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Radiolabelling of organic molecules with short-lived radionuclides (¹¹C, ¹⁸F) 1994

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RADIOLABELLING OF ORGANIC MOLECULES WITH SHORT-LIVED RADIONUCLIDES (“C, 18F) Julius Alexander Balatoni B.Sc., The University of British Columbia, 1981 M.Sc., The University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1994 © Julius Alexander Balatoni, 1994 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemistry The University of British Columbia 2036 Main Mall Vancouver, Canada Date: b. 30) jq Abstract The primary aim of this study was to develop new methods for the introduction of short- lived radionudides (11C, 18F) into organic molecules. This was accomplished by the use of organometallic intermediates: (i) i16-arene)tricarbonylchromium complexes were used to facilitate aromatic nucleophilic substitution of the attached arene for the incorporation of[11Cjcyanide anion, and (ii) vinyl-tin derivatives were employed for electrophilic fluorination reactions to produce F- and 18F-labelled vinyl fluorides. A range of simple(,6-arene)tricarbonylchroniium complexes were prepared as model systems. Fluorine was found to be the only leaving group that was readily displaced by cyanide. Non-radioactive and 11C-labelled aryl nitriles were prepared in 10 minutes by reaction of complexes 1, 4, 5, and 6 with[12Cjcyanide and[11C]cyanide in DMSO at elevated temperature. Under the reaction conditions used, the aryl nitrile product was liberated from the chromium tricarbonyl moiety thereby obviating the need for a separate oxidative decomplexation step. The reactivity of (E)-vinylstannanes 30, 31, 32, 33, and 34 was studied with elemental fluorine and acetyl hypofluorite under varied conditions. Generally, gaseous CH3OOF was found to be the most effective fluorinating agent for the electrophilic cleavage of vinyl-tin bonds. For example, 31 was fluorinated with CH3OOF, at room temperature, to yield an isomeric mixture of 39 and 40 in 41-42% yield; fluorination with F2 proceeded in 9.0% yield at best. Subsequently, 31 was radiofluorinated withCH3OO’8F in 19% radiochemical yield (based on starting[18F]F2). 11 Me Me CI I I Cr(CO)3 Cr(CO)3 Cr(CO)3 Cr(CO)3 4 5 6 Sn(Bu)3 HO R20 O/0 —Sn(Bu)3 // \c) O R’O 33 Sn(Bu)3 30 R’=R2=H 31 RMe,R=H OH 32 R’R2Me oO 0 \\ 34 HO HO MeO MeO 39 40 iii Table of Contents Abstract ii List of Tables vii List of Figures ix Acknowledgements x Dedication xi Chapter 1 GENERAL INThODUCTION 1 1.1 Background 1 1.2 Aspects of Labelling with Short-lived Isotopes 3 1.3 Objectives and Format of this Thesis 9 References 13 2 APPLICATION OF ORGANOCHROMIUM CHEMISTRY TO RADIOLABELLING 16 2.1 Introduction 16 2.2 Preparation of(76-Arene)tricarbonylchromium Complexes 21 2.2.1 Synthesis 21 2.2.2 Characterization 30 2.3 Reactions of Organochromium Compounds with Cyanide 33 2.4 Labelling Studies with11C-Labelled Cyanide 53 2.5 Summary and Conclusions 65 References 68 iv Chapter 3 DEVELOPMENT OF METHODS FOR RAPID FLUORINE LABELLING 71 3.1 Introduction 71 3.2 Synthesis of Vinyl-Tin Precursors 80 3.3 Labelling Studies with Non-Radioactive Fluorine 85 3.4 Radiofluorination Work 96 3.5 Summary and Conclusions 101 References 104 4 GENERAL CONCLUSIONS 108 References 111 5 EXPERIMENTAL 112 5.1 General Methods 112 5.2 NMR Methods and Instrumentation 115 5.3 Experimental for Chapter 2 116 5.3.1 Sources of Materials 116 5.3.2 General 116 5.3.3 Preparation of(t6-Arene)tricarbonylchromium Complexes 118 5.3.4 Substitution Reactions of Organochromium Complexes 125 5.3.5 Labelling Work with[11C]Cyanide 135 5.4 Experimental for Chapter 3 139 5.4.1 Sources of Materials 139 V Chapter 5.4.2 General 140 5.4.3 Preparation of Vinyl-Tin Substrates 142 5.4.4 Fluorination Reactions of Vinyl-Tin Compounds 148 5.4.5 Radiofluorinations with Acetyl[‘8F]Hypofluorite 153 References 156 vi List of Tables Table CHAPTER 1 1.1 Characteristics of Radionuclides Used for PET 4 1.2 Physical Properties of Some Radionucides 7 1.3 Positron Emitting Radiopharmaceuticals Used Commonly in PET Imaging Procedures 9 CHAPTER 2 2.1 Summary of Yields Obtained for Complexes Synthesized using Equation 2.9 26 2.2 Summary of Melting Points of Organochromium Complexes 31 2.3 Chemical Yields Obtained for Complex 1 39 2.4 Chemical Yields Obtained for Complex 4 41 2.5 Chemical Yields Obtained for Complex 5 42 2.6 Chemical Yields Obtained for Complex 6 43 2.7 Chemical Yields Obtained for Complex 4 using 18-Crown-6 47 2.8 Chemical Yields Obtained for Equation 2.18 using Acetonitrile as the Solvent 48 2.9 Summary of Chemical Yields 51 2.10 Summary of Radiochemical Yields Obtained for Equation 2.22 55 2.11 Summary of Radiochemical Yields Obtained for Equation 2.23 . 59 vi’ Table CHAPTER 3 3.1 Yields of (E)-Vinylstannanes.83 3.2 Summary of Yields Obtained for the Reaction of 31 with Acetyl Hypofluorite 87 3.3 Summary of Yields Obtained for the Reaction of 31 with Fluorine 89 3.4 Summary of Radiochemical Yields Obtained for the Reaction of 31 with Acetyl[18F]Hypofluorite 100 viii List of Figures Figure CHAPtER 2 2.1 Changes in arene reactivity when complexed to chromium tricarbonyl 17 2.2 Examples of carbanions which react with(i6-ha1obenzene)tricarbonyl- chromium complexes 18 2.3 Examples of carbanions which react with(6-benzene)tricarbonyl- chromium 19 2.4 Principal fragmentation pattern of(6-arene)tricarbonyl- chromium compounds 32 2.5 HPLC chromatogram obtained from the analysis of the reaction of complex 1 with cyanide at 135°C 40 2.6 Radio-HPLC chromatogram obtained from the analysis of the reaction of complex 1 with[11C]cyanide (with 0.37 equiv of carrier KCN added) at 150°C 57 2.7 Radio-HPLC chromatogram obtained from the analysis of the reaction of complex 1 with[11Cjcyanide (with no carrier KCN added) at 150°C 58 2.8 Radio-HPLC chromatograms of[‘tC]cyanide in DMSO 61 CHAPTER 5 5.1 Schematic of the fluorine gas handling system used to perform the fluorination reactions 141 x Acknowledgements I thank my supervisors, Dr. Laurie Hall and Dr. Mike Adam, for their invaluable guidance and helpful discussions. In particular, I am very grateful to Dr. Mike Adam for his invaluable assistance in all aspects of the experimental development of this work, and for his helpful advice in the preparation of this thesis. I extend my thanks to Dr. Peter Legzdins and his research group for valuable discussions and including me in their literature meetings. I am grateful to Dr. Tom Ruth and Dr. Mike Adam for the production of radio nuclides (11C, 18F) used in this work. I am also thankful for the technical assistance of Salma Jivan at TRIUMF. I gratefully acknowledge the contribution made by the Co-op students, Maurice Brulé, Dale Johnson, and Martin Dimitroff, with whom I had the pleasure of working. I greatly appreciate the service provided by the Electronics and Mechanical shops for maintenance of the NMR instrumentation, and in particular, Tom Markus, who also constructed the NMR probe. I also extend my thanks to the technical staffs of the departmental NMR, Mass Spectrometry, and Microanalytical services. I thank my friends, Dr. Jeff Coil and Dr. Sigrid Coil, for proofreading this thesis. Finally, I especially thank my supervisors, friends, and family for their incredible patience, support, and encouragement through very difficult times in which it seemed this thesis would not be completed. x Dedication To my risen Lord xi Chapter 1 GENERAL INTRODUCTION 1.1 Background Organic molecules labelled with short-lived positron emitting nuclides have had a great impact on biomedical research.’ Thus, for the first time, using positron emitting radiopharmaceuticals, quantitative in vivo measurements have been made of the human biochemical and physiological processes of the brain, heart, and other organs.2 For example, it has been shown that regional brain metabolism can be correlated with functional activity in humans under normal circumstances,3 during somatosensory stimulation,4 and also in disease states such as schizophrenia5and senile dementia.6 Other parameters of physiological function have also been measured which include blood volume, blood flow, oxygen- and glucose-metabolic rates, drug-receptor interactions, protein synthesis, amino acid transport, permeability of the blood-brain barrier, and tissue pH. These studies are intended to give a better understanding of disease states such as cancer, epilepsy, heart disease, stroke, movement disorders such as Parkinson’s disease, and mental illness.7’8 The in vivo measurement of biochemical and physiological processes, using compounds labelled with short-lived positron emitting nuclides, is based on the use of Positron Emission Tomography (PET). In this technique, a positron emitting 1 radiopharmaceutical, with the desired biological activity, is administered to a living subject. Inside the body, the radiotracer decays by emitting a positron (j3, positive electron), which travels a few millimetres, combines with an electron, its antimatter twin, and is thereby annihilated to produce two gamma-ray photons each of 511 keV. The two gamma-rays travel in opposite directions, nearly 1800 apart, penetrating the surrounding tissue and are detected by a circular array of coincidence detectors surrounding the subject being imaged. Only timed coincidence annihilation events registered by paired detectors located 180° apart are recorded. From these, the spatial distribution of the radiotracer is reconstructed by computer, and is presented as a series of cross-sectional images.7’9 The successful application of PET as a medical research tool derives from the fact that PET allows the study of physiological and biochemical processes to be done in a quantitative, non-invasive manner, within a volume element of tissue in vivo. From the technical viewpoint, a key ingredient of this process is the positron emitting radio- pharmaceutical itself.1° One of the cornerstones of modern medicine is the understand ing that all clinical symptoms result from biochemical reactions, and as a consequence, every pathology has an underlying biochemical defect.7 Thus, the radiopharmaceutical acts as a biochemical probe and, by virtue of the attached positron emitting nuclide, the fate of the radiopharmaceutical can be spatially mapped using PET. As a result, the range of studies that can be performed using PET depends on the availability of com pounds appropriate to the study, which can be labelled with positron emitting nuclides.8’1° To date, PET has been most extensively applied to problems in neurology, cardiology, 2 and oncology. As a result of the successes obtained in these studies, substantial efforts are being made to expand PET into new areas of research.11”2 Therefore, to exploit the full potential of PET, continued development of new and improved methods of radiopharmaceutical synthesis are required. 12 Aspects of Labeffing with Short-lived Isotopes The goals of radiopharmaceutical synthesis have many elements in common with tradi tional synthetic organic chemistry. Both the radiochemist and organic chemist are concerned with developing syntheses which will yield in the most direct manner, the desired compound in the largest chemical yield possible. In addition, the radiochemist is concerned with obtaining high radiochemical yields. Radiochemical yield is defined as the amount of radioactivity incorporated into the product as a percentage of the initial quantity of radioactivity used, Both the radiochemist and organic chemist require the final compound to be isolated in a chemically pure state. However, radiolabelled com pounds must also be radiochemically pure and free of other radionuclidic impurities.’3 Furthermore, organic compounds produced as pharmaceuticals and formulated for intra venous injection, whether radiolabelled or not, must also be sterile and pyrogen free.’4 In order to ensure a successful radiopharmaceutical synthesis, several additional considerations hold which are not common to synthetic organic chemistry. The key aspects that need to be addressed by the radiochemist are as follows: (i) the physical properties of the radionucide, (ii) the source and chemical form of the radionuclide, and (iii) the specific activity, stoichiometry, and reaction scale. These topics will now be 3 discussed in the above order. The most important radionuclides used in PET are ‘1C, 13N, 150, and 18F; their half- lives are listed in Table 1.1. All of these positron emitting nucides possess short half- Table 1.1: Characteristics of Radionuclides Used for PET Radionuclide Half-life % i3 Decaya Daughter (mm) 11C 20.4 99.8 B, stable ‘3N 9.96 100 13C, stable 150 2.07 99.9 15N, stable ‘8F 109.7 96.9 180, stable SOURcE: Reference 15. a13+ =positron emission. lives*,16 and emit high energy, body-penetrating radiation. These characteristics are important properties which make these radionudides suitable for medical use. However, these same properties also give rise to problems for radiolabelling development. Most significantly, the half-life of the nuclide poses a limit on the time allowable for synthesis. The total synthesis time, beginning with the generation of the radionuclide and its incorporation into the substrate, followed by any further chemical modifications (i.e., removal of protecting groups, etc.) through to the final purification of the radiopharmaceutical, should be equivalent to no more than one or two half-lives of the radioisotope.17’8 Clearly, the associated manipulative problems become particularly acute when using nudides whose half-lives are on the order of minutes. The actual * A radionucide is usually defined as short-lived when its half-life is less than 15 h. 4 imaging of the patient must in turn be completed within about three or four half-lives of the radionucide used.19 Another problem inherent in working with positron emitting nudides is the obvious radiation hazard they pose, which unfortunately, exposes the radiochemist to a potentially serious health risk. Hence, for prudent safety reasons, it is necessary to work with adequate levels of shielding, use remote operations whenever possible, properly monitor the radiation level in the work area, and to work with care while handling radioactive compounds.2° The source and chemical form in which a given radioisotope is available has a significant impact on the development of a practical synthetic strategy. The first problem is the availability of the required positron emitting nuclide. In general, positron emitting nucides are produced by nuclear reactions performed with a charged particle accelerator, generally a cyclotron.21 Some positron emitters are available from a nuclide generator system, such as the68Ge/a (half-life of 68Ga is 68.1 mm) generator. Although such generators are very convenient as they allow shipment of radionuclides for long distances from the production site, unfortunately, few such generator systems exist for positron emitting isotopes.22 Therefore, it is generally mandatory that accelerator produced nuclides be made on site, or within relatively short travelling distance (i.e., time) from the radiolabelling facilities. The chemical form of a given radionuclide is the second problem that has to be considered. Radioisotopes, as obtained from a cyclotron or generator, are usually available in a limited range of chemical forms. Although the preparation of a 5 radioisotope is dependant on the nuclear reaction used, target design, and cyclotron characteristics, the chemical form of the radioisotope is determined by a number of factors—the most crucial factor is the chemical composition of the target and the energy deposition in the target;17 this area of study is referred to as target chemistry. Therefore, if the reagent form of the radionuclide is inappropriate for a given radiolabelling procedure, then additional chemical manipulations are required to obtain the desired form of reagent. Since this increases the synthesis time and adds complexity to the experimental procedure, the best approach, whenever possible, is to directly prepare the radioisotope in the specific chemical form required for the radiolabelling step. The last topic of this section is the issue of specific activity, stoichiometry, and reaction scale; these three subjects are closely related. Specific activity is defined as the quantity of radioactivity present, generally expressed in curies, per mole of compound. One curie of activity produces 3.70 x 1010 Bq (disintegrations per second); the maximum specific activity of a radionuclide depends on its half-life (see Table 1.2), and is only attainable when no other isotope of the same element (i.e., carrier) is present; this ideal state is referred to as the carrier-free (CF) state. For some radioisotopes, this CF state can be approached only to within an order of magnitude so that some carrier is unavoidably present in most cases. For example, it is reported that in the production ofH11CN, the ratio of 11C to ‘2C is approximately 1:3000? In light of this problem, additional terminology is needed for specifying the extent of dilution present in a radiopharmaceutical product. The no carrier-added (NCA) state, as applied to an element or compound, means that no carrier of the same element or compound has 6 Table 1.2: Physical Properties of Some Radionuclides Nucide Half-life Decay Maximum Range” Maximum Modea energy (mm) specific (MeV) activity (Ci/mol) 11C 20.4 mlii i3(99+%) 0.96 4.108 9.22 x iO 150 207 mm 3(99 9%) 172 82 908 x 10” ‘3N 9.96 mm I3(100%) 1.19 5.39 1.89 x 1010 ‘8F 109.7 nun i3(97%) 0.635 2.39 1.71 x iO 3H 12.35 y f(l00%) 0.0186 0.0072 2.90 x 10 ‘4C 5730 y i31100%) 0.155 0.359 62.4 SouRcE: Reference 25. aDecay modes: r =positron emission, 3 = beta particle emission. “Maximum linear range in water. been added during its preparation. The carrier-added (CA) state means a known amount of carrier has been added to the element or compound during its preparation.’7 Especially when dealing with short-lived radiopharmaceuticals near their maximum specific activity, the mass of the product is not detectable by ordinary chemical or spectroscopic means. To ifiustrate this point, consider the mass of 1 mCi of 11C as compared to ‘4C, which is 1.5 pg (6.53 x 1010 atoms) as opposed to 0.22 mg (9.59 x 10’s atoms) of 14C.26 Therefore, the production of high specific activity radiopharmaceuticals in the CF or NCA states is highly desirable because the mass of the radiopharmaceuti cals is then so small that when administered in vivo it is usually below the threshold where any physiological response is invoked, yet there is adequate radioactivity present (in the order of 0.1 to 0.5 Ci/g of tissue in the case of PET instruments) to be detected 7 with statistical significance?4 Thus, even highly toxic molecules can be used for studies if adequate specific activities can be achieved.’7 Working with small amounts of high specific activity radionuclides usually leads to problems regarding stoichiometry and reaction scale. When a labelling reaction is performed, in which both labelling reagent and substrate are used in approximately one to one ratio, everything may work well. However, when the concentration of the labelling reagent is reduced by several orders of magnitude, as during a high specific activity radiosynthesis, very different results may be observed. Since NCA radiosyntheses are performed on a very small scale, the amount of impurities present in the reagents and solvents may be comparable to, or even exceed, the quantity of the radionuclide used in the synthesis. These impurities may compete with the radiolabelling reagent in a given reaction, leading either to unwanted side-products, or even to complete prevention of the formation of the desired radiolabelled product.27 If one of the impurities present in the synthesis is carrier, this will lower the specific activity of the radiolabelled product. This can be a very serious problem when very high specific activities are required, and as a result great care must be taken to exclude carrier from all possible sources, such as solvents, reagents, and substrates.28 In spite of the various difficulties and challenges involved in the area of radiopharmaceutical synthesis, much progress has actually been made. A variety of positron emitting radiopharmaceuticals have been developed and are currently being used in PET imaging, and some of the commonly used radiopharmaceuticals for PET 8 Table 1.3: Positron Emitting Radiopharmaceuticals Used Commonly in PET Imaging Procedures Radiopharmaceutical Application [18F]fluorodeoxyglucose Cerebral glucose metabolism Myocardial glucose metabolism [18F]fluorodopa Dopa uptake studies 18F]spiperone Dopamine receptor binding [18F]-N-methylspiperone Dopamine receptor binding [18F]-16a-fluoro-17fl-estradiol Estrogen receptor binding [‘1C]carbon dioxide Tissue pH 11C]-1-butanol Cerebral blood flow [11C]methionine Amino acid metabolism 11Cjpalmitate Myocardial metabolism [11C]acetate Myocardial metabolism [11C]glucose Cerebral glucose metabolism 11C]-N-methylspiperone Dopamine receptor binding [1501 oxygen Cerebral oxygen extraction and metabolism [15ojcarbon monoxide Cerebral blood volume Myocardial blood volume [150]water Cerebral blood flow Myocarclial blood flow [13Nlammonia Myocardial blood flow SouRcEs: References 7,8,10,29. are summarized in Table 1.3. 1.3 Objectives and Format of this Thesis With the continuing development of PET, new and innovative synthetic methodologies 9 are needed to produce the required radiopharmaceuticals for future PET applications. The primary objective of this study was the exploration of new methods for the incorporation of positron emitting radionucides—specffically 11C and‘8F—into organic compounds. Increasingly, the use of organometa.llic intermediates are providing new avenues to rapidly label organic molecules. The organic derivatives of some main group metals (B,3° Si,31’2 Ge,33 Sn,34’5 Hg,36 TI,37) have been studied and utilized for radiolabelling. Generally, their prime application has been to prepare compounds that are radiolabelled on the aromatic ring. Boron3°and silicon32 derivatives have also been successfully used for the preparation of radiolabelled alkyl halides. The common feature of these reactions is the exploitation of the reactivity of the polarized carbon-metal bond, in which the metal possesses a partial positive charge and the attached carbon possesses a partial negative charge; as a result, an organometallic compound is susceptible to electrophilic attack. This is important because the electrophilic cleavage of organometallic precursors can potentially be performed under mild conditions with short reaction times. Therefore, radionuclides that can be prepared in electrophilic reagent form, can in turn be used for the radiolabelling of organometallic precursors, in a regioselective manner. Previous studies in this laboratory focussed on the development of labelling vinyl-tin derivatives with radioactive bromine(82Br) and iodine (1231 and 1311).38 The use of vinyl- tin reagents was found to be very successful. This experience prompted our interest in extending the utility of vinyl-tin reagents to radiofluorinations with 18F. 10 The organic derivatives of transition metals, however, exhibit very different patterns of reactivity as compared to those of the main group metals.39 The fundamental reason for this is the presence of partly filled d or f orbitals. This leads to a variety of bonding interactions with organic ligands.4° Transition metals are able to form complexes in which the metal is bonded to unsaturated organic molecules such as ethylene, cyclobutadiene, or beuzene. The normal pattern of reactivity of unsaturated organic molecules when coordinated to transition metals is changed, whereby the unsaturated moieties can be attacked by a wide range of nucleophiles. The more electron- withdrawing the metal centre, the more facile is the nucleophific addition.4’ This mode of reactivity could have significant potential for the application of radiolabelling. It was decided to explore the labelling of aromatic rings with ‘1C using(i16-arene)tricarbonyl- chromium complexes as synthetic intermediates. The format of this thesis is as follows. Chapter 2 is devoted to the evaluation of arene)tricarbonylchromium compounds as synthetic intermediates for the incorporation of ‘1C, in the form of [“C]cyanide, onto aromatic rings. At the beginning of the chapter, some introductory background information is presented regarding organochromium chemistry. Then, the preparation of(6-arene)tricarbonylchromium complexes used for this study will be described. This will be followed by an examination of the reactivity of the prepared chromium tricarbonyl complexes with non-radioactive cyanide. Lastly, radiolabelling studies with “C-labelled cyanide will be presented. Chapter 3 is devoted to the development of electrophilic fluorination methodology that is applicable to vinyl-tin compounds with both non-radioactive fluorine and radiofluorine. 11 The chapter begins with an introduction to selective fluorination of organic molecules. This will be followed by a presentation of the synthesis of the vinyl-tin derivatives employed for this present study. Next, fluorination studies of the vinyl-tin precursors with elemental fluorine and acetyl hypofluorite will be described. Finally, radio- fluorination with 8F of a selected vinyl-tin derivative will be presented. In Chapter 4, the general conclusions developed from the studies described in Chapters 2 and 3 will be presented, along with suggestions for future work. In Chapter 5, the experimental details are given for the work performed for this thesis. The general methods are described first, followed by the specific experimental descriptions for Chapters 2 and 3, respectively. 12 References 1. Positron Emission Tomography; Reivich, M., Alavi, A., Eds.; Alan R. Liss: New York, 1985; Positron Emission Tomography andAutoradiography: Principles andApplications for the Brain and Heart; Phelps, M. E., Mazziotta, J. C., Schelbert, H. R., Eds.; Raven: New York, 1986. 2. Kellersohn, C. Brit. J. Radio!. 1981, 54, 91-102; Wagner, H. N. J. Am. Med. Assoc. 1986, 256, 2096-2097. 3. Reivich, M.; Kuhl, D.; Wolf, A. P.; Greenberg, J. H.; Phelps, M. E.; Ido, T.; Casella, V.; Fowler, J. S.; Hoffman, E. J.; Alavi, A.; Som, P.; Sokoloff, L. Circ. Res. 1979, 44, 127-137; Phelps, M. E.; Huang, S. C.; Hoffman, E. J.; Selin, C.; Sokoloff, L.; Kuhi, D. E. Ann Neurol. 1979, 6, 371-388. 4. Alavi, A.; Reivich, M.; Greenberg, 3. H.; Hand, P.; Rosenquist, A.; Wolf, A. P. Semin. Nuci. Med. 1981, 11, 24-31; Greenberg, J. H.; Reivich, M.; Alavi, A.; Hand, P.; Rosenquist, A.; Rintelman, W.; Stein, A.; Tusa, R.; Dann, R.; Christman, D.; Fowler, J. S.; MacGregor, B.; Wolf, A. P. Science 1981, 212, 678-680. 5. Farkas, T.; Reivich, M.; Alavi, A.; Greenberg, J. H.; Fowler, J. S.; MacGregor, R. R.; Christman, D. R.; Wolf, A. P. In Cerebral Metabolism and Neural Function; Passonneau, J. V., Hawkins, R. A., Lust, W. D., Welsh, R. A., Eds.; Williams and Wilkins: Baltimore, 1980; pp 403-408. 6. Ferris, S. H.; deLeon, M. J.; Wolf, A. P.; Farkas, T.; Christman, D. R.; Reisberg, B.; Fowler, 3. S.; MacGregor, R.; Goldman, A.; George, A. E.; Rampal, S. Neurobiol. of Aging 1980, 1, 127-131. 7. Ter-Pogossian, M. 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Liss: New York, 1985; p 65. 16. Vaalburg, W.; Paans, A. M. J. In Radionuclides Production; Helus, F., Ed.; CRC: Boca Raton, 1983; Vol. 2, p 48. 17, Fowler, J. S.; Wolf, A. P. The Synthesis of Carbon-il, Fluorine-i8, and Nitrogen-13 Labeled RadiotracersforBiomedicalApplications; Nuclear Science Series, Monograph NAS-NS-3201; Technical Information Center, U.S. Department of Energy: Springfield, VA, 1982. 18. See reference 16; Vol. 2, Chapter 2. 19. Adam, M. J., TRIUMF, Vancouver, BC, personal communication. 20. Hesslewood, S. R. In Textbook of Radiophannacy. Theory and Practice; Sampson, C. B., Ed.; Gordon and Breach Science: New York, 1990; Chapter 6. 21. Wolf, A. P.; Schlyer, D. J. In Nuclear Imaging in Drug Discovery, Development, and Approval; Burns, H. D., Gibson, R. E., Dannals, R. F., Siegi, P. K. S., Eds.; Birkhäuser: Boston, 1993; Chapter 3. 22. Robinson, G. D., Jr. In Positron Emission Tomography; Reivich, M., Alavi, A., Eds.; Alan R. Liss: New York, 1985; Chapter 4. 23. Short, M. D. In Textbook of Radiopharmacy: Theory and Practice; Sampson, C. B., Ed.; Gordon and Breach Science: New York, 1990; p 19. 24. See reference 17; pp 28-29. 25. Fowler, J. S.; Wolf, A. P. In Positron Emission Tomography and Autoradiography: Principles and Applications for the Brain and Heart; Phelps, M. E., Mazziotta, 3. C., Schelbert, H. R., Eds.; Raven: New York, 1986; p 393. 26. See reference 17; p 39. 27. See reference 17; pp 29-30. 14 28. Wilbur, D. S.; Garcia, S. R.; Adam, M. J.; Ruth, T. J. J. Labelled Compd. Radiopharm. 1984, 21, 767-779. 29. Radiophannaceuticals for Positron Emission Tomography: Methodological Aspects; Stöcklin, G., Pike, V. W., Eds.; Kiuwer Academic: Boston, 1993. 30. Kabalka, G. W. Acc. Chem. Res. 1984, 17, 215-221. 31. Wilbur, D. S.; Anderson, K. W.; Stone, W. E.; O’Brien, H. A., Jr. J. Labelled Compd. Radiopharm. 1982, 19, 1171-1188. 32. Wilbur, D. S.; Svitra, Z. V. J. Labelled Compd. Radiopharm. 1983, 20, 619-626. 33. Moerlein, S. M.; Coenen, H. H. J. Labelled Compd. Radiophann 1984, 21, 1076- 1077. 34. Adam, M. J.; Ruth, T. J.; Pate, B. D.; Hall, L. D. J. Chem. Soc., Chem. Commun. 1982, 625-626. 35. Coleman, R. S.; Seevers, R. H.; Friedman, A. M. J. Chem. Soc., Chem. Commun. 1982, 1276-1277. 36. Bo-Li, L.; Yu-Tai, J.; Zhong-Yum, P.; Maeda, M.; Kojima, M. J. Labelled Compd. Radiopharm. 1982, 19, 1089-1096. 37. Gilliland, D. L.; Basmadjiam, G. P.; Marchand, A. P.; Hinlcle, G. H.; Earlywine, A.; Ice, R. D. J. RadioanaL Chem. 1981, 65, 107-113. 38. Balatoni, J. A. M.Sc. Thesis, The University of British Columbia, Dec. 1985. 39. Coates, 0. E.; Green, M. L. H.; Powell, P.; Wade, K. Principles of Oiganometallic Chemistiy; Chapman and Hall: London, 1979. 40. Coliman, J. P.; Hegedus, L. S. Principles and Applications of Organotransition Metal Chemistiy; University Science Books: Mill Valley, CA, 1980. 41. Davies, S. 0. Organotransition Metal Chemistiy: Applications to Organic Synthesis; Perganion: Toronto, 1982; Chapter 4. 15 Chapter 2 APPLICATION OF ORGANOCHROMRJM CHEMISTRY TO RAIMOLABELLING 2.1 Introduction The first(76-arene)tri.carbonylchromium complex was reported in 1957, by Fischer and Ofele.1 Since that time, literally hundreds of such complexes have been prepared, characterized, and studied with respect to the structure and reactivity of the arene ring while complexed to the metal centre.2’3(,6-Arene)tricarbonylchromium compounds are air-stable, diamagnetic, crystalline solids, generally yellow to orange in colour. They are soluble in a variety of solvents, readily characterized by spectroscopic methods, and are easily purified by chromatographic and recrystallization techniques. As a result of the w-coordination of an arene to chromium, its reactivity is significantly altered.4 These changes in arene reactivity are summarized in Figure 2.1. The most significant change in arene reactivity is the increased ability of the coordinated arene to undergo nucleophilic aromatic substitution. It is this reaction which has been most studied and extensively employed in synthetic chemistry.5 To a lesser degree, the steric effect of the attached chromium tricarbonyl moiety has been used for the stereoselective modifications of the aromatic side chain.6 The other changes in arene reactivity have received less attention regarding their potential use for 16 Enhanced Nucleophilic EnhancedSubstitution /1 Solvolysis Enhanced Acidity HC—C— Steric H Enhanced Hinderance co Acidity Figure 2.1: Changes in arene reactivity when complexed to chromium tricarbonyl. (Adapted from reference 7.) synthesis.8’9 During early studies of the reactivity of(,76-arene)tricarbonylchromium compounds, Nicholls and Whiting found that i-chlorobenzene)tricarbonylchromium readily underwent substitution of chlorine by methoxide in very high yield.1° Uncomplexed MeOH K—OMeI + NaOMe I + NaC1 (2.1)Cr(CO)3 65°C Cr(CO)3 chlorobenzene is unreactive toward methoxide under the same conditions. Later investigations have shown other nucleophiles to react similarly, such as sodium phenoxide, aniline,11 and 2-methyl-2-propanethiol.’ (,76-Fluorobenzene)tricarbonyl- chromium also exhibits reactivity toward a variety of nucleophiles. Successful reactions with alkoxides, amines, thiolates, and cyanide have been reported.12”3 It has been shown that fluorine is more readily displaced than chlorine, during aromatic nucleophilic substitution.4 Of greater interest for organic synthesis, is the reactivity of(6-arene)tricarbony1- 17 chromium complexes with carbanions. Indeed, a variety of carbanions have been successfully used to alkylate the chromium tricarbonyl complexes of chlorobenzene and fluorobenzene; see Figure 2.2 for examples. Nucleophilic substitution for hydrogen can _____ [01 /\I I RCr(CO)3 25°C Cr(C0)3 where X = Cl or F R = C(CH3)2N, t(CH3)2COEt, C(CH3)20,tH(CO2Me), 902Me .C, Figure 2.2: Examples of carbanions which react with(6-halobenzene)tricarbonyl- chromium complexes. also occur directly on thei6-arene ring, which opens up additional synthetic pathways. (i76-Benzene)tricarbonylchromium, for example, reacts with a number of carbanions as shown in Figure 2.3. Furthermore, if the crude reaction intermediate is treated with excess strong acid, prior to oxidative demetallation, substituted 1,3-cyclohexadienes are obtained.5 Unfortunately, not all synthetically important carbanions work well with (fl6-arene)- tricarbonylchromium complexes. Grignard reagents, organocuprates, and alkylmercuric chlorides fail to react at low temperatures, while unidentified decomposition products are obtained upon heating. When strongly basic anions are used, such as methyl- or 18 R [01 /\ RCr(C0)3 0°C Cr(C0)3 where R = t(CH3)2CN, CH2N, tH2COR, CH(CH3)CO2R, C(CH3)20R, SPh CH s CN -dH-sph t—CH3, CH C—-- , -CH3 CH3 S Figure 2.3: Examples of carbanions which react with(6-benzene)tricarbonyl- chromium. butyllithium, proton abstraction results; this produces the lithiated,76-arene complex. However, this can be a useful result, as the lithiated species can then be treated with different electrophiles, thus providing an additional route for ring modifications.5 An important feature of(,76-arene)tricarbonylchromium chemistry has been the development of several methods for both complexing the chromium tricarbonyl group onto aromatic rings and being able to remove that same group efficiently when desired. A number of approaches have been developed to u--complex an arene with chromium tricarbonyl. Typically, chromium hexacarbonyl is either heated in neat arene, or with excess arene in a solvent (e.g., 1,4-dioxane, di-n-butyl ether), which affords the corresponding arene complex. After the desired chemistry has been accomplished, the final arene product can be readily recovered by mild oxidation. A variety of reagents have been successfully used for oxidative demetallation; aqueous Ce(IV), iodine, and exposure to sunlight and air, being the most common.5’6”4This is a vital consideration, if(6-arene)tricarbonylchromium complexes are to be useful in synthesis. The 19 introduction and subsequent removal of activating groups in organic chemistry presents difficult problems, not easily solved. Classical aromatic nucleophilic substitution suffers from this deficiency, as activating groups like nitro require drastic conditions for nitration, and lack mild and direct methods to remove the riitro group once its activating function is completed.’5 As a result, classical aromatic nucleophilic substitution has found limited application in complex organic synthesis.(6-Arene)tricarbonylchromium complexes potentially offer a significant improvement as synthetic intermediates since the chromium tricarbonyl moiety can be readily attached in most cases, then decomplexed in high yield once synthetic operations are done. The synthesis of radiopharmaceuticals labelled with short-lived positron emitting nucides requires special synthetic methods, due to the considerations presented earlier in Chapter 1. Prompted by the unique properties and reactivity of(76-arene)tricarbonyl- chromium complexes, we decided to explore the potential utility of these compounds for radiolabelling. There are about five reasons that would commend these organo chromium compounds for radiolabelling work. Firstly, most organic pharmaceuticals, either of potential interest for labelling or being currently used in nuclear medicine today, contain arene rings in their structures. Examples include 6-fluoro-3,4-dihydroxyphenylalanine (6-fluorodopa), the butyro phenone neuroleptics, the estradiol class of steroids, benzodiazepines, and benzamides such as Raclopride (S-(-)-3,5-dichloro-N-[( 1-ethyl-2-pyrrolidinyl)]methyl-2-hydroxy-6- methoxybenzamide).’6”7Therefore, the presence of an aromatic ring provides a possible labelling site for attaching the radionuclide of interest. Secondly, medically useful 20 radioisotopes are generally produced in anionic, nucleophilic form.18 Labelling reactions which can directly use the nucleophilic form of the radionucide are advantageous, because this eliminates additional chemical manipulations and time to modify the reagent form of the radionuclide. Thirdly,(i6-arene)tricarbonylchromium complexes facilitate nucleophffic substitution reactions on the aromatic ring. This established reactivity could allow the facile incorporation of nucleophilic radioisotopes onto aromatic rings. Fourthly, methods for complexing most arenes to the chromium tricarbonyl moiety, then later removing same, have been well established. This is in contrast to the use of traditional activating groups for nucleophilic aromatic substitution, such as nitro, which cannot be readily removed under mild conditions. Finally, the other modes of reactivity (see pp 1-2) exhibited by(,76-arene)tricarbonylchromium compounds, adds additional synthetic options when designing a radiopharmaceutical synthesis. In this chapter, the synthesis of a number of(ij6-arene)tricarbonylchromium complexes will be described. This will be followed by studies regarding the reactivity of the prepared chromium tricarbonyl compounds with cyanide. Lastly, the reactions of “C- labelled cyanide with some chromium tricarbonyl compounds will be presented. 2.2 Preparation of(q6-Arene)tricarbonylchromium Complexes 2.2.1 Synthesis The objective for this study was to prepare a range of simple (i6-arene)- tricarbonylchromium complexes in order to study their suitability for radiolabelling. To accomplish this, commercially available arenes were selected to produce the 21 corresponding chromium tricarbonyl complexes using literature methods. Five general methods have been developed for the synthesis of(i76-arene)tricarbonyl- chromium compounds. The first method, as outlined in equation 2.2, is the direct Arene + Cr(CO)6 snt (q6-Arene)Cr(CO)3 + 3 CO (2.2) reaction of arene with Cr(CO)6under thermolysis conditions. This reaction has been carried out with various solvents ranging from neat arene to non-polar (e.g., decalin) to polar (e.g., diglyme*) solvents. The reaction must be conducted at relatively high temperatures to achieve decarbonylation of the metal carbonyl and obtain ‘7r-complexed product. The majority of(i76-arene)tricarbonylchromium complexes have been prepared by this method. The second method involves a two-step process, in which during the first step Cr(CO)6has three carbon monoxide ligands replaced by more thermally labile ligands (L) such as pyridine, 4-methylpyridine, CH3N, or NH3,as shown in equation 2.3. 3 L + Cr(CO)6 heat Cr(CO)3L + 3 CO (step 1) (2.3) Arene + Cr(CO)3L heat (q6-Arene)Cr(CO)3 + 3 L (step 2) solvent In the next step, the prepared complex, Cr(CO)3Lis allowed to react with excess arene, using significantly lower temperatures, to obtain the desired product. The milder conditions of this methodology has allowed the preparation of chromium tricarbonyl complexes not obtainable by the first method described above. The third method is the *Formally named bis(2-methoxyethyl) ether. 22 direct reaction of arene with Cr(CO)6via photolysis. Irradiation of the reaction mixture at room temperature accomplishes the desired decarbonylation.(i6-Benzene)tr carbonyl- chromium and other complexes have been prepared using equation 2.4. The fourth Arene + Cr(CO)6 light (?76-Arene)Cr(C0)3 + 3 CO (2.4) solvent method is arene exchange, which relies on the observation that (fl6-arene)- tricarbonylchromium complexes undergo exchange reactions in the presence of another arene at elevated temperatures (ca. 200°C), as outlined in equation 2.5. The use of donor solvents has allowed these exchange reactions to be conducted at lower 6 heat( -AreneA)Cr(CO)3+ AreneB (q -AreneB)Cr(CO)3+ AreneA (2.5) temperatures (ca. 140°C). However, the synthetic utility of this method has been limited by the high temperatures required, and by the low yields obtained. The final method is the reaction of aikynes with chromium pentacarbonyl carbenes, as illustrated in equation 2.6. A chromium tricarbonyl complex is formed from the condensation of an (CO)5r R2 CR3 2R2 + CO (2.6) Cr(CO)3 Cr(CO)3 alkyne with a metal carbene. This novel method requires no arene starting material. A range of interesting 7r-complexed naphthalene and phenanthrene derivatives have been prepared by this route.’4’19 23 Given the various synthetic options available, it was desirable to choose the simplest, most direct synthetic procedure to prepare the desired(j6-arene)tricarbonylchromium complexes. The choice was made to prepare(,7-fluorobenzene)tricarbonylchroniium 1 by adapting the method of Mahaffy and Pauson,2°and to prepare(6-chlorobenzene)- tricarbonylchromium 2 by the method of Nicholls and Whiting.’° + Cr(CO) reflux /48 h + 3 CO (2.7) ‘ji 6 (n-Bu)2OiTHF Cr(CO)3 1 + Cr(CO)6 reflux /17 h + 3 CO (2.8)diglyme Cr(CO)3 2 Although(6-arene)tricarbonylchromium compounds are quite air-stable in solid form, their solutions are relatively air-sensitive. All preparations of these complexes, including workup and isolation, were conducted under inert atmosphere. Compound 1 was prepared in 68% yield according to equation 2.7 using an 8:1 mixture of di-n-butyl ether and tetrahydrofuran (THF). Mahaffy and Pauson reported a 90% yield for the synthesis of 1 under similar conditions.2°Alternatively, 2 was obtained in an isolated yield of 47% (equation 2.8), based on the amount of Cr(CO)6 consumed. This result is comparable to that of Nicholls and Whiting who reported a yield of 52%, after a reaction time of 3 h.’° When examining these initial experimental results, it became clear that working with 24 diglyme was less desirable, since it was very difficult to remove during workup. The butyl ether/THF mixture was readily removed to dryness, making workup much easier. More importantly, the method of Mahaffy and Pauson exhibited a potential for significantly better yields of chromium complexed product. As a consequence, it was decided to use the Mahaffy and Pauson reaction conditions for the preparation of other (6-arene)fficarbonylchromium compounds. A number of chromium tricarbonyl complexes were successfully synthesized according to the general equation 2.9. The results obtained are summarized in Table 2.1. With reflux +Arene + Cr(CO)6 (n-Bu)20/THF Complex + 3 CO j (2.9) additional work and experience, the preparative yield of 1 was improved to 80% (see Table 2.1). The reaction (equation 2.9) was found to be very sensitive to any traces of oxygen present in the reaction mixture. Regardless of how carefully the reaction apparatus, reagents, and solvents were handled under inert atmosphere, it was vital to employ freeze-pump-thaw degassing as a final step to ensure an oxygen-free environment for the reaction. The major drawback of the Mahaffy and Pauson procedure was the slowness of the reaction, thus requiring prolonged heating of the reactants (1-2 days) which can cause some decomposition of product complex. Unfortunately, this decomposition could result in further autocatalytic decomposition of any chromium tricarbonyl product formed. Some investigators have recently reported that the prime reason for decomposition of(76-arene)tricarbonylchromium during the reaction (equation 2.9) was due to the presence of impurities in the starting materials and 25 Table 2.1: Summary of Yields Obtained for Complexes Synthesized using Equation 2.9 Arene Complex Reaction I Yield Time C—F ‘ 48h 80%Cr(CO)3 1 Br CBr I 44h 19%Cr(CO)3 3 MeMe ,—F 48h 89%Cr(CO)3 4 Me Me F 16.5h 85%Cr(CO)3 5 Cl CVCF 23h 12%Cr(CO)3 6 Me0?— /MeO N I 21h 70% e Cr(CO)3 7 26 solvents used, and traces of atmospheric oxygen over the reaction mixture.21 A common problem with the direct reaction of arenes with Cr(CO)6 is the volatility of Cr(CO)6 itself, which results in sublimation of the metal carbonyl from the reaction mixture. Anticipating this problem, a special reaction apparatus was constructed* that allowed any sublimed Cr(CO)6 to be mechanically returned to the reaction mixture. However, it was found that the use of di-n-butyl ether with 10-20% of THF present, effectively washed back sublimed Cr(CO)6 to the reaction mixture. A regular Liebig condenser could then be used with conventional glassware to perform the synthetic reactions (see Experimental for details). For our purposes, a paradoxical limitation of the Mahaffy and Pauson procedure, is that while electron-donating groups on the arene helps the reaction, electron- withdrawing groups slow the reaction.22 It was found that fluorobenzene, 2-fluoro- toluene, and 4-fluorotoluene were successfully complexed, producing 1, 4, and 5 in high yield (80-89%). However, the corresponding complex of 4-chlorofluorobenzene (6) was made in only 12% yield. Persistent decomposition accompanied the preparation of 6, thereby limiting the reflux time possible—allowing the reaction to proceed longer than overnight results in progressive decomposition and in little or no product formed. In addition, during the synthesis of 6, a significant amount of 1 was obtained as a byproducttwhich had to be separated using column chromatography. *The design of this reaction vessel was obtained from Dr. Peter Legzdins of the Chemistry Department, U.B.C., and is described in the Experimental. 1This would appear to result from a reductive dehalogenation process in which the fluorobenzene complex 1 is formed from 4-chlorofluorobemzene, probably via some 27 In addition to 6, the preparations of(i76-l,4-difluorobenzene)tricarbonylchromium 8 and(,7-4-fluoroanisole)tricarbonylchromium 9 were attempted. In these two cases, only very small quantities (tens of milligrams) were obtained at best. Extensive F—’—F MeO—F Cr(CO)3 Cr(CO)3 8 9 decomposition also accompanied these reactions. As a consequence of these disappointing results, other synthetic approaches were examined that would be more suitable for complexing arenes with electron-withdrawing substituents. Limited trials of other synthetic methods also did not yield satisfactory results. It became clear that a sustained study would be required to resolve this problem and this was beyond the scope of time available for this work. Therefore, the chromium tricarbonyl complexes that could be prepared in adequate quantity were settled upon for the desired radiolabelling studies. The direct reaction of benzonitrile with Cr(CO)6 does not yield the corresponding chromium tricarbonyl complex.’°’ However, Mahaffy and Pauson were able to synthesize(6-bemzonitrile)tricarbonylchromium 10 indirectly by allowing the fluorobenzene complex 1 to react with a large excess of cyanide overnight in acetonitrile at 50°C. They obtained a 44% conversion of 10 from starting 1. Semmeihack has also chromium species in solution. Similar results have been reported by Hudeèek and Toma.21 28 reported, in a 1976 review, the successful reaction of cyanide anion with 1 at 25°C, obtaining a 94% yield.5 Unfortunately, the experimental details of this work have never been published to the best of this author’s knowledge. We were able to prepare 10 in 69% yield using different conditions, as outlined in equation 2.10. 25°C123h I + NaCN I + NaF (2.10) Cr(CO)3 DMSO Cr(CO)3 1 10 Of additional interest was the desire to prepare chromium tricarbonyl complexes in which the attached arene possessed groups of greater mobility toward aromatic nucleophilic substitution than halogen. Prompted by reports of radiolabelling studies using aromatics with dimethylsulfonium and trimethylammonium leaving groups,25’6 it was considered whether an analogous chromium tricarbonyl complex could be made. Bunnet and Herman successfully methylated(6-thioanisole)tricarbonylchromium with trimethyloxonium tetrafluoroborate to obtain -phenyldimethy1sulfonium)tricarbonyl- chromium tetrafluoroborate in 89% yield.27 However, they found that (6-N,N- dimethylaniline)tricarbonylchromium 7 could not be methylated by trimethyloxonium tetrafluoroborate.27 In our efforts, we found that complex 7 can be methylated using methyltrifluoromethanesulfonate (commonly called methyl triflate) according to equation 2.11.(,6-N,N,N-Trimethylanilinium)tricarbonylchromium trifluoromethanesulfonate 11 was produced in 59% yield. In order to compare the behaviour of this new complex 11 with the corresponding uncomplexed arene, N,N,N-trimethylanilinium trifluoromethane 29 /Me + + 25°C/48 h CF3S0 (2.11)MelCH3SOF CH21 Cr(CO)3 JCr(CO)37 L sulfonate 12 and N,N,N-trimethylanilinium iodide 13 were also synthesized as shown in equation 2.12. Compound 12 was prepared in 83% yield, while 13 was obtained in 56% yield. Me +CH3SOF/C21 25°C/30 mm [N_Me CF3SO Me 12 /Me (2.12) CH3I/CH21 25°C/24 h Me 13 2.2.2 Characterization All of the(j6-arene)tricarbony1chromium complexes, except for 11, are known compounds. Their melting points were recorded and are presented in Table 2.2. The chromium tricarbonyl complexes tended to decompose to some degree during melting point determinations. The degree of decomposition would vary with the heating rate thereby making the melting points difficult to determine in some cases. 30 Table 2.2: Summary of Melting Points of Organochromium Complexes Complex Observed mp Literature mp Reference (°C) (°C) 1 117 116-117 27 2 100-101 101-102 20 3 101-105 120 28 4 71-72 73-74 29 5 59-60 6 1-62 29 6 61-62 — none found 7 137- 138 144 20 11 120-121 — none found The identification of the chromium tricarbonyl complexes was accomplished by mass spectrometry (MS). The principal fragmentation patterns of simple (6-arene)- tricarbonylchromium compounds using electron impact MS have been well established and are shown in Figure 2.4.°’’ The fragmentation patterns exhibited by the synthesized chromium tricarbonyl complexes were consistent with the scheme depicted in Figure 2.4. Also, Muller and Göser reported that all(6-halobenzene)tricarbony1- chromium compounds gave the Cr-halogen ion on fragmentation (i.e., [(C6H5X) rj —* [CrX] + + C6H5 )? This ion was observed for each of the chromium tricarbonyl complexes with halogen-substituted aromatic rings. These characteristic features of the mass spectra, exhibited by the chromium tricarbonyl compounds, made product identification rapid and straight forward. Complex 11 was previously an unknown compound and was characterized by elemental analysis and 1H NMR spectroscopy. Compounds 12 and 13 were identified 31 [(Arene)Cr(CO)3] [(Arene)Cr(CO)2f + -Cr(CO) normal[(Arene)Cr(CO)] [Arenef’ Arene fragmentation -Cr normal[(Arene)Cr]+ [Arene] Arene fragmentation -Arene [Cr] Figure 2.4: Principal fragmentation pattern of(6-arene)tricarbonylchromium compounds. by 1H NMR spectroscopy. MS could not be employed for analysis of these salt complexes (11, 12, 13). A satisfactory elemental analysis for C, H, N, and S was obtained for 11. The 1H NMR spectrum of 11 exhibited the aromatic proton resonances at 5.77 (H-3,5), 6.07 (H-4), 6.69 (H-2,6) ppm, and a singlet for the trimethylammonium group protons at 3.56 ppm. The 1H NMR spectral data for 12 and 13 were found to be 32 the same; the aromatic protons were observed at 7.58-7.68 (H-3,4,5) and 7.97 (H-2,6) ppm, and the trimethylammoriium group protons at 3.61 ppm. These results showed the expected upfield shifts in proton resonances due to complexation with chromium tricarbonyl. This is a characteristic feature of the ‘H NMR spectra of chromium tricarbonyl complexes when compared to the uncomplexed arene.4 2.3 Reactions of Organochromium Compounds with Cyanide These studies began with investigating the reactivity of cyanide with(6-halobemzene)- tricarbonylchromium complexes 1, 2, and 3. All substitution reactions were performed I I I Cr(CO)3 Cr(CO)3 Cr(CO)3 1 2 3 under an argon or nitrogen atmosphere, using dried solvents. Complex 1 was allowed to react with excess sodium cyanide (ca. 10 equiv) in DMSO at 1500C* for 30 minutes. During heating, the initial yellow colour of the reaction mixture became dark red. After cooling, the reaction mixture was diluted with water, extracted with diethyl ether, then dried. The dried ether extracts were colourless. The ether extracts were concentrated and analyzed by gas chromatography (GC). GC analysis confirmed the presence of benzonitrile 14 as the only product obtained. This reaction was repeated under slightly different conditions, where complex 1 was treated with about 2 equivalents of sodium *Reaction temperatures always refer to the oil bath temperature used. 33 cyanide at 155°C in DMSO for 20 minutes. After workup, GC analysis of the ether extracts exhibited two prominent peaks, a major peak due to 14 and a minor peak due to fluorobenzene. For comparison, uncomplexed fluorobeuzene was allowed to react with sodium cyanide under the same conditions. GC analysis showed that only unreacted fluorobenzene was present. These initial results clearly demonstrated that the fluorobenzene complex 1 underwent successful aromatic nucleophilic substitution with cyanide and that the reaction was rapid under the conditions employed. The control experiment with uncomplexed fluoro benzene confirmed that free fluoroberizene failed to react with cyanide, and that intact chromium tricarbonyl species underwent the substitution reaction. The reactivity of the chlorobemzene complex 2 was examined next. Complex 2 was treated with sodium cyanide (ca. 0.5 equiv) in DMSO at 160°C for 15 minutes. After workup, the ether extracts were yellow in colour. GC analysis showed only the presence of chlorobenzene. The ether extracts were subsequently treated with iodine to decomplex any intact chromium tricarbonyl components, and the GC analysis was repeated. Chlorobenzene remained the only compound detected. For comparison, the reaction of 1 with sodium cyanide was repeated under the same conditions as used for 2. GC analysis showed two significant peaks, one peak due to 14 and the other due to fluorobenzene. In like manner, the ether extracts were again treated with iodine to decomplex any chromium tricarbonyl components present. GC analysis gave the same results as observed prior to oxidative decomplexation. Unfortunately, these results showed that 2 is either unreactive toward cyanide or is 34 possibly undergoing significant decomplexation under the reaction conditions used. However, 1 readily underwent substitution with cyanide using the analogous conditions employed for the reaction trial with 2. Though disappointing, this observation was complimentary to previous studies that have shown that fluorine is more easily displaced during aromatic nucleophilic substitution than chlorine.4 Therefore, these results suggested that chlorine is an inappropriate leaving group to use in future studies, and was thus abandoned. Lastly, the potential of bromine as a leaving group was investigated using the bromobenzene complex 3. Complex 3 was allowed to react with 0.5 equivalents of potassium cyanide in DMSO at 135°C for 10 minutes. After cooling, high pressure liquid chromatography (HPLC) was employed to directly examine the reaction mixture. HPLC analysis confirmed the absence of the desired product 14. In contrast, 1 was treated with cyanide under identical conditions, and benzonitrile 14 was produced. HPLC analysis not only confirmed the presence of 14, but the yield was also determined—these results will be presented and discussed later in this section. These results also indicated that 3 is either unreactive toward cyanide or is possibly decomplexing under the reaction conditions employed. Bromine, as a consequence, would also appear to be an unsuitable leaving group for aromatic nucleophilic substitution. Therefore, the only useful halogen leaving group for reactions with cyanide was determined to be fluorine. In order to further characterize the benzonitrile product 14, obtained from the reaction of 1 with cyanide, isolated products from several small scale reactions were 35 combined. This combined product sample (dissolved in diethyl ether) was subjected to GC analysis, and only one component was observed with almost 99% purity. The ether was evaporated and the product sample was dissolved in deuterated chloroform for NMR spectroscopy. The room temperature 1H NMR spectrum was recorded at 80 MHz.* In addition, the 1H NMR spectrum of authentic 14 (commercially obtained) was recorded under the same conditions. The spectrum of the product sample exhibited a multiplet centered at 7.57 ppm that was the same as that observed for authentic 14. Therefore, the 1H NMR spectrum clearly identified the product sample as benzonitrile 14. This is in addition to GC and HPLC studies which readily identified 14 by comparison of its retention time with that of authentic standard. Also, during HPLC studies, an aliquot of reaction mixture was taken and standard 14 was added to see if the assigned product peak would correspondingly increase in size. This was observed, and deemed as further evidence that the chromatographic assignment was correct. Furthermore, it was noted during the workup of the reactions of 1 with cyanide that the characteristic odour of 14 was present. A number of general observations were made during the early studies regarding the reaction of 1 with cyanide. Reactions employing approximately 2 or more equivalents of cyanide for 15-30 minutes exhibited essentially a quantitative conversion of 1 to 14, as determined by GC analysis. The temperatures used for these reactions were 150- 155°C. Additional experiments were performed in which the reaction mixtures were *These spectra were obtained on a departmental Bruker WP-80 spectrometer with tetramethylsilane used as an external standard. 36 heated for as little as one to three minutes and 14 was still produced, as observed by GC and HPLC analyses. Other reactions conducted used a large molar excess (up to 17- fold) of complex 1 relative to cyanide. Again, benzonitrile 14 was obtained, according to GC and HPLC analyses. Lastly, decomplexation of the aromatic nitrile takes place during the reaction under the conditions employed. This was demonstrated by examining a product mixture by GC (after workup) which showed no change in benzonitrile 14 concentration after treatment with iodine. Subsequently, this observation was further confirmed with additional experimental experience, which shall be presented at later stages of this discussion. As a consequence, no separate oxidative decomplexation step was needed to liberate the organic product. This result was not too surprising as it has been reported that(t6-arene)tricarbonylchromium complexes can undergo displacement of the aromatic ring in donor solvents, such as pyridine, at elevated temperatures.32 These results further indicated just how facile and rapid the substitution reaction of 1 with cyanide actually is. Since the intent was to ultimately apply this chemistry for the incorporation of[11C]cyanide into arenes, the studies to follow were designed to model this application. With excess cyanide and longer reaction times, excellent reaction yields could be assured. The half-life of 11C, however, is 20.4 minutes, which places a premium on using the shortest reaction time possible. Also, it is highly desirable that the reaction be eventually compatible with high specific activity [“Cjcyanide, for the reasons discussed earlier in Chapter 1. Therefore, the decision was made to limit subsequent cyanide substitution reactions to 10 minutes and to use cyanide as the limiting reagent 37 (0.5 equiv). Within these parameters, the reactivity of the chromium tricarbonyl complexes 1, 4, 5, and 6 was examined. The general procedure used is as follows. An aqueous solution Me—-—F Cl -—F Cr(CO)3 Cr(CO)3 Cr(CO)3 Cr(CO)3 1 4 5 6 of cyanide, containing a known amount of KCN, was dispensed into a reaction vessel, and was carefully dried under a fast flow of inert gas. (This was done to model the handling necessary in using[‘1C]cyanide, which is obtained as an aqueous solution after cyclotron production.) The(6-arene)tricarbonylchromium complex, dissolved in 1 mL of DMSO, was added to the dried cyanide. The mixture was heated for 10 minutes. Upon cooling, the reaction mixture was quantitatively transferred to a volumetric flask and diluted to a known volume with DMSO. This solution was analyzed by HPLC. The extent of product formation was determined using a calibration curve constructed using HPLC absorbance values of standard solutions of expected aryl nitrile product. The chemical yields were calculated using KCN as the limiting reagent. In order to optimize the results obtained for each of the complexes studied, the reaction temperatures were varied and the corresponding chemical yields were determined. Complex 1 was treated with cyanide, as shown in equation 2.13, using reaction 0.5 equiv KCN2 Cr(CO)3 DMSO /10 • K—CN + KF + 1 (2.13) 38 temperatures ranging from 105-150°C. The results obtained are summarized in Table 2.3. The optimum temperature found for equation 2.13 was 135°C, giving a best yield of 41%. A representative HPLC chromatogram is shown in Figure 2.5. The chromato gram shows the presence of the desired product 14 (peak A), along with residual starting complex 1 (peak C) and free fluorobenzene (peak B) which is visible only as a shoulder of an unidentified peak. Injection of a solution, consisting of fluorobemzene standard added to an aliquot of reaction mixture, exhibited an increase of this shoulder (peak B) into a distinct peak at the same retention time, thereby confirming the assignment of peak B. Another control experiment was performed with uncomplexed fluorobenzene using 0.5 equivalents of cyanide at 135°C. Once again, only unreacted fluoroberizene was observed in the HPLC chromatogram. Table 2.3: Chemical Yields Obtained for Complex 1 Temperature Yield no. of Average (°C) runs Yield 150 23-33% 2 28% 135 40-41% 2 41% 120 31% 1 31% 115 32% 1 32% 105 12% 1 12% Next, the reactions of complexes 4, 5, and 6 with cyanide were investigated. The reactivity of 4 (equation 2.14) was examined over a temperature range of 95-135°C. The results obtained are shown in Table 2.4. The best yields (41-43%) were observed at 105- 115°C. The control reaction done with 2-fluorotoluene (uncomplexed) using 0.5 equiva 39 z C I B TIME (mm) The compounds are: (A, 5.4 mm), 14; (B, 10.5 mm), fluorobenzene; (C, 16.8 mm), 1. The HPLC conditions are: Waters 10 JLm C-18 reverse phase RCM column; eluent: isocratic methanol/water, 1:1; flow rate, 2.5 mL/min; UV detection set at 280 nm. Figure 2.5: HPLC chromatogram obtained from the analysis of the reaction of complex 1 with cyanide at 135°C. The yield of 14 was determined to be 40%. 40 2i Cr(CO)3 4 0.5 equiv KCN DMSO/lOmin ,Me CN + KF + 4 15 (2.14) lents of cyanide at 135°C gave no reaction; unreacted 2-fluorotoluene was the only compound observed in the HPLC chromatogram. Two reaction trials (equation 2.14) were performed using only 1.5 mg of 4, this being about a tenth of the usual quantity of complex used per reaction trial. This results in a five-fold excess of cyanide being Table 2.4: Chemical Yields Obtained for Complex 4 Temperature Yield no. of Average (°C) runs Yield 135 29-36% 4 32% 125 28-29% 2 28.5% 115 41% 1 41% 105 41-43% 2 42% 95 26% 1 26% 135k 58% 1 58% 143& 58% 1 58% aOnly a tenth of the usual quantity of 4 was used, giving a stoichiometric ratio of 5:1, of KCN to 4. present in these reactions. Yields of 58% were obtained at both 135 and 143°C. These results were significantly better than those obtained using 0.5 equivalents of cyanide (see Table 2.4). The reaction of 5 with cyanide, as summarized in equation 2.15, was studied over the temperature range of 105-150°C. The chemical yields obtained are outlined in Table 41 Cr(CO)3 5 0.5 equiv KCN DMSO/ 10mm - Me<)—CN + KF + 5 (2.15) 2.5. The best yields (26-29%) were obtained at 115°C, while the next best results (22- 26%) were seen at 135°C. The control experiment performed using uncomplexed 4- Table 2.5: Chemical Yields Obtained for Complex 5 Temperature Yield no. of Average (°C) runs Yield 150 21% 1 21% 135 22-26% 3 24% 125 21% 1 21% 115 26-29% 2 27.5% 105 11% 1 11% fluorotoluene (0.5 equiv of KCN; 135°C) exhibited no reaction. HPLC analysis showed only the presence of unreacted 4-fluorotoluene. The reaction of 6 with cyanide (equation 2.16) was examined at 115 and 135°C. These results are summarized in Table 2.6. The best yields (26-34%) were obtained at Cl -—F Cr(CO)3 6 0.5 equiv KCN DMSO/l0min Cl + KF + 6 (2.16) the reaction temperature of 115°C. The control reaction done with 4-chiorofluoro- benzene (uncomplexed) using 0.5 equivalents of cyanide at 135°C exhibited no reaction; unreacted 4-chlorofluorobenzene was the only compound observed in the HPLC chroma togram. One reaction trial (equation 2.16) was heated (115°C) for five minutes, and gave 42 Table 2.6: Chemical Yields Obtained for Complex 6 Temperature Yield no. of Average (°C) runs Yield 135 21% 1 21% 115 26-34% 3 30% 115k 17% 1 17% a reaction time of 5 mm was used. only about half the yield observed for a 10 minute reaction time (see Table 2.6). This indicates that reaction times shorter than 10 minutes would result in a significant sacrifice in chemical yield. In the process of examining the chemical yields obtained for complexes 1, 4, 5, and 6, it would be interesting to discover any trends or systematic patterns of chemical behaviour. However, inspecting the results presented in Tables 2.3-2.6 indicates that there are no such patterns observable. What emerges is that each chromium tricarbonyl complex studied exhibits its own distinct pattern of reaction yields. The mechanism for nucleophiic substitution reactions of(,76-arene)tricarbonylchromium systems is thought to proceed by a two-step mechanism (equation 2.17),’ analogous to classical aromatic “Co Co +Y step 1 KX Cr, OCj “CO CO -x step 2 Cr,, OCj “CO CO (2.17) 43 nucleophilic substitution.*,34 The first step is addition of the nucleophile (Y) onto the aromatic ring, on the side opposite the chromium tricarbonyl moiety—this results in the exo-substituted, anionic5-cyclobexadienyl complex. The second step is expulsion of the halide leaving group (X), giving the final substitution product. If this mechanism is valid, it would be anticipated, from comparison to classical aromatic nucleophilic substitution, that electron-withdrawing groups would make the substitution reaction with cyanide more facile, while electron-donating groups would hinder same.35 Using the fluorobenzene complex 1 as the baseline standard, it may be expected that the presence of the additional methyl group in 4 would hinder the reaction with cyanide, relative to 1, and may lead to lower substitution yields during the short reaction time used. The best average yield for 1 was 41% obtained at 135°C, while 4 exhibited its best average yield of 42% at 105°C. As a result, 4 essentially equalled the best yield obtained by 1 at 30 degrees lower temperature. On the other hand, the results obtained by 5 were much more surprising. Since 4 and 5 are simply ortho- and para-isomers, respectively, similar chemical yields with cyanide might be anticipated for both complexes. However, 5 gave unexpectedly low yields, with a best average yield of 27.5% obtained at 115°C. In fact, the chemical yields obtained by 4 readily surpassed those of 5 at all temperatures investigated (see Tables 2.4 and 2.5). Complex 6, which bears an extra chlorine substituent (relative to 1), would be expected to produce the highest chemical yields with cyanide—yet this was not observed. A best average yield of 30% was exhibited at 115 °C. tThe mechanism being specifically referred to here is addition-elimination, also called SNAr by March. 44 Unfortunately, circumstances did not permit the opportunity to conduct reactions at temperatures below 115°C. It is possible that higher chemical yields could be obtained for 6 at lower temperatures. Nonetheless, equal or better yields were obtained from 1 and 4 at both 115 and 135°C. These results may be explained in part by the relative thermal stability of the complexes. Complex 6 was clearly more prone to decomposition in solution; this was particularly evident during its synthesis (see pages 27-28). Due to the presence of the chlorine substituent, in addition to the fluorine, there would be less electron density available on the aromatic ring to complex with the chromium tricarbonyl moiety, and the resulting complex would be anticipated to be less thermally stable relative to 1. Therefore, the lower chemical yield for 6 at 135°C, compared to that of 1, could stem from greater thermal breakdown of 6 at that temperature. The yields resulting from 1 and 6 at 115°C are quite comparable. As a result, 1 exhibits better yields at 135°C than 6 simply because it is likely the more thermally robust complex, and not from any greater intrinsic reactivity. On the other hand, both 4 and 5 would be expected to be somewhat more thermally stable than 1. Complexes 4 and 5 were both prepared in higher yield than 1, and were found to be as well behaved during storage and handling. However, as presented earlier, 4 exhibited better yields at lower temperatures as compared to 1, but 5 exhibited significantly lower chemical yields at all reaction temperatures employed when compared to those of 4. Since it would be reasonable to assume that both 4 and 5 would be of equivalent thermal stability, the reason for this marked difference in chemical yields obtained by 4 and 5 is unknown. For each 45 chromium tricarbonyl complex studied, the chemical yields obtained were found to be quite sensitive to reaction temperature, and hence must be optimized for each complex individually to achieve the best results possible. In order to improve upon the chemical yields obtained thus far, it was decided to investigate the use of crown ethers. The presence of crown ethers with many ionic reagents have shown increased solubility and anion reactivity in aprotic organic solvents. Therefore, by employing a crown ether with potassium cyanide, it would be anticipated that the nucleophilicity of the cyanide anion would be enhanced.36’7 Previously, 18- crown-6 (1,4,7,1O,13,16-hexaoxacyclooctadecane) has been successfully used to help convert benzyl halides38 and alkyl halides39 to their corresponding nitriles in high yield. As a result, 18-crown-6 was chosen as the crown ether to use, to examine its potential benefit on the reaction of 4 with cyanide, as shown in equation 2.18. The same general 0.5 eqv KCN2 Cr(CO)3 18-Crown-6 / 10 mm + KF + 4 (2.18) procedure was followed as described earlier, except for the addition of approximately 1.2 equivalents of 18-crown-6 to the reaction mixture prior to heating. The reactions were conducted over a temperature range of 95-135°C and the chemical yields were determined by HPLC analysis as before. The results obtained are summarized in Table 2.7. The best yields (40-44%) were now observed to occur throughout the temperature range of 105-135°C. These results did not surpass the previous best yields of 41-43%, obtained at 105-115°C, in the absence of 18-crown-6. When the reaction was repeated 46 at 105°C using approximately three equivalents of 18-crown-6, a slightly improved yield of 46% was obtained. However, the use of 18-crown-6 did significantly improve upon earlier reaction yields that were obtained at 95, 125, and 135°C (see Table 2.4)—increases of about 30-50% over earlier average chemical yields were exhibited. Table 2.7: Chemical Yields Obtained for Complex 4 using 18-Crown-6 Temperature Solvent Yield no. of Average (°C) runs Yield 135 DMSO 41% 1 41% 125 p 42% 1 42% 115 40% 1 40% 105 40-44% 2 42% 105a 46% 1 46% 95 35% 1 35% 80 CH3N 3% 1 3% 95b 4% 1 4% aAbout 3 equiv of 18-crown-6 was used. bAt this oil bath temperature, the CH3N was observed to be refluxing. In addition to these results, a couple of reaction trials (equation 2.18) were conducted using acetonitrile as the solvent with one equivalent of 18-crown-6 added; these were done using oil bath temperatures of 80 and 95°C. As shown in Table 2.7, very poor yields were obtained. After chromatographic (HPLC) analysis, these reaction mixtures were simply set aside without any further manipulations. The following day these mixtures were reexamined by HPLC, which showed a significant increase in the concentration of 2-tolunitrile 15 (see Table 2.8). These reaction mixtures were again set 47 aside for about two weeks. Then HPLC analysis was performed again and a further increase in the concentration of 15 was observed (see Table 2.8). Initially, the results obtained using CH3N as the reaction solvent looked wholly unimpressive, but unexpectedly, good yields of 15 were produced with the passage of time. Experience with Table 2.8: Chemical Yields Obtained for Equation 2.18 using Acetonitrile as the Solvent Temperature Yield Time elapsed after (°C) initial HPLC analysis 80 3% 0 80 21% --18h 80 50% —13.5 d 95 4% 0 95 21% --16h 95 46% —13.5 d DMSO as the reaction solvent has shown that after the reaction has been performed and the reaction mixture analyzed by HPLC, no further changes in nitrile product concentration was observed with subsequent reanalyses. Due to the elevated temperatures used for the reactions (equation 2.18) done in CH3N, it was expected that the chemical yields, determined initially, represented the total nitrile product formed (and decomplexed) during the 10 minute reaction time. What is not clear is whether the improved yields shown in Table 2.8 were due to the reaction continuing to occur at a slower rate at room temperature (during storage), or to the fact that the initially formed (6-2-to1unitrile)tricarbony1chromium species only decomplexed to a small extent at first, 48 then continued to slowly decomplex while being stored.* Unfortunately, this work was not followed up at the time. The preliminary results obtained using 18-crown-6 shows promise for the use of crown ethers in the substitution reactions of chromium tricarbonyl complexes. In fact, other crown ethers and cryptands are available for potential use.36’4° It is anticipated that some experimentation would be needed to find the optimum macrocycle to function in these substitution reactions with cyanide. The identification of the aryl nitrile products formed from the reactions of complexes 4,5, and 6 with cyanide was accomplished using chromatographic (HPLC) studies. Each of the nitrile products (2-tolunitrile 15, 4-tolunitrile 16, 4-chlorobenzonitrile 17) were readily identified by comparison of their respective retention times with that of authentic standard. To further confirm the identity of the nitrile products 15, 16, and 17, a separate set of reaction trials was conducted to isolate the organic products and analyze these by gas chromatography-mass spectrometry (GC-MS). These reactions were performed as described previously in the general procedure used for the earlier cyanide reactions (see page 38). Upon cooling, however, the reaction mixtures were diluted with water, then extracted with diethyl ether. The ether extracts were cooled to 0°C, then treated with iodine for two hours to oxidatively decomplex any chromium tricarbonyl species present. The treatment was quenched with the addition of aqueous sodium thiosulfate solution. The ether layer was further washed (aqueous Na2SO3and saturated *The reaction mixtures were kept in small, stoppered volumetric flasks, but these mixtures had been exposed to air during HPLC work. As a result, any chromium tricarbonyl species present could gradually undergo oxidative decomposition. 49 NaC1 solutions), then dried. The ether solution (concentrated to 1 mL) was analyzed first by GC and HPLC, then by GC-MS. Mixtures of authentic standards were prepared from the uncomplexed starting arene and corresponding aryl nitrile product (dissolved in ether) and were also analyzed by GC-MS for direct comparison to the reaction products obtained above. Comparison of the mass spectra obtained from the reaction products with those of the authentic standards confirmed the identity of the starting fluoroaromatics (2-fluoro- toluene, 4-fluorotoluene, 4-chlorofluoroben.zene) and the resulting nitrile products (15, 16, 17). However, the reaction mixture containing 4-chlorofluorobenzene and 17 also contained a third minor product which was identified as benzonitrile 14 by its mass spectrum. The formation of 14 as a side-product from the reaction of 6 with cyanide was due to the presence of a small quantity of 1 contaminating the starting complex 6. Unfortunately, the chromatographic purification of 6, during its original preparation, did not completely remove 1 as an impurity. As a consequence, 1 also underwent substitution with cyanide as a side-reaction, affording benzonitrile 14. In addition, the chemical yields for this set of reaction trials were estimated from the GC analyses using the standard mixtures for calibration. These results are shown in Table 2.9. The key feature of these results is that none of the yields surpassed the values reported earlier, which were determined without subjecting the reaction mixtures to oxidative decomplexation. This further establishes that all the aryl nitrile formed during the substitution reactions becomes decomplexed under the reaction conditions 50 Table 2.9: Summary of Chemical Yields Starting Nitrile Temperature Yield Complex Product (°C) 4 15 120 —32% 5 16 135 —26% 6 17 115 —24% used. Other leaving groups apart from halogen have been successfully used in classical aromatic nucleophilic substitution.41 Hence, it was of additional interest to our study to examine other leaving groups that could possess greater mobility toward nucleophilic substitution for(i6-arene)tricarbony1chromium systems. An obvious choice would be to examine nitro as a leaving group. Unfortunately, the attempts made to prepare (6- nitrobenzene)tricarbonylchronfiumwere unsuccessful. Chromium tricarbonyl complexes with arene rings bearing a nitro substituent have been unknown until recently.”42 However, prompted by radiolabelling studies using aromatics with dimethylsulfonium24 and trimethylammonium’26leaving groups for nucleophilic substitution reactions, we found that we were able to synthesize(,76-N,N,N-trimethylanilinium)tricarbonylchromium trifluoromethanesulfonate 11. Preliminary experiments were conducted in which complex 11 was allowed to react with cyanide as shown in equation 2.19. The same general procedure was used, as previously described for the earlier reactions (see page ‘The successful synthesis of(6-2,4,6-trinitrotoluene)tricarbonylchromium, using(CH3CN)r(CO) as the precursor for complexing the arene, has been recently reported. This represents the first chromium tricarbonyl complex of a nitroaromatic. 51 +CF3S0 No Reaction (2.19) L Cr(CO)3 ]11 38). The reactions were done at 100 and 120°C. Disappointingly, HPLC analysis showed the absence of the desired product 14. The HPLC chromatograms of the reaction mixtures exhibited two new prominent peaks, a large peak and a much smaller peak with a longer retention time; these peaks could not be identified. Complex 11 was then heated in DMSO for 10 minutes at 100°C. After cooling, the HPLC chromatogram of this solution showed the presence of a single new peak. The retention time of this peak was very close to that of the large peak observed from the reactions above. These results seem to suggest that 11 is undergoing some kind of transformation or decomposition from the heating in solution. For comparison, a trial reaction was done with uncomplexed N,N,N-trimethylanilinium trifluoromethanesulfonate 12 and cyanide at 100°C (equation 2.20). No reaction was observed, as evidenced by HPLC analysis. [KMe CF3SO No Reaction (220) Unfortunately, the preliminary reactions conducted with 11 failed to produce any 14. These results suggested some chemical breakdown of complex 11 while being heated. Additional time to study this problem, and the potential reactivity of 11, was simply unavailable. 52 2.4 Labelling Studies with‘1C-Labelled Cyanide In this section, the substitution reactions of complexes 1, 4, 5, and 6 with radioactive [11C]cyanide will be described. Due to the short half-life of ‘1C (20.4 mm), this radionuclide must be produced at or very close to the site where the radiolabelling chemistry is to be performed. Fortunately, 11C is produced regularly at the TRIUMF facility for the ongoing PET program at U.B.C. The small TRIUMF/Nordion CP-42 cyclotron is used to generate positron emitting nucides for PET, as well as other radioisotopes for commercial sale. Carbon-li was produced as 11C02 by proton irradiation of N2 gas via the nuclear reaction 14N(p,a)1Cat 15 MeV. The[11C]cyanide was produced by sequential catalytic conversion of 11C02 according to equation 2.21. l)H2 l)NH3 HCO CH HCN (2.21)2)Nicatalyst/450°C 2)PtIl000 C The H”CN was trapped in an aqueous solution of NaOH (0.1 M) to generate Na11CN for labelling use. Although the maximum specific activity of “C is 9.22 x 106 Ci/mmol, the ubiquitous presence of ‘2C in nature invariably dilutes the specific activity of any “C reagent to some value less than the maximum possible specific activity. The amount of dilution of “C by 12C can vary widely depending on the production conditions used. Specific activity values for H11CN that can be practically obtained are in the order of 2 x i0 Ci/mmol.43 Unfortunately, the specific activity of the H11CN produced at TRIUMF has not been determined, but the specific activity value is thought to be no lower than 0.5 Ci/mmol. Therefore, the specific activity of the [‘1C]cyanide used for this work 53 can possibly range somewhere between 0.5-2000 Ci/mmol (most likely in the lower values of this range). Since the quantity of actual “C-labelled product is so small,* standard chemical and spectroscopic methods of characterization, such as ‘H and “C NMR, cannot be used for direct product identification. Therefore, chromatographic methods (e.g., HPLC and GC), using non-radioactive analogues as standards, provides the best means of product identification available. The best suited chromatographic method for this purpose is HPLC.4’ For this work, product identification and analysis was performed with HPLC instrumentation that was equipped with both a UV detector and a radioactivity detector connected in series. The radiolabeffing studies began with investigating the reactivity of complex 1 with [“C]cyanide using different amounts of added KCN (i.e., carrier). The general procedure used for the radiolabelling reactions with [“Cjcyanide is as follows. After the was trapped in aqueous NaOH solution, a portion of this solution was removed and its radioactivity measured. The time at which this measurement was taken was recorded and designated as the start of synthesis (SOS). A known amount of KCN was added to the [“C]cyanide solution, then this carrier-added (CA) solution of [“C]cyanide was transferred to a reaction vessel and dried under a fast flow of inert gas. The arene)tricarbonylchromium complex, dissolved in 1 mL of DMSO, was added to the dried [“Cjcyanide and the mixture was heated for 10 minutes. Upon cooling, 25-50 L of the reaction mixture was subjected to radio-HPLC purification, and the peak was discussed in Chapter 1, this applies only to “C and other short-lived radionuclides. For example, compounds labelled with ‘H can be analyzed by ‘H NMR. 54 corresponding to the[11C]nitrile was collected and assayed for radioactivity. An equal volume of reaction mixture was also assayed at the same time, thereby determining the percentage of radioactivity in the reaction mixture contributed by the [“C]nitrile product. The radiochemical yield was then calculated. The radiochemical yields obtained have been decay corrected back to SOS to eliminate the variation of time taken for synthesis and chromatography. Therefore, the radiochemical yields can then be compared with one another, and any differences would reflect the relative efficacy of the reaction conditions used. Complex 1 was treated with[11C]cyanide as outlined in equation 2.22. The results / DMSO __ll4 (2.22)Cr(CO)3 150°C/lOmin 1 18 obtained are summarized in Table 2.10. The first four entries (Table 2.10) represent CA Table 2.10: Summary of Radiochemical Yields Obtained for Equation 2.22 Amount of 1 Amount of Radiochemical used (mol) KCN added Yield 65 0.49 equiv 34% 41 0.37 equiv 36% 43 0.35 equiv 41% 65 0.11 equiv 21% 65 0 aTrace product was observed, but its activity was too low to count in the Capintec well counter. 55 reactions, while the last entry represents a no carrier-added (NCA) reaction trial. These initial results demonstrated that some cyanide carrier must be present to achieve successful labelling with[‘1C]cyanide, under the reaction conditions used. For the CA reactions, the addition of 0.35 equivalents of KCN afforded the highest radiochemical yield (41%). A representative radio-HPLC chromatogram of a CA reaction is shown in Figure 2.6. The chromatogram exhibits the presence of the product, [“C CN]benzonitrile 18 (peak B), and unreacted[11C]cyanide (peak A). The radio-HPLC chromatogram of the NCA reaction trial is shown in Figure 2.7. This chromatogram shows the presence of 18 as only a small peak, indicating a very low radiochemical yield was obtained. The time taken for synthesis and chromatography—the synthesis time—was about 30-60 minutes (measured from SOS) depending on experimental circumstances. Most typically, the synthesis time was 40-45 minutes. Additional CA[‘1C]cyanide reactions were then performed with complexes 1, 4, 5, and 6. Each of these complexes were treated with [“C]cyanide, using 0.5 equivalents of carrier, as shown in equation 2.23. The results obtained are summarized in Table 2.11. F 0.5 equiv KCNR ___________________ a —11CN 223Cr(CO)3 DMSO/ 135°C/lOmin R The radiochemical yields parallel fairly closely the chemical yields obtained with non radioactive cyanide under similar reaction conditions (see Tables 2.3-2.6 for comparison). Initially, H”CN was trapped in aqueous 0.005 M NaOH solution for the first two reaction trials in order to minimize the amount of hydroxide present in the [“Cjcyanide 56 z C C A TIME (mm) The compounds are: (A, 2.3 mm), [‘1C]cyanide; (B, 6.5 mm), [“C-CN]bemzonitrile 18. The radio-HPLC conditions are: C-18 reverse phase Whatman Partisil 10 ODS-3 column, 25 cm x 9 mm; eluent: isocratic methanol/water, 1:1; flow rate, 5.0 mL/min. Figure 2.6: Radio-HPLC chromatogram obtained from the analysis of the reaction of complex 1 with [‘1C}cyariide (with 0.37 equiv of carrier KCN added) at 150°C. The radiochemical yield of 18 was determined to be 36%. 57 C,) z C /KZ / 0T65 I I I I I 0 5 10 15 20 TIME (mm) The radio-HPLC conditions are: C-18 reverse phase Whatman Partisil 10 ODS-3 column, 25 cm x 9 mm; eluent: isocratic methanol/water, 1:1; flow rate, 5.0 mL/min. Figure 2.7: Radio-HPLC chromatogram obtained from the analysis of the reaction of complex 1 with[11C]cyariide (with no carrier KCN added) at 150°C. Only a trace of 18 was produced, for a radiochemical yield of <1%. 58 Table 2.11: Summary of Radiochemical Yields Obtained for Equation 2.23 Starting Complex ‘1C-Labelled Nitrile Radiochemical Product Yield ‘CN Cr(CO)3 1 18 1 34% Cr(CO)3 4 19 Me’F 31% Cr(CO)3 5 20 19% Cr(CO)3 6 21 reactions. After these early experiments, it was observed that the radioactivity of the trapped[1tCjcyanide was being lost during the drying procedure. In subsequent experiments, 0.1 M NaOH solution was used for trapping H’1CN. However, examination of the radiolabelling results of 1 with NCA[‘1Cjcyanide and CA [“C]cyanide (using 0.11 equiv of KCN) showed that in these cases the[11C]cyanide 59 underwent nucleophulic substitution in low yield. It was thought that perhaps hydroxide was interfering with the reactivity of[11C]cyanide when 11CN is present in low concentration (little or no carrier used). Typically, a volume of 0.5 mL of 0.1 M NaOH (containing trapped 11CN) was used—this introduces 50 mol of 0H into the radio- labelling reaction. As a result, some experiments were done using different concentra tions of NaOH solution to trap H11CN, and 0.025 M NaOH was the least concentrated solution that efficiently trapped and retained the[11Clcyanide upon drying. Next, the behaviour of [“C]cyanide alone in DMSO solution was examined by radio HPLC. The chromatograms obtained are shown in Figure 2.8. Chromatogram A was obtained from the analysis of a solution of NCA[11C]cyanide (originally trapped in 0.025 M NaOH, then dried as usual) in DMSO. Note the extra peaks, apart from the main peak (retention time, 4.1 mm), that are present. With the addition of 25 /Lmol of KCN to this[1tCjcyanide solution, radio-HPLC analysis was repeated and chromatogram B was obtained. The extra peaks were significantly reduced in size, but not eliminated. A second batch ofH11CN was trapped in 0.05 M NaOH solution and dried, followed by the addition of 1 mL of DMSO. This solution of NCA[‘1C]cyanide was heated at 150°C for 10 minutes. The colourless solution became amber in colour during heating. Radio HPLC analysis of the NCA[11C]cyanide solution, after cooling, exhibited chromatogram C. The effect of heating resulted in a dramatic change in the appearance of the radio HPLC chromatogram relative to earlier results. When chromatogram A is compared with chromatogram C, the small extra peaks observed in A have become the dominant peaks observed in C. These results suggested that 11CN (in low concentration) may be 60 11CN 0 z 0 B 11CN C) flME (mm) The radio-HPLC chromatograms represent the following: (i) chromatogram A was obtained from a solution of NCA[11C]cyanide in DMSO, (ii) chromatogram B resulted from the addition of carrier KCN (25 mol) to the [‘1C]cyanide solution used for chromatogram A, and (iii) chromatogram C was obtained from a second batch of NCA [‘1C]cyanide that was heated at 150°C for 10 mm in DMSO. The retention time for ‘1CN’ was 4.1 mm in each chromatogram. Figure 2.8: Radio-HPLC chromatograms of[11C]cyanide in DMSO. 61 changing into a different chemical form, in the presence of hydroxide, that cannot undergo nucleophilic substitution. Two radiolabelling trials were done with 6, in which hydroxide concentration was reduced. For the first trial, a batch of CA[11C]cyariide was prepared using H”CN that was trapped in 0.025 M NaOH, followed by the addition of 0.11 equivalents of KCN, and was dried as usual. Complex 6 was treated with the CA[11Cjcyanide at 150°C as described in the general procedure.[11C-CN]-4-Chlorobenzonitrile 21 was obtained in a radiochemical yield of 21%. For the second trial, the H”CN was trapped in a different manner to eliminate the presence of hydroxide from the [“C]cyanide reagent. A second production run of H11CN was trapped in a glass loop that was emersed in a CC14/C02 (-23°C) cooling bath. Any ammonia gas, from the conversion of ‘1CH4 (equation 2.21), was swept through the glass ioop with helium transfer gas. Then the glass ioop was removed from the cooling bath and the H11CN was slowly added to a reaction vessel containing a mixture of 6, carrier KCN (ca. 0.4-0.8 equiv*), and 1 mL of DMSO. This mixture was heated for 10 minutes at 125-130°C. Radio-HPLC analysis of the cooled reaction mixture showed that 21 was obtained in 29% radiochemical yield. These two results suggested that some improvement of radiolabelling efficiency with [11C]cyanide is possible by limiting the hydroxide quantity in the reaction mixture. The identification of the[11C]nitrile products,[11C-CN]benzonitrile 18, [‘1C-CN]-2- tolunitrile 19,[11C-CN]-4-tolunitrile 20, and11C-CN]-4-chlorobenzoriitrile 21, was *Two small crystals of KCN were used, which were not weighed, thus the quantity indicated was estimated. 62 accomplished using radio-HPLC studies. The chromatographic behaviour of the [11C]nitriles was consistent with that observed for the related non-radioactive aromatic nitriles (14, 15, 16, 17). For the reaction of complex 1 with CA[‘1C]cyanide, additional product identification was performed with GC. The reaction mixture of a reaction trial was analyzed by GC (injection temperature, 250°C; oven temperature, 90°C; N2 carrier gas flow, 2.0 mL/min) and found 18 to be present at a retention time (RT) of 8.1 minutes. This was confirmed by the addition of standard (non-radioactive) benzonitrile 14 to an aliquot of reaction mixture and the assigned product peak (RT = 8.1 mm) would correspondingly increase in size. Moreover, another portion of reaction mixture was subjected to radio-HPLC purification and the peak containing 18 was collected. The radio-HPLC fraction was analyzed by GC and the peak due to 18 exhibited the same retention time as standard 14. The radiolabelling results obtained in this study are still preliminary, but they demonstrated that aromatic nucleophilic substitution reactions with CA[‘1C]cyanide can be successfully performed with(6-arene)tricarbonylchromium compounds. However, the use of high specific activity NCA[11Cjcyanide gave a disappointing result. The reasons for this are not clear, but this is not an uncommon problem when labelling with radionudides; the general reasons for this phenomenon were discussed in Chapter 1. In this instance, it was thought that the presence of hydroxide, which was absent in the model labelling studies with non-radioactive cyanide, could be the problem. The hydroxide could affect the radiolabelling results in three different ways. The first possibility is that the hydroxide could be chemically changing the[11C]cyanide into a 63 different form, thereby reducing the already low quantity of 11CN available for reaction. Evidence for this possibility was suggested by the radio-HPLC studies of[‘1C]cyanide alone in DMSO. It was observed that 11CN underwent change to unidentified radio- products upon heating. This observation warrants additional study to determine what is actually happening to the 11CN. The second possibility is that the hydroxide is a competing nucleophile with 11CN. Since about 50 mol of hydroxide is present, and the actual quantity of 11CN is about one to three orders of magnitude less, the hydroxide is necessarily in significant excess. Therefore, reaction with hydroxide may become the dominant process, even if hydroxide is less reactive toward chromium tricarbonyl complexes than cyanide. The final possibifity is that hydroxide could be hydrolysing the [“C]nitrile product to the corresponding[11C]amide and [‘1Cjcarboxylic acid. Some water would need to be present in the reaction mixture for hydrolysis to occur. However, given the very small amount of[11C]nitrile product formed, very little water would be necessary. It is quite possible that the drying of the trapped [“C]cyanide solution is not totally complete, thereby leaving sufficient moisture to allow hydrolysis to proceed. Standards of the anticipated hydrolysis products, benzarnide and benzoic acid, were subjected to HPLC analysis (using the same HPLC conditions as for the reaction mixtures) and exhibited retention times of 3.2 and 2.2 minutes, respectively. These retention times are consistent with those of the unidentified radio-peaks observed in the chromatograms presented in Figures 2.6 and 2.7. 64 2.5 Summary and Conclusions The objective of this study was to explore the potential of(6-arene)tricarbony1chromium compounds as intermediates for the radiolabelling of arene structures. The results of this investigation have clearly demonstrated that chromium tricarbonyl complexes can be used to prepare aryl[11C]nitriles in fair radiochemical yield. As a result, this study represents an important first step in the development of chromium tricarbonyl complexes for radiolabelling. A range of simple(i76-arene)tricarbonylchromium complexes were prepared in 12-89% yield from the reaction of free arene with Cr(CO)6 in a refluxing mixture of di-n-butyl ether and THF. Arenes bearing electron-withdrawing groups gave much lower yields of chromium tricarbonyl product in comparison to arenes with electron-donating groups. The reactivity of(6-halobenzene)tricarbonylchromium complexes 1, 2, and 3 with cyanide anion was investigated and only 1 was found to undergo aromatic nucleophilic substitution successfully. Hence, both chlorine and bromine were determined to be ineffective leaving groups for reactions with cyanide. Therefore, the reactivity of fluorine-substituted, chromium tricarbonyl complexes 1, 4, 5, and 6 were studied in detail. Complex 1 was allowed to react with cyanide (0.5 equiv) in DMSO over a temperature range of 105-150°C and gave beuzonitrile 14 in 12-41% yield. The best yield (41%) was obtained at 135°C. Control experiments with uncomplexed fluorobenzene confirmed that no fluorine displacement by cyanide occurred with free fluorobenzene under the reaction conditions used for 1. 65 Complex 4 was treated with cyanide (0.5 equiv) in DMSO over a temperature range of 95-135°C, which afforded 15 in yields of 26-43%; the best yields (41-43%) were produced at 105-115°C. Complex 5 was allowed to react with cyanide (0.5 equiv) in DMSO over a temperature range of 105-150°C and obtained yields of 11-29% of 16. The best yields (26-29%) were exhibited at 115°C, while the next best results (22-26%) were observed at 135°C. The reaction of 6 with cyanide (0.5 equiv) in DMSO was studied at temperatures of 115 and 135°C only. The best yields of 17 (26-34%) were obtained at 115°C. The reaction time was kept to 10 minutes for these studies. When the reaction time was reduced to five minutes, the chemical yield was significantly reduced also. The cyanide substitution reaction of 4 was also studied in the presence of 18-crown-6. These reactions were conducted over a temperature range of 95- 135°C, and improvements in chemical yield, over earlier studies, were observed throughout the temperature range examined. The best yield of 15 (46%) was obtained at 105°C using three equivalents of 18-crown-6. Clearly, the use of crown ethers shows promise toward maximizing the yields obtained from the substitution reactions of cyanide with chromium tricarbonyl complexes. Substitution reactions with 1, 4, 5, and 6 were performed using[11C]cyanide, and the corresponding aryl[11C]nitriles 18, 19, 20, and 21 were obtained. Some CA reactions were done (at 150°C) with 1 which indicated that 0.35 equivalents of carrier (KCN) was required to afford the best radiochemical yield (41%). Unfortunately, the NCA reaction trial with 1 gave only a trace of 18. These initial results suggest that some carrier must 66 be present to achieve successful radiolabelling with [“C]cyanide, however, continued development of this work could well enable NCA radiolabelling to be accomplished in the future. The aryl[11C]nitriles 19, 20, and 21 were produced in 34%, 31%, and 19% radiochernical yields, respectively, using 0.5 equivalents of carrier at 135°C. The synthesis times, as measured from SOS, were typically 40-45 minutes in length, and are acceptable for radiolabelling with ‘1C. 67 References 1. Fischer, E. 0.; Ofele, K. Angew. Chem. 1957, 69, 715; Chem. Ber. 1957, 90, 2532- 2535. 2. Zeiss, H.; Wheatley, P. J.; Winkler, H. J. S. Benzenoid-Metal Complexes: Structural Determinations and Chemistry; Ronald: New York, 1966. 3. Silverthom, W. E. Adv. Organomet. Chem. 1975, 13, 47-137. 4. Coilman, 3. P.; Hegedus, L. S. Principles and Applications of Organotransition Metal Chemistry; University Science Books: Mill Valley, CA, 1980; Chapter 14. 5. Semmeihack, M. F. .1. Organomet. Chem. Libr. 1976, 1, 36 1-395. 6. Jaouen, G. Ann. N. Y. Acad. Sci. 1977, 295, 59-78. 7. See reference 4; p 653. 8. Pearson, A. J. Metallo-organic Chemistry; Wiley Interscience: New York, 1985; pp348-362. 9. McQuillin, F. 3.; Parker, D. G.; Stephenson, G. R. Transition Metal Organometallics for Organic Synthesis; Cambridge University: Cambridge, 1991; pp 182-208. 10. Nicholls, B.; Whiting, M. C. I. Chem. Soc. 1959, 551-556. 11. Rosca, S. I.; Rosca, S. Rev. Chim. 1974, 25, 46 1-465. 12. Alemagna, A.; Buttero, P. D.; Gorini, C.; Landini, D.; Licandro, E.; Maiorana, S. J. Am. Chem. Soc. 1983, 48, 605-607. 13. Mahaffy, C. A. L.; Pauson, P. L. J. Chem. Res., Synop. 1979, 128. 14. Harrington, P.3. Transition Metals in Total Synthesis; Wiley Interscience: New York, 1990; pp 317-319. 15. Caiy, F. A.; Sundberg, R. J. Advanced Organic Chemistry—Part B: Reactions and Synthesis; Plenum: New York, 1977; Chapter 7. 16. Kilbourn, M. R. Fluorine-18 Labeling of Radiopharmaceuticals; Nuclear Science Series, Monograph NAS-NS-3203; National Academy: Washington, DC, 1990. 17. Radiopharmaceuticals for Positron Emission Tomography: Methodological Aspects; Stöcklin, G., Pike, V. W., Eds.; Kiuwer Academic: Boston, 1993. 68 18. Radionuclides Production; Helus, F., Ed.; CRC: Boca Raton, 1983; Vol. 2. 19. Davis, R.; Kane-Maguire, L. A. P. In Comprehensive Organometallic Chemistiy: The Synthesis, Reactions and Structures of Organometallic Compounds; Wilkinson, G., Stone, F. 0. A., Abel, E. W., Eds.; Perganion: Toronto, 1982; Vol. 3, pp 1001-1021. 20. Mahaffy, C. A. L.; Pauson, P. L. Inorg. Synth. 1979, 19, 154-158. 21. Hudeek, M.; Toma, . .1. Organomet. Chem. 1990, 393, 115-118. 22. See reference 19; p 1001. 23. Mahaffy, C. A. L.; Pauson, P. L. J. Chem. Res., Miniprint 1979, 1776-1794. 24. Maeda, M.; Fukumura, T.; Kojima, M. Chem. Pharm. Bull. 1985, 33, 1301-1304; .1. Labelled Compd. Radiopharm. 1986, 23, 1104-1105. 25. Angelini, G.; Sperauza, M.; Wolf, A. P.; Shiue, C-Y. .1. Fluorine Chem. 1985,27, 177- 191. 26. Haka, M. S.; Kilbourn, M. R.; Watkins, 0. L.; Toorongian, S. A. J. Labelled Compd. Radiopharm. 1989, 27, 823-833. 27. Bunnet, J. F.; Hermann, H. .1. Org. Chem. 1971, 36, 408 1-4088. 28. Ofele, K. Chem. Ber. 1966, 99, 1732-1736. 29. Mahaffy, C. A. L. J. Organomet. Chem. 1984, 262, 33-37. 30. Gilbert, J. R.; Leach, W. P.; Miller, J. R. .1. Organomet. Chem. 1973, 49, 219-225. 31. Muller, J.; Göser, P. Recent Dev. Mass Spectrosc., Proc. mt. Confi Mass Spectrosc. 1969 (Pub 1970), 1175-1180. 32. Carganico, G.; Buttero, P. D.; Maiorana, S.; Riccardi, G. J. Chem. Soc., Chem. Commun.. 1978, 989-990. 33. See reference 4; pp 653-661. 34. March, J. Advanced Organic Chemistty: Reactions, Mechanisms, and Structure, 3rd ed.; Wiley Interscience: New York, 1985; Chapter 13. 35. See reference 34; pp 584-586. 36. See reference 34; pp 77-79, 321-322 and references contained therein. 37. Kriipe, A. C. .1. Chem. Ed. 1976, 53, 6 18-622. 69 38. Zubrick, J. W.; Dunbar, B. I.; Durst, H. D. Tetrahedron Lett. 1975, 71-74. 39. Cook, F. L.; Bowers, C. W.; Liotta, C. L. .1. Org. Chem. 1974, 39, 3416-3418. 40. Gokel, G. W.; Durst, H. D. Synthesis 1976, 168-184. 41. Miller, J. Aromatic Nucleophilic Substitution, Elsevier: New York, 1968; Chapter 5. 42. Azam, K. A.; Kabir, S. E.; Kazi, A. B.; Molla, A. H.; Ullah, S. S. Chem. Abstr. 1988, 109, 23086r. 43. Meyer, G.-J. Radiochim. Acta 1982, 30, 175-184. 44. Ruth, T. J., TRIUMF, Vancouver, BC, personal communication. 70 Chapter 3 DEVELOPMENT OF METHODS FOR RAPID FLUORINE LABELLING 31 Introduction In the early 1950s, the pioneering work of Fried and Sabo led to the first significant successful application of selective fluorination to modify the biological activity of an organic molecule.1 The fluorosteroid, 9a-fluorohydrocortisone acetate 22, that was OAc 0 prepared by the Squibb Company researchers, showed approximately an 11-fold increase in glucocorticoid activity over that of cortisone acetate. This report stimulated much new research into developing ways of selectively introducing fluorine at specific sites in compounds of potential biological interest, in order to modify their biological activity. Since the landmark work of Fried and Sabo, the study of selectively fluorinated 71 molecules has resulted in a variety of useful applications in biochemistry and medicine.2’3 These applications include the use of fluorine-containing organic pharmaceuticals as anticancer and antiviral agents, antiinflammatory drugs, antibiotics, antifungal and antiparasitic agents, general anesthetics, and therapeutic drugs for mental illness. In addition, pharmaceuticals labelled with radioactive fluorine (18F) play an important role in PET imaging. Perfluorinated hydrocarbons are no longer the major focus of study in fluorine chemistry. Currently, interest in selectively fluorinated compounds is continuing to increase, along with the recognition of the importance of selective fluorination methodology.4’5 The attractiveness and utility of fluorine as a substituent in biologically active molecules are due to the unique properties of fluorine that can induce profound and unexpected effects on biological activity. Firstly, fluorine is the most electronegative of all elements, with an electronegativity (Pauling scale) of 3.98 as compared to 3.44 for oxygen, 3.16 for chlorine, or 2.96 for bromine. It is this property which produces pronounced electronic effects in a molecule after the introduction of a fluorine substituent. Secondly, fluorine, with its small van der Waals radius (1.35 A), closely resembles hydrogen (van der Waals radius 1.20 A) in size. As a result, the fluorinated analogue will usually closely resemble the non-fluorinated molecule in size when a fluorine is substituted for hydrogen. This allows, for instance, fluorinated analogues to still meet steric requirements at enzyme receptor sites. Thirdly, fluorine forms a stronger bond with carbon than does hydrogen—the carbon-fluorine bond energy is 456- 486 kJ/mol, while carbon-hydrogen bond energy varies from 356 to 435 kJ/mol. 72 Therefore, carbon-fluorine bonds exhibit increased thermal and oxidative stabifity over that of carbon-hydrogen bonds. Lastly, fluorine, when replacing hydrogen in a molecule, usually enhances lipophilicity which increases the rate of absorption and transport of the fluorine-containing compound in vivo. In many cases, this characteristic may be the most important in improving pharmacological activity.6’7 An additional feature of fluorine is the availability of the artificially prepared radionuclide, 18F, which decays by positron emission. Fluorine-18 (half-life, 109.7 mm) is one of the four key radionuclides used in PET. The other conimorily used positron emitting nuclides (11C, ‘3N, 150) possess very short half-lives (—‘2-20 mm) in comparison to ‘8F. The longer half-life of ‘8F allows for more complex or multistep radio- pharmaceutical syntheses to be conducted, and the resulting compounds can be transported over moderate distances for use at different locations. In addition, the study of relatively slow biological processes can be performed with‘8F-labelled agents, which would not be feasible with agents using nuclides with shorter half-lives. Furthermore, 18F-labelled compounds have the potential to produce PET images of superior resolution due to the relatively low energy positron (maximum 0.635 MeV) emitted by 18F (see Table 1.2 for comparison)—this factor will be of increasing importance as PET instrumentation improves.8’9 The utilization of 18F-labelled pharmaceuticals with PET imaging has enabled a number of human biochemical and physiological processes to be investigated in vivo, as was presented in Chapter 1. More recently, however, the application of PET is being extended beyond the study and diagnosis of disease to the area of drug discovery, 73 development, and approval of new pharmaceuticals. Drug candidates can be labelled with positron emitting nuclides, such as 11C and 18F, to provide information regarding drug absorption, distribution, metabolism, and elimination in vivo (human or animal subjects) using PET imaging. These studies can complement information obtained using 3H- and‘4C-labelled analogues in animals. Alternatively, established PET imaging protocols can be used to monitor the biological behaviour of drug candidates in vivo to determine the therapeutic potential or efficacy of such compounds. For disease states where no animal models exist, PET becomes a unique tool that still enables drug assessment to be carried out. Also, given the interest in fluorine-containing biologically active molecules, it would be possible—in principle, at least—to study the in vivo biochemistry of the 18F-labelled analogues via PET.1° Underlying the continued development and application of fluorinated compounds is the ongoing need for new and improved methods to selectively introduce fluorine into polyfunctional molecules at specific sites. However, the mild and selective introduction of fluorine into organic molecules has been and continues to be of considerable challenge to organic chemistry. Although elemental fluorine was first prepared by Moissan in 1886,11 the organic chemistry of fluorine developed slowly in comparison to the other halogens. It was quickly discovered that the reaction of fluorine with organic compounds was highly exothermic, and often resulted in explosions. These observations understandably discouraged further research with fluorine for decades after Moissan’s time. In the 1930s, Bockemüller demonstrated that fluorine, when diluted with inert gas (usually nitrogen), could be used for selective fluorination of organic substrates under 74 controlled conditions.’2 Since that time, new fluorinating agents and reactions have been developed making possible the synthesis of the many organofluorine compounds available today.’3”4 Currently, the range of methods for introducing fluorine into organic compounds is based on the use of either elemental fluorine, hydrogen fluoride, inorganic fluorides, or various organofluorine reagents. These fluorinating agents can be broadly characterized as sources of either nucleophilic or electrophilic fluorine. With these fluorinating agents, many methods have been developed to effect the transformation of different organic functional groups to organofluorine derivatives, and are catalogued in multiple books’5”6 and reviews.4’137”8 However, many of these methods, though successful in conventional synthetic chemistry, are not compatible with the requirements of radiolabelling with 18F.8’9 A primary limitation of radiofluorination methodology is the limited range of useful synthetic precursors that can be produced in‘8F-labelled form. The only reagent available for nucleophilic fluorination is [‘8F]fluoride anion.’9 However, [‘8F]fluoride has seen increasing application because of continuing investigations performed studying the different variables involved in optimizing the reaction conditions for its use with structurally diverse substrate molecules. Investigators have studied the influence of reaction solvent, source of ‘8F, fluoride counterion, catalyst (e.g., crown ethers), ‘9F carrier levels, reaction vessel material, substrate concentration, leaving group, and temperature on the radiochemical yields obtained with [‘FJfluoride.9” Reaction conditions have been developed such that a number of aliphatic and aromatic 75 compounds have now been successfully radiolabelled with 18F •9,20 As a result, [18F]fluoride has gained increased importance for radiofluorination work. In contrast to nucleophilic fluorination, a number of electrophilic fluorinating agents have been produced in‘8F-labelled form, using [‘8F]F2 as the source of 18F in each case. A list of the various fluorinating agents prepared with 18F is presented below. However, Fluorinating Agents Labelled with 18F9’2° fluorine (F2) chlorine monofluoride (C1F) perchioryl fluoride (C1O3F) trifluoromethyl hypofluorite (CF3OF) xenon difluoride (XeF2) trifluoroacetyl hypofluorite (CF3COOF) nitrosyl fluoride (NOF) acetyl hypofluorite (CH3COOF) N-fluoro-2-pyridone N-fluoropyridinium trifiates N-fluoro-N-alkylsulfonamides (RSO2NFR ‘) N-fluoro-bis(trffluoromethylsulfonyl)imide ((CF3SO2NF) most of these reagents have not actually been evaluated as to their scope and utility for radiolabelling with 1F.9 In practical experience, the vast majority of electrophilic fluorinations are performed with [18F]F2 and CH3OO18F.21’2 Acetyl hypofluorite is a relatively new reagent that was originally developed by Rozen and co-workers in 198 1,23 and then was prepared in‘8F-labelled form in 1982? Acetyl hypofluorite has been found to be a milder and more selective electrophilic fluorinating agent, in comparison to elemental fluorine, for a variety of substrate molecules.25’6 Moreover, the development of a simple on-line gas-solid phase synthesis of acetyl hypofluorite27 has significantly increased its utility for radiofluorinations. The other 18F-labelled fluorinating agents listed previously are not trivial to produce on a routine basis, and as 76 a result, have not engendered serious interest by PET research groups as yet.21 Clearly, much additional research needs to be done in order to develop the full potential of electrophilic fluorination methodology for radiolabelling. The requirement to develop methods to prepare‘8F-labelled aromatic compounds for the PET program at U.B.C. prompted Adam and co-workers28’9to examine the reacti vity of main group organometallic derivatives with electrophilic fluorinating agents. It was essential that the fluorination reaction be rapid, the fluorinating agent be readily available in‘8F-labelled form for routine use, and be efficient in the incorporation of radiofluorine. Also, it was desired that the reaction conditions be mild and the experimental manipulations be as simple as possible to perform to accommodate future automation. It was known that the electrophilic halogenation of organometallic derivatives such as those of tin30 and mercury was well established. However, very little study had been done regarding the reactivity of carbon-metal bonds with electrophilic fluorinating agents. As a result, the reactivity of aryl-tin, (then later) aryl silicon, -germanium, -lead, -mercury, and -thallium bonds, with elemental fluorine was investigated.28’9Fluorobenzene was successfully produced from phenyltri-n-butyltin (48- 70% yield), tetraphenyltin (15-56% yield), tetraphenyllead (48% yield), and diphenyl mercury (26-36% yield). The other organometaffic derivatives studied gave only poor yields (2.4-12%), or no detectable product in the case of the organothallium derivative. These studies represent the first reports of the cleavage of aryl-metal bonds by electrophilic fluorination. Since the reports of Adam et al.,28’9 the fluorination of organometallic compounds has 77 been under study by other researchers as well. As a result, various simple aromatic compounds have been radiolabelled with 18F utilizing organotin,29’323 organo silicon,33’45 organogermanium,33and organomercur6’37derivatives. In addition, aryl lithium38’39 and Grignard39’4°reagents have also been successfully radiofluorinated. More significantly, the strategy of electrophiic fluorination of organometallic derivatives have been applied to the preparation of18F-labelled pharmaceuticals for PET. Aryl-tin precursors were used to prepare both 3-O-methyl-6-[18F]fluorodopa41and 6-[’8F]fluoro- dopa42 by reaction with CH3OO18F(20% radiochemical yield) and[18F]F2 (25% radio- chemical yield), respectively. 6-[’F]Fluorodopa has also been prepared from an aryl silicon derivative using [18F1F2 (5-10% radiochemical yield)43 and an aryl-mercury derivative using CH3OO18F(—40% radiochemical yield).” 6-[18FjFluorometarami- no145 and4-[18F]fluoro m-tyrosine6were successfully prepared from organomercury substrates by reaction withCH3OO’8Fin approximately 30% and 25-30% radiochemical yields, respectively.6-[18F]fluoroveraldehyde was readily prepared in 30% radiochemical yield from an organotin precursor using[18F]F2,but interestingly could not be obtained from the corresponding organomercury derivative.47 This case reaffirms the need for various synthetic options to be available to successfully synthesize a desired18F-labelled compound. The application of electrophilic fluorination via organometallic intermediates has been almost exclusively focussed on introducing fluorine onto aromatic rings. This is understandable because of the many organic compounds of biological interest that contain aromatic rings in their structures. Nonetheless, other potential applications also 78 exist. Some alkyl fluorides have been prepared via organometallic derivatives, such as cyclohexyl fluoride48 and n-tetradecyl fluoride49 from the corresponding Grignard reagents, in low yield. Benzyl[18F]fluoride was prepared from potassium benzylpenta fluorosilicate in 6% radiochemical yield.35 Generally, however, primary and secondary alkyl fluorides have been readily obtained using nucleophilic substitution with fluoride anion. Alternatively, a number of biologically interesting molecules exist which contain a vinyl function in their structures.50’12 The vinyl functionality provides a potential site for labelling with fluorine. Moreover, some important biomolecules specifically contain the fluorovinyl grouping,7’534and may be of interest for 18F-labelling. These potential applications prompted interest in extending the strategy of electrophilic fluorination of organometaffic derivatives as a general methodology to prepare fluorovinyl compounds, and is suitable for radiofluorinations with 18F. Upon review of the fluorination studies of aryl-metal systems, it seemed that the organotin reagents gave the highest chemical and radiochemical yields, although organo mercury and -silicon derivatives gave good results also. The electrophilic fluorination reactions of aryl-tin reagents are rapid. Previous work55 with vinyl-tin compounds have demonstrated that they are sufficiently stable to be prepared in bulk, and then stored for use as needed. Vinyl-tin compounds can be readily prepared by reduction of the corresponding acetylenic compounds with tin hydride reducing agents.56’57 In addition, other methods of preparation of vinyl-tin derivatives are also available.58 These factors suggested to us that vinyl-tin substrates offered the best potential as reagents for fluorination studies. 79 In this chapter, the preparation of the vinyl-tin derivatives employed for this work will be described. This will be followed by fluorination studies of the vinyl-tin substrates with elemental fluorine and acetyl hypofluorite. Lastly, the successful radiofluorination of a vinyl-tin derivative of a steroid will be presented. 3.2 Synthesis of Vinyl-Tin Precursors The vinyl-tin derivatives were prepared by hydrostannylation of the corresponding acetylenic precursors, and most of the results obtained have been described previously in the author’s M.Sc. Thesis.55 The acetylenic starting materials were either obtained commercially or prepared using literature methods, as summarized in Schemes 3.1 and 3.11. The acetylenic compounds, 17a-ethynyl- 1,3,5( 10)-estratriene-3, 1713-diol 23, 3-methoxy- 17a-ethynyl- 1,3,5(10)-estratriene- 17i3-ol 24, 3, 17i3-dimethoxy-17a-ethynyl-1,3,5( 10)- estratriene 25, 7,8-dideoxy-1,2:3,4-di-O-isopropylidene-D-glycero-a-D-galacto-oct-7-yno- pyranose 28, and 7,8-dideoxy-l,2:3,4-di-O-isopropylidene-L-glycero-a-D-galacto-oct-7- ynopyranose 29, were hydrostannylated (see Schemes 3.111 and 3.IV) by an adaptation of literature procedures.57 The acetylenic precursors were treated with approximately two equivalents of tri-n-butyltin hydride and a catalytic amount of AIBN (2,2 ‘-azobis(2- methylpropionitrile)), followed by heating the mixture at 95°C overnight. The (E) vinyistannylated products were isolated by chromatographic workup of the reaction mixtures, and the chemical yields are summarized in Table 3.1. Each of the (E) vinyistannanes was characterized by their physical properties (optical rotation values, 80 Scheme 3.! 1) KOHIDMSO 2) Mel Scheme 3.11 Cr03-2 py CH21 (py = pyridine) OH HCCMgBr ThF HO_ // 28 + L-OH / 23 24 + 25 81 Scheme 3.111 2 R’O 23 R’=R2=H 24 R’Me,R=H 25 R’R2Me (n-Bu)3SnH AIBN/heat R20 R1 -Sn(Bu)3 30 R’=R2=H 31 Me,R=H 32 R’R2Me Scheme 3.W / 0 (n-Bu)3SnH AIBN/heat (n-Bu)3SnH AIBN/heat Sn(Bu)3 Sn(Bu)3 _0H o\ 34 82 melting points of crystalline products), elemental analysis, and spectral data (1H NMR, mass spectrometry). The AIBN catalyzed hydrostannylation reaction was found to be quite successful for all the acetylenic precursors used. However, higher yields of (E)-vinylstannylated Table 3.1: Yields of (E)-Vinylstannanes Starting Material Product Chemical Yield 23 30 59% 24 31 90% 25 32 94% 28 33 61% 29 34 59% product were obtained from the steroidal acetylenic compounds, 24 and 25, in compar ison with the carbohydrate acetylenic substrates (28, 29). It is known that the hydrostan nylation reaction can produce three different isomers when using terminal acetylenes,59 as shown in equation 3.1. As a consequence, the chemical yield of (E)-vinylstarmane will — (Bu)3SnH (Bu)3Sn,,H ÷ H\H + H\,,Sn(Bu)3 R — H R H R Sn(Bu)3 R H (3.1) regioisomer Z-isomer E-isomer vary depending on the proportion of (Z)-vinylstamiane and “regioisomer” which are produced. It was observed that the hydrostarinylation reactions of 28 and 29 produced greater proportions of alternative isomeric products, thus giving consistently lower yields of (E)-vinylstannanes. The lower hydrostannylation yield of 17a-(E)-tributylstannylvinyl- 83 1,3,5(10)-estratriene-3,17fl-diol 30 from 23 was due to significant protonolysis of vinyl-tin product formed during the reaction;* this was most probably due to the presence of the unprotected phenolic hydroxyl group. During a more recent preparation of 3-methoxy- 17a-(E)-tributylstamiylvinyl- 1,3,5(10)- estratriene-1713-ol 31, the isomeric product, 3-methoxy-17a-(Z)-tributylstaunylvinyl- 1,3,5(10)-estratriene-17j3-ol 36, was also isolated (— 10% yield). Compound 36 has not HO MeO’1’ Sn(Bu)3 been fully characterized as yet. However, in the preparation of (E)-7,8-dideoxy-1,2:3,4- di-O-isopropylidene-8-C-tributylstannyl-L-glycero-c-D-galacto-oct-7-enopyranose34, both alternative isomeric products, (Z)-7,8-dideoxy- 1,2:3,4-di-O-isopropylidene-8-C-tributyl- Sn(Bu)3 (Bu)3Sn..— stannyl-L-glycero-cx-D-galacto-oct-7-enopyranose 37 and 7,8-dideoxy- 1,2:3,4-di-O-isopro- pylidene-7-C-tributylstannyl-L-glycero-a-D-galacto-oct-7-enopyranose 38, were isolated as *A significant amount of 17a-vinyl- 1,3,5(10)-estratriene-3, 17-diol 35 was recovered from the hydrostannylation of 30; see Experimental for details. 84 a 3:2 mixture. The chemical yields of 37 and 38 were estimated to be 15% and 10%, respectively. Compounds 37 and 38 were identified by their ‘H NMR spectral data. 3.3 Labelling Studies with Non-Radioactive Fluorine Prior to this study, there were some reports of vinylmetallated compounds having been used for the synthesis of fluorovinyl compounds. There was an early report in which the direct fluorination of a vinyl-tin compound (1,2,3,4,7,7-hexafluoro-5,6-bis(trimethyl- stannyl)bicyclo[2.2.ljhepta-2,5-diene) with F2 was studied, but the desired product was afforded in <5% yield.6° Since then, Di Raddo and Diksic6’prepared4-[’8Fjfluoroanti- pyrine using [‘8F]F2 in 18% radiochenilcal yield from 4-(trimethylsilyl)antipyrine. Lee and Schwartz62 prepared various simple vinyl fluorides in 71-88% yield using vinyl- lithium reagents and N-tert-butyl-N-fluorobenzenesulfonamide as the source for electrophiic fluorine. Interestingly, Flanagan et al.5’ attempted to fluorinate 6- chloromercuricholest-5-en-3j3-ol with elemental fluorine, acetyl hypofluorite, and xenon difluoride under various conditions, but without success. Our study examined the reactivity of the (E)-vinylstannanes, 30, 31, 3,17(3-dimethoxy- 17a-(E)-tributylstannylvinyl-1,3,5(10)-estratriene 32, (E)-7,8-dideoxy-1,2:3,4-di-O-isopropy- lidene-8-C-tributylstannyl-D-glycero-a-D-galacto-oct-7-enopyranose 33, and 34, with elemental fluorine and acetyl hypofluorite. The fluorination of 31 was initially studied in small scale experiments using excess F2 or gaseous CH3OOF, and the resulting crude reaction mixtures were analyzed by TLC and 19k’ NMR It was evident from TLC analysis that both fluorinating agents produced 85 multiple products, but the 17 NMR spectra indicated that the reaction of 31 with CH3OOF produced significantly more fluorovinyl product than with F2. It was also observed that neither fluorinating agent consumed all of the starting material. As a result, 31 was fluorinated with CH3OOF (Scheme 3.V) on a larger scale in the following manner. Compound 31 was treated with approximately 1.3 equivalents of gaseous CH3OOF in CFC13 at room temperature. This procedure was conducted with six portions of 31 in order to employ a sufficient amount of starting material for fluorination. The crude reaction mixtures were combined and subjected to column chromatography. The fractions containing 3-methoxy- 17a-(E)-fluorovinyl- 1,3,5( 10)-estra- triene- 1713-0139 and 3-methoxy-l7cx-(Z)-fluorovinyl-1,3,5(10)-estratriene-1713-ol 40 were subjected to additional purification by HPLC, whereby 39 and 40 were isolated in 29.5% and 3.8% yield, respectively. Compounds 39 and 40 were readily characterized by 1H and ‘9F NMR. Scheme 3.V HO HO MeO I T > CH3OOF _J.. L/ + CFCI3 HO MeO 31 86 MeO Having successfully isolated the fluorovinyl products 39 and 40, the opportunity emerged to quantitatively study the fluorination reactions of 31 under different conditions. A number of small scale reactions with 31 were conducted using the following general procedure. A solution of 31 (in a chosen solvent) was treated with a small excess (ca. 1.2-1.4 equiv) of fluorinating agent (F2 orCH3OOF). The solvent was evaporated and the reaction mixture was dissolved in a known volume of chloroform. This solution was analyzed by HPLC to determine the combined yield of both fluorovinyl products 39 and 40. (A solution of 39 was used as an external standard.) Firstly, 31 was allowed to react with gaseous CH3OOF (Scheme 3.V) at both -78°C and room temperature. The results are summarized in Table 3.2. The best yields of fluorinated product were obtained at room temperature, although fewer side-products were observed (by HPLC) when the reaction was performed at -78°C. Secondly, 31 was Table 3.2: Summary of Yields Obtained for the Reaction of 31 with Acetyl Hypofluorite Solvent Temperaturea Yieldb no. of Average runs Yieldb CFC13 -78°C 26-31% 2 29% CFC13 r.t. 41-42% 2 41% CH3O r.t. 14% 1 14% CH3N r.t. 24% 1 24% TIIF r.t. 9.3% 1 9.3% ar.t. = room temperature. bRefers to the combined yield of 39 and 40. treated with CH3OOF in alternative solvents, namely dried methanol, acetonitrile, and 87 tetrahydrofuran, at room temperature. The yields are summarized in Table 3.2. None of these solvents provided improved yields of fluorinated product. Clearly, CFC13proved to be the best solvent tested for the fluorination reaction of 31. Finally, the reaction of 31 with elemental fluorine (Scheme 3M) was studied at both -78°C and room temperature. The results are summarized in Table 3.3. It was evident by both analytical Scheme 3.VI MeO’ —Sn(Bu)3 0.1% F2/Ne CFC13 MeO F + MeO HO7 HO MeO + MeO 88 Table 3.3: Summary of Yields Obtained for the Reaction of 31 with Fluorine Temperaturea Yield of 39 and 40 Yield of 24 Yield of 41 -78°C 4.2% 2.2% 14.5% r.t. 9.0% 5.4% 7.2% ar.t. = room temperature. TLC and HPLC that 31 is less reactive toward F2 than CH3OOF as less starting material is consumed by F2. However, reaction with F2 does result in the formation of two identified non-fluorinated side-products, 24 and 3-methoxy-17a-vinyl-1,3,5 (10)- estratriene-17/3-ol 41. The identity of these compounds was established via HPLC; by comparison of their respective retention times with those of authentic standards. The identity of 41 was further confirmed by ‘H NMR spectroscopy of a preliminary reaction trial done with F2 at -78°C. The reaction of 31 with F2 clearly gave the lowest yields of fluorovinyl product (39 and 40) in comparison to the reactions performed with CH3OOF. The reactivity of 30 with fluorine and acetyl hypofluorite was investigated. Compound 30 was treated with approximately 0.8 equivalents of dilute (0.1%) F2 in Ne gas mixture in CFC13 at -78°C. The TLC chromatogram of the reaction mixture exhibited the formation of a new, more polar component, along with other minor components. This dominant new component, as observed by TLC, was isolated by column chromatography for identification by NMR. The 270 MHz ‘H NMR spectrum revealed that the isolated material was 17a-vinyl-1,3,5(10)-estratriene-3,173-diol 35. No trace of fluorovinyl product was observed in the NMR spectrum. Alternatively, 30 was treated with 89 HOJ5 -Sn(Bu)3 approximately 1.2 equivalents of gaseous CH3OOF in CFCI3 at -78°C. TLC analysis of the crude reaction mixture indicated the total consumption of starting material and the formation of an extremely complex mixture. The 19f? NMR spectrum of the reaction mixture exhibited several weak signals of fluorinated products which could not be assigned. The corresponding 1H NMR spectrum could not provide any useful information due to its extremely complex pattern. Unfortunately, no fluorovinyl product could be obtained from 30 using either F2 or CH3OOF as fluorinating agents. Since the 3-O-methylated (E)-vinylstannane 31 was successfully fluorinated as described earlier, the difficulty in fluorinating 30 is most likely due to the presence of the unprotected phenolic hydroxyl group. Therefore, this functionality must be protected with an easily removed protecting group, such as tefl butyldimethylsilyl, in order to develop a synthetic route to 17a-(E)-fluorovinyl-1,3,5(1O)- estratriene-3, 17i3-diol. The reactivity of 32 was also studied with fluorine and acetyl bypofluorite. The use of elemental fluorine was investigated first. Four small scale reaction trials were performed in which 32. was allowed to react with approximately 0.9 equivalents of dilute fluorine at -78°C (2 trials), 0°C (1 trial), and room temperature (1 trial), in CFC. The 90 MeO11 —Sn(Bu)3 TLC analysis of each reaction trial exhibited similar results; the TLC chromatograms showed the consumption of some starting material and the formation of a dominant, slower migrating component amongst several other minor components. In order to isolate the dominant new component observed by TLC, the reaction mixtures were combined and subjected to column chromatography on silica gel. Half of the original amount of 32 used for all four trials was recovered in one portion; then the dominant new component was isolated with continued elution. The 270 MHz 1H NMR spectrum of this isolated material revealed that a mixture of several compounds were present. The compounds were identified as 25, 3, 173-dimethoxy- 17cr-vinyl-1,3,5(10)-estratriene 42, and 3,17/3-dimethoxy-17a-(E)-fluorovinyl-1,3,5(10)-estratriene 43 in an approximate ratio of 71:18:11, plus another component that could not be identified was present. The use of acetyl hypofluorite, prepared in two different forms, was investigated next. Compound 32 was added to a slight excess of CH3OOF prepared in a solution of glacial acetic acid and aninionium acetate by the method of Rozen et al?3 at room temperature. The reaction proceeded for about 15 minutes before the acetic acid was removed. The crude reaction mixture was analyzed by TLC, which revealed the con sumption of some starting material and the formation of a new, more polar component 91 Me07- MeO MeO MeO plus some other minor components. The reaction mixture was further analyzed by 1H and 9F NMR spectroscopy. The ‘9F NMR spectrum exhibited a few weak signals due to unidentified products, whereas the 1H NMR spectrum indicated the presence of 25, 32, and 42, but not fluorovinyl product 43. Compound 32 was then treated with excess gaseous CH3OOF in CFC13 at -78°C. The TLC chromatogram of the reaction mixture exhibited only a small degree of conversion of starting material to a new, slower migrating component, along with other minor components. The 270 MHz 1H NMR spectrum of the reaction mixture revealed largely unreacted starting material and a very small amount of 25 present. However, the 9F NMR spectrum showed the presence of 43. These studies of the reactivity of 32 indicate that this derivative is relatively unreactive toward CH3OOF in comparison to 31. No fluorovinyl product 43 could be obtained 92 with the use of CH3OOF in acetic acid, whereas only a minute amount of 43 could be produced with gaseous CH3OOF. The reaction of 32 with elemental fluorine produced some 43 which could be identified in a mixture of products. These results suggested that fluorine was the most effective fluorinating agent used with 32. However, a larger scale fluorination of 32 utilizing F2 was not pursued due to the low potential yield and anticipated separation problems. Next, the reactivity of 33 toward fluorine and acetyl hypofluorite was examined in several small scale experiments. In the first experiment, 33 was allowed to react with approximately 0.9 equivalents of dilute fluorine in CFC13 at -78°C. The TLC chromato Sn(Bu)3 gram of the reaction mixture revealed the formation of a new, more polar component in addition to a few minor components. The dominant new component, as observed by TLC, was isolated by column chromatography and subjected to 1H NMR spectroscopy. This material consisted of four different compounds that were identified as 28, 7,8- dideoxy- 1,2:3,4-di-O-isopropylidene-D-glycero-a-D-galacto-oct-7-enopyranose 44, (E)-8- C-fluoro-7,8-dideoxy-1,2:3,4-di-O-isopropylidene-D-glycero-a-D-galacto-oct-7-enopyranose 45, and (Z)-8-C-fluoro-7,8-dideoxy-1,2:3,4-di-O-isopropylidene-D-glycero-cy-D-galacto-oct- 7-enopyranose 46, in which 44 was the dominant component. 93 HO HO /°KL o\ 28 44 F For the second experiment, 33 was added to approximately 0.8 equivalents of CH3OOF prepared in a solution of glacial acetic acid and ammonium acetate at room temperature. The reaction was allowed to proceed for about 10 minutes, then the acetic acid was removed. The crude reaction mixture was analyzed by TLC, which showed the formation of a dominant, slower migrating component plus a few minor components. Column chromatography of the reaction mixture resulted in the isolation of the dominant new component observed by TLC. This material was examined by ‘H NMR spectroscopy, which revealed that a mixture of 44, 45, and 46 was present and 44 was the major component. In the third experiment, 33 was treated with approximately 1.2 equivalents of gaseous CH3OOF in CFC13 at -78°C. Analytical TEC of the reaction mixture revealed the 94 formation of a dominant, more poiar component and a few minor components. The dominant new component, as observed by TLC, was isolated via preparative TLC (silica gel), and analyzed by 1H and 19F NMR spectroscopy. This material was found to be largely a mixture of 45 and 46, with some unidentified impurities present. The fluorination studies of 33, though qualitative in nature, showed that gaseous CH3OOF was the most effective fluorinating agent used. This agent gave the highest degree of conversion of 33 to fluorovinyl product (45 and 46). The use of F2 or CH3OOF in acetic acid solution produced 44 as the main product. Unfortunately, the supply of 33—as starting material—was virtually all consumed so that further larger scale fluorination work could not be conducted. Therefore, fluorination studies were continued using the L-glycero-a-D-galacto epimer 34 instead. Compound 34 was fluorinated with CH3OOF (Scheme 3.VII) in the following Scheme 3.VII Sn(Bu)3 F [—OH CH3OOF [—OH [—oH / CFC13 / + / maimer. Compound 34 was treated with approximately 1.3 equivalents of gaseous CH3OOF in CFC13 at room temperature. This procedure was performed with two portions of 34 to increase the reaction scale. The crude reaction mixtures were 95 combined and subjected to column chromatography. (E)-8-C-Fluoro-7,8-dideoxy-1,2:3,4- di-O-isopropylidene-L-glycero-a-D-galacto-oct-7-enopyranose 47 was isolated in the first chromatography fraction, and then two fractions were collected that contained mixtures of 47, (Z)-8-C-fluoro-7,8-dideoxy-1,2:3,4-di-O-isopropylidene-L-glycero-a-D-galacto-oct- 7-enopyranose 48, and 7,8-dideoxy-1,2:3,4-di-O-isopropylidene-L-glycero-c-D-galacto-oct- 7-enopyranose 49. The ratio of components in each chromatography fraction was 0 oc measured by ‘H NMR spectroscopy. The isolated yields of 47 and 48 were determined to be 36% and 10%, respectively. Furthermore, an additional fraction was subsequently collected which was found to contain a single compound, identified as (E)-8-C-acetoxy-7- deoxy-1,2:3,4-di-O-isopropylidene-L-glycero-a-D-galacto-oct-7-enopyranose 50 by ‘H NMR. Compound 47 was readily characterized by ‘H and ‘9F NMR. 3.4 Radiofluorination Work In this section, the radiofluorination of (E)-vinylstannane 31 using acetyl[‘8Fjhypofluorite will be presented. The production of 18F, like 11C, is also routinely performed at the TRIUMF facility. Fluorine-18 can be produced either using the TRIUMF 500 MeV 96 cyclotron, or more efficiently with the smaller TRIUMF/Nordion CP-42 cyclotron via the2°Ne(p,2pn)18Fnuclear reaction. For this work, circumstances required that the TRIUMF 500 MeV cyclotron be utilized. Radiofluorine was produced by proton irradiation of a gas mixture of natural Ne* with approximately 0.1% F2 present using the 20Ne(p,Spall)18Freaction at 500 MeV. The radiofluorine is obtained in the form of 18F- labelled F2. Unfortunately, the production of [‘8F]F2 is necessarily carrier-added. Since the cyclotron target is constructed from nickel-based alloy, it must be passivated with F2. This process creates a thin layer of nickel fluoride within the interior surface of the cyclotron target, and provides a source of carrier fluorine to be present. Moreover, a small percentage (0. 1-2.0%) of molecular fluorine must be present in the neon target gas for[18F]F2production. However, if the amount of carrier F2 is reduced too much in the target gas mixture in order to significantly increase specific activity, low yields and poor recoveries of[18F]F2are obtained.9 As a result, the practical upper limit on the specific activity of[18F1F2 that can be achieved is reported to be around 12 Ci/mmol.63 Therefore, all electrophilic fluorinations performed with18F-labelled F2, and reagents derived from [18F]F2, result in products of low specific activity.TM Whenever a radiofluorinated product must be produced in high specific activity, the only available labeffing reagent is[18Fjfluoride anion.65 Nonetheless, there has been discussion in the literature about producing high specific activity[18F]F2. The key idea is to exploit the stabifity of the negative fluorine ion in the gas phase and attract the gaseous anionic ‘8F *Natural neon consists of 90.92% of 20Ne, 0.26% of 21Ne, and 8.82% of 22Ne. 97 to an electrode, then perform an electrochemical oxidation to generate[18F]F2.9 To date, there are no reports of anyone exploiting this approach, but such a development would represent a true breakthrough for high specific activity radiofluorinations. Another aspect regarding the use of18F-labelled F2 is that the maximum radiochemical yield possible is 50%. Due to the need of carrier F2 to be present in the neon cyclotron target, it would be a statistical improbability that both fluorine atoms in the {18F]F2 produced would be fluorine-18 (i.e., 18F—’8). Therefore, only one fluorine-18 atom is expected to be present in a 18F-labelled F2 molecule (i.e., 18F—’9). As a result, if an electrophilic fluorination reaction proceeded in 100% maximum chemical yield, the corresponding radiofluorination procedure would be expected to give 50% radiochemical yield at best, since there is only a 50% probabffity of the fluorine-18 atom becoming incorporated rather than the fluorine-19 atom. For reactions using fluorination reagents such as CH3OO’8F,which contain only one fluorine atom, the maximum radiochemical yield continues to be 50% because all these reagents are prepared from[18F]F2. This is an unfortunate disadvantage of all radiofluorination methods that utilize[18F]F2 as the source of fluorine-18.9 The general procedure used for the radiofluorination reactions of 31 is as follows. The 500 MeV cyclotron gas target was filled with the desired amount of 1% F2/Ne gas mixture, then pure Ne was added to dilute the F2 gas content to approximately 0.1% F2 in Ne. The target gas mixture was irradiated with the 500 MeV proton beam. (Typical 18F production parameters were 10 mm of target irradiation at 69 A of beam current.) When irradiation of the cyclotron target was stopped, the time was noted and designated 98 as the end of bombardment (EOB). After target irradiation was completed, the contents of the target were passed through a potassium acetate/acetic acid column to produce gaseous acetyl[‘8F]hypofluorite and was added to a solution of 31 (110-120 mol) in CFC13 (20 mL). The reaction mixture was assayed for radioactivity (before and after evaporation of CFC13), along with the ammonium acetate/acetic acid column. After dissolution in some chloroform, a small aliquot of reaction mixture was subjected to radio-HPLC purification and the peak corresponding to‘8F-labelled fluorovinyl product was collected, then assayed for activity. An equal volume of reaction mixture was also assayed at the same time in order to determine the percentage of product that was present in the reaction mixture. Based on the total activity of ‘8F produced at EOB, the radiochemical yield was then determined. The resulting radiochemical yields were decay corrected back to BOB. Compound 31 was radiofluorinated with CH3OO’8F,as outlined in Scheme 3.VIIL Scheme 3 VIII HO HO CH3OO18F MeO CFC13 HO MeO 31 MeO3 The results obtained are summarized in Table 3.4. Compound 31 was kept in excess relative to fluorinating agent in order to minimize side-reactions and to maximize the efficiency of incorporation of radiofluorine. Using excess fluorinating agent would help maximize the chemical yield, but could produce lower radiochemical yields. The radiofluorination reaction of 31 gave the highest radiochemical yields (19%) when conducted at room temperature. The radiolabelled product that was isolated for yield determinations was a mixture of 3-methoxy-17-a-(E)-[8F]fluorovinyl-1,3,5( 10)-estra- Table 3.4: Summary of Radiochemical Yields Obtained for the Reaction of 31 with Acetyl[18FjHypofluorite Amount of Temperaturea Radiochemical CH3OO18Fused Yieldb —0.74 equiv r.t. 19% —0.50 equiv r.t. 19% —0.77 equiv -78°C 9.7% —0.50 equiv -78°C 5.0% ar.t. = room temperature. bRefers to the combined yield of 51 and 52; the radio- chemical yield was determined from the initial activity of[18FJF2produced at EOB. triene-17-o1 51 and 3-methoxy-17a-(Z)-[8F]fluorovinyl-1,3,5( 10)-estratrien.e-17i3-ol 52. The specific activity of this product mixture was not determined. However, in a separate experiment, the specific activity of CH3OO’8F generated using 18F production parameters described earlier was determined to be about 190 mCi/mmol. Therefore, the isolated product mixture would be of quite low specific activity, as expected. The identification of the[18F]fluorovinyl products 51 and 52 was accomplished using 100 radio-HPLC studies. The chromatographic behaviour of 51 and 52 was consistent with that observed for the related non-radioactive fluorovinyl compounds (39 and 40). In addition, the leftover reaction mixtures (after radio-HPLC analysis), obtained from the radiofluorinations done at room temperature (first two entries of Table 3.4), were allowed to decay to zero activity. These mixtures were then analyzed by ‘9F NMR spectroscopy. The 19J NMR spectra readily confirmed the presence of 39 and 40, thereby confirming the successful radiofluorination of 31. In this study, the fluorination reaction of 31 with gaseous acetyl hypofluorite was successfully extended to accomplish the 18F-labelling of 31. The radiofluorination reaction is essentially instantaneous upon addition of fluorinating agent. The time taken from EOB to the isolation of crude reaction mixture was in the order of 15-17 minutes. The additional time required thereafter was for the radio-HPLC purification work. Clearly, the synthesis time is quite short and well suited for radiolabelling with ‘8F. 3.5 Summary and Conclusions The purpose of this study was to develop a general methodology to directly prepare fluorovinyl compounds from vinyl-tin intermediates, and to utffize this synthetic approach for radiofluorinations with ‘8F. This objective was indeed fulfilled to a large degree, as most of the vinyl-tin substrates studied were successfully fluorinated using gaseous acetyl hypofluorite. In addition, one of the vinyl-tin derivatives was radiofluorinated with acetyl [‘8F]hypofluorite in respectable radiochemical yield. The vinyl-tin substrates were readily obtained from the AIBN catalyzed hydrostamiyl 101 ation of the corresponding acetylenic precursors, with tri-n-butyltin hydride, in 59-94% yield. The reactivities of (E)-vinylstannanes 30, 31, 32, 33, and 34 were studied with elemental fluorine and acetyl hypofluorite under varied conditions. The most effective fluorinating agent used to fluorinate 31 was gaseous acetyl hypofluorite, at room temperature, which afforded yields of 4 1-42% of 39 and 40 as an isomeric mixture (yields determined via HPLC analysis). In contrast, fluorination of 31 with elemental fluorine gave 9.0% yield at best. Furthermore, (E)-fluorovinyl 39 and (Z)-fluorovinyl 40 were prepared and isolated in 29.5% and 3.8% yields, respectively, from the reaction of 31 with CH3OOF. Compound 30 could not be directly fluorinated using either F2 or CH3OOF. This result is most likely due to the presence of the unprotected phenol function of 30. By protecting this group, it would be anticipated that 30 can be successfully fluorinated in an analogous manner to 31. Alternatively, 32 was found to be relatively unreactive toward fluorination as compared to 31. Reaction of 32 with gaseous CH3OOF produced only a trace of (E)-fluorovinyl product 43. Better results were observed employing elemental F2 as some 43 could be obtained as a minor component in a mixture of products. Compound 33 was readily fluorinated using gaseous CH3OOF. Treatment of 33 with CH3OOF (prepared in acetic acid solution) or with F2 also generated fluorinated product, but gave 44 as the main product. However, the stock of 33 had been virtually all consumed, so that further larger scale reactions with gaseous CH3OOF could not be performed. Therefore, fluorination studies were continued with L-glycero-a-D-galacto 102 epimer 34 instead. Compound 34 was allowed to react with gaseous CH3OOF (at room temperature) and (E)-fluorovinyl 47 and (Z)-fluorovinyl 48 were obtained in 36% and 10% yield, respectively. Compound 31 was radiofluorinated using gaseous acetyl[18F]hypofluorite in 19% radiochemical yield. The radiolabelled product consists of a mixture of (E) [‘8F]fluorovinyl 51 and (Z)-[18Fjfluorovinyl 52 of low specific activity. The reaction times were of the order of 15-17 minutes (measured from EOB) which is well suited for radiolabelling with 18F. 103 References 1. Fried, 3.; Sabo, E. F. .1 Am. Chem. Soc. 1954, 76, 1455-1456. 2. Ciba Foundation Symposium in Carbon-Fluorine Compounds: Chemistiy, Biochemistiy and Biological Activities; Elsevier: New York, 1972. 3. BiomedicinalAspects ofFluorine Chemistiy; Filler, R., Kobayashi, Y., Eds.; Kodansha Ltd.: Tokyo, 1982. 4. Furin, G. G. Soy. Sci. Rev., Sect. B: 1991, 16, 1-140. 5. Selective Fluorination in Organic and Bioorganic Chemistiy; Welch, J. T., Ed.; ACS Symposium Series 456; American Chemical Society: Washington, DC, 1991. 6. Filler, R.; Naqvi, S. M. In Biomedicinal Aspects of Fluorine Chemistiy; Filler, R., Kobayashi, Y., Eds.; Kodansha Ltd.: Tokyo, 1982; Chapter 1. 7. Welch, J. T. In Selective Fluorination in Organic and Bioorganic Chemistiy; Welch, J. T., Ed.; ACS Symposium Series 456; American Chemical Society: Washington, DC, 1991; Chapter 1. 8. Fowler, J. S.; Wolf, A. P. The Synthesis of Carbon-li, Fluorine-i 8, and Nitrogen-i3 LabeledRadiotracersforBiomedicalApplications; Nuclear Science Series, Monograph NAS-NS-3201; Technical Information Center, U.S. Department of Energy: Springfield, VA, 1982. 9. Kilbourn, M. R. Fluorine-i8 Labeling of Radiopharmaceuticals; Nuclear Science Series, Monograph NAS-NS-3203; National Academy: Washington, DC, 1990. 10. Nuclear Imaging in Dnsg Discoveiy, Development, and Approval; Burns, H. D., Gibson, R. E., Dannals, R. F., Siegi, P. K. S., Eds.; Birkhäuser: Boston, 1993. 11. Moissan, H. Compt. Rend. 1886, 102, 1543-1544; 103, 202-205. 12. Bockmüller, W. Liebigs Ann. Chem. 1933, 506, 20-59. 13. Purrington, S. T.; Kagen, B. S.; Patrick, T. B. Chem. Rev. 1986, 86, 977-1018. 14. Banks, R. E.; Tatlow, J. C. In Fluorine: The First Hundred Years (1886-1986); Banks, R. E., Sharp, D. W. A., Tatlow, J. C., Eds.; Elsevier Science: New York, 1986; Chapter 4. 15. Hudlicky, M. Chemistiy of Organic Fluorine Compounds: A Laboratoiy Manual with Comprehensive Literature Coverage, 2nd ed.; John Wiley and Sons: New York, 1976. 104 16. New Fluorinating Agents in Organic Synthesis; German, L., Zemskov, S., Eds.; Springer-Verlag: Berlin, 1989. 17. Sharts, C. M.; Sheppard, W. A. Org. React. (N Y.) 1974, 21, 125-406. 18. Gerstenberger, M. R. C.; Haas, A. Angew. Chem., mt. Ed. Engl. 1981, 20, 647-667. 19. Nickles, R. J.; Gatley, S. J.; Votaw, J. R.; Kornguth, M. L. mt. J. App!. Radial. Isot. 1986, 37, 649-66 1. 20. Berridge, M. S.; Tewson, T. J. mt. .1. App!. Radiat. Isot. 1986, 37, 685-693. 21. Adam, M. J., TRIUMF, Vancouver, BC, personal communication. 22. See reference 9; pp 70-90. 23. Rozen, S.; Lerman, 0.; Kel, M. .1. Chem. Soc., Chem. Commun. 1981, 443-444. 24. Fowler, J. S.; Shiue, C.-Y.; Wolf, A. P.; Salvador, P. A.; MacGregor, R. R. J. Labelled Compd. Radiopharm. 1982, 19, 1634-1636. 25. Rozen, S.; Lerman, 0.; Kol, M.; Hebel, D. I. Org. Chem. 1985, 50, 4753-4758. 26. Adam, M. J. .1. Chem. Soc., Chem. Commun. 1982, 730-73 1; Adam, M. 3.; Pate, B. D.; Nesser, J.-R.; Hall, L. D. Carbohydr. Res. 1983, 124, 215-224. 27. Jewett, D. M.; Potocki, 3. F.; Ehrenkaufer, R. E. J. Fluorine Chem. 1984,24,477-484. 28. Adam, M. J.; Pate, B. D.; Ruth, T. 3.; Berry, 3. M.; Hall, L D. .1. Chem. Soc., Chem. Commun. 1981, 733. 29. Adam, M. J.; Berry, 3. M.; Hall, L. D.; Pate, B. D.; Ruth, T. 3. Can. .1. Chem. 1983, 61, 658-660. 30. Ingham, R. K.; Rosenberg, S. D.; Gilman, H. Chem. Rev. 1960, 60, 459-539. 31. Larock, R. C. Tetrahedron 1982, 38, 1713-1754. 32. Adam, M. J.; Ruth, T. J.; Jivan, S.; Pate, B. D. .1. Fluorine Chem. 1984, 25, 329-337. 33. Coenen, H. H.; Moerlein, S. M. J. Fluorine Chem. 1987, 36, 63-75. 34. Di Raddo, P.; Diksic, M.; Jolly, D. J. Chem. Soc., Chem. Commun. 1984, 159-160. 35. Speranza, M.; Shiue, C.-Y.; Wolf, A. P.; Wilbur, D. S.; Angelini, G. J. Chem. Soc., Chem. Commun. 1984, 1448-1449. 105 36. Visser, G. W. M.; Halteren, B. W. v.; Herscheid, J. D. M.; Brinlcman, G. A.; Hoekstra, A. .1. Chem. Soc., Chem. Commun. 1984, 655-656. 37. Visser, G. W. M.; Bakker, C. N. M.; Halteren, B. W. v.; Herscheid, J. D. M.; Brinkman, G. A.; Hoekstra, A. .1. Org. Chem. 1986, 51, 1886-1889. 38. Ehrenkaufer, R. E.; MacGregor, R. R. mt. .1. Appi. Radiat. Isot. 1983, 34, 613-615. 39. Satyamurthy, N.; Bida,, G. T.; Barrio, J. R.; Phelps, M. E. J. Nucl. Med. 1984, 25, P23. 40. Oberdorfer, F.; Hofman, E.; Maier-Borst, W. J. Labelled Compd. Radiopharm. 1988, 25, 999-1005. 41. Adam, M. J.; Lu, J.; Jivan, S. J. Labelled CompcL Radiopharm. 1994, 34, 565-570. 42. Namavari, M.; Bishop, A.; Satyamurthy, N.; Bida, G.; Barrio, J. R. mt. J. Appi. Radiat. Isot. 1992, 43, 989-996. 43. Diksic, M.; Farrokhzad, S. J. Nuci. Med. 1985, 26, 1314-1318. 44. Luxen, A.; Barrio, 3. R.; Bida, G. T.; Satyamurthy, N. J. Labelled Compd. Radiopharin. 1986, 23, 1066-1067. 45. Mislankar, S. G.; Gildersleeve, D. L.; Muiholland, G. K.; Massin, C. C.; Toorongian, S. A.; Wieland, D. M. .1 Nucl. Med. 1987, 28, 624. 46. Barrio, 3. R.; Perimutter, M. M.; Luxen, A.; Melega, W. P.; Grafton, S. T.; Huang, S. C.; Hoffman, J. M.; Van Moffaert, G.; Mazziotta, J. C.; Phelps, M. E. J. Nuci. Med. 1989, 30, 752; Gildersleeve, D. L.; Van Dort, M. E.; Rosenspire, K. C.; Toorongian, S.; Wieland, D. M. .1. Nucl. Med. 1989, 30, 752. 47. Luxen, A.; Perimutter, M. M.; Barrio, J. R. J. Labelled CompcL Radiophwm. 1989, 26, 1-2. 48. Purrington, S. T.; Jones, W. A. J. Fluorine Chem. 1984, 26, 43-46. 49. Barnette, W. E. .1. Am. Chem. Soc. 1984, 106, 452-454. 50. Knapp, F. F., Jr.; Goodman, M. M.; Kabalka, G. W.; Sastry, K. A. R. J. Med. Chem. 1984, 27, 94-97; Penman, M. E.; Watanabe, K. A.; Schinazi, R. F.; Fox, J. J. .1. Med. Chem. 1985, 28, 74 1-748. 51. Flanagan, R. 3.; Charleson, F. P.; Synnes, E. I.; Wiebe, L. I.; Theriault, Y. X.; Nakashima, T. T. Can. J. Chem. 1985, 63, 2853-2860. 106 52. Franke, L. A.; Hanson, R. N. J. Nucl. Med. 1984, 25, 1116-1121. 53. Bey, P.; McCarthy, 3. R.; McDonald, I. A. In Selective Fluorination in Organic and Bioorganic Chemistry; Welch, J. T., Ed.; ACS Symposium Series 456; American Chemical Society: Washington, DC, 1991; Chapter 8. 54. Ailmendinger, T.; Felder, E.; Hungerbuehler, E. In Selective Fluorination in Organic and Bioorganic Chemistiy; Welch, J. T., Ed.; ACS Symposium Series 456; American Chemical Society: Washington, DC, 1991; Chapter 13. 55. Balatoni, J. A., M.Sc. Thesis, The University of British Columbia, Dec. 1985. 56. van der Kerk, G. J. M.; Noltes, J. C. J. App!. Chem. 1959, 9, 106-113. 57. Jung, M. E.; Light, L. A. Tetrahedron Left. 1982, 23, 385 1-3854; Ensley, H. E.; Buescher, R. R.; Lee, K. .1. Org. Chem. 1982, 47, 404-408. 58. Omae, I. J. Organomet. Chem. Libr. 1989, 21, 1-341. 59. Poller, R. C. The Chemzstiy of Organotin Compounds; Academic: New York, 1970; pp 112-113. 60. Banks, R. E.; Haszeldine, R. N.; Prodgers, A. J. Chem. Soc., Perkin Trans. 1 1973, 596-598. 61. Di Raddo, P.; Diksic, M. mt. J. App!. Radiat. Isot. 1985, 36, 953-956. 62. Lee, S. H.; Schwartz, J. J. Am. Chem. Soc. 1986, 108, 2445-2447. 63. See reference 9; p 5. 64. See reference 9; pp 22-24. 65. See reference 9; p 37 and 43. 107 Chapter 4 GENERAL CONCLUSIONS The investigation presented in Chapter 2 demonstrated that(6-arene)tricarbony1- chromium complexes can be used as synthetic intermediates for the radiolabelling of the attached arene ring with “C via nucleophilic substitution with[11C]cyanide. This result represents the first application of chromium tricarbonyl complexes for radiolabelling with short-lived nucides. From a broader perspective, however, this accomplishment could be the first step to a range of new radiolabelling methods based on the utilization of organotransition metal complexes. A number of transition metal systems facilitate the addition of various nucleophiles onto unsaturated organic molecules while complexed to the metal centre.’ This pattern of reactivity needs to be explored further with nucleophilic forms of medically useful radioisotopes,2such as ‘8F, 75Br, 77Br, and 123j The application of chromium tricarbonyl complexes could be significantly advanced with additional studies. A further exploration of reaction conditions should be conducted to improve radiochemical yields, particularly with NCA [“Cjcyanide. This could include an investigation of various crown ether and other catalysts, employment of microwave drying of the [“C]cyanide solution and microwave heating of the radiolabeffing reaction, and the use of smaller volumes of reaction solvent to concentrate the reactants. Also, a study of alternative ways of trappingH11CN in the absence of hydroxide may result in 108 significantly improved labelling yields. Furthermore, other potentially effective leaving groups should be explored to complement or supersede fluorine. Lastly, the substitution reactions of F-, Br, and 1 with chromium tricarbonyl complexes warrant study because of the importance of radiohalogens in nuclear medicine.2 The investigation described in Chapter 3 revealed that vinyl-tin derivatives can be directly fluorinated with gaseous acetyl hypofluorite, in most cases, to produce stable and ‘8F-labelled vinyl fluorides. This result represents another extension of the use of organotin compounds for electrophilic fluorinations, including radiofluorinations with‘8F. Very recently, other researchers have reported using vinyl-tin compounds to prepare vinyl fluorides. Tius and Kawakami3fluorinated a number of simple vinyl-tin derivatives with XeF2 (in the presence of AgPF6)in 24-52% yield, however, reaction times ranged from 3-18 hours. Hodson and co-workers4used cesium fluoroxysulfate to fluorinate some vinyl-tin compounds which produced vinyl fluorides in 25-56% yield, and in some cases, a-fluoroketones were unexpectedly made in 47-75% yield (reactions were run overnight). Matthews et al.5 reported the electrophilic fluorination of several (fluorovinyl)stannanes with 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2joctane bis(tetrafluoroborate) affording difluoro olefins in 35-74% yield (30 mm reaction time at 80°C). Each of these reports provides new methods for fluorinating vinyl-tin compounds which is very positive, however, each of these methods, for various reasons, would appear to be incompatible for radiolabelling with ‘8F. What is gratifying is that several years after choosing vinyl-tin intermediates for our fluorination studies, other research groups are also selecting tin reagents for the preparation of vinyl fluorides. 109 Undoubtedly, the application of organotin compounds will continue to be extended as interest in selectively fluorinated organic molecules remains. Nonetheless, the following suggestions for future work can be made. The exploration of alternative fluorinating agents for use with vinyl-tin substrates would be very prudent. Also, the reactivity of fluorine and acetyl hypofluorite should be further examined with a wider range of structurally diverse vinylstannylated derivatives. Moreover, the fluorination of vinyl-silicon and -mercury derivatives warrant some additional study. In retrospect, it is pleasing to see that although a few years have unfortunately elapsed since the experimental work was performed and the writing of this thesis completed, the results obtained are still new and currently relevant, and have not been duplicated in the literature. Even though various aspects of the specific experimental studies could have been done differently, the studies pursued for this thesis were well worthwhile and exhibit significant future potential. The application of organometallic compounds as synthetic intermediates for radiolabelling is only in the early stages of development still, but will continue to expand with future research. 110 References 1. Davies, S. G. Organotransition Metal Chemistiy: Applications to Organic Synthesis; Pergamon: Toronto, 1982; Chapter 4. 2. Nozaki, T. In Radionuclides Production; Helus, F., Ed.; CRC: BocaRaton, 1983; Vol. 2, Chapter 3. 3. Tius, M. A.; Kawakami, J. K. Synth. Commun. 1992, 22, 1461-1471. 4. Hodson, H. F.; Madge, D. 3.; Widdowson, D. A. Synlett 1992, 831-832. 5. Matthews, D. P.; Miller, S. C.; Jarvi, E. T.; Sabol, J. S.; McCarthy, J. R. Tetrahedron Lett. 1993, 34, 3057-3060. 111 Chapter 5 EXPERIMENTAL 5.1 General Methods Solutions were concentrated under reduced pressure with a Büchi rotary evaporator, except for solutions of organochromium complexes, which were concentrated in vacuo using a high vacuum, rotary vacuum pump assembly. Melting points were taken in capillaries (in air) using a Büchi 510 oil bath melting point apparatus, or were obtained on a Fisher-Johns melting point apparatus, and are uncorrected. Optical rotations were determined using a Perkin-Elmer model 141 polarimeter. Tetrahydrofuran (THF) and diethyl ether were distilled from calcium hydride or sodium benzophenone ketyl.’ Dimethyl sulfoxide (DMSO) was distilled under reduced pressure from calcium hydride, then carefully stored under nitrogen. Di-n-butyl ether was distilled from sodium under nitrogen, while hexane, dichioromethane, and acetonitrile were distilled from calcium hydride. Methanol was distified from magnesium methoxide prepared in situ by reaction of methanol with magnesium turnings. For high pressure liquid chromatography (HPLC) work, singly distilled water, HPLC grade acetonitrile and methanol, and reagent grade hexanes and diethyl ether were used, which were filtered (0.45 m Millipore brand Durapore membrane) before use. All other solvents used were of spectro or reagent grade, and were used without further treatment. 112 Thin-layer chromatography (TLC) was performed on pre-coated silica gel plates (Baker-flex Silica gel 1B2-F or E. Merck Silica gel 60, No. 5534). Visualization was effected either by (a) spraying with 30% sulfuric acid in ethanol, then heating, (b) with short-wavelength UV light, or (c) by visual inspection (for coloured, organochromium compounds). The TLC solvent systems used were very similar to those listed for the column chromatography of the individual compounds. Column chromatography was performed using Silica gel 60 (E. Merck, 70-230 mesh). Flash chromatography was performed using Silica gel 60 (E. Merck, 230-400 mesh) by the method of Still et al.2 HPLC was done with a system consisting of a Spectro-Physics SP 8700 solvent delivery system, a Rheodyne 7126 injector, an ISCO (model V4) variable wavelength UV detector operated at 254-280 nm, and a Spectro-Physics SP 4270 integrator recorder, using one of the following columns, either (a) a Waters 10 Lm C-18 reverse phase RCM column (column A), (b) two Waters 10 m C-18 reverse phase RCM columns connected in series (column B), (c) a C-18 reverse phase Whatman Partisil 10 ODS-3 column, 25 cm x 9 mm, equipped with a Waters C-18 guard column (column C), or (d) normal phase Phenomenex Ultremex 5 Silica column, 25 cm x 10 mm (column D). The following solvent mixtures were used for HPLC: (i) methanol/water, 1:1 (solvent A), (ii) water/acetoriitrile, 1:1 (solvent B), (iii) water/acetonitrile, 3:2 (solvent C), and (iv) hexanes/diethyl ether, 4:1 (solvent D). The following gradient solvent programs were also used for HPLC: (i) from 0 to 10 mm, an isocratic mixture of methanol/water (85:15) was used, followed by a systematic increase to 100% methanol during 10 to 12 mm, and a constant composition of 100% methanol was maintained from 12 to 20 mm 113 (solvent program A), and (ii) from 0 to 18 mm, an isocratic mixture of methanol/water (75:25) was used, followed by a systematic increase to 100% methanol during 18 to 21 mm, and a constant composition of 100% of methanol was maintained from 21 to 30 mm (solvent program B). Radio-HPLC analysis and purification was performed with the same HPLC system fitted with a NaI(Tl) scintillation detector system and a dual channel strip chart recorder. Radioactive samples were assayed with either a Capintec well counter (model CRC-543X) or a Beckman 8000 scintillation counter. Analytical gas chromatography (GC) was performed with a Hewlett-Packard model 5840A GC, equipped with an FID detector, using a 30 m x 0.75 mm i.d. wide bore (Supelco SPB-1) capillary column. Unless otherwise stated, GC analyses were performed isothermally using the following conditions: injection temperature, 200°C; oven temperature, 120°C; N2 carrier gas flow, 2.0 mt/mm. Low resolution electron impact mass spectra were recorded with either a Kratos/AEI MS902 or a Kratos/AEI MS5O mass spectrometer. An ionization potential of 70 eV was used for the mass spectra obtained. Spectra are quoted as m/z values, while selected ion fragmentations are reported as percentages of the base peak. High resolution mass measurements were determined using the Kratos/AEI MS5O mass spectrometer. Gas chromatography-mass spectrometry (GC-MS) was performed using a Delsi Nermag RiO 1OC quadruple mass spectrometer interfaced with a Varian 6000 GC or a Kratos MS8O coupled to a Carlo Erba 4160 GC. All microanalyses were performed by Mr. P. Borda, Microanalytical Laboratory, University of British Columbia. 114 5.2 NMR Methods and Instrumentation 1H NMR spectra were measured at room temperature at 270 and 400 MHz. The 270 MHz spectra were obtained with a home-built spectrometer based on a Bruker WP-60 console, a Nicolet 1180 computer (32K), a Nicolet 293B pulse programmer, a one Megabyte Diablo disk drive (model 31), and an Oxford Instruments Superconducting solenoid magnet. In addition to this spectrometer, a separate data processing system, consisting of another Nicolet 1180 computer (32K), one Megabyte Diablo disk drive (model 31) and a digital plotter, was available for processing and plotting of NMR data. Standard NTCFTB software was used on the spectrometer and data station. Furthermore, some of the 270 MHz data was transferred from the Nicolet 1180 data station to a newer Nicolet 1280 computer, for processing with increased computer memory and digital plotting. The 400 MHz spectra were obtained with a Bruker WH-400 high-resolution spectrometer equipped with an Aspect 3000 computer. Along with this spectrometer, a separate Bruker data processing system, comprised of an Aspect 3000 computer, digital plotter and dot matrix printer, was available for data processing and plotting. Factory Bruker software was used on the spectrometer and data station. ‘9F NMR spectra were measured at 188 and 254 MHz, also at room temperature. The 188 MHz spectra were obtained with a Bruker AC-200E spectrometer. The NMR data from this spectrometer was transferred to and processed on the Bruker data station, described above. The 254 MHz spectra were obtained using the previously described home-built spectrometer (controlled by the Nicolet 1180 computer) with a home-built, 115 19F-tuned probe.* 5.3 Experimental for Chapter 2 5.3.1 Sources of Materials Chemicals and reagents were purchased from suppliers as follows. Chromium hexacarbonyl was obtained from Pressure Chemical Co. Fluorobenzene, 2- and 4- fluorotoluene, 4-chiorofluorobeuzene, 4-chlorobenzonitrile, methyltrifluoromethane sulfonate, and 18-crown-6 were purchased from Aldrich Chemical Co. Eastman Kodak Co. supplied the benzonitrile and 4-tolunitrile, and ICN Pharmaceuticals Inc. supplied the 2-tolunitrile. HPLC grade methanol and acetonitrile were obtained from Fisher Scientific Ltd., or BDH Chemicals. 5.3.2 General All manipulations for the preparation and purification of the(6-arene)tricarbonyl- chromium complexes were performed so as to maintain all chemicals under an atmosphere of nitrogen or argon using conventional bench-top techniques for the manipulation of air-sensitive compounds.3 The(6-arene)tHcarbonylchromium complexes, that were synthesized from Cr(CO)6, were prepared either using a 200-mL, round-bottom, three-neck flask fitted with a nitrogen inlet, a magnetic stir bar, and a condenser equipped with a mineral oil-bubbler The probe construction and electronics was done by Tom Markus (Electronics shop, Chemistry Department, U.B.C.). 116 (glassware A), or a specially constructed reaction apparatus (glassware B) that consists of a 250-mL, round-bottom flask fused to a 3 in. long condenser which is joined to an additional 3 in. long glass tube (1 in. o.d.) equipped with a central ST* 24 ground glass joint and two angled ST 19 ground glass joints. This glassware is fitted with a glass rod that is placed through the central ST 24 joint and reaches to a dimple at the bottom of the flask and has a small paddle near the bottom of the flask (to stir the reaction mixture) plus a 3 in. long screw paddle which closely fits the interior wall of the condenser region (to scrap and return sublimed Cr(CO)6to the reaction mixture). The glass rod is rotated by a variable speed, overhead stirrer motor. The assembled glassware is also equipped with a nitrogen inlet and mineral oil bubbler. With either set-up, the glassware was wrapped with aluminum foil to protect the reaction from light. Silica gel 60 (E. Merck, 70-230 mesh) was used for the filtration of organochromium solutions to remove any decomposition. All substitution reactions were carried out in oven-dried Pierce Reactivials® (Rockford, IL) equipped with magnetic stir bars and screw caps that were fitted with Teflon-lined, silicone septa.t H”CN was produced via the catalytic conversion of “CO2. The “CO2 was produced on the TRIUMF/Nordion CP-42 cyclotron using the‘4N(p,a)11Creaction at 15 MeV. The “CO2 in the N2 target gas was converted to “CH4 by mixing the target gas with *sT denotes standard taper. 1These septa provided an excellent seal to maintain the exclusion of air and moisture, and to prevent the loss of volatile components. 117 H2(g) then passing the mixture over a Ni cata]yst at 450°C. Thereafter, the 11CH4 was combined with NH3(g) and passed over Pt at 1000°C, thus obtaining H”CN.4 The H’1CN was trapped in an aqueous solution of NaOH (1 mL, 0.1 M) to produce Na”CN. 5.3.3 Preparation of(,6-Arene)tricarbonylchromium Complexes Preparation of -fluorobenzene)tricarbonylchromium 1. Chromium hexacarbonyl (1.0 g, 4.54 mmol) and fluorobenzene (5.0 mL, 53 mmol) were dissolved in a mixture of (n-Bu)20/THF (80 mL/10 mL) in glassware A. The reaction mixture was degassed by performing three freeze-pump-thaw cycles. Upon reintroducing a nitrogen atmosphere into the reaction vessel, the stirred reaction mixture was heated to reflux for 48 h. The reaction mixture was cooled to room temperature and filtered through a short pad of silica gel to remove any greyish-green decomposition. The bright yellow filtrate was evaporated to dryness in vacuo. The residue was dissolved in diethyl ether, and the resulting solution was transferred by canula filtration into a Schlenk tube. The undissolved residue was then washed with some hexane and these washings were likewise added to the ether solution. Solvent was slowly removed under reduced pressure until yellow crystals began to appear. The ether/hexane mixture was warmed gently until all crystals redissolved, then was placed in the freezer for crystallization. Bright yellow crystals were isolated and dried under a flow of N2, initially, then under a high vacuum overnight. Two additional crops of crystals were obtained from the mother liquor, affording a total yield of 0.84 g (80%) of 1, mp 117°C {lit.5 mp 116-117°C}. Mass spectrum, m/z: 232 (M, 38), 204 (M-CO, 3), 176 (M 118 2(CO), 9), 148 (M-3(CO), 54), 96 (M-Cr(CO)3,21), 71(33), 52 (100). Preparation of(,6-ch1orobenzene)tricarbony1chromium 2. Chromium hexacarbonyl (1.86 g, 8.45 mmol) and chlorobenzene (25 mL, 0.25 mol) were added to diglyme (30 mL) in glassware B. The stirred reaction mixture was heated to reflux for 16.5 h. The reaction mixture was cooled to room temperature, then filtered through a short pad of Celite using some diethyl ether to rinse and facilitate the transfer of the reaction vessel contents. The yellow filtrate was concentrated via distillation under reduced pressure, then some petroleum ether was added to induce crystallization. Even with cooling, no crystals had formed. Therefore, some ethyl acetate was added to this solution and was then evaporated in vacuo. This process was repeated several times and the volume of diglyme was successfully reduced. Once again, petroleum ether was added to the concentrated solution till a small amount of precipitation occurred, then was placed in a fridge for cooling. A single large, yellow crystal was obtained, which was removed and washed with cold petroleum ether, then dried under a flow of argon. The filtrate was heated and filtered hot, to remove some greenish decomposition, then was placed back in the fridge for crystallization. Yellow crystals (400 mg) were isolated and dried in vacuo. The large single crystal, obtained initially, was recrystallized from hot petroleum ether. This afforded 265 mg of additional yellow crystals, which gave a total yield of 47% (665 mg) of 2 (based on Cr(CO)6 consumed*), mp 100-101°C {lit.6 mp *Unreacted Cr(CO)6remaining in the reaction vessel was recovered by sublimation under high vacuum; this gave 0.60 g of recovered Cr(CO)6. 119 101-102°C}. Preparation of(6-bromobenzene)tricarbony1chromium 3. Chromium hexacarbonyl (4.00 g, 18.2 mmol) and bromobenzene (10.0 mL, 95.2 mmol) were added to a mixture of (n-Bu)20/THF (100 mL/10 mL) in glassware B. The reaction mixture was degassed by performing a single freeze-pump-thaw cycle. Upon reintroducing an N2 atmosphere over the reaction mixture, it was heated, with stirring, to reflux for 44 h. The cooled reaction mixture was filtered through a short pad of silica gel and the yellow filtrate was reduced to dryness in vacuo. Compound 3 was obtained as dark yellow crystals, in a yield of 992 mg (19%), mp 101-105°C {lit.7 mp 120°C}. Mass spectrum, m/z: 294 (81Br: M, 31), 292 (79Br: M, 34), 266 (81Br: M-CO, 3), 264 (79Br: MtCO, 3), 238 (81Br: M-2(CO), 6), 236 (79Br: M-2(CO), 6), 210 (81Br: M 3(CO), 26), 208 (79Br: Mt3(CO), 28), 158 (81Br: M-Cr(CO)3,5), 156 (79Br: M Cr(CO)3,4), 133 (81Br: 5), 131 e9Br: 7), 52 (100). Preparation of(i6-2-fluorotoluene)tricarbonylchromium 4. Chromium hexacarbonyl (1.0 g, 4.54 rninol) and 2-fluorotoluene (5.8 mL, 53 mmol) were dissolved in a mixture of (n-Bu)20/THF (80 mL/10 mL) in glassware A. All subsequent steps were essentially identical to the preparation of compound 1. Compound 4 was obtained as yellow crystals, in a total yield of 0.99 g (89%), mp 71- 72°C {lit.8 mp 73-74°C}. Mass spectrum, m/z: 246 (M, 15), 218 (M-CO, 1), 190 (M 2(CO), 4), 162 (Mt3(CO), 25), 110 (M-Cr(CO)3,26), 109 (44), 71(12), 52 (100). 120 Preparation of(i76-4-fluorotoluene)tricarbonylchromium 5. Chromium hexacarbonyl (4.00 g, 18.2 mmol) and 4-fluorotoluene (10.0 mL, 91 mmol) were added to a mixture of (n-Bu)20/THF (100 mL/10 mL) in glassware B. The reaction mixture was degassed by performing a single freeze-pump-thaw cycle. Upon reintroducing an N2 atmosphere over the reaction mixture, it was heated, with stirring, to reflux for 16.5 h. The cooled reaction mixture was filtered through a short pad of silica gel and the yellow filtrate was reduced to dryness under reduced pressure. After further drying in vacuo, compound 5 was obtained as bright yellow crystals, in a yield of 3.79 g (85%), mp 59-60°C {lit.8 mp 61-62°C}. Mass spectrum, m/z: 246 (M, 11), 218 (M-CO, 1), 190 (M-2(CO), 3), 162 (M-3(CO), 23), 110 (M-Cr(CO)3,17), 109 (31), 71(16), 52 (100). Preparation of (q’-4-chlorofluorobenzene)tricarbonylchromium 6. Chromium hexacarbonyl (4.00 g, 18.2 mmol) and 4-chlorofluorobenzene (10.0 mL, 93.9 nimol) were added to a mixture of (n-Bu)20/THF (100 mL/10 mL) in glassware A. The stirred reaction mixture was heated to reflux for 23 h. Upon cooling to room temperature, the reaction mixture was concentrated to about 10 mL under reduced pressure and a plug of sublimed Cr(CO)6(1.1 g) was recovered from the condenser. The concentrated reaction mixture was chromatographed on neutral alumina (Fisher, 80-200 mesh) with hexane as the eluent. A single yellow band was collected and the solvent was removed in vacuo. TLC analysis, on alumina (eluent: hexane), of the yellow residue showed the presence of two components. Column chromatography was repeated on the 121 two-component mixture using silica gel with hexane/ether (5:1). The first fraction, after solvent removal, afforded 0.42 g of 6 as yellow crystals, mp 6 1-62°C {lit. mp: none found}. TLC analysis on silica gel (eluent: hexane/ether, 1:1), indicated the presence of only one component. Mass spectrum, m/z: 268 (37C1: M, 6), 266 (35C1: M, 17), 240 (37C1: M-CO, 2), 238 (35C1: M-CO, 3), 212(37C1: M-2(CO), 2), 210 (35C1: M-2(CO), 5), 184 (37C1: M-3(CO), 9), 182 (35C1: M-3(CO), 25), 132 (37C1: M-Cr(CO)3,10), 130 (35C1: M-Cr(CO)3,29), 95 (22), 89 (37C1: 3), 87 (35C1: 9), 71 (14), 52 (100). A second fraction (0.12 g) was collected, which contained a mixture of 1 and 6, with 6 being the dominant component as determined by ThC analysis (silica gel; eluent: hexane/ether, 1:1). This material unfortunately underwent partial decomposition and was discarded. A final fraction was collected, and after the eluate was evaporated to dryness in vacuo, 0.11 g of yellow crystals were isolated, mp 97-100°C. TLC analysis (silica gel; eluent: hexane/ether, 1:1) showed this material to be a mixture of 1 and 6, with 1 being the major component present. Mass spectrometric analysis of this mixture gave the following results; mass spectrum, m/z: compound 1, 232 (M, 22), 204 (M-CO, 3), 176 (M 2(CO), 5), 148 (Mt3(CO), 27), 96 (M-Cr(CO)3,40); compound 6, 268 (37C1: M, 0.6), 266(35C1: M, 2.0), 240(37C1: MtCO, 0.1), 238(35Cl: M-CO, 0.3), 212(37C1: M-2(CO), 0.2), 210(35C1: M-2(CO), 0.4), 184(37C1: M-3(CO), 1.1), 182(35C1: M-3(CO), 3.7), 132 (37Cl: MtCr(CO)3,1.8), 130 (35C1: M-Cr(CO)3,6.9). The isolated yield of 6 (obtained from the first fraction) was 12%, based on the amount of Cr(CO)6consumed. 122 Preparation of(q6-N,N-dimethylaniline)tricarbonylchromium 7. Chromium hexacarbonyl (4.00 g, 18.2 mmol) and N,N-dimethylaniline (20 mL, 0.16 mol) were dissolved in a mixture of (n-Bu)20/THF (60 mL/5 mL) in glassware A. The stirred reaction mixture was heated to reflux for 21 h. After cooling to room temperature, the volume of the reaction mixture was concentrated under reduced pressure and a large crop of yellow crystals deposited. The remaining supernatant was then canula transferred to another flask. The crystals were collected on a glass frit and washed repeatedly with cold hexane, then dried in vacuo. Compound 7 was isolated as yellow crystals, for a yield of 3.30 g (70%), mp 137-138°C {lit.6 mp 144°C}. Preparation of ((-benzonitri1e)tricarbony1chromium 10. DMSO (2 mL) was added to compound 1 (44.8 mg, 0.193 mmol) and NaCN (21.4 mg, 0.437 minol) contained in a 5-mL Reactivia1® under argon. The reaction mixture was stirred for 23 h at ambient temperature. The reaction mixture was then added to water (20 mL) and subsequently extracted with diethyl ether (3 x 15 mL). The ether extracts were washed with aqueous, saturated sodium chloride solution and dried over anhydrous magnesium sulfate. The dried ether extracts were filtered through a short pad of silica gel and the solvent wasjaken to dryness under reduced pressure. Compound 10 was isolated as yellow crystals, in a yield of 32 mg (69%). 123 Preparation of(6-N,N,N-trimethy1aniIinium)tricarbony1chromium trifluoromethane sulfonate 11. Compound 7 (1.00 g, 3.89 rnmol) was added to CH21 (25 mL) and stirred at room temperature until dissolved. Methyltrifluoromethanesulfonate (1.1 mL, 9.7 rnmol) was added by syringe and the mixture was stirred overnight. Yellow crystals had deposited and the supernatant was canula transferred to another flask. This solution was stirred overnight. The yellow crystals were dried in vacuo, then dissolved in acetonitrile. The resulting solution was transferred by canula filtration into a Schienk tube. The volume of CH3N was reduced by a third under reduced pressure, then diethyl ether was added until the solution became slightly cloudy. This mixture was placed in the freezer overnight. The previous solution, in which the reaction was allowed to continue, afforded another crop of yellow crystals. These were isolated and recrystallized as described for the first batch of crystals. Both batches of crystals were isolated, and dried under a flow of N2 initially, then under high vacuum. Compound 11 was obtained as yellow crystals, in a total yield of 0.97 g (59%). An analytical sample was obtained by performing two sequential recrystallizations of a portion of 11 from acetonitrile/diethyl ether, mp 120- 121°C (dec.).* Anal. calcd. forC,3H4rFNO6S:C 37.06, H 3.35, N 3.32, S 7.61; found: C 37.40, H 353, N 3.49, S 7.44. ‘H NMR (270 MHz, DMSO-d6)& 3.56 (s, 9H, N(CH3),5.77 (t, 2H, H-3,5), 6.07 (t, 1H, H-4), 6.69 (d, 2H, H-2,6). *Performed in capillaries that were packed and sealed under nitrogen. 124 Preparation of N,N,N-trimethylanilinium trifluoromethanesulfonate 12. N,N-Dimethylaniline (1.00 mL, 7.89 mmol) and methyltrifluoromethanesulfonate (1.1 mL, 9.7 mmol) were added sequentially by syringe to CH21 (25 mL) while under a nitrogen atmosphere. The reaction mixture was allowed to stir at room temperature. After 30 mm, TLC analysis indicated that all the N,N-dimethylaniline present was consumed. The copious quantities of white crystals that deposited, were collected and dried in vacuo. These crystals were recrystallized from dichloromethane/diethyl ether, which afforded a total yield of 1.87 g (83%) of 12, mp 82-83°C {lit. mp: none found}. 1H NMR (270 MHz, DMSO-d6)&: 3.61 (s, 9H, N(CH3),7.58-7.67 (m, 3H, H-3,4,5), 7.97 (d, 2H, H-2,6). Preparation of N,N,N-trimethylanilinium iodide 13. N,N-Dimethylaniline (1.00 mL, 7.89 mmol) and iodomethane (0.54 mL, 8.67 mmol) were added to CH21 (25 mL) under a nitrogen atmosphere. The reaction mixture was allowed to stir overnight at room temperature. The resulting white crystals which had deposited were collected, washed with diethyl ether and dried under reduced pressure. Compound 13 was obtained in a yield of 1.16 g (56%). 1H NMR (270 MHz, DMSO-d6) & 3.61 (s, 9H, N(CH3),7.58-7.68 (m, 3H, H-3,4,5), 7.97 (d, 2H, H-2,6). 5.3.4 Substitution Reactions of Organochromium Complexes Substitution reactions with stable cyanide. General Procedure: An aliquot (200 L, 10 mg/mL) of aqueous KCN (32 mo1) was 125 injected into a 5-mL Reacti-vial under inert atmosphere. The water was evaporated to dryness, using a block heater, under a rapid flow of nitrogen or argon. The (,- arene)tricarbonylchromium complex (65 mol) was taken up in DMSO (1 mL), then added by syringe to the Reacti-vial®. The stirred mixture was heated at the desired temperature in a thermostated silicone oil bath for 10 minutes. After cooling (ice/water bath) to room temperature, the reaction mixture was quantitatively transferred to a volumetric flask and diluted to a known volume with DMSO. This solution was analyzed by HPLC, which was standardized with known solutions of expected aryl nitrile product, to determine the extent of product formation. The chemical yields were calculated using KCN as the limiting reagent. A separate set of reactions were performed to isolate the organic products and identify these by GC-MS, therefore, the workup of these reaction mixtures, after cooling, was conducted as follows. The reaction mixture was diluted with water or saturated NaC1 solution (3 mL), and extracted with diethyl ether (1 x 6 mL, 2 x 3 mL). The combined ether extracts were washed with water or saturated NaC1 solution (2 x 3 mL). The stirred ether solution was treated with iodine (146 mo1) at 0°C for 2 hours to ensure complete decomplexation. The treatment was quenched with the addition of aqueous sodium thiosulfate solution (5 mL, 0. 1M) and the ether layer was washed further with sodium thiosulfate solution (4 mL, 0.1M), then with saturated NaC1 solution (2 x 4 mL) and was dried over anhydrous magnesium sulfate. The dried ether layer was filtered and concentrated under reduced pressure to 1 mL. The concentrated ether solution was analyzed initially by GC and HPLC, then by GC-MS. 126 Reaction of 1 with cyanide. A typical reaction trial was performed as follows. Compound 1 (15.6 mg, 67.21Lmol) was allowed to react with KCN (2.10 mg, 32.2 mol), at 135°C, as described in the general procedure above. The final DMSO mixture was analyzed by HPLC (column A; eluent: solvent A; flow rate, 2.5 mL/min; UV detection, 280 nm) and it was determined that 1.34 mg of benzonitrile 14 (RT* = 5.4 mm) was present, for a yield of 40%. Other reaction trials were conducted at 105, 115, 120, and 150°C, which gave yieldst of 12, 32, 31, and 33%, respectively. Reaction of 4 with cyanide. A typical reaction trial was performed as follows. Compound 4 (16.2 mg, 65.8 mol) was allowed to react with KCN (2.10 mg, 32.2 jmol), at 105°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (column B; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 rim) and it was determined that 1.62 mg of 2-tolunitrile 15 (RT= 12.1 mm) was present, for a yield of 43%. Other reaction trials were conducted at 95, 115, 125, and 135°C, which gave yieldst of 26, 41, 29, and 36%, respectively. Two reaction trials were performed using 1.5 mg (6.1 1Lmol) of 4 with KCN (2.00 mg, 30.7 mol), at 135 and 143 °C, using the general procedure. In each case, 0.41 mg of 15 ‘RT denotes retention time. 1These results represent the best chemical yields obtained where more than one reaction trial was performed. 127 was obtained, for a yield of 58% based on 4 as the limiting reagent. A separate reaction trial was done to identify the organic products by GC-MS. Compound 4 (16.2 mg, 65.8 mol) was allowed to react with cyanide, at 120°C, as described in the general procedure. The concentrated ether solution was analyzed by GC and only two significant peaks were observed, which were identified as 2- fluorotoluene (RT=3.2mm) and 2-tolunitrile 15 (RT=5.7 mm). This sample was further analyzed by HPLC (column A; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 nm) and found that 15 eluted first (RT=7.3 mm), followed by 2-fluorotoluene (RT 16.2 nun). Identification of the above products was accomplished by the comparison of the GC and HPLC retention times with those of authentic samples. The identity of the products was further confirmed by GC-MS, using for comparison the mass spectra (acquired under very similar instrumental conditions) obtained from a premade solution of authentic samples. Mass spectra, m/z: 2-fluorotoluene, 110 (M, 48), 109 (M-H, 100), 108 (M-2H, 15), 89 (3), 83 (15), 74 (12), 59 (15), 57 (6); compound 15, 117 (M, 100), 116 (M4-H, 85), 115 (M4-2H, 13), 90 (44), 89 (47), 63 (16). Reaction of 5 with cyanide. A typical reaction trial was performed as follows. Compound 5 (16.1 mg, 65.4 ILmol) was allowed to react with KCN (2.10 mg, 32.2 /Lmol), at 115°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (column B; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 nm) and it was determined that 1.11 rng of 4-toluriitrile 16 (RT= 12.2 mm) was present, for a yield of 29%. 128 Other reaction trials were conducted at 105, 125, 135, and 150°C, which gave yields* of 11, 21, 26, and 21%, respectively. A separate reaction trial was done to identify the organic products by GC-MS. Compound 5 (16.5 mg, 67.0 mo1) was allowed to react with cyanide, at 135 °C, as described in the general procedure. The concentrated ether solution was analyzed by GC and only two prominent peaks were observed, which were identified as 4- fluorotoluene (RT=3.2mm) and 4-tolunitrile 16 (RT=6.3 mm). This sample was further analyzed by HPLC (column A; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 mm) and found that 16 eluted first (RT=7.mm), followed by 4-fluorotoluene (RT= 15.9 mm). Identification of the above products was performed by the comparison of the GC and HPLC retention times with those of authentic samples. The identity of the products was further confirmed by GC-MS, using for comparison the mass spectra (acquired under very similar instrumental conditions) obtained from a premade solution of authentic samples. Mass spectra, m/z: 4-fluorotoluene, 110 (M, 66), 109 (M-H, 100), 89 (2), 83 (21), 57 (3); compound 16, 117 (M, 100), 116 (M-H, 52), 90 (38), 89 (30), 63 (7). Reaction of 6 with cyanide. A typical reaction trial was performed as follows. Compound 6 (18.2 mg, 68.3 1mol) was allowed to react with KCN (2.10 mg, 32.2 Mmol), at 115°C, as described in the ‘These results represent the best chemical yields obtained where more than one reaction trial was performed. 129 general procedure. The final DMSO mixture was analyzed by HPLC (column B; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 urn) and it was determined that 1.53 mg of 4-chlorobenzonitrile 17 (RT= 12.5 mm) was present, for a yield of 34%. A reaction trial was also conducted at 135°C, which gave a yield of 21%. An additional reaction trial was performed at 115°C that was heated only for 5 mm (instead of 10 mm) and gave a chemical yield of 17%. A separate reaction trial was done to identify the organic products by GC-MS. Compound 6 (17.7 mg, 66.3 mo1) was allowed to react with cyanide, at 115°C, as described in the general procedure. The concentrated ether solution was analyzed by GC and two major peaks were observed, which were identified as 4-chlorofluorobenzene (RT = 3.5 mm) and 4-chlorobenzonitrile 17 (RT = 7.1 mm), and also a smaller unknown peak was observed that was identified eventually (by GC-MS) as benzonitrile 14 (RT 4.5 mm). This sample was further analyzed by HPLC (column A; eluent: solvent A; flow rate, 2.5 niL/mill; UV detection, 270 nni) and found that 14 eluted first (RT = 4.6 mm), followed by 17 (RT = 7.6 mm) and an unidentified peak (RT = 10.1 mm), then finally 4- chlorofluorobenzene (RT = 18.3 mm) appeared last. Identification of the principal products was performed by the comparison of the GC and HPLC retention times with those of authentic samples. The identification of the products was completed by GC MS, using for comparison the mass spectra (acquired under very similar instrumental conditions) obtained from a premade solution of authentic samples in the case of the principal products, and by inspection of the mass spectrum obtained for 14. Mass spectra, m/z: 4-chlorofluorobenzene, 132(37C1: M, 32), 130 (35C1: M, 100), 95 (M.-Cl, 130 58), 94 (M-HCl, 11), 69 (13), 68 (13), 65 (8), 51(11), 50 (36); compound 17, 139 (37C1: M, 31), 137 (35C1: M, 100), 112 (37C1: M-HCN, 1), 110(35C1: M-HCN, 3), 102 (M Cl, 36), 76 (14), 75 (25), 74 (11), 68 (4), 51(19), 50 (35); compound 14, 103 (M, 100), 102 (M-H, 2), 77 (M-CN, 8), 76 (M-HCN, 40), 63 (4), 52 (9), 51 (21), 50 (26). Reaction of 4 with cyanide in the presence of 18-crown-6 and DMSO. A typical reaction trial was performed as follows. Compound 4 (16.0 mg, 65.01Lmol) was allowed to react with KCN (2.10 mg, 32.2 1Lmol), at 105°C, as described in the general procedure except that about 1.2 equivalents of 18-crown-6 (9.8 mg, 371.mol) was added to the reaction mixture before heating. The final DMSO mixture was analyzed by HPLC (column B; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 nm) and it was determined that 1.65 mg of 2-tolunitrile 15 (RT= 14.3 mm) was present, for a yield of 44%. Other reaction trials were conducted at 95, 115, 125, and 135°C, which gave yields of 35, 40, 42, and 41%, respectively. An additional reaction trial was performed using 16.2 mg (65.8 1.mol) of 4 with KCN (2.10 mg, 32.2 mol), at 105°C, which used approximately 3 equivalents of 18-crown-6 (26 mg, 98 1Lmol) and afforded a chemical yield of 46%. Reaction of 4 with cyanide in the presence of 18-crown-6 and CH3N. Two reaction trials were performed as follows. In the first trial, 4 (16.2 mg, 65.8 1Lmol) was allowed to react with KCN (2.10 mg, 32.2 Mmol), at 80°C, as described in the 131 general procedure except that 1 equivalent of 18-crown-6 (8.5 mg, 321mol) was added to the reaction mixture and CH3N was used as the reaction solvent. The final CH3N mixture was analyzed by HPLC (column B; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 nm) and it was determined that 0.094 mg of 2-tolunitrile 15 (RT= 13.8 mm) was present, for a yield of 3%. This mixture was stored and reanalyzed approximately 18 hours later by HPLC; it was found that 0.81 mg of 15 was now present, which represents a yield of 21%. The mixture was stored again, then reanalyzed 13.5 days after the initial HPLC analysis. At this time, 1.90 mg of 15 was determined to be present, for a final yield of 50%. The second reaction trial was performed in the same way as the first, except that an oil bath temperature of 95°C was used (it was noted that the reaction mixture was refluxing). A chemical yield of 4% was obtained, as determined by HPLC. The mixture was stored, and upon reanalysis about 16 hours later, the yield was found to be increased to 21%. This mixture was stored again, then reanalyzed 13.5 days after the initial HPLC analysis. The final yield of 15 was found to be 46%. Attempted reaction of 11 with cyanide. Two reaction trials were performed as follows. In the first trial, 11 (27 mg, 64 mo1) was allowed to react with KCN (2.10 mg, 32.2 mo1), at 100°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (column A; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 nm) and the chromatogram exhibited a large peak (RT=7.9 mm) and a much smaller peak (RT= 18.2 mm), but 132 neither peak could be identified. Under these HPLC conditions, benzonitrile 14 was exhibiting a retention time of 4.4 mm. Therefore, HPLC analysis confirmed the absence of any desired 14 in the product mixture. The second reaction trial was performed in the same way as the first, except the reaction temperature used was 120°C. The same essential results were obtained as reported in the first trial above. Heating of 11 in DMSO without cyanide present. Compound 11 (27 mg, 64 mol) was heated in DMSO (1 mL) for 10 mm at 100°C, in the same fashion as described in the general procedure; the addition of aqueous KCN and its associated drying was not done. The resulting DMSO solution was analyzed by HPLC (column A; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 nm) and the chromatogram exhibited a single large peak (RT=8.1 mm) which could not be identified. Attempted reaction of fluorobenzene with cyanide. Fluorobenzene (5.7 1.L, 60 mo1) was allowed to react with KCN (2.00 mg, 30.7 !mo1), at 135°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (column A; eluent: solvent B; flow rate, 2.5 mL/min; UV detection, 270 urn) and the chromatogram exhibited a single peak (RT = 4.2 mm) which was identified as fluorobenzene. Under these HPLC conditions, benzouitrile 14 had a retention time of 2.8 mm, thereby confirming the absence of 14 in the product mixture. 133 Attempted reaction of 2-fluorotoluene with cyanide. 2-Fluorotoluene (6.6 L, 60 mol) was allowed to react with KCN (2.00 mg, 30.7 mol), at 135°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (column A; eluent: solvent B; flow rate, 2.5 niL/mm; UV detection, 270 nm) and the chromatogram exhibited a single peak (RT = 7.2 mm) which was identified as 2-fluorotoluene. Under these HPLC conditions, 2-tolunitrile 15 had a retention time of 3.9 miii, thereby confirming the absence of 15 in the product mixture. Attempted reaction of 4-fluorotoluene with cyanide. 4-Fluorotoluene (6.6 L, 60 mo1) was allowed to react with KCN (2.00 mg, 30.7 ,Lmol), at 135°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (column A; eluent: solvent B; flow rate, 2.5 mL/min; UV detection, 270 mn) and the chromatogram exhibited a single peak (RT = 7.3 mm) which was identified as 4-fluorotoluene. Under these HPLC conditions, 4-tolunitrile 16 had a retention time of 4.1 mm, thereby confirming the absence of 16 in the product mixture. Attempted reaction of 4-chiorofluorobeuzene with cyanide. 4-Chlorofluorobenzene (6.4 L, 60 mol) was allowed to react with KCN (2.00 mg, 30.7 mol), at 135°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (column A; eluent: solvent B; flow rate, 2.5 mL/min; UV detection, 270 mm) and the chromatogram exhibited a single peak (RT=7.6 miii) which was identified as 4-chlorofluorobenzene. Under these HPLC conditions, 4- 134 chlorobenzonitrile 17 had a retention time of 4.6 mm, thereby confirming the absence of 17 in the product mixture. Attempted reaction of 12 with cyanide. Compound 12 (18 mg, 63 .4mol) was allowed to react with KCN (2.10 mg, 32.2 pmol), at 100°C, as described in the general procedure. The final DMSO mixture was analyzed by HPLC (colunm A; eluent: solvent C; flow rate, 2.5 mL/min; UV detection, 270 am) and no product peaks were observed in the chromatogram (12 is not observable by UV detection at 270 mu). Under these HPLC conditions, benzonitrile 14 had a retention time of 4.1 mm, thereby confirming the absence of 14 in the product mixture. 5.3.5 Labelling Work with [“C] Cyanide Substitution reactions with [“C] cyanide. General Procedure: After the H11CN was generated and trapped in aqueous NaOH solution (1 mL, 0.1 M), 0.5-1.0 mL of this radioactive stock solution was taken (the solution was counted at this stage and the time was notedD and a known amount of non-radioactive KCN (carrier) was added, then this mixture was added to a 5-mL Reacti vial® (which contained an inert atmosphere). The [“C]cyanide solution was rapidly evaporated to dryness (using a block heater) under a fast flow of nitrogen or argon. A solution of(,6-arene)tricarbonylchromium (40-65 mol) in DMSO (1 mL) was added by syringe to the Reacti-vial®. The stirred mixture was heated at the desired temperature This point in time was designated as the start of synthesis (SOS). 135 in a thermostated silicone oil bath for 10 minutes. Upon cooling (ice/water bath) to ambient temperature, a small portion of the reaction mixture was subjected to radio HPLC purification and the peak corresponding to the[11C]nitrile product was collected and counted to determine the decay corrected radiochemical yield. Reaction of 1 with [“C]cyanide. A representative reaction trial was performed as follows. Compound 1 (9.5 mg, 41 mol) was treated with a mixture of 27.8 mCi (SOS) of[11C]cyanide and 0.37 equivalents of carrier KCN (0.98 mg, 15 mol), then heated at 150°C, as described in the general procedure. HPLC analysis (column C; eluent: solvent A; flow rate, 5.0 mL/min; UV detection, 254 mn) of the reaction mixture determined that 9.93 mCi (decay corrected to SOS) of[11C-CN]benzonitrile 18 (RT = 6.5 mm) was produced for a radiochemical yield of 36%. Other reaction trials were conducted at 150°C which used varying amounts of carrier KCN (0.11, 0.35, 0.49 equiv) and gave 21%, 41%, and 34% radiochemical yields, respectively. An additional reaction trial was performed at 135 °C, which used 0.51 equivalents of carrier KCN, and afforded a radiochemical yield of 35%. Reaction of 1 with no carrier-added [11Cjcyanide. Compound 1 (15 mg, 65 mol) was treated with 22.8 mCi (SOS) of [“Cjcyariide (with no carrier KCN added), then heated at 150°C, as described in the general procedure. 136 HPLC analysis (column C; eluent: solvent A; flow rate, 5.0 mL/min; UV detection, 254 nm) of the reaction mixture determined that only a trace of [“C-CN]benzonitrile 18 (RT = 6.5 mm) was present—the activity of the collected product fraction was too low to be counted with the Capintec well counter. Reaction of 4 with [“C] cyanide. Compound 4 (14.8 mg, 60.1 mol) was treated with a mixture of 8.50 mCi (SOS) of [“Cjcyanide and 0.51 equivalents of carrier KCN (2.00 mg, 30.7 mol), then heated at 135 °C, as described in the general procedure. HPLC analysis (column A; eluent: solvent B; flow rate, 2.5 mL/min; UV detection, 270 urn) of the reaction mixture determined that 2.93 mCi (decay corrected to SOS) of[11C-CN]-2-tolunitrile 19 (RT = 4.0 mm) was produced for a radiochemical yield of 34%. Reaction of 5 with [“C]cyanide. Compound 5 (14.8 mg, 60.1 mol) was treated with a mixture of 7.84 mCi (SOS) of [‘1C]cyanide and 0.51 equivalents of carrier KCN (2.00 mg, 30.7 mol), then heated at 135 °C, as described in the general procedure. HPLC analysis (column A; eluent: solvent B; flow rate, 2.5 mE/mm; UV detection, 270 mu) of the reaction mixture determined that 2.39 mCi (decay corrected to SOS) of[‘1C-CN]-4-tolunitrile 20 (RT=4.l mm) was produced for a radiochemical yield of 31%. 137 Reaction of 6 with [“C]cyanide. Compound 6 (12.3 mg, 46.1 mol) was treated with a mixture of 16.73 mCi (SOS) of [‘1C]cyariide (in this case the H11CN was trapped in 0.025 M NaOH solution) and 0.11 equivalents of carrier KCN (0.33 mg, 5.0 mol), then heated at 150°C, as described in the general procedure. HPLC analysis (column C; eluent: solvent A; flow rate, 3.0 mL/min; UV detection, 254 mn) of the reaction mixture determined that 3.54 mCi (decay corrected to SOS) of[11C-CN]-4-chlorobenzonitrile 21 (RT = 12.0 mm) was produced for a radiochemical yield of 21%. Another reaction trial was performed at 135°C, which used 0.51 equivalents of carrier KCN, and afforded a radiochemical yield of 19%. Reaction of 6 with [‘1C]cyanide in the absence of base. An experiment was done to eliminate the presence of base (both ammonia and hydroxide) from the labelling[11C]cyauide reagent. TheH11CN/NH3gas stream was passed through a glass loop, which was emersed in a CC14/C02(-23°C) cooling bath, and trapped out the H11CN, while sweeping away the NH3 with helium gas flow. The glass ioop was removed from the cooling bath and H’1CN was slowly purged into a Reacti vial® which contained a mixture of 6 (10.0 mg, 37.5 mo1), carrier KCN (1-2 mg, 15-31 mol*), and DMSO (1 mL) under N2 atmosphere. When the H11CN transfer was complete, the radioactive mixture was counted (3.87 mCi, SOS). This mixture was Two small crystals of KCN were used, which were not weighed, thus the quantity indicated was estimated. 138 heated for 10 minutes at 125-130°C. After cooling, HPLC analysis (column C; eluent: solvent A; flow rate, 3.0 mL/min; UV detection, 254 nm) of the reaction mixture determined that 1.11 mCi (decay corrected to SOS) of [“C-CN]-4-chlorobenzonitrile 21 (RT= 14.9 mm) was produced for a radiochemical yield of 29%. 5.4 Experimental for Chapter 3 5.4.1 Sources of Materials l7cr-Ethynylestradiol 23 was obtained from Sigma Chemical Co. Tri-n-butyltin hydride was purchased from the Aldrich Chemical Co. and Alfa Products, and 2,2 ‘-azobis- (2- methylpropionitrile), commonly referred to as AIBN, was supplied by Aldrich Chemical Co. The O-methylated estradiols, 3-methoxy- l7cr-ethynyl- 1,3,5( 10)-estratriene- 17f3-ol 24 and 3, 17fl-dimethoxy-17a-ethynyl- 1,3,5( 10)-estratriene 25, were prepared by adapting the method of Johnstone and Rose.9 Compound 23 was treated with powdered potassium hydroxide in DMSO, followed by the addition of iodomethane. Flash chromatography of the crude product mixture on silica gel with hexanes/diethyl ether (2:1) afforded 24 and 25 in 67% and 31% yields, respectively. 3-Methoxy-l7cr-vinyl-1,3,5(10)-estratriene- 17fl-ol 41 was also prepared by an adaptation of the procedure of Johnstone and Rose.9 A mixture of 23 and 17a-vinyl-1,3,5(10)-estratriene-3,17f3-diol 35, obtained from the hydrostarinylation of 23 (see Subsection 5.4.3), was treated with potassium hydroxide in DMSO, and then with iodomethane. Flash chromatography on silica gel with hexanes/ diethyl ether (1:1) gave pure 41 in 52% yield (based on the amount of 35 used). 139 The acetylenic sugars, 7,8-dideoxy- l,2:3,4-di-O-isopropylidene-D-glycero-a-D-galacto-oct- 7-ynopyranose 28 and its L-glycero-a-D-galacto epimer 29, were prepared according to published procedures in two steps. First, 1,2:3,4-di-O-isopropylidene-a-D-galacto- hexodialdo- 1,5-pyranose 27 was prepared by oxidation of 1,2:3,4-di-O-isopropylidene-a-D- galactopyranose 26 (Koch-Light Laboratories Ltd.) using either chromium trioxide dipyridine complex (in 52% yield) as described by Arrick and co-workers,’° or the chromium trioxide-pyridine complex in the presence of acetic anhydride (in 68% yield) according to Garegg and Samuelsson.” Then 27 was treated with ethynylmagnesium bromide according to the procedure of Hems et al.12 and a mixture of 28 and 29 was obtained (85% yield) in a ratio of 62:38 (determined by ‘H NMR), respectively. This mixture was separated using column chromatography on silica gel with dichloromethane/ hexanes/diethyl ether (6:2:1). Research grade Ne and 1% F2 in Ne gas mixture were specially prepared in ultra-high purity (suitable for 18F production) by either Matheson Gas Products (Edmonton, AB) or Canadian Liquid Air Ltd. (Vancouver, BC). Freon-li (CFC13)was purchased from Matheson Gas Products. 5.4.2 General Fluorine gas is toxic, highly corrosive, and generally dangerous, and therefore requires handling with great care.’3”4 Hence, a specialized fluorine gas handling system was used to perform all studies involving elemental fluorine and acetyl hypofluorite, and is shown in Figure 5.1. This gas handling system was constructed from components, such 140 CYCLOTRON SHIELDING vent metering ( valve pressure pressure transducer transducer reaction Figure 5.1: Schematic of the fluorine gas handling system used to perform the fluorination reactions. as stainless steel and Teflon, that are compatible with reactive fluorine gas. The fluorine tank and gas handling system are stationed in a fume hood specifically designed for use with radionucides. The gas handling system is connected via 1/8 in. o.d. stainless steel tubes to the gas targets used on the TRIUMF/Nordion CP-42 cyclotron and the larger TRIUMF 500 MeV cyclotron. The cyclotron gas targets are essentially gas-tight cylinders, fabricated from nickel (for the CP-42 cyclotron)’5or Inconel 600, a nickel- based alloy (for the 500 MeV cyclotron).’6 The gas handling system was connected to the reaction vessel with Teflon tubing (1/16 or 1/8 in. o.d.) using Swagelok fittings 141 (Crawford Fitting Co., Salon, OH) and Teflon ferrules. Gaseous CH3OOF was produced by passing a dilute mixture of F2 (—0.1%) in Ne through a column containing a solid mixture of potassium acetate/acetic acid in a molar ratio of 2:3. TheCHOOK/CHOH mixture was prepared according to the method of Jewett et al.17 The quantification of the amount of F2 or CH3OOF used for fluorination experiments was accomplished by iodometric titration.18”9 Prior to performing actual fluorination reactions with vinyl-tin substrates, one or two “dummy” trials would be done in which the fluorinating agent was added to an aqueous solution of excess KI, and the liberated 12 was then titrated with standardized 0.1 M sodium thiosulfate solution. [‘8F1F2was produced by the20Ne(p,Spall)’8Freaction with a target gas mixture of 0.1% F2 in natural neon that was irradiated with 500 MeV protons from the TRIUMF cyclotron.16 Gaseous CH3OO’8Fwas prepared by venting the [‘8F]F2 produced after target irradiation through the solidCH3OOK/CHOHcolumn17that was described earlier. 5.4.3 Preparation of Vinyl-Tin Substrates General procedures for hydrostannylation of acetylenic compounds. Procedure A: Under an N2 atmosphere, a solution of acetylenic compound in 1,4- dioxane was prepared. To this stirred solution, five equivalents of tri-n-butyltin hydride was added by syringe, then the mixture was refluxed overnight. The solvent was removed and the crude mixture was chromatographed on a silica gel column to obtain 142 the stannylated product. Procedure B: Under an N2 atmosphere, a mixture of acetylenic compound, a catalytic amount of AIBN, and two equivalents of tri-n-butyltin hydride were combined, and the stirred mixture was heated overnight (thermostated silicone oil bath, 95°C). After cooling, the entire reaction mixture was chromatographed on a silica gel column to isolate the stannylated product. General: In all cases, the vinyl-tin products after chromatographic isolation were thoroughly dried under high vacuum, then stored in vacuo over sodium hydroxide pellets. Vinyl-tin compounds are sufficiently air-stable so that they can be easily handled in the open under normal conditions, however, prolonged exposure to the atmosphere will result in gradual decomposition. Proper storage, therefore, is very important. Preparation of l7cr-(E) -tributylstannylvinyl-1,3,5(10) -estratriene-3,1713-diol 30 and l7a- vinyl-1,3,5(10)-estratriene-3,17j3-diol 35. Method A: Compound 23 (1986 mg, 6.70 mmol) in 1,4-dioxane (10 mL) was hydro stannylated as described in procedure A. Flash chromatography of the crude mixture was performed on silica gel (3.5 cm x 15 cm) with dichloromethane/hexanes/diethyl ether (6:2:1). The first fraction contained 30 as the dominant product plus two unidentified byproducts, while further elution isolated a mixture of 23 and 35 (1090 mg) in a ratio of 29:71 as determined by ‘H NMR. Therefore, 774 mg of 35 was estimated to be present in the second fraction for a yield of 38%. The first fraction was further purified by column chromatography on silica gel (150 mg) with dichloromethane/ 143 hexanes /ether (6:4:1) which afforded 780 mg of 30 (20% overall isolated yield) as a very viscous syrup, and upon drying overnight in vacuo, crystallized as an off-white solid. An analytical sample was obtained by flash chromatography on silica gel with dichioromethane/hexanes/ether (6:4:1), mp 86-87.5°C, [a]4 + 19.00 (c 1.7, 1,4-dioxane). Anal. calcd. forC32H5O2Sn: C 65.43, H 8.92; found: C 65.60, H 8.78. 1H NMR (270 MHz, CDC13)& 0.81-0.97 (m, 18H, 18-CH3andSn(CHCH3),1.20-2.37 (several m, 26H, 17-OH, Sn(CH2CH3)CH and CH2 of steroid nucleus), 2.80 (m, 2H, CH2 of steroid nucleus), 5.30 (s, 1H, 3-011), 6.07 (d,J2021 = 19.4 Hz, 2JSn,H 70 Hz, 1H, H-21), 6.20 (d, 3JSn,H = 66 Hz, 1H, H-20), 6.57 (d, 24 = 2.9 Hz, 1H, H-4), 6.62 (dd, J1,2 = 8.8 Hz, 1H, H-2), 7.13 (d, 1H, H-i). Mass spectrum, m/z: 531(120Sn: M-C4H9,100). In an attempt to separate 35 from 23 for characterization, column chromatography of a portion of the mixture of 23 and 35 was performed on silica gel with dichloromethane/ hexanes/ether (6:4:1). Unfortunately, complete separation was not achieved as 23 co eluted with all fractions containing 35, with the largest percentage of 23 being present in the early fractions. Therefore, a sample was obtained by combining several of the late fractions that contained the lowest percentage of 23. This sample was recrystallized from benzene/hexanes, then analyzed by HPLC (column C; eluent: methanol/water, 3:1; flow rate, 6.0 mL/min; UV detection, 280 nm) and found that a ratio of 23 (RT=.6mm) to 35 (R.=5.0 mm) of 19:81 was present. As a result, to effectively isolate 35 an HPLC separation was carried out by injecting this sample (dissolved in THF) in several portions onto column C and eluting 35 using the same conditions (flow rate was changed to 3.0 mL/min) employed for HPLC analysis. The peaks corresponding to 35 were collected, 144 but due to a minute amount of 23 still present, the isolated material was subjected to additional HPLC purification. The material obtained from the second HPLC purification was recrystallized from benzene/hexanes, which afforded 35 as white crystals, mp 169.5-170°C, [a15 + 58.5° (c 1, 1,4-dioxane). Anal. calcd. forC20H60:C 80.50, H 8.78; found: C 80.54, H 8.72. ‘H NMR (270 MHz, CDC13)ô: 0.95 (s, 3H, 18- CH3), 1.24-2.37 (several m, 14H, 17-OH, CH and CH2 of steroid nucleus), 2.80 (m, 2H, CH2 of steroid nucleus), 4.68 (hr s, 1H, 3-OH), 5.15 (dd,J2021a 10.8 Hz, ‘21a,21b 1.2 Hz, 1H, H-21a), 5.20 (dd,2O,lb = 17.3 Hz, 1H, H-21b), 6.11 (dd, 1H, H-20), 6.56 (d, J24 = 2.8 Hz, 1H, H-4), 6.62 (dd, J12 = 8.2 Hz, 1H, H-2), 7.14 (d, 1H, H-i). Mass spectrum, m/z: 298 (M, 51), 280 (M-H2O, 15). Exact mass calcd. forC20H60:298.1934; found: 298.1934. Method B: Compound 23 (1.00 g, 3.37 mmol) was hydrostannylated as described in procedure B. Column chromatography on silica gel (100 g) with dichloromethane/ hexanes/diethyl ether (6:4:1) afforded in one portion 1.17 g (59%) of 30. Physical and spectral (‘H NMR) properties of this material were identical with those reported earlier. TLC analysis of the reaction mixture confirmed the presence of 35, but the compound was not eluted off the column. Preparation of 3-methoxy-17o&(E)-tributyIstanny1vinyI-1,3,5(10)-estratriene-173-o1 31. Compound 24 (492 mg, 1.59 mmol) was bydrostannylated as described in procedure B. Column chromatography on silica gel (100 g) with hexanes/diethyl ether (5:1) yielded in the first fraction 114 mg of crude 3-methoxy-17a-(Z)-tributylstannylvinyl-1,3,5(10)- 145 estratriene-17fl-ol 36, and in the following two fractions, 860 mg of 31. Compound 31 was isolated as a colourless oil in 90% yield, [Qf]4 + 17.1° (c 1.2, 1,4-dioxane). Anal. calcd. forC33H54O2Sn: C 65.90, H 9.05, 0 5.32; found: C 65.83, H 9.00, 0 5.28. Mass spectrum, m/z: 602 (‘20Sn: M, 0.2), 545 (‘20Sn: M-C4H9,100). Preparation of 3,173-dimethoxy-17o-(E)-tributy1stanny1viny1-1,3,5(1O)-estratriene 32. Compound 25 (1.00 g, 3.08 minol) was hydrostannylated as described in procedure B. Column chromatography on silica gel (200 g) with hexanes/diethyl ether (20:1) yielded in the first fraction 0.23 g of 32 plus a minute amount of unidentified byproduct, and in the second fraction 1.56 g of pure 32. Compound 32 was isolated in an overall yield of 94% as an oil, and after drying in vacuo for 15-30 minutes, crystallized as a white solid. The material from the second fraction exhibited mp 50-52.5°C, [a]4 +44.1° (c 1.2, CHC13). Anal. calcd. forC34H56O2Sn: C 66.35, H 9.17, 0 5.20; found: C 66.37, H 9.18, 0 5.35. ‘H NMR (270 MHz, C6D) ô: 0.93 (t, J 7.3 Hz, 9H, Sn(CHCH3), 1.01 (m, 6H, Sn(CHCH3),1.12 (s, 3H, 18-CH3), 1.21-2.29 (several m, 25H, Sn(CH2CH3),CH and CH2 of steroid nucleus), 2.63-2.86 (m, 2H, CH2 of steroid nucleus), 3.23 (s, 3H, 17-OCH3),3.39 (s, 3H, 3-OCH), 6.23 (d,J2021 = 19.7 Hz, 2JSn,H = 75 Hz, 1H, H-21), 6.37 (d, 3Sn,H = 69 Hz, 1H, H-20), 6.68 (d, J2,4 = 2.6 Hz, 1H, H-4), 6.75 (dd, J,,2 = 8.6 Hz, 1H, H-2), —7.16 (d, 1H, H-i). Mass spectrum, m/z: 616(120Sn: M, 1), 601(120Sn: M-CH3,5), 559 (‘20Sn: M-C4H9,66). 146 Preparation of (E)-7,8-dideoxy- 1,2:3,4-di-O-isopropylidene-8-C-tributylstannyl-n-glyce- ro-a-D-galacto-oct-7-enopyranose 33. Compound 28 (884 mg, 3.11 mmol) was hydrostannylated as described in procedure B. Column chromatography on silica gel (200 g) with dichioromethane/hexanes/diethyl ether (6:6:1) afforded in one fraction 1093 mg (6 1%) of 33. Compound 33 was isolated as a colourless syrup, [a]3 -36.1° (c 1, CHC13). Anal. calcd. forC26H48O6Sn: C 54.28, H 8.41; found: C 54.21, H 8.52. 1H NMR (270 MHz, CDC13) & 0.81-0.97 (m, 15H, Sn(CH2CH3),1.21-1.55 (2 m, 24H, 2 x C(CH3)2and Sn(CH2CH3), 2.81 (d,J6,OH = 7.0 Hz, 1H, 6-011), 3.68 (dd, J5,6 = 6.5 Hz, J4,5 = 1.7 Hz, 1H, H-5), 4.32 (dd, J1,2 = 5.0 Hz,J23 = 2.3 Hz, 1H, H-2), 4.34 (m, J6,7 = 4.4 Hz, J6,8 = 1.4 Hz, 1H, H-6), 4.45 (dd, J3,4 = 8.0 Hz, 1H, H-4), 4.61 (dd, 1H, H-3), 5.59 (d, 1H, H-i), 6.18 (dd, J78 = 19.2 Hz, 3Sn,H = 64 Hz, 1H, H-7), 6.38 (dd, 2Sn,H = 70 Hz, 1H, H-8). Mass spectrum, m/z: 561 (120Sn: M-CH3,3), 519(120Sn: M-C4H9,100). Preparation of (E)-7,8-dideoxy-1,2:3,4-di-O-isopropylidene-8-C-tributylstannyl-L-glyce- ro-oc-D-galacto-oct-7-enopyranose 34. Compound 29 (898 mg, 3.16 mmol) was hydrostannylated as described in procedure B except that after 19 hours of heating, 0.50 mL of tri-n-butyltin hydride (0.54 g, 1.86 nimol) was added and heating was continued for another three hours. Column chromatography on silica gel (200 g) with hexanes/diethyl ether (4:1) yielded in the first fraction 444 mg of a mixture of (Z)-7,8-dideoxy-1,2:3,4-di-O-isopropylidene-8-C- tributylstanny1-L-glycero-c-D-galacto-oct-7-enopyranose 37 and 7,8-dideoxy- 1,2:3,4-di-O- 147 isopropylidene-7-C-tributylstannyl-L-glycero-cr-D-galacto-oct-7-enopyranose 38 in a ratio of 3:2 as determined by 1H NMR. As a result, 266 mg of 37 and 178 mg of 38 was estimated to be present in the mixture, for chemical yields of 15% and 10%, respectively. Further elution afforded 1076 mg of 34 in a second fraction, for a 59% yield. Compound 34 was isolated as a colourless syrup, [a]4 -37.0° (c 1.3, CHC1). Anal. calcd. forC26H48O6Sn: C 54.28, H 8.41, 0 16.68; found: C 54.57, H 8.47, 0 16.55. Mass spectrum, m/z: 561 (120Sn: M-CH3,2), 519(120Sn: M-C4H9,100). 5.4.4 Fluorination Reactions of Vinyl-Tin Compounds Fluorination of vinyl-tin substrates with elemental F2 or gaseous CH3OOF. General Procedure: A solution of vinyl-tin compound (60-200 1mol) was prepared (different solvents were employed) and placed in a glass reaction vessel (oven dried) under inert atmosphere. A stream of inert gas was passed through the vinyl-tin solution via a 1/16 in. o.d. Teflon tube, positioned at the bottom of the solution, which was controlled by the fluorine gas handling system (see Figure 5.1). To conduct fluorinations at 0 or -78°C, the vinyl-tin solution was cooled with either an ice/water (0°C) bath or aC02/2-propanol (-78°C) cooling bath. The desired quantity of 1% F2/Ne gas mixture (60-230 mol F2) was loaded into either the CP-42 or 500 MeV gas target using the fluorine gas handling system, and then pure Ne was added to dilute the F2 concentration to approximately 0.1% in Ne. To perform fluorinations with F2, the dilutedF2/Ne gas mixture was then added directly to the vinyl-tin solution after the inert gas flow was turned off. However, to perform 148 fluorinations with gaseous CH3OOF, the dilutedF2/Ne gas mixture was instead passed through a solidCH3OOK/CHOH column and the effluent added to the vinyl-tin solution. Both fluorinating agents (F2 or CH3OOF) were added at a flow rate of - 50 mL/min to the vinyl-tin solution. After the addition of fluorinating agent was completed, the reaction mixture was transferred to a round-bottom flask and the solvent was removed in vacuo. The residue was analyzed by TLC, then worked up as considered appropriate. Preparation of 3-methoxy-17a-(E)-fluorovinyl-1,3,5(1O) -estratriene-1713-ol 39 and 3- methoxy-17a-(Z)-fluorovinyl-1,3,5(10)-estratriene-173-ol 40. Compound 31 (92.7 mg, 154 mol) was dissolved in CFC13 (20 mL) and added to a glass reaction vessel (2.0 cm o.d. x 10 cm length). This solution was treated with approximately 1.3 equivalents of CH3OOF, at room temperature, as described in the general procedure. Five additional fluorinations of compound 31 were conducted in the same manner, employing a total of 554.6 mg of 31 (922 mol). The reaction mixtures obtained were combined, then subjected to column chromato graphy on silica gel (100 g) with hexanes/diethyl ether (4:1) as the eluent. The first fraction (55 mg) collected contained 39 in 96% purity as indicated by HPLC analysis (column D; eluent: solvent D; flow rate, 3.5 mL/min; UV detection, 280 nm). With continued elution, a second fraction (66 mg) was obtained which contained a mixture of 39 (RT= 17.8 mm) and 40 (RT=26.2 mm) in a ratio of 79:21 by HPLC analysis. Both of the isolated chromatography fractions were subjected to further purification via HPLC. 149 Each fraction was dissolved in a minimum of ether, and then was injected in several portions onto the HPLC silica gel column and eluted using the same conditions employed for HPLC analysis. The peaks corresponding to 39 and 40 were collected, whereby all of 39 was isolated in one batch (90.0 mg) and all of 40 was isolated in another batch (11.5 mg). The isolated yields of 39 and 40 were 29.5% and 3.8%, respectively. Quantification of yields for the reaction of 31 with gaseous CH3OOF. Compound 31 (76.4 mg, 127 mol) was dissolved in CFC13 (20 mL) and added to a glass reaction vessel (2.0 cm o.d. x 10 cm length). This solution was treated with approximately 1.3 equivalents of CH3OOF, at room temperature, as described in the general procedure. After solvent removal, the residue was dissolved in a known volume of CHC13. The product mixture was analyzed by HPLC (column C; solvent program A; flow rate, 6.0 mL/min; UV detection, 280 urn) using a standard solution of 39 as an external standard. It was determined that 17.0 mg of 39 and 40 (both co-elute, RT=S.O mm) was present, for a yield of 41% based on the amount of 31 used. Additional fluorination trials were conducted using alternative reaction solvents. Compound 31 (74.1 mg, 123 mo1) was dissolved in dried CH3O (20 mL) and treated with approximately 1.3 equivalents ofCH3OOF as described above. It was determined by HPLC analysis that 5.59 mg of 39 and 40 was obtained, for a yield of 14%. Compound 31 (80.7 mg, 134 mol) was dissolved in dried CH3N (20 mL), which required about 0.5 mL of CHC13 to help solubilize 31, and was treated with 150 approximately 1.2 equivalents of CH3OOF as described above. It was determined by HPLC analysis that 10.4 mg of 39 and 40 was obtained, for a yield of 24%. Compound 31 (71 mg, 118 mo1) was dissolved in dried THF (20 mL) and treated with approximately 1.4 equivalents ofCH3OOF as described above. It was determined by HPLC analysis that 3.62 mg of 39 and 40 was obtained, for a yield of 9.3%. Quantification of yields for the reaction of 31 with elemental F2. Compound 31 (103.2 mg, 172 mol) was dissolved in CFC13 (20 mL) and added to a glass reaction vessel (2.0 cm o.d. x 10 cm length). This solution was treated with approximately 1.25 equivalents of F2, at room temperature, as described in the general procedure. After solvent removal, the residue was dissolved in a known volume of CHC13. The product mixture was analyzed by HPLC (column C; solvent program A; flow rate, 6.0 mL/min; UV detection, 280 nm) using a standard solution of 39 as an external standard. It was determined that 5.10 mg of 39 and 40 (both co-elute, RT=4.97 mm) was present, for a yield of 9.0% based on the amount of 31 used. In addition, two side-products that were present in significant amounts were identified as 3-.methoxy-l7a- ethynyl- 1,3,5 ( 10)-estratriene- 173-ol 24 (RT = 4.39 mm) and 3-methoxy- 17a-vinyl- 1,3,5(10)- estratriene-173-ol 41 (RT = 5.70 mm). Standard solutions of 24 and 41 were prepared for use as external standards. HPLC analysis indicated that 2.89 mg of 24 and 3.85 mg of 41 were present, for yields of 5.4% and 7.2%, respectively. An additional fluorination experiment was done using 95.9 mg (159 ILmol) of 31 in CFC13 (20 mL), which was treated with approximately 1.35 equivalents of F2 at -78°C, 151 as described above. It was determined by HPLC analysis that 2.23 mg of 39 and 40 was obtained, for a yield of 4.2%. Furthermore, the side-products 24 and 41 were obtained in 2.2% (1.11 mg) and 14.5% (7.23 mg) yields, respectively. Preparation of (E)-8-C-fluoro-7,8-dideoxy-1,2:3,4-di-O-isopropylidene-L-glycero-a-D- galacto-oct-7-enopyranose 47 and (Z)-8-C-fluoro-7,8-dideoxy-1,2:3,4-di-O-isopropyli- dene-L-glycero-a-D-galacto-oct-7-enopyranose 48. Compound 34 (83.6 mg, 145 mol) was dissolved in CFC13 (20 mL) and added to a glass reaction vessel (2.0 cm o.d. x 10 cm length). This solution was treated with approximately 1.3 equivalents of CH3OOF, at room temperature, as described in the general procedure. A second fluorination of compound 34 (80.9 mg, 141 mol) was performed as outlined above. The reaction mixtures obtained were combined, then subjected to column chromatography on silica gel (60 g) with dichloromethane/hexanes/ diethyl ether (6:2:1) as the eluent. The first fraction (29.5 mg) collected contained 47 in —98% purity as indicated by ‘H NMR. The second fraction (4.0 mg) contained a mixture of 47, 48, and 7,8-dideoxy- 1,2:3,4-di-O-isopropylidene-L-glycero-a-D-galacto-oct-7-enopyranose 49 in a ratio of 43:36:21 as determined by ‘H NMR. The third fraction (9.0 mg) contained a mixture of 47, 48, and 49 in a ratio of 6:83:11. The final fraction (5.4 mg) contained a single compound that was identified as (E)-8-C-acetoxy-7-deoxy-1,2:3,4-di-O-isopropylidene-L- glycero-a-D-galacto-oct-7-enopyranose 50. The chemical yield of 47, as contained in the first three chromatography fractions, was 152 determined to be 36% (32 mg). The yield of 48, as contained in the second and third fractions, was determined to be 10% (9 mg). A larger scale synthesis of 47 and 48 was carried out as follows. Compound 34 (81.9 mg, 142 mol) was dissolved in CFC13 (20 mL) and added to a glass reaction vessel (2.0 cm o.d. x 10 cm length). This solution was treated with approximately 1.35 equivalents of CH3OOF, at room temperature, as described in the general procedure. Three additional fluorinations of compound 34 were performed in the same manner, employing a total of 326.5 mg of 34 (567 mol). The reaction mixtures were combined, then subjected to column chromatography on silica gel (140 g) with dichioromethane/hexanes /diethyl ether (6:2:1) as the eluent. 5.4.5 Radiofluorinations with Acetyl[18F]Hypofluorite General procedure for radiofluorination with gaseous CH3OO’8F. A solution of vinyl-tin compound (110-120 mol) in CFC13 (20 mL) was prepared in a glass reaction vessel (2.0 cm o.d. x 12.5 cm length) under inert atmosphere. The 500 MeV gas target was filled with 4 or 6 psi (0.27 or 0.41 atm) of 1% F2/Ne gas mixture, then pure Ne was added until 100 psi (6.8 atm) was reached. For a typical ‘8F production run, the target gas mixture was irradiated for 10 minutes at 69 1A. When the irradiation of the target gas was stopped, this time was noted and designated as the end of bombardment (EOB). After irradiation, the radioactive gas mixture was passed through a solidCH3OOK/CHOH column and into the reaction vessel at a flow rate of —50 mL/min. With the addition ofCH3OO18Fcompleted, the reaction mixture 153 was transferred to a round-bottom flask, then assayed for radioactivity. TheCH3OOK/ CH3OOH column was also assayed for activity. The reaction mixture was evaporated to dryness in vacuo and reassayed for activity, then dissolved in a small amount of CHC13. An aliquot of this mixture was subjected to radio-HPLC purification and the peak corresponding to the18F-labelled product was collected, then counted to determine the percentage of product present in the reaction mixture. The decay corrected radiochemical yield was calculated by dividing the total activity due to product (in the reaction mixture) with the total activity of 18F produced in the cyclotron target at EOB. Reaction of 31 with CH3OO18Fat room temperature. Compound 31 (72.1 mg, 120 mol) was radiofluorinated using approximately 0.74 equivalents ofCH3OO’8F(produced with 6 psi of 1%F2/Ne), at room temperature, as described in the general procedure. HPLC analysis (column C; solvent program A; flow rate, 6.0 mL/min; UV detection, 254 mn) of the reaction mixture determined that 3.68 mCi (decay corrected to EOB) of 3-methoxy-l7cy-(E)-[’8F]fluorovinyl-1,3,5( 10)-estra- triene- 173-o1 51 and 3-methoxy-17a-(Z)-[’8Fjfluorovinyl-1,3,5 ( 10)-estratriene- 17i3-ol 52 (both co-elute, RT=5.5 mm) was produced for a radiochemical yield of 19%. An additional radiofluorination trial was conducted using a greater excess of vinyl-tin 31. Compound 31 (71.0 mg, 118 1Lmol) was radiofluorinated using approximately 0.5 equivalents of CH3OO’8F(produced with 4 psi of 1%F2/Ne), at room temperature, as described in the general procedure. HPLC analysis (column C; solvent program B; flow rate, 6.0 mL/min; UV detection, 254 mu) of the reaction mixture determined that 154 3.79 mCi (decay corrected to EOB) of 51 and 52 (both co-elute, RT 11.0 mm) was produced for a radiochemical yield of 19%. Reaction of 31 with CH3OO18Fat -78°C. Compound 31 (69.0 mg, 115 1mol) was radiofluorinated using approximately 0.77 equivalents of CH3OO’8F(produced with 6 psi of 1%F2/Ne), at -78°C, as described in the general procedure. HPLC analysis (column C; solvent program A; flow rate, 6.0 mL/min; UV detection, 254 am) of the reaction mixture determined that 1.72 mCi (decay corrected to EOB) of 51 and 52 (both co-elute, RT=S.4 mm) was produced for a radiochemical yield of 9.7%. An additional radiofluorination trial was conducted using a greater excess of vinyl-tin 31. Compound 31 (71.7 mg, 119 mo1) was radiofluorinated using approximately 0.5 equivalents of CH3OO18F(produced with 4 psi of 1%F2/Ne), at -78°C, as described in the general procedure. HPLC analysis (column C; solvent program B; flow rate, 6.0 mL/min; UV detection, 254 nm) of the reaction mixture determined that 1.65 mCi (decay corrected to EOB) of 51 and 52 (both co-elute, RT = 10.7 mm) was produced for a radiochemical yield of 5.0%. 155 References 1. Gordon, A. 3.; Ford, R. A. The Chemist’s Companion: A Handbook ofPractical Data, Techniques and References; Wiley Interscience: New York, 1972; p 439. 2. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923-2925. 3. Shriver, D. F.; Drezdzon, M. A. The Manipulation ofAir-sensitive Compounds, 2nd ed.; Wiley Interscience: Toronto, 1986. 4. Adam, M. J.; Grierson, J. R.; Ruth, T. J.; Pedersen, K.; Pate, P. D. J. Nuci. Med. 1987, 28, 1599-1603. 5. Bunnet, J. F.; Hermann, H. J. Org. Chem. 1971, 36, 408 1-4088. 6. Mahaffy, C. A. L.; Pauson, P. L. Inorg. Synth. 1979, 19, 154-158. 7. Ofele, K. Chem. Ber. 1966, 99, 1732-1736. 8. Mahaffy, C. A. L. .1. Organomet. Chem. 1984, 262, 33-37. 9. Johnstone, R. A. W.; Rose, M. E. Tetrahedron 1979, 35, 2169-2173. 10. Arrick, R. B.; Baker, D. C.; Horton, D. Carbohydr. Res. 1973, 26, 441-447. 11. Garegg, P. J.; Samuelsson, B. Carbohydr. Res. 1978, 67, 267-270. 12. Hems, R.; Horton, D.; Nakadate, M. Carbohydr. Res. 1972, 25, 205-216. 13. Hudlick, M. Chemistiy of Organic Fluorine Compounds: A Laboratoiy Manual with Comprehensive Literature Coverage, 2nd ed.; John Wiley and Sons: New York, 1976; pp 13-14. 14. Matheson Gas Data Book, 5th ed.; Matheson Gas Prducts: East Rutherford, NJ, 1971; pp 261-265. 15. Ruth, T. J. mt. .1. Appl. Radiat. Isot. 1985, 36, 107-110. 16. Ruth, T. J.; Adam, M. J.; Burgenjon, J. J.; Lenz, J.; Pate, B. D. mt. J. AppL Radiat. Isot. 1985, 36, 93 1-933. 17. Jewett, D. M.; Potocki, J. F.; Ehrenkaufer, R. E. J. Fluorine Chem. 1984,24, 477-484. 18. Skoog, D. A.; West, D. M. Fundamentals of Analytical Chemistiy; Holt, Rhinehart and Winston: New York, 1965; pp 462 and 485-490. 156 19. Casella, V.; Ido, T.; Wolf, A. P.; Fowler, J. S.; MacGregor, R. R.; Ruth, T. 3. .1. Nuci. Med. 1980, 21, 750-757. 157

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