 3275 (N-H); 3096 (C-H^); 2940 (C-H); 1716 (C=O i ); 1655 1  2  a  ( 0 = 0 ^ ) ; 1487 (N-O  asym  d  ); 1362 (N-O ). TI N M R (300 MHz, d -acetone): 5 8.13 (s, sym  6  IH, -CHO), 7.80 (br. s, IH, -NH-), 7.50 (s, IH, lm-H ), 7.14 (s, IH, lm-H ), 5.25 (s, 2H, 5  4  -C# -CO-), 4.21 (t, 2H, -CH -0), 3.55 (q (dt), 2H, -NH-Cr7 -). 2  2  2  1 3  C N M R (75 M H z ,  de-acetone): 5 166.7 (-CO-NH-), 161.8 (-CHO), 128.7 and 128.2 (lm-C and J), 62.8 4  (-CH -CO-), 52.5 (-NH-CH -), 39.0 (-CH -0). 2  2  2  18  3.2.5.2  CycF3 (19) Conditions similar to those for the synthesis of 18 were used, except that D M F  was not used. Following addition of ( C F S 0 ) 0 , the slurry became a clear, bright yellow 3  2  2  solution; stirring was continued for 1 h at 0 °C and 2 h at r.t. during which a yellow slurry was formed. The mixture was filtered and the filtrate was reduced in volume to ~5 mL. before E t 0 (25 mL) was added to yield a white precipitate. The solid was filtered off and 2  the yellow filtrate was reduced in volume to ~2 mL before being column chromatographed (CH C1 -> CH C1 :acetone, 4:1). The first eluted band (R = 0.85, yellow) yielded 19, a 2  2  2  2  f  brilliant yellow powder (0.135 g, 36 %). Crystals suitable for X-ray analysis were obtained from slow evaporation of a solution of the compound in a 1:1 mixture of CH C1 : acetone. 2  Anal. calc. for  C8H7N4O5SF3:  2  C, 29.28; H , 2.15; N , 17.07; found: C, 29.15; H , 2.22; N ,  86  References on page 129  Chapter 3  17.42. L R - M S [DCI(+)]: 329 (TVf + H). IR (v, cm ): 3155 ( C T ^ ) ; 2980 (C-H ); 1  ta  2918 (C-H); 1710 (C-O); 1485 (N-O  asym  ); 1362 (N-O ); 1203 (S=0). U V - V i s (MeOH): sym  314 (4.3). H N M R (300 M H z , d -acetone): 5 7.58 (d, I H , lm-H ), 7.17 (d, I H , Im-H ), X  6  5  6.78 (s, -CH=C-), 4.64 (t, 2H, -CH -0-\ 2  4.36 (t, 2H, -N-Cff -)2  1 3  4  C N M R (75 M H z , de-  acetone): 5 146.5 (-CH=C-), 128.6 and 127.8 (Im-C,wj), 120.9 (q, -CF ), 89.0 (-CH=C3  ), 68.8 (-CH2-O-), 49.9 (-N-CH2-). F{TI} N M R (188 M H z , dg-acetone): 5 0.88 (s, 19  19  3.2.6  Reactions with RSU1111  3.2.6.1  Synthesis of (20), a pentafluorinated derivative of RSU1111 The same reaction procedure to synthesize RevEF5 (synthesis 3) was used to  synthesize 20. C F C F C 0 H (105 uL, 1.00 mmol) was dissolved in THF (15 mL) and 3  2  2  N M M (110 uL, 1.05 mmol) and iBuClFrm (150 uL, 1.15 mmol) were added. To the resulting white slurry were added RSU1111 (0.206 g, 1.07 mmol) and N M M (125 p L , 1.19 mmol). Stirring was continued at r.t. for 20 h before the yellow precipitate that formed was filtered off, washed with THF ( 3 x 5 mL) and the filtrate purified via column chromatography (CH2CI2:acetone, 20:1). Two bands eluted, and the minor band with Rf = 0.60 was isolated to yield 20 as a colourless oil (0.0155 g, 5 %). L R - M S [DCI(+)]: 350 (TVf + NH4), 333 O T + H). H R - M S [DCI(+)] calc. for CgHjoOaN^: 333.06221; found 333.06198. T I N M R (300 M H z , d -acetone): 8 8.76 (br. s, I H , -NH-), 7.54 (s, I H , 6  lm-H ), 7.09 (s, I H , Im-H ), 4.90 (d, I H , -OH), 4.78 (dd, I H , Im-CMH)-), 4.38 (dd, I H , 5  4  lm-CH(H)-), 4.22 (m, I H , -Cr7(0H)), 3.50 (m, 2H, -Gr7 -NH-). F{ B} N M R (188 l9  x  2  M H z , de-acetone): 5 -6.77 (t, -CF ), -46.35 (q, -C7v). 3  87  References  on page 129  Chapter 3  e e  O  r=\  NH3C1  'N'  N.  OH  OH  N02  H  20  RSU1111 3.2.6.2  Synthesis of (21), a dibromo derivative ofRSUllll Reaction conditions including stoichiometrics identical to those for the synthesis  of compound 20 were used except CBr C02H was used instead of CF3CF2CO2H. The 3  white precipitate that formed was filtered off, washed with THF ( 3 x 5 mL) and the filtrate was purified using the C T R O N (Et 0:acetone, 1:1). Two bands eluted and the second 2  band (Rf = 0.65) gave a yellow oil. Addition of E t 0 yielded a white precipitate that was 2  isolated via filtration and dried in vacuo (0.0184 g, 5 %). Anal. calc. for CgHiolSUO^^: C, 24.89; H , 2.61; N , 14.51; found: C, 25.60; H , 2.65; N , 13.54. L R - M S [EI(+)]: 387 (M+l), 340 O f - N 0 ) , 156 Ovf -CH NHCOCBr H), 114 (2N0 Im), 80 (Br). H R - M S 2  2  2  2  [EI(+)] calc. for C Hio0 N Br2 ( C H i O N B r B r ) [ C H O N 81  8  4  81  4  8  0  4  79  4  8  10  4  79 4  B r ] : 388.91061 2  (386.91266) [384.91470]; found 388.91604 (386.91227) [384.91401]. *H N M R (300 MHz, CD3OD): 5 7.46 (s, IH, lm-H ), 7.13 (s, I H , lm-H ), 6.21 (s, I H , -CBr #), 4.67 5  4  2  (dd, I H , Im-Ci7(H)-), 4.29 (dd, IH, Im-CH(7i)-), 4.05 (m, I H , -C#(OH)-), 3.38 (dd, 2H, -C# -NH-). C N M R ( 7 5 MHz, CD OD): 5 151.7 (-C=0), 129.1 and 128.2 ( I m - C w j ) , 1 3  2  3  4  66.9 (-CH(OH)-), 54.3 (-CH -NH-), 44.8 (Im-CH -), 37.2 (-CBr H). 2  2  2  O  21 3.2.6.3  Synthesis of (22), a terminal nitrile derivative ofRSUllll This procedure was adapted from a synthesis reported by Fortier and McAuley  describing the synthesis of a chelating ligand from an amine and an acrylonitrile. ' In a 23 24  88  References on page 129  Chapter 3  50 mL flask, CH C1 (5 mL) was added to RSU1111 (0.102 g, 0.531 mmol) to give a 2  2  slurry. Acrylonitrile (5 mL, excess) and K C 0 (0.150 g) were then added and the reaction 2  3  mixture was refluxed for 24 h. T L C analysis revealed the presence of numerous products (~ 19). The two major bands observed were isolated via preparative T L C (band 1 (22), Rf = 0.33; band 2 (23), R = 0.55 for CH Cl :MeOH, 20:1) to yield colourless oils (0.0136 g f  2  2  and 0.0334 g, respectively). IR (22, v, cm' ): 3376 (OH); 3139 (C-Hjm); 2929, 2847 1  (C-H); 2247 (CsN); 1631 (C-OH); 1487 (N-O  ); 1363 (N-O ). H N M R (200 M H z , X  asym  sym  ds-acetone, 22): 5 7.49 (s, I H , Im-i^), 7.09 (s, IH, Im-H ), 4.73 (dd, I H , Im-C#(H)-), 4  4.46 (dd, I H , Im-CH(//)-), 4.10 (m, IH, -C#(OH)-), 2.93 (td, 2H, -NH-Gr7 -), 2.80 (dd, 2  IH, -C#(H)-NH-), 2.71 (dd, I H , -CH(//)-NH-), 2.64 (t, 2H, -C# -CN); integrations 2  support the addition of only 1 moi acrylonitrile per RSU1111, while peak assignments were made with the aid of 2D COSY; (d -acetone, 23): 5 7.52 (s, I H , lm-H ), 7.11 (s, I H , 6  s  lm-H ), 4.83 (dd, I H , Im-Gr7(H)-), 4.60 (dd, I H , Im-CH(#)-), 3.98 (m, I H , -CH(04  CH -)-), 3.81 (dt, IH, -0-Cr7(H)-), 3.60 (dt, IH, -0-CH(i/)-), 2.80 (dd, I H , -C#(H)2  NH -), 2.76 (dd, I H , -CH(#)-NH -), 2.64 (dd, 2H, -Gr7 -CN). 2  2  2  1 3  C N M R (75 M H z ,  ds-acetone, 22): 6 128.5 and 127.9 (Im-C „d s), 100.6 (-CN), 69.7 (-CH(OH)-), 53.9 4a  (-CH -NH-), 52.8 (-NH-CH -), 46.0 (Im-CH -), 18.8 (-CH -CN). 2  2  2  2  The reaction was repeated as described above, except E t N (165 pL) was 3  substituted for K C 0 and the reaction was performed in neat acrylonitrile; the resulting 2  3  white slurry became an orange solution after 24 h at reflux. The solvent was then removed and acetone was added to the residual oil to generate a tan-coloured, microcrystalline solid that was filtered off. The filtrate was taken to dryness to give a yellow-brown oil; T L C revealed 2 major bands, one of which was 22, that was purified via column chromatography  ( C H C l : M e 0 H , 20:1). Two colourless oils corresponding to 22 2  2  (0.0409 g) and a new product (24) (0.0196 g) were isolated. L R - M S [DCI(+)]: 283 ( M + NH4 - CN), 267 ( M + H - CN), 240 ( M - 2CN). *H N M R (300 MHz, d -acetone): 6 7.50 6  (s, I H , Im-H ), 5  7.09 (s, I H , Im-H ), 4.81 (dd, I H , Im-C#(H)-), 4.44 (dd, I H , 4  Im-CH(77», 4.19 (m, I H , -C#(OH)-), 3.00 (t, 4H, -N-C# -), 2.86 (m, I H , -C# -N-), 2  2  2.71 (t, 4H, -Cr7 -CN); integrations support the addition of 2 moi acrylonitrile per 2  89  References  on page 129  Chapter 3  RSU1111, while peak assignments made with the aid of 2D COSY.  1 3  C N M R (75 M H z ,  de-acetone): 5 128.4 and 127.9 (Im-C a„d s), 100.7 (-CN), 69.2 (-CH(OH)-), 58.0 4  (-CH -N-), 54.1 (-N-CH2-), 50.7 (Im-CH -), 16.6 (-CH -CN). 2  2  3.2.7  2  Reactions of Nitroimidazoles with Bu NF»H 0 4  2  The syntheses of fluorinated nitroimidazoles via exchange of F" with other halogens on the side-chain were attempted, with the goal being to develop a useful procedure for obtaining fluorinated PET precursors. 3.2.7.1  Reaction of EBrl with Bu NF»H 0 4  2  EBrl (10 mg) and B u N F * H 0 (40 mg, 4.2 equiv.; this compound must be 4  2  handled quickly in air as it is extremely hygroscopic) were added to a Schlenk tube and the system was evacuated at r.t. (~1 h). Dry d6-dmso (1 mL) was added via syringe and the mixture was stirred for 3 h; within 1 min the initially colourless solution immediately changed to blue, then green, green-brown, orange and finally to yellow-orange. T L C analysis revealed the formation of a new product that was isolated via preparative T L C (CH Cl :MeOH, 20:1) as a colourless oil (25). UV-Vis (dmso): 372, 264 nm. T i N M R 2  2  (200 M H z , de-dmso): 7.58 (s, lm-H ), 5  7.01 (s, Im-# ), 5.11 (s, -C# -CO-), 4.15 (t, 4  -CH BT), 3.46 (t, -NH-C#2-), 2.11 (m, -C# -CH Br). 2  2  2  19  2  F N M R (188 M H z , d -dmso): 5 6  -69.85 (s). Identical observations were made when the nitroimidazole used was EBrl(-l); however, when the fluoride source used was KF/2,2,2-Kryptofix with either EBrl or EBrl(-l) there was no blue intermediate observed.  90  References on page 129  Chapter 3  H  F  25 3.2.7.2  Reaction of EBrl with Bu NF«H 0 and CH C0 H 4  2  3  2  The above procedure (see 3.2.7.1) was repeated except acetic acid (1.2 equiv.) was also used, its function being to perhaps stabilize the nitroimidazole ring and hence promote fluoride exchange on the side-chain (C-Br bond)  25  Again, the final solution was  yellow, and T L C analysis revealed two bands that were isolated via preparative T L C (CH Cl :MeOH, 20:1). Band 1 (26, R = 0.15) was isolated to yield a white solid, while 2  2  f  band 2 (Rf = 0.55) was a colourless oil, its analytical data matching those of compound 25. Data for 26: L R - M S [EI]: 229 (IVf + H), 211 (TVf - OH), 182 (M* - N 0 ) . TI N M R (200 2  M H z , de-acetone): 6 7.62 (br s, IH, -NH-), 7.40 (s, IH, lm-H ), 7.00 (s, I H , Im-H ), 5.11 s  4  (s, 2H, -C7f -CO-), 3.60 (t, 1H, -OH), 3.46 (q, 2H, -C# OH), 3.23 (q (dt), 2H, 2  2  -NH-CrY -), 1.54 (p, 2H, -C# -CH OH); peak assignments made with the aid of 2D 2  2  2  COSY. These data suggest that the presence of acetic acid leads to hydrolysis of the C-Br bond. H  N02  26  3.2.7.3  Reaction of SR2508 with Bu NF»H 0 4  2  Reaction conditions identical to those for the reaction of fluoride with EBrl (section 3.2.7.1) were used, except the solvent was MeCN. The major band observed for T L C analysis was isolated to give a yellow oil that was purified using the C T R O N (CH Cl :MeOH, 10:1) to yield a colourless oil (27) (0.0252 g, 27 %). LR-MS[EI(+)]: 185 2  2  91  References  on page 129  Chapter 3  (IVf - H), 167 (TVT - F), 137 (Ivf - F - CH OH), 123 (TVT - F - C H C H O H ) , 108 2  2  2  (Im-CH CO-). IR (v, cm- ): 3403 (N-H); 2924, 2851 (C-H); 1735 (C=0); 1588, 1377, 1  2  1331, 1059; no v o bands were observed. UV-Vis (dmso): 370 (s was -70 % of that for N  SR2508). rl N M R (200 MHz, d -acetone): 5 7.00 (d, I H , lm-H ), 6.73 (s, I H , lm-H ), x  6  5  4  4.77 (br s, I H , -NH-), 4.56 (s, 2H, -Cr7 -CO-), 3.83 (m, 4H, -Cr7 OH + -NH-C# -), 3.52 2  2  2  (t, I H , -OH). F N M R (188 MHz, d -acetone): 5 -74.72 (s). 19  6  H  F  27  3.2.7.4  Reaction of « N 0 I m (« = 2, 4, 5) with B u N F « H 0 2  4  2  The title reaction was carried out in d6-dmso and the analytical data are summarized in Table 3.1. The results from this study were all obtained from the in situ reaction mixtures, and are used for the qualitative comparison of the data obtained from the reactions with fluoride in the previous sections (3.2.7.1-3). For the reaction with 2Me5N0 Im, the major band from the T L C analysis was isolated [IR (v, cm" ): 3406 1  2  (N-H); 2959, 2872 (C-H); 1523, 1460, 1376, 1254, 1206, 1028, 878; no v  N 0  bands at  1501 and 1380 cm were observed.] -1  92  References  on page 129  Chapter 3  Table 3.1:  Summary of the Tf N M R , F{Ti} N M R and UV-Vis data for « N 0 I m compounds and for their reaction with Bu4NF«H 0 19  2  2  T i N M R (5)  Sample  19  F{TI} N M R (6) a  U V - V i s (nm)  Imidazole Positions l 2N0 Im 2  2N0 Im + F 2  2  4N0 Im + F" 2Me5N0 Im 2  2Me5N0 Im + F 2  a  -  4  5  ~  7.35  7.35  ~  328  ~  ~  6.70  6.70  -68.95  374  ~  2  2  14.40  13.25  4N0 Im  b  b  8.32  ~  7.84  ~  302  7.70  —  7.04  -67.94  364  12.95  2.32  8.20  ~  —  314  —  2.15  7.60  —  -67.80  376  The F signal observed in each spectrum is rather broad. The N-H signal is not observed in the *H NMR spectrum of the reaction mixture because of the addition of H 0 with the fluoride source 0Bu4NF«H O) which leads to rapid proton exchange with the N-H proton. 1 9  2  2  93  References  on page 129  Chapter 3  3.2.8  Cyclic Voltammetry of Nitroimidazoles The cyclic voltammograms for the nitroimidazoles were obtained using the  procedure described in Chapter 2 (Section 2.2.6) using the cell depicted in Figure 2-2 which contains the A g reference electrode. The reduction potentials of the one-electron reduction of the nitro group are reported in Tables 3.2-3.4 versus the SCE, the standard reference used to report these values in the literature. The listed conversion factor is detailed in Chapter 2, Section 2.2.6.  Table 3.2: Summary of reduction potentials for the 2-nitroimidazoles vs. SCE  Conversion Factor  FeCp E m  Ep„ E : Ei/2 (avg.)  (avg.) vs. Ag  vs. Ag  SR2508  479  -953,-1088:-1021  -56  -1077  EF5  408  -933,-1053:-993  +16  -977  EF3  336  -1060, -1163; -1112  +88  -1024  EF3(-1)  474  -910, -1020; -965  -50  -1015  EF2Br  471  -905,-1045;-975  -47  -1022  E=F2  520  -843,-1027;-935  -96  -1031  EF1  507  -899, -1009; -954  -83  -1037  EFl(-l)  620  -857, -981; -919  -196  -1115  ECU  565  -836,-954;-895  -141  -1036  ECll(-l)  595  -883,-1013;-948  -171  -1119  EBrl  489  -893,-1053;-973  -65  -1038  EBrl(-l)  650  -815, -965; -890  -226  -1116  Compound  2  p a  94  26  (424  - El/2 F e C p 2 )  E l * vs. SCE  (mV)  References on page 129  Chapter 3  Table 3.3: Summary of reduction potentials for the 2-methyl-5-nitroimidazoles vs. SCE Compound  FeCp E 2  1 / 2  E , E pc  p a  : E i / (avg.) 2  Conversion Factor  26  E i / vs. SCE 2  (mV)  (avg.) vs. Ag  vs. Ag  Metronidazole  393  -1039,-1207;-1123  +31  -1092  MF5  363  -1031,-1225;-1128  +60  -1068  MF3(-1)  379  -1026,-1212;-1119  +45  -1074  MFl(-l)  338  -1139, -1331; -1235  +85  -1150  MCll(-l)  504  -999, -1167; -1083  -80  -1163  MBrl(-l)  649  -856,-1008;-932  -225  -1157  (424  - El/2 F e C p 2 )  Table 3.4: Summary of reduction potentials for the 2-methyl-4-nitroimidazoles vs. SCE  FeCp Ei/2  E , Epa: Ei/2 (avg.)  Conversion Factor  (avg.) vs. Ag  vs. Ag  (424-E /2FeCp2)  (mV)  2M4NF5  575  -1078,-1220;-1149  -151  -1300  2M4NF3(-1)  628  -1060,-1220;-1140  -204  -1344  2M4NF1(-1)  629  -1134, -1286; -1210  -205  -1415  2M4NC11(-1)  660  -1090,-1216;-1153  -236  -1389  2M4NBrl(-l)  707  -1029,-1201;-1115  -283  -1398  Compound  2  pc  95  26  1  Ei/2  vs. SCE  References on page 129  Chapter 3  3.3  Results and Discussion  3.3.1  Synthesis of the Nitroimidazole Side-Chains Nitroimidazoles themselves have been shown to exhibit some activity in  biological systems (e.g. 2N0 Im or azomycin is a natural antibiotic and antiprotozoal 2  agent). However, because of their relatively low solubility in aqueous systems their use is 27  limited. Various groups have reported the addition of numerous side-chains onto the N l position of the nitroimidazole ring ' " 16 28  and the efficacy of such derivatives as  31  hypoxia-selective cytotoxins or radiosensitizers seems to depend on the partition coefficient (P); as P increases the lipophilicity increases and thus the compound's ability to cross the cell membrane also increases.  32  The major problem with most of these  nitroimidazole compounds is that addition of the side-chain leads to increased cytotoxicity, specifically neurotoxicity which is the major factor that limits clinical dose. ' A relatively 33 34  new compound with a pentafluorinated side-chain (EF5) seems to be one of the best candidates for use as a hypoxia-selective species. ' The original synthesis of EF5 (Section 2 5  3.2.2.2, synthesis 1) required a number of reaction steps that resulted in a relatively low yield (33 %), so a new method was developed in which the side-chain could be added in a single, high-yield step. The perfluoropropylamine side-chain for EF5 (section 3.2.2.2, synthesis 1) was originally synthesized via a 3-step reaction from perfluoropropionic acid (Scheme 3-1). '  35 36  The synthesis of compound 7 required two steps (see Scheme 3-9, p. 102) while the coupling of 7 and pentafluoropropylamine to form the amide linkage took place in the final  Scheme 3-1:  Synthesis of petafluoropropylamine from perfluoropropionic acid.  step (total of six steps). In the present work, linking the amine with iodoacetic acid to form IF5 (Scheme 3-2), two of the steps are eliminated which gives a higher overall yield  96  References on page 129  Chapter 3  of E F 5 .  17  IF5 is easily purified via sublimation, however the C-I bond is mildly reactive H  o r  OH  / \ /CFa H N' ^ F f «HC1  +  2  II  O  W  I  N -Me  5 ^A.  ^ \  "  1^  ^  ^CF ' ~"CF 2>  '  3  II  IF5 Scheme 3-2: Synthesis of IF5, the precursor to EF5. and if the compound is not stored in the absence of light, it will slowly decompose. Changing the halogen from iodine to chlorine produces a more stable side-chain (C1F5), as the C-Cl bond is more resistant to cleavage by U V radiation. A n interesting feature of 37  C1F5 is that it is only visible on the TLC plate in the presence of U V light, behaviour opposite to that of EF5. The use of CIF5, although stable upon exposure to U V light (as monitored by *H N M R spectroscopy) did not, however, give a higher yield for the synthesis of EF5. Therefore, from a practical sense, C1F5 may be more useful than IF5. Kagiya and others " 28  30  have investigated reduction of the amide side-chain to the amine  using the relatively mild reducing agent borane-THF which selectively reduces the amide ' without affecting the nitro moiety. The reduction of IF5 using BH »THF was 38 39  3  successful in forming the corresponding amine (3) (see Scheme 3-3), but its stability was much lower than that of IF5. A n N M R sample of the initially pale yellow solution of 3, stored at r.t. under ambient light, turned dark red-brown over a period of several days. T I N M R spectra recorded every 2 d revealed a decrease in the intensities of the signals for 3 and the appearance of new signals. Chromatographic separation of a 10-day old N M R sample (via preparative TLC) gave three bands with the first one assigned as compound 4; this was initially colourless but became yellow on the TLC plate upon exposure to U V light. Analysis of the *H N M R data indicated that 3 underwent cyclization to form compound 4 (Scheme 3-3). The instability of 3 is in accordance with the weak C-I bond; the loss of iodine is confirmed from the UV-Vis data which show that both I and I " are 2  3  formed (presumably by the photolysis of HI). The liberation of I from HI in the presence 2  of U V light is well known. ' Of note, 4 has an aziridine ring analogous to that in R S U 8 37  97  References on page 129  Chapter 3  1069 (formed from the bromoethylamino derivative R B 6145).  R S U 1069 is used as a  D N A alkylating agent, suggesting that 4 may also serve the same purpose; however, because there is no nitroimidazole moiety attached 4 will not be hypoxia-selective.  +  HI  H  3  Scheme 3-3:  4  Formation of aziridine ring upon exposure of 3 to U V light.  Because of the interest in using halogen exchange reactions to form radiolabelled species for use in PET imaging of hypoxia, a monobromo derivate of IF5 41  was synthesized (IBr) (see Scheme 3-4). This compound is extremely reactive as it contains two reactive C - X ( X = Br, I) bonds. IBr was only stable at < 10 °C in the dark, while exposure to heat and/or U V light resulted in decomposition to two new species (Scheme 3-4). The decompostion was monitored by H N M R spectroscopy. The peak at 5 X  7.90 for IBr, typical of N H amides, disappears and a new peak appears at 5 2.51, in the range typical for cyclic amine N H protons.  42  The other signals at 8 4.81 (a methylene  triplet shifted slightly downfield due to the proximity of the C H to an O atom), 8 3.74 (a 2  H  1  IBr Scheme 3-4:  2  Cyclization of IBr in the presence of heat and/or UV-radiation.  98  References on page 129  Chapter 3  triplet adjacent to the amine N H , showing no coupling to the N H proton) and 8 2.37 (a pentet, presumably resulting from an overlapping triplet of triplets) together support the formation of a cyclic species with the proposed structure 1. Analogous cyclization products have been suggested in the literature, but no spectroscopic data has been reported to support these hypotheses. ' For the reaction of SR2508 with T f 0 a similar 43 44  2  cyclized product was observed in this thesis work, as confirmed by an X-ray structure (Figure 3-14, p. 123). The H-atoms adjacent to an amide linkage are more acidic (activated) because the amide exists in two tautomeric forms, while removal of electron density from the C - H bonds also increases their acidity. Although the bond energy of C-I is lower than that of C-Br, reactivity of the C-I appears to be limited by its proximity to the amide moiety. This prevents the formation of any cyclization products resulting from the cleavage of the C-I bond. The formation of a species corresponding to 2 (Scheme 3-4) is also supported by the H N M R spectrum. Because the four-membered ring is torsionally J  strained this species is short-lived and eventually rearranges to 1. Most of the "side-chain compounds" were either purchased from Aldrich or received as kind donations from Prof. W. Dolbier at the University of Florida. The only fluorinated amine side-chain synthesized was 3-fluoropropylamine hydrochloride (5), the precursor for the synthesis of EF1. Obtaining 5 was imperative for comparison of the reaction products from the halogen exchange reaction between E B r l and F", and the authentic E F 1 compound. A previously reported synthesis required four steps to obtain the parent amine of 5, and the yield was necessarily small (Scheme 3-5). ' For this thesis 45 46  \ ^ /  0  SOC12 77%  H  II ,NH  2  <P  *  t0 /H 2  V Y  F  F 2  \ ^  18%  C  1  /x^^  _  P0  3  J  a  NH 52%  j  N H  *  0  2  71  o  5  /o  Scheme 3-5: Synthesis of 3-fluoropropylamine reported by Pattison et al.  45  99  References on page 129  Chapter 3  work, two different syntheses of 5 were attempted. Synthesis 1 used the fluorinating agent diethylamino-sulfur trifiuoride (Et NSF , DAST), a useful reagent for converting alcohols 2  3  to their corresponding fluorides in high yield.  47  Previous results have suggested that  D A S T has less carbonium-type rearrangements and less dehydration side-reactions than occur with other fluorinating agents (e.g. H F , SeF^pyridine ). Reaction of D A S T with 48  49  3-hydroxy-propylamine did indeed yield the desired fluorinated product, 3-fluoropropylamine; however, other less volatile side-products formed which were not vacuum transferred from the reaction mixture. 3-Fluoropropylamine and diethylamine (as their HC1 salts) were the only two species detected in the T i N M R spectrum of the isolated solid. The presence of diethylamine (a side-product of the DAST reaction, Scheme 3-6) as the major product (Et NH : 5 « 6) suggested that all of the DAST had reacted, but not 2  DAST  O H M  -  9  F  +  HF + Et NSF 2  H0 9  Et NH»HF + SO,  HJSL  Scheme 3-6:  2  Reaction of 3-hydroxypropylamine with DAST.  selectively to form 5. A n explanation for the low yield is that the HF formed during the synthesis of 5 produced the hydrofluoride salt of the fluorinated product, which remained in the solid residue after vacuum transfer of the volatile components. Unfortunately, the X  H N M R spectrum of this residue was not obtained. The second method to synthesize 5  proved to be much more successful. Incorporation of a primary amino group onto an alkyl chain was easily done using the Gabriel synthesis; reaction of potassium phthalamide 13  with the dihaloalkyl species, l-bromo-3-fluoropropane, produced the 3-fluoropropylphthlamide (6) by S N substitution at the reactive C-Br bond (Scheme 3-7). The major 2  advantage of the use of the phthaloyl group for protecting the nitrogen atom is its susceptibility to facile removal by means of hydrazine.  100  14  Another advantage of this  References  on page 129  Chapter 3  reaction is that the amine can be easily separated by extraction with H C 1 from the phthaloyl hydrazide formed during the hydrazinolysis. In this case, the hydrochloride salt was actually the desired product, 5 (Scheme 3-7). One other synthesis of 5 exists in which the reaction intermediate is the azide species, although the yield is much lower than via 10  the chemistry illustrated in Scheme 3-7.  O Scheme 3-7: High yield Gabriel synthesis of 3-fluoropropylamine hydrochloride.  3.3.2  Nitroimidazoles Nitroimidazoles are useful compounds for targeting the hypoxic regions of  cancerous tumours; the nitro moiety is readily reduced in the presence of nitroreductases within the cells to form macromolecular adducts. Various nitroimidazoles  (2, 4  derivatives) have been synthesized previously for use as radiosensitizers " 50  52  and  5N0  2  and their  efficacy was up to three times greater in areas of hypoxia. Because the nitroimidazole, EF5, was found to be non-toxic when compared to other compounds of similar composition, the focus of this thesis work was on variation of the fluorinated side-chain 2  and the position of the nitro group about the imidazole ring. Most of the nitroimidazoles were synthesized via the mixed anhydride species generated from reaction of an acid moiety with isobutylchloroformate (Scheme 3-8). The carbonyl group nearest the imidazole is the more reactive of the two, the isobutyl carbonate being the better leaving group on reaction with an appropriate  101  RiNH  References  2  amine,  on page 129  Chapter 3  this leading to formation of an amide bond. N-Methylmorpholine (NMM) was used to "mop up" free ET in solution (Scheme 3-8).  H NRi 2  /R5  yR5  , O H  -HCI  Y  R2  R  Y  H(X  ^O.  2  o  o  +  o  R =N02, R4=R =H R =N0 , R =Me, R4=H R4=N0 , R =Me, R =H 2  5  5  2  2  2  2  MR1  (Ri= halogenated side-chain)  2M4NR-I  5  Scheme 3-8: Reaction mechanism for addition of side-chain via an amide linkage. Synthesis of the nitroimidazole acetic acid derivatives was achieved by chemically altering existing nitroimidazoles. For 2-nitroimidazole derivatives, the hypoxia selective radio sensitizer SR2508, obtainable in large amounts from Dr. C. Koch (Univ. of Perm.), was treated with FfTMeOH. This treatment cleaved the amide bond to yield the methyl ester (8) which was subsequently worked up with NaOH/HCl to yield the corresponding acid (7) (Scheme 3-9). The major problem with this procedure is that  OH  Dowex (H+) MeOH  OMe 1  N.  N O  2  0  OH  NaOH HCI N0  2  °  SR2508  Scheme 3-9: Synthesis of 7, via 8 from SR2508.  102  References on page 129  Chapter 3  increasing the acidity of the solution (with HC1) to a pH of ~4, to ensure the formation of 7, results in concomitant protonation of the imidazole ring at N3. Subsequent extraction with EtOAc thus produced 7 in significantly low yield because most of it remained in the aqueous layer as the hydrochloride salt (as judged by T L C and T i N M R spectroscopy). The protonated species proved to be useable for the synthesis of the desired halogenated nitroimidazoles, the procedure (Scheme 3-8) requiring an additional equivalent of N M M ; however, the yields were typically half of those obtained when the pure acid was used. For  the  2Me5N02lm  compounds,  the  hypoxia-selective  cytotoxin  metrodinazole, which is used in the treatment of anaerobic bacterial infections, was 53  oxidized with Jones' reagent converting the alcohol to the acid functional group (Scheme 3-10). The yield obtained was slightly higher than reported and, according to T L C , some 20  of the metronidazole was only partially oxidized to the aldehyde species. O f particular note, when additional Jones' reagent was added to convert the remaining aldehyde to the acid, a lower yield was obtained. NO  N0  2  OH N  Me  NO,  2  H  •Tone's Reagent  /  \  OH  acetone, r.t Me  Me  13  Metronidazole  Scheme 3-10: Oxidation of metronidazole with Jones Reagent to yield the carboxylic acid derivative, 13. For the 2Me4N02lm compounds, the acid species had an extra methylene in the link between the imidazole ring and the acid functional group. Treatment of 2-methyl-4nitro-l-imidazolepropionitrile (Aldrich) with sulfuric acid resulted in hydrolysis of the nitrile to the carboxylic acid after several days at high temperature. Formation of the amide is facile, nevertheless, further hydrolysis to the acid requires relatively harsh conditions, mainly because of the extra resonance capability of the nitrogen lone-pair (Scheme 3-11).  103  References  on page 129  Chapter 3  NH R—C=N  H 0 3  R'X  NH OH  ©  X  NH  R  2  Tautomer of amide  OH H,0  2  -NH  R-  O  OH ©  2  2  •H © OH  x O  NH4  +  NH  3  OH  +  RR^^OH  R""^OH  carboxylic acid  OH  © -NH3  OH  +H  -NH  R-  2  OH  Scheme 3-11: Acid hydrolysis of 2-methyl-4-mtro-l-imidazolepropionitrile (abbreviated as RCN). The title compound (EF5) on which most of this work was based on has been approved for clinical trials by the National Cancer Institute (US) through support of large scale synthesis, formulation and toxicological studies. The improved synthesis (Scheme 312)  17  of EF5 from IF5 will be useful, especially if the compound is approved for use in  CF, NO,  Cs CQ 2  O  CF-  T N0  IF5  2^CF,  3  DMF, 5CPC  O 2  EF5  Scheme 3-12: New synthetic route to EF5. humans as either a radiosensitizer or a hypoxia-selective imaging agent. Other 2-nitroimidazole compounds in this series (EF4Br, EF3, EF3(-1), EF2Br, EFl(-l), ECU, ECll(-l), EBrl, EBrl(-l), EIAA, EPrA) were all isolated in relatively high yield (up to 83 %) and could 'be synthesized via the alternate route (i.e. synthesis of the complete side-chain via reaction of the appropriate amine with iodoacetic acid, followed by coupling with 2N02lm, see Scheme 3-2 and Scheme 3-12) with the exception of the bromo derivatives which are too reactive (see Section 3.2.1.2). The high reactivity of the bromo derivatives is also indicated by their poor elemental analyses as the C-Br bond is readily hydrolyzed by H2O (in the air).  104  References on page 129  Chapter 3  The monofluorinated  species  (EF1,  see  Scheme 3-13) was the only  2-nitroimidazole compound in the series that could not be isolated in a yield higher than 15 %. The required monofluoropropylamine hydrochloride (or hydrofluoride) reagent was extremely  hygroscopic  which  may  explain  why 2-nitro-l-H-imidazol-1-yl-ethyl  isobutanoate (11) is the major product (Scheme 3-13). The presence of H 0 in this 2  reaction  could result  in the  formation of isobutylcarbonate  via "reaction with  isobutylchloroformate. The isobutylcarbonate would also be formed as a co-product of the reaction  between  3-fluoropropylamine  isobutylcarbonate could lose C 0  2  and  isobutylmethylimidazoylcarbonate.  The  to form isobutanol, which is the next strongest  nucleophile present after the amine. Because of the formation of HCI the amine would remain as its hydrochloride salt, thus decreasing its nucleophilicity and therefore allowing the isobutanol to react with the acyl carbonate group to form 11. The synthesis of ECll(-l) via reaction of TsCl with SR2508 was unexpected. The desired product was the tosylate species that could be used as a reactive intermediate for the formation of EFl(-l) (Section 3.2.7). Unfortunately, the tosylate could not be isolated because the chloride ions readily react to form the monochloro species ECll(-l). The reaction of phenylmethanesulfonyl fluoride using the same reaction conditions did not yield the sulfonate intermediate and consequently the desired EFl(-l) species was not formed. The highly reactive tosylate intermediate was also exploited for the synthesis of compound 16, the chlorinated derivative of metronidazole (Section 3.2.3.7). The ability to alter the biological activity of a compound by changing the composition of an associated side-chain prompted the attempted synthesis of a pentafluoro species analogous to EF5, but with the amide linkage reversed (Scheme 3-14). Because of the difficulty in forming the reverse amide linkage for RevEF5, three different syntheses were attempted. Synthesis 2 uses conditions identical to those of synthesis 1 for EF5 (Scheme 3-8), the major difference being that the pentafluoro group is introduced as pentafluoropropionic acid. This acid species is highly reactive with H 0 from the air, as 2  apparent from substantial fuming when the sample bottle is opened, but does readily react  105  References  on page 129  Chapter 3  EFl  Scheme 3-13: Formation of the isobutylester (11) in the presence of F£ 0. 2  with /'so-butylchloroformate to form the mixed anhydride. The problem with this reaction arises when 10 (the 2-nitroimidazole ethylamine product from synthesis 1, step 2) is added to the reaction mixture. The amine preferentially reacts with the carbonyl group associated with the isobutylformate to yield a species similar to 11 as the major product (as shown by H N M R ) , analogous to that formed during the synthesis of E F l (see X  Scheme 3-13), although a different mechanism must be involved. This result can be explained accordingly: the electron-withdrawing fluorine atoms stabilize the negative charge  of the  pentafluoropropionate,  making it  a better leaving group  than  isobutylcarbonate. To avoid the problem encountered in synthesis 2, the peptide coupling agent S T M l > B F was reacted with pentafluoropropionic acid (synthesis 3). Addition of 4  base (Et N) in the first step leads to formation of tetramethylurea and (N-succinimidyl)3  pentafluoropropionate (according to T L C data). Once again, addition of 10 results in a reaction; however, neither of the major products was RevEF5, which was observed in low  106  References on page 129  Chapter 3  concentration via TLC. Synthesis 1 allowed the isolation of RevEF5 in high yield using two different synthetic approaches for the third step of the reaction (steps 3 a and 3b). The first route, a well known procedure for the formation of an amide bond using D C C (synthesis 1, step 3 a) was employed, leading to the formation of a reaction intermediate analogous to that in synthesis 3. This reaction (29 % yield), however, was more successful than synthesis 3 (< 1% yield), and may be attributed to the greater stability of the sideproduct formed, dicyclohexylurea (DCU). The presence of BF " in synthesis 3 may also 4  hinder the reaction although it is not obvious how. An advantage of synthesis 1 (step 3 a) is that the D C U (because of its low solubility in DMF) can be readily separated from the product mixture via filtration. For the second route (step 3b), the amine on the side-chain of  10 (nucleophilic) was reacted  with the highly reactive  perfluoroanhydride  (CF CF CO) 0 to give RevEF5 in a somewhat higher yield (32 %). 3  2  2  P  S ^  F /  P  1  = \  T N0  \  s t e  P  = /  2  2)6MHC1  \ / Y N0  2  © 0 . N H CI  \  3  10  2  Step 3 a I  H \x^\^N.  y NO  2  1) DCC, NHS, C F C F C 0 H 2) NMM 3  ^.CFx CF3 O RevEF5  Y  2  2  1)NMM ' 2) (CF CF CO) 0 3  2  2  Step 3b  Scheme 3-14: The 3-step synthesis of RevF5 from 2-bromoethylphthalamide. Another 2-nitroimidazole species with a side-chain terminating with an amino group is RSU1111. This compound contains a side-chain one methylene unit longer than in 10, and also contains a secondary hydroxyl group similar to that of the radiosensitizer misonidazole. Analogous to synthesis 2 of RevEF5, the major product formed was the 54  isobutylformate-linked amide species, while 20 was isolated in low yield (5 %). The incorporation of the pentafluoropropyl acyl group is confirmed by H R - M S and more importantly, by F{ H} N M R spectroscopy, which gives signals at 8 -6.77 and -46.35 19  J  107  References  on page 129  Chapter 3  corresponding to the CF3 and C F groups, respectively (cf. RevEF5: 8 -6.79 and -46.43). 2  In the *H N M R spectrum the protons on the methylene groups adjacent to the chiral -CH(OH)- centre are all magnetically inequivalent (diastereotopic protons). A n interesting aspect of this spectrum is that the -CH -NH- protons have essentially the same chemical 2  shift while the Im-CH - proton chemical shifts are well separated (AS 0.40). This may 2  suggest that the O H group on the adjacent carbon is interacting with one of the protons of the methylene group nearest the nitroimidazole. The same chemical shift distribution is seen in the TI N M R spectrum of 21; the lm-CH - proton chemical shifts have nearly the 2  same separation as above (A5 0.38). The observation of a signal at 8 6.21 is in accordance with the H R - M S results which suggest that one of the Br-atoms of the C B r C 0 H 3  2  precursor has been displaced by a hydrogen. The anticipated product was the tribromo species, but presumably because of the bulkiness of the Br-atoms and the relatively weak C-Br bond, the dibromo species is isolated. The C N M R spectrum of 21, compared with 1 3  that of RSU1111, has extra signals at 8 151.7 and 37.2, confirming the presence of -C=0 and - C B r H units. 2  The coordination chemistry of nitroimidazoles is also of interest because these do not typically form strong interactions with the metal centres of interest (e.g. Pt, Ru), unlike imidazoles which form more stable species. '  The metal complexes may be used  55 56  as nitroimidazole delivery agents to the D N A by coordinating to the D N A s nitrogen bases, hence bringing the nitroimidazoles in close proximity to the site of action.  57  Synthesis of a nitroimidazole with a side-chain containing a chelating moiety would ensure stronger interaction with the metal centre, while maintaining the functionality of the hypoxia active nitroimidazole. Reaction of acrylonitrile with an amine is a useful method for adding two C H C H C N groups to the existing amine, followed by reduction of the 2  2  nitriles to yield the tridentate species R N ( C H C H C H N H ) . ' 23  2  2  2  2  2  24  Addition of acrylonitrile  to RSU1111 proved to be much more difficult than anticipated. Even when the reaction was done in neat acrylonitrile, the major product was the mono-substituted species 22, while the bis-substituted species (24) was formed only half as much. A small quantity of a compound corresponding to the additon of acrylonitrile at the hydroxyl group (23) was  108  References on page 129  Chapter 3  also isolated and characterized by TI N M R spectroscopy. A n interesting property of compounds 22 and 23 is that not only do they exhibit inequivalent methylene *H signals for the C H bound to the 2N02lm moiety, but they also have inequivalent methylene 2  proton signals for a C H bound to the N H and N H group, respectively. This is not 2  2  observed for compound 24. The difference between the chemical shifts of the two protons is not as pronounced for the methylene group bound to either the N H or N H groups (e.g. 2  5 2.80 and 2.71 for 22 vs. 5 4.73 and 4.46 for the 2N0 Im-Gr7 - protons), suggesting a 2  2  weaker intramolecular interaction. This finding is puzzling and tends to suggest that the newly added propylnitrile arm in 22 has a conformation which interacts with one of the methylene  protons  nearest to  the  N H group,  consequently inducing magnetic  inequivalence. The presence of the two pendant arms in 24 possibly makes the species bulky enough to prevent such an interaction. Reduction of the nitrile groups of 24 was unsuccessful using a number of different reducing agents (e.g. L A H , NaH, H ) including 2  B H6, 2  which has been reported to reduce C N groups in the presence of an N 0 group. In 29  2  all cases, the nitro group was reduced, either partially or completely, making further pursuit of this chelating compound futile. Comparison of the H N M R spectra for the 2-nitroimidazole compounds with l  variation in number and type of halogen atoms present reveals some interesting results. For the most part, for each of these compounds the chemical shifts of the protons of the 2 N 0 I m - C H - entity fall in the same range, displaying that the composition of the side2  2  chain has little effect on this component of the molecule (lm-H : 5 7.49 —» 7.66; Im-H*. 8 5  7.08 -> 7.21; Gr7 CO: 8 5.15 2  5.36). There is a larger variation for the chemical shift of  the N H proton (8 7.52 - » 9.05) as its position shifts with changes in concentration of H 0 2  in the N M R solvent due to proton exchange between the two species. Removal of the 42  N0 -group, however, does effect the chemical shifts of the imidazole protons and C H 2  2  bound to the imidazole at N l . For ImF5, the compound identical to EF5 less the nitro group, the chemical shifts are significantly upfield of those for EF5 (e.g. Im-H^. 8 7.66 (EF5) -> 7.16 (ImF5)). The electron-withdrawing N0 -group decreases electron density 2  within the imidazole ring. The *H N M R signals for the C H adjacent to the CF CF3 group 2  109  2  References  on page 129  Chapter 3  for these two compounds are not effected by the presence or absence of the N0 -group on 2  the imidazole ring. The chemical shift(s) of the C H group(s) in the side-chain bound to the amide 2  N H are dependent on the atoms on either side of them. For instance, the chemical shift for the C H of E F 5 (bound to N H and CF ) is 4.06 ppm, which matches exactly that of 2  2  EF3(-1) whose only difference is that the C F group is now C F . Comparison of the 8 2  3  values for E F 5 versus E F 3 and EFl shows an upheld shift for the -NH-C# - moiety (8 2  4.06 —» 3.50 —» 3.38, respectively), exhibiting the effect of the proximity and number of electonegative F-atoms. The absence of halogen atoms on the side-chain (e.g. as in EPrA) results in a 8 of 3.20, even further upfield. The most pronounced effect of the fluorine atom can be seen for EFl (Figure 3-4) and EFl(-l) (Figure 3-5) as the protons for C H F 2  are shifted downfield to 8 ~ 4.50. This group is also easily identified in the *H N M R spectrum as a doublet of triplets due to the large  2  JHF  value  (66  Hz and  48  Hz,  respectively). The adjacent C H also has a strong interaction with the fluorine atom, as 2  indicated by  3  JHF  values  (42  Hz and  30  Hz, respectively). The N H signal for EFl is not  observable at the magnification presented in Figure 3-4; however, the signal is present at 8 7.64 if the vertical scale is expanded. The N H signal is more pronounced for the EFl(-l) spectrum at 8 7.87 (Figure 3-5) and may be related to the increased amount of H 0 2  evident in this spectrum; the broad signal is a result of slow exchange of the N H proton, and the electrical quadrupole moment of the nitrogen nucleus that induces moderately efficient spin relaxation.  42  Comparison of the compounds with identical composition, except for variation in the halogen atom terminating the side-chain, demonstrates the direct correlation between electronegativity and chemical shift. The 8 values for the C H X protons (EFl: 8 2  4.51; ECU: 8 3.66; EBrl: 8 3.50) decrease almost proportionally to the decrease in electronegativity (EN) of the X atom ( E N for F, CI and Br is 4.0, 3.0 and 2.8, respectively); the trend is as expected. The same effect is observed for the EXl(-l) compounds. Analysis of the  1 3  C N M R spectra for these compounds for the C atom  110  References  on page 129  Chapter 3  directly bound to X also exhibits the dependence of 6 on E N ( E F l ( - l ) : 5 84.70; E C l l ( - l ) : 5 43.46; E B r l ( - l ) : 5 37.88).  N 0  2  acel one <  b a  ..  ' 3.0  1 1 1  1  1 1  ' 1 1  1 1 1 1  8.5  1  1 1 1  6.0  '1  .. 1  1 1 1  7.5  F i g u r e 3-4:  d  e  f  1  7.0  1 1 1 1  1  1 1 1 1  G.5  1  1 1 1 1  6.0  1  1 1  1 1  5.5  1  1 1 1 1  5.0  11  1  1 1 1 1  1  1 1  4.5  1,  ''1 4.0  ppm  3.5  ... 1 . J 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 3.0  2.5  2.0  1.5  1.0  0.5  *H N M R spectrum of E F l in d6-acetone .  d  H; N0  ace one  2  C  b  a  f  d 3.0  ... |  8.5  8.0  Figure 3-5:  e  11J „ i U l J 4  , J  | . . . . | . . . . | . . . . | . . . . | . . | . . . . | . . . . . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .. . 7.5  7.0  6.5  6.0  5.5  5.0  4.5  4.0  ppm  3.5  3.0  2.5  2.0  1.5  1.0  0.5  ^ N M R spectrum of E F l ( - l ) in d -acetone. 6  Analysis of the F N M R spectra for the fluorinated compounds reveals some 19  interesting and unpredictable results. For example, comparison of the chemical shifts of  111  References  on page 129  Chapter 3  the C F groups of EF5 and EF3, 5 -4.34 and 10.84 respectively, shows results opposite to 3  those expected. The signal for EF5, which has an electron-withdrawing CF2-group adjacent to the CF , is further upheld than for EF3 which has a C H bound to the C F . 3  2  3  Also surprising is the downfield shift of the fluorine signal for EF2Br (5 32.71) when compared to that for EF3. The two compounds are identical with the exception of substitution of one of the F atoms by Br. The lower electronegativity of Br should have shifted the signal further upfield; however, the opposite is true. The C F of EF5 comes at 2  8-41.31 while the C H F h a s a signal at 8 -142.50 showing that as the number of F-atoms 2  on a single carbon decreases the signal is shifted further upfield, following the expected electronegativity trend. Examination of the  X  H and  19  F N M R spectra for the 4-nitro- and 5-  nitroimidazole compounds reveals the same trends observed for the 2-nitroimidazole species. In the T i N M R spectra, the closer proximity of the nitro group to the imidazole proton leads to a downfield shift of the signal (e.g. MF5: Im-H 8 7.93 vs. EF5: Im-H 8 4  4  7.21). For both MXl(-l) and 2M4NX1(-1) (where X= F, CI and Br), chemical shift differences are observed analogous to those for the EXl(-l) compounds, following the trend that increased E N leads to a downfield shift in the C H X proton signal. 2  Other analytical techniques used to characterize these compounds included mass spectrometry, and UV-Vis and IR spectroscopies. The UV-Vis spectra did not reveal much information other than the presence of a nitro group on the imidazole ring (the energy band of the major chromophore was very destinctive of the nitro group position; 316, 310 and 300 nm for 2 N 0 , 5 N 0 , and 4 N 0 , respectively). Mass spectrometry was 2  2  2  used to characterize the compounds for which acceptable elemental analysis results were not obtainable. For the most part, the compounds with the brominated side-chains were problematic due to their highly reactive C-Br bond. The M S spectra typically contained a very weak parent peak and a base peak which corresponded to Wf - N 0 , suggesting that 2  the bond between the imidazole ring and the nitro group is easily cleaved. IR spectroscopy, next to N M R spectroscopy, was the most diagnostic tool for characterizing the nitroimidazole compounds. The major bands of interest were the stretching bands for  112  References  on page 129  Chapter 3  the N - H and C=0 groups of the side-chain and the N - 0 of the N0 -group associated with 2  the imidazole ring. A comparison of the IR data for these compounds shows that there is some electronic influence, albeit weak, from the substituents of the side-chain on the N - H and C=0 stretching frequencies. For the most part the N0 -group was too far removed 2  from the substituents on the end of the side-chain to be affected. The asymmetric and symmetric VNO values were two of the more dominant bands observed in the spectrum of each nitroimidazole and typically spanned the ranges, VNOsym  VNOasym  (1484 - 1506 cm" ) and 1  (1361 - 1400 cm" ). The 4 N 0 group had bands at slightly higher wavenumbers 1  2  than those of the 2 N 0 group, while the values of the 5 N 0 group were lower. These 2  2  findings may result from the proximity of the N0 -group to the side-chain. The 4 N 0 2  2  group is the furthest removed and therefore the possibility of interaction with the side-chai