U OF L RPMS ITEM

SYNTHESES OF SCME AND 18F-IAEEEJUED CCMPOCINT)K

FOR CONICAL PET STUDIES

A THESIS submitted in part fulfilment for the degree of DOCTOR OF PHILOSOPHY at the University of London Royal Postgraduate Medical School by

Sajinder Kaur Luthra BSc (Hons), C.Chem. MRSC

MRC Cyclotron Unit Hammersmith Hospital Ducane Road London W12 OHS February 1989 To Ajay, mum and dad

"An investment in knowledge pays the best interest" Benjamin Franklin

2 ABSTRACT A method for the photosynthesis of [1:LC] glucose from cyclotron- produced cartoon dioxide was improved with respect to [^C] cartoon dioxide entrapment, pH of photosynthesis, product separation and, for radiation safety, amenability to remote control. [13C/1:LC] Co-labelling and subsequent 13C-NMR spectroscopy established that 11C entered all positions of the glucose molecule. By this improved procedure adequate activities of [1:LC] glucose were prepared routinely and safely for human intravenous injection and PET studies of glycogen repletion in ischaemic myocardium. For the purpose of labelling opiate receptor antagonists, potential methods for the preparation of [l-11C]cyclopropane- carboxaldehyde, [ l-^C] cyclopropylmethyl iodide and [ l-^C] cyclopro- panecarbonyl chloride from [11C]cartoon dioxide were investigated. The cartoonation of cyclopropanemagnes ium bromide with [1:LC] carbon dioxide followed by treatment with phthaloyl dichloride provided a novel and efficient route to no-carrier-added (n.c.a) [l-^C]- cyclopropanecarbonyl chloride. This method was extended to the efficient preparation of other n.c.a [11C]acid chlorides (R11C0C1; R = Me, Et, Pr, c.Bu, furanyl). These [i:LC]acid chlorides were shown to be useful agents for preparing [^^C] amides from secondary amines e. g. 1,2,3,4-tetrahydroisoquinoline (THIQ). One product, N- [ 11C ] cyclopropanecarbonyl-THIQ, was reduced successfully to N- [-^C]cyclopropylmethyl-THIQ with lithium aluminium hydride. Rapid purification of this amine was shown to be possible by Sep-pak treatment and HPLC. Reference amides and amines were synthesised from THIQ and then fully characterised by mass spectrometry and %- and 13C-NMR spectroscopy. Spectroscopy revealed dynamic processes e.g. inversion in the heterocyclic ring, inversion through nitrogen, and in amides restricted rotation about the Ccg-N bond. A detailed NMR study was carried out in order to elucidate the nature of these processes and allow confirmation of structure. Reaction of [ l-^C] cyclopropanecarbonyl chloride with N- (descyclopropylmethyl)diprenorphine followed by reduction with

3 lithium aluminium hydride gave [1:LC]diprenorphine, a novel radioligand for the opiate receptor system. The position of label was unambiguously determined by 13C/11C co-labelling and 13C-NMR spectroscopy. This chemistry coupled with rapid HPLC purification, was adapted to remote control, so enabling high activities (> 37.2 GBq) of high specific activity [11C]diprenorphine to be prepared rapidly (< 60 min) for human intravenous injection and in vivo studies of opiate receptors with PET. Buprenorphine, a structural congener of diprenorphine, was similarly labelled. [2-1]-C]Furoyl chloride was prepared for labelling the a y adrenoreceptor antagonist, prazosin. The 5-fluoromethyl and 5-/3 -fluoroethy 1 analogues of the NMDA receptor antagonist, MK-801 {(+) 5-methyl-10, ll-dihydro-5H-dibenzo [a, d ] cyclohepten-5,10-imine}, were labelled with 18F by nucleophilic displacements at appropriate cyclic sulphamates and purified by Sep- pak treatment and HPLC. These [ 18F] fluoroalkyl analogues have potential in the study of the NMDA receptor system in man by PET.

4 ACKNOWLEDGEMENTS Without the following people this thesis would not have been completed on time, so my thanks to a ll of them. At the MRC Cyclotron Unit Chemistry Section my thanks to Dr V.W. Pike for reading and correcting the numerous drafts and for his guidance and assistance, Dr D.J. Silvester for helping me to get started and providing useful commentary, J.C. Clark and the late P.L. Horlock for help in automating systems, and especially Dr F. Brady and D.R. Turton for a ll their help and assistance in developing my id e a s . My gratitude to a ll those in the unit who have helped in so many ways on innumerable occasions. Deserving of special mention are Dr M.J. Kensett whose ties provided endless entertainment, and S. Osman whose friendship and typing skills have been invaluable. Mr K.Dowsett (Argonaut) who generously took time out from Ribena drinking to poke fun at my oily brown products and Dr C. Pascali the human dictionary. I would also like to thank Dr P. Landais for proof re a d in g . Thanks also to Dr J.L. Cheetham, P. Bloomfield and J. Heather for their considerable help with computing and to Dr G. Taylor, Dr D. Watson (RPMS) and Dr S. Waters for help with the mass spectra. I am grateful to the staff of Kings College, Queen Mary College, North London Polytechnic and City of London Polytechnic for the NMR s p e c tra . Finally I am indebted to my fam ily, especially my husband, for their continual support and encouragement.

5 OCMTEMES 1 GENERAL lOTRODUCrXCN 9 1.1 Preamble 10 1.2 Positron Emission and PET 12 1.3 Production of Positron-Emitting Isotopes 17 1.4 Specific Activity Consideration for Radiotracer 26 Application 1.5 Primary [ 11C] Precursors, Derived Labelling Agents and 28 labelled compounds 1.6 Primary [ 18F] Precursors, Derived Labelling Agents and 36 labelled Compounds 1.7 Strategy for labelling 46 1.8 References 48 2 HCTOSYNIHEITC PREPARATION OF [1:LC] GLUCOSE AND ANALYSIS 60 2.1 Introduction 61 2.2 Discussion 68 2.3 Experimental and Results 76 2.3.1 Cultivation of Algae 76 2.3.2 Production of [^C] Carbon Dioxide 80 2.3.3 Trapping of [13-C]Carbon Dioxide 81 2.3.4 Optimization of Buffer pH 84 2.3.5 Separation of Algae Cells 84 2.3.6 Hydrolysis of [^C]Sugars - Optimisation of 85 Hydrolysis time 2.3.7 Neutralisation of Radioactive Hydrolysate 86 2.3.8 Final Procedure for the Preparation of 86 [1:LC] Glucose 2.3.9 Preparation of [13C/11C]Glucose 93 2.4 References 97 3 PREPARATION OF [ 1:LC] DIPRENORPHINE AND [ BUPRENORPHINE 100 3.1 Introduction 101 3.1.1. Strategy for labelling 110 3.2 Discussion 116 3.2.1 Approach A: Reductive Y-Alkylation 116 3.2.2 Approach B: N-Alkylation 118

6 CONTENTS (continued) 3.2.3 Approach C: -Acylation Followed By Reduction 127 3.2.4 Carbon-11 Labelling of Diprenorphine and 139 Buprenorphine 3.3 Experimental and Results 148 3.3.1 Stable Chemistry 148 3.3.2 Carbon-11 Chemistry 160 3.4 References 189 4 mEPARATICN OF [^CJACID CHLORIDES AS IABELLENG AGENTS 198 4.1 Introduction 199 4.2 Discussion 201 4.2.1 Section A: Preparation of [13-C]Acid Chlorides 201 4.2.2 Section B: Preparation of f1-^] Prazosin with 208 [2-11C]Furoyl Chloride as Labelling Agent 4.2.3 Section C: Assignment of % - and 13C-NMR 211 Spectra of N-Acyl-THIQ Compounds 4.3 Experimental and Results 242 4.3.1 Stable Chemistry 242 4.3.2 Carbon-11 Chemistry 257 4.3.3 Description of % - and 13C-NMR Spectra 269 of N-Acyl-THIQ Compounds 4.4 References 324 5 NUClEOfflmC SUBSTITUTION WITH [18F]FLUORIDE 330 5.1 Introduction 331 5.2 Discussion 340 5.3 Experimental and Results 346 5.3.1 [18F]Fluoride Production 346 5.3.2 Preparation of Tetrabutylammonium [18F]Fluoride 346 5.3.3 Preparation of 5-[ 18F]fluoromethyl and 5-/3- 346 [ 18F] fluoroethyl analogues of MK 801 5.3.4 [ 18F]Fluorination of p-Nitrobenzaldehyde and 353 o-Nitrobenzaldehyde and its 4,5-Dimethoxy and 4,5-Dibenzyl Derivatives 5.4 References 356 APPENDIX 360

7 GLOSSARY CAN Ceric (IV) Ammonium Nitrate

DABCO 1,4-Diazabicyclo [2.2.2] octane

EMF N, N-Dimethyl formamide

EMSO Dimethyl Sulfoxide

DTBP 2,6-Di-tert-butylpyridine

NBPN N— (descyclopropylmethyl) buprenorphine

NDFN N- (descyclopropylmethyl) diprenorphine

PDC Fhthaloyl Dichloride

THF Tetrahydrofuran

THIQ 1,2,3,4-Tetrahydroisoquinoline

THQ 1,2,3,4-Tetrahydroquinoline

*PLEASE NOTE - Figures and Tables which are not presented in the discussions are in the experimental and results sections.

8 CHAFFER 1

GENERAL INTOODOCTICW

9 CTNER&L HraCTOCCTCN 1.1 Preamble The radiotracer principle - that the fact that a given isotope may be radioactive does not in anyway affect its chemical (or biological) properties1 - was developed by George Von Hevesy and its importance recognised by his receipt of the Nobel Prize in 1943. Since then the application of this principle has proved essential to the investigation of basic chemical and functional processes in biology. In medicine, emission tomography has evolved from the radiotracer principle to be an important imaging, investigational and research technique. Emission tomography enables the spa ial distribution of any source of 7-rays within a subject to be determined and allows direct in vivo measurements of regional tissue function, often non- invasively. In such studies, tracers labelled with appropriate radioisotopes are introduced into the human body and the emitted 7 - rays are detected by a scanner or tomograph. Computer analysis reconstructs the distribution of the radiotracer from the detected 7 - rays. Data are presented as a series of image slices through transverse or transaxial planes. There are two types of emission tomography, single photon emission tomography (SPEC!)2 '3 and positron emission tomography (PET).4-7 SPECT uses tracers labelled with radioisotopes that decay by emitting one 7 -ray per nuclear disintegration (e.g., 123I, 99m rc),8 A primary limitation of these radioisotopes is the difficulty of determining the direction and depth within the body of the origin of any detected 7-ray. Moreover it is seldom possible to label a compound for SPECT without perturbing its biological activity. By contrast PET relies on the use of compounds labelled with positron-emitting radioisotopes (e.g. 11C , 13N, 150, 18p) .9-11 Positrons emitted from such radioisotopes annihilate with electrons to give externally detectable pairs of 7-rays.4"7 This technique can give a quantitative spatial distribution of the administered radioisotope. Moreover the use of 11C, 13N or 150 enables biologically active compounds (either tracers of metabolism or

10 ligands for particular target sites and receptors) to be labelled isotopically (i.e. without altering biological activity). Moreover 18F can sometimes serve as a useful substitute for hydrogen, since fluorine has a similar Van der Waal' s radius to hydrogen12 and therefore causes little steric perturbation.13 For these reasons PET has became an increasingly powerful technique in medical research, especially for relating biochemical changes in vivo to particular diseases. A particularly important property shared by the most useful positron-emitting radioisotopes is short half-life C1^, 20.4 min; 13N, 9.96 min; 150, 2.30 min; 18F, 109.8 min). Consequently these radioisotopes need to be produced in close proximity to the PET scanner. A particle accelerator, nearly always a cyclotron, is used to generate the positron-emitting radioisotopes. Only a limited number of primary chemical forms ( e.g. 11C02 / 18F2, 18f_) can he generated. Radiosyntheses with these radioisotopes has to be limited in complexity and duration, usually to not more than three to four half-lives. Often high initial radioactivities must be used, therefore necessitating, for radiation safety, the use of shielded and remotely controlled apparatus. These constraints pose special difficulties in attempts to label compounds with positron-emitting radioisotopes. Primarily this thesis concerns improvement to a method for preparing [1:LC]glucose for metabolic studies, the development of new 11C-labelling agents and their application to labelling the opiate receptor ligands, diprenorphine and buprenorphine, and methods of labelling analogues of the NMDA receptor antagonist, MK 801, with 18F. The remainder of this chapter gives a more detailed account of positron emission and PET, of 11C and 18F production and of radiochemistry with 11C and 18F.

11 1.2 Positron Emission and PET The decay of neutron rich-radioactive nuclei by the emission of a , p or 7 particles14 are familiar processes to most chemists in view of the widespread exploitation of such nuclei (e.g. 14C, 3H) in chemistry and biology. Less familiar is the decay of neutron- deficient (or proton rich) nuclei which decay by one of two main mechanisms, namely electron capture or positron emission.14'15

A) Electron capture (EC): In this process a proton becomes a neutron by capturing an orbital electron, causing energy to be released as X-rays:

p + e“ ------► n

An example is provided by the decay of 123I

123I + e“ ------► 123Te

B) Positron emission: In this process a proton changes into a neutron and ejects a positron (a particle with the same mass as an electron but with a positive charge). In this case the number of protons in the nucleus (Z) decreases by one:

p ------► n + p +

An example is provided by the decay of carbon-11:

1:LC ------► 11B + 8+ 6 6

The characteristics of common radioisotopes used to label compounds for PET studies are shown in Table 1.1.

12 Table 1.1. Ocmnon positron-emitting radioisotopes

Radioisotope Half-life %£+ decay Daughter (min)

Carbon-11 20.4 99.8 11B, stable

Nitrogen-13 9.96 100 13C, stable

Oxygen-15 2.03 99.9 15N, stable

Fluorine-18 109.8 96.9 180 , stable

Gallium-68 68.3 90 68Zn, stable

Bromine-75 98.0 76 75Se,radioactive 118.5 days

{Reproduced from Wolf and Fcwler (1985) }9

Most of the light proton-rich radioisotopes decay almost exclusively by positron emission with very short half-lives. Electron capture becomes an important process at higher Z. After emission the positron travels a distance dictated by its initial energy and the "stopping power" of the surrounding matter. In matter of unit density this distance is of the order of a few mm .6 When the positron has lost nearly all of its kinetic energy, it interacts with an electron (Fig 1.1A) and for a short period the two particles exist as a transition state called "positronium" (Fig 1.IB).6 Positronium then annihilates.5 '6 In this process, the masses of both the electron and the positron are converted into electromagnetic radiation in the form of two 7-rays of equal energy (511 keV) that travel in almost exactly opposite directions (to conserve energy and linear momentum; Fig 1.1C). The deviation (ca. 0 .6°) of the angle between the two photons from 180° is due largely to the thermal motion of particles before annihilation.16

13 A. B.

^ e‘

Fig 1.1. (A) Positron approaches an electron; (B) the two particles interact to form a "positronium"; (C) on annihilation, the masses of both particles are converted into electromagnetic radiation in the form of two photons having an energy of 511 keV and travelling nearly collinearily {reproduced from Ter-Pogossian (1985)}.5

It is this annihilation radiation that is detected externally by a PET scanner, a device allowing a subject to be surrounded by one or more rings of sensitive photon detectors. {The MRC Cyclotron Unit scanner has 8 rings, each containing 512 bismuth germanate detectors (Fig 1.2a or b)}. Detection of the simultaneous arrival of two photons travelling in opposite directions allows the location of positron annihilation (and hence positron emission) to be established (Fig 1.3). Use of a computer algorithm on such emission data can reconstruct the spatial distribution of positron-emitting radioisotope in a chosen section of a subject injected with a labelled compound. Data can be accumulated over short intervals (20 s) to provide kinetic information on the distribution of radioactivity.

14 Fig 1.2a radiation detectors

H Ul

coincidence lines

Fig 1.2a and 1.2b Detector configuration of the Hammersmith PET scanner (ECAT V). It consists of eight rings of bismuth germanate detectors (512 per ring) with removable inter-ring septa and retractable transmission ring source for each ring. EMISSION scan, where positron annihilation radiation (two gamma rays) from an administered radiotracer is detected by coincidence detectors. TRANSMISSION scan, where annihilation radiation from an external source of positron emitting radioisotope is measured through the body. Fig 1.3. Illustration of the electronic coincidence configuration for collimation of annihilation radiation (reproduced from Ter-Pogossian (1985)}.5

It should be noted that although the emitted 7-rays penetrate tissue relatively easily, loss of signal due to tissue absorption takes place and must be corrected for. This process is called tissue attenuation correction.6 Attenuation correction can be made by measuring the actual attenuation through the subject using a "transmission" scan (Fig 1.2b). Curing a transmission scan, an external ring source of a positron-emitting radioisotope (68Ga, /3+ 90%, t]_/2 = 68*1 min, daughter of 68Ge, EC 100%, = 287 days) is placed around the subject. The ratio of the coincident events registered from the ring source with no subject in the field of view to that with the subject in the field of view gives the tissue attenuation correction. Ihis correction factor is used to attenuate the "emission" scan (Fig 1.2a) of the same subject. Thus, a principal intrinsic advantage of positron-emission is that it permits

16 the detection of radioisotopes in absorbing medium with sensitivity independent of depth in the absorber. The spatial resolution (i.e. the accuracy of the localization of a radioisotope) of a PET scanner is mainly dependent on the physical size and cross-sectional geometry of the detectors and this subject has been investigated extensively.6 '17'18 Typically, in very general terms, state-of-the-art PET devices can attain a spatial resolution of less than 10 mm .5 In the MRC Cyclotron Unit PET scanner (ECAT V) a spatial resolution of approximately 8 mm is usually attained in a human clinical study.19 In summary, PET is capable of delivering quantitative information on the spatial and kinetic distribution of compounds labelled with positron-emitting radioisotopes - it can perhaps be regarded as in vivo quantitative autoradiography. The present state to which PET is developed, permits in vivo measurements of, for example, blood flow, glucose metabolism, fatty acid metabolism and receptor distribution. PET provides a unique technique for many of these measurements.

1.3 Production of Positron-Emi tti ng Isotopes Since PET only locates the annihilation of positrons, tracers must be labelled with positron-emitting isotopes, such as 11C, 13N or 18F. Generally, these radioisotopes are produced in targets attached directly or indirectly (via a beam transport system) to cyclotrons contained in shielded enclosures e.g. concrete vaults (Fig 1.4). The Scanditronix MC 40 MK II cyclotron at the MRC Cyclotron Unit, consists of a powerful electromagnet between whose poles is a vacuum chamber containing two semi-circular electrode boxes called "dees" and an arc ion source (Fig 1.5). Positive ions are produced by the ion source and they accelerate toward the dee which is at a negative potential. Once within the dee box, the ions do not accelerate any further but follow a semi­ circular path due to the constraints imposed by the magnetic field.

17 4 f Cyclotron . Ion source

Deflector

Vacuum box

Dees

North pole

Fig 1.5. Open view of a cyclotron (reproduced from Comar and Crouzel (1986)}.20

If the accelerating potential difference between the dees changes with a frequency such, that the electric field reverses direction precisely when the ions next reach the gap between the dees, the ions are accelerated further. With increasing velocity and hence increasing energy, the ions follow an ever increasing spiral path towards the periphery of the electromagnet. When the desired energy has been reached, they are extracted electrostatically by a negatively-charged deflector into the beam transport system. This uses precisely aligned magnets to direct and focus the extracted beam onto one of a series of targets, situated on an actuator outside the cyclotron vault (Fig 1.4. shows the MRC cyclotron set-up). The current MRC cyclotron is capable of accelerating a, 3He, p and d particles.

19 1.3.1 Production of -^C and 18F Radioisotopes are produced by bombarding stable isotopes with energetic particles in a target system (e.g. Fig 1.6 or 1.7) .-*-5 For nuclear reactions of protons or deuterons yielding the commonly used positron-emitting radioisotopes (^C, ^50, -*-•% or 18F) the minimum energy of the particle (proton or deuteron) required to overcome the Coulomb barrier is below 5 MeV .2 0 '21 However the projectile energy that gives the desired radioisotope in maximal yield with acceptable radioisotopic purity is generally higher than the threshold value. Theoretical maximal radioisotope yields from most of the commonly used nuclear reactions are given in Table 1.2. Factors that should be considered in the production of radio­ isotopes are the excitation function of the appropriate nuclear reactions, the energy and intensity of available particle beams, the quantity of radioisotope that could be prepared from a particular nuclear reaction, radioisotopic purity, target design and construction (i.e. dissipation of heat during bombardment and radioisotope recovery from the target) and target chemistry. Extensive data dealing with these subjects have already accumulated and have been reviewed quite recently.22-24 Target chemistry controls the chemical form in which the produced radioisotope is obtained after bombardment. Here a number of factors are important, including the target window material (usually a thin metal foil, which is therefore susceptible to thermal damage, pressure stress, corrosion or radiation damage), the material used for the interior surface of the target body and target content (e.g. gas composition). The proceedings of a recent workshop reveal the current status of targetry and target chemistry in detail.25 In the section that follows, a brief description of only the most general and useful methods of producing carbon-11 and fluorine-18 are presented.

20 Table 1.2. Saturation yields3 of seme positron-emitting radioisotopes

Particle energy and yield

Nuclear reaction 8 MeV p 16 MeV p 8 MeV d 10 MeV d GBq//iA GBq//iA GBq/fiA GBq//iA

180(p,n)18F 4.09 8.63 20ife(d,Q )l8F 1.90 2.57

1.49 6.36 10B(d,n) i:k P 1.60 1.97 i:LB(p,n) 11Cc 2.94 11.23

a These yields are taken from the best current data. The data for 1:LC and 18F are ± 5%. The yields listed are for a thicktarget of the element, the energy being that impinging on the target (not on the target window) b The calculation is based on enriched (100%) boron-10 c The calculation is based on natural-abundance (80.2%) boron-11 {Data reproduced from Wolf and Fowler (1985)).10

Production of -^C The early history of the production of 11C can be found in an excellent review by Wolf and Redvanly.26 Currently, the most convenient method for producing 11C is in a nitrogen gas target (Fig 1.6 ) using the 14N(p,a)11C nuclear reaction.27”29 The advantages of this target include high yield (Table 1.3), low maintenance requirement, simplicity of design and simple operation. The chemical form of the 11C obtained after bombardment is dependent on target gas composition. For example, most sources of high purity nitrogen contain a trace of oxygen (in ppm) which is sufficient to oxidize the 1XC to 11CX>2 mainly with [^C] carbon monoxide as minor impurity. If it is necessary to optimise the 11C02 yield, the target gas is passed over CuO to oxidise [^C]carbon monoxide.30 The presence of any carbon containing impurity in the target gas will reduce the specific activity of the 11C02 produced. It should be noted that 11C02 is always contaminated with [13N]nitrogen produced by the 14N(p,pn)13N reaction. 21 Fig 1.7 fl schematic representation of the MRC Cyclotron Unit ,#0-enriched tuater target for the production of [1#F]fluoride.

22 A nitrogen-hydrogen gas mixture can be used as target material for the direct production of [^C] hydrogen cyanide,31 although an alternative combination of target chemistry with chemical processing3 0 '32 is more reliable. The addition of hydrogen (5%) to nitrogen, with the target run at high beam current, produces [13C]hydrogen cyanide (plus 11C=N=M)33 followed by proton-induced radiolysis of [13C] hydrogen cyanide (plus 1:k>^=N and any 11C0 present) to give [13C]methane in situ.32 Small amounts of ammonia are also produced (Scheme 1.1). Passage of this mixture of [11C]methane and ammonia over Pt wool at 1273 K gives f1^]hydrogen cyanide in almost quantitative yield. The preferred method for the production of f1^ ] methane is from 11CC>2 exterior to the target. Reduction of 11C02 over Ni-catalyst with hydrogen at 673 K provides [13C]methane in good yield.

N 2 (H 2) ------► [11C]*

Scheme 1.1 The radiosynthesis of [11C]hydrogen cyanide

Since the generated 11C is always diluted with a large volume of target gas, pre-concentration of activity is often necessary before use. In the case of 11C0 2 , the target gas mixture is passed either into a molecular sieve (4 A°) trap or into a cryogenic trap consisting of a stainless steel loop Immersed in liquid argon.34 [i:LC] Methane is usually pro-concentrated by passage into Forapak Q at 53 K. In each case the concentrated activity is recovered in a small

23 volume of nitrogen (or helium) by heating {a stainless steel trap to 293-373 K, a molecular sieve trap to > 493 K, or a Porapak Q trap to ambient temperature}. These traps deliver insignificant amounts of any [13N]nitrogen impurity.

Production of 18 F Unlike 11C production, 18F targetry design varies considerably with the chemical form of the 18F required. To meet current radiotracer demands, 18F is required to undergo both electrophilic and nucleophilic reactions.

a) Electrophilic 18F 18F-Labelled elemental fluorine is most often produced by the 20Ne(d,a)18F nuclear reaction. A passivated nickel target containing neon with carrier fluorine (ca. 0 .1% v/v) is irradiated with deuterons. Careful yield studies35”37 have shown that target construction (including the material selected and its machining) and gas composition (carrier levels) are critical in the optimization of [18F] fluorine recovery from the target. Target gas impurities such as nitrogen, C02 or organics cause a drastic reduction in the yield of [18F]fluorine.38 However adequate amounts of [18F]F2 can be routinely produced from high beam currents (50 nA) of low energy deuterons (10 MeV)39 or more often, from lower beam currents of high energy deuterons (Table 1.3).35

b) Nucleophilic 18F Production of [18F]fluoride using the 180(p,n)18F reaction on 180 -enriched water was first reported by Carlson et al.40 At that time, due to the large volume of the target, this method was considered too expensive for routine production of [18F] fluoride. Therefore, a production method employing natural water, using the 160(3He,p) 18F reaction was developed.41 '42 This method can produce useful levels of exchangeable carrier-free [18F]fluoride ion {i.e. 18F” (H20)n, Table 1.3), for incorporation into tracers for PET studies.

24 Table 1.3. Production parameters for and 18F

Nuclear Threshold Main Typical Specific Dilution reaction energy product yield activity with (MeV) (GBq) (G B q/im ol) carrier

14N(p,a) 1ZC 3.13 1X002 93a 150 2 x 103 on N2

14N(p,a) 11C 3.13 i :lc h 4 67b 130 3 x 103 on N2 & H2 (5%)

20Ne(d,a)18F 0 18F—F 13.6a ca.0.2 3 x 105 on Ne & F2 (0 .1%)

160(3He,p) 18F 0 18F"(H20) n 14.8d 138 5 x 102 on H20

180(p.n) 18F 2.5 18f -(h 20 )n 44e 370 2 x 102 on H2i80

a) 19 MeV, 30 /xA, 30 min; b) 18 MeV, 30 /xA, 40 min2 7 ; c) 14 MeV, 15 /xA, 120 min35; d) 36 MeV, 40 /xA, 60 min43; e) 15 MeV, 20 /xA, 60 min44 (reproduced from Pike (1988)}.11

Recently with the development of small volume targets45 '46 and efficient recovery systems, most groups have taken to the proton irradiation of 180 -enriched water as their most convenient source of high levels of aqueous [18F]fluoride (Table 1.3). Figure 1.7, shows a stainless-steel 180-enriched water target (ca. 20% enrichment v/v) now in use at the MRC Cyclotron Unit. This target uses 1.8 ml of 180-enriched water, which can be recovered after irradiation. The conservation of 180 -enriched water has been approached in several ways by different groups. Some groups employ distillation to recover the 180-enriched water from 18F. Others use ion-exchange columns4 7 '48 to trap the [18F]fluoride and efficiently recover the total volume of 180-enriched water. Electrolytic separation49 of

25 [18F] fluoride freon 180-enriched water is also possible; under very mild conditions, 97% of 18F” can be transferred into natural water. Hie radioisotopic purity and the reactivity of the [18F] fluoride is dictated by the target materials used. The leaching of cations from 180 -enriched water targets has received considerable attention.25'4 4 '50 Cations, such as Ca2+, Al3+ are undesirable since they probably produce unreactive metal [18F] fluorides {CaF4*, A1F(H20) 52+} which cause problems in subsequent nucleophilic reactions. Suitable target chamber and foil materials include, Ni, Ag, 316 stainless, Ti, Pt and E h .51

1.4 Specific Activity Consideration for Radiotracer Application An essential consideration in the preparation and use of precursors for radiotracer synthesis is the specific activity which is expressed as radioactivity per mass of compound (e.g. in GBq//imol) ,52 More simply this term identifies the extent of dilution of the radiotracer with non-labelled compound or carrier. The theoretical maximum specific activity of a radioisotope is given by Equation 1.

A — = (In 2/ti/2) ...... Eq 1 N

= (0.693/tV 2 )

where, A is the radioactive decay rate (disintegration per unit time), tjy2 is the half-life and N is the number of atoms of the radioactive element (6.023 x 1023 atoms/mol).

Terminology expressing the extent of dilution of a radiolabelled compound with unlabelled compound has been proposed5 3 '54 as follows:

i) Carrier-free (CF) refers to the state where a radiolabelled compound is known not to be diluted with corresponding stable compound (carrier).

26 ii) No-carrier-added (NCA^ refers to the state where carrier has not been intentionally added daring the preparation and all precautions have been taken against dilution from external sources of carrier

iii) carrier added (CA) refers to the state where a known amount of carrier has been added to the labelled compound.

In the case of labelled tracers, a truly CF state is seldom achieved, i.e. maximum specific activity is rarely attained (Table 1.3). For example, in the production of 11002 and in its subsequent conversion into other compounds such as, formaldehyde or [11C]iodamethane, traces of 12002 from the target, experimental set­ up and reagents, are unavoidably introduced into the synthetic procedure even when stringent precautions are taken. As a consequence, any radiotracer prepared from these precursors generally contains multi-nanomoles of stable product. The amount of carrier is particularly critical in PET applications with highly toxic or receptor directed radiolabelled compounds. Consideration to the route giving the highest specific activity is often therefore important before developing a synthetic procedure. Since the positron-emitting radioisotopes are short-lived, quantities of radioactivity, specific activity and radiochemical yields (r.c.y) must be related to a reference time point. In this thesis, all such values are related to either the end of radioisotope production (end of bombardment or EOB) or the end of radiosynthesis (EOS) at time t. Correspondingly, all radiochemical yields are corrected to EOB or are quoted at EOS. In addition, since the specific activity is related to the activity at time t, this value will vary with the half-life of the radioisotope. Sometimes it has been more convenient to state the carrier content (a constant) in the system (nanomoles or micromoles), rather than the specific activity.

27 1.5 Primary r RRci Precursors. Derived labelling Agents and Tahpllpd Occixxinds Carbon-11 is incontestably the most widely used positron- emitter for the labelling of compounds for biomedical research and PET. Incorporation into the structure of a molecule necessitates that cyclotron-produced RRc be in a highly reactive form. ^Re­ labelling can be achieved by using either a primary precursor such as

RRg o 2 or a derived labelling agent such as [11C]iodamethane. The subject of [ 1:LC]precursors, [1:LC]labelling agents and 1:LC-labelled compounds has been addressed extensively in a number of recent reviews.9"11'26'55 Therefore, this section addresses the synthesis of the most commonly used primary precursors and derived labelling agents and refers to some of the important publications that have appeared in 1988. In addition applications of [1:LC] precursors or [Relabelling agents have been selected to emphasise the variety of compounds that can be labelled with carbon-11. Carbon-11 is generally recovered from the target in one of two forms: RRco2 and [RRc]methane. From these two simple primary precursors a variety of more complex and, in some cases, more reactive [RRc] labelling agents have been prepared (Fig 1.8). Whereas, most applications of RRco2 and its principal labelling agents involve [RRc]carboxylation and [RRc]alkylation (Fig 1.8), the principal labelling agent derived from [RRc]methane, [RRc]hydrogen cyanide, is employed in a variety of displacement or addition reactions (Fig 1.8). Most syntheses with RRc have involved the formation of a C-C, C-N, C-S or C-O bond. For example, one of the most facile reactions for C-C bond formation is the carboxylation of a Grignard reagent.9'56 This approach has been used to label a number of fatty acids, including [1-RRc] acetate,57 [ 1-RRc] palmitate,58 and /3- methyl[l-RRc]heptadecanoate.59 Both [1-RRc] acetate and [1- 21C]palmitate have been extensively used to study the myocardial metabolism of natural fatty acids in humans; the analogue £-methyl [1- X1C] heptadecanoate was designed to be trapped metabolically thereby simplifying PET studies of myocardial metabolism. [ RRc] Carbonation

28 11ch 3,no 2

11c h 4------11co

Fig 1.8 [11C]Labelling agents derived from [11C]carbon dioxide of Grignard reagents has also led to a variety of [ ^C] alcohols, such as, [13C]methanol60 and [l-1^ ] butanol,61 for the investigations of blood-brain barrier permeability, and to [^C]aldehydes, such as [ 1:LC]benzaldehyde,62 [11C]veratraldehyde,63 as labelling agents in the preparation of [3-^C]phenylalanine^ and [l-^-CJDopa.64 Racemic carboxyl-labelled [13C] amino acids , such as alanine, phenylalanine phenylglycine and Dopa, have all been prepared via the carboxylation of a-lithiO'isocyanides (Scheme 1.2).65-67

i) Base NH2 ii) 11C 02 I R— CH2— NC ------R’— CH — 11COOH iii) H+

Scheme 1.2 Carboxylation of a -lithiosocyanides to give [11C]amino acids

The resolution of racemic [^C] amino acids has been performed by enzymic amino-oxidation68, chiral hplc69'70 or by selective binding to human serum albumin immobilised on Sepharose gel.7-1* [13C]Amino acids have been used in studies of human protein synthesis72'73 and in same cases, as precursors to neurotransmitters.74 An interesting carboxylation reaction is that of a benzcphenone dianion (A), generated by the addition of powdered lithium.75 Hydrolysis and esterification of the [-^-k^benzylic acid (C) with 3- quinucl idinol in the presence of carbonyl diimidazole and cuprous chloride yields [13C]QNB (quinuclidinyl-benzylate), a radioligand for the muscarinic receptor (Scheme 1.3).

30 o

OLi 11 uox Vc. I CO. \ / ^ O L i c 6h 5- - c- ^ 6^5 C I / \ . OLi h5c 6 ^ 6^5 (A) (B)

[11C]QNB (C)

Scheme 1.3 Preparation of [11C]QNB

[1:LC] Carbon dioxide has also been used directly in the photosynthesis of [^C]sugars (see Ch. 2, Sec 2.1). However, the vast majority of syntheses with carbon-11 require a suitable labelling agent. The versatility of [i:LC]iodamethane has been demonstrated by its extensive use in methylation reactions, predominantly to form bonds in amines and amides.9 [ 12C] Iodomethane is conveniently prepared from 11002 by reduction with lithium aluminium hydride (LiAlH4), followed by treatment of [1:LC]methanol with constant boiling hydroiodic acid (Scheme 1.4).

i) LAH HI 11C 0 2 11c h 3o h 11c h 3i ii) H20

Scheme 1.4 Preparation of [11C]iodomethane

31 The synthesis was first described by Comar76 and has been improved by several groups,77-79 to minimise the introduction of any source of carrier (e.g. 12002) into the system. Recommendations referring to the preparation and handling of solutions of LiAlH4 in tetrahydrofuran (THF) under inert atmosphere, reagent concentration and reaction volume have been suggested to achieve high specific activities.34'80 Wagner et al81 produced [^-k]iodomethane by a one step recoil synthesis using N2/HI mixture as target gas. More recently, diphosphorous tetraiodide82 has been used to convert [xk ] methanol into [ -*-k] iodomethane, thus avoiding HI and its unstable and corrosive properties. [ -^k ] Methylation of a primary or a secondary amine is a well- established procedure in ^k-chemistry.9 '83 For example, [tf-methyl^k] nomifensine, a marker for pre-synaptic dopaminergic re­ uptake sites84'85 has been synthesised by //-alkylation of the nor- compound using [ -^k]iodomethane in dimethylsulfoxide (EMSO) /dimethylformamide (EMF),86 Since the starting amine (ca. 20 /iitiol) is always in vast excess over iodomethane (stable plus active), most reactions of primary amines with [ Ik] iodomethane give the mono- substituted //-[11C]methylamine. However, the formation of the dimethyl product87 may be noticed when RNHCH3 is significantly more reactive than the starting amine, R-NH2. Ctoampeting reactions of the labelling agent with solvent (e.g. -^-kl^I with EMSO), traces of degraded solvent or contaminants, can also hinder the formation of the desired compound. In addition to //-[-^kjmethylations, several 0-[11C] methylations have been reported. [11C]Raclopride (E), a selective dopamine (E^) receptor antagonist, has been prepared by O-alkylation of the corresponding phenol (D) with [-^k] iodomethane (Scheme 1.5).88 A recent example of O-'-kl^ bond formation is in the synthesis of enantiomerically pure neurotransmitter precursors:89 L-[3-1]-C] tyrosine, L-[3-1:LC]Dopa, L-[3-1:k] tryptophan and hydroxytryptophan. These precursors are prepared from D,L-[3- xk ] alanine by multi-enzymatic syntheses. Racemic [ 3--^k] alanine is prepared initially by direct alkylation of a protected glycine

32 derivative, N - (diphenylmethylene) glycine-tert-butyl ester, with [ ^C ] iodcmethane.90

OH 9

c 2h5

11CH3I NaOH in DMSO

Scheme 1.5 Preparation of [11C]raclopride

[iic] Formaldehyde, in reductive [1:LC]methylation, is an alternative to [i:k:]iodamethane for N - [ ] methylat ion. The synthesis of [1:LC] formaldehyde involves the oxidation of [1:LC]methanol over silver wool catalyst at elevated temperature (ca. 623 K) .78,91,92 The reproducibility of this synthesis is poor because it is difficult to control activation of the catalyst. Consequently, this labelling agent is less frequently used, except for selective [ 1]-C] methylat ions on sensitive substrates.93 Since alkylation reactions with [ 11C] iodomethane have proved to be extremely valuable for labelling compounds with carbon-11, this approach has been extended to the use of other [13-C] a lk y l and [1:LC]benzyliodides as labelling agents. These include [l-1-^:]ethyl iodide,94”96 [l-13-C]propyl iodide,96 [l-13C]butyl iodide,96 [1- 13C] isobutyl iodide,96 [1-1:LC] isopropyl iodide,97 [l-i:LC] benzyl

33 iodide98 and [1-1:LC]substituted benzyl iodides.99 Synthesis of the unbranched [1:LC] iodides involves [ 11C ] carbonation of an appropriate Grignard reagent, reduction of the [^-^C]adduct with IlAlH^ and treatment with HI (Scheme 1.6).

11 CO. Li AIK HI RMgBr R11COOMgBr ------^ r 11c h 2o h - R 11C H 2I

R = ^ , c h 30 ^ ^ >

CHgO^

■

Scheme 1.6 Synthesis of [11C]alkyl iodides

1:LC-Labelled substituted benzyl iodides have potential use in the synthesis of [ 3-11C] tyrosine and L-Dopa. [2-i :lC] Isopropyl iodide, prepared via the [1:LC]carbonation of methyllithium has been usefully applied to the synthesis of £-adrenergic receptor ligands,100 such as [1:LC]atenolol (F), [11C]metoprolol (G) and [1:1-C]propanolol (H) (Scheme 1.7).

CH / 3 DMF/DMSO RNHp + I — 1CH ------R— N v CH. \ CH3 11CH \ c h 3

OCH 2C H C H 2- iHf\ _ / c = o OH I NHo (F) O H (G) O H (H)

Scheme 1.7 Preparation [11C]atenolol(F), [11C]metoprolol (G) and [11C]propanolol (H)

34 A common approach to the labelling of p -adrenergic receptor ligands101”103 has been reductive alkylation with [2-11C]acetone. [2-11C]Acetone can be prepared by [ 1:LC] carbonation of msthyllithium (Scheme 1.8) ,104

2MeLi r 11 *1 H+ 11C02 ------|Me211C(OLi)J ------^ Me211CO

Scheme 1.8 Preparation of [11C]acetone

The molar ratio of methyllithium to 11C02 is critical to the purity of [2-11C]acetone produced, since excess methyllithium results in the formation of [2-11C]tert-butanol as the major by-product (Scheme 1.9).

r- -i MeLi [Me2' 1C(OLi) 2j ------Me3"COH

Scheme 1.9 Formation of [2-11C]tert-butanoi

As described earlier, the principal labelling agent derived from [13€]methane is hydrogen cyanide. A large number of compounds, mostly 11C-labelled amines,105-107 have been prepared via the Bucherer-Strecker synthesis, utilising f1^]hydrogen cyanide as the labelling agent (Scheme 1.10).

H H 11CN** OH" I RCHO ------R—C -1C = 0 ------^ r — c — 11 COO" (NH4)2C03 I / \ +nh 3 HN (D,L)

O Scheme 1.10 Preparation of [11C]amines with [11C]hydrogen cyanide

35 Catalytic chlorination of [^-C]methane at 583 K on pumice stone impregnated with cupric chloride gives f1^ ] chloroform. This can be converted into [1:LC]diazamethane of high specific activity, by reaction with hydrazine and potassium hydroxide .108 [ 1:LC] Diazamethane has only had application in the synthesis of a [i:LC]methyl ester108 and requires to be explored as an alternative to [ 11C] iodamethane for [i:iC]methylations. Recently [1:LC] phosgene has also been prepared from [1:LC]methane. Chlorination of [1:LC]methane gives [1:LC]carbon tetrachloride, which can be catalytically oxidized over iron fillings to t1^]ph o s g e n e.109 [1:LC]Phosgene has been used to label the /3-adrenergic receptor ligand, 4- (3-tert-butylamino-2- hydroxypropoxy)-benzimidazol-2-one (OGP 12177}.110

1.6 Primary r 18F1 Precursors, Derived labelling Agents and labelled Ccampounds Recently there has been growing interest in organic compounds labelled with fluorine-18, due partly to its longer half-life (Table 1 .1 ) and the higher specific activity achievable with fluorine-18 (Table 1.3) compared to carbon-11. The longer half-life allows comparatively long synthesis as well as the study of relatively slow biological processes. In addition, fluorine-18 decays to give a relatively low energy positron with a short range (2.4 mm)111 in material of unit density. This range is less than the best resolution achievable in the current generation of PET scanners; resolution in the PET study is not therefore limited by positron range as it is for other positron-emitting radioisotopes. In many organic syntheses, 18F is used to replace H in the target molecule because their Van der Waal's radii12 are similar. In molecules where conformational recognition is important minimal steric disturbance by a substituent is especially significant. Once introduced, the high energy of the carbon-fluorine bond12 renders it relatively resistant to metabolic transformations. Replacement of H by F can have a profound effect on the chemical and biochemical properties of the parent compound.112 Fluorine is highly electronegative and can therefore influence the

36 electron distribution in the molecule, thereby affecting the basicity, acidity or reactivity of neighbouring functional groups. As a consequence of the available electron density, fluorine can participate in hydrogen bonding with H. Replacement of H by F also changes 1 ipophilicity. The interplay of these factors m y therefore alter the bio-distribution of the fluorinated analogue from that of the unsubstituted molecule. The synthesis of 18F-labelled organic compounds has been addressed in a number of reviews^""11' 118' ll2^ and this section only describes the synthesis of the cammonly used fluorinating agents (e.g. molecular [18F]fluorine, [18F]fluoride and labelling agents derived from them) for electrophilic and nucleophilic reactions.

"Electrophilic11 Fluorinating Agents [18F]Fluorine gas is the original "electrophilic" fluorinating agent and is the source of other labelling agents such as acetyl [ 18F]hypofluorite, xenon [18F] difluoride and perchloryl [18F] fluoride (Fig 1.9). ------Xe18F2

20Ne(d,a)18F ------18F-F ------CH3C0018F

------CI0318F Fig 1.9 [18F]Labelling agents derived from [18F]F2

Molecular fluorine is well known to be highly reactive. It is a strong oxidizing agent that will react with almost any organic compound, usually exothermically. In addition fluorine tends to form radicals which react indiscriminately with organic molecules leading to mixtures of products. Dilution of fluorine with an inert gas, such as neon, provides more control and selectivity.115'-1-16 Typical bombardments for producing [18F] fluorine gas require fluorine at concentrations of 0.1-0.2% in neon. Consequently, the specific activity of [18F] fluorine eluted from the target is low and mainly

37 determined by the target volume and operational pressure. The formation of a C-F bond by "electrophilic" reaction requires the fluorinating agent (e.g. F2 , XeF2 or CH3COOF) to possess a degree of polarization such that the fluorine carries some positive charge enabling reaction with an electron-rich substrate, such as an unsaturated molecule, a carbanion and in some cases, a C-H single bond. Before 1982 [18F] fluorine was used extensively to produce a number of 18F-labelled radiotracers,9 including 5-fluorouracil,117 4- fluoroantipyrine118 and 2-deoxy-2-fluoro-D-glucose (FDG) .119 The reaction of 3,4,6-tri-0 -acetyl-D-glucal with elemental fluorine gives c is addition across the double bond but is not particularly selective as to which face of the molecule addition occurs. Consequently, both gluco and manno-2 -deoxy-2-fluoropyranosyl fluorides are produced.120 Today, many syntheses employ the milder electrophilic labelling agent, acetyl [18F]hypofluorite. The preparation of acetyl hypofluorite was first described by Rozen et al121 and subsequently two general methods have been developed for the direct production of a c e t y l [1 8 F]hypofluorite from [1 8 F]fluorine. Acetyl [18F] hypofluorite may be generated in solution by passing [18F] fluorine into glacial acetic acid containing sodium acetate,122'123 ammonium acetate124 or rubidium acetate.125 Recently procedures have been developed for the production of acetyl [18F]hypofluorite in the gas phase, by passing [18F] fluorine into a column containing sodium acetate trihydrate126, supported sodium acetate127 or an acetic acid-alkali metal (Na or K) acetate complex.128 All these methods give almost a theoretical radiochemical yield (50%) of acetyl [18F] hypofluorite from [18F] fluorine. Acetyl [18F] hypofluorite was initially applied to the preparation of [18F]FDG via addition to 3,4,6-tri-0 -acetyl-D-glucal. This method increases the ratio of [18F]FDG to 2-deoxy-2- [ 18F] fluoro- mannose (FEM) to 95:5 and doubles the r.c.y. of [18F]FDG to 20% . 1 2 6 '129 In general, it has been shown that acetyl

38 [18F] hypof luorite has different stereoselectivity to [18F] fluorine12 6 and that the selectivity of both reagents is a function of solvent129•120 and substrate.121 »122 Acetyl [18F]hypofluorite adds across a double bond to give an unstable intermediate, which loses acetic acid in the presence of base to form an 18F-labelled vinyl fluoride (Scheme 1.11).

H 18t OAc 18Fn Ac0 18F base

Scheme 1.1 l Formation of [18F]vinyl fluoride

This approach has been used in the preparation of 4-[18F]fluoro- antipyrine133 a blood flow marker, 5-[18F]fluorouracil134 and 5- [18F]fluoro-2'-deoxyuridine135 for tumour studies by PET. Recently, acetyl [ 18F]hypofluorite has been used to label L-6- [ 18F] fluoro-Dopa, an analogue of the anti-Parkinsonian drug, Lr-Dopa (L-3,4-dihydroxyphenylalanine) . The reaction of acetyl [18F] hypof luorite with N-acetyl-3-0 -methyl-4-0 -acetyl-Ir-Dopa in acetic acid, followed by hydrolysis, produces L-2- and L-6- [ 18F]fluoro-Dopa in equal amounts.136 The isomer required for PET studies of dopamine turnover in Parkinson's disease,137'138 and other movement disorders,139 is L-6- [ 18F]fluoro-Dopa. This can be isolated by preparative hplc in 3-4% r.c.y at EOS, based on acetyl [18F] hypof luorite.136 In contrast, the reaction of [18F] fluorine with L-Dopa in liquid HF, gives a mixture of L-2-, 5-, and 6- [ 18F] fluoro-Dopa in the ratio of 7:1:12 and the pure 6-isomer is isolated by recycle hplc.140 This method is less convenient for routine production since it requires special materials for handling the potentially dangerous HF and fluorine safely. A c e t y l [1 8 F]hypofluorite also reacts with aryl- mercurials,141'142 aryl-tri-n-butyltins,143 trimethylsilanes and pentafluoro-silicates,144'145 to give substituted aryl fluorides. O t h e r [18 F] labelling agents, such as xenon [18F] difluoride (Xe18F2)146 or perchloryl fluoride (C10318F )147 are seldom used due to the difficulties involved in their preparation.

39 Nucleophilic Fluorination Reactions Currently fluorination by nucleophilic substitution with [18F]- fluoride or reactions of hydrogen [18F] fluoride are the only available methods offering no-carrier-added syntheses with maximal incorporation (100%, theoretical) of the available activity. Although gaseous hydrogen [18F] fluoride is potentially versatile for organic synthesis, there are serious disadvantages in its production, including serious loss148 of anhydrous carrier-free [18F] fluorine to the wall of the target chamber, tubing and chemical reaction vessel. Addition of fluorine gas to improve the recovery of hydrogen [18F] fluoride results of course in carrier added product. For many radiotracer applications of 18F (e.g. receptor ligands) an almost carrier-free product is required. While, aqueous [18F]fluoride can be easily produced in high yield and high specific activity from an 180-enriched water target,149'150 the recovered activity cannot be used directly in syntheses. It is well known that water decreases the reactivity of fluoride (fluoride ions are capable of forming strong H bonds to a variety of electron-acceptors i.e. protic solvents) and that aqueous fluoride is an extremely poor nucleophile at carbon.151 In the absence of a more powerful H-bond electron acceptor, water will solvate and effectively mask the fluoride anion. This is particularly true for n.c.a reactions, where the relatively small amount of [18F] fluoride ions (< 1 nmol from most target systems) can be completely solvated in the presence of only trace water, thus rendering the 18F anions unreactive in nucleophilic reactions. Generally, the base strength of an ionic fluoride is dependent on the amount of water that is present, the solvent in which it is dissolved and on the countercation.151 For nucleophilic substitution reactions essentially an unsolvated or "naked" fluoride anion is required. Dipolar aprotic solvents are considered to be superior, owing to their poor anion and high cation solvating properties. A supporting salt, consisting of a large, easily dehydrated cation, (such as, tetraethylammonium (TEA+)) and a basic , poorly nucleophilic anion (such as hydroxide) which is soluble in polar

40 aprotic solvent, leads effectively to an unsolvated fluoride anion with enhanced reactivity. The nuclear reaction of choice for [18F]fluoride production is 180(p,n)18F characterized by a high yield at comparatively low proton energy. Therefore, the first stage of any 18F-labelling reaction involves the removal of bulk water. This results in the concentration of involatile contaminants produced in the target during irradiation, such as, metal ions (Ca2+, Al3+) which bind [18F] fluoride tightly or anions (Cl", N03") which compete with [18F]fluoride in nucleophilic substitution.51 A recent report has shown that the amount of anions (F", N02", NO3", Cl") present in the target water increases as a function of the dose accumulated on the target.152 Generally, the production of involatile contaminants is minimised by good target design.

[ 18f ] Fluoride obtained from the target is usually used without further purification. However, this approach has led to inconsistency in yields reported for the same reaction by different groups. Recently [ 18F] f luorotrimethylsilane153 •154 has been employed as a highly reactive and reproducible source of [18F] fluoride for systematic radiochemical investigations.155 [ 18F] Trimethyl- fluorosilane is a useful volatile intermediate for the purification of [18F] fluoride since it is easily formed from aqueous [18F] fluoride by reaction with trimethylchlorosilane and is easily cleaved after distillation by base to regenerate [18F] fluoride (yields approach 100%). A number of methods have been used for the generation of reactive [18F] fluoride from aqueous [18F]fluoride. For example, macrocyclic polyethers and aminopolyethers in the form of their alkali complexes (K+-18-crown-6,156'157 K+-2,2,2- aminopo I y.ether,158'159) bulky cations (Rb+, Cs+ ),1 6 0 '161 tetraalkylammonium hydroxides (I^N+OH",162 normal-B^lF^OH"163) and dipolar aprotic solvents have all been employed with the aim of improving fluoride solubility and of suppressing the hydration/solvation of the fluoride ion. A common strategy is to

41 remove the water by distillation from added base (e.g. Rb2C03 or normal-Bu4NOH) and the residual [18F] fluoride salt is dried by azeotropic distillation with acetonitrile. Loss of [18F] fluoride can be substantial if only weak base is present. The dried residue is then resolubilised in a dipolar aprotic solvent (e.g EMSO or acetonitrile) containing the substrate for nucleophilic attack. It has been suggested that resolubilisation of [18F] fluoride depends on the vessel material164-166 used and that glass vessels (borosilicate glass) are not suitable. A systematic study employing THF as resolubilising solvent has indicated that commercially available siliconised glass (Vacutainer) is preferable to platinum, borosilicate glass or pre-treated siliconised borosilicate glass.164 However, at the MRC Cyclotron Unit, similar results have been obtained for pre-treated (siliconised) and untreated borosilicate glass, with platinum crucibles giving least [ 18F]fluoride adsorption. It should be noted that resolubilisation is more difficult from all vessel types if a high degree of dryness has been achieved. The influence of carbonate/polyether concentration with regard to the amount of radioactivity adsorbed onto the walls of Wheaton glass vessels was examined by Block et al.167 These investigators showed that small amounts of [K** 2 .2 .2 ]2C03 lead to a drastic decrease of fluorine-18 adsorption although the extent of adsorption remains constant after the addition of more than 0.2 mmol. Probably the combination of reaction vessel, the concentration of cationic species and solvent type, all determine the solubility of [ 18F] fluoride for a particular reaction. Water can apparently be tolerated in relatively large amounts without severe adverse effects in some nucleophilic fluorinations.168-170 A systematic study has shown that no inhibition of [18F] fluoride incorporation into cyclic sulphate or 2 ,4-dinitrochlorobenzene was apparent until the amount of water reached one half the concentration of the organic compound and TEA+.155 As mentioned earlier, nucleophilic substitution reactions are best performed in dipolar aprotic solvents.151 For example, 18-

42 crown-6 successfully solubilises potassium salts (fluoride or acetate)171'3-72 in acetonitrile producing extremely reactive anions ("naked" anions: F”, O^OOCf) presumably because of the weak anion solvation forces in acetonitrile and the complete dissociation of the electrolyte. In general, the choice of solvent for [18F]- fluorination depends on the type of reaction, the cationic species involved and the desired reaction temperature. In so far as direct comparisons have been made, acetonitrile has became established as a convenient solvent for aliphatic substitution reactions168 and EMSO is favoured in aromatic substitutions161'178 where high reaction temperatures are often required. However, EMSO is prone to decompose below its boiling point and is an oxidising agent in its own right. Other solvents that have been used in reactions with [18F] fluoride include, acetone ,174 chloroform ,175 THF ,164 DMF ,165 dichloromethane,168 O-dichloro-benzene168. Block et al159 have studied the exchange of 1,2-ditosyfoxyethanewith n.c.a. APE/K18F as a function of solvent polarity. They showed that the yield of [ 18F] fluorotosyloxyethane increased with solvent polarity in the order of acetonitrile > dichloromethane > chloroform > 1,4-dioxane. An exception was found to be acetone which in spite of its rather higher polarity and hence solubilisation of the salt complex, gave small substitution yields. The solubility and reactivity of the fluoride anion are highly dependent on the accompanying cation. Only large and "soft" alkali cations (K4 , Rb4 , Cs+) are effective, since their polar interactions with the "hard" anion are weaker than those of the "hard" cations (Li4 or Na4). This effect is more valid for polyether assisted reaction systems e.g. K4 / 2.2.2. APE and tetraalkylammonium systems (R4N4 ).167 At present, there is no basis for preferring K4 / 2.2.2. APE or R 4N4 as cationic species for 18F incorporation into the many classes of compounds studied, such as, sugar derivatives (cyclic sulphate or triflate),155 fatty acid derivatives (iodo-methyl esters),155 disubstituted alkanes {X(CH2)nX for n = 1-3, X = Br, 0 - mesylate or 0 - t o s y l a t e}1 5 8 '168 or aromatics (fluoro or chlorodinitro-benzenes).155'176 However, caesium or rubidium cations

43 are usually employed for difficult aromatic substitutions160'161 since tetra-alkylammonium fluorides have low thermal stability.177 A variety of aliphatic [18F]fluorides and aryl [18F] fluorides have been prepared by reaction of the appropriate substrate with [18F] fluoride. Alkyl halides have been exploited widely as substrates for 18F exchange . Classically, the reactivity of halogens as leaving groups decrease in the order of I" > Br- > Cl” > F". This is so in the gas phase preparation of alkyl [18F] fluorides from the corresponding alkyl iodides, bromides and chlorides.178 However, the difference between halogens (especially bromo and iodo167'179) is small when compared to differences caused by other parameters of the reaction and differences between halogens and other leaving groups (X = Br, I, 0 -methanesulphony 1, p-toluene- sulphonyl) .150'163 [18F]Fluoroalkyl halides have been prepared in relatively good yield from the corresponding alkyl bromides and iodides166'179-181 and have been shown to be good IV-alkylating reagents.179 The well known organo-sulphonyl groups, especially p- toluenesulphonyl (tosyl) , methanesulphonyl (mesyl) and trifluoromethanesulphonyl (triflyl) have featured prominently as leaving groups in aliphatic nucleophilic substitution reactions with [18F] fluoride. The leaving ability of these groups decreases in the order of "triflyl > me syl > tosyl > halogens.159 For example, the leaving ability of triflyl group is approximately 40,000 times greater than that of tosyl and in some molecules its presence may lead to elimination rather than to substitution. Nevertheless, displacement of triflyl by 18F has resulted in a wide variety of labelled compounds, including 3-[1 8 F] fluoro-3-deoxy*-D-glucose182, [18F]FDG158'162 and a series of estrogenic compounds184. The mesyl group has found some applications, particularly in the 18F-labelling of steroids157, fatty acids174'184 and dopamine receptor ligands (N- [18F]fluoroalkyl-spiperones)186. A second approach to labelling N- fluoroalkyl spiperones179'186 has involved the synthesis of [18F]fluoroalkylating agents, followed by //-alkylation of spiperone. Apart from u>-[ 18F] fluoroalkyl halides (Br,I)179, w- [ 18F] fluoroalkyl tosylates (prepared by 18F displacement of bis-tosyloxyalkanes)186

44 have also been used for this purpose. A recent application of the displacement of tosyl by [18F] fluoride is the synthesis of the progesterone receptor ligand, 16a-ethyl-21-[18F]fluoro-19- norprogesterone.187 This radioligand might allow the measurement of estrogenic receptor density in patients with breast tumours with a view to elevating the response to hormone therapy. Aromatic nucleophilic substitution with [18F] fluoride is only feasible when the aromatic ring is sufficiently activated. The structural requirement is an o r th o or p a ra relationship between the

l e a v i n g group (F, Cl, I, N02) and an electron withdrawing activating group (N02, CN, COR, CHO). The nitro group behaves both

as a n excellent leaving group for fluoride and as an activating group when in the o rth o or pa ra position. For example, the activating effect of the substituent group X in the fluorodenitration of C6H 4 (N02)X in EMSO at 423 K increases in the order m-N02 « p-CN ca.

= o-N02 < o-CN < p-N02 .161 Korguth et al155 have shown that both fluoride-for-fluoride and fluoride-for-chloride exchanges are favoured over fluoride-for-nitro exchange when both nitro and halogen are present in the same molecule. In addition they found that the hetero-halide exchange is much slower than isotope exchange; 2,4- dinitrochlorobenzene reacts almost as rapidly as the aliphatic triflates and cyclic sulfate. It should be noted that fluoro for fluoro substitution reactions are facilitated by the cancellation of solvation effects, making this a viable labelling approach where low specific activity is acceptable. Aromatic nucleophilic substitution has been used for the synthesis of both carrier added (18F for 19F) and no-carrier-added (18F for N02) [18F]haloperidol and [18F]spiperone in low yields.188”190 Recently, a procedure has been reported191 that may have distinct advantages over the azeotropic removal of water in the presence of base for the generation of "activated" fluoride. The method utilises a polymeric quaternary ammonium hydroxide resin for both trapping and nucleophilic activation of [18F] fluoride from target water. In addition radiofluorination reactions are conducted

45 by passing a solution of substrate back and forth through the heated [18F]resin bed. The authors claim that base sensitive substrates are labelled in acceptable yield due to the intermittent and brief contact with the heated resin. Reasonable yields have been reported for halogen exchange (^8F for Br or I), nitro displacement and triflate displacement. In addition [ 1:LC]cyanations have also been performed on the same resin (13C for Br or N02).

1.7 Strategy far labelling It is apparent from the above brief review that a variety of labelling precursors/agents and methods are available for both the 11C- and 18F- labelling of radiotracers and radioligands. However, it is of utmost importance that a great deal of consideration be given to radiotracer or radioligand design before a particular synthetic route is approached. Ihe selection of a molecular structure for labelling requires intimate interaction between many specialised disciplines, including clinicians, chemists, biochemists, pharmacologists and physiologists. Primarily, an appropriate tracer or ligand needs to be identified for the clinical question being asked. An essential requirement of PET in measuring the rate of any biochemical process or in describing a phenomenon quantitatively is that the molecular fate of the radiolabel be known, bearing in mind that tissue sampling is precluded in PET studies on humans. This entails a thorough examination of the biochemistry, pharmacology, biodistribution and metabolism of candidate compounds in small animals. For instance extension of principles developed with [14C] 2-deoxy-D-glucose192 to PET studies of glucose metabolsism requires the labelling of 2-deoxy- D-glucose with ^C. Studies have shewn that 11C labelled 2-deoxy- D-glucose is transported competitively with glucose but undergoes only the first step of phosphorylation. This results in "metabolic trapping" of the 11C as labelled 2-deoxy-D-glucose-6-phosphate.194 Consequently, one is certain that the PET image is formed from positrons originating from the labelled 2 -deoxy-D-glucose- 6 - phosphate.193

46 Another consideration is that the transport of the labelled compound to the target site and its subsequent accumulation must be sufficiently high to provide adequate count rate for imaging. Radiotracer/radioligand targeting has been approached in a number of ways, including the use of labelled inhibitors for enzymes,194 labelled ligands for receptors195-197 and metabolic trapping.193'198 Once a priority structure for labelling has been identified, factors such as the choice of label, the position of label and the required specific activity became important. To a certain degree the biological process being investigated influences the choice of label; the half-life of the radioisotope should be sufficiently long to measure the physiological process of interest. Often, the position as well as the choice of label is dictated by the structure of the molecule and the labelling routes that may be envisaged for introducing the radioisotope. However, if non-isotopic labelling (e.g. displacement of H by 18F) is employed then the choice for the position of the label must not mask the desired characteristics of the parent molecule. Sometimes, non-specific labelling at a particular position can provide a valuable radiotracer for PET studies. For example L-Dopa converts into dopamine in dopaminergic neurons. In principle this process can be followed with labelled L- Dopa. Labelling with fluorine-18 is best achieved in the 6- position rather than the 2- or 5- position, otherwise biological behaviour is seriously perturbed.199 High specific activity is essential when highly toxic compounds are considered for labelling e.g. the powerful neurotoxin MPTP which has been labelled with carbon-11 for study in Rhesus monkeys.200 In the case of radioligands for receptor studies, achievable specific activity is an important criterion in radioligand design. Essentially, a high specific activity has to be achieved otherwise stable ligand will occupy the small number of target receptors at the expense of radioligand. It should be noted that radioligands must possess additional properties to be useful for PET studies and these will be considered in detail in Chapter 3 (Sec 3.1).

47 1.8 REFERENCES

1. Friedlander G. , Kennedy J.W. and Miller J.M., "Nuclear and Radiochemistry", 2nd Ed. John Wiley and Sons,Inc., N.Y. , Ch. 7, 191 (1964).

2. Keyes J.W.Jr., "Computed Emission Tomography", Ed. Ell P.J and Holman B.J., Oxford University Press, Oxford, Ch. 7, 243 (1982).

3. B udinger T.F., "Positron Emission Tomograpy", Ed. Reivich M. and Alavi A., Alan R. Liss, Inc., N.Y., Ch. 12, 227 (1985).

4. M u e h lle h n e r G. and C o ls h e r J.G., "Computed Emission Tomography", Ed. Ell P.J and Holman B.J., Oxford University P r e s s , O xford, Ch. 1, 3 (1982).

5. Ter-Pogossian M.M., "Positron Emission Tomography", Ed. Reivich M. and A la v i A ., A lan R. Liss, Inc., N.Y., Ch. 2, 43 (1985).

6. Hoffman E.J. and Phelps M.E., "Positron Emission Tomography and Autoradiography: Principles and Applications for the Brain and Heart", Ed. Phelps M.E., Mazziotta J.C. and Schelbert H.R. , Raven Press, N.Y., Ch. 6, 237 (1986).

7. P h elp s M.E., Hoffman E.J., M u llan i N.A. and Ter-Pogossian M.M. , J. Nucl. Med., 16, 210 (1975).

8. Stocklin G. and Kloster G. , "Computed Emission Tomography", Ed. Ell P.J and Holman B.J., Oxford University Press, Oxford, Ch. 10, 299 (1982).

9. Fow ler J.S. and W olf A.P., "Positron Emission Tomography and Autoradiography: Principles and Applications for the Brain and Heart", Ed. Phelps M.E., Mazziotta J.C. and Schelbert H.R. , Raven Press, N.Y., Ch. 9, 291 (1986).

10. W olf A.P. and Fowler J.S., "Positron Emission Tomography", Ed. Reivich M. and Alavi A., Alan R. Liss, Inc., N.Y. , Ch. 3, 63 (1985).

11. P ik e V.W., "Isotopes: Essential Chemistry and Applications", Ed. Jon es J.R., The Royal Society of Chemistry, London, Ch. 1, 1 (1988).

12. Pauling L., "The Nature of the Chemical Bond", 3rd Ed., C o rn ell University Press, Ithaca, 82 (1960).

13. Goldman P ., Science, 164. 1123 (1969).

14. Friedlander G., Kennedy J.W. and Miller J.M., "Nuclear and Radiochemistry", 2nd Ed., John Wiley and Sons, Inc., N.Y., Ch.

48 2 , 18 (1964).

15. C lark J.C. and Buckingham P.D., "Short-lived Radioactive Gases for Clinical use", Butterworth, London, (1975).

16. Harper P.V., Int. J. Appl. Radiat. Isot., 28, 5 (1977).

17. Hoffman E.J., Huang S.C., Plummer D. and Phelps M.E. , J. Comput. Assist. Tomography, 6, 987 (1982).

18. Hoffman E.J. and Phelps M.E., Comput. B io l. Med., 345 6, (1976).

19. Spinks T.J., Jones T. , Gilardi M.C. and Heather J.D., IEEE Trans. Nucl. Sci., 3_5, 721 (1988).

20. Comar D. and C rouzel C ., Nucl. Med. Biol., 13, 101 (1986).

21. Krasnov N.N., Dmitriyev P.P., Dmitriyeva S.P., Konstantinov I.O. and Molin G.A. , "The uses of cyclotrons in Chemistry, Metallurgy and Biology", Ed. Amphlett C.B., Butterworths, London, 341 (1970).

22. Qaim S.M., Radiochim. Acta, .30, 147 (1982).

23. F e r r ie r i R.A. and W olf A.P., Radiochim. Acta, 34, 69 (1983).

24. Qaim S.M. and Stocklin G., Radiochim. Acta, 34, 25 (1983).

25. "Proceedings of the First Workshop on Targetry and Target Chemistry", Ed. Helus F. and Ruth T.J., DKFZ P ress Dept., Heidelberg, W. Germany (1987) .

26. W olf A.P. and Redvanly C.S., Int. J. Appl. Radiat. Isot., 28., 29 (1977).

27. C a s e lla V.R., Christman D.R., Ido T. and W olf A.P., Radiochim. Acta, 25, 17 (1978).

28. B ida G.T. , Ruth T.J. and W olf A.P., Radiochim. Acta, 27., 181 (1980).

29. Ephevre M. and Seide C., Phys. Rev. C., 3, 2167 (1971).

30. Christman D.R. , Finn R.D., Karlstrom K. and Wolf A.P., In t. J. Appl. Radiat. Isot., 26., 435 (1975).

31. Lamb J.F., James R.W. and Winchell H.S., Int. J. Appl. Radiat. I s o t ., 22, 475 (1971).

32. Finn R.D., Boothe T.E., Vora M.M., H ild n er J.C., Emran A.M. and K oth ari P.J., Int. J. Appl. Radiat. Isot., 35., 323 (1984).

49 33. Ache H.J. and W olf A.P., Radiochim. Acta, 6, 32 (1966).

34. Crouzel C. , Langstrom B. , Pike V.W. and Coenen H.M. , Appl. Radiat. Isot., 3j8, 605 (1987).

35. Casella V. , Ido T. , Wolf A.P., Fow ler J.S., MacGregor R.R. and Ruth T.J., J. Nucl. Med., 21, 750 (1980).

36. W ieland B.W., S ch ly er D.J., Ruth T.J. and W olf A.P., J. Label. Compd Radiopharm., 18, 27 (1981).

37. Ferrieri R., Schlyer R., Wolf A., Wieland B., Fowler J., MacGregor R. and Rosenthal S., Proceedings of the 3rd World Congress of Nuclear Medicine and Biology,2130 (1982). 2,

38. Bida G.T., Ehrenkaufer R.L., W olf A.P., Fow ler J.S., MacGregor R.R. and Ruth T.J., J. Nucl. Med., 21, 758 (1980).

39. Diksic M. and Toda Y., Can. J. Chem., 61, 661 (1983).

40. C arlson C.H., Singer L. , Service D.H. and Armstrong., I n t. J. Appl. Radiat. Isot., 4, 210 (1959).

41. Tilbury R.S., Dahl R . , Mamacos J.P. and Laughlin J.S., In t. J. Appl. Radiat. Isot., 21, 277 (1970).

42. Fitschen J., Beckman R. , Holm V. and Nevert H. , Int. J. Appl. Radiat. Isot., 28., 781 (1977).

43. Knust E.J., M achulla H-J. and Roden W. , Appl. Radiat. Isot., 37, 853 (1986).

44. Kilbourn M.R., Hood J.T. and Welch M.J., Int. J. Appl. Radiat. Is o t., 35, 599 (1984).

45. W ieland B.W. and W olf A.P., J. Nucl. Med., 24, 122 (1983).

46. K ilb ou rn M.G. and Welch M.J., J. Nucl. Med., 24, 120 (1983).

47. S c h ly e r D.J., Bastos M. and Wolf A.P., J. Nucl. Med., 28., 764 (1987) .

48. Jewett D.M., Toorongian S.A., Mulholland G.K., W atkins G.L. and K ilb ou rn M.R., Appl. Radiat. Isot., 39, 1109 (1988).

49. A le x o f f D. , S ch ly er D.J. and W olf A.P., Appl. Radiat. Isot., 40, 1 (1989).

50. "Proceedings of the Second Workshop on Targetry and Target Chemistry", Ed. Ruth T.J., McQuarrie S.A and Helus F. , Deutsches Krebsforschungszentrum, Insstitut ftlr Nuklearmedizin, Heidelberg, West Germany , Sept. (1987).

50 51. Nickles R.J., G a tley S.J., Votaw J.R. and Kornguth M.L. , A ppl. Radiat. Isot., 37, 649 (1986).

52. Friedlander G. , Kennedy J.W. and Miller J.M., "Nuclear and Radiochemistry", John Wiley and Sons,N.Y., Ch. 12, 405 (1964).

53. W olf A.P., J. Label. Compd Radiopharm., 18, 1-2 (A b str) (1981); {see also, Wolf A.P. Reply., J. Nucl. Med., 22, 392 (1981)}.

54. W olf A.P. and Fowler J.S., Radiopharmaceuticals S o c II,ie t y o f Nuclear Medicine, N.Y., 73 (1979).

55. Comar D. , Berridge M. , Maziere B. and Crouzel C. "Computed Emission Tmography", Ed. Ell P.J.and Holman B.L., Oxford University Press, Oxford, Ch. 2, 42 (1982).

56. W instead M.B., Lamb J.F. and Winchell H.S., J. Nucl. Med., 14, 747 (1973).

57. P ike V.W. , Eakins M.N. , A lla n R.M. and Selwyn A.P., I n t. J. Appl. Radiat. Isot., 33., 505 (1982).

58. P ike V.W., Eakins M.N., A lla n R.M. and Selwyn A.P., J. Radioanal. Chem., 64, 291 (1981).

59. L iv n i E. , Elm aleh D.R. , Levy S., Brownell G.L. and Strauss W.H., J. Nucl. Med., 23, 169 (1982).

60. R a ic h le M.E., E ic h lin g J.O., Straatman M.G., Welch M.J., L arson K.B. and Ter-Pogossian M.W., Am. J. Physiol. 230. 543 (1976).

61. D isc h in o D.D., Welch M.J., K ilb ou rn M.R. and Raichle M.E., J. Nucl. Med., 24, 1030 (1983).

62. Tang D.Y. , Lipman A., Meyer G.J., Wan C.N. and W olf A.P., J. Label. Compd Radiopharm., 435 (1979). 16,

63. Halldin C. and Langstrom B. , Acta Chem. Scan. Ser.B., 38, 1 (1984).

64. Halldin C. and Langstrom B., Int. J. Appl. Radiat. Isot., 3 5 . 779 (1984).

65. Vaalburg W. , Beerling-van der Molen H.D., R e if f e r s S.K., Rijskamp A., Woldring M.G. and Wynberg H. , Int. J. Appl. Radiat. Isot., 30, 151 (1979).

66. R e if f e r s S.K., Beerling-van der Molen H.D., Vaalburg W. , Ten- Hoeve W. , Paans A.M.J., Korf J., Woldring M.G. and Wynberg H. Int. J. Appl. Radiat. Isot., 28, 955 (1977).

67. Langstrom B. and Stridsberg B. , Int. J. Appl. Radiat. Isot.,

51 30, 151 (1979).

68. Digenis G.A. , Casey D.L. , W esner D.A. , Washburn L.C. and Hayes R.L., J. Nucl. Med., 20, 662 (1979).

69. Washburn L.C. , Sun T.T., Byrd B.L. and Callahan A.P., J. Nucl. Med., 23, 29 (1982).

70. B o ls te r J.H., Vaalburg W. , Van Veen W. , Van Dijk T. , Van Der Molen H.D., Wynberg H. and Woldring M.G., Int. J. Appl. Radiat. Isot., 34, 1650 (1983).

71. Wu J.H.C., Harper P.V. and Lathrop K.A., J. Nucl. Med., 74 (1981).

72. Henze E., Schelbert H.R., B a rrio J.R., E gbert J.E., Hansen H.W. and MacDonald N.S., J. Nucl. Med., 23., 671 (1983).

73. B arrio J.R., P helps M.E., Keen R.E. and MacDonald N.S., Proceeding of the 3rd World Congress of Nuclear Medicine and Biology, 2, 2135 (1983).

74. G arn ett E.S. , Firnau G. and Nahmais C ., Nature, 305, 137 (1983).

75. Prenant C. , Barre L. and C rouzel C ., J. Label. Compd Radiopharm., 26, 199 (1988) Abs.

76. Comar D. , M aziere M. and C rouzel c . , Radiopharm. and Label. Compd, 1, 461 (1974).

77. Langstrom B. and Lundquist H ., Int. J. Appl. Radiat. Isot., 27. 357 (1976).

78. Marazano C ., Maziere M., Berger G. and Comar D . , Int. J. Appl. Radiat. Isot. , 28, 49 (1977) .

79. Iw ata R. , Ido T. , Saji H. , Suzuki K. , Yoshikawa K. , Tamate K. and Kasida Y ., Int. J. Appl. Radiat. . Iso t., 30, 194 (1979).

80. Iwata R. , Ido T., Ujuce A., Takahashi T. , Ishiwata K. , Hatano K. and Sugahara M., Appl. Radiat. Isot., 39, 1 (1988).

81. Wagner H.N.Jr., Stocklin G. and Schaak W. , J. Label. Compd Radiopharm. , 18, 1557 (1981).

82. Oberdorfer F ., Hanisch M., Helus F. and Maier-Borst W., Int. J. Appl. Radiat. Isot., 36^, 435 (1985).

83. Langstrom B. , Antoni G., Dannals R.F., Halldin C., Nagren K. , R avert H.T. and Svard H. , "Biomedical Imaging", Ed. Hayashi 0. and Torizuka T ., Academic Press, 387 (1986).

52 84. Leenders K.L., Aquilonius S.M., Bergstrom K. , Bjurling P., Crossman A.R., Eckernas S.A., Gee A.D., Hartvig P., Lundquist H., Langstrom B ., Rimland A. and Tedroff J., Brain Res., (1987) in p r e s s .

85. Aquilonius S.M., Bergstrom K., Eckernas S.A., Hartvig P., Leender K.L. , Lundquist H. , Antoni G. , Gee A.G., Rimland A., Ulin J. and Langstrom B., Acta Neurol. Scand., 76, 283 (1987).

86. Ulin J., Gee A.D., Malmburg P. and Langstrom B. , J. Label. Compd Radiopharm., 26, 409 (1988) Abs.

87. Lasne M.C., P ike V.W. and Turton D.R., Br. J. Radiology, (1988) in p r e s s .

88. Ehrin E. , Gawell L. , Hugberg T. , De Paulis T. and Strom P. , J. Label. Compd Radiopharm., 24, 931 (1987).

89. Bjurling P. , Watanabe Y. , Malmburg P. and Langstrom B. , J. Label. Compd Radiopharm. , 26i, 243 (1988) Abs.

90. Antoni G. and Langstrom B., J. Label. Compd Radiopharm. , 24, 125 (1987).

91. Berger B. , Maziere M. , Sastre J. and Comar D. , J. Label. Compd Radiopharm. , 11_, 59 (1980) .

92. Christman D.R., Crawford E.J., Friedkin M. and Wolf A.P., Proc. Natl. Acad. Sci. USA., 69, 988 (1971).

93. Pike V.W., Palmer A.J., H orlock P.L., Perun T.J., F re ib e r g L.A., Dunnigan D.A. and L is s R.H., J. Chem. Soc. Chem. Commun., 173 (1982).

94. Ehrin E. , Farde L. , De Paulis T. , Eriksson L. , Greitz T. , Johnstrom P. , Litton J.E., N ils s o n J.L.G., Sedvall G. , Stone- Elander S. and Ogren S.O., In t. J. Appl. Radiat. Iso t., 36, 269 (1985).

95. Siegers G., Sambre J., Goethals P. , Vandecasteele C. and Van Haver D . , Appl. Radiat. Isot., 37, 279 (1986).

96. Langstrom B., Antoni G. , Gullberg P., Halldin C., Nagren K. , Rimland A. and Svard H. , Appl. Radiat. Isot., 37., 1141 (1986).

97. Antoni G. and Langstrom B., Appl. Radiat. Isot., 38, 655 (1987) .

98. Antoni G. and Langstrom B. , J. Label. Compd Radiopharm. , 24, 125 (1987).

99. F asth K.J., Malmburg P. and Langstrom B., J. Label. Compd

53 Radiopharm. , 26, 251 (1988) .

100 Antoni G. , Malmborg P., Ulin J. and Langstrom B. , J. Label. Compd Radiopharm., 2JL> 208 (1988).

101. Prenant C., Sastre J., Crouzel C. and Syrota A., J. Label. Compd Radiopharm., 24, 277 (1987).

102. Berger G. , Maziere M. , Prenant C., Sastre J., Syrota A. and Comar D., J. Radioanal. Chem., 74, 301 (1982).

103. Berger G. , Prenant C. , Sastre J., Syrota A. and Comar D. , Int. J. Appl. Radiat. Isot., 34, 1556 (1983).

104. Berger G. , Maziere M. , Prenant C. and Comar D. , Int. J. Appl. Radiat. Isot., 31, 577 (1980).

105. Washburn L.C., Wieland B.W., Sunn T.T., Hayes R.L. and Butler T.A., J. Nucl. Med., 19, 77 (1978).

106. Sambre J., Vandecasteele C. , Goethals P., Rabi N.A. , Van Haver D. and Siegers G. , Int. J. Appl. Radiat. Isot., 36., 275 (1985).

107. Hayes R.L., Washburn L.C., Wieland B.W., Sunn T.T., Turtle R.R. and Butler T.A. , J. Nucl. Med., 17., 748 (1976).

108. Crouzel C., Amano R. and Fournier D. , Appl. Radiat. Isot., 38, 297 (1987).

109. Landais P. and Crouzel C., Appl. Radiat. Isot., 38, 297 (1987).

110. Boullais C., Crouzel C. and Syrota A., J. Label. Compd Radiopharm., 23> 565 (1986).

111. "Stopping Powers for Electrons and Positrons", International Commision on Radiation Units and Measurements", Report 37, 258 (1984).

112. "Biomedical Aspects of Fluorine Chemistry", Ed. Filler R. and Kobayashi Y., Elsevier Biomedical Press, N.Y. (1982).

113. Palmer A.J., Clark J.C. and Goulding R.W., Int. J. Appl. Radiat. Isot., 2_8, 53 (1977).

114. Welch J.T., Tetrahedron, 43, 3123 (1987).

115. Barton D.H.R., Hesse R.H., Markwell R.E., Pecket M.M. and Toh H.T., J. Am. Chem. Soc., 98, 3034 (1976).

116. Barton D.H.R. , Hesse R.H., Toh H.T. and Pecket M.M. , J. Org. Chem., 37, 329 (1972).

54 117. Vine E.N. , Young D. , Vine W.H. and Wolf A.P., I n t. J. Appl. Radiat. Isot., .30, 401 (1979).

118. Shiue C-Y. and Wolf A.P., J. Label. Compd Radiopharm., 18, 1059 (1979).

119. Ido T. , Wan C-N., Casella V., Fowler J.S., Wolf A.P., Reivich M. and Kuhl D.E., J. Label. Compd Radiopharm., 14, 175 (1978).

120. Ido T., Wan C-N., Fowler J.S., J. Org. Chem., 42, 2341 (1977).

121. Rozen S., Lerman 0. and Kol M. , J. Chem. Soc. Chem. Commun., 443 (1981).

122. Shiue C-Y., Salvadori P.A., Wolf A.P., Fowler J.S. and MacGregor R.R., J. Nucl. Med., 23, 108 (1982).

123. Diksic M. and Jolly D., Int. J. Appl. Radiat. Isot., 34, 893 (1983).

124. Shiue C-Y., Salvadori P.A.and Wolf A.P., J. Label. Compd Radiopharm., 20, 157 (1983).

125. Palmer A.J., MRC Cyclotron Unit (London), unpublished observation.

126. Bida G.T., Styamurthy N. and Barrio J.R., J. Nucl. Med., 25., 1327 (1984).

127. Padgett H.C., Cook J.S. and Barrio J.R., J. Nucl. Med., 24, 121 (1983).

128. Jewett D.M., Potocki J.F. and Ehrenkaufer R.E., Synth. Commun., 14, 45 (1984).

129. Shiue C-Y., Fowler J.S., Wolf A.P., Alexoff D. and MacGregor R.R. , J. Label. Compd Radiopharm., 22., 503 (1985).

130. Bida G.T., Styamurthy N. , Padgett H.C. and Barrio J.R., J. Label. Compd Radiopharm., 21, 1196 (1984).

131. Van Rijn C.J.S., Herscheid J.D.M., Visser G.W.M. and Hoekstra A., Int. J. Appl. Radiat. Isot., 36., Ill (1985).

132. Ehrenkaufer R.E., Potocki J.E. and Jewett D.M., J. Nucl. Med., 25, 333 (1984).

133. Shiue C-Y., Kutzman R.S. and Wolf A.P., Eur. J. Nucl. Med., 10. 278 (1985).

134. Diksic M. , Farrokhzad S., Yamamoto L.Y. and Feindel W., In t. J. Nucl. Med. B io l.,11, 141 (1984).

55 135. Shiue C-Y., Wolf A.P. and Friedkin M., J. Label. Compd Radiopharm., 19, 1395 (1982) .

136. Adam M.J., Ruth T.J., Grierson J.R., Abeysekera B. and Pate B.D., J. Nucl. Med., 27, 1462 (1986).

137. Garnett E.S., Nahmias C. and Firnau G., Can. J. Neurol. S ci., 11, 174 (1984).

138. Garnett E.S., J. Neurol. Sci., 69, 223 (1985).

139. Leenders K.L., "Movement Disorders: a study with positron emission tomography", PhD Thesis, Rudopi, Amsterdam (1986).

140. Firnau G., Chirakal R. and Garnett E.S., J. Nucl. Med., 25. 1228 (1984).

141. Visser G.W.M., Van Halteren B.W., Herscheid J.D.M., Brinkman C. and Hoekstra A., J. Label. Compd Radiopharm., 2_1, 1185 (1984) Abs.

142. Luxen A., Perlmutter M. and Barrio J.R., J. Label. Compd Radiopharm., 26., 1 (1988) .

143. Adam M.J., Ruth T.J., Jivan S. and Pate B.D., J. Flourine Chem., 25, 329 (1984).

144. Speranza M. , Shiue C-Y., Wolf A.P., Wilbur D.S. and Angelini G., J. Label. Compd Radiopharm., 21, 1189 (1984) Abs.

145. Speranza M. , Shiue C-Y. and Wolf A.P., J. Nucl. Med., 25., 126 (1984) Abs.

146. Schrobilgen G. , Firnau G., Chirakal R. and Garnett E.S., J. Chem. Soc. Chem. Commun., 198 (1981).

147. Ehrenkaufer R.E. and MacGregor R.R. , J. Label. Compd Radiopharm., 19, 1637 (1982) .

148. Helus F. , Maier-Borst W. , Sahm V. and Wiebe L.I., Radiochem. Radioanal. Lett., 38. 395 (1979).

149. Iwata R. , Ido T., Brady F. , Takahashi T. and Uj ice A., Appl. Radiat. Isot., 38., 979 (1987).

150. Kilbourn M.R. , Jerabek P.A. and Welch M.J., Int. J. Appl. Radiat. Isot., 36., 327 (1985).

151. Clark J.H., Chem. Rev., 80, 429 (1980).

152. Bergman J., Aho K., Haaparanta M., Reissel A. and Solin 0., J. Label. Compd Radiopharm., 26., 143 (1988).

56 153. Rosenthal M.S., Bosch A.L., N ic k le s R.J. and Gatley S.J., In t. J. Appl. Radiat. Isot., 36., 318 (1985).

154. Hutchins L.G., Bosch A.L., Rosenthal M.S., N ic k le s R.J. and G a tley S.J., Int. J. Appl. Radiat. Isot., 36., 318 (1985).

155. Korguth M.L., DeGrado T.R., Holden J.E. and Gatley S.J., J. Label. Compd Radiopharm., 25, 369 (1988).

156. Irie T. , Fukushi K. , Inone 0., Yamasaki T. and Kasida Y. , J. Label. Compd Radiopharm., 19, 1645 (1982).

157. Irie T. , Fukushi K. , Ido T. , Nozaki T. and Kasida Y. , In t. J. Appl. Radiat. Isot., .33., 1449 (1982).

158. Hamacher K. , Coenen H.H. and Stocklin G. , J. Nucl. Med., 2 7. 235 (1986).

159. Block D., Coenen H.H. and Stocklin G., J. Label. Compd Radiopharm., 24, 1029 (1987).

160. Shiue C-Y., Watanabe M. , Wolf A.P., Fowler J.S. and Salvadori P.A., J. Label. Compd Radiopharm., 21, 5353 (1984).

161. Attina M. , Cacace F. and Wolf A.P., J. Label. Compd Radiopharm., 20, 501 (1983) .

162. G a tley S.J.and Shaughnessy W.J., Int. J. Appl. Radiat. Isot., 31, 339 (1980).

163. Chi D.E., K ilbourn M.R. , Katzenellenbogen J.A., Brodack J.W. and Welch M.J., Appl. Radiat. Isot., 37, 1173 (1986).

164. Brodack J.W., K ilbourn M.R., Welch M.J. and Katzenellenbogen J.A., Appl. Radiat. Isot., .37.> 217 (1986).

165. Irie T., Fukushi K. , Ido T. and Nozaki T. , J. Label. Compd Radiopharm., 1£, 9 (1981) .

166. Coenen H.H., Schuller M. and Stocklin G., J. Label. Compd Radiopharm., 21, 1197 (1984).

167. Block D., Klette B. and Knochel A., J. Label. Compd Radiopharm., 23., 467 (1986) .

168. G a tle y S.J., Int. J. Appl. Radiat. Isot., 3J3, 255 (1982).

169. Ballinger J.R., Bowen B.M., Firnau G., Garnett E.S. and Teare F.W., Int. J. Appl. Radiat. Isot., 35, 1125 (1984).

170. Kilbourn M.R., Brodack J.W., Chi D.Y., Dence C.S., Jerab ek P.A., Katzenellenbogen J.A., P a tr ic k T.B. and Welch M.J., J.

57 Label. Compd Radiopharm., 23., 1174 (1984).

171. Liotta C.L. and Harris H.R. , J. Am. Chem. Soc. , 96, 2250 (1974).

172. L io t t a C.L., H arris H.P., McDermott M. , Gonzales R. and Smith K ., Tet. Lett., 28, 2417 (1974).

173. B errid g e M.S., C rou zel C. and Comar D . , J. Label. Compd Radiopharm. , 22., 687 (1985) .

174. Van der Ley M., J. Label. Compd Radiopharm., 20, 453 (1983).

175. Irie T. , Fukushi K. , Ido T. , Nozaki T. and Kasida Y. , I n t. J. Appl. Radiat. Isot., .35, 517 (1984).

176. Hamacher K. , Coenen H.H. and Stocklin G. , J. Label. Compd Radiopharm. , 23., 1047 (1986) .

177. Sharma R.K. and Fry J.L., J. Org. Chem., 48, 2112 (1983).

178. Yagi M. , Murano Y. and Izawa G. , Int. J. Appl. Radiat. Isot., 33, 1335 (1982).

179. Shiue C-Y. , Bai L-Q. , Teng R-R. and W olf A.P., J. Label. Compd Radiopharm., 24, 55 (1987).

180. Shiue C-Y., Bai L-Q., Teng R-R., W olf A.P. and Arnett C.D., J. Nucl. Med., 27, 906 (1986) Abs.

181. Coenen H.H., Colosimo M., Schuller M. and Stocklin G., J. Label. Compd Radiopharm., 23., 587 (1986).

182. Levy S., Elmaleh D.R. and L iv n i E. , J . Nucl. Med., 23., 918 (1982).

183. Kiewsewetter D.O., K ilb ou rn M.R., Landvettes S.W., Heimen D.F., Katzenellenbugen J.A. and Welch M.J., J. Nucl. Med., 25, 1212 (1984).

184. Berridge M.S., Tewson T.J. and Welch M.J., Int. J. Appl. Radiat. Isot., 34, 727 (1983).

185. Kiesewetter D.O., Finn R.D. and Eckelman W.C., J. Label. Compd Radiopharm., 23., 1040 (1986) .

186. Block D., Coenen H.H., Laufer P. and Stocklin G. , J. Label. Compd Radiopharm., 23., 1042 (1986) .

187. De Grout T.J., Zijlstra S., Daemen B.J.G. and Vaalburg W. , J. Label. Compd Radiopharm., 26., 450 (1988).

58 188. Kilbourn M.R., Welch M.J., Dence C.S., Tewson T.J., Saji H. and Maeda M. , Int. J. Appl. Radiat. Isot., 3j>, 591 (1984).

189. Shiue C-Y., Fowler J.S., W olf A.P., McPherson D.W., A r n e tt C.D. and Zecca L. , J. Nucl. Med., 27., 226 (1986).

190. Farrokhzad S. and Diksic M. , J. Label. Compd Radiopharm., 22, 721 (1985).

191. Mulholland G.K., Mangner T.J., J e w e tt D.M. and Kilbourn M.R. , J. Label. Compd Radiopharm. , 26., 378 (1988).

192. Sokoloff L. , "Positron Emmision Tomography and Autoradiography: Principles and Applications for the Brain and Heart", Ed. Phelps M.E., Mazziotta J.C. and Schelbert H.R., Raven Press, N.Y., Ch. 1, 1 (1986).

193. Reivich M. , Alavi A., Wolf A.P., Greenberg J.H., Fowler J.S., Christm an D. , MacGregor R. , Jones S.C., London J. , Shiue C-Y. and Yonekura Y ., J. Cereb. Blood Flow Metab. , 2, 307 (1982).

194. Fowler J.S., W olf A.P., MacGregor R.R., Dewey S.L, Logan J . , Schlyer D. and Langstrom B. , J. Label. Compd Radiopharm., 2 6 . 236 (1988).

195. Eckelman W.C., Reba R.C., G ibson R.E., Rzeszotarski W.J., Vieras F ., Mazaitis J.K. and Francis B., J. Nucl. Med., 20, 350 (1979).

196. G ibson R.E., Eckelman W.C., Rzeszotarski W.J. et al., In: "Principles of Radiopharmacology", Ed. Columbetti L.G., CRC press, Buca Raton, FL., 2, 17 (1979).

197. Krohn K.A., J. Nucl. Med., 21, 593 (1980).

198. Gallagher B.M., Ansari A., Atkins H. , Casella V., Christman D.R. , Fow ler J.S., Ido T. , MacGregor R.R. , Som P., Wan C.N., W olf A.P., Kuhl D.E. and Reivich M. , J. Nucl. Med., 18., 990 (1977).

199. Firnau G. , Garnett E.S., Chirakal R. , Sood S., Nahmias C. and Schrobilgen G., Appl. Radiat. Isot., 37., 669 (1986).

200. Hartvig P. , Larsson B.S., L indberg B.S., O reland L. , G u llb erg P. , Langstrom B. , Rimland A., Lundqvist H. , Malmborg P. and L in d q u ist N.G., Acta Neurol. Scand. , 74, 10 (1986).

59 C H A P T E R 2

FH7IOSYNIHETTC VPEPKBKFICN OF

r^irn-noDSE a n d a n a l y s i s

60 2.1 I17ER0D0CTICN Glucose is a highly important substrate for metabolism within living organisms. For example, the brain under normal conditions depends exclusively upon glucose for energy production and thereby the maintenance of its functional and structural integrity. In the brain greater than 85% of glucose is metabolised aerobically to carbon dioxide and water via the tricarboxylic acid cycle (aerobic glycolysis).1"^ Ihe remainder is metabolised to lactate (anaerobic glycolysis). In the heart, though free fatty acids (FFA) are the preferred substrates for oxidative energy metabolism, under anaerobic conditions the myocardium resorts to using glucose.1 '2 '5 '6 in these circumstances glucose is taken up by the heart muscle cell and rapidly phosphorylated to glucose-6-phosphate (G6P), a reaction mediated by the enzyme, hexokinase. G6P is further metabolised along several distinct routes (Fig 2.1) to meet the requirements of the cell.

Fig 2.1 Metabolism of glucose in the myocardial cell {Reproduced from Taegtmeyer (1986) }6

61 These systems include (a) glycolysis (degradation via a series of steps to pyruvate, through to acetyl CCA and then entrance into the citric acid cycle), (b) conversion into glycogen (an intracellular glucose store) and (c) metabolism via the pentose-phosphate pathway (yielding ribose and NADEH). The latter route is relatively inactive in normal myocardium. In myocardial ischaemia, oxygen demand and supply are no longer in balance and oxidative metabolism of free fatty acid is severely affected.7-9 The onset of ischaemia is usually associated with enhanced glycolysis. As a result glycogen breakdown to glucose-1- phosphate is stimulated to compensate for increased glucose demand10'1-1- (Fig 2.2).

UDP Glycogen

Pi Giycoaen Synthase D (G-S-P't)

Uridine-diP-glucose Phosphorylase a

PPi Glucose-1-P UTP

UTP - Uridine triphosphate PPj_ - Inorganic pyrophosphate P-j_ - Inorganic phosphate Fig 2.2 Degradation of glycogen in the myocardium (Reproduced from TaegtmeyernPaev-ri-mowor* (1986)}

62 Taegmeyer6 reports that during myocardial ischaemia, glycogen stores are depleted 50% within 5 min and 70% within 20 min. Consequently, it was proposed (by Drs P. Camici and L. Araujo, MRC Cyclotron Unit, Hammersmith Hospital) that it might be possible to use [^CJ glucose to reveal glycogen repletion in human myocardium during recovery from transient ischaemia. For this reason the preparation of [^^C] glucose for myocardial PET studies was undertaken. Prior to 1983, the procedures listed in Table 2.1 had been reported for the labelling of glucose with carbon-11. These include both chemical and biosynthetic methods. Specifically labelled D- [ 1-X1C] glucose had been prepared by the Kiliani-Fischer synthesis with [1XC] hydrogen cyanide as labelling agent.15'16 In the biosynthetic methods, 11002 was photosynthetically incorporated into Swiss chard leaves,12 Broad bean leaves12 or algae.14'17 The method reported by Ehrin et al17 was considered to have distinct advantages. First the use of unicellular green algae provides for experimental reproducibility. Secondly, unlike all the other methods, it produces radiochemically pure [12-C] glucose. However, preliminary investigation of this procedure revealed a number of difficulties. Firstly the method of concentrating 11C02 as [ 11C ]carbonate was found to be inefficient. Secondly, at the low pH at which photosynthesis was performed, much of the added

[11C]carbonate was lost as 1:lC02. Finally, the separation of the algae after photosynthesis involved centrifugation; this necessitated extensive handling of the apparatus, resulting in prolonged radiation exposure. Thus, the initial aim of the project was to modify the various stages of the procedure to obtain greater efficiency and permit adaptation to remote control. The chemistry and operations that are essential to the photosynthetic preparation of [^C] glucose are outlined in Scheme 2.1.

63 Table 2.1: Reported procedures for the preparation of It-gluoose labelled with carbon-11.

P ro ced u re Reagents Illumination Rad iochem ica1 P r o d u c t(s ) R e fe r e n c e Yield at EOB (Y ear) (min) (%)

Biosynthetic Swiss chard 20 27 D-f^C] glucose 1 2 (1 9 7 1 ) leaves & [H c ]002 D-f^C] fructose

Biosynthetic Broad bean 30 4 3 -4 6 D-W c] glucose 1 3 (1 9 7 3 ) leaves & [11C]CD2 D- [11C ]fructose

Biosynthetic Scenedesmus 3 5 0 -7 0 D- [l^C]glucose 1 4 (1 9 8 0 ) obtusiusculus Chod. D-[13C]fructose & [i :lC]C032_

C h em ical D-Arabinose - - D- [1-11C]glucose 1 5 (1 9 8 1 ) & H [n C]CN D - [ l - ^ C ] m annose

C h em ical D-Arabinose - 30-45 D-[l-11C]glucose 1 6 (1 9 8 2 ) & H [i: lC]CN D -[1-11C]mannose

Biosynthetic Scenedesmus 5 25 D-[^C] glucose 1 7 (1 9 8 3 ) obtusiusculus Chod. & [11C]0032~ CHEMISTRY OPERATION N2 gas

14N (p,a)l:lC --- Radioisotope production i :lO 02 Formation HC02/N2

KDH aq. --- 1^002 Trapping

X1C 032- aq.

algae Fhotosynthes is hv under N2

Intracellular Separation of algae (X1C) compounds

HC1 Hydrolysis ▼ Extracellular (1:LC) sugars Neutralisation Rotary evaporation Preparative HPLC Adjustment to isotonicity Sterilisation u D-(i:LC)GIUOOSE

Scheme 2.1. Chemistry and operations involved in the photosynthetic preparation of glucose

The incorporation of 002 (into sugars or other compounds) occurs in four phases (Fig 2.3):18 i) Carboxylation phase: C02 adds to rihulose biphosphate to form two molecules of phosphoglyceric acid (PGA). ii) Reduction phase: PGA is reduced to phosphoglyceraldehyde (or triose phosphate) by utilising NADIH2 and ATP. iii) Regeneration phase: ribulose biphosphate is regenerated for further 2 fixation, by a complex series of reactions involving 3-, 4-, 5-, 6- and 7-carbon sugar phosphates. iv) Product synthesis phase: sugars, carbohydrates, fats, free acids, amino acids and organic acids are synthesised. 65 RuBP

i) Garboxviation phase PGA

ii) Reduction Triose P phase

Hexose P Triose P Triose P Tetrose P iii) Regeneration Pentose P phase Triose P Heptose P RuP R uB P =

Sugars iv) Product Carbohydrates synthesis Fats_____ phase Fatty Acids Amino Acids Carboxylic Acids

Fig 2.3. Summary ot reactions of photo synthetic C02 fixation {Reproduced from Hall and Rao}18

66 Carbon-14 tracer studies have been used to examine the pathways and end-products of photosynthesis. ^ “25 The distribution of the label within a product molecule depends on the photosynthetic conditions used. Therefore, the second phase of this project was to establish the position (s) of the ^-^C-label in the [^C] glucose produced. It was envisaged that co-inclusion of ^C-enriched carbonate into the 13C-photosynthetic procedure and examination of the isolated [1:k: / 13C] product by 13C-NMR spectroscopy might provide information about the distribution of the 13C-label (and by analogy 11C-label) and the level of incorporation into a particular carbon in the same molecule. This information would assist the mathematical modelling of the metabolism of [1:LC] glucose for future studies of brain energy metabolism by means of PET.

67 2.2 n r s m g n m Hie use of green algae in preference to higher plants (i.e. Swiss chard or broad bean leaves) for the photosynthesis of [1 1 C]glucose has become an important procedure in radiochemistry, 25 -jhis can be mainly attributed to the fact that a statistically large number of organisms are exposed to the same experimental protocol and so any deviation by a particular algal cell has a negligible effect u p o n the overall experimental outcome. In addition, these organisms are microscopic and aquatic. Therefore they can be readily subjected to uniform and non-varying conditions of illumination and nutrition.19 However, a major concern in using these organisms is keeping them free from bacterial contamination. Three types of bacterial contamination were observed in the originally acquired cultures of Scenedesmus obtusiusculus chod. Radiolabelled products produced for human intravenous injection must be sterile and apyrogenic. It was envisaged that unknown bacterial contamination could be a source of pyrogens in the C11^]glucose preparations. Therefore several techniques including filtration, antibiotic treatment and ultra-violet irradiation, were examined with a view to eliminating bacterial contamination in the cultivated algae. Filtration, eliminated large proportions of all types of bacterial cells. Antibiotics, such as gentamicin, cefotaxime or streptomycin, at lew concentrations had no effect on bacterial cell growth, whereas at high concentrations both bacterial and algae cell growth were affected (Table 2.2). Algae cells appeared to be inflated and decreased in number with cultivation time. Similar results were obtained using ultra-violet radiation. Short periods of exposure caused no bacterial cell damage while longer periods affected both algae and bacterial cells. In contrast, a "sub-culturing" technique produced a bacteria- free culture. This was achieved by cultivating an isolated colony of algae cells consecutively on agar plates. As described in Section 2.1, the radiosynthesis of [1:LC]glucose involves the trapping of cyclotron-produced 11CQ2' photosynthesis, separation of algae, hydrolysis and hplc purification. These stages

68 of the preparation will new be discussed. In the published procedure, 11002 was trapped by bubbling the gas through a solution of potassium hydroxide (Fig 2.7). This trapping procedure was found to be 65-70% efficient. To concentrate 11C02 efficiently, a novel trapping system (Fig 2.8) was developed. This consisted of a plastic syringe packed with glass wool and wetted with potassium hydroxide solution. The parameter flow rate of 11C02 and volume of potassium hydroxide solution were optimised for maximum trapping efficiency. The trapping efficiency of 11002 increased as the volume of KDH increases from 35 ^1 and reached a plateau at 350 /zl (Fig 2.9). Flow rates of 11C02/N2 between 50 to 265 ml min"1 gave trapping efficiencies of 95% or greater (Table 2.3). At higher flow rates (> 400 ml min"1) a decrease was observed (to < 80%) because KDH solution was swept from the trap. In addition low flow rates (< 100 ml min"1) required an unacceptably long time (9-10 min) to dispense the activity from the target. Thus, the optimal parameters for 11C02 trapping were 300 p i of KDH and a flow rate of 250 ml min"1, giving a trapping efficiency of greater than 95% in a recovery time of 6-7 min. These conditions were used routinely. Typically between 12.7- 14.5 GBq (350-400 mCi) of f1^]potassium carbonate were obtained for routine productions of t11^]glucose for clinical PET studies. This trapping system can be used for other applications in radiochemistry. For example, it was possible to prepare alkaline solutions containing high levels of radioactivity for PET phantom studies. Amino acids, such as Lr-aspartic acid have been labelled with carbon-11 by enzymatic syntheses in aqueous media.26 This trapping procedure may prove to be a useful method for concentrating 11O02 for such syntheses. The next stage in the radiosynthesis of t1^]glucose, involves the addition of [ 11C]carbonate to algae cells in buffer at pH 5.0. The final pH of the buffer is important for two reasons. Firstly, the effect of buffer pH on the equilibrium reaction of carbon dioxide in aqueous solution must be considered i.e. Eq 2.1

69 H20 + CO2 H2003 = ^ ^ Y& + HC03“^ = S H + + 0032- . .Eq 2.1 pKa (H2003) = 6.37 pKa (HC03~) = 10.33

In the reported procedure,17 carbonate in potassium hydroxide (10 ml, 0=1 M) is added to algae cells in a buffer at pH 5.0. This results in a final buffered pH of 5.9. It is clear from aquation 2.1, that at this pH the equilibrium lies to the left and the carbon-11 label is lost as 11O02. Investigations of the overall buffer pH showed, that at pH < 6.2, only 55-65% of the added [^C]activity was retained by the algae buffer (Table 2.4), whereas in the pH range of 6 .9-7.2 greater than 80% of the added radioactivity was retained. Secondly, the buffer pH should not adversely affect the photosynthetic ability of the algae cells. At pH < 7.0 algae were found to incorporate 80-95% of the available activity whereas at pH > 7.2 they were found to incorporate < 30% (Table 2.4). Consequently, the optimal pH, in terms of solution retention of [1:LC] activity and of incorporation into algae was determined to be 6.9. It should be noted that, pre-illumination of algae cells in buffer at pH 7.0 rather than pH 5.0, lowered the incorporation of the label to 60-70%. Once the radioactivity has been incorporated by photosynthesis, the algae cells must be separated from the buffer. This was achieved in the published procedure by centrifugation. This procedure was not easily amenable to remote control and involves extensive handling, thus entailing high radiation exposure. Consequently, a technique was developed to circumvent these problems. It involved transfer of the algae suspension from the phcrtosynthetic reaction vessel under a pressure of nitrogen onto a filter membrane (Fig 2.10). Algae cells retained by the filter membrane were recovered by back-washing the filter with hydrochloric acid for hydrolysis. Hydrolysis of [ carbohydrates to [1:LC] glucose with dilute hydrochloric acid was found to be optimal using 7 min at 413 K. Hydrolysate was neutralised on a column of ion-retardation resin and the [1:LC] glucose isolated by preparative hplc as reported by Ehrin

70 et al.17 Isolated product was found to be radiochemically and chemically pure by analytical tic and hplc. Hie developed procedure provided glucose in 14-19% r.c.y from 1:LC02 within 50 min from EOB. Routinely, 206-595 MBq (or 7-16 mCi) of [13C]glucose was prepared containing 0.5-1.0 mg of carrier glucose for PET studies of ischemic myocardium c Hie position (s) of label in the [^C] glucose is an important criterion for the mathematical modelling of tracer distribution in man. Several techniques are available for the examination of the position(s) of the label in a molecule. A classical approach to the elucidation of biosynthetic pathways uses a carbon-14 labelled precursor to trace an appropriate biosynthetic process. For example, the uptake of 14C02 by land plants and algae has been extensively studied. 18~22,27,28 Algae such as Chlorella pyrenoidosa or Scenedesmus obliquus have been exposed to 14C02 for varying periods of time under a wide variety of conditions.19'20 '22 Hie photosynthetic products were isolated by chromatography23 and extensive degradative procedures applied to elucidate the labelling pattern of the carbon skeleton.20”22 This approach is extremely time-consuming and provides information only on the percentage of label at a particular carbon, from which the distribution of the label in a particular molecule is then deduced. Nevertheless useful information has been obtained. All of the carbons in fructose-1,6-diphosphate are labelled in the early stages of photosynthesis with 14C02, but to different extents.2 0 '2 9 '30 Originally, Calvin and Mass ini2 9 showed that after short exposure times (1 to 5 s) carbon atoms C3 and C4 are the first to became labelled, followed considerably later (30 to 60 s exposure time) by C]_ and C2 and then C5 and C6 (Fig 2.4).

71 ?i *?2 *

Fructose-1,6-diphosphate

Fig 2.4 Distribution of 14C-label in fructose-1,6-diphosphate

Bassham et al22 have reported that after fixation of 14C02 f°r a period of 15 s, there is equality of labelling at C-j_ and C2 in phosphoglyceric acid (PGA) and furthermore that in the fructose-1,6- diphosphate formed, there was not only equality of labelling between C3 and C4 but also between Cj and C6 and between C2 and C5. Kandler and Gibbs30 assayed all six carbon atoms independently and found that C4 was always labelled to a greater extent than C3 and also and C2 to a greater extent than C5 and C6. With respect to the discrepancy between C3 and C4, Bassham has noted that Gj_, C2 and C3 of fructose-1 ,6-diphosphate are derived from dihydroxyacetone phosphate and C4, C5 and C6 from phosphoglyceraldehyde. Trebst and Fieldler31 extended these observations and proposed that the discrepancy between C3 and C4 arises in the reactions forming PGA from carbon dioxide. They suggested that this would be consistent with the view that carboxylation in vivo gives rise to one molecule of PGA released immediately from the enzyme complex and a second molecule released only after reduction on the enzyme site. This would preferentially give rise to two pools of triose phosphate which will differ in their average level of radioactivity. In the case of the observation that and C2 were labelled to a greater extent than C5 and C6, Bassham32 suggested that this may be due to the fact that and C2 atoms of both fructose-1,6-diphosphate

72 and sedoheptulose are involved in transfers of "active glycoaldehyde" catalysed by transaldolase and transketolase in the photosynthetic cycle. There may be sane equilibration between the two-carbon moiety transferred in these reactions and and C2 atoms of fructose-1,6- diphosphate. In the case of biosynthetic procedures involving carbon-13 labelled precursors, the labelled products can be detected by mass spectrometry.33 However, proof of the exact location of the enriched site(s) is problematic since total enrichment of each fragment ion is measured and assignment depends on the proposed fragmentation pattern which may not be completely discerned. In contrast, 13C-NMR spectroscopy offers a number of advantages when applied to biosynthetic studies.34”3*7 This technique can potentially give information about the percentage label at a particular carbon. In addition the distribution of the labelled carbon (s) in the same molecule can be deduced from 13C-13C coupling patterns. This direct method obviates the need for chemical degradation as in the case of 14C studies. This method was selected to examine label distribution in photosynthesised [1:LC]glucose. Thus, carbon-13 enriched precursor was co-included in the radiosynthesis of [1:LC]glucose, by replacing potassium carbonate used as carrier in the [^C]glucose preparations, with the same weight of 13C-enriched potassium carbonate (91 atom %, 6.5 /xmol). The use of higher amounts of 13C-precursor was avoided so as not to disturb the photosynthetic pathway of carbon-11 and the distribution of the label in the desired product. Consequently, the radiosynthesis was performed repeatedly to obtain enough [13C/llc]glucose for study by proton-decoupled 13C-NMR spectroscopy. For comparison, the 13C-NMR spectrum of natural abundance D- glucose was also recorded. The spectrum obtained at about 30 min after sample preparation was mainly due to a-D-glucose. The same solution examined at 20 h displayed clear signals from a - and £-D- glucose as a result of mutarotation in E^O. In water at 298 K the a ifi ancmeric composition of D-glucose is close to 2:3.38 The chemical shift assignments of the spectrum of D-glucose obtained at

73 20 h are recorded in Table 2.6. Hie assignments have been well established by previous studies.39“41 The 13C-NMR spectrum of photosynthetically prepared 13C- enriched glucose (the total amount of 13 C-enr iched product was estimated to be in the range of 396-630 /jg and the quantity of natural abundance glucose was estimated to be < 20 mg) was initially acquired at 22.5 MHz using 40,000 pulses in a 16 h experiment. This spectrum exhibited chemical shifts of natural abundance glucose (Table 2.6) with satellite peaks attributed to 13C-13C coupling, just visible above the noise level. The formation of natural abundance glucose in the ^3 C-enriched preparations was minimised by pre-illuminating the algae under nitrogen before photosynthesis, where intracellular pools of reducible substances were consumed. However, the existence of endogenous glucose within the algae cells would still be expected to lead to 12C-carrier. The 13 C-enr iched sample was re-examined at 100.62 MHz and 23000 pulses were used to obtain a spectrum (Fig 2.14). This clearly displays satellites due to 13C-13C spin-spin coupling. Spin-spin interaction between adjacent 13C-nuclei in the same molecule results in satellite bands appearing on either side of the main carbon-13. signal. For exanple, the outer components of the signal at 5 95.7 (C]^) arises from coupling to an adjacent 13C-labelled C2/3. Conversely, the satellites of the signal at 5 74.0 (C2^) was a result of coupling to 13C-labelled C ^ and C3^. The central intense feature of each triplet (signal at S 95.7 or 74.0) was attributed to 13C-labelled C^g (or C2^) adjacent to unlabelled 12C2^ (or 12C-^ and/or -^C^) on the same glucose molecule. It is apparent from the 13C-NMR spectrum (Fig 2.14) that all of the carbons in the 13c- enriched sample appear to exhibit 13C-satellites although in the case of C3£, C5£ and C^, C6^, the signals overlap and the satellites are difficult to distinguish from the major central components. Consequently, the signal pattern indicates that the 13 C-enr iched label (and by analogy 11C) was incorporated into all positions of the glucose molecule. An unidentified singlet at 5 62.4 was observed, probably due to a minor inpurity.

74 Measured values of 13C-13C coupling constants (Table 2.6) are in good agreement with published values.4 2 '43 The heavy atom effect upon the chemical shifts (isotope shift) of 13C bonded to in relation to 13C banded to 13C was calculated for C-j_, c2 , C3 and C4 carbons. The values obtained for (0.06), C2p (0.03), (0 .0), ^ (0.018) were small and in agreement with previous estimates for 13C-enriched glucose.43 The ratio of the integrated intensity of the central resonance of the triplet to the integrated intensity of the outer doublet assigned to a particular carbon, should give a measure of the degree of 13C-enrichment at the adjacent carbon in the same molecule. At the field strength (100.6 MHz) at which the 13C-enriched product was examined, the components of the various triplets were not completely resolved. Consequently, only a very approximate value for 13C- enrichment at an adjacent carbon atom could be derived. For example, in the case of an average figure of 26% enrichment at C2 carbon was calculated (Table 2.6). This result was based on the assumption that the integrated intensity of the central resonance of the triplet assigned to or was solely due to the 13C-signal of natural abundance glucose in the 13C-enriched sample. Therefore, the percentage of enrichment at C2 was probably under estimated since a certain percentage of the central resonance was expected to be from 13C-enriched atom adjacent to a 12C2 carbon where a singlet would be obtained. It should be noted that the value calculated from the triplet assigned to C2^ (31%) probably measures the level of enrichment at both and C3^ carbons. The values obtained indicated that to a first approximation the degree of 13 C-enrichment at each of the carbons in the synthesised glucose was similar. In principle the 13C-NMR study has demonstrated the fate of 13G02 (and by analogy 11C02) under the photosynthetic conditions utilised. Clearly, the biosynthetic pathway undergoes a series of complicated steps and therefore requires an extensive controlled analysis to establish precisely the isotopic distribution pattern and the extent of labelling at each position in the same molecule and this was beyond the current investigation.

75 2.3 EXPERIMENTAL AND RESDIIES 2.3.1 Cultivation of Algae A strain of the uni-cellular green alga, Scenedesmus ob tusiu scuiu s Chod was kindly provided by Dr E. Ehrin (Karolinska Hospital, Stockholm, Sweden). Synchronised cultures of the uni-cellular algae were grown as described by Kylin et al .44 A schematic representation of the apparatus used is shewn in Fig 2.5.

Fig 2.5 Schematic representation of the apparatus used for the cultiuation of Scenedesmus obtusiusculus Chod.

76 Culture flasks (500 ml) equipped with a gas dispersion tube were sterilised before use, by either heating the flasks in an oven at 473 K overnight or autoclaving at 100 kN m "2 (15 psi) for 15 min at 393 K. A flask containing sterile culture medium (250 ml, for composition see Appendix 1) was inoculated under aseptic conditions with a sample of the algae suspension and set in a glass water-bath at 303 K. A constant stream of air mixed with 2.5% C02 was passed via a millipore filter (0.22 /im, Millex GS) through the suspension at a flow rate of approximately 100 ml min"1. The flasks were illuminated with two fluorescent lamps (Thom T 20W/29) placed 6-8 cm from the glass wall to give a light intensity of 100-120 /jw m "2 at the outer surface of the water bath. The lamps were operated via an automatic switch set for 15 h of light and 9 h of darkness. In this way a synchronous culture was obtained with cell division occurring during the dark period and cell growth during the light period. On average the algae grew to maximum density in about one week, after which they gradually lost viability. Hence, new culture media was inoculated each week to ensure good cell viability.

Examination of algae culture for bacterial contamination As a means of monitoring the algae culture for bacterial contamination, nutrient broth no.2 solution (Appendix 2) and nutrient broth no. 2 agar plates (Appendix 3) were inoculated and incubated at 298 K while exposed to the 15 h light and 9 h dark cycle. A few days later, the nutrient broth solution was found to be milky. Microscopic examination revealed three different bacterial contaminants: i) small, circular and static; ii) small, circular and fast moving; and iii) rod shaped with circular motion. Examination of the nutrient broth agar plates also showed the presence of three types of bacterial colonies: (a) different sized creamy^white (large number); (b) similar sized bright-yellow (less in number); and (c) small orange (very few). The creamy-white colonies gave a green fluorescence when viewed under a fluorescent light and a positive result to an oxidase test (filter paper dipped in 1% N,N,N' ,N'- tetramethyl-1 ,4-phenylenediamine, streaked with one bacterial colony;

77 bacteria forming indophenol oxidase give bright violet colouration) indicative of Pseudomonas species. The other two types of colonies gave negative results in both tests and were not further identified. The following techniques were applied in an attempt to eliminate the bacterial contamination in the cultivated algae culture.

(a) Filtration S in ce, there is a five to ten fold difference in sizes between algae (> 5 ^m) and bacterial cells (0.1-0.5 ^m), the contaminated algae suspension (5 ml) was passed onto a sterile filter (5 pore size, Millex-GS) under aseptic conditions. Algae cells retained on the filter surface were washed continuously with water for injection (50 ml) and then backwashed off the filter with sterile culture medium (10 ml) and cultivated as described in Sec 2.3.1. This procedure reduced the bacterial contamination dramatically but not completely. This was probably due to entrapment of bacterial cells within the filter body.

(b) Antibiotics Antibiotics* such as gentamicin, streptomycin and cefotaxime were added at various concentrations (Table 2.2) to the cultivation media (250 ml). The media was then inoculated with the contaminated algae and cultivated as described in Sec 2.3.1. Aliquots were removed every 12 h and monitored on nutrient broth agar plates (Table 2 .2 ). The addition of antibiotics to the cultivating media at low concentrations, initially resulted in the reduction of bacterial growth (Table 2.2). However, after 3-4 days of cultivation, the contaminating bacteria appeared (under microscope) to be growing as originally. At higher concentrations of antibiotics, bacterial species were found to be damaged (i.e. only cell debris floating). The algae cells also appeared to be inflated and decreased in number with cultivation time (> 72 h).

78 Table 2.2. Effect of antibiotics cn algae growth.

Antibiotic Concentration (/ig/mL)

200 100 50 25 12.5 10 5

Gentamicin S2 S2 S2 S1 S1 RR

Cefotaxime - - s2 S1 R RR

Gentamicin & ———- R R R Cefotaxime

Streptomycin - - R R R R R

S1 - bacterial species appeared to be sensitive to these concentrations initially but became resistant with time of cultivation (> 72 h)

S2 - bacterial species sensitive but severe retardation in algae growth

R - bacterial species resistant to the antibiotic

*preliminary results of inoculated blood-agar plates showed that the contaminating bacteria were sensitive to these antibiotics

(c) Ultra-violet radiation Samples of contaminated algae (10 ml) were placed in Petri- di^hes under aseptic conditions. These were exposed to ultra-violet radiation (A = 254 nm, intensity 440 /xW cm-2) for different periods. Cultivating media (250 ml) was then inoculated with these samples and the algae grown as described in Sec 2.3.1. Microscopic analysis of the cultivated media showed that short exposures (2 and 5 min) to ultra-violet radiation caused no bacterial cell damage virile longer periods (10, 15 and 20 min) affected both bacterial and algae cell growth.

79 (d) ” Sub-culturina” Petri dishes containing ionagar (1.5%, Oxoid) with essential electrolytes (see Appendix 1) and sodium bicarbonate (10 g per 500 ml electrolyte medium) were inoculated and streaked sequentially at right-angles, thereby covering the entire surface of the agar plate. The petri dishpg were then incubated at ambient temperature- under a cycle of 15 h light and 9 h dark (light intensity described in Sec 2.3.1) for one week. After this incubation period small colonies of algae were observed on the agar plates. A single colony was isolated (on the basis of apparently lowest contamination by aid of a microscope) and transferred to sterile cultivating media (10 ml, Appendix 1). A fresh agar plate was then inoculated with this solution and incubated as described above. This procedure was repeated until an apparently bacteria-free culture was produced (examined through a microscope). This culture was further tested under aerobic and anaerobic conditions, using both nutrient agar and blood agar plates by the Bacteriology department (Royal Postgraduate Medical School, Hammersmith Hospital). These investigators confirmed that the algae culture was bacteria-free.

2.3.2 Production of f 11C'lCarbcn Dioxide The target system used for the production of 11O 02 is depicted in Fig 2.6. The target (volume 1.4 1 at STP) is constructed of aluminum with a titanium foil (0.013 mm) beam entry window. Nitrogen (99.999%) was used as target and sweep gas. The target was pressurised to 50 kN ni”2 (7.5 psi) and bombarded with a 7.6 MeV proton beam (40 ^A beam current) produced on the original MRC cyclotron. The 11O02 was produced (0.2 mCi ml”1) in > 98% radiochemical purity. Other radioisotopic impurities were 1:LO 0 (< 1%), 13N 2 (< 0.1%) and 150 2 (< 0.1%) at BOB.45

80 Hot-cell containing trap for P’CJCO, collection Beam ------from ______c y c l o t r o n P r o t o n s (7.6 MeU) ▲

Titanium foil FIouj 5meter Regulator Target/ siueep gas (N2)

Fig 2.6 Schematic representation of target system used for the production of P’ClCOj.

2.3.3 Trapping of ri:LC1Carbon Dioxide The apparatus used for trapping 11G02 in the published procedure is depicted in Fig 2.7. Target gas (100 ml min”1) was bubbled through potassium hydroxide solution (10 ml, 0.1 M) over a period of 6-7 min and the untrapped activity collected on a soda-lime trap connected at the vent. This trapping procedure was found to be between 65-70% efficient. mco2/n 2 \/

Flotu meter

Uent Photosynthetic uesset

K0H (0.1 M)

Fig 2.7 Trapping of "C02 in potassium hydrouide solution

81 Hie alternative system developed for the concentration of 1-kx>2 as [ 11C]carbonate is shewn in Fig 2.8. Hie apparatus consists of a plastic syringe (1 ml, Plastipak) packed tightly with glass wool (BEH product no. 33056) onto which potassium hydroxide solution was Injected. A soda-lime trap was connected at the vent.

Optimisation of trap performance i) Hie volume of KDH soln. (0.5 M) injected onto the glass wool trap was varied between 50 and 400 /il. In each experiment' 11002/N2 was passed at a flow rate of 150 ml min"1 through the glass wool trap impregnated with a known volume of KDH soln. (Fig 2.9).

ii) Hie flow of 11C02/N2 through the glass wool trap containing KOH soln. (350 /il, 0.5 M) was varied between 50 to 520 ml min"1. In each experiment total radioactivity from the target was dispensed and the time determined (Table 2.3).

Table 2.3. Trapping efficiency of 11Q02 at various flow rates of 11CJ02/n2 through glass wool trap containing KDH (300 /il, 0.5 M)

Flow rate of Time to empty Trapping efficiency

target gas of 11O 02

(ml min"1) (min) (%)

50 10 98.3

90 9 99.4

135 8 97.0

200 7 98.3

265 6 94.5

400 5 81.8

520 4 76.4

82 Bq. "COj2* aq. KOH (9.5 ml) to algoe

Soda lime

’’COj/N j in 11C02 collection 11C0j2_ dispensed

Fig 2.8 Glass-wool trap containing aq. KOH for trapping cyclotron produced "C02.

vot. KOH (0 -5 M )//jl Fig.2*9 Trapping efficiency of i C ] CO 2 a g a i n s t v o l . K O H ( 0 - 5 M )

83 Operation of glass wool trap At the end of bombardment (BOB), the target gas was passed through the glass wool trap (Fig 2.8) containing KDH soln. (300 yjl, 0.5 M) at a flow rate of 150 ml min-1. Typically the radioactivity trapped as [^c]potassium carbonate was 12.7-14.5 GBq (350-400 mCi), decay-corrected to BOB. The Tinni-mum volume of aqueous solution required to elute the activity from the trap was found to be 0.75 ml.

2.3.4 Opt-imi?aticn of Buffer p H Carbon dioxide trapped as [ 11C] carbonate on glass wool impregnated with KDH solution (300 y.1, 0.5 M) was eluted with a solution (9.5 ml) containing potassium hydroxide (known quantity) and potassium carbonate (0.9 mg). The radioactivity was dispensed into vessel A (Fig 2.10) containing algae cells in buffer at pH 5.0 (for conditions see Sec 2.3.8). The resulting suspension was illuminated for 5 min and the algae cells separated. Radioactivity incorporated by algae cells, in the supernatant and in the soda-lime connected at the vent of vessel A was measured. The pH of the supernatant was also determined (Table 2.4).

2.3.5 Separation of Algae* fleHs The algae suspension under a pressure of nitrogen (67 kN m“2 or 10 psi) was transferred from vessel A (see Fig 2.10) to an on-line filter (0.22 ^m, Millex GS, Millipore Inc.) over a period of 5 min. The trapped algae cells were back-washed off the filter membrane by hot hydrochloric acid (8 ml, 1.5 M) into vessel B. It was found that this volume of HC1 was required to remove most of the cells from the filter membrane. When the volume of HC1 used was reduced (i.e. 4 ml, 1.5 M) only 50% of radioactive algae cells was released from the filter surface.

84 Table 2.4. Optimisation of buffer pH

KDH Supernatant^ Radioactivity Photosynthesis0 retained by buffer (M) P« (%) (%)

0.06 5.65 58 94

0.15 6.13 64 89

0.20 6.38 72 91

0.23 6.58 70 89

0.26 6.75 73 87

0.28 6.92 82 88

0.29 7.02 89 81 r- CO CO 0.30 7.20 81, 90 VO

0.31 7.33 88 43

0.32 7.60 84, 94 18, 23

a) Final pH of buffer after the addition of radioactivity in a solution containing appropriate molarity of KDH (9.5.ml) and K2C03 (0.9 mg) b) Percentage of radioactivity retained by the buffer used to suspend the algae for photosynthesis c) Percentage of radioactivity incorporated by algae cells

2.3.6 Hydrolysis of r-^O Sugars - (Xtimisaticn of Hydrolysis ti-rre After photosynthesis and filtration, hot hydrochloric acid (8 ml, 1.5 M) was used to remove trapped algae cells from the filter membrane into vessel B (Fig 2.10). The algae cells were then boiled at 413 K and stirred with a flew of nitrogen. Aliquots of the radioactive hydrosylate were removed at 1 min intervals and analysed by tic {system 1: Polygram Ionex 25 SA Eb+ ; eluant, EtQH:H20 (25:75 v/v) see Sec. 2.3.8 for Rf values}. It was found that the conversion

85 of [^C] sugars into glucose was optimal for 7 min hydrolysis at 413 K.

2.3.7 Neutralisation of Radioactive Hydrolysate Hie volume of HC1 (8 ml, 1.5 M) used for hydrolysis necessitated the use of a anion exchange column (40 x 20 mm i.d., Bio-rad AG 11A3, resin bed packed in EtCH (10 ml) and washed with H20 (30ml)}. For neutralisation the radioactive hydrolysate was loaded onto the ion exchange column under a flow of nitrogen (see Fig 2.10). The activity retained on the column was eluted with H20 for injection into vessel C (see Fig 2.10). It was found that 6 ml of H 20 was required to elute the radioactivity efficiently (ca. 90%) from the column bed as a neutral solution.

2.3.8 Final Procedure for -the Preparation of rGlucose a) Preparation of algae for photosynthesis The cultivated algae was transferred into several sterile vials (20 ml, Sterilin) under aseptic conditions. The suspension was then centrifuged (900 g, MSE-MISTRAL 6L) for 5 min and the cultivating, media decanted. The pellets of cells were resuspended in H20 for injection and centrifuged as above. This procedure was repeated three times. Finally, the pellets of cells were resuspended into a known volume of buffer (vol. calculated as shown below) of morpholinoethanesulphonic acid (100 mM), calcium chloride (0.5 mM) and magnesium chloride (5 mM): the buffer was adjusted to pH 5.0 by the addition of KCH (1 M).

Determination of the amount of cultivated alaae required for photosynthesis: The density of the cultivated algae was determined spectrophcfcametrically at 525 nm. The reference cuvette (1 cm) contained H20 for injection. The number of ml (X) of cultivated algae required for radiosynthesis was calculated by the formula,

86 X = JU8 X 100 A where A is the absorbance measured for cultivated algae. For each experiment, X (ml) of centrifuged algae cells were resuspended in the above buffer (35 ml) corresponding to ca. 2 x 108 algae cells ml”1. b) Pre-illumination The algae ral in buffer (35 ml, cell density ca. 2.8 x 108 ml"1, pH 5.0 ) were placed into vessel A (see Fig 2.10) and a stream of nitrogen (25 ml min"1) was passed through the suspension. The cell suspension was then illuminated (250 W, Osram HWL-lamp at 28-30 cm distance, giving 18,000-20,000 lux at the surface of the reaction vessel A) for 45 min at 303 K. c) Photosynthesis f1^ ] Carbon dioxide was trapped as [ 11C] carbonate onto a glass wool trap containing a solution of KDH (300 ^1, 0.5 M, CONVOL, BEH). The f11^activity was eluted from the trap by a solution of KDH (9.5 ml, 0.26 M) containing K2CO3 (0.9 mg). This solution was dispensed into vessel A (Fig 2.10) containing pre-illuminated algae in buffer at pH 5.0. The addition of radioactivity in an alkaline solution, resulted in an increase of buffer pH to 6.9. The algae suspension was further illuminated for 5 min under nitrogen. The cells were then separated from the buffer by passing the suspension under a pressure of nitrogen (67 kN in"2 or 10 psi) onto an on-line filter (0.22 ^m pore size, Millex GS). A layer of algae cells was formed on the filter membrane which were removed by back washing the filter with hot hydrochloric acid (8 ml, 1.5 M ) . The suspension was collected in vessel B and heated for 10 min at 413 K. The resulting mixture was then passed through a loop (FIFE, 0.75 mm i.d? 5 turns of 30 mm diameter) submerged in an ice-water bath, onto an ion-exchange column (40 x 20 mm i.d., Bio-rad AG 11A8, packed in EtOH (10 ml) and eluted with H2O (30 ml)}. The column was eluted with H20 for injection (6 ml) and the neutralised radioactive eluate 87 nq. K2nC032 in Filtrate out

Fig 2.1 0 Schematic representation of the apparatus used for the photosgnthetic preparation of ("Clglucose. collected in vessel C. This solution was then passed under a pressure of nitrogen (67 kN m"2 ) through a millipore filter (0.22 ^m, Millpy GS) into a rotary evaporator. The solution was evaporated to a volume of 1.2-1.4 ml and injected onto a preparative bplc column (5 particle size, Aminex HPX-87P Heavy metal, 300 x 7.8 m m i.d.) equipped with pre-column (Micro guard system, Aminex HPX-85) and temperature controller (Model 7930, Anachem) set at 323 K. The column was monitored continuously with a Nal (Ti) scintillation detector and a differential refractrameter (R 401, Waters) for radioactive and stable material response, respectively. The radioactive peak eluting at the same retention time as D-glucose (9.2-11 min) was collected (Fig 2.11).

d) A n a ly s is i) HPLC An aliquot of the radioactive fraction collected from the preparative hplc column was injected onto a reverse-phase hplc column ('V “ Bondapak-NH2n/ 5 /un particle size, 300 x 3.9 mm i.d.,) eluted with a mixture of acetonitrile and water (95:5 v/v) at a flow rate of 2 ml min"1. The eluant was monitored continuously for radioactivity and refractive index. The hplc chromatogram revealed a single radioactive peak with the same retention time as authentic D-glucose (10.8-13.0 min; Fig 2.12).

ii) TDC System 1 Strong cation exchange resin supported on plastic sheets (Polygram Ionex-25 SA Na+ ; CAMLAB) were converted into the Pb2+ form, by immersing the plastic sheets in deionised water for 1 h and then transferring them to a solution of lead acetate (5%) for 2 h. The plates were then washed several times with deionised water and left to dry in air for 24 h.

89 A Refractive Index - Radioactivity (log cps) i 21 Peaaie eaain f "] lcs b HPLC by glucose ["C] of separation Preparative 2-11 Fig Hie radioactive product collected from the preparative hplc column and reference D-[1-14C] sugars ( purchased from Amersham International, pic) as reference compounds (see below) were applied onto a tic sheet (Eb2+ form) and developed in a mixture of ethanol and water (25:75 v/v). The stable material was visualised by spraying the plastic sheet with a ceric sulphate reagent {ceric sulphate (1 g) and ammonium molybdate (2.5 g) dissolved in a solution of sulphuric acid (10%)} and heating the plate for 3 min at 373 K. Radioactivity was detected by placing the developed plate onto an X- ray film (50 AFW, AGFA-GEVAEKT, KODAK) in the dark and exposing the film for 12 h. The film was developed by immersion in developing medium (10% solution of LX 24, KODAK) for 5 min, followed by water and then into a solution of fixer (10% solution of FX 40, KODAK) for 5 min. Finally, the film was thoroughly washed with H20 and left to dry in air.

System 2 A cellulose pre-coated plastic sheet (0.1 mm, Merck) was spotted with radioactive product from the hplc column and reference D-[1-14C] sugars and developed with a mixture of pyridine, n-butanol and water (1:1:1 v/v). This was then exposed as in system 1. The radioactive product co-migrated with D-[1-14C] glucose on each system (Table 2.5). A typical tic autoradiograph of system 1 is shown in Fig 2.13.

Table 2.5. Rf values for [^C]glucose and standards.

Plates Developing Rf values Solvent [14C]G1u [14C]Man [14C]Gal [11C]Glu

Polygram EtOH & Ionex 25 H 20 (25:75) 0.7 0.2 0.5 0.7 SA-Pb+

Pyridine Cellulose n-Butanol 0.54 0.49 0.59 0.54 H 20 (1:1:1)

91 Fig. 2*12 An analytical hplc chrom atogram o f l ” C ] g lu c o s e

r 1 1) [" C ] glucose i SOLVENT 2) l C ] glucose FRONT 1 [14C ] glucose 3 ) []UZ ] g l u c o s e

H* 4) (1^C ] m annose 5) (1/fC I galactose i

I ORIGIN L J 1 2 3 4 5 Fig. 2-13 A tic autoradiograph o f [MC ] glucose, l14C] glucose and other [,4C] sugar

92 e) Formulation for intravenous injection Hie radioactive product obtained fran the preparative hplc column was diluted with H2O for injection and sterilised by filtration (0.22 /Jin pore size, Millex-GS). All randomly selected preparations of [^C] glucose passed independent (Safepharm Ltd) tests for apyrogenicity and sterility. f) Radiochemical yield of F ^ € 1 glucose The developed procedure, routinely produced radiochemically and chemically pure [1;1C] glucose within 50 min from BOB and in 14-19% radiochemical yield from 11002 (decay-corrected). Typically 206-595 MBq (7-16 mCi) of glucose was obtained from 12.7-14.5 GBq (350- 400 mCi) of [^C]potassium bicarbonate at the end of synthesis (EOS). The specific activity of glucose was estimated to be in the range of 74-185 MBq//xmol (2-5 mCi /xmol”-*j corresponding to 0.4- 1.0 mg (2.2-5.5 /xmol) of stable glucose. This was determined by comparison of peak area of "carrier" glucose in the -^-preparations with peak area of known amounts of reference material.

2.3.9 Preparation of r^c/^Ol Glucose Exactly the same procedure as that described for the radiosynthesis of [^C] glucose was followed except that 13c- enriched potassium carbonate (91% atom enrichment) was co-included with [^C]potassium carbonate. In each experiment, 11G02 collected on the glass wool trap (containing KDH, 300 /xl, 0.5 M) was eluted with a solution, of potassium hydroxide containing 13C-enriched potassium carbonate (0.9 mg, 6.5 /xmol). The isolated 13C- and i:Lc- labelled product was filtered (0.22 /xm , Millex-GS) under aseptic conditions into a sterile vial and stored at 253 K. This procedure was repeated for twenty preparations. The resulting products were combined and evaporated to dryness. An estimation of the quantity of ^C-enriched product in the final sample was based on the assumption that [13C] potassium carbonate was incorporated by photosynthesis in the same manner as [X1C] potassium carbonate and that the same yields (10-16 %) of both

93 products were obtained. In addition it was assumed that the glucose molecule was labelled in all six positions (maximum incorporation of 13C-precursor). Therefore the amount of 13C-enriched glucose in the final sample was estimated to be in the range of 2.2-3.5 /xmol (396- 630 pg). The quantity of stable glucose was based on the "carrier" added [13C/Hc]glucose preparations, where the amount of natural abundance glucose was found to vary between 0 .4-1.0 mg in an individual preparation. Hence, the maximum amount of stable glucose in the 13C-enriched sample was estimated to be < 20 mg. The 13C-enriched product was examined in D2O by 13C-NMR spectroscopy at 22.5 MHz and 100.62 MHz at ambient temperature. The 13C-NMR spectrum obtained at 100.62 MHz is shown in Fig 2.14. The chemical shifts of natural abundance D-glucose at 22.5 MHz and 13c- enriched glucose both at 22.5 and 100.62 MHz are presented in Table 2.6. This table also includes calculated 13C-13C coupling constants.

94 4a,/3

i. -»------1------1------1_____ i____ L t -i----- 1_____ L J___ L___i. 90 0 -«---- -L ji_____ I___ 80.0 PPM 70.0 60 0

Fig 2.14. PFT 13C{ 1H)NMR spectra of photosynthetically prepared D-[i:LC/13C]glucose at 100.62 MHz; sanple contained 396-630 /ig of 13C-enriched D-glucose, in D 2O at 303 K. Table 2 .6. ^ O N M R data of ^C-enricbed and natural abundance D-glucose.

CARBON CHEMICAL SHIFT (<5ppn) COUPLING CONST,

No. D-Glucosea ^C-Enriched Glucose*3 kj (13C-13C) Hz

22.5 MHz 22.5 MHz 100.62 MHz Litc Exp

la 93.2 93.3 91.9 45c 45

2a 72.6 72.6 71.3e - 39

3a 73.9 74.0 72.6 45c ,39d 39

4a 70.7 70.8 69.5 39d 39

5a 72.5 72.6 71.3 - -

6a 61.7 61.8 60.5 43 -

W f 97.0 97.1 95.7 47c 45

2(3 75.2 75.4 74.0e 47c 40

3/3f,5/3 77.0 77.0 76.6 39d -

4/3 70.7 70.8 69.5 39c 39

5/3,3/3f 76.8 77.1 76.4 43c 39

6/3 61.9 62.0 60.7 43 -

a) PFT 13C{1H}NMR spectra of natural abundance D-glucose in D2O at 303 K b) PFT 13C{1H}NMR spectra of photosynthetically prepared [ 11C/13C] glucose ? sairple contained 396-630 n q of 13C-enriched D-glucose in D2O at 303 K c) Fran Breitmaier et al.42 d) From Cohen et al.43 e) Percentage of enrichment at C2 (26%) calculated from signals assigned to Cja and Cjft by ratio of the integrated intensity of the central resonance to the outer resonance of the triplets f) Value calculated from C^S measures enrichment both at Cjfi and C ^ (31%)

96 2 .4 REFERENCES

1. Lassen N.A., Physiol. Rev., 39, 204 (1959).

2. "Metabolic Pathways", Ed. Greenberg D.M., 3rd Ed., Academic Press, Inc., N.Y., 1, Ch. 3, 4 (1967); 2, Ch. 8, 9 (1968).

3. Alavi A., Reivich M. , Greenberg J.H. and Wolf A.P. , "Computed Emission Tomography", Ed. Ell P.J. and Holman B.L., Oxford University Press, Ch. 4, 134 (1982).

4. Sokoloff L. , "Positron Emission Tomography and Autoradiography; Principles and Applications for the Heart", Ed. Phelps M.E., Mazziotta J.C. and Schelbert H.R., Raven Press, N.Y., Ch. 1, 1 (1986).

5. Schelbert H.R., "Computed Emission Tomography", Ed. Ell P.J. and Holman B.L., Oxford University Press, Ch. 3, 91 (1982).

6. Taegtmeyer H., "Positron Emission Tomography and Autoradiography; Principles and Applications for the Heart", Ed. Phelps M.E., Mazziotta J.C. and Schelbert H.R., Raven Press, N.Y., Ch. 4, 149 (1986).

7. Opie L.H. , Am. Heart J. , 76., 685 (1968).

8. Hillis L.D. and Braunwald E. , New Engl. J. Med., 296. 971-8; 1034-41; 1093-6 (1977).

9. Liedtke J.A., Prog. Cardiovasc. Dis., 23, 321 (1981).

10. Cornbleth M. , Randle P.J., Parmeggiani A. and Morgan H.E., J. Biol. Chem., 238, 1592 (1963).

11. Scheuer J. and Stezoski S.W., Circ. Res., 27, 835 (1970).

12. Lifton J.F. and Welch M.J., Radiat. Res., 45, 35 (1971).

13. Goulding R.W. and Palmer A.J., In t. J. Appl. Radiat. Isot., 24, 7 (1973).

14. Ehrin E. , Westman E. , Nilsson S-O. and Nilsson J.L.G., J. Label. Compd. Radiopharm. , 12, 453 (1980).

15. Shiue C-Y. and Wolf A.P., J. Nucl. Med., 22, 58 (1981) Abs.

16. Hara T. and Nozaki T. , J. Label. Compd. Radiopharm., 19., 1631 (1982).

97 17. Ehrin E. , Stone-Elander S., Nilsson J.L.G., Bergstrom M., Blomqvist G., Brismar T ., Eriksson L ., Greitz T ., Jansson P.E., Litton J-E. , Malmborg P. , af Ugglas M. and Widen L. , J. Nucl. Med., 24, 326 (1983).

18. Hall D.O. and Rao K.K. , "Photosynthesis: Institute of Biology's, Studies in Biology no. 37", 2nd Ed., Arnold, London (1 9 7 2 ).

19. Bassham J.A. and Calvin M. , "The path of Carbon in Photosynthesis", Prentice-Hall, Inc., New Jersey, Ch. 5, 34 (1 9 5 7 ).

20. Gibbs M. and Kandler 0., Proc. Nad. Acad. Sci., 43., 446 (1 9 5 7 ); Plane Physiol., 31, 411 (1957).

21. Benson A.A., Calvin M. , Hass V.A., Aronoff S., Hall A.G., Bassham J.A. and Weigl J.W., "^Cin Photosynthesis, In: Photosynthesis in Plants", Ed. Franck J. and Loomis W.E., Iowa, Iowa State College Press, 381 (1949).

22. Bassham J.A., Benson A.A., Kay L.D., H arris A.Z., W ilson A.T. and Calvin M. , J. Am. Chem. Soc. , 76., 1760 (1954).

23. Benson A.A., Bassham J.A., Calvin M. , Goodale T.C., Haas V.A. and Stepka W., J. Am. Chem. Soc., 72, 1710 (1950).

24. Benson A.A. and Calvin M., J. Exp. Bot., 1, 63 (1950).

25. Ruben S. and Kamen M.D. , Phys. Rev., 549 (1940); J. Am. Chem. Soc., 62, 345 (1940).

26. Barrio J.R., Egbert J.E., Henze E. , Schelbert H.R and Baumgartner F.J., J. Med. Chem., 25., 93 (1982).

27. Bassham J.A. and Calvin M., "Currents in Biochemical Research", Ed. Green D.E., Wiley Interscience, N.Y., 29 (1958).

28. Bassham J.A. and Buchanan B.B., "Photosynthesis: Development, Carbon Metabolism and Plant Productivity", Ed. Govindjee, Academic Press, Inc., N.Y., 2, Ch. 6, 141 (1982).

29. Calvin M. and Massini P. , Experienda, 8, 445 (1952).

30. Kandler 0. and Gibbs M. , PI. Physiol. Lancaster, 31., 411 (1956).

31. Trebst A. and Fieldler F., Z. Naturf., B, 17, 553 (1962).

32. Bassham J.A., A. Rev. PI. Physiol., 15., 101 (1964).

33. Bender M.M., Am. J. Sci. Radiocarbon Suppl., 10, 468 (1968).

98 34. Sequin U. and Scott I.A., Science, 186. 101 (1974).

35. Mclrmes A.G. and Wright J.L.G., Acc. Chemical Res., 8, 313 (1 9 7 5 ).

36. London R.E., Kollman V.H. and Matigoff N.A., J. Am. Chem. Soc., 97, 3565 (1975).

37. Simpson T.J., Chem. Soc. Rev., 4, 497 (1975).

38. P e r lin A.S. and Casu B ., Tet. Lett., 2921 (1969).

39. P e r lin A.S., Casu B. and Koch H.J., Can. J. Chem., 48, 2596 (1970).

40. Koch H.J. and Perlin A.S., Carb. Res., L5, 403 (1970).

41. Dorman D.E. and Roberts J.D., J. Am. Chem. Soc., 92, 1355 (1970).

42. Breitmaier E. and Voelter W. , "Carbon-13 NMR Spectroscopy", Verlag Chimie (1974).

43. Cohen S.M., Ogawa S. and Shulman R.G., Proc. Natl. Acad. Sci., 76, 1603 (1979).

44. Kylin A., Sundberg I. and Tillberg J-E., Physiol. Plant, 27. 376 (1972).

45. C lark J.C. and Buckingham P.D., "Short-lived Gases For Clinical Use", Butterworths, London (1975).

99 CHAPTER 3

PREPARAlTOf OF r^lDimENORFHINE AND r 1XC1EUFPENQRFHINE

100 3 .1 iimajpocrrcN The extract of the opium poppy has been used for medicinal purposes since cla ssica l Greek times. The major components of th is extract, morphine and its analogues, produce a variety of pharmacological responses, the most important of which are analgesia, euphoria and addiction.1-3 One of the characteristics of opiate drugs is that they produce pharmacological effects at extremely low chemical concentrations. This observation led to the hypothesis that opiates may be acting at specific binding sites or "receptors’'. Receptors consist of protein complexes and are often located on the surface of cells (Fig 3.1). They are capable of recognising and selectively interacting ("binding") with drugs, hormones or neurotransmitters. Neurotransmitters are released by nerve endings, diffuse across the synaptic cleft and modulate the firing of other nerve c e lls or neurons (Fig 3.1). Receptor activation results in the transduction of a message (through altering molecular configuration, enzyme activation, or ion permeability etc.) that initiates a series of events which ultim ately produce the physiological response associated with the drug or neurotransmitter.4'5 Drugs that mimic the action of the natural transmitter at the receptor site are termed "agonists" and drugs that do not elicit any pharmacological effect as a result of binding but inhibit agonist action by blocking receptors are termed "antagonists".6 The existence of receptors for opiates in mammalian nervous tissue was first established independently by Pert and Synder,7 Simon and co-workers8 and Terenius9 in 1973. In vitro binding of tritiated opiates to membranes was initially used to demonstrate opiate receptors in rat brain.7 '9 Studies of dissected homogenised brain regions10”12 and microscopic autoradiography13-15 on thin slices of rat brain following in vitro and in vivo labelling, have demonstrated that opiate receptors are concentrated in caudate nucleus/basal ganglia, thalamus and prefrontal/ frontal cortex with undetectable binding in the cerebellum. In contrast, determination of the distribution of opiate receptors in human brain had until

101 commencement of this work been limited to in vitro studies of post­ mortem tissue.3

Pre-synaptic Post-synaptic

Key: Endogenous : Transmitter

Exogenous : > Receptor ligand (agonist) Receptor ligand (antagonist) , Re-uptake site ligand

Fig. 3.1. Synaptic complex.

Several opiate receptor subtypes have been discriminated in binding studies. It is now believed that at least three predominant

subtypes exist designated as the /j , 8 and k receptors.16”19 /* Receptors are involved in pain perception and analgesia and are concentrated in areas such as the thalamus, parietal cortex, peri­ aqueductal grey, whereas 8 receptors are more concentrated in limbic

areas, caudate nucleus and the pons.20'21 k Binding sites are more prominent in the cerebral cortex, the dentate nucleus, the

102 substantia nigra and the amygdala.21'22 An ability to measure the distribution and concentration of endogenous opiate receptors in living man would enable study of the physiological role of the opioid system in brain and the relationship of this system to pain control, narcotic addiction and neurological as well as psychiatric disorders. Several types of neurotransmitter receptors in the living brain and the heart have been investigated by PET. For example, type 2 dopamine receptors (D2) in the brain have been characterised by using a potent dopamine antagonist, spiperone, and its derivatives labelled with 1:LC, {3-N-[1:LC-:meth.yl] spiperone},23'24 18F, {[18F] s p i p e r o n e 2 5 '26 a n d [ 18F]W-methylspiperone27'28} 76 Br, {[76Br]bromospiperone}29 and 77Br {[77Br]p-bromospiperone} .30 More recently, a more selective dopamine (D2) receptor antagonist, [11C] raclopride, has been used to visualise these receptors.31'32 In the heart, muscarinic cholinergic receptors have been characterised by using [11C]methyl-QNB33 and the peripheral type benzodiazepine receptor by using [11C]PK 11195.34 Propanolol,35 practolol36 and CGP 1217737 have all been labelled with 11C for studying (5- adrenergic receptors. In addition, the uptake of [18F]estradiol into estrogen-receptor rich breast tumours has been shown by Mathias et a l .38 Therefore, PET has potential for the examination of the opiate receptor system in vivo, provided that a suitable ligand can be identified. For receptor-ligand interactions to be successful in PET, ligands considered must meet specific criteria: (a) high affinity

(i.e. low Kq ) ; (b) high selectivity? (c) low non-specific binding; (d) penetration of blood brain barrier (BBB); (e) ease of rapid labelling with positron-emitting isotope; (f) little or no metabolism of active ligand; (g) saturability of binding; and (h) adequate preclinical data from biochemical, kinetic and anatomical quantitative methods to characterise the pharmacology of the ligand. Evaluation of the ligand-binding characteristics using autoradiographic techniques is a fundamental step in the selection of ligands for in vivo studies. For example, in vivo

103 autoradiographic studies can provide insight into (a) the kinetic behaviour and anatomical distribution of specific ligands; (b) estimation of the BBB permeability and (c) the basis for the evaluation and analysis of various tracer kinetic models.6'39 Of primary importance in the selection of a ligand is that it must have high affinity for the receptor. In vivo autoradiographic studies40-42 have demonstrated that localisation of radioligands in areas with high concentration of receptors is only feasible with very high affinity radiolabelled compounds (Kq < 10“9 M). It has been shown that application of a high affinity ligand in low concentration favours specific binding (binding that is associated with the receptor) over non-specific binding (binding not associated with the receptor). For example, the uptake of [^C]raclopride

(affinity of [3H] raclopride for E^: Kd = 1*1 nM)32 in regions known to have high concentration of dopamine receptors (caudate, putamen) is high in comparison to that in dopamine poor (cerebellum) regions.32'43 In healthy human subjects, [1:LC]raclopride binding in the caudate nucleus and the putamen was found to be 4-5 fold greater than non-specific binding in the cerebellum32 and this is the highest ratio that has been observed for the dopamine receptor system by PET. In summary, the specific:non-specific binding ratio determines in large part, the degree to which, regions of the brain that contain different densities of receptors can be quantitatively discriminated by PET. Another important factor to be considered in the selection process is the binding selectivity for the receptor class of interest. Cross-interaction with non-target receptors is exemplified by the binding of the butyrophenone neuroleptic, spiperone. Although, spiperone has high affinity for dopamine D2 receptors, it also binds to serotonin (5 HT2) receptors and less weakly to dopamine receptors, cholinergic, muscarinic and histamine receptors.44-46 However, the differential localisation of both dopamine (i.e. in basal ganglia)40 and serotonin (i.e. in frontal cortex)47 receptors in brain simplifies the modelling and interpretation of binding data to a certain extent. Cross-binding is not a problem for ligands such

104 as [i:LC]raclopride and [18F] estradiol which bind selectively to dopamine (D2)32,43 and estrogen38 receptors, respectively. The kinetics of the 1 igand-receptor interaction (i.e. the rates of association and dissociation of ligand) are equally important in the choice of ligands for in vivo studies. For example, in the case of [i:LC]raclopride, peak activity in the caudate/putamen32 was reached within 20-30 min, whereas for spiperone, the maximal ratio of striatal to cerebellum activity was not reached until several hours after injection.48 Therefore, 18F-labelled spiperone is preferred to W-[ 11C-methyl] spiperone for PET studies since radioactivity decay time and the turnover time of the radioligand in tissue are within the time window of the PET technique. In addition to demonstrating that high affinity ligands have specific binding, it is also necessary to corroborate that this specific binding is both saturable and displaceable by agonists and antagonists having affinity for the same receptor. This is one of the criteria required for claiming that a ligand interacts with a specific receptor site. For example, the binding of N- [ 11C-methyl]Ro 15-1788 to the benzodiazepine receptor can be displaced by injecting flunitrazepam as the competing ligand.49 When high doses of haloperidol (2-3 mg/kg) were injected after [ 1:LC] raclopride, the binding of the ligand in the striatum was reduced to the cerebellar level within 20 m i n .32 A property of a potential ligand that must be kept in mind is whether it is an agonist or an antagonist. In general, antagonists are favoured for in vivo studies since antagonists accumulate much more readily than their agonist analogues in sodium-rich environments.50 Synder51 has shown that dramatic differences in the behaviour of opiate agonists and antagonists appear when sodium ions are present. In vitro studies have demonstrated that concentrations of sodium that are found normally in the body enhance the binding of opiate antagonists and greatly diminish the binding of agonists. Snyder51 postulated that the opiate receptor can exist in two different conformations: a sodium-binding form (i.e. in vivo) with a high affinity for antagonists and a non-sodium form (i.e. in vitro)

105 with a high affinity for agonists. For example, [3H]-6-deoxy-6/3- fluoro-oxymorphone, an opiate agonist, has a high in vitro (i.e. sodium-free) affinity in the 1-2 nM range52 but fails to accumulate in vivo (caudate:cerebellum = 1). FRR permeability is an important criterion in the selection process. Potential ligands should exchange rapidly between blood and brain tissue, thus permitting fast transit to the receptor site of interest. Several studies have suggested that the diffusion of tracers across BBB obeys a structure-activity relationship.53-55 It has been inferred that the octanol/saline partition coefficient of potential ligands should be used to predict BBB permeability before proceeding to in vivo studies.53 However, physiological pH has to be taken into account since a ligand containing ionizable functional groups will have an octanol to saline partition coefficient much lower than the non-ionized form. For most ionizable ligands, the neutral species has far greater BBB permeability and the contribution of the charged form to diffusive transport may be negligible. Ligands having high partition coefficients bind to albumin, lipoproteins and blood cells56'57 and thus contribute to non­ saturable non-specific binding. For example, pimozide, a neuroleptic butyrophenone, has a binding affinity for D2 receptors similar to spiperone58 but, because of a higher hydrophobicity, has a slower clearance from non-specific sites.59 As a result, pimozide presents a lower striatum to cerebellum binding ratio and is not ideally suited for in vivo receptor studies. An additional consideration in the selection process is ligand metabolism. Metabolic stability can influence both the choice of position to label and which radioisotope to utilise. A common method for 1-^-labelling is //-alkylation (amino or amide) with [^C] iodomethane. For same radioligands e.g. N- [ 11C-methyl]Ro 15- 1788,49 w-demethylation occurs rapidly in vivo. N-Methyl-spiperone which has been labelled with both 18F and 13€, gives different sets of radiolabelled metabolites for the two radioligands. Significant ligand metabolism has important implications in the PET study, since

106 these radiolabelled metabolites can potentially contribute to specific as well as non-specific binding. Preliminary studies have shown that peripherally formed metabolites of [18F]n - methylspiperone do not accumulate in the target (striatum) or non­ target (cerebellum) regions of the brain.28 Specific activity is a major concern in receptor-ligand studies. The low number and concentration of receptors in vivo (1-20 pmol/g wet tissue)58'60 coupled with the sometimes acute pharmacological effect of extremely low doses of receptor-binding drugs dictates a need for high specific activity. In receptor studies, it is better sometimes to choose a synthetic strategy leading to the less toxic antagonists rather than the highly toxic agonists. Through in vitro binding studies, several investigators have demonstrated that the opiate receptor antagonist, diprenorphine, (1) labelled with tritium, has extremely high affinity (Kq = 0.22 nM)61 and binds with equal affinity to the \i, S and k opiate receptor subtypes.62-64 [%]Diprenorphine has also been utilised for in vivo labelling of opiate receptors in the rat15 and monkey brain.65 However, it has been suggested that diprenorphine may be binding predominantly to n receptors in vivo, since [ 3H] diprenorphine exhibits a pattern of "clusters" and "streaks" in the striatum15'66 which corresponds to a ^ pattern as opposed to the uniform pattern observed with S receptors.20 Both Pert et al12 and Frost et al67 have shown that after injection of low doses of [3H] diprenorphine, the activity clears rapidly from non-specific regions (i.e. cerebellum) and becomes selectively associated with specific opiate receptor regions (i.e. thalamus, striatum). Maximum striatum to cerebellum ratios for a number of [3H]opiate ligands are presented in Table 3.1. In general, Frost et al67 have observed that a high specific to non-specific binding ratio (diprenorphine exhibits a striatum to cerebellum ratio of 8.7) in vivo is obtained when the specifically bound ligand has slow clearance from the brain, as in the case of diprenorphine. The inhibition of specific binding of [3H]diprenorphine has been demonstrated by co-injecting unlabelled

107 opioid drugs,68 whereupon the specific binding of [3H]diprenorphine reduces to the level of non-specific binding. Perry et al69 have showed that although metabolites of diprenorphine appear in the blood, no metabolites are detected in the brain for up to 1 h after injection.

T a b le 3 .1 . In-Vivo Binding to Opiate Receptors3

3H-Labelled Maximum Striatum/ Ligand Cerebellum Ratio

Diprenorphine 8.7

Buprenorphine 4.5

Etorphine 3.0

Bremazocine 4.1

Naltrexone 4.5

Naloxone 2.9

Lofentanil 6.0

Ethylketocyclazocine 1.0

SKF-10047 1.0

Morphine 1.0

Dihydromorphine 1.0

Oxymorphone 1.0

a) Male ICR mice were injected with approximately l^g/kg of the 3H- labelled opiate and were killed at various times post-injection. The striatum, thalamus, cerebral cortex, and cerebellum were dissected, weighed and counted using standard methods. The striatum/cerebellum ratio is expressed according to milligrams of striatum and cerebellum. (Reproduced from Frost et al).67

108 On the basis of the above mentioned criteria the opiate receptor antagonist, diprenorphine (1) has been suggested as the candidate of choice,67'70 for clinical PET investigation of the opiate receptor system. Buprenorphine (2) which is a structural congener of diprenorphine, is a mixed agonist-antagonist,71'72 with high affinity (Kg = ca. 0.1 nM)73 for opiate receptors. Although, buprenorphine has a similar binding affinity to diprenorphine, it possesses a different profile of affinities for the various opiate receptor subtypes. Buprenorphine has a higher affinity for the n and k subtypes in vivo74 and by subtracting sequential diprenorphine studies, specific 5 binding could be calculated. Therefore, buprenorphine was chosen as the other candidate for the investigation of the opiate system by PET. Since both diprenorphine and buprenorphine appear to possess the essential properties required for a PET ligand, it was decided to consider how to label these compounds with carbon-11, at high specific activity with high radio-chemical yield and in high chemical and radiochemical purity for intravenous injection.

109 3.1.1 Strategy for labelling Examination of the molecular structure of diprenorphine (1) reveals three positions at which labelling with carbon-11 could be envisaged, namely, C2q / c 22 or c23 (F^ 3 -2)

H3Cb— c — oh 2 0 i 1 9 I I R R = CH3; Diprenorphine (1) R = C(CH3)3; Buprenorphine (2) Fig 3.2 Structure of diprenorphine and buprenorphine

Literature referring to the labelling of other opiates or structurally related compounds of interest with carbon-11 is presented in Table 3.2.

110 T a b le 3 .2 . [^C ]Labelled Opiates

C om pound P r im a r y L a b e l l i n g R e f e r e n c e s P r e c u r s o r A g e n t (Y e a r )

[N-me thyl -^ C ] Morphine “ 0 0 2 H1 :lCHO 75 (1979)

[N - me thyl -11C] E t o r p h in e 1 :lO 02 Hi : l CHO 76 (1981)

[N - me thyl -^-^-C] M o r p h in e U 2 1:lC H 3I 77 (1979) 78 (1982) 79 (1982)

[N - me thyl - 1:LC ] H e r o in X10 0 2 1:LC H 3 l 77 (1979)

[N- methyl--^-C]Codeine irLc o 2 ^ ^ h 3 i 79 (1982)

[N - me thyl - 1 1 C ] P e t h i d i n e 1 2 C 02 1:LC H 3 l 78 (1982)

Two predominant methods had been used to label opiates with 1^C: (a) reductive [^-k^m ethylation with [ 11C] formaldehyde and (b) [1:LC] alkylation using [ iodomethane. The opiate receptor agonist, etorphine (J),76 is closely related in m olecular structure to both diprenorphine and buprenorphine. This compound has been successfully labelled with carbon-11, by treatm ent o f N— (desm ethyl)-etorphine (or nor-etorphine) ( I ) w i t h [ -^C] formaldehyde follow ed by reduction with sodium cyanoborohydride

(S c h e m e 3.1).

Scheme 3.1 Preparation of [N-11C-methyl]etorphine from nor-etorphine

111 The opiate morphine (L) has been labelled by both reductive alkylation75 and N -alkylation reactions77"'79 on nor-morphine (K ).

Reductive [11C]methylation involves essentially the same synthetic route as for [N-me thyl -11C] etorphine. In the case of the N- alkylation reaction the labelled product (L) is obtained by the reaction of nor-morphine w ith [11C]iodomethane (Scheme 3.2) .

H 11CH3

(K) (L)

Scheme 3.2 Preparation of [N-11C-methyl]morphine from nor-morphine

Acetylation of [N-me th yl-11C]morphine yields N-methyl [ - ^C] heroin (M) ,77 Both, [N-methyl-^C l]codeine (N)79 and [N-methyl- ^ C l ] pethidine (O) ,78 have also been labelled by an N--alkylation reaction of the appropriate nor-compound with [11C]iodomethane.

O

CH3CO 11ch 3

o [N-11 C-methyl]heroin [N-11 C-methy Ijcodeine [N-11C-methyl]pethidine A caramon feature in all of these labelled opiates is the n — methyl group which is easily labelled with 11C. It should be noted that, in both diprenorphine and buprenorphine, replacement of the N- cyclopropylmethyl substituent by an N-methyl group or by another substituent (i.e. alkyl, allyl or cyclabutylmethy1 ) would be expected to drastically effect the pharmacological activity of the parent compound.80 For example, in the case of another opiate, morphine, it has been shown that substitution of the N-methyl group by a larger alkyl group, such as, allyl or cyclopropylmethyl, gives compounds with potent specific antagonistic activity against morphine and related compounds.81 In general, N- (cyclopropylmethyl) compounds are antagonists, while the N- (cyclobutylmethyl) series show more agonist action.82 In addition, structure-activity studies at peripheral sites of diprenorphine have demonstrated that the size of the alkyl groups in the tertiary alcohol function and the configuration of C^g have a profound effect on intrinsic biological activity.80 Therefore, alterations to the basic structure of diprenorphine or buprenorphine would lead to compounds not having the desired pharmacological activity. B u m s et al83 have labelled a morphinan (Q) with carbon-11, by reacting [11C]methyllithium with a ketone precursor (P) (Scheme 3.3), and claimed, that a similar strategy could be applied to the preparation of [1:LC]diprenorphine.

Scheme 3.3 Reaction of [11C]methylithium with a ketone precursor to give 11C-labelled morphinan

113 However, the required ketone precursor for diprenorphine would involve a multi-stage synthesis and due to the time constraint involved, this route was considered impractical for labelling diprenorphine. Similarly, labelling at the C22 c a r ^DOn would require a protracted multi-stage synthesis of a protected precursor most probably from thebaine, for subsequent [ 1:LC ] methylation with [ 1:LC] iodcsmethane followed by deprotection. In contrast, labelling at the C23 carbon could be achieved from the precursors, N- (descyclopropylmethyl)diprenorphine (3) or N-(des- cyclopropylmethyl) buprenorphine (4) which were available as gifts from Reckitt and Colman in small but useful quantities (1-2 g). The approaches taken by other workers to label opiates by reductive-alkylation and N-alkylation reactions were considered for the labelling of target compounds (1) and (2) (Fig 3.2). Reductive alkylation (approach A; Scheme 3.4) of (3) or (4) would require the development of [ 1-1:LC] cyclopropanecarboxaldehyde (5), whereas N- alkylation (approach B; Scheme 3.4) of (3) or (4) would require the development of [ l-i:LC] cyclopropylmethyl iodide (6) as labelling agents. In addition to these approaches, a novel route to the introduction of a [ 11C ] cyclopropylmethyl group into target molecules (3) and (4) was considered (approach C; Scheme 3.4) . This approach was based on the original syntheses of cold diprenorphine and buprenorphine.84 It would involve the formation of a [Re] amide derivative, by the reaction of [l-11C]cyclopropanecarbonyl chloride (7) with the appropriate precursor (3) or (4), followed by reduction to give [ 11C]diprenorphine (8) or [Re] buprenorphine (9). For each approach, the labelling agent would have to be prepared efficiently from cyclotron-produced [Rc] carbon dioxide. This chapter specifically addresses the development of these R e ­ labelling agents (5,6 and 7) for the labelling of diprenorphine and buprenorphine via reaction with precursors (3) and (4), respectively.

114 where R = CH3; N-(descyclopropylmethyl)diprenorphine (3) R = C(CH3)3; N-(descyclopropylmethyl)buprenorphine (4) R = CH3; [11C]diprenorphine (8) R = C(CH3)3; [11C]buprenorphine (9)

Scheme 3.4 Carbon-11 labelling approaches to [11C]diprenorphine and [11C]buprenorphine

115 3 .2 DISCUSSION Each of the three possible approaches considered for the [-^C]- labelling of diprenorphine (1) and of buprenorphine (2), i.e. A) reductive N-alkylation B) N-alkylation C) N-acylation followed by reduction required the development of a new labelling agent, i.e. i) [ 1-11C] cyclopropanecarfooxaldehyde (5) ii) [l-11C]cyclopropylmethyl iodide (6) iii) [l-11C]cyclopropanecarbonyl chloride (7), respectively.

3.2.1 Approach A: Reductive ^-Alkylation The attempted preparation of [ 1-11C] cyclopropanecarboxaldehyde (5) from 11C02 (Scheme 3.5) can be compared with the synthetic route reported for the formation of [ -^C] formaldehyde (S)85-87 (Scheme 3.6).

( 10)

II + III

Scheme 3.5 i) Cyclo C3H5M gBr in Et20 ; ii) LiAlH4 in Et20 ; iii) H 20 ; iv) oxidizing agent: Ag wool or 2(NH4)2Ce(N03)6

H11CHO

(R) (S)

Scheme 3.6 i) LiAIH4 in THF; ii) H20 ; iii) Ag wool catalyst

116 In both cases, the final step in the synthesis involves the oxidation of the [^C] alcohol to the respective aldehyde, (5) or (S). [ 11C] Cyclopropanemethanol (U.) itself was prepared from 11C02 by [ 11C] carbonation of cyclopropanemagnesium bromide (r.c.y of [1:LC] adduct (10) in > 90%) followed by reduction and hydrolysis (r.c.y of [1 ] cyclopropanemethanol 70-80% from CO 2, decay corrected).

In the simpler case of the reduction of ^ - 0 ^ 2 methanol (R), 1:L002 is trapped in a solution of LiAlH4 in THF, then hydrolysed. A r.c.y of greater than 90% has been achieved.85 However, the yield of the dehydrogenation of [^C] methanol to [ 1:LC] formaldehyde over silver wool catalyst varies in the range, 50-75%.85-87 In the analogous cyclopropyl system, it was found that, on a macroscale, cylopropanemethanol catalytically dehydrogenates to cyclopropanecarboxaldehyde over heated silver wool. Yields were in the range 30-56%, depending on the efficiency of the activation of the silver wool catalyst. Attempts to translate this procedure to 11C chemistry at the no-carrier-added level resulted in the formation of [ 1ILC]cyclopropanecarboxaldehyde (5) in poor r.c.y (2-8%). Several factors contributed to this poor yield. In particular, it was found necessary to pre-activate the silver wool with an air- cyclopropanemethanol mixture to obtain any conversions at the macroscale and no-carrier-added level. This process can reduce the specific activity of the f1-^] product if the alcohol used to activate the catalyst is not completely removed from the system. In manipulations involving 11C the furnace was heated often and "activated" an hour before the passage of cyclopropanemethanol (11). Curing this period the catalytic surface of the silver may have changed, causing a decrease in the percentage of dehydrogenation. Additionally, at the n.c.a level quantities of "carrier" are small (0.2-0 .5 /unol) and losses occur readily due to adsorption of activity onto the apparatus used. Since water is generated during the dehydrogenation process and has a similar boiling point to cyclopropanecarboxaldehyde, it may pose a problem

117 in the next step of synthesis. However, this route to [11C]cyclopropanecarboxaldehyde cannot be ruled out since in the case of [1:LC]formaldehyde, high r.c.y at high specific activity have been achieved under optimum conditions. It is envisaged that at optimum conditions, similar yields for [^^Cjcyclopropanecarboxaldehyde could be obtained. Another oxidising agent, ceric ammonium nitrate was also investigated for its suitability to convert cyclopropanemethanol into cyclopropanecarboxaldehyde. Young and Trahanovsky88 have reported that this conversion occurs readily under mild conditions. When tried the macroscale synthesis gave yields of 68%, in good agreement with those reported (64%).88 However, when this procedure was transferred to the n.c.a level with 11C, r.c.y in the range of 8-10% were obtained. A major problem encountered was that at the n.c.a level further oxidation to [i:LC]cyclopropanecarboxylic acid occurred readily. This procedure might lend itself to selective oxidation to the desired aldehyde by manipulation of conditions.

3.2.2 Approach B: iY-Alkylatian The synthetic route used for the preparation of [l-^C]- cyclopropylmethyl iodide (6) is shown in Scheme 3.7. The preparation can be compared with the synthetic route reported for the formation of [^-kzjiodomethane (T) (Scheme 3.8) The preparation of [ 11C] iodomethane involves a two step synthesis. 11002 ls reacted with LiAlH4 in Et20 or THF and the radioactive adduct is hydrolysed to [1:LC]methanol. Conversion of this into [1:1-C]iodamethane is achieved by treatment with hydroiodic acid. Radiochemical yields in the range of 60-90% have been reported.89'90 In the corresponding reaction of cyclopropanemethanol (12) with hydroiodic acid, the expected91-95 rearrangement occurred and the identity of the products (13), (14), (15) (Scheme 3.9) was confirmed by ^-NMR spectroscopy.

118 Schem e 3.7 i) Cyclo C3H5Br in Et20 ; ii) LiAIH4 in Et20 ; iii) H20 ; iv) HI or P2I4 or (C H ^SiC I and Nal

11co 2 ' 11ch3oh------<- 11c h 3i (S) (T)

Scheme 3.8 i) LiAIH4 in Et20 or THF; ii) H20 or HI; iii) HI

Scheme 3.9 Reaction of cyclopropanemethanol with hydroiodic acid

%-NMR analysis of the final product also showed, that the major component was the desired compound (13) with a maximum yield of 78% (see Table 3.3). The two rearranged products, allylcarbinyl iodide (14) and cyclobutyl iodide (15) varied between 22-38% . Roberts and Mazur91 found in a similar reaction regime that the action of hydrobromic acid on cyclopropanemethanol gave a mixture of bromides corresponding to Scheme 3.9, with cyclopropylmethyl bromide (65%) as the major constituent.

119 The reactions of cyclopropanemethanol with various haloacids have been studied extensively.91'95'96 Roberts and Mazur91 were the first to report that under acidic conditions, the generated cyclopropylmethyl cation (13a) (formed when water is eliminated) undergoes facile interconversion between cyclopropylmethyl (13a), cyclobutyl (15a) and allylcarbinyl (14a) cations, via a common cationic intermediate, Scheme 3.10.

CH2 CH2---- CH+ cationic ^CH — CH + intermediate / ch 2 CH0 CH, (13a) (15a)

ch 2=ch -ch 2-ch 2+ (14a) Scheme 3.10 Interconversion via cationic intermediate

Much experimental and theoretical work has been devoted to the nature of the cationic intermediate involved in these interconversions.9^“" Roberts and Mazur100 described this species as a "non-classical" cation (a structure intermediate between a cyclopropylmethyl cation and a cyclobutyl cation, suggested to be more stable than either of these classical cations92). These investigators initially proposed a tricyclobutonium ion (16) and in subsequent work revised their view and favoured a rapidly equilibrating set of non-classical bicyclobutonium cations101 (Scheme 3.11), instead of a single non-classical species.

CH

CH 2

CH? + * "tricyclobutonium ion" (16) 120 Scheme 3.12 Interconversion of "bisected" cyclopropylmethyl cations cyclopropylmethyl 3.12 "bisected" Scheme of Interconversion

121 Whereas the majority of the experimental evidence on C^E.j+ indicates that the species is a non-classical cation, controversy continues regarding the cationic intermediate (s) involved in the equilibration process.98'99'102"104 At present opinion is divided, with some favouring the rapidly interconverting bicyclobutonium cations (16a-c? Scheme 3.11) and others the "bisected" cyclopropylmethyl cations (17a-c; Scheme 3.12). In contrast the rapid equilibration of the methylene carbons (*CH2) in the cylopropylmethyl, cyclobutyl and allycarbinyl systems has been well established.92'95'98'99'101 For example, once a cyclopropylmethyl cation is formed, it can rearrange to two other cyclopropylmethyl cations. This rearrangement probably occurs through the non-classical intermediates described above and accounts for the scrambling which is completely stereospecific.105'106 Isotopic labelling techniques have been used to demonstrate this p h e n o m e n o n .9 3 - 9 5 '101 Specifically, the reaction of [ 1-14C] cyclopropanemethanol with dilute hydrochloric acid has been studied by 14C-degradation.95 These investigators found a third of the total 14C-activity on each of the three methylene groups for each of the rearranged products examined. However, it should be noted that the distribution of the isotopic label and the exact composition of the products obtained can depend on the attacking reagent and reaction conditions. Thus, for example, the deamination of [ 1-14C] cyclopropyf methyl amine (U) results in the formation of cyclopropanamethanol (V) and cyclobutanol (W) with the 14C-label distributed as shown in Scheme 3.13.101

24% 35.8%

Scheme 3.13 Deamination of [1-14C]cyclopropanemethylamine 122 Recent 13C-NMR studies93'94 on the treatment of [1-13C]- cyclopropanemethanol with SbF5~S02ClF-S02F2 at 148 K, have shown that the 13C-label is distributed randomly between the methylene and methine positions of the C ^y * cation. These studies indicate that the 11C label in [1:LC]- cyclopropylmsthyl iodide would be distributed mainly among the methylene groups of the cyclopropylmethyl system. In addition, the rearranged products ([13-C] cyclobutyl iodide and [ i:k:] allylcarbinyl iodide) would be expected to have scrambled labels. The complexity involved in the formation of cyclopropylmethyl iodide via a delocalised carbocation intermediate, might limit the use of this molecule as a labelling agent. Specifically, the rearranged products might compete with cyclopropylmethyl iodide in N-alkylation reactions. This limitation was not considered sufficient to impede the development of cyclopropylmethyl iodide as a labelling agent. Cyclobutyl iodide was not expected to undergo an iV-alkylation reaction with (3) or (4) since it gave no reaction with nor-morphine, probably due to steric hindrance. Also isolation of the desired [1:LC]amine (8) or (9) from the N-allylcarbinyl derivative might be achievable by chromatographic techniques. In addition, scrambling was considered not to be too critical in a PET study since metabolism might be expected to proceed with N-C cleavage rather than C-C cleavage in the cyclopropyl ring. Consequently, other synthetic routes which may generate cyclopropylmethyl iodide as the major product were investigated and are now discussed briefly. The preparation of cyclopropylmethyl iodide (13) reported by Denis and Krief,107 is shown in Scheme 3.14.

+ inorganic products

( 12) (13)

Scheme 3.14 Reaction of cyclopropanemethanol with diphosphorous tetraiodide

123 The reported procedure was adapted to yield the desired product (13) in short reaction tines (Table 3.4). Optimum conversion of cyclopropanemethanol with diphosphorous tetraiodide was achieved within 5 min at 378 K. The isolated product contained cylopropylmethyl iodide (13) (ca. 80% purity) and the two rearranged products (14 and 15). At higher temperatures and longer reaction times (15 min) the relative proportion of the rearranged impurities increased (ca. 30-50%), even with the addition of ETBP. When this reaction was performed at ambient temperature with short reaction times (5 min), low yields (< 20%) of the product were obtained. The published method reported a yield of 71% at low temperature (293 K) using a much longer reaction time (20 h). However, in 1:LC chemistry these long reaction times are impractical due to the short half life, where reaction times of a few minutes are desired. In addition, the stoichiometry of reactions is an important factor to consider since all reagents in ^-^e-reactions are in vast excess in comparison to the [1:LC]-precursor. For example, an excess of P2I4 was always used in order to mimic the conditions required in 1 ^C-chemistry. Synthetic routes for the conversion of alcohols into iodides10®'109 under mild conditions were adapted for the preparation of cyclopropylmethyl iodide (Scheme 3.15 and 3.16, respectively).

CH20H + (H3C)3SiCI 0 base (12)

(H3C)3SiCI + Nal CH20-Si(CH3)3 CH2I + [(H3C)3Si]20 + NaCI CH3CN (1 8 ) D >(1 3 )

Scheme 3.15 Preparation of cyclopropylmethyl iodide via silyl ether

124 (H3C)3SiCI + Nal ch 2oh >CH2I + (CH3)3SiOH + NaCI CH3CN ( 12) (13)

Scheme 3.16 Preparation of cyclopropylmethyl iodide from cyclopropanemethanol

The synthesis was conducted either by a two-step procedure , where the trimethylsilylether derivative (18) was isolated or this species was generated in situ as described by Olah et al.109 The results are presented in Sec 3.3.1.2 and Table 3.5. The two step procedure gave the desired compound (13) in 72% yield and rearranged products (14,15) were not observed. However, for technical reasons it was considered that the "one-pot" procedure was more suitable for application to 11C. The results show that the overall yield of cyclopropylmethyl iodide decreased from 75 to 47% as the reaction time was reduced from 20 to 5 min. In contrast, the purity of the product (13) did not vary from 85-90%, the contaminants being the two rearranged by-products (14 and 15). It should be noted that as the temperature of the reaction is decreased (340 to 273 K), the overall yield drops to 25%, where the purity of the product (13) was found to be between 90-95%. Since this synthetic route under certain conditions gave a relatively pure product (> 85%) and in high yield (> 70%), the synthesis is presently being transferred to 11C chemistry. Recently Langstrom et al110 have published the synthesis of [l-^C]-labelled alkyl iodides, such as ethyl, propyl, butyl and isobutyl. The method involves the reaction of 11C02 with an appropriate Grignard reagent, reduction of the formed [-^C]adduct and treatment with hydroiodic acid (Scheme 3.17) .

i) 11C02 ii) LiAIH4 RMgBr ------R11CH?I iii) HI 2 R = ch 3, ch 3ch 2, ch 3ch 2ch 2, (CH3)2CH

Scheme 3.17 Preparation of [11C]alkyl iodides 125 These investigators found that this approach gave [1:LC] alkyl iodides having 65-95% radiochemical purity, with [l-1-^:] isobutyl iodide being least pure (65%). More recently, Rimland and Langstram,111 have reported the synthesis of [l-11C]cyclopropylmethyl iodide by a four step procedure (Scheme 3.18). This involves the [-^Cjcarbonation of cyclopropanemagnesium bromide, followed by reduction with LiAlH4 and treatment with toluenesulphonyl bromide to give [iicjcyclopropylmethyl bromide. This is converted into the corresponding (^C] iodide by reaction with sodium iodide in acetone (Scheme 3.18). A radiochemical yield of 13% and radiochemical purity of 80% was reported.

j) 11C02 ii) LiAIH4 TsBr + heat

Nal in (CH3)2CO heat Scheme 3.18 Synthesis of [11C]cyclopropyimethyl iodide

This procedure does not appear to have distinct advantages over the "one-pot" trimethylsilyl method described above, since the latter procedure has the potential to produce higher purity product.

126 3.2.3 Approach C: //-Acylation Followed By Reduction The most common method for the synthesis of acyl halides is from the corresponding acid.112'113 Mazur et al101 reported the preparation of [ 1-14C] cycloprcpanecartooxylic acid from 14C02 . This synthetic route was adapted to the formation of [1 - llc]cyclopropanecarboxylic acid (20; Scheme 3.19).

HCI C00H

(19) (20) Scheme 3.19 Preparation of [11C]cyclopropanecarboxylic acid

The [11(2]acid (20) was obtained in 80-90% r.c.y, having a radiochemical purity of > 95%. Cyclopropanecarbonyl chloride has been prepared from cyclopropanecarboxylic acid by the action of thionyl chloride. 114,115 This reagent has the advantage that gaseous by-products are formed (Scheme 3.20).

RCOOH + SOCI2 ------RCOCI + HCI + S02 Scheme 3.20

Several irivestigatorsll2 '116' H 7 have suggested that the mechanism involves the initial formation of the mixed anhydride (chlorosulphite; Scheme 3.21),

which is converted into the acyl chloride, either by an internal electron shift (i) or by an SN2 displacement mechanism (ii), (Scheme 3.22).

127 o (ii) 11 RCOCI + S09- — L^— RC-^-OSOCI c ) Scheme 3.22 Chlorosuiphite converts into an acid chloride

The kinetics of the reaction between thionyl chloride and carboxylic acids have been studied116 and the reaction has been shown to be first-order with respect to each reactant. It has also been noted that as the reaction proceeds, the rate slows down due to the competing reaction (formation of X), which accounts for the relatively low yields obtained for acid chlorides by this method.

RCOCI + RCOOH------R(C0)20 + HCI (X)

Nakatsuka et al118 have reported the preparation of [1-14C]- cyclopropanecarbonyl chloride from 14CC>2 , where the [14C]acyl chloride was generated by the reaction of [1-14C]cyclopropane- carboxylic acid with thionyl chloride. Consequently, the synthetic route shown in Scheme 3.23, was investigated for the preparation of [l-11C]cyclopropanecai±x)nyl chloride (7).

Scheme 3.23 Preparation of [11C]cyclopropanecarbonyl chloride with thionyl chloride as the chlorinating agent

128 At the n.c.a level, a convenient means of verifying the formation of [11C]acyl chloride is by its reaction with an appropriate amine to produce an identifiable [^C]amide ((21); Scheme 3.23). The amines (Scheme 3.24) chosen, contained a chromophoric group with high extinction coefficient, thus enabling easy detection and determination of the specific activities of their derivatives, by means of an ultraviolet spectrophotometer linked to hplc. The feasibility of the reaction between cyclopropanecarbonyl chloride (22) and an amine such as, aniline (23), 4-benzylpiperidine (24), 1,2,3,4-tetrahydroquinoline (THQ, 25) and 1,2,3,4- tetrahydroisoquinoline (THIQ, 26) was investigated on the macroscale.

where HNR is,

(25) (26) Scheme 3.24 Amines chosen for reaction with cyclopropanecarbonyi chloride

The formation of N -cyclopropanecarbonyl -aniline (27) occurred readily (ca. 80% yield). In the case of 4-benzylpiperidine, commercial samples of this product were found to be impure by analytical hplc and spectroscopy. Purification (> 85%) was achieved by preparative hplc, however the amine was found to be unstable even at 273 K. N-Cyclopropanecarbonyl-4-benzylpiperidine

129 (28) was obtained in 65% yield. Hie reaction of THQ (25) or THIQ (26) with cyclopropanecarbonyl chloride (22) also occurred readily, giving N -cycloprcpanecarbonyl-THQ (29) in 53% yield and N- cyclopropanecarbonyl-THIQ (30) in 80% yield, respectively. The formation of [l-11C]cycloprqpanecarbonyl chloride (7) from 1±CQ2 was investigated by two different pathways (a) and (b) (Scheme 3.23). In both cases, cyclcpropanemagnesium bromide (19) was carbonated with 11CQ2 form the [llc] adduct (10). In approach (a), this adduct was hydrolysed to [ 1:LC] cyclopropanecarboxylic acid (20) with HC1 and then thionyl chloride was added. In approach (b) thionyl chloride was added directly to the [-^C] adduct (10). The work up involved the removal of excess thionyl chloride (and HC1 in approach (a)} and reaction solvent. The formed [11C]acyl halide (7) was converted into t1-^] amide by reaction with amine 23, 24 or 26 (Scheme 3.23) either by a "one-pot" method or a "two-pot" method (Fig 3.8). In the "one-pot" method the amine was added directly to the [11C]acyl halide, whereas in the "two-pot" method, the [1:LC]acyl halide was isolated from the reaction mixture by distillation and then reacted with the amine in pot 2 (Fig 3.8). The results are presented in Table 3.6.

F o r at-[ l-1 1 C]cyclopropanecarbonyl-aniline (31), N-[ 1- 1 1 C] cyclopropanecarbonyl-4-benzylpiperidine (32) and N-[ 1- 1-k?]cyclopropanecarbonyl-THIQ (33), r.c.y varied between 10-25%. Small improvement in the r.c.y (5-10%) was observed when approach (a) was used and when the synthesis was carried out in "one-pot". However, large amounts of the amine 23, 24 or 26 (0.15-0.5 mmol) were used in these preparations. This was found necessary, since the unreacted thionyl chloride and the formed HC1 could not be completely removed at 373 K from the reaction mixture. Probably, small quantities of thionyl chloride became physically entrapped in the radioactive residue, tubing, 3-way taps etc. At, higher temperature loss of [11C]acyl chloride was observed. In addition, this synthetic route (both approaches (a) and (b)) involves the removal of reaction solvent as well as thionyl chloride and HC1. This operation leaves a solid residue containing [11C]acyl chloride.

130 Consequently, the isolation of the [ 11C] acyl chloride from this residue is difficult and therefore poor yields are obtained. Concentration would also promote the formation of the unwanted anhydride. To obtain essentially quantitative yields in the reaction of [1- 11C]cyclopropanecarbonyl chloride with an amine necessitates the complete isolation of this [i:LC]acyl chloride (7). A further aspect of the procedure is to achieve complete removal of the excess acid halide used for the conversion of the f1^] adduct (10) into the desired [11C]acyl halide and the produced hydrogen chloride. This would allow the use of a minimal quantity of starting amine (2-5 mg) as required (for the projected syntheses of 11C-labelled diprenorphine and buprenorphine the starting precursors (3) and (4) were available in only very limited supply). Thus in subsequent approaches these issues are confronted in order to establish optimal conditions for [11C]cyclopropanecarbonyl chloride formation and its conversion into [i:LC]amide. A milder reagent than thionyl chloride for the conversion of carboxylic acid into acyl halide is oxalyl chloride.112'119 This reagent has the advantage that the acid by-product (Y) that is formed in reaction with a carboxylic acid is unstable and decomposes to gaseous products which can be removed from the reaction mixture. Thus reaction is driven to produce the desired acyl halide (Scheme 3.25).

COCI C00H RCOOH + RCOCI + COCI COCI (Y)

CO + C02 + HCI Scheme 3.25 Reaction of a carboxylic acid with oxalyl chloride

This synthetic route was adapted to prepare [ 1—1:1G] — cyclopropanecarbonyl chloride (Scheme 3.26).

131 11C02 MgBr

(19)

i) (COCI)2 ii) DTBP

THIQ .11COCI

(7) Scheme 3.26 Preparation of [11C]cyclopropanecarbonyl chloride with oxalyl chloride as the chlorinating agent.

In this synthesis, oxalyl chloride was added to the [1:LC] adduct (10) formed in the reaction of cyclopropanemagnesium bromide (19) with 11C02. In the previous approach distillation was used to remove HC1 from the reaction mixture and found to be inefficient. Here a hindered base, 2,6-di-tert-butylpyridine (DEBP) was also added to fulfil two functions; firstly to remove HC1 by formation of the salt of the acid and secondly to allow reactants to be contained in homogeneous solution. It was found that the addition of DTBP facilitated the removal of unreacted oxalyl chloride and by-products (HC1, CD and C02). However, small amounts of oxalyl chloride were carried over with [l-11C]cyclopropanecarbonyl chloride (7) to the second reaction vessel containing THIQ (Fig 3.8). As a result greater than 20 mg of THIQ was required for [^C] amide (33) formation. Radiochemical yields in the range of 15-25% (Sec 3.3.2.5) were obtained. Pames et al120 have reported a method for the microscale synthesis of volatile 14C-labelled acid chlorides. These investigators used excess oxalyl chloride to generate the [14C]acyl chlorides and employed vacuum line transfer techniques to isolate the desired product. However, it should be noted that although the

132 labelled acyl chlorides were characterised as their corresponding anilides, no mention of the amount of starting amine that was required in the preparation was made. In addition the sequence of operations takes 5 h for completion. The two approaches considered above for the formation of [ l-1^ ] cyclcprcpanecarbonyl chloride both necessitate the removal of the chlorinating agent prior to the distillation of the desired acyl chloride. The technical aspects of the apparatus used for the preparation makes this task impractible. As a consequence large amounts of the starting amine (> 20 mg) are consumed in the preparation. Hence, the results of these findings prompted the use of a high boiling acid chloride as a chlorinating agent, thus avoiding its removal from the f1^ ] reaction mixture. The use of a higher boiling acid chloride R 1C0C1 for the preparation of a lower boiling acid chloride R00C1 was first reported by Brown.121 The reaction of an acid chloride with a carboxylic acid probably involves an anhydride intermediate ((Z); Scheme 3.27).

R1C0CI + R2COOH

(Z) A

R1COOH + R2COCI Scheme 3.27 Reaction of an acid chloride with a carboxylic acid

The continuous removal of the lower-boiling acid chloride displaces the equilibrium to the right, hence high yields of the desired acid chloride are obtained. This approach has been applied to the preparation of a number of acyl halides. For example, benzoyl chloride has been used to generate acetyl and propionyl chloride in 80-90% yield from the corresponding carboxylic acid.121 Similarly, acryloyl chloride (70%),122 propionyl chloride (80%)123 and isovaleryl chloride124 have been prepared. Fhthaloyl dichloride (PDC) has been employed in the preparation of acetyl chloride125 and fumaroyl chloride.126

133 It was considered that PDC was an excellent candidate for the required application. Firstly, the large difference in the boiling points of the two acyl chlorides (phthaloyl, 542-544 K; cyclopropanecarbonyl, 392 K) would facilitate their separation. Secondly, the chances of transferring PDC to the second reaction vessel containing the starting amine are la*/. Consequently, [l-1^ ] cyclopropanecarbonyl chloride (7) was generated either from [ l-^C] cyclopropanecarboxylic acid (20) or the [l^C] adduct (10), by the addition of PDC. In both cases, the [11C]acid chloride (7) was isolated from the reaction mixture (Fig 3.8) within 10 min and reacted with IHIQ (26) to give the [11C]amide (33) (Scheme 3.28). Parameters such as the amount of PDC and THIQ needed were varied and the results are presented in Table 3.7. Formation of [-^CJacyl chloride (7) from the [-^-k^acid (20) gave r.c.y of [^^C]amide in the range of 15-45% depending on the quantities of PDC and THIQ used. The estimated yield of [^CJacyl chloride did not change drastically from 50-70% if the amount of PDC was reduced from 700 to 100 mg. However, the r.c.y of [1:LC]amide decreased by 10-15% when the quantity of THIQ was reduced from 30 to 10 mg. This was probably due to residual HC1 in the system protonating the starting amine, thus lowering the amount available for reaction.

Scheme 3.28 Preparation of [11C]cyclopropanecarbonyl chloride with phthaloyl dichloride as the chlorinating agent 134 Interestingly, the direct conversion of the [13C] adduct (10) into the [11C]acyl chloride (7) gave similar r.c.y as obtained via the [1:LC]acid (20). In addition, the minimal amount of starting amine required for optimal conversion into amide (33) was found to be 5 mg. Further reduction in starting amine resulted in lower conversion. [13€] Amide preparation, including preparative hplc purification, was achieved in 45 min from EOB. The [1:LC] amide formed by this approach was found to be radiochemically and chemically pure both by normal and reverse phase hplc and tic. The product was also examined by mass spectrometry. The mass spectrum exhibited a parent peak at Vi/Z = 201 and fragments characteristic of the N -cyclopropanecarbonyl-THIQ system (Sec 3.3.1.3). In order to establish the position of the carbon-11 label in the labelled amide (33), 13C-enriched CO2 (90 atom%) was co-included in the radiosynthesis. The isolated mixture of ^ c - and 13C-labelled amide was examined by 13C-NMR spectroscopy and the spectrum exhibited a singlet at 5 172.34 (see Fig 4.34 and Table 4.9 for 13C- NMR chemical shifts of reference compound 30). This peak corresponds to the carboxamido-carbon of N-cyclopropanecarbonyl-THIQ (natural abundance) as described in Sec 4.3.3.3. This observation corroborates the findings of Mazur et al.101 They reported that, in the reduction of [l-14C]cyclopropanecarboxylic acid to the corresponding [14C]alcohol, the 14C-label is not scrambled. Furthermore, the preparation of [ 1-14C]cyclopropanecarboxylic acid from cyclopropanemagnesium bromide similarly shows no scrambling of the 14C-label.101 This procedure provided N- [ 1-11C] cyclopropanecarbonyl-THIQ (33) in good r.c.y, high radiochemical purity and without the deliberate addition of carrier.

135 3.2.3.1 Reduction of T^ CIAmide The principal reducing agent employed in the reduction of amides is LiAlH4 .127-129 Bentley and Hardy84 also utilised this reagent for reduction of the amide, formed by the reaction of cyclopropanecarbonyl chloride with N- (descyclopropylmethyl) - diprenorphine (3) as a synthesis of diprenorphine. The feasibility of this reaction was investigated for the reduction of N- cyclopropanecarbonyl-THQ (29) and N-cyclopropanecarbonyl-THIQ (30; Scheme 3.29).

Reduction of (29) resulted largely in the formation of the starting amine THQ (25), whereas (30) gave the desired amine (34) in 80% yield (Scheme 3.29). The mechanism of tertiary amide reduction is not well understood. However, several theories have been proposed. In summary, the mechanism can be considered as shown in Scheme 3.30.128

136 OM RCONR'2 + HM ^ — R — C — NR’2 H

(AA)

MH RCH2NR'2 + M20 R’2NH + RCHO ------RCH2OH + R'2NH (BB) (CC) (DD)

Scheme 3.30 Mechanism of tertiary amide reduction

It is likely that an O-aluminate complex (AA) is formed which may subsequently react by way of three routes; (a) nucleophilic attack by the hydride reagent on the carbon oxygen bond to form the tertiary amine (BB) ; (b) hydrolysis to convert the intermediate (AA) into the aldehyde (OC) which on further reduction gives the alcohol (DD); (c) nucleophilic attack by the hydride reagent on the carbon- nitrogen bond with subsequent hydrolysis results in the formation of the alcohol (DD). Therefore, reductive deamination to the aldehyde competes with amine formation. It has been suggested that bulky N- substituents favour aldehyde formation by inhibiting lone pair nitrogen electron delocalization and thereby increasing the susceptibility of the carbonyl group to nucleophilic attack.129 This factor may account for the loss of the cyclopropanecarbonyl group in the THQ derivative (29). Micovic and Mihailovic130 have reported that in the case of N-benzoyl-THQ, reduction by HAIH 4 resulted in the formation of the corresponding amine (37-39%), benzyl alcohol (49-53%) and THQ (44-47%). They attributed their findings to steric hindrance on the nitrogen atom.

137 In contrast, under the conditions employed, amine (34) formation was favoured in the reduction of tf-Gyclopropanecarbonyl-THIQ. Thus these conditions were translated to the carbon-11 level. Similarly, it was found that the reduction of N-[l-11C]cyclopropanecarbonyl- THIQ (33) with L1AIH4 resulted in the formation of ^-[l-1^]- cyclopropylmethyl-THIQ ((35); Scheme 3.31).

Scheme 3.31 Reduction of N-[11C]cyclopropanecarbonyl-THIQ with LiAIH4

Optimal conversion of the [1:LC] amide (33) into amine (35) was achieved when the reaction mixture was heated for 3 min and r.c.y's in the range of 15-20% were obtained from 11002* Important to note is that under the reaction conditions employed, the carbon-11 label is not expected to scramble. This expectation is supported by findings of Mazur et al .101 They found no scrambling in the preparation of [ 14C] cyclopropanemethylamine by LiAlH4 reduction of [l-14C]cyclopropanecarboxamide. As a direct consequence of these promising developments, the next stage, labelling of the target molecules (8 ) and (9), was undertaken.

138 3.2.4 Carbnn-n Tahe>1liner of Diprenorphine (8 ) and Buprenorchine (9) The above discussion details the development of the labelling agents (5), (6) and (7) considered for the labelling of target molecules (8 ) and (9) with carbon-11. Here, the synthetic routes leading to these labelling agents are briefly compared, in terms of r.c.y, ease of preparation, reprcducibility and position of the carbon-11 label in the molecule. Under the experimental conditions investigated, poor r.c.y's were obtained for [ 1-^C] cyclopropanecarboxaldehyde (5). In addition, the procedural operations were complicated and further oxidation to the corresponding acid occurred. In the case of cyclopropylmethyl iodide, the extent of side-reactions leading to rearranged product were difficult to control and the label would be scrambled. However, if this labelling agent can be produced in high radiochemical purity, this approach has the advantage that the target product can be prepared in a single step reaction. The most successful approach was the synthesis of [1-1:LC]- cyclopropanecarbonyl chloride (7). The procedure applied is short, the labelling agent is obtained in high r.c.y with good specific activity and in high radiochemical purity. In addition, the cyclopropanecarbonyl system is specifically labelled in the 1- position. Consequently, [1-^C]cyclopropanecarbonyl chloride was chosen for the labelling of the target molecules (8 ) and (9).

3.2.4.1 r-*^lDiprenorphine (8) T h e synthesis of [^CJdiprenorphine from N-(des- cyclpropylmethyl) diprenorphine (which here for simplicity we shall call NDHJ (3)) is shown in Scheme 3.32. [1-^^C]Cyclopropanecarbonyl chloride (7) was generated from the adduct (10) by the addition of PDC as described Sec 3.3.2.5. The reaction of this [-^--kljacyl chloride with NDRT (3) occurred readily and the [1:LC]amide (36) was obtained in 55-65% yield. However, since the starting amine contains a phenolic hydroxy group, formation of the [^C]ester (37) was noticed as a side reaction. Generally the radiochemical yield of f1-^]ester was 20%. The addition of a hindered base (i.e. DTBP)

139 along with NDFN in the second reaction vessel (Fig 3.9) always gave [i^jester as major product (> 60%); therefore NDFN (ca. 2.5 mg) was used as its own base in this reaction.

o

Scheme 3.32 Reaction of [1-11C]cyclopropanecarbonyl chloride with NDPN followed by reduction with LiAIH4

The formation of the [^<2]amide (36) was established by 13C-NMR study. Thus the radiosynthesis was performed with [11C/13C]CX)2 (90 atom %) and the product purified by preparative hplc. The

140 radioactive peak having similar retention time to a structurally related compound(38) (Fig 3.3) was collected (reference amide was not available, therefore retention time was estimated by inference) and examined by 13C-NMR spectroscopy. The spectrum exhibited a single peak at 8 172.0. Chemical shifts of carboxamido-carbons in N- (cyclopropyl)amides are very similar (Fig 3.3). Therefore by comparison the singlet at 8 172.0 was assigned to the carboxamido- carbon in the amide derivative of diprenorphine (36). Additionally, this confirms the position of the 13C-label (by analogy 11C) to be on the carbonyl carbon. The reduction of amide (36) with IiiAIH4 generally resulted in the formation of [1:LC] amine (8 ). However, [ l-1-^] cyclopropane- methanol (11) was found to be produced in this reaction. Several sources are available for the formation of this species, for example, the reduction of any formed [^^C] ester (37). More importantly, as described in Sec 3.2.3.1, aldehyde formation and subsequent reduction to the [ 1:LC] alcohol is expected to be prevalent when the IV- substituents are bulky.139'130 Gaylord139 has commented that if deficient amounts (i.e. less than 0.5 equivalent) of LiAlH4 are used at low temperature (203-273 K) and if the hydride is added to the amide rather than vice versa, aldehyde formation is favoured. In N- heteroaromatic amides it has been noted that reductive deamination to a mixture of aldehyde and alcohols occurs under these conditions.139 Optimal conditions for the formation of the [13C] amine (8 ) were found to be the addition of an excess amount of LiAlH4 (0.32 mmol) and heating the reaction mixture for 3 min at 338 K. longer heating resulted in lowering of the f1^]amine yield. It should be noted that even under optimal conditions reduction of f1^] amide (36) sometimes resulted in the formation of [11C]cyclopropanemethanol (11) as major product. As a consequence other reducing agents such as borane and borane-methyl sulphide were investigated in the reduction of the model amide (30). Although both reducing agents gave the desired amine (34), other impurities were found in the product and difficulties were observed in the removal of excess agent. However time did not allow their full evaluation.

141 COMPOUND s (ppm) (amido-carbon)

R = cyclo Pr 172.4

(30)

R = Ph 172.5 RNH CO R = p-N02Ph 172.4

HCL ^

'N -C O < ^| 172.4

Me°TMe' ✓ (38) s c \ HO Me

172.0

Fig 3.3 Data from FTB{1 H} NMR (22.5 MHz; CDCI3 at ambient temp, with TMS as internal standard.

142 Isolation of the [1:LC] amine (8 ) from the reaction mixture was achieved by decomposing the excess L 1AIH4 by the addition of dry methanol. Decomposition with methanol had the advantage that a homogeneous solution was obtained at the end of reaction, which was then passed through a series of silica gel Sep-paks to remove the inorganic salts formed. Inis allows easy adaptation to remote handling. To be able to achieve this manipulation was extremely important since clinical preparations would require high starting activity of H c o 2 (> 3.7 x 1010 Bq). Ihe rate at which activity was passed through the Sep-pak had to be finely controlled to ensure sufficient contact between silica gel and the reaction mixture and thereby efficient removal of inorganic salts. Final purification of the [^C] amine (8) was achieved by normal- phase preparative hplc. Normal and reverse phase tic and normal phase hplc demonstrated that the radioactive product obtained from this synthetic route co - migrated with authentic diprenorphine (1) and was both radiochemically and chemically pure. The position of the carbon-11 label was determined by performing the radiosynthesis with [iic/13C]C02 and the isolated product was examined by 13C-NMR spectroscopy. The spectrum exhibited a single peak at 5 59.9 attributable to a methylene group (multiplicity was established by an appropriate spin-echo gated decoupling technique). This chemical shift is characteristic of the N-methylene group of the cyclopropylmethyl system in diprenorphine and similar derivatives, where the chemical shift range is 5 59.1-59.8 (Fig 3.4). By analogy, the singlet at 5 59.9 was assigned to the methylene group at C23 (^CH^R1; where R'= cyclopropyl) in diprenorphine (Fig 3.2). It should be noted that when the cyclopropyl moiety is replaced by a different substituent (i.e. R'= H or CH=€H2; Fig 3.4) the chemical shift of the =N-CH2R ’ carbon is displaced upfield. Consequently, the singlet at 5 59.9 confirms that the carbon-13 label (and by analogy carbon-11) is positioned on the ^-O^R' carbon, where R' is the cyclopropyl ring. The next stage in the development of this labelled ligand was the transfer of the chemistry to a remote handling system where high

143 COMPOUND CH2R' S (ppm)

R' = CH = CH2 57.52 CH2R' 59.12

CH2R' 59.42

OH

R = H, R’ = 59.82 R = Me, R' = H 43.42

[13C] Diprenorphine R = H, R' = 59.91,3

Fig 3.4 1) Data from FT 13C {1H} NMR (22.5 MHz; CDCI3) at ambient temp. with TMS as internal standard; 2) Data obtained as in (1) but at 25.03 MHz. From Carroll et al131 .3) Shown to be a carbon atom of CH2 -group by an appropriate spin - echo gated decoupling technique. 144 starting activities of 11G02 could be used, thus providing sufficient amounts of [ ^-C ]diprenorphine for human PET studies. Due to the renewal of the MRC cyclotron and PET scanner, this work was carried out on visits to Commissariat a l'Energie Atomique, Service Hospitalier Frederic Joliot (Orsay, France). To adapt the procedure to remote control further required the design of apparatus that could be easily used to transfer reactants and reagents without manual manipulation. This necessitated the scaling down of the quantities of reagents used in the preparation. A schematic representation of the "semi-automated system" used for the preparation of [11C]diprenorphine is shown in Fig 3.10. The apparatus was installed (by P.L Horlock) into a 2 inch lead hot cell and the sequence of operations was manually operated by a central control unit comprising of three rotary switches. The "semi-automated" system provided [ ^C] diprenorphine in 10-30% r.c.y in 57-63 min from EOB. The samples were routinely analysed by hplc and tic to ensure radiochemical and chemical purity. The purified sample was formulated for human intravenous injection. Typically, 0.56-1.7 GBq (15-47 mCi) of [1:LC] diprenorphine was produced from 44-55 GBq (1.2-1.5 Ci) of 11002. The specific activity of [ ^C] diprenorphine in twenty preparations was found to be in the range of 37.2-85.6 GBq//zmol (1.0-2.3 Ci//imol) at EOB or 5.6-11 GBq//jmol (150-300 Ci/^mol) at EOS. The amount of carrier (stable diprenorphine) in these preparations varied between 47 and 606 nmol (20 and 258 fig). The level of "hot" and "cold" diprenorphine permitted for a single intravenous injection into a human subject was set in the range of 0.19-0.37 GBq (5-10 mCi) and 0.3-0.5 fig/kg, respectively. The above procedure provided adequate levels of activity and acceptably low amounts of carrier for initial investigations of the opiate receptor system in man by feh?.132'133 Subsequent to this work Lever et al134 have reported the preparation of [^C]diprenorphine. The procedure involves the selective ’ 0 -alkylation of 3- (o-t-butyldimethylsilyl) -6- (O-desmethyl) diprenorphine (39) with [ 11C] iodomethane, followed by desilylation.

145 This provides [ 1]-C]diprenorphine labelled at the 6-methoxy position, in 10% r.c.y (Scheme 3.33).

(39) Scheme 3.33 Selective O-alkylation of (39) with [11C]iodomethane

Although the radiosynthesis is a single-step process, the formation of 3-(O-t-butyldimethylsilyl)-6- (O-desmethyl) diprenorphine (39) requires nine-step synthesis from thebaine. Additionally this method does not yield as high a degree of carbon-11 labelling as the [l-^C]cyclopropanecarbonyl chloride route.

146 3.2.4.2 fi:1ClBuprsnon]hine (9) The synthetic route developed for the preparation of [^C]- diprenorphine was also applied to the synthesis of [ 1:LC] buprenorphine (Scheme 3.34).

N11CH2

(4) (40) (9)

Scheme 3.34 Synthesis of [11C]buprenorphine with [1-11C]cyclopropanecarbonyl chloride as the labelling agent

T h e preparation involved the reaction of N (des- cyclopropylmethyl) buprenorphine (NBFN, 4) with [ l-^C] cyclopropane- carbonyl chloride (7) to give the [^C]amide (40). Subsequent reduction with LiAlH4 resulted in the formation of [^C]buprenorphine (9). Chemical and radiochemical purity of the isolated product (separated by preparative hplc) was established by analytical hplc and tic. The product was found to be radiochemically pure and only minor chemical contamination was detected by hplc. The preparation was carried out in the "semi-automated system" developed for [11C]diprenorphine. [H-c] Buprenorphine was produced for i.v. injection in 57 min from EOB and in ca. 20% r.c.y. The specific activity of [ 11C] buprenorphine was dictated by the specific activity of the labelling agent, [l-11C]cyclopropanecarbonyl chloride. Therefore, it was envisaged that [H-c]buprenorphine could be produced routinely with a specific activity comparable to [ He ] diprenorphine.

147 3.3 EXPERIMENTAL AND RESULTS 3 .3.1 Stable Chetnigtrv

3.3.1.1 Preparation of Cylccaxpanprarbnxald^ivde Procedure A : silver-catalysed oxidation (adapted from Pike et al)135 A mixture of cyclpprcpanemathanol vapour (0.5 g, 6.9 mmol; generated by distilling at 438 K), and air was passed (flow rate 80 ml min-1) into an electric furnace containing silver wool catalyst (1 g; Prolabo) distributed over 3.5 cm in a quartz tube (length 10 cm; i.d. 0.7 cm) heated to 643 K. Before each manipulation, the catalyst was "activated" by exposure to cyclopropanemethanol (0.5 g) in a current of air (80 ml min-1) at 643 K. The products formed were collected in a reaction vessel containing either dichloromethane (2 ml) or water (2 ml) both at 273 K. The dichloramethane fraction was further worked up by drying over anhydrous magnesium sulphate followed by distillation. A colourless liquid (yield 0.26 g, 56%) was obtained; b.p. 371-374 K (760 mm Hg); Lit. 370-372 (760 mm Hg),114 370-373 K (740 m m Hg) ,88 XH-NMR analysis showed the presence of minor impurities (cyclopropanemethanol and dichloramethane, < 10%).

Characterisation - -^H-NMR (60 MHz, CDC13): S 1.0 (2H, H ^ ) , 1.05

(2H, H 2b ) t 1*8 (1H, m, %), 9.0 (IH, d, CHO). Values in agreement with Lit.116

Dimedone D erivative The crude product collected in water (2 ml) was added to an alcoholic solution of dimedone {5,5-dimethyl-l,3-cyclohexadione; 2.0 g, 14.3 mmol; dissolved in methanol (4 ml) and diluted with water (16 ml)). The mixture was heated at 373 K for 10 min, cooled and filtered. A white crystalline solid was obtained; m.p. 447-449 K, Lit 449-449.5 K .114

148 Procedure B: oxidation with CAN (adapted from Young and Trahanovsky)88 Cyclopropanemethanol (0.8 g, 11.1 mmol) was added to a solution of ceric ammonium nitrate (CAN, 13.5 g, 24.6 mmol) in water (25 ml). The resulting deep red solution was heated on a steam bath until colourless (10-15 min). The mixture was cooled, saturated with sodium chloride and transferred to a separating funnel. Water (40 ml) was then added and the crude product extracted into CH2Cl2 (4 x 25 ml). The combined CH2Cl2 fractions were dried over a mixture of magnesium sulphate and sodium bicarbonate. The filtered solution was then reduced to 10 ml by rotary evaporation. This residue was then distilled and the product collected into a receiver cooled in an ice- acetone bath. A colourless liquid (0.53 g, 68%) was obtained (b.p. 371-373 K at 760 mm Hg, Lit 370-372 K at 760 mm Hg114), which contained CH2C12 (< 8%) as shown by %-NMR.

Characterisation - %-NMR (80 MHz, CDC13): 8 1.04 (2H, H ^ ) , 1.12

(2H, H2B), 1.81 (1H, m, H 3), 8.92 (1H, d, CHO; 3Jc h O C H = 5 -6 Hz) and 5.32 ppm (CH2C12 impurity). 13C-NMR (^-H-decoupled, 20.12 MHz,

CDCI3 ) : 8 7.34 ( C ^ + b ), 22.72 (Cx), 201.73 (CHO). Values in agreement with Lit.137

3.3.1.2 Preparation of cvclocaxpvlmethvl iodide (13) Procedure A To cyclopropanemethanol (1.8-3.6 g, 25-50 mmol), constant boiling hydroiodic acid (4.7-12.7 g, 37-100 mmol of 57% solution in water) was added with stirring. Reaction temperatures and conditions are given in Table 3.3. On completion of reaction, the reaction mixture was worked-up in the following manner. To ease separation, water was added and the crude product was extracted into chloroform (2 x 20 ml). The combined CHCI3 extracts were further washed with hydrochloric acid (2 M, 2 x 20 ml), water and then successively with 10% solution of sodium thiosulphate and water. The resulting CHC13 extract was dried over anhydrous magnesium sulphate and the solvent carefully removed by rotary evaporation. The residue was distilled

149 giving a pale orange liquid (b.p. 403-405 K at 760 ram Hg) in quantitative yield. The results are presented in Table 3.3.

Characterisation - %-NMR spectra (60 MHz, CDCI3) showed a broad multiplet at 5 0.2-1.7 (5H, cyclopropyl protons) and a sharp doublet at 5 3.05-3.15 (CH2-I). Chemical shifts of isomeric iodides were also present at 8 1.95-2.30 (m), 8 2.35-2.9 (m) and 8 4.3-4.8 (quintet) due to cyclobutyl iodide and at 8 4.9-6.2 (complex multiplet) due to vinyl protons. These values were in agreement with literature.138

Table 3.3. Ratio of products obtained in the reaction of cyclapropanemethanol and HI.

Compound HI Reaction Reaction Product Composition3 (12) temp. time (13) (14) + (15) (mmol) (mmol) (K) (min) (%) (%)

50 100 408 5 62 38

25 37 298 10 73 27

25 37 298 5 78 22

25 37 273 25 65 35

25 37 267 5 77 23

a) Percentages estimated from %-NMR spectra (60 MHz, CDCI3) by integrated signal intensities. Errors estimated as ± 10%.

Procedure B: treatment with P2I4 (adapted from Denis et al)107 Cyclopropanemethanol (100 mg, 1.40 mmol) was added to a stirred solution of diphosphorous tetraiodide (3.2 g, 5.6 mmol) in carbon disulfide at 273 K under nitrogen (P2I4 was recrystallised

150 and washed with small quantities of CS2 before use; product stored under nitrogen). The reaction mixture was allowed to stir at ambient temperature for 12 h during which period an orange precipitate was formed. A saturated solution of potassium carbonate was then added and the crude product extracted into diethyl ether. After drying over magnesium sulphate and filtration, the filtrate was reduced to a volume of ca. 5 ml by rotary evaporation. The product was isolated by distillation under nitrogen (yield 57%; b.p. 406-409 K at 760 mm Hg; Lit 361-363 K at 150 mm Hg138).

Characterisation - -^H-NMR (60 MHz, CDCI3) : 5 0.2-1.5 (5H, m, cyclopropyl protons), 3.15 (2H, d, CH2-I). Isomeric impurities (ca. 15%) at 5 4.5, 2.1, 2.65.

In a number of experiments, cyclopropanemethanol (50 /xl, 0.62 mmol) was added to a reaction vessel containing P2I4 (1.4 g, 2.48 mmol) alone or suspended in DTBP (50-250 /xl, 0.22-1.1 mmol) under nitrogen at 273 K. The reaction vessel was sealed and heated for several minutes (see Table 3.4) with stirring. The product under a flow of nitrogen (25 ml min”1) was distilled into a second reaction vessel containing CHCI3 at 273 K. The collected product was analysed on a reverse phase hplc column (30 x 0.39 cm i.d., 10 /xm particle size, 'V Bondapak-C18n, Waters Associates) eluted at 2.0 ml min”1 with a mixture of methanol and water for injection (50:50 v/v). Cylopropylmethyl iodide (13) eluted between 6 .9-7.7 min whereas allylcarbinyl iodide (14) and cyclobutyl iodide (15) had retention times of 7.8-8 .4 and 8 .6-9.3 min respectively. The percentage distribution of isomers is presented in Table 3.4.

151 Table 3.4. Ratio of products obtained in the reaction of cyclcprcpaneaiiethanol and P2 I4 -

Temperature Reaction Amount Product composition0 of vessel time of DTBP (13) (14) (15) (K) (min) (Ml) (%) (%) (%)

423a 15 - 57 20 23

423a 15 50 48 29 23

423a 10 - 65 14 21

423b 10 - 67 18 15

418a 15 200 65 22 13

418a 10 200 67 20 13

393a 15 - 66 17 17

393a 10 250 80 11 9

378a 10 - 79 14 7

378a 5 - 81 12 7

a . P2I4 recrystallised and washed with CS2 before use b. p2I4 used directly without purification c. Estimated from peak area of HP1£ chromatogram

Procedure C : via silyl ether (adapted from Morita et al)108 1. Preparation of trimethylsilyl ether (18): A mixture of cyclopropanemethanol (7.2 g, 0.10 mol) and dry- pyridine (19 g, 0.24 mol) were added dropwise to chlorotrimethylsilane (13.04 g, 0.12 mol) in dry benzene (50 ml) with stirring at ambient temperature. The reaction mixture was heated at reflux temperature (353-363 K) for 2 h, cooled and then filtered to remove the formed precipitate. This was further washed with dry- ether and the combined fractions distilled twice to give a colourless liquid (8.34 g, 58%; b.p. 393-399 K at 760 mm Hg). %-NMR analysis showed the presence of the desired compound (18) contaminated with 152 pyridine (ca. 14%).

Characterisation - ^H-NMR (60 MHz, CDCI3) : 8 0.2-1.3 (m, cyclopropyl ring protons), 3.14 (d, cycloprapylmethylene protons). Inpurity at 8 7.0-7.7 (4H, m), 8.75 (1H, broad doublet).

2. Conversion of trimethylsilyl ether (18) into iodide (13). Without further purification, the prepared trimethylsilyl ether (18) (2.88 g, 0.02 mol) was added to the mixture of chlorotrimethylsilane (3.26 g, 0.03 mmol) and anhydrous sodium iodide (4.5 g, 0.03 mmol) in dry acetonitrile (7.5 ml). The resulting mixture was refluxed (352-353 K) for one hour and then poured with stirring into a saturated solution of sodium bicarbonate (50 ml) followed by deiodonization with sodium hydrogen sulphite. The resultant organic layer was extracted with ether (3 x 25 ml) and the combined extract dried over anhydrous sodium sulphate. The filtrate was then reduced in volume ca. 5 ml by rotary evaporation and this residue distilled to give a pale orange liquid (2.62 g, 72%; b.p. 405-408 K at 760 mm Hg). %-NMR gave expected 8 valves, as described in procedure B.

3. "One-pot" preparation of iodide (13): (adapted from Olah et al)109 To a mixture of cyclopropanemethanol (0.89-1.78 g, 12.3-24.7 mmol) in dry acetonitrile (20 ml), chlorotrimethylsilane (5.4-10.8 g, 0.05-0.1 mol) was added with vigorous stirring. Reaction temperatures and times are given in Table 3.5. The reaction was quenched by the addition of water (20 ml). Ihe crude product was extracted into CHCI3 (2 x 20 ml) which was further washed with water (2 x 20 ml), followed by a 10% solution of sodium thiosulphate (3 x 20 ml) and water (2 x 20 ml). The resulting chloroform extract was dried over anhydrous magnesium sulphate and the solvent carefully removed by rotary evaporation. A pale orange residue was generally obtained which required no further purification (distillation did not Improve the purity of the final product).

153

10

10-15 10-15

andNal. 3 Product(s)a (%) (%) (%> (l3) (l3) + (14 15) 90-95 5-10 85-90 ca.90 85-90 10-15 85-90 (%) 60 65 25 90-95 5-10 75 75 yield Overall ) by) integrated signal intensities. 2 2 3 10 10 20 (min) time Reaction (K) temp. 340-343 5 47.5 340-343 340-343 296-298 273-275 Reaction 3 0.1 0.05 323-326 0.1 0.1 (mol) ClSiMe 25.3 25.3 0.05 25.3 25.3 0.05 25.3 25.3 Nal (mmol) Errors estimated as ± 10%. 12.3 24.7 12.3 24.7 24.7 12.3 a) a) Percentages estimated from %-NMR spectra MHz, (60 CDCL (mmol) Table 3.5. Ratioof products obtained inthe reaction of cyclcprcpanemethanol andClSiMs -methanol Cyclopropane

154 The purity of the compound was estimated from %-NMR by integrated signal intensities. The values were in agreement with those obtained from the analytical reverse phase hplc chromatography (see procedure B for conditions). The overall yields and the percentages of isomeric iodides are presented in Table 3.5.

3.3.1.3 Preparation of N-Cvcloprocanecarfacnvl-THIO (30) Freshly distilled cyclopropanecarbonyl chloride (4.54 ml, 5.22 g, 0.05 mol) in dry diethyl ether (50 ml) was added dropwise to a stirred solution of THIQ (12.5 ml, 13.3 g, 0.1 mol) in diethyl ether under nitrogen. An immediate formation of a white precipitate was observed. On completion of addition, the mixture was refluxed for 1 h, cooled and then successively washed with dilute hydrochloric acid (2 N), water, sodium bicarbonate solution (10%), water and a saturated solution of sodium chloride. The resulting ethereal solution was dried over anhydrous magnesium sulphate and evaporated giving a light orange liquid. The product was isolated by vacuum distillation as a colourless oil, which crystallised overnight at 269 K as a white crystalline solid (yield 7.83 g, 80%; b.p. 420-421 K at 1 mm Hg, m.p. 314-315 K ) .

Characterisation - Found: C, 76.74; H, 7.70; N, 6.77; C^H^NO requires: C, 77.58; H, 7.51; N, 6.96. IR (liq) 1635 cnT1 (broad C=C, stretch). Vi/Z 201 {V&, 100%}, 200 {[M-l]+ , 55%}, 132 ([M-C3H 5C=0]+ , 65%}, 117 {[M-C4PI6NO]'f, 23%), 104 {[ M - C ^ N O ] * , 34%}. For details of % (Fig 4.20 and 4.26) and 13C (Fig 4.34) NMR spectra, see Tables 4.8 and 4.9, respectively.

3.3.1.4 Preparation of N-Cvclcprrx^anecarbcnvl-THD (29} The synthetic procedure used was essentially the same as that described for the preparation of compound (30) . Cyclopropanecarbonyl chloride (4.54 ml, 0.05 mol) in diethyl ether was added dropwise to a stirred solution of THQ (25) (13.3 g, 0.1 mol) in diethyl ether under nitrogen. The product was isolated by vacuum distillation as a colourless liquid (yield 5.3 g, 53%; b.p.

155 430-432 K at 1 mm Hg).

Characterisation - Found: C, 77,31; H, 7.65; N, 6 .8 6 ; C ^ H ^ N O requires: C, 77.58; H,7.51; N, 6.96. TR (liq) 1645 cm-1 (broad G=0 stretch). Hi/Z 201 {M+, 46%), 133 {[M-C2H 3CH=C=0]+ , 100%), 132 {[M-C4H50]+ , 51%), 117 {[M-C5H80]+ , 14%), 104 {17%}, 85 {33%}, 83 {49%}, 69 {C3H 5CO+, 46%}. for and 13C-NMR spectral data, see Tables 4.12 and 4.13, respectively.

3.3.1.5 Preparation of N-C^lopropylrnethvl-'IHIO (34) Procedure A W-Cyclopropanecarbonyl-JIHIQ (30) (5.0 g, 25 mmol) in diethyl ether was added to an ethereal suspension (75 ml) of LiAlH4 (1.42 g, 38 mmol) under nitrogen, at such a rate that the reaction mixture refluxed gently at ambient temperature. It was further refluxed for 1 h at 348 K. Aliquots were taken out every 15 min and analysed by tic: silica gel plates, eluant, hexane and ethyl acetate (80:20 v/v), amide (30) Rf = 0.14; amine (34) Rf = 0.68. On completion of reduction, the reaction mixture was cooled and the excess LiAlH4 decomposed by dropwise addition of ethyl acetate. The resulting mixture was then poured into excess ice-cold dilute sulphuric acid and the suspension made alkaline by the addition of sodium hydroxide (2 N). The crude product was extracted into ethyl acetate, washed successively with water and saturated solution of sodium chloride. The ethyl acetate solution was then dried over anhydrous magnesium sulphate and the solvent removed by evaporation. The product was isolated by vacuum distillation as a colourless liquid (yield 3.27 g, 70%; b.p. 378-380 K at 2.0 mm Hg).

Characterisation - Found: C, 83.26; H, 8.95; N, 7.58; C13H 17N requires: C, 83.42; H, 9.09; N, 7.49. IR (liq) 2700-3070 cm“l (aromatic and alicyclic C-H stretches; 0=0 stretch absent). E/Z i87 {Mt, 57%}, 186 {[M-l]+ , 88%}, 146 {[M-C3H5 ]+ , 100%}, 132 {[M-C4H7 ] + , 32%), 104 {[M-C5H9N]+ , 36%). For XH- and 13C-NMR spectral data, see Tables 4.14 and 4.15, respectively.

156 Procedure B A solution of cyclopropylmethyl bromide (2.78 g, 20 mmol) in ethanol (10 ml) was added to THIQ (26) (2.46 g, 18 mmol) in ethanol containing sodium bicarbonate (1.6 g, 20 mmol). The reaction mixture was refluxed for 2 h, cooled and filtered. Excess ethanol was removed by evaporation and the residue was taken up in water. The pH of the solution was adjusted to 11-12 with c. ammonia (d = 0 .88). The crude product was extracted into CHCI3 and successively washed with water and a saturated solution of sodium chloride. After drying and removal of solvent, the product was isolated by vacuum distillation as a colourless liquid, yield 2.1 g (62%); b.p. 400-402 K at 5 mm Hg.

Characterisation - Found: C, 83.26; H, 9.35; N, 7.29; C13H 17N requires: C, 83.42, H, 9.09; N, 7.49. % - and 13C-NMR spectral data identical to that for the product obtained by procedure A.

Procedure C (adapted from lane)139 A solution of borane in THF (1 M, 25 ml, 25 mmol) was added dropwise to N-cyclopropanecarbonyl-THIQ (30) (1 g, 4.97 mmol) in THF 925 ml) with stirring under nitrogen at 273 K. The resulting solution was refluxed for 1 h. curing this period, formation of a white gel-like material was observed. The reaction was followed by tic (conditions as in procedure A). On completion of reduction , the reaction mixture was cooled and excess borane decomposed by the slow addition of methanol (50 ml). Anhydrous hydrogen chloride was then bubbled through the reaction mixture at 273-277 K, until a pH of ca. 2 was obtained. The resulting green-yellow solution was further refluxed for 1 h, cooled and the solvent removed by evaporation. The yellow residue was then taken up in water (required warming) and the pH adjusted to ca. 10 by the addition of sodium hydroxide pellets. The aqueous solution was then saturated with sodium chloride and the crude product extracted into ether. After drying and evaporation of solvent, the product was isolated by vacuum distillation as a colourless liquid; yield 520 mg (56%); b.p. 399-401 K at 5 mm Hg.

157 For assignment of % - and 13C-NMR signals see Tables 4.14 and 4.15, respectively.

Characterisation: %-NMR (80 MHz, CDCI3), 8 0.05-0.7 (m, Hlla+b), 0.7-1.2(m, H10), 2.42 (d, Hg), 2.65-3.05 (m, H 3 + H4), 3.72 (s, H5- H8); 1.4-2.0 (m, impurity, 8%), 3.3-3.65 (m, impurity, 6%). 13C-NMR (20.12 MHz, CDCI3), 8 3.96 (Clla+b), 8.57 (C10), 28.89 (C4), 50.89 (C3), 56.01 (C-^), 63.39 (Cg), 125.6-134.83 (aromatic carbons); impurities present at 8 29.05, 29.93 and 44.85.

Procedure D (adapted from Lane)-1-39 The synthetic procedure used was essentially the same as that described in procedure C. A solution of borane-methyl sulfide in CH2CI2 (1 M, 25 ml, 25 mmol) was added dropwise to N-cyclopropane- carbonyl-THIQ (1 g, 4.97 mol) with stirring under nitrogen at 273 K. During reflux, the appearance of a white gel-like material occurred although much later than in procedure C. Attempts to isolate the product after work-up, by vacuum distillation resulted in the formation of a white solid, thus only a fraction of the entrapped liquid could be distilled. Colourless liquid was obtained, 290 mg (31%); b.p. 396-397 K at 5 mm Hg.

Characterisation - %-NMR (80 MHz, CDCI3), 8 0.05-0.7 (m, % i a+b) / 0.7-1.3 (m, H10), 2.4 (d, Hg), 2.6-3.05 (m, H3 + H4), 3.7 (s, Hx), 7.05 (s, H 5-H3 ); 1.35-1.95(m, impurity, 9%), 3.15-3.6 (m, impurity, 13%). 13C-NMR (^-decoupled, 20.12 MHz, CDC13), 8 4.03 (Clla+b), 8.65 (C10), 28.97 (C4), 50.99 (C3), 56.09 (Cx), 63.49 (Cg), 125.71-134.9 (aromatic carbons); impurities present at 5 29.15, 30.03, 44.96, 58.58, 61.76 and 62.31.

158 3.3.1.6 Preparation N-Ovcloprocr/Impthyl-TTO The synthetic procedure used was essentially the same as that described for the preparation of compound (34) (procedure A). N- cyclopropanecarfoonyl-JITiQ (29) (5.0 g, 25 mmol) in diethyl ether was added to an ethereal suspension of LiAlH4 (1.42 g, 38 mmol). The reaction mixture was worked-up as described for compound {(34); procedure A}. %-NMR analysis of the isolated product showed the presence of THQ (25) (Table 4.16) only.

159 3.3.2 Carbcn-11 Chemistry 3.3.2.1 Preparation of c^lopropaTipmrxin^siuin bromide (19) The reaction vessel (labelled as 5 in Fig 3.5), air condenser (labelled as 3) and magnesium turnings were left in an oven at 393 K for approximately 12 h and assembled while hot as shown in Fig 3.5. Air was purged from the system by a continuous flow of dry nitrogen (20 ml min'"1) for half an hour. Flow was then reduced to 2-3 ml min-1. To reaction vessel 5 (Fig 3.5) containing magnesium turnings (50 mg, 2.0 mmol), a solution of cyclcpropyl bromide (121 mg, 10 mmol) and a crystal of iodine in sodium dried diethyl ether (6 ml) was added under nitrogen. The reaction mixture was allowed to stand for 5 min (allows etching of magnesium turnings) and on stirring an immediate loss of iodine colouration was observed. The resulting mixture was further stirred for 1 h at 310 K to allow completion of cyclopropanemagnesium bromide formation. At the end of this period, reaction vessel 5 was cooled and the product sealed under nitrogen (by K-75 three way taps, labelled as 2 in Fig 3.5). In all preparations this reagent was filtered (Millex PG, pore size 0.22 ^m) before use.

3.3.2.2 Preparation of r11ClcvclopropanecarbQxvlic acid (20) The apparatus used for this synthesis was similar to that shown in Fig 3.6 for the preparation of [ 11C] cyclopropanemethanol. Cyclopropanemagnesium bromide (0.5 mmol) in diethyl ether (3 ml) was placed into vessel A under nitrogen. Cyclotron produced 11002 in N2 was trapped onto a molecular sieve trap at a flow rate of 500 ml min”1 at ambient temperature (the trap was always pre-conditioned before use by keeping it under vacuum at 673 K in order to remove "carrier"). The activity was released in nitrogen (5 ml min”1) by heating the molecular sieve to 513 K. The Grignard reagent was carbonated for 2 min at ambient temperature. The f1^]adduct (10) was then hydrolysed with hydrochloric acid (0.8 ml, 6 M) with vigorous stirring. The two phases were allowed to separate and the aqueous layer was withdrawn from vessel A. The t1^]product was

160 > N2 out

> O > < - o Mg(CI04)2

f f l m

Fig 3.5 . Schematic representation of apparatus used for the preparation of cyclopropanemagnesium bromide: 1. needle ualue; 2. K75 threeway nylon taps; 3. air condenser; 4. liquid paraffin; 5. reaction uessel containing Mg turnings; 6. H20 bath; 7. stirrer-heater; 8. hamilton syringe containing cyclopropyl bromide, crystal of l2 in Et20. PTFE tubing (o. 4 mm) used for all connections. extracted as the sodium salt by the addition of solution of sodium bicarbonate (1.26% w/v; 5 ml) to vessel A. Hie isolated aqueous layer containing the [i:LC]acid (20) was further heated at 373 K for 5 min under a stream of nitrogen (to drive off any 11C02), cooled and analysed by hplc and tic. The preparation of [ 11C] cyclopropanecarboxylic acid (20) takes 8-10 min from the end of radioisotope production and gives a r.c.y of ca. 80-90% from 11002 (decay corrected).

Analysis of f 11C1 cvclooropanecarboxvlic acid (20) The radioactive product was analysed by reverse-phase hplc (30 x 0.39 cm i.d. ? Partisil 10-SAX) eluted at 2.0 ml min”1 with sodium phosphate buffer (pH 3.85). The eluant was monitored continuously for radioactivity and refractive index. A single radioactive peak, with the same retention time (11.95 min) as authentic cyclopropanecarboxylic acid was obtained. The radioactive product was also analysed by two different tic systems: system A, KC1SF reverse phase plates (Whatman) developed in Et0H:H20 (80:20 v/v), Rf = 0.77? system B, cellulose plates developed in CH3C N :H 20 :c .NH40H (8:1:1 v/v), Rf = 0.19 or (CH3)2C0:H2O:c.NH4OH (8:1:1 v/v), R f = 0.39.

3.3.2.3 Preparation of T11C1 cvclcoropanemethanol (11) The apparatus used for the preparation of [ 11C] cyclopropane- methanol is shown in Fig 3.6. A filtered solution of cyclopropanemagnesium bromide (0.5 mmol) in diethyl ether (3 ml) was placed into vessel A under nitrogen. The trapped 11002 (see Sec 3.3.2.2 for trapping of 11002 onto molecular sieve trap) in nitrogen was dispensed into the Grignard reagent at a flow rate of 5 ml min”1 for 2 min at ambient temperature. L1AIH4 (38 mg, 1 mmol) in diethyl ether (1 ml) was added to vessel A followed by neat diethyl ether (500 /il). The reaction was quenched by the addition of water (1 ml) or hydrochloric acid (1 ml, 6 M). The two phases were allowed to separate and the aqueous layer was withdrawn from vessel A. A solution of sodium bicarbonate (1.26% w/v, 5 ml) in water for

162

Soda lime cgclo C3H5MgBr Et20 in o 3 ualue Mg(CI04)2 Needle I N2 m N2 Reaction oessel R t Soda lime Poppet Paine 1) Cyclotron produced 11C02 collected Cyclotron 1) onto produced 11C02 molecular sieue trap; 3) Addition of LiAIH4 in Et20, 2min, Et20, follouiedin by HCI; 3) of Addition LiAIH4 2) Trapped nC02 dispensed Trapped 2) 2min; nC02 Et20, in into cyclo.C5H5MgBr 4) Withdrawal of ether and aqueous phase. 2 n 2/ N N jn Ualue position for each operation indicated by number. / x 3.6 Fig Apparatus used for the preparation of P’Clcyclopropanemethanol co from target 11

163 injection was then added to vessel A with vigorous stirring. The phases were allowed to separate and the aqueous layer was withdrawn from vessel A. This procedure was repeated twice using water for injection (2 x 5 ml). The residual ether layer containing [ 11C] cyclopropanemethanol (11) was then measured and further analysed by hplc and tic. The preparation of [i:LC] cyclopropanemethanol (11) takes 10-12 min from EOB and gives ca. 70-80% r.c.y from 1:lC02 (decay corrected).

Analysis of r 11C1 cyclopropanemethanol (11) Aliquots of the ether layer containing the radioactivity were analysed on a reverse-phase hplc column {30 x 0.4 cm i.d; Partisil 10-SAX, eluted at 2.0 ml min'”1 with sodium phosphate buffer (pH 3.85)}. The eluant was monitored continuously for radioactivity and refractive index. Analysis showed a single radioactive peak eluting at the same retention time as reference cyclopropanemethanol (4.5 min). The radioactive ethereal layer was also analysed by two different tic system: system A, KC18F r e v e r s e phase plates (Whatman), developed in EtOH:H20 (80:20 v/v), Rf = 0.79; system B, cellulose plates developed in Q^Qh^Ckc.NH^H (8:1:1 v/v), Rf = 0.95.

3.3.2.4 Preparation of r 11C1 cyclooirpanecarbaxaldehvde (5) Procedure A: silver catalysed oxidation (adapted from Pike et al)135 The apparatus used for this preparation is depicted in Fig 3.7. [ 11C] Cyclopropanemethanol (11) was prepared as described in Sec 3.3.2.3. This was released from the formed [11€]adduct (10) by the addition of water (350 /xl). Vessel A (Fig 3.7) was then heated to ca. 338 K for 5 min to remove excess diethyl ether under a flow of nitrogen (20 ml min”’1) and was then transferred to an oil bath at 438 K. [ 11C] Cyclopropanemethanol vapour (together with H 20) was carried in a stream of nitrogen (80 ml min-’1) through a column of "Porapak P" (60-80 mesh) and calcium chloride, then onto the silver wool catalyst (1.0 g) at 653 K. The catalyst had been pre-activated

164

11C02/N2 in 11C02/N2 3. 3. 338K; 4. 438K Fig 3.7 Fig Apparatus used for the preparation of P’Clcyclopropanecarbonaldehyde. Ualue position for each operation indicated by number. dispensed 2min,2min, nC02 1) 293K; Et02, followed 2) in Addition of UAIH*, into3) Distillation cyclo.C3H5MgBr of at by 338K; H20; Et20 4) ["CjCyclopropanemethanol distilled at 438K ouerwool Hg catalyst at 653k.

165 at 643 K by exposure to an air-cyclopropanemethanol mixture formed by distilling the alcohol (0.5 g) in a current of air (80 ml min”1). The system was then thoroughly flushed with a stream of N2. The labelled products were collected in vessel B containing water (0.5 ml) at 273 K. The radiochemical yield of [11C]cyclopropanecarboxaldehyde (5) was evaluated after the addition of "carrier" (stable product, 0.1 g, 1.43 mmol) by selective precipitation with dimedone as described in Sec 3.3.1.1. The r.c.y's were found to be in the range of 2-8% from 11C02/ corrected for decay.

Procedure B: oxidation with ceric ammonium nitrate [11C]Cyclopropanemethanol (10) was prepared in situ as described in Sec 3.3.2.3. To this a solution of ceric ammonium nitrate (0.66 g, 1.2 mmol) and cyclopropanemethanol as "carrier" (40 mg, 0.55 mmol) in water (1.5 ml) was added. The red-orange reaction mixture was heated to ca. 353 K for 5 min and a colourless solution was obtained. The vessel was cooled, CH2C12 (5 ml) was added followed by a saturated solution of sodium chloride with vigorous stirring. The two phases were allowed to separate and the aqueous layer withdrawn from the vessel. The procedure was repeated twice more with water for injection (2 ml). The CH2C12 fraction containing the radioactivity was gently heated under a flow of nitrogen to remove solvent. The radiochemical yield of [11C]- cyclopropanecarboxaldehyde (5) was determined by either the formation of a dimedone or 2 ,4-dinitrophenylhydrazone derivative.

Dimedone Derivative The radioactive residue left after evaporation of CH2C12 was taken up in water (0.5 ml) and an alcoholic solution of dimedone (210 mg, 1.5 mmol; dissolved in methanol (500 fil) and diluted with water (2 ml ) } was added with stirring. The formed precipitate (due to the addition of carrier) was filtered and the activity measured. The [^C]dimedone derivative was formed in 6-10% r.c.y (decay-corrected from 11002) .

166 2,4-DinitrqphenyIhydrazcne Derivative A solution of 2 ,4-dinitrcphenylhydrazine {200 mg, l mmol; dissolved in methanol (5 ml) and C.H2SO4 (0.5 ml)} was added to the radioactive fraction with stirring. Hie resulting mixture was filtered and the solid washed with methanol (2 ml). The radioactivity in the resulting precipitate was measured showing a r.c.y of 8-10% (decay-corrected from 11CC>2) •

3.3.2.5 Preparation of fcvclopropanecarbcnvl chloride

Procedure A: thionyl chloride used to generate (7) Cyclopropanemagnesium bromide (19) was prepared as described in Sec 3.3.2.1. A filtered solution of (19) (0.5 mmol) in diethyl ether (3 ml) was placed into vessel A (Fig 3.8) under nitrogen. The trapped 1-LCC>2 ^ nitrogen was then passed through the reagent at a flow rate of 5 ml min'"1 for 2 min at ambient temperature. Thionyl chloride (120 mg, 1.38 mmol) alone or containing hydrogen chloride (0.52 mmol; generated by the addition of water (5 ^1) to thionyl chloride} in diethyl ether (0.9 ml) was added to the f1^]adduct (1 0 ). The reaction mixture was allowed to stir for 5 min under nitrogen at 298 K. Vessel A was then transferred to an oil bath to remove excess thionyl chloride, HC1 and diethyl ether, either between 363-373 K under a flow of nitrogen or under reduced pressure (10 mm Hg) at 313 K. The residue containing the [1:LC]acid chloride (7) was treated in the following manner. a) "One-pot” synthesis: To vessel A (Fig 3.8) aniline (23), 4-benzylpiperidine (24) or THIQ (26) (0.15-0.5 mmol) in diethyl ether (5 ml) was added and the resulting mixture warmed (328 K) for 5 min to promote f1^] amide {(31), (32) or (33)} formation. Hplc solvent {Na2HP04 (0.025 M) in a mixture of methanol and water (60:40)} was then added with stirring and the phases separated for measurement and analysis.

167

Fig 3.8 Fig Rpparatus used under for the or (C0CI)2, preparation Ualue position3a) E120 Remoual & ofof for ["Cjcyclopropanecarbonylor eachPDC; eacess operation MCI or (C0CI)2 Et20 chloride in S0CI2, indicated 2) HCI by & dispensed number. or 2min, S0CI2 [uC]C02 additionEt20 in 1) in Et20, 293K; into of cyclo.C3H5MgBr S0CI2 N2 or 3b) ReducedN2 pressure; 4) [nC]Cyclopropanecarbonyl chloride distilled into uessel under or reduced C N2 pressure.

168 b) 1'Two-pot" synthesis: Vessel A (Fig 3.8) was placed into an oil bath (438 K) and the [11C]acid chloride (7) was distilled under a flow of nitrogen (20-25 ml min”1) or under reduced pressure (2.5 mm Hg) at 313 K into vessel C containing an amine (0.15-0.37 mmol) in THF at 203 K. The resulting mixture was then heated for 10 min at 328 K and analysed as described below.

Analysis of f1^ ! amide The radioactive products (32) and (33) obtained by the "one-pot" or "two-pot" procedure were analysed on reverse phase hplc ('V- Bondapak" C18, 30 x 0.39 cm i.d., Waters Associates) eluted in the case of 4-benzyl-piperidine derivative (32) at 1.5 ml min”1 with disodium hydrogen phosphate buffer (0.025 M in a mixture of methanol and water (60:40)) and for N-[l-11C]cyclopropaneca2±)onyl-THIQ (33) at 2.0 ml min”1 with a mixture of methanol and water (50:50). Retention times of products are given in Table 3.6. The radioactive products (31) and (32) were also analysed by tic, silica gel 60 F254, developed in a mixture of chloroform, ethyl acetate and glacial acetic acid (15:5:1). Rf values are given in Table 3.6.

Procedure B: oxalyl chloride used to generate (7). The [^C] adduct (10) was prepared as described in procedure A using the apparatus shown in Fig 3.8. Oxalyl chloride (145 mg, 1.14 mmol) in diethyl ether (0.9 ml) was then added to (10) and the reaction mixture allowed to stir for 2 min at 298 K. DTBP (256 mg, 1.33 mmol) in diethyl ether (0.7 ml) was then added with stirring. Vessel A was then placed into an oil bath at 373 K to remove excess oxalyl chloride and diethyl ether. To distil the [11C]acid chloride (7) vessel A was transferred to an oil bath at 438 K. The activity was carried in a flow of nitrogen (20-25 ml min”1) into vessel C containing THEQ (26) (20-30 mg, 0.15-22 mmol) in THF at 203 K. The resulting mixture was then heated for 10 min at 238 K to promote f1^]amide (33) formation. The radioactive product obtained, was

169 analysed by reverse phase hplc as described in procedure A. A radioactive peak having the same retention time as reference (30) (5 .2-6.5 min) was observed. The r.c.y's were found to be in the range of 15-25% from corrected for decay.

Table 3.6. Retortion times and Rf values of [-^C]amides.

Amine used [1]-C] amide R.C.Y HPLC TDC (%)a R.T. (min) Rf

Aniline (23) (31). ,, (31) (31) 10-25“ 'a 0.65

4-Benzyl piperidine (24) (32) . (32) (32) 15-2 5c'01 15.2- 17.2 0.59 10-15c 'e 15.2- 17.2 0.59

TKEQ (26) (33) (33) (33) 10-15c 'e 5.0- 6 .6 15-20c 'e 'f 5.0- 6 .6

a) r.c.y estimated from 1-L002 (corrected for decay) b) r.c.y determined from peak area of tic scan c) r.c.y determined from fractions collected from hplc column d) "one-pot" procedure e) "two-pot" procedure f) via [l-1-^]cyclopropanecarboxylic acid

Procedure C: phthaloyl dichloride used to generate (7). i) Via [^^Joyclopropanecarboo^lic acid (20): The [^C] adduct (10) was prepared as described in procedure A using the apparatus shown in Fig 3.8. In a number of experiments anhydrous hydrogen chloride (18-50 mg, 0.5-1.37 mmol) was passed through vessel A containing the adduct (10) over a period of 2 min with stirring. PDC (141-700 mg, 0.7-3.5 mmol) in diethyl ether (0.5-0.9 ml) was then added and the reaction mixture stirred for 2 min under nitrogen. One of a number of different bases such as 170 hexamethylphosphoramide, proton sponge {1 ,8-bis (dimethylamino) - naphthalene}, 2 ,4 ,6-collidine and DTBP (0.7-3.8 mmol) in diethyl ether were added to vessel A to remove excess HC1. Any remaining HCl (e.g. trapped in FIFE lines) and diethyl ether were removed by heating the reaction mixture from 333 K to 393 K over a period of 5 min under a flow of nitrogen (10-15 ml min"1). Vessel A was then transferred into an oil bath at 438 K and the formed [^-^Cjacid chloride (7) was carried in a flew of nitrogen (20-25 ml min"1) into vessel C containing THIQ (10-30 mg, 0.075-0.225 mmol) in THF (2 ml) at 203 K. The resulting mixture was then heated for 5 min at 333 K and the t1^ ] product analysed by tic and hplc. Radiochemical yields and analytical results are given in Table 3.7.

ii) Ocnpound (7) generated directly fron (10) [11C]Acid chloride (7) was generated by the addition of PDC (100-300 mg, 0.49-1.48 mmol) in diethyl ether (0.5-0.9 ml) to vessel A (Fig 3.8) containing the f1^]adduct (10). DTBP (134-670 mg, 0.7-3.5 mmol) in diethyl ether was then added and the reaction mixture allowed to stir for 2 min at 298 K. Diethyl ether was then removed at 333 K and the [11C]acid chloride (7) was carried in a flow of nitrogen (20-25 ml min"1) into vessel C containing THIQ (2.5-10 mg, 0.02-0.08 mmol) in THF (2 ml) at 203 K. The resulting mixture was then heated for 5 min at 333 K and THF removed under a flow of nitrogen at 358 K. Vessel C was cooled in an ice-water bath (273 K) and the radioactive residue was taken up in hplc solvent (CH2CI2 , 1 m l ) . The t1^]activity was injected onto a hplc column (’V-PorasilM, particle size 10 /jm, 30 x 0.7 cm., Waters Associates). The column was eluted at 5.0 ml min"1 with CH2Cl2 and the eluant was monitored continuously for radioactivity and for absorbance at 254 nm. The radioactive fraction having the same retention time as reference compound (30) (15.4-16.8 min) was collected. The radioactive fraction collected was further analysed to determine its chemical and radiochemical purity.

171 Analysis i) HPLC Aliquots of the collected radioactive fraction were analysed by both normal phase and reverse phase hplc. Normal phase system: 'V~ Porasil" column (30 x 0.39 cm i.d., Waters Associates) eluted at 2.0 ml min”1 with CH2CI2 containing 0.1% of a mixture of ethanol, water and triethylamine (100:2:2 v/v). Reverse phase system: 'V- Bondapak C18" coiunm (10 /im particle size, 30 x 0.39 cm i.d., Waters Associates) eluted at 2.0 ml min”1 with a disodium hydrogen phosphate buffer (0.025 M) in a mixture of methanol and water (40:60). On both systems, a single radioactive peak with the same retention time as the reference compound (30) was obtained, the results are presented in Table 3.7. Apart from the reference material no other stable compound was detected. ii) TLC Aliquots were also analysed by tic on two systems. System A, silica gel plates eluted with a mixture of hexane and ethyl acetate (7:3 v/v): system B, silica gel plates eluted with CH2C12 containing 0.5% of a mixture of EtOH:H20:Et3N (100:2:2 v/v). Radioactivity was detected by autoradiography and stable material by exposure to UV light or to iodine. On both tic systems the radioactive product camigrated with authentic N-cyclopropanecarbonyl-THIQ (30). See Table 3.7 for Rf values. iii) Mass spectroscopy The radioactive fraction collected from the preparative hplc column after decay was evaporated to dryness. The residue was examined by mass spectrometry. The spectra revealed a parent peak at M/Z 201, peaks at M/Z 132 ([M-C3H 5C=0]+ , 66%), 117 {[M-C4H 6NO]+ , 25%) and 104 {[M-C^yNO]-1", 33%) which are characteristic fragments of N— cyclopropanecarbonyl-THIQ (30).

172

(min) 45-50 45-50 45-50 30-351 45-50 30-351 Prep. Time R f 0.393 0.633 0.393 0.633 0.393 0.633 0.611 0.393 0.63k 0.611 30-351 0.611 TIC 4.45h 4.45h 4.45h 4.45h (min) 15.39 15.39 15.39 15.39 15.39 15.39 15.39 HPLC R.T. . — — 1.5-2.2 1.5-2.2 (GBq/imvol) activity^ Specific

15-30d 31-40d 37-45d 20-30e (33) (33) (%) [^C]amide R.C.Y of

containing 0.5% of EtOH:H20:Et3N (100:2:2); 1) without hplc 2 CI 2 64-70 50-70 20-35e 1.5-2.2 50-70 20-35e 58-68 50-70 of [11C]acid Estimatedyield chloride (7) (%)

90-95 50-70 92-96 90-95 90-95 tiona (%) [^CJcarbona- Efficiencyof 5 10 90-95 (mg) THIQ (mg) 100 350 20 100 2.5 90-95 50-70 10-25e 1.5-2.2 700 30 PDC (7) (7) and its reactionwith TEHQ (26) togive N-[l-^^]cyclopropanecarbonyl-OHIQ (33). C (i) C (i) C (i) 141 C (ii) 300 10 90-98 C (ii) 100 10 C (ii) C (ii) Procedure Table 3.7. Conditionsused and the efficiencyof various steps inthe preparation of [3dC]cyclcpropanecarbonyl chloride [-^C]amide (33) fraction collected from the preparative hplc column; f) starting activity of -^OC^, 372-930 MBq; g) a) a) determined from 11C02 activity dispensed into cyclopropanemagnesium bromide; b) determined fromthe percentage ofactivity that distilled fromvessel A into vessel C; c) r.c.y and specific activity measurements are based on 1^002 trapped; d) r.c.y based on the [1:LC]amide (33) fraction collected from the analytical reverse-phase hplc column; e) r.c.y based on the analytical reverse-phasesilica hplc; plates, h) analytical Hex:EtOAC normal-phase hplc; (7:3); i) k) tic system: silica silica plates, plates, CHC^iEtOAciAcOH CH (15:5:1); j) puri f ication.f puri

173 iv) N— [ ^C / 1^ ] cycd^rqpanecarix^^l-'IHIQ In one experiment, ^-3C—enriched OO2 (90 atom %, 35 /jmol) was co— included in the radiosynthesis and the purified product examined by 13C-NMR spectroscopy (CDC13, 22.5 MHz). The spectrum exhibited a single peak at 8 172.34 {see Table 4.9 for 13C-NMR chemical shifts of reference compound (30)} assigned to the carboxamido-carbon of N- cyclopropanecarbonyl-THIQ (30).

3.3.2.6 Preparation of N-rc^loorocy/Imeftdivl-lHIO (35) N- [ 11C] cyclopropanecarbonyl-TFilQ (33) was prepared as described in Sec 3.3.2.5, procedure C (ii). Vessel C (Fig 3.8) containing [■^-^CJamide (33) was cooled to ca. 298 K and LiAlH4 (23.7 mmol, 1.0 M) in THF (1.0 ml) was added with stirring under nitrogen. The resulting mixture was heated for 3 min at 333 K. Vessel C was then submerged into a cold bath at 203 K for one min and the excess LiAlH4 destroyed by the slow addition of water with stirring. The radioactivity was extracted into hplc solvent {mixture of chloroform, ethanol and c.ammonia (100:1:0.1 v/v)}. Aliquots were then injected onto a silica gel hplc column ('V-Forasil", 30 x 0.39 cm i.d., Waters Associates) eluted at 1.0 ml min-1 with the same solvent. The eluant was monitored continuously for radioactivity and for absorbance at 254 nm. The radioactive fraction having the same retention time as reference material (34) (5.0-5.8 min) was collected. N- [ cyclopropanecarbonyl-THIQ (33) eluted between 3.15-4.0 min under the same hplc conditions. N- [ 1^C] Cyclopropylirethyl-THIQ (35) comigrated with authentic N- cyclopropylmethyl-THIQ (34) on two different tic systems using silica gel plates. System A: eluant CHCl3 :Et0Ac:c.NH40H (3:7:1), Rf = 0.68; System B: eluant Et20:Et0Ac:Ac0H (15:5:1), Rf = 0.26. The preparation requires ca.40 min (excluding hplc purification) from the end of radioisotope production and gives an overall r.c.y of 15-20% (based on 11002 used and corrected for radioactive decay).

174 3.3.2.7 Preparation of r^CIDiprenortiiine (8) Procedure A "Manual Manipulation11 The apparatus used for the synthesis was assembled while hot, as illustrated in Fig 3.9. Air was then purged from the system by a continuous flew of dry nitrogen (15-20 ml min”-*-) for half an hour prior to use. A filtered solution of freshly prepared cyclopropanemagnesium bromide (19) (0.5 mmol) in diethyl ether (3 ml) was placed into vessel A under nitrogen, followed by further addition of diethyl ether (1 ml). Cyclotron produced 112 trapped on the molecular sieve trap was then released into the reagent in a flow of nitrogen (5 ml min"1) over a period of 2 min at ambient temperature. PDC (710 mg, 3.5 mmol) and DTBP (344 mg, 1.8 mmol) in diethyl ether (1.0 ml) were then added and the reaction mixture stirred for 2 min. Vessel A was then placed into an oil bath at 338 K to remove the excess diethyl ether and then transferred to an oil bath at 438 K. A regulatory valve (labelled as C in Fig 3.9) was opened to allow the formed [11C]acid chloride (7) to be carried in a stream of nitrogen (20-25 ml min"1) into a solution of NDHM (3) (2-3 mg, 4.7-7 /imol) in THF (5 ml; the solution was sonicated to solubilise the amine) under nitrogen at 203 K. Vessel B was then heated at 338 K for 3 min to promote f1^] amide (36) formation. Aliquots were removed and analysed by tic and hplc (see below for conditions). Rf values are given in Table 3.8 and a typical hplc chromatogram is shown in Fig 3.10. Vessel B was cooled to ambient temperature and LiAlH4 (16.7 mmol, 1.0 M) in THF (0.7 ml) was added. The resulting mixture was heated at 338 K for 3 min and then cooled. Dry methanol (5 ml) was then added slowly with stirring. The resultant solution was removed from vessel B and loaded onto a series of three silica gel "Sep-paks11 (2.25 g) that had been pre-conditioned with THF (20 ml). These were then eluted at 5 ml min"1 with THF (10 ml) and the radioactive eluate rotary evaporated to dryness. The residue was taken up in hplc solvent (1.4 ml of a mixture of chloroform, ethanol and c.ammonia, 100:1:0.1 v/v) and injected onto a silica gel hplc column ('V~ Forasilu, particle size 10 pm, 30 x 0.7 cm i.d.,) eluted at 4.0 ml

175 Receiver to collect N2 in Et20 "C02/H2 from target Soda Needle \ y lime value

Mg(CI04)

L

Molecular 2 sieue trap

I Mg(CI0+)2 H '•J o\

cyclo.C3H5MgBr ^ 7 Flow in Et20 meter

Needle value

NDPN in THF N2 in liessel R UesselB

Fig 3.9 "Manual" apparatus used for the preparation of [11C]diprenorphine. Halve (K75-tap) position for each operation indicated bg number. 1) Cgclotron-produced 11C02 collected onto molecular sieve trap; 2) Trapped "COa dispensed into cyclo.C3H5MgBr in Et20; 3) Addition of PBC O' DTBP; 4) Removal of Et20; 5) f ’CjCyclopropanecarbonylchloride distilled from uessel fl into uessel B; 6) Promote I11Cjamide formation; 7) Addition of LiflIH* inTHF; 8) Addition of MeOH; 9) Removal of I^Cjreaction mixture from vessel B. min-1 with the same solvent. UV-Absorbance and radioactivity were monitored simultaneously. The radioactive fraction having the same retention time as reference material (1) (8.4-9.5 min, Fig 3.11) was collected. An aliquot was taken and further analysed by tic and hplc (see below). The remaining fraction was formulated for human intravenous injection as described below.

Procedure B "semi-automated system" A schematic representation of the "semi-automated system" used is presented in Fig 3.12 and the sequence of operations for the preparation of [11C]diprenorphine are detailed in Appendix-4. Vessels A, B and reagent lines I, II were tested for leaks and flushed with dry nitrogen for 30 min prior to use. CXiring this period both vessels A and B were kept in a Woods alloy baths at 448 K. Cyclotron produced 1 1 CC>2 was trapped into a stainless steel loop (2 cm diameter, 0.75 mm i.d.) immersed in liquid argon. The activity was then released in nitrogen (by plunging the loop into warm water) at 1.5 ml min-1 into vessel A containing cyclopropanemagnesium bromide (0.17 mmol) in diethyl ether (1 ml) at ambient temperature under nitrogen. After 2 min, [11C]ca3±>onation was quenched by the addition (via line I) of PDC (285 mg, 1.4 mmol) plus DTBP (268 mg, 1.4 mmol) in diethyl ether (0.5 ml). Vessel A was then heated at 338 K for 3 min, under a flow of nitrogen (5-10 ml min-1) to remove solvent. Ihe radioactive residue was then transferred to a Woods alloy bath at 448 K. The released [^--^CJacid chloride (7) was carried by a stream of nitrogen (15 ml min”1) into vessel B containing a solution of NDFN (3) (2-3 mg, 4.7-7 /imol) in THF (1 ml) kept under nitrogen at 203 K. The transfer of t1^ ] activity to vessel B was monitored by a GM-tube mounted close to this vessel. Generally, the transfer of activity was achieved within 4 min. Vessel B was then transferred to a bath at 338 K and the solution heated for 3 min while stirred with a flow of nitrogen. LiAlH4 (0.88 M) in THF (360 /zl) was then added (via line II) to vessel B and the resultant mixture heated for 3 min at 338 K, under

177 ) m n 4 5 2 ( E C N A B R O S B A V U * Time Time (min.) Fig 3.10 Fig chromatogram Hplc [iiC]amideof (36)

178 179 Fig 3.11 3.11 Fig Preparative chromatogramhplc of [11C]diprenorphine (8) RADIOACTIVITY 180 nitrogen. Vessel B was then transferred to a cold bath at 203 K for 30 sec. Dry methanol (1.5 ml) was then added slowly while stirring with nitrogen (10 ml min-1). Under a pressure of nitrogen (15-20 ml min""1, via valve 8 and 5N), the resulting mixture was then loaded onto silica gel "Sep-pak" (two connected in series, 1.5 g) that had been pre-conditioned with THF (10 ml). These were then eluted (via line IV) over 2 min with THF (7.0 ml) and the radioactive eluate collected into vessel C. The solvent was removed under nitrogen by vacuum distillation at 338 K. Dichloromethane (1.5 ml) was then added (via line III) and the radioactive residue removed from the sides of vessel C by stirring with nitrogen. The resulting solution was loaded onto an injector (labelled as 12) via suction through line V and injected onto a silica gel hplc column (conditions as for procedure A). The radioactive fraction eluting between 8.4-9.5 min was collected, an aliquot was analysed and the major fraction formulated as described below.

Analysis of r^CIDiprenorohine i) HPLC A) The radioactive fraction collected from the preparative hplc column (procedure A and B) was routinely analysed by normal phase hplc using a 'V-Porasil" column (30 x 0.39 cm i.d.) eluted with a mixture of chloroform, ethanol and c.ammonia (100:2:0.2 v/v) at a flow rate of 2.0 ml min-1. The eluant was monitored continuously for radioactivity and for absorbance at 280 nm. A single radioactive product having the same retention time as authentic diprenorphine (4.0 min) was obtained and diprenorphine (1) was the only stable compound detected.

B) Also reverse phase hplc was used to analyse the radioactive fraction collected. A "/i-Bondapak C^g" column (30 x 0.39 cm i.d.) was eluted with a mixture of disodium hydrogen phosphate (0.025 M), methanol and water (40:60, adjusted to pH 7 with phosphoric acid) at a flow rate of 2.0 ml min-1. [ 11C]Diprenorphine (8) eluted at the same retention time (11.3 min) as authentic diprenorphine (1).

181 ii) TDC The radioactive fraction collected was analysed by three different tic systems, two normal phase and one reverse phase. System A: silica gel plate, eluant CHC^iEtQAcic.NH^H (3:7:1); system B: eluant CHCl3 :MeOH:c.NH4QH (18.2:1.6:0.2) ; system C: RPC18 plate, eluant Me0H:H20 (8:2). The stable compound was observed by exposure to iodine vapour or UV irradiation. A typical autoradiograph of the radioactive reaction mixture before and after hplc purification is shewn in Fig 3.13a and 3.13b, respectively. As can be seen from Fig 3.13b, one radioactive product was observed after hplc purification which co-migrated with authentic diprenorphine (1) and no other chemical contamination was detected. Rf values of [ ^C] diprenorphine (3) on each tic system are given in Table 3.8.

Table 3-8. Revalues of [^C]diprenorphine (8 ) and other [-^C]products observed in its synthesis.

[ Compounds System A System B System C

[i:LC]Acid (20) 0.68 0.93 -

[1:LC]Alcohol (11) 0.55 0.74 -

[1:LC]Amide (36) 0.21 0.48 0.61

[1:LC]Ester (37) origin 0.28 0.88

[ 1:LC] Diprenorphine (8) 0.49 0.41 0.24

iii) [ Diprenorphine In one experiment, 13C-enriched C02 (90 atom %, 47 /unol) was co­ included in the radiosynthesis and the purified product was examined by broad-band proton-decoupled FT 13C-NMR spectroscopy (CDCI3 , 22.5 MHz). The spectrum exhibited a peak at S 59.86 in accord with the chemical shift assigned to C23 in diprenorphine (Fig 3.2). See Table 3.9 for chemical shift assignment of reference diprenorphine (1). Multiplicity was established by spin-echo gated decoupling

182 Fig 3.13b Fig Fig 3.13aFig before hplc hplc before purification Fig 3.13a and 3.13b Autoradiographs of [iiCJDiprenorphine

[n C JE ste r [n C JA m id e [nC]Diprenorphine CJCyclopropanecarbinol [11

183 technique. The spectrum also exhibited two other peaks at 5 25.5 and 68.0. These were attributed to THF by comparison of the 13C-NMR chemical shift data with literature.140 Additionally, the 1H spectrum of the enriched sample exhibited two multiplets centered at 5 3.75 and 1.85 in accord with THF proton chemical shifts.141

Table 3.9. [^CJChemical shifts of Diprenorphine (1)

Carbon Chemical Carbon Chemical Number Shift Number Shift 5 (ppm) 6 (ppm)

1 119.34 14 47.06

2 116.66 15 35.35

3 137.58 16 43.70

4 145.60 17 17.50

5 97.05 18a 'c 29.55

6 80.42 19 74.47

7 47.69 20b 24.77

8 32.14 CM 29.79

9 35.94 22 52.52

10 58.33 23a 59.79

11 22.77 24 9.11

12 127.54 25 3.99

13 132.17 26 3.31

Assignment by analogy with published spectrum but multiplicities confirmed by DEFT/SEGD. a) Shown to be CH2 by DEPT/SEGD. b) Shown to be CH3 by DEFT/SEGD alternative assignments. c) Assignment of this carbon by Carroll et al131 was ambiguous and alternative with C20 and C21, but here chemical shift confirmed by DEFT/SEGD.

184 Evidence for the formation of the amide (36) During the 13C/11C co-labelling experiment, an aliquot was removed before reduction, chromatographed and the main radioactive peak (Fig 3.10) was collected. The 13C-NMR spectrum (0 x 13, 22.5 MHz) exhibited a singlet at 8 171.95. This peak was attributed to the carboxamido-carbon of compound (36) by comparison with carbonyl chemical shifts exhibited by similar amides shown in Fig 3.3.

Formulation of r 11C1diprenorphine for human intravenous injection The [ 11C ]diprenorphine collected (Fig 3.11) from the preparative hplc column (procedure A and B) was evaporated to dryness. The radioactive residue was then dissolved in aqueous acetic acid (500 /xl, 1% v/v) diluted first by the addition of sodium citrate solution (6.0 ml, 3.8% w/v) and then by water for injection (4.0 ml). The product was sterilised by filtration (0.22 /jm pore size, Millex-GS). All randomly selected preparations passed independent tests for apyrogenicity and sterility.

Determination of the specific activity of r^^Cl diprenorphine The normal phase analytical hplc (system A, Sec 3.3.2. ) was used to measure the specific activity of [ ^C] diprenorphine (8). For this purpose the response of the absorbance detector at 280 nm was pre-cal ibrated with respect to mass by measuring the peak areas of known masses of reference diprenorphine (1) injected onto the column. Response was found to be linear over the range 0.6-6.2 ^g (1.4-14.6 nmol). All test samples contained stable diprenorphine within this range. For each determination the [^C]diprenorphine that eluted from the analytical column was collected and measured for total activity (e.g a GBq) in a calibrated well-counter at a known time (t min) from the end of radiosynthesis. The area of the uv absorption peak corresponding to stable diprenorphine (1) in this sample was calculated and on the basis that peak area is proportional to concentration, the quantity of stable diprenorphine in the collected radioactive fraction was calculated (b /imol). The specific activity of [ ^-k:] diprenorphine (8 ) at the end of radiosynthesis (S) was then

185 calculated according to the formula:

S = (a/b) e+ 0 *034xt GBq /xmol

fi:LCl Diprenorphine: preparation time. radiochemical yield and specific activity The "semi-autcmated" procedure described in Sec 3.3.2.7 (procedure B) produced 0.56-1.7 GBq (15-47 mCi) of [13C]diprenorphine in an injectable form, in 57-63 min from EOB. In twenty preparations the radiochemical yield was found to vary between 10-30% (decay- corrected from 11C02) starting from 44-55 GBq (1.2-1.5 Ci) of 11C02- The specific activity of [1 ] diprenorphine in these preparations was in the range of 37.2-85.6 GBq//jmol (1.0-2.3 Ci//zmol) at EOB, corresponding to a specific activity range of 5.6-11.2 GBq//zmol at EOS. Consequently the amount of carrier (stable diprenorphine) was found to vary between 47 and 606 nmol (or 20-258 /ig).

3.3.2.8 Preparation of [^ClBuprenorphine (9) The synthetic route used for the preparation of [-^C] buprenorphine (9) was similar to that described for the preparation of [ 13C ] diprenorphine (8). The "semi-automated" system shown in Fig 3.12 was assembled as described previously and the sequence of operations detailed in Appendix 4 were used to prepare (9). Cyclopropanemagnesium bromide (0.2 mmol) in diethyl ether (1.0 ml) was placed into vessel A under nitrogen. Cyclotron produced i :lc o 2 was then trapped into a stainless steel loop in liquid argon. The radioactivity in nitrogen (1-2 ml min”1) was then dispensed into the reagent at ambient temperature over a period of 2 min. PDC (285 mg, 1.4 mmol) and DTBP (268 mg, 1.4 mmol) in diethyl ether (0.5 ml) were then added (via line I) to vessel A and the radioactive mixture stirred for 2 min. Vessel A was then transferred to an oil bath at 338 K and the solvent removed under a flow of nitrogen (5-10 ml min”1) over 3 min. The radioactive residue was then transferred to a Woods alloy bath at 448 K. The flew of nitrogen was then increased

186 to 15 ml min-1 to distill the [11C]acid chloride (7) into vessel B containing a solution of NBFN (4) (2 mg, 4.3 mmol) in THF (1 ml) kept under nitrogen at 203 K. LiAlH4 (1.0 M) in THF (320 /il) was then added (via line II) to vessel B and the resultant mixture heated at 338 K for 3 min under nitrogen. The solution was then cooled by placing vessel B into a cold bath at 203 K for 30 sec. Methanol (1.5 ml) was then added (via line II) slowly with stirring. The resultant solution under a pressure of nitrogen was loaded onto a silica gel "Sep-pak" (two connected in series, 1.5 g) that had been pre-conditioned with THF (10 ml). The retained activity was eluted (via line IV) over 2 min with THF (7.0 ml) and the eluate collected into vessel C. The solvent was removed by vacuum distillation and the radioactive residue was taken up into dichloromethane. The crude product was injected onto a silica gel hplc column (Partisil M9 50/10, 50 X 0.7 cm i.d., Whatman Ltd) eluted at 6.0 ml min-"1 with dichloramethane containing 3.5% of a mixture of ethanol, water and triethylamine (100:2:2 v/v). The radioactive fraction having the same retention time as reference buprenorphine (2) (6 .2-7.6 min) was collected. Unchanged amine (4) was retained on the column.

Analysis of Buprenorphine i) HPDC Aliquots of the radioactive fraction collected from the preparative column were analysed by normal phase hplc using a 'V- Porasil" column (30 x 0.39 cm i.d.) eluted at 2.0 ml min"1 with a mixture of chloroform, ethanol and c. ammonia (100:1.5:0.15 v/v). The eluant was monitored continuously for radioactivity and for absorbance at 280 nm. A single radioactive peak with the same retention time (5.05 min) as reference buprenorphine (2) was obtained. Together with stable buprenorphine, minor chemical contamination was also detected in the [ 11C]buprenorphine sample.

187 ii) TIC Aliquots of the radioactive fraction collected were also analysed by two normal phase tic systems using silica gel plates. System A: eluant CHC^iMeOHic.NI^OH (18.2:1.6:0.2 v/v); system B: eluant CHCI3 :EtQAc:c .NH40H (3:7 v/v). Radioactivity was detected by autoradiography and the stable compound was cbserved by exposure to iodine vapour or UV radiation. A single radioactive product with Rf values (system A, Rf = 0.62; system B, Rf = 0.59) as reference buprenorphine was observed. No chemical contamination was detected by either tic systems.

Formulation of r-^C 1 buprenorphine for human intravenous injection The radioactive fraction collected was evaporated to dryness. The residue was then solubilised in aqueous acetic acid (500 ^ 1 , 1% v/v) diluted with sodium citrate solution (6.0 ml, 3.8% w/v) followed by water for injection (4.0 ml) and finally sterilised by filtration (0.22 /un pore, Millex GS). f 1 Buprenorphine: preparation time, radiochemical yield and specific activity The preparation of [ 11C ]buprenorphine (9) required 57 min from EOB and provided an injectable solution in ca. 20% r.c.y (based on ^^C02 used and corrected for radioactive decay). Thus useful activities (> 370 MBq or > 10 mCi) of buprenorphine with specific activities similar to those obtained in the preparation of [ 11C]diprenorphine (> 6 GBq//imol at EOS) can be prepared for PET studies.

188 3 .4 REFERENCES

1. Frederickson R.C.A. and Haynes M.D., "Drugs in Central Nervous System Disorders", Ed. Horwell D.C., Marrel Deckker, N.Y. (1985) .

2. Lewis J.W., Bentley K.W. and Cowan A., Ann. Rev. Pharmacol., 11, 241 (1971).

3. Simon E.J. and Hill J.M., Ann. Rev. Pharmacol. Toxiocol., 1 8 . 371 (1978).

4. Cooper J.R., Bloom F.E. and Roth R.H., "The Biochemical Basis of Neuropharmacology", 4th Ed., Oxford University Press, N.Y., 109 (1982).

5. Kandel E.R. and Schwartz J.H., "Principles of Neural Science", Elsevier/North-Holland, N.Y. (1981).

6. Young A.B., Frey K.A. and Agranoff B.W., "Positron Emission Tomography and Autoradiography: Principles and Applications for the Brain and Heart", Ed. Phelps M.E., Mazziotta J.C. and Schelbert H.R., Ch. 2, 73 (1986).

7. Pert C.B. and Snyder S.H., Science, 179. 1011 (1973).

8. Simon E.J., Hiller J.M. and Edelman I., Proc. Natl. Acad. Sci., 70, 1947 (1973).

9. Terenius L., Acta Pharmacol. Toxicol., 317 32., (1973).

10. Kuhar M.J., Pert C.B. and Snyder S.H., Nature, 245. 447 (1973).

11. Hillman J.M., Pearson J. and Simon E.J., Res. Commun. Chem. Path. Pharmacol., 6, 1052 (1973).

12. Pert C.B. and Snyder S.H., L ife S ci., 16, 1623 (1975).

13. Harkenham M. and Pert C.B., J. Neuroscience, 1129 2, (1982).

14. Young W.S III and Kuhar M.J., Brain Res., 179, 255 (1979).

15. Pert C.B., Kuhar M.J. and Snyder S.H., L ife S ci., 16, 1849 (1975).

16. Chang K.J. and Cautrecasas P. , J. B io l. Chem., 2 5 4 . 2610 (1979) .

17. Chang K.J., Hazum E. and Cautrecasas P., Proc. Natl. Acad. Sci. USA, 78, 4141 (1981).

189 18. Pfeiffer A. and Herz A., Biochem. Biophys. Res. Commum., 1 0 1 . 38 (1981).

19. Snyder S.H. and Goodman R.R. , J. Neurochem. 35., , 5 (1980).

20 . Goodman R.R., Snyder S.H., Kuhar M.J. and Young W.S.III., Proc. Natl. Acad. Sci. USA., 77, 6239 (1980).

21 . Pfeiffer A., Pasi A., Mehraein P. and Herz A . , Brain Res., 2 4 8 . 87 (1982).

22. Goodman R.R. and Snyder S.H., Proc. Natl. Acad. Sci. USA, 7 9 . 5703 (1982).

23. Leenders K.L., Herold S. and Brooks D.J., Lancet, 2, H O (1984).

24. Wagner H.N.Jr., Burns H.D., Dannals R.F., Wong D.F., Langstrom B. , Duelfer T. , Frost J.J., Ravert H.T., Links J.M., Rosenblum S.B., Lukas S.E., Kramer A.V. and Kuhar M.J., Science, 2 2 1 . 1264 (1983) .

25. Welch M.J., Kilbourn M.R., Mathias C.J., Mintun N.A. and Raichle M.E., L ife S ci., 33, 1687 (1983).

26. Arnett C.D., Shiue C-Y., Wolf A.P., Fowler J.S., Logan J. and Watanabe M . , J. Neurochem., 44, 835 (1985).

27. Arnett C.D., Wolf A.P., Shiue C-Y., Fowler J.S., MacGregor R.R. , Christman D.R. and Smith M.R., J. Nucl. Med., 2J_, 1878 (1986).

28. Arnett C.D., Fowler J.S., Wolf A.P., Shiue C-Y. and McPherson D.W., L ife S ci., 36, 1359 (1985).

29. Maziere B. , Loch C., Baron J.C., Sgouropoulous P. , Duquesnoy N. , D ' a n t o n a R. a nd C a m b o n H. , fur. J. Pharmacol., 1 1 4 . 267 (1985) .

30. Crawley J.C.W., Smith T., Veall N., Zanelli G.D., Crow T.J. and O w e n F . , Lancet, 2, 975 (1983).

31. Ehrin E., Farde L. , De Paulis T. , Eriksson L. , Greitz T. , Johnstrom P., Litton J-E., Nilsson J.L.G., Sedvall G. , Stone- Elander S. and Ogren S-O., Int. J. Appl. Radiat. Isot., 36., 269 (1985).

32. Farde L. , E h r i n E. , E r i k s s o n L. , Greitz T. , H a l l H. , H e d s t r o m C-G., Litton J-E. and Sedvall G., Proc. Natl. Acad. Sci. USA, 82, 3863 (1985).

190 33. Syrota A., Comar D. , Paillotin G. , Davy J.M., Avmont M.C., Stul z a f t 0. and M a z i e r e B . , Proc. Natl. Acad. Sci. USA, 82, 584 (1985) .

34. Charbonneau P., Syrota A., Crouzel C., Vallois J.M., Prenant C. and Crouzel C., Circulation, 7_3, 476 (1986).

35. Berger G. , Maziere M. , Prenant C. , Sastre J., Syrota A. and Comar D. , J. Radioanal. Chem. 301, TJ±, (1982).

36. Berger G., Prenant C., Sastre J., Syrota A. and Comar D., In t. J. Appl. Radiat. Isot. 34, , 1556 (1983).

37. Boullais C., Crouzel C. and Syrota A., J. Label. Compd Radiopharm., 565 23, (1986).

38. Mathias C.J., Welch M.J., Katzenellenbogen J.A., Brodack J.W., Kilbourn M.R. , Carlson K.E. and Kiesewetter D.O., Nucl. Med. B io l., 14, 15 (1987).

39. Barrio J.R., "Positron Emission Tomography and Autoradiography: Principles and Applications for the Brain and Heart", Ed. Phelps M.E., Mazziotta J.C. and Schelbert H.R., Ch. 10, 451 (1986).

40. Kuhar M.J., Mur r in L.C., Malouf A.T. and Klemm N. , L ife S ci., 22, 203 (1978).

41. L a d u r o n P .M. , Janssen F.M. and L e y s e n J.E. , Biochem. Pharmacol., 27. 307 (1978).

42. L a d u r o n P .M. , Janssen F.M. and L e y s e n J .E. , Biochem. Pharmacol., 27. 317 (1978).

43. Farde L. , Hall H. , Ehrin E. an d Sedvall G ., Science,231. 258 (1986) .

44. Enna S.J. and Coyle J.T., Neuroleptics In: "Neuroleptics: Neurochemical Behavioural and Clinical Perspectives", Ed. Coyle J.T. and Enna S.J., Raven Press, N.Y., 1 (1984).

45. Snyder S.H., Science, 224. 22 (1984).

46. Peroutka S.J. and Snyder S.H., Am. J. Psychiatry, 1 3 7 . 1518 (1980).

47. Pedigo N.W., Yamamura H.I. and Nelson D.L., J. Neurochem., 36, 220 (1981).

48. Welch M.J., Kilbourn M.R., Mathias C.J., Mintun M.A. and Raichle M.E., L ife S ci., 33, 1687 (1983).

191 49. Comar D. , Maziere M. , Godot J.M., Berger G. and Soussaline F., Nature, 280. 329 (1979).

50. Pert C.B., Snyder S.H. and Kuhar M.J., "Tissue Responses to Addictive Drugs", Ed. Ford D.H. and Clovet D., Spectrum, Holliswood, N.Y., 89 (1976) .

51. Snyder S.H., S c i. Am., 236, 44 (1977).

52. Rothman R.B., Danks J.A., Jacobson A.E., Burke T.R., Rice K.C. and Pert C.B., Neuropeptides, 4, 311 (1984).

53. O l d e n d o r f W . H . , Proc. Soc. Exp. Biol. Med., 147, 813 (1974).

54. Raichle M.E., Eichling J.O., Straatmann M.G., Welch M.J., Larson K.B. and Ter-Pogossian M.M., Am. J. Physiol., 2 3 0 . 543 (1976) .

55. Rapoport S.I., Ohno K. and Pettigrew K.D., Brain Res., 1 7 2 . 354 (1979).

56. Kragh-Hansen U., Pharmacol. Rev., 33., 17 (1981).

57. Wilkinson G.R., Drug Metab. Rev., 14, 427 (1983).

58. Creese I., Neuroleptics In: "Neuroleptics: Neurochemical, Behavioural and Clinical Perspectives", Ed. Coyle J.T. and Enna S.J., Raven Press, N.Y., 183 (1983).

59. Laduron P. and Leysen J., Biochem. Pharmacol., 26., 1003 (1977).

60. Frost J.J., "Receptor Binding Radiotracers", Ed. Eckelman W.C., CRC Press, Boca Raton, FL., 25 (1982).

61. Rosenbaum J.S., Holford N.H.G. and Sadee W., J. Pharmacol. Exp. T h e r., 233, 735 (1985).

62. Chang K.J., Hazum E. and Cautrecases P., Proc. Natl. Acad. Sci. USA, 78, 4141 (1981).

63. Childers S.R., Snowman A.M. and Snyder S.H.. Fur. J. Pharmacol., .55, 11 (1978).

64. Chang K.J., Hazum E. and Cautrecasas P., Proc. Natl. Acad. Sci. USA, 77, 4469 (1980).

65. Wamsley J.K., Zarbin M.A., Young W.S.III. and Kuhar M.J., Neuroscience, 595 2, (1982).

66. Atweh S.F. and Kuhar M., Brain Res., 134. 393 (1977).

192 67. Frost J.J., Dannals R.F., Duelfer T., Burns H.D., Ravert H.T., Langstrom B. , Subramanian V.B. and Wagner H.N.Jr., Ann. Neurol., 15, S85-S92 (1984).

68. Rosenbaum J.S., Holford N.H.G., Richards M.L., Aman R.A. and Sadee W. , Mol. Pharmacol., 25.> 242 (1984).

69. Perry D.C., Mullis K.B., Oie S. and Sadee W. , Brain Res., 1 9 9 . 49 (1980).

70. Jones A.K.P., Attached to the Medical Research Council, Cyclotron unit, Hammersmith Hospital.

71. Hovell B.C. and Ward A.E., J. Int. Med. Res., 5, 417 (1977).

72. Heel R.C., Brogden R.N., Speight T.M. and Avery G.S., Drugs, 17, 81 (1979).

73. Sadee W., Rosenbaum J.S. and Herz A., J. Pharmacol. Exp. Ther., 223. 157 (1982).

74. Sadee W. , Richards M.L., Grevel J. and Rosenbaum J.S., Life Sci., 33, 187 (1983).

75. Allen D.R. and Beaumier P.L., J. Label. Compd Radiopharm. , 1 6 . 61 (1979).

76. Maziere B., Godot M. , Prenant C. and Comar D., J. Radioanal. Chem., 62, 279 (1981).

77. Kloster G., Roder E. and Machulla H.J., J. Label. Compd Radiopharm., 16, 441 (1979) .

78. Svard H. , Nagren K. , Malmborg P., Sohn D. , Sjoberg S. and L a n g s t r o m B. , J. Label. Compd Radiopharm. , 19., 1519 (1982).

79. L a n g s t r o m B. , A n t o n i G., H a l l d i n C. , Sva r d H. a nd B e r g s o n G. , Chemica Scripta., 20, 46 (1982).

80. Lewis J.W., "Advances in Biochemical Phychopharmacology", Ed. Braude M.C., Harris L.S, May E.L., Smith J.P and Vallarreal J.E., Ravern Press, N.Y., 8, 123 (1974).

81. May E.L., "Agonist and Antagonist Actions of Narcotic Analgesic Drugs", Ed. Kosterlitz H.W., Collier O.J. and Villerreal J.E., University Park Press, Baltimore, 17 (1973).

82. Kotick M.P., Leland D.L., Polazzi J.O. and Schut R.N., J. Med. Chem., 23, 166 (1980).

193 83. Burns H.D., Lever J.R., Dannals R.F., Frost J.J., Wilson A.A., Ravert H.T., Subramanian B. , Zemyan S.E., Langstrom B. and Wagner H.N.Jr., J. Label. Compel Radiopharm., 21, 1167 (1984).

84. Bentley K.W. and Hardy D.G. , J. Am. Chem. Soc. , 89., 3281 (1967).

85. Marazano C. , Maziere M. , Berger G. and Comar D. , Int. J. Appl. Radiat. Isot., 28., 4 9 (1977).

86. Berger G. , Maziere M. , Sastre J. and Comar D., J. Label. Compd Radiopharm. , 17., 59 (1980) .

87. Christman D.R., Crawford E.J., Friedkin M. and Wolf A.P., Proc. Natl. Acad. Sci. USA, 69, 59 (1980).

88. Young B. and Trahanovsky W.S., J. Org. Chem., 32, 2349 (1967).

89. Marazano C., Maziere M. , Berger G. and Comar D. , Int. J. Appl. Radiat. Isot., 28., 49 (1977).

90. L a n g s t r o m B. and L u n d q v i s t H. , Int. J. Appl. Radiat. Isot., 27., 357 (1976).

91. Roberts J.D. and Mazur R.H. , J. Am. Chem. Soc., 13_,2509 (1951).

92. Breslow R. , "Molecular Rearrangements", Ed. De Mayo P., Interscience Publisher, N.Y., Part 1, 233 (1963).

93. Staral J.S., Yavari I., Roberts J.D., Prakash G.K.S., Donovan D.J. and Olah G.A., J. Am. Chem. Soc., 100, 8016 (1978).

94. Staral J.S. and Roberts J.D., J. Am. Chem. Soc., 1 0 0 . 8018 (1978) .

95. Lee C.C. and Cessna A.J., Can. J. Chem., 58., 1075 (1980).

96. Caserio M.C., Graham W.H. and Roberts J.D., Tetrahedron, 1 1 . 171 (1960).

97. Olah G.A., Jevell C.L., Kelly D.P. and Porter R.D. , J. Am. Chem. Soc., 94, 146 (1972).

98. Richey G., "Carbonium Ions", Ed. Olah G.A. and Schleyer P.v.R., Wiley-Interscience, N.Y., 3, Ch. 25 (1972).

99. Wiberg K.B., Hess B.A.Jr. and Ashe A.J.III., "Carbonium Ions", Ed. Olah G.A. and Schleyer P.v.R., Wiley-Interscience, N.Y. , 3., Ch. 25 (1972).

194 100. Roberts J.D. and Mazur R.H. , J. Am. Chem. Soc. , T3, 3542 (1951).

101. Mazur R.H., White W.N., Semenow D.A., Lee C.C., Silver M.S. and Roberts J.D., J. Am. Chem. Soc., 81, 4390 (1959).

102. Kelley D.P., Underwood G.R. and Barron P.F., J. Am. Chem. Soc., 98, 3106 (1976).

103. Olah G.A., Spear R.J., Hiberty P.C. and Hehre W.J., J. Am. Chem. Soc., 98, 7470 (1976).

104. Kirchen R.P. and Sorensen T.S., J. Am. Chem. Soc., 99, 6687 (1977).

105. Wiberg K.B. and Szeimies G., J. Am. Chem. Soc., 90, 4195 (1968); 92, 571 (1970).

106. Majerski Z. and Schleyer P.von R. , J. Am. Chem. Soc., 93., 665 ( 1 9 7 1 ) .

107. Denis J.N. and Krief A., J. Chem. Soc.Chem. Commun., 229 (1983).

108. Morita T. , Yoshida S., Okamoto Y. and Sakurai H. , Synthesis, 379 (1979).

109. Olah G.A., Narang S.C., Gupta B.B.G. and Malhotra R. , J. Org. Chem., 44, 1247 (1979).

110. Langstrom B., Antoni G., Gullberg P., Halldin C., Nagren K. , R i m l a n d A., a nd S v a r d H. , Appl. Radiat. Isot., 37., 1141 (1986).

111. Rimland A. and Langstrom B. , Appl.Radiat. Isot., 3_8, 949 (1987).

112. Ansell M.F., "The chemistry of acyl halides", Ed. Patai S., Interscience publishers, John Wiley and Sons, London, Ch. 2, 35 ( 1 9 7 2 ) .

113. Buehler C.A. and Pearson D.E., "Survey of Organic Synthesis"., Wiley Interscience, N.Y., 1, 863 (1970).

114. Smith L.I. and Rogier E.R. , J. Am. Chem. Soc., 73., 4047 (1951).

115. Jeffery G.H. and Vogel A.I., J. Chem. Soc., 1804 (1948).

116. Beg M.A. and Singh H.N., Physik. Chemie., 237, 129 (1968); 227. 272 (1964); Fette un Seifen, 70, 640 (1968).

117. Gerrard W. and Thrush A.M., J. Chem. Soc., 2117 (1953).

195 118. Nakatsuka I., Kawahara K. , Kamada T. and Yoshitake A., J. Label. Compd Radiopharm., 14, 133 (1978).

119. Fieser L.F. and Fieser M. , "Reagent for Organic Synthesis", John Wiley and Sons, Inc., 1, 767 (1967).

120. Parnes H. and De Keczer S., J. Label. Compd Radiopharm. , 2 2 . 287 (1984).

121. Brown H.C., J. Am. Chem. Soc., 60, 1325 (1938).

122. Stempel G.H.Jr., Cross R.P. and Mariella R.P., J. Am. Chem. Soc., 72, 2299 (1950).

123. Forrest J., Liddel D.A. and Tucker S.H., J. Chem. Soc., 454 (1946).

124. Finan P.A. and Fothergill G.A., J. Chem. Soc., 2723 (1963).

125. Bubner M. and Schmidt L.H. , Pharmazie, 18., 668 (1963).

126. Kyrides L.P., Org. Syn. Col., 3, 422 (1955).

127. Fieser L.F. and Fieser M. , "Reagent for Organic Synthesis", John Wiley and Sons, Inc., JL, 581 (1967).

128. Challis B.C. and Challis J.A., "The Chemistry of Amides" Ed. Zabicky J., Interscience Publishers, John Wiley and Sons, London, Ch. 13, 731 (1970).

129. Gaylord N.G., "Reduction with Complex Metal Hydrides", Interscience publishers, John Wiley And Sons, N.Y., 544 (1956).

130. Micovic V.M. and Mihailovic M.L.J., J. Org. Chem., 18, 1190 (1953).

131. Carroll F.I., Moreland C.G., Brine G.A.and Kepler J.A., J. Org. Chem., 41, 996 (1976).

132. Jones A.K.P., Luthra S.K., Pike V.W., Herold S. and Brady F. , Lancet, 2, 8456 (1985).

133. Jones A.K.P., Luthra S.K., Maziere B. , Pike V.W., Loch C., Crouzel C., Syrota A. and Jones T. , J. Neurosci. Methods, 23., 121 (1988).

134. Lever J.R., Dannals R.F., Wilson A.A., Ravert H.T. and Wagner H . N . J r . , Tet. Lett., 28, 4015 (1987).

135. Pike V.W. , Palmer A.J., Horlock P.L., Perun T.J., Freiberg L.A. , Dunnigan D.A. and Liss R.H. , J. Chem. Soc. Chem. Commun. , 173 (1982).

196 136. Wiberg K.B. , Barth D.E. and Schertler P.H. , J. Org. Chem. 3, 8 . 378 (1973).

137. Chalchat J.C., Garry R.Ph., Lacroix B. and Michet A., Mag. Res. Chem., 23, 977 (1985).

138. Lansbury P.T. and Pattison V.A. , J. Am. Chem. Soc. , 85., 1886 (1963).

139. Lane C.F., Aldrichimica Acta, 8., 20 (1975).

140. Stothers J.B., "Carbon-13 NMR Spectroscopy", Ed. Blomquist A.T. and Wassermar H. , Academic Press, Inc., 270 (1972).

141. Williams D.H. and Fleming I., "Spectroscopic Methods in Organic Chemistry", 2nd Ed., McGraw-Hill Book Company (UK) Ltd., Maidenhead, 139 (1973) .

197 CHAHL'KR 4

PKEPKRKEICN OF r^CIACID CHTORIDRS AS TARFTUNG AGENTS

198 4 .1 INTROCOCTICN The lose of [ l-1-^] cyclopropanecarbonyl chloride as a labelling agent was described in Chapter 3. An important extension of this work would clearly be to develop other [11C]acid chlorides for labelling compounds (particularly amides and amines) of potential use in PET studies. Examples of such compounds are prazosin (40) (an a ^-adrenoreceptor ligand),1 melatonin (41) (a neurohormone),2'3 acetazolomide (42) (a carbonic anhydrase inhibitor),4 carfentanil (43) (an opiate receptor agonist)5 and naloxone (44) (an opiate receptor antagonist)6 (Fig 4.1).

N------N ch 3o

nh 2so2—C c — nh *cII - ch 3 o S Melatonin (41) Acetazolamide (42)

Ov CH3

Fig 4.1 Structures proposed for the carbon-11 labelling with [11C]acid chlorides

The work described in this chapter therefore had two aims. The first was to extend the method developed for the preparation of [1- 11C] cyclopropanecarbonyl chloride to other simple aliphatic [i:LC]acid chlorides. (Such [l-i:LC]acid chlorides had hitherto been neglected as potentially useful labelling agents). The second was to

199 evaluate the procedure for preparing more elaborate [11C]acid chlorides, such as [2-11C]furayl chloride and [l-1-LC]acryloyl chloride, which might serve as labelling agents for particularly interesting target compounds e.g. prazosin and naloxone. Thus the -^C-labelling of acid chlorides, namely RQ0C1, where R = Me (45), Et (46), Pr (47), c.Bu (48), vinyl (49), Hi (50) furanyl (51) and thiophenyl* (52), was investigated. In the original development of n.c.a [l-11C]cyclopropanecarbonyl chloride (7), THIQ (26) was found to be useful for trapping the acid chloride (7) as an easily detectable amide, tf-[ 1- cyclopropanecarbonyl-THEQ (33). I t was decided to apply THIQ (26) similarly in the development of other [11C]acid chlorides. For this purpose the following reference compounds were prepared: N- acetyl-THIQ (53), N-propionyl-TKEQ (54), N-butyry1-THIQ (55), N- cyclobutanecarbonyl-THIQ (56), N-acryloyl-THIQ (57), iV-benzoyl-THIQ (58), N- (furan-2-carbonyl) -THIQ (59) and N- (thiophene-2-carbonyl) - THIQ (60). Mass spectrometry and %- and 13C-NMR spectroscopy were used to characterise these compounds. THIQ derivatives are known to exhibit dynamic processes in solution (e.g. inversion in the heterocyclic ring, inversion through nitrogen and in amides restricted rotation about the C^-N bond."7-10 These can affect the NMR spectra profoundly. As a means of assisting the interpretation of the %- and 13C-NMR spectra of THIQ- amides (30, 53-60), some compounds were examined at different temperatures. In addition the correlation of carbon-proton chemical sh ifts was studied (in 2 dimensions) on N-propionyl-THIQ (54) in order to allow assignment of the spectra. In support of the above NMR studies, N-acetyl-THQ (61), AT-propionyl-THQ (62), W-butyryl-THQ (63), N - (thiophene-2 -carbonyl) -THQ (64), N-ethy1-THIQ (65), N-propyl- THIQ (66), N- (3-butene) -THIQ (67), AHbenzyl-THIQ (68), W-furfuryl- THIQ (69), N-propyl-THQ (70) and JV-butyl-THIQ (71) were synthesised and examined by NMR spectroscopy. I t was envisaged that a comparative study of these compounds might assist in understanding the dynamic processes occurring in the N-acyl-THIQ (30, 53-60) series.

200 The nomenclature thiophenyl used throughout the text refers to the thienyl group. 4 .2 n rs c n s s iC N 4.2.1 Section A; Preparation of r^CIAcid Chlorides The potential of [11C]acid chlorides as labelling agents for a number of ligands of interest has been described in Sec 4.1. Synthetic routes analogous to the formation of [l-11^] cyclopropane- carbonyl chloride were examined further with a view to preparing other n.c.a [l-11C]acid chlorides (Scheme 4.1).

11 CO, RMgBr jj311C00MgBrj

PDC + DTBP

THIQ R11COCI

0

where R = Me, Et, Pr, c Bu, Vinyl and Ph Scheme 4.1 Formation of [11 CJacid chlorides and their conversion to [11C]amides

Alkyl halides react directly with certain metals to give organometallic compounds, the most commonly used metal being magnesium for the preparation of Grignard reagents.11' 12 The order of halide reactivity is iodide > bromide > chloride.12 However, bromides are generally preferred for the preparation of Grignard reagents since they are more stable than iodides and have a higher reactivity than chlorides. The structure of Grignard reagents in solution has aroused much detailed study and discussion over the years.13'14 Schlenk and Schlenk15 proposed that in solution an equilibrium exists between the alkylmagnesium halide and a dimeric species (Scheme4.2).

201 2RMgX -c.—■'rTr- R2Mg + MgX2 ^ ^ R2Mg.Mg.X2

Scheme 4.2 Structure of Grignard reagents in solution

Extensive studies new demonstrate that the Schlenk equilibrium ex ists and that the point of equilibrium depends on R, X, solvent, concentration and temperature.16-18 The degree of association depends on the concentration of Grignard reagent. Grignard reagents prepared from alkyl bromides and iodides are monomeric in THF at all concentrations and also in diethyl ether at low concentrations (< 0.5 M) i.e. dimeric species are not present in measurable amounts and only part of the Schlenk equilibrium operates.19-21

2RMgX R2Mg + MgX2

However, Grignard reagents prepared from alkyl bromides or iodides in diethyl ether at higher concentrations (0.5-1.0 M) contain dimers, trimers and higher polymers.19'20 Those prepared from alkyl chlorides in diethyl ether are dimeric at all concentrationsi.e. the complete Schlenk equilibrium operates.19'20 For aryl Grignard reagents in diethyl ether the prominent species is ArMgX, whereas in THF both ArMgX and Ar2Mg are significant.22'23 In addition it has been shown that which ever form predominates also coordinates with two molecules of diethyl ether (Scheme 4 . 3 ) .24'25

ORo ORp ORp

R-----Mgl — X R-----Mg l — R X-----Mg l — X

ol. r 2 t.o r 2 t.o r 2 Scheme 4.3 Coordination of Grignard reagent in diethyl ether 202 Alkyll ithiums do not e x ist as monomeric species in hydrocarbon solvents or diethyl ether.26'27 Alkyllithiums are also often more reactive than Grignard reagents and under the conditions applied in "no-carrier-added" 11C-chemistry often yield a mixture of radioactive products on reaction with ^-kx^.28 Their use was not investigated here. In the lig h t of the above considerations, a ll the Grignard reagents studied (KMgX; R = Me, Et, Pr, c.Bu vinyl andFh), except vinyl bromide, were prepared in diethyl ether and at low concentrations (0.2-0.5 M). THF was used as solvent for the preparation of vinylmagnesium bromide to prevent elimination and coupling reactions.29 An important side reaction to consider in the preparation of organametallics is Wurtz-type coupling to give a symmetrical alkane of higher carbon number.12'30 This side reaction can became important i f reagents are stored. A ll Grignard reagents were therefore freshly prepared before use. [ 11C] Carbonation of a Grignard reagent generally gives a t1-^] adduct. Ashby e t a l31 proposed that the addition of Grignard reagents to carbon dioxide occurs by rapid coordination of the magnesium atom with the oxygen atom of carbon dioxide, followed by a rate-determining nucleophilic attack by the R group (Scheme4.4)

OEt, c = o 1 5~ 5+ * r r— Mg — X + o = c = O X---- YM g fr R

OEtg __ OEL,

O II Et,0 + XMgO— C — R

Scheme 4.4 Reaction of Grignard reagent with carbon dioxide Carbonation of a Grignard reagent and subsequent hydrolysis yields a carboxylic acid.11'32'33 These reactions are well characterised; control of temperature, concentration of reagent and reaction time are important for obtaining maximum yields.11'32'33

203 Refluxing conditions (i.e. high reaction temperature: > 298 K), high concentration of Grignard reagent and long carbonation times promote the formation of by-products (e.g. ketones and tertiary alcohols).11/32 The entrapment of cyclotron produced 11C02 by the prepared Grignard reagents was found to be greater than 85%, except for cyclobutylmagnesium bromide (Table 4.6). In this case incorporation of H c o 2 was found to be about 70% and then only if the bromide was redistilled just before use. Conversion of the [He]adduct into the corresponding [Hc]acid chloride was achieved by the addition of phthaloyl dichloride. The ease of isolation of the generated [Hc]acid chloride depended on boiling point. For volatile [Hcjacid chlorides 45, 46 or 47, vessel A (Fig 4.19a) containing the reaction mixture was heated to the appropriate temperature and the [Hc]acid chloride was transferred by a flow of nitrogen (pre-determined for optimum yield) without difficulty. However, the isolation of [1- llc] cyclobutanecarbonyl chloride (48) (b.p. 413 K) was more difficult; it was found necessary to heat the FIFE tubing between vessel A and B with a hot air blower to obtain efficient transfer. The r.c.y of these [Hcjacid chlorides were estimated to be in the range of 40-60% (Table 4.6) based on transfer of radioactivity. Subsequent reaction of these [11C]acid chlorides (45-48) with THIQ (26) gave the expected f1-^]amides. These were obtained in 15- 45% overall r.c.y, with N- [ l-^C] acetyl-THIQ (72) having the highest yield. The specific activity of the [^C]amides, where R is Me (72), Et (73) and Pr (74), was found to vary between 1.1 and 2.8 GBq/^mol corresponding to the presence of carrier in the range of 8-30 /ig (see Table 4.6). Lower specific activity was obtained for N- [1- ^CJcyclobutanecarbonyl-THIQ (75) (0.38-0.93 GBq/^mol, 20-80 fxq). The specific activity of -^-^C-labelled compounds depends on the amount of carrier source (e.g. C02) inadvertantly introduced into the system. The main variable in the applied synthetic route was the Grignard reagent since other factors affecting specific activity (carrier from target lines, traps etc.) were the same for all

204 [11C]acid chlorides. Therefore the lower specific activity of N-[l- 11C] cyclobutanecarbonyl-THIQ (75), might relate to problems encountered in the formation of cyclobutylmagnesium bromide. For example there might have been an ingress of air into the apparatus. t1-^]Amides where R = Me (72), Et (73), Pr (74) and c.Bu (75) were found to be radiochemically and chemically pore by analytical hplc and tic (Table 4.7). In addition the residue of carrier in each [1:LC] amide (72-75) after radioactive decay was examined by mass spectroscopy. The obtained mass spectra correspond to those of reference amides (53-56). All compounds in this series gave spectra with intense peaks for the molecular ions (Table 4.7) except where R is c.Bu and Fh. All of the compounds (53-60) also display a peak at M/Z = 132. Baldwin et al34 have reported that in the case of N- acetyl-TKEQ (53), the fragment M/Z = 132 is formed by the loss of acetyl, followed by a 1-2 hydrogen shift. The M/Z = 132 peak for all compounds was assigned analogously. However this mode of fragmentation was less important for benzoyl (58) and heterocyclic derivatives 59 and 60. In these compounds the charge is considered to be localised on the carbonyl oxygen atom and homolytic cleavage results in the formation of an acylium ion (EE) (Scheme 4.5).35

0 II - NR'R" + R ------C — NR'R" ------> R-----CEE== 0 (EE) Scheme 4.5 Formation of an acylium ion

The prominence of fragment (EE) in the mass spectra of compounds 58-60 reflects the efficient stabilisation of the positive charge in fragments of these compounds. A feature present, common to all of the mass spectra is the signal at M/Z = 104. This corresponds to a loss of the nitrogen atom and the C3 carbon atom together with substituents at these positions. This has been attributed to a reverse Diels-Alder type reaction leading to fragment ion (FF) (Scheme 4.6).34 Substantial evidence has accumulated from high resolution mass measurements and

205 studies on deuterium labelled derivatives to confirm the retro Diels- Alder reaction exhibited by THIQ and its derivatives.36'37

Scheme 4.6 Reverse Diels-Alder type reaction observed in N-acyl-THIQ componds

Isolation was unsuccessful for [l-11C]acryloyl chloride (49) and for [l-1^ ] benzoyl chloride (50). To establish the formation of [1- -^C]benzoyl chloride, 13C-enriched carbon dioxide was co-included in the synthesis and the preparation was performed in a single vessel (Fig 4.19c). A large excess of THIQ (26) (133 mg) was used in the synthesis and the isolated product was examined by 13C-NMR and mass spectroscopy. The 13C-NMR spectrum exhibited a single peak at 172.13 ppm in accord with the chemical shift assigned to the carboxamido- carbon of N-benzoyl-THIQ (58) of natural abundance (5-^yis = 171.97 ppm; Table 4.9) (see Section 4.2.3 for discussion on the assignment of % - and 13C-NMR spectra of N-acyl-THIQ (53-60)).

206 The mass spectrum shows a parent peak at M/Z = 238 (natural abundance N-benzoyl-THIQ (58) gives a parent peak at M/Z = 237) and the characteristic fragmentation pattern described above. Hence, spectroscopy confirmed the formation of N- [ 1-^3C] benzoyl-THIQ (and by analogy the corresponding 11C compound) in the one-pot synthesis. However, since benzoyl chloride has a high boiling point (466 K), isolation of [1-1:LC] benzoyl chloride (50) from the radioactive reaction mixture by distillation was difficult to achieve, even under reduced pressure (PDC also distilled over). Formation of [1-13C] benzoyl chloride (50) might be best achieved using a volatile reagent, such as thionyl chloride or oxalyl chloride, for the chlorination reaction. This could then be boiled off leaving [11C]acid chloride to react for example with excess amine, in a labelling procedure. Further investigation is required to establish the formation of [l-11C]acryloyl chloride (49), under the conditions applied. It is envisaged that these studies will involve the use of 13C-enriched carbon dioxide and subsequent NMR spectroscopic and mass spectrometric analysis. In addition an alternative route to the formation of this [i:1C]acid chloride should not be ruled out. This might involve the [ 11C] carbonation of vinyllithium rather than of a Grignard reagent. Machulla et al38 have used this approach in the formation of pyridine-2- [ 11C] carbonyl chloride, the only other [11C]acid chloride reported prior to the work described here. The successfully prepared n.c.a [l-13C]acid chlorides promise to serve as very useful labelling agents in 13G-chemistry. Already [1:LC] acetyl chloride (45) has been used as a labelling agent for a number of ligands. The neurohormone, melatonin (41) and its halogen derivative 6-fluoromelatonin have both been labelled with carbon-11 utilising [^C]acetyl chloride as the labelling agent.39'40 In addition acetazolomide, a carbonic anhydrase inhibitor, has been labelled with carbon-11 using the same approach. 41

207 4.2.2 Section B. 1he Preparation of r11C1Prazosin with r2~-u ClFurovl Chloride as labelling Agent Labelling of the a ^adrenoreceptor antagonist, prazosin, with -^C might be considered in the 7-methoxy group (with [11C] iodomethane) or in the furoyl moiety. The furoyl group is rapidly cleaved off from the piperazine ring in vivo, giving piperazine derivatives with some antihypertensive activity.1 Presumably these piperazine type derivatives could interact with the a adrenoreceptor or with non-specific sites. If labelled, these derivatives might degrade the quality of signal (specific receptor binding of [^C]prazosin) that might be obtained in PET. Therefore it was of interest to label in the furoyl position to avoid metabolism to labelled piperazine derivatives (Scheme 4.7).

N CH30 N N H CH30 (74) nh 2

COCI

Scheme 4.7 Carbon-11 labelling of prazosin with [2-11C]furoyl chloride

Two possible routes were investigated for the formation of 2- furylmagnesium bromide. The first utilised 2-iodofuran. This

208 compound was synthesised from 2-chloromercurifuran as reported by Gilman et al.42 However this approach was found to be unsatisfactory due to the instability of 2-iodofuran, which on standing converted into an insoluble resin.43 The second approach required the synthesis of 2-bromofuran.4 3 This was achieved by decarboxylation of 5-bromofuroic acid with copper chromite as catalyst in quinoline. The reported procedure43 used copper powder as the catalyst and required higher temperature (503-508 K) for decarboxylation. At these higher temperatures traces of quinoline are transferred with the 2-bromofuran thus preventing the formation of the Grignard reagent. The use of copper chromite enabled the decarboxylation to be achieved at 473 K. To reduce the decomposition of 2-bromofuran anhydrous diethyl ether was added immediately after distillation and the solution was stored over sodium wire under nitrogen at 273-277 K. 2-Furylmagnesium bromide was synthesised by the reaction of 2-bromofuran with magnesium turnings in diethyl ether. Carbonation of 2 -fury lmagnes ium bromide with [12C/13C]carbon dioxide followed by hydrolysis gave 2-[12C/13C]furoic acid as shown by % - and 13C-NMR spectroscopy. The prominence of the carbonyl signal in the 13C-spectrum indicated that the molecule was labelled in this position. The preparation of [2-11C]furoyl chloride (51) was analogous to the preparation of the other [11C]acid chlorides described in Sec 4.2.1. (This work was done in collaboration with Dr E. Ehrin at Commissariat a l'Energie Atomique, Service Hospital ier Frederic Joliot, Orsay, France). [2-11C]Ruroyl chloride (51) has a boiling point of 447 K and trace amounts of PDC transferred during distillation thus competing for the amine (76). Thus the minimum amount of PDC (2 fil) that gave optimal yield of [2-11C]furoyl chloride was added to the carbonated Grignard reagent. To achieve efficient transfer of the generated [2- 11C]furoyl chloride, the vessel containing the radioactive reaction mixture and the tubing connecting the two vessels (apparatus similar to Fig 4.19a) were heated with an electric heater (613 K ) .

209 The reaction between [2-i:LC]furoyl chloride (51) and 2- (piperazin-l-yl)-4-amino-6 ,7-diirethoxy-quinazoline (76) (gift from Pfizer) gave [11C]prazosin (77) (Scheme 4.7). [2-11C]Furoyl chloride reacts preferentially at the secondary amino group rather than at the primary amino group. Possibly the primary amino group is rendered less reactive by partial delocalisation of the lone pair electrons over the quinazoline ring. [1:LC] Prazosin was obtained in 30-40% overall r.c.y with specific activity in the range of 26-37 GBq//xmol (0.7-1.0 Ci//imol) at EOS. The samples were found to be radiochemically and chemically pure by normal and reverse phase hplc and tic. In addition the position of the carbon-11 label was confirmed by 13C-NMR study of 13C labelled material. The 13C-NMR spectrum exhibited a single peak at 8 158.6 in accord with the chemical shift assigned to the carbonyl atom in authentic prazosin.

210 4.2.3 Section C. Assignment of 1H- and -^C-NMR Spectra of ?/-Acvl- THIO Ocppounds 4 .2.3.1 Pnp>1 imirwryrto^rvatlons in the %-NMR spectra of N - a c v l - m io Normally in the %-NMR spectra of N-acyl THIQ's, a singlet would be expected for the isolated C1-CH2 and two triplets for the adjacent C3 and C4-CH2 protons. Compounds 59 (R = furanyl) (Fig 4.29) and 60 (R = thiophenyl) (Fig 4.30) exhibited the expected pattern although in 59, C1-CH2 was broad. In contrast, the low field (80.9 or 89.6 MHz) %-NMR spectra of the N-acyl-THIQ series where R = Me (53), Et (54), Pr (55), c.Pr (30), c.Bu (56), vinyl (57) and Hi (58), were found to exhibit either two resonances or broad and featureless signals for the nitrogen ring C^, C3 and C4 methylene protons. Sharp signals were only observed for the aromatic ring and the R substituent protons. A typical example is the %-NMR spectrum (80.9 MHz) of N-cyclopropanecarbonyl-THIQ (30) at ambient temperature (Fig. 4.20). In NMR spectroscopy, broadening and doubling of signals has been related to time-dependent processes44”46 and in particular to compounds which interconvert between distinct forms. These dynamic processes can often be elucidated by variable temperature NMR- spectroscopy.44-46 Consequently two compounds of interest, 30 (R = c.Pr) and 60 (R = thiophenyl) were chosen for variable temperature NMR studies which are reported in Sec 4.3.3. The dynamic processes that might give rise to the "doubling-up11 effect {i.e. the two resonances observed for a particular proton (s) or carbon (s)) observed in the N-acyl-THIQ series (30, 53-60) will now be discussed briefly.

4.2.3.2 EVnamic processes For the N-acyl-THIQ series three dynamic processes7-10 can be considered, namely i) restricted rotation of the acyl group about the C^-N bond ii) inversion of the saturated ring iii) inversion through nitrogen.

211 These processes m y occur in combination.

Restricted rotation about the C^q -N bond Simple amides such as N,N-dimethylfomamide,4 7 '48 N,y-dimethyl- acetamide47'48 and N ,N-propionamide48 exhibit a doublet for the N- methyl protons at 302 K (Fig 4.2).

R. CH< CH< \ / ” \ / C ------N C ------N

S ' \ f o ' c h 3 O \ CHr (GG) (HH) Fig. 4.2 Two possible conformers of N,N-dimethyformamide This doublet must arise because the magnetic environments of CH3A and CH33 are non-identical even though the two methyl groups are chemically indistinguishable. Exchange of CH3A in forms (GG) and (HH) takes place via rotation about the C^-N bond.47'48 The C^-N bond displays partial double­ bond character49 because a lone pair of electrons on nitrogen can delocalise into the 71--system of the carbonyl group, thus generating a 1,3-dipole with nitrogen bearing a partial positive charge and oxygen a partial negative charge (Fig 4.3).

nr->1 ------^r* 7' 0 n 1, c £ ------N N+-

=3 ( ^ ) R3 R2

Fig 4.3 Delocalisation of the lone pair of electrons on the nitrogen

X-ray diffraction studies50 of crystalline amides have shown that the amide structure is planar with bond lengths approximately midway between those expected in the two contributing canonical forms (JJ) and (KK). Therefore it is reasonable to assume that the resonance hybrid (LL) (Fig 4.4) will have limited rotation about the

Cq -j-N bond.

212 o

R C : N Rc / R* (LL)

Fig 4.4 Resonance hybrid

A consequence of lack of free rotation about the C^-N bond is the existence of conformational isomers (M l) {E or cis} and (NN) (Z or trans } (Fig 4.5). In conformer (M l) the nitrogen substituent R2 is cis to the oxygen atom while the nitrogen substituent R3 is trans and vice-versa for conformer (NN) .

O O /•* R 1 C : R2 R3 \\/ N

(MM) (NN) R"

Fig 4.5 Conformers assigned cis or trans

The two substituents on the nitrogen, experience different electronic environments and give signals with distinct chemical shifts, provided that rotation about the C^-N bond is too slow to average out the signal on the NMR time-scale.

The concept of limited rotation about the Cc q -N bond in simple amides can also be applied to the W-acyl-THIQ series (30, 53-60); this series of compounds structurally resembles the simple amides already discussed. In these compounds a cyclic nitrogen system, THIQ, is adjacent to a carbonyl group bearing an R substituent (Fig 4.6). If we consider that the W-acyl group is a handle by which

rotation about the C^q -N bond can occur, it is reasonable to assume that the lone pair of electrons on the nitrogen will be in plane with the carbonyl group to generate partial double-bond character. The degree to which this delocalisation occurs in these amides warrants further investigation and is not in the scope of this thesis.

213 A lack of free rotation about the Cq q -N bond in this series of compounds would yield two distinct conformers (I and II) (Fig 4.6) as for simple amides.51 Conformers are designated with respect to the Cj_-carbon and the R substituent. When the R substituent is cis to the C-L-carbon the conformer is referred to as E (I) and when the R substituent is tr a n s to the C^-carbon it is referred to as the Z (II) conformer.

Fig 4.6 Two possible conformers of N-acyl- THIQ compounds due to restricted rotation about the C ^-N bond

R i m inversion It is known that in alicyclics such as cyclohexane, and many of its derivatives, that the ring is puckered and free from angle strain.52 Though conformers interconvert, cyclohexane and many of its derivatives exist predominantly in a chair conformation in which steric repulsion is minimised. A double bond in a six-membered ring, such as that in cyclohexene, can cause the ring to assume a half-chair conformation (00)12 (Fig 4.7), in which, C4 and C5 lie on opposite sides of a plane containing C5-C1-C2-C3 and the double bond.53 On each carbon one bond is axial and the other is equatorial. The cyclic systems are non-rigid and chair-chair interconversions occur easily.53 Ha

Ha Fig 4.7 Cyclohexene : half-chair conformation 214 Ring reversal in piperidine has been studied extensively.54'55 The process of chair-chair ring reversal (Fig 4.8) converts the ring into its mirror image and interchanges the axial or equatorial nature of all substituents. Thus different chemical shifts may be obtained for the axial and equatorial substituents at a particular carbon.

Fig 4.8 Chair-chair ring reversal in piperidine

In the case of THIQ (26), Booth56 reported that the half-chair conformation would generally be adopted by the heterocyclic ring in preference to a half-boat conformation owing to the lower repulsive non-bonded interactions involved in the former. He concluded that the heterocyclic ring is not a rigid half-chair but a rapidly inverting half-chair (Fig 4.9).

Fig 4.9 Half chair ring reversal in 1,2,3,4-tetrahydroisoquinoline

Sugiura et al^ have studied ring inversion processes in N- alkyl-THIQ derivatives. They propose that in these compounds ring reversal is related to those in piperidines. Similarly the //-acyl-THIQ (30, 53-60) series reported in this thesis might be compared with the already studied piperidine and THIQ and //-alkyl-THIQ systems. By analogy it is reasonable to assume that the heterocyclic ring in the N-acyl-THIQ series can adopt half-chair conformations capable of ring reversal equilibria (III and IV) as shown in Fig 4.10.

215 COR

in IV

Fig 4.10 Two possible conformers of N-acyl-THIQ compounds due to ring inversion

Nitrogen inversion N-Cyclic systems containing a trivalent nitrogen with a lone pair of electrons are known to undergo nitrogen inversion.44 The lone pair of electrons oscillate from one side of the XYZ plane to the other thus converting the molecule from one conformer to another.

N Z X N

Several examples of nitrogen inversion have been cited for piperidine and its derivatives44'54'55 (Fig 4.11). Sugiura et al7 have suggested that nitrogen inversion in N-alkyl-THIQ occurs similarly.

N R N

Fig 4.11 Nitrogen inversion in piperdine and derivatives

Since the heterocyclic ring in the N-acyl-THIQ (30, 53-60) series contains a nitrogen atom with a lone pair of electrons,

216 nitrogen inversion can be considered for these compounds. In one conformation (V) the N-acyl group (ROC) will be axial and in the other conformation (VI) it will be equatorial to the nitrogen ring (Fig 4.12).

COR

Fig 4.12 Two possible conformers of N-acyl-THIQ compounds due to nitrogen inversion

To asses which of the above processes occurs in the N-acyl-THIQ (3 0 , 53-60) series, it was necessary to determine at which atoms changes occur and the rates of interconversion of conformers from the NMR data. These data can also be used to calculate free energies of activation, AG* , for interconversions in these molecules which might then be compared with values known for each type of dynamic process.

Determination of rate constant and free energy of activation The % - and 13C-NMR spectra of the N-acyl-THIQ series indicate that these compounds interconvert between two unequally populated states, A and B (assumed to be in equilibrium), with unequal populations, PA (the fraction of molecules in state A) and Pg (the fraction of molecules in state B), respectively.

217 represents the rate constant for conversion of conformer A into conformer B and kB represents the rate constant for the reverse process i.e. at equilibrium k^P^ = kgPg Interconversion between states A and B are noticed by NMR experiments if the process is sufficiently slow, i.e. lie within the NMR time-scale.44'45 The rate of interconversion can be varied for the NMR experiment by changing temperature. For instance if a broad signal is observed for the molecule at ambient temperature an increase of temperature will tend to sharpen the signal to a singlet whereas decrease of temperature gradually resolve the broad signal into two sets of resonances. The absolute rate theory developed by Eyring57'58 allows the free energy of activation AG* of a transition (e.g. A to B or B to A) to be calculated from Equation 4.1

fcBT k = k --- exp (-AG*/RT) Eq 4.1 h

where k is the first-order rate constant for the transition, k is the transmission coefficient (the fraction of all molecules in the transition state that convert; this value depends on the nature of the process but is often taken as unity). kB is Boltzmann1 s constant, h is Plancks constant, R is the universal gas constant and T is absolute temperature. In principle, the first order rate constant k can be determined from variable temperature NMR experiments since the rate of interconversion from state A to state B depends on absolute temperature.44'59 Where two sets of signals are observed the rate of interconversion between A and B is said to be slow2'17'18, i.e. the life-time (r) of each state is long. At intermediate lifetimes the signals are broad and intermediate rates of exchange are expected. In the case where the life-time of each state is exceptionally short, (i.e. the rate of interconversion is fast) then the spectrometer can 218 only distinguish an average environment and this will give rise to one signal with an averaged chemical shift i/av, which is given by Equation 4.2.

uav = PA UA + PB UB Eq 4.2

The averaged coupling constant J av is then given by Equation 4.3.

Jav = P^ J^ + Pg Jg Eq 4.3

The temperature at which the two resonances merge and no observable valley between the two signals exists (Fig 4.40) is known as the coalescence temperature (Tc) ,44'45 If it is assumed that the populations of the A and B states are equal (i.e. PA = Pg) then from the coalescence point, the rate constant, kc (which will equal kA or kB) of the exchange at the coalescence temperature can be determined by Equation 4.4.

pc k~ = ------Au Eq 4.4 1.414 where Au is the difference in chemical shifts of the two states, i.e. Hz at Tc anc^ Pc is the population of the A or B state.

If kc and the numerical values of all constants are substituted in Eyring's Equation (4.1), expression 4.5 is obtained. This allows the free energy of activation AG* c to be calculated.

AG*c = aTc {9.972 + log (^/A v)) Eq 4.5

a = 1.914 x 10"2 (AG* c in kJ mol-1)

Equation 4.5 applies only to systems where the populations of the A and B states are equal.44'59 For exchanging systems with unequal populations, where AG* for A to B (AG*^) or B to A (AG* g) are

219 not equal, two approximate methods exist for obtaining free energies of activation for the exchange. For the first of these methods, Mannschreck60 stated that at coalescence, where two unequally populated signals merge, the lineshapes depend not only on the difference in chemical shifts (Aw) but also on the equilibrium constant (K^) and the line-width resulting from exchange broadening. For the JV-acyl-THIQ series, the equilibrium constant and the line- widths were unknown at the coalescence temperature and therefore this method was not applicable. The second approximate method, developed by Shanan-Akidi and Bar-Eli,61 involves a graphical treatment. They proposed that Equation 4.6 applies at coalescence:

3 /2 P h - P B = AP Eq 4.6 3 X

where PA and PB are the populations (which correspond to the intensities of NMR signals) of states A and B and X = 27rrcAv where rc is the mean lifetime of the two states at the coalescence temperature i.e l/rc = l/rA + l/rB (rA is the lifetime of state A at the coalescence temperature and rB is the lifetime of state B at the coalescence temperature). The authors obtained a graphical relationship (Fig 4 .1 3 ) between calculated values of t^Aw and AP at coalescence. From known values of Aw and AP, r c can be obtained. For example, when AP = 0 .4 , t q A u = 0 .3 4 7 and for a given A w , r c can be calculated. The rate constants kA and kB of the exchange for A to B and B to A can then be obtained from Equations 4.7 and 4.8.

2 kA = --- (1 - AP) Eq 4.7 2 Tc

1 kB = --- (1 + AP) Eq 4.8

2 t c 220 When the rate constant /cA or &B and Tc are known Equation 4.9 can be used to calculate AG* A or AG* B (see Appendix 5 for derivation of Eq 4.9 from Eq 4.1).

AG*a = aTc [10.319 + log (Tc/kA)] Eq 4.9

a = 1.914 x 10“2 (AG* A is in kJ mol”1)

The accuracy of the derived AG* values for state A or B depends on the accuracy with which the coalescence temperature (Tc) is measured and to a lesser extent on the error in the rate constant determination. An error in Tc of ± 2 K corresponds to an error in AG* of ± 0.5 kJ mol-1 whereas there would have to be an error of ca. 20% in k to have the same effect.44 The accuracy of the Tc values reported here were ± 5 K.

AP = Pa - P b Fig 4.13 Relationship between A? and at coalescence (Reproduced from Oki}44 221 For N -^^clcpropanecarbonyl-THIQ (30) (Fig 4.40c) and N- {thiophene-2-carixinyl} -THIQ (60) variable temperature proton spectra were used to determine the coalescence point of the protons. Integrated signal intensities for C1-CH2 protons were used to determine the fraction of conformers. The difference in fraction of conformers (AP = P^-Pg) was then used to obtain a value for (from Fig 4.13) from which rc was determined (see Table 4.1). Equations 4.7 and 4.8 were used to determine kA (the rate constant for A to B) and kg (the rate constant for B to A) and Equation 4.9 was used to calculate the free energy of activation for state A to B (AG* A) and for state B to A (AG* g). The results are presented in Table 4.1.

Table 4.1. Conformational parameters for oonpounds (30) and (60)a'b

Cmpd TC s (%) AP T&U 'cc kA kB AG* ^ AG* g Ci -CH2 (K) (ppm) (Hz) (ms) (S-1) (kJ mol-1)

(30) 303 4.87 4.55 0.2 0.3 10.5 38.2 57.4 65.1 64

(60) 263 4.93 4.85 0.18 0.29 14.7 27.8 40.7 56.9 56

a) all parameters are quoted as being at the coalescence temperature

b) the coalescence temperature is defined for these experiments as the temperature at which the two signals are just resolvable. In this respect the determined AG* A and AG* g are in error. However, the difference between AG* A and AG* g is of the same order of magnitude for an error of ± 5 K. For example for compound 30 if T*-. were 298 K then AG* A = 63.95 kJ mol-1 and AG* B = 62.94 kJ mol . This gives a difference of 1010 J mol-1, similar to the difference at 303 K i.e. 900 J mol"1. Therefore the differences in AG* A and AG* g for compounds 30 and 60 suggest that state A converts less readily to state B than state B converts to state A

c) the mean lifetime of the two states at the coalescence temperature 222 Comparison of reported and calculated values for energy barriers to rotation about the C ^ - N bond, ring inversion and nitrogen inversion

The energy barriers to rotation about C^q -N bonds (hereafter referred to as barriers to rotation) in seme simple and heterocyclic amides are reported to lie within the range 50-90 kJ mol”1 (Table 4.2). The AG* values for ring inversion in piperidine and in some of its alkyl derivatives are in the 40-50 kJ mol”1 range, as is that for cyclohexane (Table 4.3). Reported barriers to nitrogen inversion in six-membered heterocyclics lie in the 25-40 kJ mol”1 range (Table 4.4)

Table 4.2. Barriers to rotation about C^q -N bond in selected amides

Compound AG* TC Solvent Ref (kJ mol"1) (K)

N, N'-dimethyl formamidea 87.45 392 Neat 62

N, N -dimethylacetainidea 76.15 298 63

N, N -dimethylbenzoylamide3 62.76 285 CD2Br2 48 l-acetyl-4-methylpiperidine 68.62 330 CDCI3 64

1-benzoylpiperidine 62.00 292 CDCI3 64 iV-formtylindoline 78-79 65,66

N-acetylindol ine 63-64 65-67

N-formyl-THQ 75-76 308 EXlSO-d^ 65,68

//-acetyl-THQ 50-51 236 CDCI3 65,68

W-acetyl-l-methyl-THTQ^'c 60.4 298 CDCI3 10

a) No ring inversion possible b) AG* value calculated (from Eq 4.5) using chemical shifts and coalescence temperature reported in the publication10 c) AG* values calculated (from Eq 4.5) for other //-acyl-l-methyl-THQ were 60.5 ± 0.5 kJ mol”1 at 298 K when R O O = CH2CH3 , n-C3Hy, i-K^Hy. However when R O O = t-K^Hg, AG* = 48.3 kJ mol”1 at 233 K 223 Table 4.3. Barriers to ring inversion

Compound AG* Tc Solvent Ref (kl mol”1) (K)

Cyclohexane3 43.09 203-253 Neat 69

Piperidine3 43.51 210 CD3OD 54,70,71

W-methylpiperidinek 50.21 245 CD3OD 54,70,71

N-t-butylpiper idine*3 46.86 233 CD3OD 54,70,71

a) Ring inversion only b) Ring inversion and/or nitrogen inversion

Table 4.4. Barriers to nitrogen inversion

Compound AG* Tc Solvent Ref (kJ mol-1) (K)

Piperidine 25.52 131 CHFC12-CF2c1 72

1 ,2 ,2 ,6-tetramethyl- 38.07 213 c f 2c i 2 73 piperidine

N-methyl-4-piperidone 36.02 180 c h f c i 2 74

In summary examination of the literature AG* values for six- membered heterocyclics suggest that the barrier is highest for

restricted rotation about the C^q -N bond, indicating that the rate of interconversion between two states is slower in comparison to ring and nitrogen inversion and also that the rate of ring inversion is slower to nitrogen inversion. 224 Assessment of the dynamic process involved in N-acvl-THIO series The rate constants kA and kg and the free energies of activation AG*A and AG* B calculated for compounds 30 and 60 are shown in Table 4.1, together with the difference between AG*A and AG*B. There is a difference of approximately 900 J mol”1 between AG* A and AG* B for conpound 30 and a difference of 1100 J mol-1 between AG* A and AG* B for compound 60. These are broadly similar to those calculated from published data for N-acyl-l-methyl-JIHIQ's10 (Table 4.5).

Table 4.5. Rate constants and free energy of activation calculated for ^-acyl-l-methyl-THIQ' s.a

Acyl group Tc kA kB AG* a a g * b AG* a — AG* B

(K) s”1 kJ mol”1 J mol”1

c h 3g =o 298 88.9 139.1 61.9 60.7 1200

i C3H7C=0 298 58.6 119.1 62.9 61.1 1800

t C4H9O O 233 18.2 64.5 50.9 48.5 2450

a) All values calculated from data published by Potapov et al10

The AG* A and AG* B values determined for compounds 30 (R = c.Pr) and 60 (R = thiophenyl) (Table 4.1) can be compared with literature values for restricted rotation (Table 4.2), ring inversion (Table 4.3) and nitrogen inversion (Table 4.4). The published values for restricted rotation about the C^-N bond were calculated in the majority of cases by application of Equation 4.5 where differences in conformer populations is not considered. Thus errors in the estimated AG* c values will exist. The AG* A and AG* B values obtained for compound 30 is within the range observed for energy barriers to restricted rotation about the

Cc q -N bond in related compounds. However, the AG* A and AG* B values for compound 60 (R = thiophenyl) are lower and close to those for

225 ring inversion. Contribution from nitrogen inversion was probably negligible in compounds 30, 53-60 since the range of barriers g I

in Table 4.4 are considered to be below that detectable by % - l

13C-NMR spectroscopy3 '54'72 at ambient temperature. This process i not therefore considered any further for the N-acyl-THIQ series (30, 53-60). To asses which of the two possible processes (i.e. ring inversion or restricted rotation) was being observed in the N -acyl- THIQ series, all of the 1H- and 13C-NMR spectra of these compounds were examined together. If we consider the Cy-C&2 proton resonances in the ^-H-NMR spectra of compounds 30, 53-60 and the and C3 carbon signals of these compounds in the 13C-NMR spectra, the signal patterns observed at ambient temperature are those shown in Fig 4.14.

Substituent ^-250 MHz -^C-62.9 MHz R CpCH2 Slow Long C, and C3 i A

Me, Et.Pr

c.Pr, vinyl

Ph

Furanyl

Thiophenyl V Fast Short Fig 4.14 Signal patterns observed for the Cr CH2 and C, and C3 carbons in the ’H- and ,3C-NMR spectra of N-acyl-THIQ series

226 It is apparent frcan the proton signal pattern that the rates of exchange (k) depend on the R substituent. Two resonances were observed for R = Me, Et, Pr, c.Pr and vinyl Implying slow interconversion between two discrete states of long life-time (r). Broad and partially resolved signals, implying intermediate rates of exchange and lifetime, were obtained for R = Eh. Only singlet behaviour was observed for compound 60 (R = thiophenyl), indicating fast rate of exchange and short life-time. Studies on a series of acyclic amides3 (R00N(013)2 / v^iere R = alkyl, phenyl and vinyl) have indicated that the energy barrier to rotation decreases as the bulk of R substituent increases. This alteration in the barrier to rotation has been ascribed to a combination of steric and electronic effects which vary with the nature of the substituent.3 '64'75 An R group may exert an effect sterically by physically hindering rotation or electronically (by influencing the nature of the C^-N bond). Steric effects may also indirectly influence the character of the C^-N bond. For example, near planarity in the amido group relative to the heterocyclic ring is required for the formation of a partial double bond; conceivably this may be sterically hindered. It has been shown that the barrier to rotation in //,//- dimethylbenzamide is much lower than that in //,//-dimethyl - isobutyramide.75 Since the phenyl group is inductively electron withdrawing it should enhance the barrier to rotation about the Cc q -N bond. However conjugation between the phenyl ring and the carbonyl group reduces the double bond character of the C^-N bond through the contribution of canonical forms (Fig 4.15). This leads to lowering of the barrier to rotation. This effect may explain the enhanced rate of interconversion in compound 58 (R = Ph). A similar explanation might apply to compounds 59 (R = furanyl) and 60 (R = thiophenyl). In the case of compound 57 (R = vinyl) where the extent of conjugation would be expected to be less, the energy barrier was expected to be between that of compound 58 and that of compounds 53, 54 and 55.

227 CH / 3 C— N c= N \ / \ / \ ch3 o ch3 o ch3

Fig 4.15 Canonical structures of N.N-dimethylbenzamide

The changes in the 13C signals for C^_ and C3 carbons appear to mirror those observed in the %-NMR spectra, although they were slightly offset due to the different operating frequencies. Examination of the complete 13C-NMR spectra of the N-acyl-THIQ series (Fig 4.31-4.39) revealed that the carbons in these compounds undergo differential broadening. This was particularly apparent in spectra for compounds 58-60 (Fig 4.37-4.39). Here the signals for the C± and C3 carbons were very broad for compound 58 and extremely broad for compounds 59 and 60. The C4 carbon signals were also broad but in 59 and 60 were much less so than the and C3 carbon signals. In contrast, the signals from the rest of the molecule in these compounds 58-60 were extremely sharp and not doubled up. This suggested that the C]_, C3 and C4 carbons were being influenced by a process other than restricted rotation about the C^-N bond, since this process would be expected to affect all carbons equally, as in compounds 53-57. Probably the C^, C3 and C4 carbons in compounds 58-60 were being affected by ring inversion. As can be seen from Fig 4.10, ring inversion causes major disturbance at the and C3 carbons and to a lesser extent at the C4 carbon.7 This effect was clearly reflected by the differential broadening observed for those carbons in the 13C-NMR spectra of compounds 58-60. As noted earlier the AG* A and AG* B values (56.9 and 56.0 kJ mol”1) determined for compound 60 were close to the range (40-50 kJ mol”1, Table 4.3) previously reported for ring inversion. Free energies of activation were not determined for the simple alkylamides (53-55). Based on the appearance of the % - and 13C-

228 NMR spectra (i.e. linewidths) for the C1-CH 2 group and the variation between compound 30 (R = c.Pr) and compound 60 (R = thiophenyl) (ca. 10 kl mol-1) the simple alkyl amides were expected to have AG* values fairly close to that for compound 30. The effects observed in the JV-acyl-THIQ series may be interpreted as follows. In compounds 53-57, probably both restricted rotation about the C^q -N bond, ring inversion and nitrogen inversion occurs. The two latter processes are most probably too rapid to be observed under the NMR conditions used. It seems therefore that the observed process under these conditions is restricted rotation about the C^-N bond. In compounds 58-60 ring inversion may be sufficiently slow due to the increased bulk of the R substituent to be manifested as signal broadening in the 13C-NMR spectra of these compounds. Also the barrier to restricted rotation might be so low that its influence on the appearance of the spectra would not be observed at ambient temperature. The differential broadening of the C^, C3 and C4 carbons may result from ring inversion only. Rapid ring inversion accounts for the absence of an AB pattern in the %-NMR spectra for the C^-C^ protons in all of the iv-acyl- TTCEQ compounds examined. Even when the rate of ring inversion was slowed down to manifest itself in the 13C-NMR spectra of compounds 58-60, the proton spectra were unaffected. Indeed, the %-NMR spectrum of compound 60 at 213 K did not reveal an AB pattern and a much lower temperature might be needed for its observation. AB coupling was unlikely to be fortuitously zero for all the compounds and conditions studied. However a temperature of 213 K was sufficient to slow down the rotation about the C^-N bond to allow observation of the two conformers in both and 13C-NMR spectra (Fig 4.42a-g). As the temperature was lowered the contribution (to all carbons) from rotation increased as did that from ring inversion (mainly to C^, C3 and C4). As a result of two processes (restricted rotation and ring inversion) occurring at rates which are beginning to approach and possibly overlap, the situation was complex and

229 difficult to interpret {especially for different observational ( % and 13C) frequencies}. To obtain further information on the dynamic processes occurring in the w-acyl-THIQ series, the effect on the NMR spectra of changing the position of the nitrogen in the heterocyclic ring (position 1) and also the nitrogen ring substituent was examined. A number of N-acyl-THQ (61-64), N-{CH2R}-THIQ (65-69) and W-{CH2R}- THQ (70, 71) derivatives were synthesised and their % - and 13C-NMR spectra examined. It was anticipated that in the IV-acyl-THQ series introduction of a bulky substituent on the carbonyl carbon atom might prevent rotation about the C^-N bond and thus allow the other two processes, ring inversion and nitrogen inversion, to be observed in the NMR spectra. In addition it was considered that in the w-{CH2R}- THIQ and N-{CH2R}-THQ compounds, where rotation about the C^-N is absent and only ring and/or nitrogen inversion can occur, useful information might be obtained on how these processes appear in NMR. The %-NMR chemical shifts of the N-acyl-THQ series 61-64 and 29 {where R = Me (61), Et (62), Pr (63), c.Pr (29), thiophenyl (64)} at ambient temperature in CDCl3 at 250 MHz are given in Table 4.12. The ^H-NMR spectra of N- {thiophene-2-carbonyl} -THQ (64) is shown in Fig 4.43. Sharp proton signals were observed for all compounds and no indication of broadening or doubling up of resonances was present. The 13C chemical shifts of these compounds in CDCI3 at 62.9 MHz are given in Table 4.13. The 13C-NMR spectra of compounds 61 and 64 are shown in Figs 4.45 and 4.46, respectively. Several interesting features were exhibited by compound 61. Very broad signals were observed for the C2 and C8a carbons whereas an extremely broad signal just visible above the noise level, was observed for C4a. In contrast the other carbon signals were sharp and displayed no doubling up effect. In the case of compounds 62 (R = Et) and 63 (R = Pr), C2 and C8a carbons were broad but less so than in compound 61, whereas C4a was still extremely broad. Again the other carbon signals were sharp. All of the carbons in compound 29 (R = c.Pr) and 64 (R = thiophenyl) exhibited sharp singlets.

230 In order to observe the dynamic processes occurring in the N- acyl^IHQ series, compounds 61 and 64 were examined at lower temperatures. The ^H-NMR spectrum of compound 61 at 213 K (Fig 4.47; chemical shifts in Table 4.12) shewed that the heterocyclic ring CH2's and the acetyl methyl resonances were all doubled up. The lower field component (8 3.83) of the C2-CH2 resonances was the more intense and vice versa for the Cg-CH^i multiplets (8 1.9-2.05). The aromatic ring multiplet also exhibited more fine structure but the signals were too complex to discern any doubling up effect. In the case of compound 64 slight broadening and loss of fine structure for all proton signals was observed on going from 273-213 K; the aromatic protons appeared to be the most affected. However the 13C-NMR spectrum of this compound at 223 K exhibited slight broadening of C2 (8 43.97) and C4a (8 132.39) carton signals only. A number of -^H-NMR studies have been reported on //-formyl, N- acetyl-THQ and their derivatives.65'68'76”79 However few 13C-NMR Spectra65,68 been reported. //-formyl-THQ (78; chemical shifts in Table 4.12) exhibited two resonances for the formyl and C g-H protons at ambient temperature.65 Compounds 29, 61-64 displayed singlet behaviour at this temperature although compound 61 exhibited two resonances for some protons at 213 K (Fig 4.47). The low field component (8 3.83) of the C2-CH2 multiplets has been assigned to the E conformer ( O O is c is to C2 carbon) and the higher field component (5 3.74) to the z conformer.65 '6 8 '80 The E conformer was found to be predominant (80%), in agreement with published data on this coirpound65'68 '80 and the //-formyl derivative.65'68'80 However the latter compound has the greater preference for this conformer (90%).65'80 This has been attributed to steric interactions between the C g -H proton and the acyl substituent.68'79'80 In conformer E, the acetyl group is able to deviate from the plane of the aromatic ring due to flexibility in the heterocyclic ring. Consequently conjugation of the lone pair of electrons on the nitrogen with the n electrons of the carbonyl group decreases. Thus rotation about the CgQ-N bond is less restricted. The 13C-NMR spectra of the N-acyl-THQ series (29, 61-64) showed

2 3 1 that the C2, C8a and C4a carbon signals were sharpening on going from conpound 61 through to 29 to 64 (whilst the other carbon signals remained unchanged). It seems most likely that the rate of rotation about the C^-N bond is reduced as the bulkiness of the acyl substituent is increased (thus allowing a compound with a bulky group to exist in one conformation). This concept is supported by the low temperature % - and 13C-NMR study performed on conpound 64 where only slight broadening of C2 and C4a carbon signals was observed. This indicated that the rate of ring inversion may have been reduced. Thus the singlet behaviour observed in the % - and 13C-NMR spectra of compound 64 can be ascribed to the presence of one conformer. This was in contrast with the TKEQ analogue (60) where singlet behaviour was attributed to rapid rotation and/or ring inversion processes. In the % - (Tables 4.14 and 4.16) and 13C- (Tables 4.15 and 4.17) NMR spectra of both N-{CH2R}-THIQ (34, 65-69) and W-{CH2R}-THQ (70, 71) compounds, the signals were sharp and no indication of broadening or doubling up of signals was observed at ambient temperature. Probably under the conditions used both ring and nitrogen inversion was too fast to be observed. However these observations suggest that in the spectra of the N-acyl-THIQ series (30, 53-58) the dominant process giving arise to the doubling up effect is restricted rotation about the C ^- N bond which can manifest itself at ambient temperature.

4.2.3.3 Assignment of conformers in the % - and 13C-NMR spectra of //-acvl-THIO ccmpounds The ^-H-NMR spectra of compounds 30, 53-58 in CDCI3 at ambient temperature (Figs 4.21-4.28) exhibited two singlet resonances for the C1-CH2 protons and two triplets for the C3-CH2 protons. In the case of compounds 59 and 60 the %-NMR spectra in CDCI3 showed only one singlet and one triplet for these protons at ambient temperature (Figs 4.29 and 4.30). However conpound 60 exhibited two resonances for the C^-CH2 protons at 263 K and lower temperatures (Table 4.10). The ^H-NMR spectrum of compound 59 was only obtained at ambient

232 temperature. The -^-H-NMR spectra of all of the compounds studied in CDCI3 , except (30) (R = c.Pr) and (60) (R = thiophenyl) showed that the more intense component of the C1-CH2 doublet was at a lower field relative to the less intense component. Whereas the C3-CH2 protons exhibited the more intense component at a higher field than the less intense component. In the case of compounds 30 and 60 the more intense component of the C1-CH2 protons was observed at higher field to the less intense component. The C3-CH2 signals had the more intense component at a lower field, although the signals were generally overlapped for compound 60 even at low temperatures (> 223 K). The 13C-NMR spectra of compounds 30, 53-58 (Figs 4.31-4.37) in CDCI3 at ambient temperature exhibited four signals for the and C3 carbons, although for compound 58 they were very broad and not fully resolved. The two central signals were the most intense in all cases. Compounds 59 and 60 gave extremely broad signals for these two carbons (Figs 4.38 and 4.39), which in the case of compound 60 resolved into four sharp signals at 223 K (Fig 4.42g), the central two again being the more intense components. Although the proton spectra could be easily assigned in terms of multiplicity and chemical shifts for the different proton groups, some difficulties were encountered in the assignment of 13C signals particularly pairing of signal components (and correlation to proton signal pairs). In addition a further complication which arose in the assignment of the and C3 carbons in the 13C-NMR spectra was the discrepancy that exists in the chemical shift values reported for these carbons in the THIQ system.81-83 Hughes et al81 assigned the carbon as 8 48.2 and C3 carbon as 8 43.8 whereas Singh et al83 gave an opposite interpretation. In order to obtain an unambiguous assignment and pair signal components for the C]_ and C3 carbons, a carbon-proton two dimensional chemical shift correlation study was performed on w-propionyl-THIQ (54) (Fig 4.48). This shows the proton carbon shift correlation of C^, C3 , C4 , C^Q anc* C11 carbons of compound 54.

233 Analysis of the 2D correlation spectra shows that the lower field proton signal at 8 4.72 of the C^-CI^ doublet corresponds to a carbon signal at 8 44.20 whereas the higher field proton signal at 8 4.60 correlates to a lower field carbon signal at 5 47.19. In the case of C3-CH2 protons the lower field triplet at 5 3.82 relates to the higher field carbon signal at 8 39.64 whereas the higher field triplet at 8 3.66 assigns to the lower field carbon signal at 8 43.06. It is seen that the most intense component in the -^H-NMR spectra is also the most intense component in the 13C-NMR spectra. As described previously N-acyl-THIQ compounds can exist as two conformers primarily due to restricted rotation about the C ^ - N bond. The two conformers were designated as Z or E (Fig 4.6). In the z conformer (II) the R substituent is tra n sto the C1-CH2 group whereas in the E conformer (I) it is c is to this group. In the Z conformer the C^-Cf^ protons are deshielded relative to those in the E conformer.10 This is due to the anisotropic effect of the amido group. This causes large diamagnetism (shielding) in the cone regions extending above and below the plane of the amido group.51 The regions in the plane of the amide group are paramagnetic (deshielding).84'85 Paulsen and Todt86'87 developed a model for this principle (Fig 4.16). C

Fig 4.16 Anisotropic effect of the amido group

234 If we consider that the possible positions of the protons on the nitrogen are A through to F or A 1 through to F1 then A,A1 D^D1 are in the plane of the amido group. In this plane although both A and A 1 are deshielded, A 1 is more deshielded than A. Protons in positions C and C1 are in the "out of plane region", (i.e. shielded region) where C1 is shielded more strongly than C. The positions E and E 1 are equivalent to C and C1 . If the averaging of the effects of anisotropy on all positions A-F and A 1-F1 leads to an average "out of plane" conformation for the protons, then these protons will experience an overall shielding effect.86 This is found in the case of N,N-dimethylamides,88~90 the protons of the N-methyl group c is to oxygen resonate at a higher field than those tra n sto the oxygen. In contrast in N,iV-dimethyl- amides91 and N ,iV-diisopropylamides,91 the relative shielding constants of protons in the c is and tra n s sites are reversed as compared to N, N -dimethylamide. In these compounds the N-CH2 protons average position is "in the plane" of the amido group therefore the N-CH2 protons that are cis to the oxygen resonate at a lower field than the N-CH2 protons that are tra n sto the oxygen. The cis N-ethylmethy 1 group on the other hand resonates at higher field than the tra n s methyl group. In summary the average position of AT-CH2 protons are in the plane of the amido group (deshielded) whereas N-CH3 protons are in the "out of plane" conformation (shielded). Other work tends to corroborate this line of reasoning.92^93 By analogy it is possible to compare the nitrogen ring CH2 protons of AT-acyl-THIQ series with the N-CH2 protons of N ,N- dialkylamides. It is therefore reasonable to assume that the nitrogen ring methylene protons are also deshielded by the amido group. When the C1-CH2 protons are c is to the carbonyl group (Z) they are more deshielded than when they are tra n s (E) to the carbonyl group. Assignment of C1-CH2 protons was unambiguous due to the lack of multiplicity. Hence the lower field signal of the C1-CH2 doublet in the %-NMR spectra of compounds 53-55 and 56-58 in CDCI3 was

235 assigned to the Z conformer (H) and the higher field signal was assigned to the E conformer (I). In contrast the lower field signal of the C3-CH2 resonances was assigned to the E conformer and the higher field resonance was assigned to the Z conformer. In the case of compounds 30 and 60 the ^-NMR chemical shifts of the C± and C3 methylene groups in CDCI3 were reversed to those compounds considered above (see below for explanation). Consequently the higher field signal of the C i -CH2 doublet was assigned to the z conformer and the lower field signal was assigned to the E conformer, whereas for the C3-CH2 protons the assignment was reversed. Published studies on N-acyl-indolines, 65,76,77 //-acyl- 1HQ65,68,76 N-acyl-1-methyl-THIQ10 derivatives have all assigned the lower field signal of doubled up resonances with respect to the anisotropic effect of the carbonyl group. Similarly as discussed for the -^H-NMR spectra, the low field 13C signal of the Cp-C^ doublet with the exception of compounds 30 and 60 can be assigned to the Z conformer and the high field signal to the E conformer. However, the 2D spectrum of compound 54 (Fig 4.48) clearly shows that the higher field signal of Cp carbon pairs corresponds to the lower field signal of the C1-CH2 doublet in the -^H-NMR spectrum. This observation can be explained by considering the field effect of the carbonyl group. The anisotropy of the carbonyl group causes deshielding of the nuclei lying in the cone extending from the carbonyl oxygen but shielding of nuclei lying outside this cone (Fig 4.17). Therefore the protons are deshielded but the Cp carbon is shielded.

Fig 4.17 Anisotropic effect of the carbonyl group in N-acyl-THIQ compound 236 In the z conformer is c is to the carbonyl group whereas in the E conformer it is tra n sto this group (Fig 4.6). Although is shielded in both conformers, it is more shielded in the Z conformer than in the E conformer. This results in the carbon in the Z conformer resonating at a higher field than the E conformer. The C3 carbon is more shielded in the E conformer than in the Z conformer and consequently will resonate at a higher field in the former case. This assignment of signals accords with studies by Fritz et a l .94 iheir 13c investigations shewed that the a and £ carbons of N ,N-dialkylamides (Fig 4.18) resonate at a lower field in the tra n s N-alkyl chain. trans R C H 2C H 2(CH2)nC H 3 \ N. a o CH, H 2(CH2)nC H 3 cis Fig 4.18 N,N-dialkylamides

McFarlane95 has reported that in the case of N , N -dimethyl substituted amides, the 13C resonance of the methyl group c is to the carbonyl group is at higher field to that which is tra n s . Jones and Wilkins96 commented on a reverse relationship observed for the % - and 13C-NMR resonances for a series of para-substituted N ,N - dimethylbenzamides. A similar trend was observed in N-acyl-THQ,65 the authors suggested a carbonyl shielding effect on the c is carbon. Consequently the and C3 carbons in the 13C-NMR spectra of N-acyl- THIQ were assigned in a similar manner. The higher field signal of the Cj carbon pair was assigned to the Z conformer (H) and the lower field signal was assigned to the E conformer (I). In the case of the C3 carbon pair, the lower field signal was assigned to the Z conformer and the higher field signal was assigned to the E conformer. In both the % - and 13C-NMR spectra of compounds 30, 53-60, the most intense component of signal pair was assigned to the

237 predominant conformer Z. The difference in the chemical shifts of C1-CH2 and C3-CH2 proton resonances and and C3 carbon pairs observed in compounds 30, 53-60 can be attributed to the anisotropic effect of the phenyl ring of THIQ (26) where methylene group is more deshielded than the C3 methylene group, in both the ^H- and 13C-NMR spectra. The assignment of the Z and E conformers was not discussed in terms of the anisotropy of the phenyl ring. It was assumed that for all of the compounds studied, the effect on and C3 and C4 was the same in both conformers.

4.2.3.4 Conformer populations of iY-acvl-THIO series The population of conformers Z and E of compounds 30, 53-58 and 60 was determined from the integrated proton signals of C3-CH2 and C3-CH2 protons respectively. The results are presented in Table 4.18. The z conformer was predominant in CDCI3 for compounds 30, 53-58 and 60 with values in the range 55-63%. However, for compounds 30 and 60 the C^-CII2 and C3-CH2 pair protons were assigned opposite to those of compounds 53-57 (see solvent effects for explanation). For two compounds 30 and 57, the %-NMR spectra were also obtained in toluene-dg. In both cases the Z conformer was predominant (Table 4.18). Within the temperature ranges where discrete signals could be observed for the and C3 methylene protons for compound 30 (Fig 4.40d-f) and C^-CI^ protons for compound 60, the population of conformers did not appear to differ significantly with temperature. The relative population of z and E conformers of compounds 30, 53-57 and 60 were also estimated from the 13C-NMR spectra by measurement of peak height from the spectrum. The results are presented in Table 4.19. It is apparent from the values that the relative population of conformers for compounds 30, 53-57 and 60 remained virtually unchanged for the different R substituents. In all cases the Z conformer was predominant, in agreement with the results from the %-NMR spectra.

238 Both Krivdin et al68 and Ccsnbrisson et al65 also found that the conformer populations derived from the 1H- and 18C-NMR spectra of N- acyl^IHQ and N-acetyl-pyrrole agree. The preference for the z conformer for the N-acyl-THIQ series may be explained in terms of steric interaction. In the z conformer the R substituent is the furthest away from the THIQ ring, whereas in the E conformer it is proximal to the ring and hence steric interaction is more pronounced. Similar observations were reported by Dalton et al8 for amide derivatives of 6,7-dimethoxy-THIQ. Potapov et al-1-0 also found that the Z conformer was predominant in the N-acyl-l-methyl-THIQ series.

4.2.3.5 Solvent effects The influence of solvent effects on the NMR spectra of some simple amides has been reviewed51 and it was not the purpose of this study to examine detailed solvent effects on the NMR spectra of N- acyl-THIQ series (30, 53-60). However the spectra of two compounds were by necessity obtained in two solvents (CDCI3 and toluene-dg) and the observed spectra require some comment. The -^H-NMR spectrum of compound 30 was run at ambient temperature in CDCI3 at 80.9 MHz (Fig 4.20) and also at 250 MHz (Fig 4.26). The more intense component of the doublet observed for the C1-CH2 protons was at high field to the less intense component in both spectra. However in the proton spectrum of compound 30 in toluene-dg at 303 K (and lower temperature) and 89.6 MHz (Fig 4.40d- e), the more intense component of the C^-C^ doublet was at a low field to the less intense component (a downfield shift of 8 0.14 on going from CDCI3 to toluene-dg). The less intense component on the other hand shifted upfield by 5 0.31 for the same solvent change. In contrast, the spectra of compound 57 in CDCI3 at ambient temperature and 250 MHz (Fig 4.27) exhibited the more intense component of the C^-CH2 doublet at lower field than the less intense component. On changing the solvent to toluene-dg and running the proton spectrum at ambient temperature at 89.6 MHz (Fig 4.22), the more intense component was still observed at lower field. However

239 both, signals were shifted upfield. This was 5 0.17 for the more intense signal and S 0.32 for the less intense signal. The 13C-NMR spectra of compound 30 at ambient temperature in CDCI3 at 22.5 MHz and 69.2 MHz (Fig 4.34) and in toluene-dg at 22.5 MHz always showed C1 as a doublet with the component at highfield as the more intense signal (Fig 4.41a-b). There was no significant (< ± S 0.5 ) difference in chemical shifts between the two solvents. Solvent effects (CDCI3 , EMSO) have been reported to cause changes in conformer population in //-thioacetyl-indolines.97 However in the present study it is likely that the apparent change in conformer population suggested by the %-NMR spectra on going from CDCI3 to toluene-dg is due simply to a reversal of the chemical shifts of the components of the C^-Q^ signal. This is supported by the fact that the conformer population ratios are the same in both solvents as shown by 13C-NMR spectra of compound 30. This apparent mismatch 1H-13C chemical shift was resolved in the case of compound 54 (R = Et) using a carbon proton 2D chemical shift correlation study and could be applied to compound 30. The reason for the %-NMR chemical shift changes in amides on going from chloroform to an aromatic solvent have been reviewed by Stewart et al .51 For N,N-dimethylformamiide, the //-methyl groups are more shielded in benzene compared to non-aromatic solvents with one of the //-methyl groups exhibiting a greater upfield shift than the other. Shielding effects were attributed to the anisotropy of the benzene ring in a complex formed by interaction between the n- electrons of the phenyl ring and the positively charged amide nitrogen atom.98 '99 The structure proposed for the complex is shown in Fig 4.19.

Fig 4.19 Complex proposed for the anisotropic effet of benzene on amides 240 The preferred position of the negative carbonyl oxygen is as far away from the phenyl ring as possible. Although both N-methyl groups are in the diamagnetic (shielding) region, field currents associated with the benzene ring, the W-methyl trans to oxygen experiences the greater shielding. The same effect has been reported for other N, N-dialkylamides and it has been generally accepted that in benzene that the N-alkyl protons in the E conformer are more shielded than those in the Z conformer.51 The results obtained in the proton spectra of compound 30 and 57 agree with the reported observations. In both cases the signal of the C1“CH2 protons in conformer E (I) (Fig 4.6), i.e. higher field component of compound 57 and lower field component of compound 30 (see Sec 4.2.3. for assignment) are both shifted the most going from CDCI3 to toluene-dg. This causes no difference in the chemical shift pattern of C^-CE^ protons in compound 57 but inverts the chemical shifts of these protons in compound 30. However for both compounds there was no influence in the 13C-NMR spectra by the change in solvent. This is in line with the report of McFarlane,95 that a change from a non-aromatic to aromatic solvent was sufficient to invert the position of N-methyl signals in the -^-H-NMR spectra of N ,N- dlmethylformamide and related compounds but not in the 13C-NMR spectra. This emphasises that local magnetic fields do not have effects of comparable magnitude on both and 13C-NMR chemical shifts.

241 4.3 EXPERIMENTAL AND RESDEES

4.3.1 Stable Chf=«nrigtrv 4.3.1.1 2-<^oranerciirifuran The procedure reported by Gilman et al42 was used to prepare 2- chloromercurifuran. Mercuric chloride (20 g, 0.074 mol) and sodium acetate (24.5 g, 0.3 mol) were dissolved in water (450 ml) and the resulting solution chilled to 273-277 K. An ethanolic solution (35 ml) of furan (5.02 g, 0.074 mol) was added rapidly to the reaction vessel which was then sealed immediately. After standing for 4 min a cream precipitate was formed. This was for 12 h. The precipitate was filtered off and the desired crude product extracted with hot ethanol. The filtrate on cooling gave a fine cream precipitate which was purified by crystallisation from ethanol. Product: cream powder (7.5 g, 35%, m.p. 423-425 K) .

Characterization - 3H-NMR (89.6 MHz, EMSO-d^), 5 6.43 (1H, m, H3 ;

JHg H3 = 35 H z ), 6.54 (3H, m, H4 ; JHg H4 = 26.8 Hz), 7.85 (IH, m, H5 ; JHg H5 = 40.3 Hz). 13C-NMR (22.5 MHz, EMSO-dg) , 5 190.0 (C3; JHg c3 = 146.5 Hz), 118.9 (C2 ; JHg C2 = 384.5 Hz), 145.5 (C4 ; JHg C4 = 170.3 Hz) 168.4 (Cx).

4.3.1.2 Preparation of 2-iodofuran 2-Chlorcmercurifuran (5 g, 16.5 mmol) was suspended in water (50 ml) and stirred vigorously. A solution of iodine (2.1 g, 16.5 mmol) and potassium iodide (2.75 g, 16.5 mmol) in water (15 ml) was added slowly over a period of 30 min. Sodium thiosulphate was then added and the reaction product steam distilled from a 2-litre flask. The product was extracted from the distillate with diethyl ether and then dried over anhydrous magnesium sulphate. On evaporation a light yellow liquid was obtained and this was distilled at reduced pressure under nitrogen. A pale straw coloured liquid distilled over at 316- 318 K (15 mm Hg) and was collected into a receiver at 195 K. At the first indication of decomposition (formation of a yellow solid? iodoform) in the water condenser, vacuum was released and the receiver removed. The isolated product was immediately diluted with

242 anhydrous diethyl ether.

Characterization - ^H-NMR (60 MHz, CDC13), S 6.2 (1H, m, H 4), 6.4 (1H, m, H 3), 7.4 (1H, m, H5) . tyZ 194 (M**, 100%}, 165 { [M-CHO]+, 13%}, 127 {[M-I]+ , 14%}, 44 {27%}, 39 {[M-ICHO]+ , 42%}.

4.3.1.3 Preparation of 2-brarofuran The decarboxylation procedure reported by Shepard et al43 was adapted as follows. 5-Bromofuroic acid (5.0 g, 26.2 mmol) and copper chromate (1.0 g, 3.2 mmol) as catalyst were placed in a reaction vessel equipped with a nitrogen bleed. Freshly distilled quinoline (10 ml) was then added and the apparatus lowered into a previously heated oil bath set at 473 K. Evolution of carbon dioxide was observed within 5 min of heating and the product was distilled into a receiver kept at 273-277 K. To reduce decomposition the isolated product was immediately diluted with dry diethyl ether (0.29 g/10 ml for a 0.2 M Grignard reagent) and stored over sodium wire under nitrogen at 273-277 K. Product: colourless liquid (2.5 g, 65%).

Characterization - ^ - N M R (89.6 MHz, CDCI3), S 6.26 (1H, m, H 3 ; J 3 ^4 = 3.3 Hz), 6.33 (1H, m, H4), 7.37 (IH, m, H5; J4/5 = 1.9, J 3 ,5 = 1.0 Hz). 13C-NMR (^-decoupled, 22.5 MHz, CDC13), S 111.2 (C4), 112.5 (C3), 121.9 (C2), 144.3 (C5)# IVZ 146/148 {M+, 58%}, 117/119 {[M- CHO]+ , 14%}, 39 {[M-BrCH0]+ , 100%). Chemical shift values in agreement with literature.100

4.3.1.4 Preparation of 2-f12C/13Cl furoic acid A filtered solution of 2-furoylmagnesium bromide (0.5 mM) in diethyl ether (1 ml, see Sec 4.3.2. for preparation) was added to a reaction vessel under nitrogen (apparatus similar to Fig 4.19c). [12C/13C] Carbon dioxide {13C generated from [13C] potassium carbonate (91% atom, 7.5 mmol) by the addition of sulphuric acid (250 /il, 1 M) in nitrogen was dispensed into the Grignard reagent via sodium carbonate and magnesium perchlorate traps at a flow rate of 10 ml min”1. After 2 min hydrochloric acid (0.8 ml, 6 M) was added to the

243 reaction vessel with vigorous stirring (nitrogen flow increased to 30 ml min"1). The phases were allowed to separate and the aqueous layer was withdrawn from the reaction vessel. The ethereal layer in the reaction vessel was further washed with water for injection and then dried over anhydrous magnesium sulphate. The evaporated residue was examined by 1H- and 13C-NMR.

Characterization - %-NMR (89.6 MHz, CDCI3) , 8 6.56 (1H, m, H 4 ;

JH3H4 = 3 *52 ' 7 -34 (m ' m' h 3 ; j H3H5 = °*73 Hz)/ 7 *65 (1H, m, % ; JH4H5 = 1 -76 Hz)* 13C-NMR (^-decoupled, 22.5 MHz, CDCI3 ), 8 112.28 (C4), 120.2 (C3) 143.86 (C2), 147.49 (C5), 163.85 (OO, signal enhanced).

4.3.1.5 Preparation of y-acetvl-THIO (53} Procedure A A solution of freshly distilled acetyl chloride (3.6 ml, 0.05 mol) in dry diethyl ether (50 ml) was added dropwise to a stirred solution of THIQ (26) (12.5 ml, 0.1 mol) in diethyl ether (100 ml) and continuously purged with dry nitrogen. The mixture was refluxed gently for 1 h and cooled. The white precipitate was removed by filtration and the residual solvent by rotary evaporation. An orange-red residue was obtained. The product was isolated from this residue as a pale cream crystalline solid (m.p. 317-319 K) either by vacuum distillation (yield 5.5 g, 63%) or recrystallisation (4.84 g, 55%) from ethyl acetate and petroleum ether (9:1 v/v).

Characterization - Found: C, 74.43; H, 7.52; N, 7.89; C^H^NO requires: C, 75.40; H, 7.48; N, 7.99. IR v-wax (Nujol) 1645 cm"1 (broad O O stretch). Hi/Z 175 {M+, 100%}, 160 {[M-CH3]+ , 18%}, 132 {[M-CH30 0 ] + , 78%}, 117 {[M-C2H 4NO]+ , 80%}, 104 {[M-C3H5NO]+ , 84%}. For details of % (Fig 4.23) and 13C (Fig 4.31) NMR spectral data see Tables 4.8 and 4.9, respectively. Description of ^-H-NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

244 Procedure B A mixture of THIQ (26) (6.25 ml, 0.05 mol) and acetic anhydride (4.7 ml, 0.05 mol) in dry pyridine (25 ml) was stirred overnight at ambient temperature in a stoppered reaction flask. The yellow solution was rotary evaporated to remove solvent and the yellow oily residue was taken up in ethyl acetate and washed successively with water, dilute hydrochloric acid (2 M), water, sodium bicarbonate solution (10 %) and finally water. The final organic layer was dried over anhydrous magnesium sulphate and evaporated to give a pale yellow oil. Crystallisation from ethyl acetate and petroleum ether (7:3 v/v) gave the desired product (53) (yield 4.93 g, 56%) as shown by mass spectrometry and % - and 13C-NMR spectroscopy.

4.3.1.6 Preparation of N-prooionvl-THIO (54) Freshly distilled propionyl chloride (4.45 ml, 0.05 mol) in dry diethyl ether (50 ml) was added drcpwise to THEQ (26) (12.5 ml, 0.1 mol) in diethyl ether (150 ml) with stirring under nitrogen. A white precipitate (THIQ hydrochloride) formed immediately. The mixture was refluxed for 1 h, cooled and then successively washed with dilute hydrochloric acid (2 M), water, sodium bicarbonate solution (10%), water and finally a saturated solution of sodium chloride. The organic layer was dried over anhydrous magnesium sulphate and evaporated giving a light orange liquid. The product was isolated by vacuum distillation as a colourless liquid (yield 5.9 g, 63%; b.p. 435-438 K at 6 mm Hg).

Characterization - Found: C, 76.18; H, 8.10; N, 7.36; C-j^lS^0 requires: C, 76.16; H, 7.99; N, 7.40. IR (liq) 1645 cm-1 (broad 0=0 stretch). M/Z 189 {M4*, 100%), 174 {[M-CH3]+ , 38%), 132 {[M- C2H 50 0 ] + , 74%}, 117 {[M-C3H 6NO]+ , 71%}, 104 {[M-C4H7NO]+ , 58%}. For details of % (Fig 4.24) and i3C (Fig 4.32) NMR spectral data see

Tables 4.8 and 4.9, respectively. Description of Ih -NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

245 4.3.1.7 Preparation of ff-butvrvl-THIO (55) The synthesis was essentially the same as that described for the preparation of compound 54. Butyryl chloride (5.2 ml, 0.05 mol) in diethyl ether was added dropwise to a stirred solution of THCQ (26) (12.5 ml, 0.1 mol) in diethyl ether under nitrogen. The product was isolated by vacuum distillation as a colourless liquid (yield 6.15 g, 61%; b.p. 448-449 K at 4 mm Hg).

Characterization - Found; C, 77.0; H, 8.61; N, 6.96; C^H^NO requires: C, 76.8; H, 8.43; N, 6.89. IR (liq) 1645 cm-1 (broad G O stretch). M/Z 203 {M+, 100%), 174 ([M-C2H5 ]+ , 71%), 132 {[M-

C3H7C O ] + , 59%), 117 ([M-C4H8NO]+ , 43%), 104 { [M-Cs H qNO]*, 35%). For details of (Fig 4.25) and 13C (Fig 4.33) NMR spectral data see Tables 4.8 and 4.9, respectively. Description of ^H-NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

4.3.1.8 Preparation of jV-cvclobutanEK^arixawl-THIO (56) The synthesis was essentially the same as that described for the preparation of compound 54. Cyclobutanecarboxylic acid chloride (5.2 ml, 0.05 mol) in diethyl ether was added dropwise to a stirred solution of THIQ (26) (13.3 g, 0.1 mol) under nitrogen. The product was isolated by vacuum distillation as a colourless liquid. On standing at 269 K, the product crystallised as a colourless crystalline solid (yield 4.78 g, 44.5%; b.p. 476-477 K at 8 mm. H g).

Characterization - Found; C, 77.94; H, 8.13; N, 6.43; C^H^NO requires: C, 78.10; H, 7.96; N, 6.50. IR (liq) 1640 cm”1 (broad O O stretch). M/Z 215 {M+, 58%), 186 {[M-C2H 5 ]+ , 85%), 133 {[M-

C4P%C=0]+ , 100%), 132 {[M-C4H 7C=0]+ , 93%), 117 { [M-C5H q NO]+ , 32%), 104 ([M-CgHgNO]4", 31%). For details of % (Fig 4.21) and 13C (Fig 4.35) NMR spectral data see Tables 4.8 and 4.9, respectively. Description of ^-H-NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

246 4.3.1.9 Preparation of Jf-acrvlovl-TOIQ (57) The synthesis was essentially the same as that described for the preparation of compound 54. Acryloyl chloride (2.1 ml, 0.025 mol) in diethyl ether was added drogwise to a stirred solution of THIQ (26) (6.65 g, 0.05 mol) in diethyl ether under nitrogen. The product was isolated by vacuum distillation as a colourless liquid, which crystallised on standing at 269 K as a white solid (yield 2.83 g, 61%; m.p. 300-302 K ) .

Characterization - Found: C, 76.99; H, 6.95; N, 7.49; C12H13N0 requires: C, 77.0; H, 6.91; N, 7.11. IR uT[]ax (liq) 1650 cm”1 (C=0). M/Z 187 {!/&, 100%}, 186 {[M-l]+ , 67%}, 132 {[M-C2H 3C=0]+ , 37%}, 117 {[M-C3H 4NO]+ , 39%}, 104 {[M-C4H 5NO]+ , 37%}. For details of % (Fig 4.27) and 13C (Fig 4.36) NMR spectral data see Tables 4.8 and 4.9, respectively. Description of %-NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

4.3.1.10 Preparation of N-benzovl-TOIO (58} The synthesis was essentially the same as that described for the preparation of compound 54. Benzoyl chloride (2.9 ml, 0.025 mol) in diethyl ether was added to a stirred solution of THIQ (6.65 g, 0.05 mol) in diethyl ether under nitrogen. After work-up, the resulting ethereal solution upon evaporation gave an extremely viscous residue. This was purified by column chromatography (30 x 30 cm, 200-400 mesh silica gel) eluted with dichloromethane. The resulting viscous colourless liquid, on standing at 269 K two months crystallised as a colourless crystalline solid (yield 4.36 g, 74%; m.p. 371-373 K).

Characterization - Found: C, 79.93; H, 6.44; N,5.98; C16H15N0 requires: C,81.01; H,6.33; N, 5.91. IR i'max (liq) 1630 cm”1 (bread O O stretch). M/Z 237 {M+, 77%}, 236 {[M-l]+ , 55%}, 132 {[M- C6H50 0 ] + , 32%}, 117 {[M-CYHgNO]4", 48%}, 105 {C6H5C=0+ , 100%}, 104 {[M-C8H7NO]+, 33%}. For details of % (Fig 4.28) and 13C (Fig 4.37) NMR spectral data see Tables 4.8 and 4.9, respectively. Description

247 of %-NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

4.3.1.11 Preparation of N—(furar^?-raTbcnyl)-lHIO (59) Hie synthesis was essentially the same as that described for the preparation of compound 54. Furcyl chloride (4.93 ml, 0.05 mol) in diethyl ether was added drcpwise to a mixture of THIQ (26) (6.65 g, 0.05 mol) and pyridine (4.04 ml, 0.05 mol) in diethyl ether (50 ml) with stirring, under nitrogen. After reflux for 1 h, the reaction mixture was worked-up as described for compound 54. Evaporation of the final ethereal solution gave a white solid. The product was crystallised from ethyl acetate and heptane (6:4 v/v) as a white crystalline solid (yield 6.57 g, 58%; m.p. 353-355 K).

Characterization - Found: C, 74.03; H, 5.84; N, 6.11; C^H^C^N requires: C, 73.99; H, 5.77; N, 6.16. IR (Nujol) 1610 (C-O) and 1630 cm"1 ( O O ) . M/Z 227 (M+, 100%}, 132 ([M-C4H 30C=O]+ , 23%}, 117 {[M-C5H4N02 ]+ , 23%}, 104 [M-C6H5N02 ]+ , 30%}, 95% {C4H 3OC=Of, 63%}. For details of % (Fig 4.29) and 13C (Fig 4.38) NMR spectral data see Tables 4.8 and 4.9, respectively. Description of 1H-NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

4.3.1.12 Preparation of N-(thicphene-2-carbonyl)-THIQ (60} The synthesis was essentially the same as that described for the preparation of compound 54. Thiophene-2-carbonyl chloride (5.35 ml, 0.05 mol) in diethyl ether was added dropwise to a stirred solution of THIQ (26) (13.3 g, 0.1 mol) in diethyl ether under nitrogen. After reflux for 1 h, the reaction mixture was washed with dilute hydrochloric acid (2 M) followed by water. The latter caused the precipitation of a white solid between the aqueous and organic phases. This solid was recovered and redissolved in dichloromethane and worked-up further as described in the preparation of 54. Evaporation of the resulting dichloromethane solution and recrystallisation of the residue from ethyl acetate and heptane (6:4

248 v/v) gave a white crystalline solid (yield 6.54 g, 54%; m.p. 365.5- 367.5 K).

Characterization - Found: C, 68.96; H, 5.36; N, 5.96; S, 13.02; C14H 13NOS requires: C, 69.14; H, 5.35; N, 5.76; S, 13.17. IR (Nujol) 1600 cm-1 (sharp O O stretch). Vi/ Z 243{M+ / 100%), 132 {[M- C4H 3S O O ] + , 30%), 117 {[M-C5H4NOS]+ / 47%), 111 {C4H 3S O O + , 91%), 104 {[M-C6H 5NOS]+ ). For details of % (Fig 4.30) and 13C (Fig 4.39) NMR spectral data see Tables 4.8 and 4.9, respectively. Description of %-NMR spectra is given in Sec 4.3.3.2 and 13C-NMR spectra in Sec 4.3.3.3.

4.3.1.13 Preparation of N-acetvl-TOO (61) The synthesis was essentially the same as that described for the preparation of compound (53; procedure A). Acetyl chloride (3.6 ml, 0.05 mol) in diethyl ether was added dropwise to a stirred solution of THQ (25) (12.5 ml, 0.1 mol) in diethyl ether under nitrogen. The product was isolated by vacuum distillation as a colourless liquid (yield 5.76 g, 66%; b.p. 417-419 K at 1.5 mm Hg).

Characterization - Found: C, 75.28; H, 7.63; N, 8.0; C^H^HO requires: C, 75.43; H, 7.43; N, 8.0. IR i/^x (liq) 1660 cm-1 (broad O O stretch). M\Z 175 {V&, 45%), 133 {[M-CH2=0 0 ]+ , 93%), 132 {[M- C2H 30]+ ,100%), 117 ([M-C3H60]+ ,13%), 85 (39%). For % and 13C (Fig 4.45) NMR spectral data see Tables 4.12 and 4.13, respectively. Description of ^-H-NMR spectra is given in Sec 4.3.3.8 and 13C-NMR spectra in Sec 4.3.3.9.

4.3.1.14 Preparation of ff-propionvl-'HK) (62) The synthesis was essentially the same as that described for the preparation of compound 54. Propionyl chloride (4.45 ml, 0.05 mol) in diethyl ether was added dropwise to a stirred solution of THQ (25) (13.3 g, 0.1 mol) in diethyl ether. The product was isolated by vacuum distillation as a colourless liquid. On standing at 269 K, the product crystallised as a white crystalline solid (yield 6.78 g,

249 72%; m.p. 319-322 K).

Characterization - Found ; C, 76.19; H, 8.05; N, 7.39; C^H^NO requires: C, 76.16; H, 7.99; N, 7.40; IR l667 cm”1 (broad 0=0 stretch). M/Z 189 {}/&, 33%}, 133 {[M-CH3O M > = 0 ] + / 100%), 132 {[M- C3H50]+ , 70%), 117 {[M-C4H8H0]+ , 11%), 77 {12%}, 57 {17%}. For 1H- and 13C-NMR spectral data see Tables 4.12 and 4.13, respectively. Description of •ki-NMR spectra is given in Sec 4.3.3.8 and 13C-NMR spectra in Sec 4.3.3.9.

4.3.1.15 Preparation of W-butvrvl-THO (63) The synthesis was essentially the same as that described for the preparation of compound 54. Butyryl chloride (5.2 ml, 0.05 mol) in diethyl ether was added dropwise to a solution of THQ (25) (13.3 g, 0.1 mol) in diethyl ether, under nitrogen. The product was isolated by vacuum distillation as a colourless liquid (yield 6.25 g, 62%; b.p. 426-428 K at 18 mm Hg).

Characterization - Found: C, 76.97; H, 8.58; N, 6.91; C^H^NO requires: C, 76.81; H, 8.43; N, 6.89. IR (liq) 1660 cm”1 (broad G O stretch). M/Z 203 {M+, 28%}, 133 {[M-C2H5CH=C=0]+ , 100%}, 132 {[M-C4H70]+ , 55%}, 117 {[M-C5H 10o ]+ , 10%}, 85% {59%}, 83 {90%}. For % - and 13C-NMR spectral data see Tables 4.12 and 4.13, respectively. Description of %-NMR spectra is given in Sec 4.3.3.8 and 13C-NMR spectra in Sec 4.3.3.9.

4.3.1.16 Preparation of N-(thiochene-2-carbonyl)-IHQ (64) The synthesis was essentially the same as that described for the preparation of compound 54. Thiophene-2-carbonyl chloride (5.35 ml, 0.05 mol) in diethyl ether was added dropwise to a stirred solution of THQ (25) (13.3 g, 0.1 mol) in diethyl ether, under nitrogen. Evaporation of the final ethereal solution gave a crystalline product. Recrystallisation from ethyl acetate and petroleum ether (7:3 v/v) gave large colourless crystals (yield 6.91 g, 57%; m.p. 339-340 K).

250 Characterization - Found: C, 69.34; H, 5.46; N, 5.73; S, 13.12; C14H 13NOS requires: C, 69.11; H, 5.39; N, 5.76; S, 13.18. TR (Nujol) 1600 cm"1 (sharp 0=0 stretch). Vi/ Z 243 (V&, 21%), 132 {[M- C5H3SO]+ , 7%), 111 {C4H 3SC=0+ , 100%), 83 {5%}, 43 {19%}, 39 (12%). For details of % (Fig 4.43) and 13C (Fig4.46) NMR spectral data see Tables 4.12 and 4.13, respectively. Description of -^-H-NMR spectra is given in Sec 4.3.3.8 and 13C-NMR spectra in Sec 4.3.3.9.

4.3.1.17 Preparation of ff-etfavl-THIO (65) N-acetyl-THIQ (53) (500 mg, 2.86 mmol) in dry diethyl ether (5 ml) was added to an ethereal suspension (25 ml) of lithium aluminium hydride (400 mg, 10 mmol) under nitrogen at such a rate that the reaction mixture refluxed gently at ambient temperature. The reaction mixture was further refluxed for 1 h at 348 K. Aliquots were taken every 15 min and analysed by tic, system A: silica gel plates, eluant, hexane and ethyl acetate (70: 30); compound (amide) Rf = 0.08; compound (amine) Rf = 0.57. On completion of reduction the reaction mixture was cooled and the excess lithium aluminium hydride d ec o m p o se d by dropwise addition of ethyl acetate. The reaction mixture was then poured gradually into excess ice-cold dilute sulphuric acid and the resultant suspension made alkaline by the addition of sodium hydroxide (2 M ) . The crude product was extracted into ethyl acetate and washed successively with water and a saturated solution of sodium chloride. Drying and removal of the solvent gave a pale red-orange liquid. TLC of the crude product [System B: silica gel plates, eluant, heptane and ethyl acetate containing 10% triethylamine (50:50 v/v) ] exhibited (exposure by UV or I3) one major (desired product, Rf = 0.74) and one minor component (THIQ, Rf = 0.15). The product was purified on a preparative column (System C: 10 x 3 cm bed, silica gel, 200-400 mesh) eluted with a mixture of heptane and ethyl acetate (85:15 v/v). Evaporation of the appropriate fractions gave a colourless liquid (yield 310 mg 67%).

251 Characterization - Found: C, 81.93; H, 9.46 ; N, 8.90; C3JH 15N requires : C, 81.99; H, 9.32; N, 8.7. IR i^ y (liq) 2730-3070 cm”1 (aromatic and aliphatic C-H stretches; 0=0 stretch absent). M/Z 161 {M*, 100%}, 160 {[M-l]+ , 64%}, 146 {[M-CH3 ]+ , 97%), 132 {[M-C2H 5 ]+ , 25%}, 104 {[M-C3H 7N]+ , 89%}, 89 {33%}, 77 {30%}. For % - and 13C-NMR spectral data see Tables 4.14 and 4.15, respectively. Description of %-NMR spectra is given in Sec 4.3.3.11 and 13C-NMR spectra in Sec 4.3.3.12.

4.3.1.18 Preparation of ff-propvl-THIO (66) Procedure A The synthesis was essentially the same as that described for the preparation of compound 65. N-propionyl-THIQ (54) (500 mg, 2.64 mmol) in diethyl ether was added to an ethereal suspension of lithium aluminium hydride (350 mg, 9.24 mmol). Reduction was followed by tic [System A: silica gel plates, eluant, hexane and ethyl acetate (80:20 v/v) ]; compound (amide) Rf = 0.09, compound (amine) Rf = 0 .2 2 . The reaction mixture worked-up as described for compound 65. TIC: of the crude product [System B: silica gel plates, eluent, heptane and ethyl acetate containing 10% Et3N (50:50 v/v)] exhibited one major (desired product, Rf = 0.83) and one minor (THIQ, Rf = 0.15) component. The crude product was purified by column chromatography (conditions as for compound 65, system C). A pale orange liquid was obtained (yield 290 mg, 63%).

Characterization - Found: C, 81,75; H, 9.61; N, 8.13; C12H17N requires: C, 82.09; H, 9.7; N, 8.0. IR (liq) 2730-3070 cm""1 (aromatic and aliphatic C-H stretches; O O stretch absent). E/Z 175 (E&, 13%}, 174 {[M-l]+ , 70%}, 173 {[M-2]+ , 50%}, 172 {[M-3]+ , 100%}, 146 {[M-C2H5 ]+ , 31%}, 132 {9%}, 130 {46%}, 104 {[M-^HgN]4", 8%}. For % - and 13C-NMR spectral data see Tables 4.14 and 4.15, respectively. Description of -^-H-NMR spectra is given in Sec 4.3.3.11 and 13C-NMR spectra in Sec 4.3.3.12.

252 Procedure B A solution of bromoprqpane (1.15 ml, 12.5 mmol) in ethanol (5 ml) was added to THIQ (26) (3.33 g, 25 mmol) in ethanol (25 m l ) . The reaction mixture was refluxed for 2 h and then cooled. Excess ethanol was removed by evaporation. The residue was then taken up in water and the pH of the solution adjusted to 11-12 with c. ammonia (d = 0.88). The crude product was extracted into chloroform and successively washed with water and a saturated solution of sodium chloride. The chloroform layer was dried over anhydrous magnesium sulphate and evaporated to give a light orange liquid. The latter was distilled under vacuum (yield 3.67 g; b.p. 340-341 K at 1.5 mm Hg). However, tic analysis [system B for compound 65] revealed the presence of THIQ in the distilled product. Preparative column chromatography [conditions as for compound 65, system C] was then used to purify the product (yield 2.55 g, 58%). The product was identical to that prepared by procedure A (by %-NMR and mass spectrometry).

4.3.1.19 Preparation of JY-but-3-ene-THIO (67) The synthetic procedure used was essentially the same as that described for the preparation of compound 66 (procedure B). A solution of 4-bramo-l-butene (2.66 g, 20 mmol) in ethanol was added to THIQ (26) (2.46 g, 18 mmol) in ethanol containing sodium bicarbonate (1.6 g, 20 mmol). After work-up the product was isolated by vacuum distillation as a colourless liquid (yield 2.2 g, 65%; b.p. 393-394 K at 2 mm Hg).

Characterization - Found: C, 83.51; H, 9.20; N, 7.55; C^H^N

requires: C, 83.42; H, 9.09; N, 7.49. IR vjqqx (licQ 2720-3060 cm-3- (aromatic and aliphatic C-H stretches; N-H stretch absent). E/Z 187 {M+, 6%), 186 {[M-l]+ , 17%), 146 {[M-C3H5 ]+ , 100%), 130 {16%}, 117 (44%), 104 {[M-C5H9N]+ , 18%), 91 {22%}. For % - and 13C-NMR spectral data see Tables 4.14 and 4.15, respectively. Description of %-NMR spectra is given in Sec 4.3.3.11 and 13C-NMR spectra in Sec 4.3.3.12.

253 4.3.1.20 Preparation of N-faenzvl-THIQ (68) The synthetic procedure used was essentially the same as that described for the preparation of compound 65. N-benzoyl-THIQ (58) (500 mg, 2.11 mmol) in diethyl ether was added drcpwise to an ethereal suspension of lithium aluminium hydride (280 mg, 7.4 mmol). The reduction was monitored by tic [silica gel plates, eluent, hexane and ethyl acetate (70:30 v/v) ]: compound (amide) Rf = 0.21, compound (amine) Rf = 0.76. After work-up, evaporation of the final ethyl acetate solution, gave a light brown viscous oil. TLC analysis exhibited two UV absorption bands [system B for compound 65] The product was purified by preparative column chromatography (10 x 3 cm bed, silica gel, 200- 400 mesh), eluted with a mixture of heptane and ethyl acetate (90:10 v/v). A colourless liquid was obtained (320 mg, 68%).

Characterization - Found: C, 78.77; H, 7.07; N, 6.70; C^H^NO requires: C, 78.87; H, 7.04; N, 6.57. IR (liq) 2740-3080 cm-1 (aromatic and aliphatic C-H stretches; 0=0 stretch absent). IVZ 223 (M+, 71%), 222 {[M-l]+ , 100%), 146 {[M-C6H 5]+ , 18%}, 132 {[M-C7H7 ]+ , 46%}, 104 {[M-CgHgN]*, 38%}, 91 {C7H7+ , 88%}, 65 ( C ^ * , 19%}. For % - and 13C-NMR spectral data see Tables 4.14 and 4.15, respectively. Description of %-NMR spectra is given in Sec 4.3.3.11 and 13C-NMR spectra in Sec 4.3.3.12.

4.3.1.21 Preparation of N-furfurvl-THIO (69) The synthesis was essentially the same as that described for the preparation of compound 65. N-{furan-2-carbonyl}-THIQ (59) (500 mg, 2.2 mmol) in diethyl ether was added to an ethereal suspension of lithium aluminium hydride (300 mg, 7.9 mmol). The reduction was followed by tic [silica gel plates, eluant, hexane and ethyl acetate (80:20)]: compound (amide) Rf = 0.14, compound (amine) Rf = 0.36. Evaporation of the worked-up ethyl acetate solution, gave a light orange liquid (yield 340 mg, 72%). No further purification was carried out.

254 Characterization - Found: C, 78.77; H, 7.07; N, 6.70; C^H^NO requires: C, 78.87; H, 7.04; N,6.57. IR I'max (11(5) 2720-3120 cm-1 (aromatic and aliphatic C-H stretches; C=0 absent). M/Z 213{M*, 56%), 212 {[M-l]+ , 58%), 146 {[M-C4H 30]+ , 15%), 145 (29%), 132 {[M- C5H50 ]+ , 39%), 104 {[M-C6H7NO]+ , 50%), 81 {C5H5O4-, 100%), 53 {20%}. For %- and 13C-NMR spectral data see Tables 4.14 and 4.15, respectively. Description of -4i-NMR spectra is given in Sec 4.3.3.11 and 13C-NMR spectra in Sec 4.3.3.12.

4.3.1.22 Preparation of propyl-THO (70) The synthesis was essentially the same as that described for the preparation of compound 65. N-propionyl-THQ (62) (500 mg, 2.65 mmol) in diethyl ether was added to an ethereal suspension of lithium aluminium hydride (350 mg, 9.26 mmol) under nitrogen. Reduction was followed by tic [System A: silica gel plates, eluant, hexane and ethyl acetate (80:20)]; compound (amide) Rf = 0.25, compound (amine) Rf = 0.85. After work-up, the final ethyl acetate solution was evaporated to give a light orange liquid. TIC analysis [system B for compound 65] exhibited two UV absorption bands. Preparative column chromatography [10 x 3 cm bed, silica gel, 200-400 mesh: eluant, heptane and ethyl acetate (90:10 v/v) ] was used to purify the product. A colourless liquid was obtained (yield 340 mg, 74%).

Characterization - Found: C, 82.38; H, 9.79; N, 8.03; Cf2% 7N requires: C, 82.28; H, 9.71; N, 8.0. IR (llc3) 2820-3070 cm'1 (aromatic and aliphatic C-H stretches; C=0 absent). M/Z 175 {M*, 31%), 146 {[M-C2H5 ]+ , 100%), 130 {15%}, 118 {9%}, 91 {9%}. For 1H- and 13C-NMR spectral data see Tables 4.16 and 4.17, respectively. Description of %-NMR spectra is given in Sec 4.3.3.13 and 13C-NMR spectra in Sec 4.3.3.14.

255 4.3.1.23 Preparation of M-butyl-THD (71) The synthesis was similar to that described for the preparation of compound 65. N-butyryl-THQ (63) (500 mg, 2.46 mmol) in diethyl ether was added to an ethereal suspension of lithium aluminium hydride (330 mg, 8.61 mmol) under nitrogen. The reduction was monitored by tic [system A for compound 65]: compound (amide) Rf = 0.31, compound (amine) Rf = 0.88. After work-up, the final ethyl acetate solution was evaporated and the orange residue purified by column chromatography [10 x 3 cm bed, silica gel, 200-400 mesh: eluant, heptane and ethyl acetate (90:10)]. A pale liquid was obtained (yield 300 mg 65%).

Characterization - Found: C, 82.43; H, 10.09; N, 7.60; C^HigN requires: C, 82.54; H, 10.05; N, 7.41. IR i^rax: (llc2) 2840-3060 cm”1 (C-H stretches; C=0 absent). M/Z 189 (M4*, 29%}, 146 {[M-C3H7 ]+ , 100%}, 130 {9%}, 118 {7%}, 104 {5%}, 91 (7%}. For XK- and 13C-NMR spectral data see Tables 4.16 and 4.17, respectively. Description of %-NMR spectra is given in Sec 4.3.3.13 and 13C-NMR spectra in Sec 4.3.3.14.

256 4.3.2 Carbm-li fftenristry 4.3.2.1 Preparation of Gricmard reagents Procedure A: preparation of methvlmaqnesium bromide Magnesium turnings (120 mg, 5.0 mmol) were placed in a glass reaction vessel (labelled as 5 in Fig 3.5) and left in an oven at 393 K for approximately 12 h together with the air condenser (labelled as 3 in Fig 3.5). The apparatus was assembled while hot as shown in Fig 3.5. Air was purged from the apparatus 0.5 h by a continuous flow of dry nitrogen (15-20 ml min"1). The flow rate was then reduced to 2-3 ml min-1. Diethyl ether (10 ml) containing a crystal of iodine was added to vessel 5 via syringe 8 (Fig 3.5). Bromamethane was then bubbled through the diethyl ether at a flow rate of 8-10 ml min"1. The reaction mixture was stirred and allowed to achieve reflux. To prevent a vigorous reaction, vessel 5 was cooled occasionally in an ice-water bath. Approximately one hour of refluxing was required to consume all of the metallic magnesium. At the end of the reaction, the reagent was sealed under nitrogen (by K-75 three way nylon taps, labelled as 2 in Fig 3.5) and stored at 273-277 K if not used directly. The reagent was always filtered (0.22 /im pore size, Millex-FG) before use.

Procedure B: preparation of other Grianard reagents The general procedure described below was used to prepare the following Grignard reagents (RMgBr) where R is Et, Pr, c.Bu, vinyl, Ph and [2 ]-furanyl. The apparatus used is shown in Fig 3.5. A solution of RBr (5 mmol) in diethyl ether or THF (10 ml; see Table 4.6 for Grignard solvent) containing a crystal of iodine was added to vessel 5 via syringe 8 . The solution was allowed to stand for 5 min which caused etching of the magnesium turnings. On stirring generally an immediate loss of iodine colouration was observed although in the case of the vinyl and 2-bromofuran refluxing conditions were required to initiate the reaction. The resulting mixtures were refluxed for approximately 1 h. In most cases a straw coloured solution was obtained as the final product. In the case of

257 vinylmagnesium bromide and 2-fnroylmagnesium bromide a suspension of dark green solid was obtained. On filtration (0.2 /un, Millex FG) the common straw coloured solution was obtained.

4.3.2.2 Preparation of r^Olacid chlorides A schematic representation of the apparatus used for the preparation of [i:k:]acid chlorides is shown in Fig 4.19a. Prior to the syntheses vessels A and B were kept at 393 K and the apparatus was flushed with dry nitrogen for 30 min.

Cyclotron-produced i :lC02 was trapped in a stainless steel loop (0.75 mm i.d.) immersed in liquid argon. The activity was then dispensed (loop plunged into water at 323 K) into vessel A containing a filtered (0.22 /zm, Millex FG) solution of freshly prepared Grignard reagent (0.2 mmol) in diethyl ether or THF (1 ml) at ambient temperature under nitrogen. After 2 min, the carbonation was quenched by the addition of PDC (150 /z l, 1 mmol) and DTBP (270 /z1, 1.2 mmol). On addition of these reagents the formation of a precipitate was observed which redissolved with further addition. Generally the reaction mixture was heated gently under nitrogen (5-8 ml min-1) to remove solvent (Et20 at 338K and THF at 413 K). In the case of [1XC]acetyl chloride (in diethyl ether) and [-^Cjacryloyl chloride (in THF), the solvent was also distilled into vessel B (Fig 4.19a) since the difference between the boiling points of these acid chlorides and solvent was small. After removal of solvent from vessel A, the radioactive residue was heated at higher temperature to distill off the [-^Cjacid chlorides (see Table 4.6 for the required oil bath temperature). A flow rate of 20-25 ml min-1 of N 2 was required to efficiently distill the [11C]acid chlorides into vessel B containing THIQ (26) (5 mg, 3.75 mmol) in THF (1 ml) at 195 K. The resultant mixture was allowed to warm (ca. 310 K) for 5 min to promote amide formation. THF was removed at 358 K and vessel B was cooled to ambient temperature. The radioactive residue was taken up in hplc solvent (dichloromethane) and injected via a filter (Acrodisc, Gelrnan Ltd) onto a silica gel hplc column ( "^“Porasil", particle size 10 /zm,

258

5) 5) 203 K 6) 6) 338 or 413 K THIQ in THF in THIQ Grignard reagentGrignard aflpparatus used for the synthesis of lHC]amides. 9 .i 4 Fig Ualue Ualue position for each operation are indicated by number. Cyclotron 1) producedtrapped [nC]C02 liquid Rr; in 2) 2) dispensed l"C]C02 into reagent Grignard uessel in of3) Rddition reagents; R; 4) Eucess soluent Grignard (Et20 or THF) distilled into uessel THF) chlorideor 5) ["Cjllcid distilled C; into uessei g; 6) Remoual of soluent from uessel B; 7) 7) Rddition and withdrawalsoluent. of IIPLC

259 30 x 0.7 cm i.d., Waters Associates) eluted at 5.0 ml min-1 with the same solvent. The eluant was monitored continuously for radioactivity and for absorbance at 254 nm. The radioactive fraction having the same retention time as reference material (see Table 4.7) was collected. The starting amine, TKEQ (26), was retained on the column (retention time 30.5-31.5 min) and was well separated from the collected radioactive samples. A typical preparative chromatogram, that for Af-fl^^butyryl-THIQ (74) is shown in Fig 4.19b. The collected radioactive fractions were further analysed to determine chemical and radiochemical purity.

Analysis i) HFEC Aliquots of the radioactive fractions collected from the preparative hplc column were analysed by normal phase hplc. Samples were injected onto a silica gel hplc column ('V-Porasil", particle size 10 /xm, 30 x 0.39 cm i.d., Waters Associates), eluted at 2.0 ml min-1 with dichloromethane containing 0 .1% of a mixture of ethanol, water and triethylamine (100:2:2 v/v). The eluant was monitored for radioactive and UV absorbance at 254 nm. For compounds 72-75, a single radioactive peak with the same retention time as reference compounds was obtained. Retention times are given in Table 4.7. Apart from the reference compounds no other stable compounds were detected. For a number of compounds (see Table 4.7) reverse phase hplc

column ('V-Bondapak", particle size 10 ^m, 30 x 0.39 cm i.d., Waters Associates) eluted at 2.0 ml min-1 with a mixture of methanol:water (1:1 v/v) was also utilised to establish the purity of the [1:LC]amides. In all cases a single radioactive and UV active peak with the same retention time as the appropriate reference material was observed (Table 4.7)

260 26 24 22 20 18 16 14 12 10 8 6 4 2 0

Fig.4.i9b HPLC chromatogram fcr the preparative separation of N — [11C ] butyryl - 1,2,3,4 tetrehydroisoquinoiine (74)

261 - - (W) 8-15 10-30 12-18 10-30 20-80 Stable material .5 . 8 . 2 1 - - - 1 . 1 1.7-2 0.38-0.93 (GBq/mol) activity Specific

0

- -

(%) 15-30 18-30 25-40 1.5-2 20-30 1.5-3.0 amide R.C.Y of [n C]

2

(MBq) 372-744 372-930 558-930 10-25 372-558 372-558 activity of ri:LC]G0 Starting

*3 collected at E.O.B 2

d C]00 (%) C]RG0C1 l 55-56 558-930 35-60 55-56 : 50-70 1 1 40-60 375-558 i [ Estimated yield of

(%) >95 >85 >90 > 8 8 of [n C]- Efficiency carbonation C]acid chlorides were distilled 1 1 383 413 438 443 >70 498 (K) tenpa Oil bath Et2° Et20 373 >95 Et20 Et20 Et20 THF 378 >95 d Et20 solvent Grignard 3 7 H 3 4 2 CH 3 2 5 ) c h R h c h = 2 2 CH 6 c c h Cyc-C3H5 c h (CH Substitutent Amide (73) (74) (72) (75) cyc.C (33) (80) (81) a - temperature of oil bath fromwhich [l- b - determined by the % of cactivity - corrected that distilledfor decay and from basedvessel on A the to total vessel activityB (Fig of4.19a) [ d - [Hcjacid chloride could not be isolated by distillation Table 4.6. Radiochemical parameters of [l-^CJacid chlorides and their conversion into [^C]amides

262

, 2

Cl 2

175 2 0 1 VI/7& (M+ ion)

& 0.1A% 2 Cl 2 0.63 0.62 214 0.53 189 0.58 203 0.45 Rf TIC system B^'*-

Rf TIC 0.28 0.35 0.16 systemA^'e

0 :Et3N [100:2:23]} 2 5.9 0.39 7.6 4.0 anal.hplcc R.T. (min) Reverse phase

+ 0.5% A, A = EtOH:H 2 5.3 5.4 4.0 9.1 0.46 4.7 7.8 C1 2 anal.hplcb R.T. (min) Normal phase

3 18.2-20.4 15.4-16.8 4.45 14.4-15.8 16.2-17.8 24.2-26.2 R.T. (min) C] amideC] fraction on normal phase hplc column {/i-porasil; eluant, CH Preparative hplc column C]amide fraction on reverse phase hplc column (p-Bondapak; eluant, MeQH:H20 [50:50] 1 : 1 1 1 3 5 7 H H 3 4 CH 3 2 ) R 2 c h ml min- 1 } ml min- 1 } CH2ai3 (CH eye. C 2 . 0 2 . 0 Substituent C]Amides collected from prep, hplc column after decay, analysed by electron inpact mass spectroscopy 1 1 Retention time (R.T.) of [^C]amides collected from preparative hplc silica column {/i-porasil; eluant, CH flew rate 4.0 ml min-1} Analysis of collected [ flew rate flow rate [ Radioactivity detected by autoradiography and stable compound by exposure to iodine TIC system A: silica gel plates {solvent, HexrEtOAc [7:3]} TLC system B: silica gel plates {solvent, CH Analysis of collected [ (33) (73) (75) eye. C (72) (74) t1^Amide ] Table 4.7. retention HPLC times and TIC Rf values of [-^C]amides a) f) b) e) c) d) g)

263 ii) TLC Aliquots were also analysed by tic on two systems using silica gel 60 F254 sheets (Camlab). The plates were developed either in hexane:ethyl acetate (7:3 v/v) or dichloramethane containing 0.5% of a mixture of ethanol, water and triethylamine (100:2:2 v/v). Radioactivity was detected by autoradiography and stable compounds by exposure to iodine. On both tic systems the radioactive products co­ migrated with the reference compounds (Table 4.7). No other radiochemical or chemical impurity was detected on either tic system.

iii) Mass Spectroscopy The radioactive fractions (compounds 72-75) collected from the preparative hplc column after decay were rotary evaporated to dryness. The samples were then examined by mass spectrometry in the electron impact mode. The mass spectra of all of the radioactive compounds prepared were identical to those of the reference compounds. The parents ions (M+) as well as the characteristic features, such as the M/Z 132 [M-ROO]+ , 117 [M-N0HCR]+ and the retro Diels-Alder fragment [M-CH2=NC0R]+ at M/Z 104, were observed. The mass to charge ratio (M/Z) of the parent ion (M*) observed for the "carrier" in the radiosyntheses of ccmpounds 72-75 are given in Table 4.7.

4.3.2.3 Preparation of r^-ky13 Cl benzoyl chloride The synthetic route used for the preparation of [ 11C/ 13 C] benzoyl chloride was essentially the same as that described for the preparation of [ 13-C] acid chlorides (Sec 4.3.2 .2 ). A schematic representation of the apparatus used for this synthesis is shown in Fig 4.19c. The trapped 13-002 and [13C]carbon dioxide (generated by the addition of sulphuric acid (250 /xl, 1 M) to 13C-enriched potassium carbonate (91 atom %, 7.2 jumol in 200 y.1 of H 20] were dispensed into vessel B containing a filtered solution of phenylmagnesium bromide (0.05 mmol) in diethyl ether (1 ml) at ambient temperature under nitrogen (5 ml min”1). The [11C/13C]acid chloride was then generated from the radioactive adduct by the

264 "c o 2/n 2 III \/

Mg(CI04)2

NqIICOj Poppet ualue

Soda lime

to Ch Q t ( ji 2,5

UesselB UesselB

Fig4 .19cSchematic representation of apparatus used for the sgnthesis of [13C/,,Cjbenzogl chloride. Ualue position for each operation indicated by number. 1) Trapped 1,C02 and 13C02 (generated by the addition of H2S04 (250 pi, IM) to K2,3C03) dispensed into phenyl magnesium bromide in uessel B; 2) Rddition of POC and DTBP; 3) Rddition of Til IQ in TIIF; 4) Uessel £ heated to 308 K to premote [,3C/,,Cjamide formation; 5) Remoual of reaction miuture. co-addition of PDC (7 /il, 0.05 mmol) and DTBP (100 /il, 0.45 mmol). This line was then flushed with diethyl ether (500 /il) and the reactants allowed to stir for 5 min at 328 K. THIQ (26) (133 mg, 1 mmol) in diethyl ether (1 ml) was added to vessel B and the reaction mixture heated (308 K) for 10 min to promote [1;LC]amide formation. The radioactive diethyl ether was recovered, washed with dilute hydrochloric acid (2 M) and water for injection. It was then dried over anhydrous magnesium sulphate and evaporated giving a light orange oil. This was taken up in hplc solvent (dichloromethane) and injected onto a silica gel column ("/i-Forasil", particle size 10 /m f 30 x 0.7 cm i.d., Waters Associates) eluted at 3.0 ml min-1 with the same solvent. The radioactive fraction eluting between 19-21.5 min was collected. This was evaporated to dryness and the product examined by proton-decoupled Fourier transform 13C-NMR spectroscopy (62.9 MHz, CDCI3) and mass spectrometry (electron impact, 70 eV). The 13C spectrum exhibited an enhanced singlet at 172.03 ppm in accordance with the carboxamido-carbon of compound 58 (see Table 4. 9). The mass spectrum revealed a parent peak at M/Z 238 {natural abundance iV-benzoyl-THIQ (58) gives a parent peak at M/Z 237 {M*, 76%}, a peak at M/Z 106 for the acylium ion {[M-C6H 5C=0]+ , 100%), a characteristic fragment of N-benzoyl-THIQ (58).

4.3.2.4 Preparation of r^Clprazosin The apparatus used for the preparation of [^C] prazosin was similar to that used for the synthesis of [^Cjacid chlorides (Fig 4.19a). The system was assembled while hot and flushed with dry nitrogen as described previously. A filtered solution of 2- furoylmagnesium bromide (0.02 mmol) in diethyl ether (1 ml) (prepared as described in Sec 4.3.2.2) was placed into vessel A under nitrogen. The trapped 1-L002 in nitrogen was dispensed into the Grignard reagent at a flow rate of 5 ml min-1 over a period of 2 min at ambient temperature. A mixture of PDC (2 /il, 0.014 mmol) and DTBP (100 /il, 0.45 mmol) in diethyl ether was added and the reaction mixture allowed to stir for 2 min. The reaction solvent was then removed under nitrogen (30 ml min-1) by heating the reaction mixture

266 at 348 K. The formed [2-11C]furayl chloride was transferred from vessel A to vessel B by heating vessel A (Wood's alloy-bath, 498 K) and the PTFE line connecting the two reaction vessels with a hot air blower (623 K, Leistar-Chibli). The activity was trapped into a solution of 2- (piperazin-l-yl) -4-amino-6,7-dimethoxy-quinazoline (76) (2 ng, 7 /iinoi) in THE (1 ml) under nitrogen at 273 K. The reaction mixture was then heated gently (reaction vessel B touching Wood's alloy bath at 498 K) both to promote [1:LC] amide formation and to remove reaction solvent. The radioactive residue in vessel B on cooling was taken up in hplc mobile phase {dichloromethane containing 2.5% (v/v) of a mixture of ethanol, water and triethylamine (100:2:2 v/v)} and injected onto a preparative hplc column ('V-Forasil", particle size 10 ^m, 30 x 0.78 cm i.d., Waters Associates) eluted at 4 ml min-1 with the same mobile phase. UV-absorbance (254 nm) and radioactivity were monitored simultaneously and the radioactive fraction having the same retention time as the standard (7.5-9 min) was collected. This fraction was then evaporated to dryness and the residue dissolved in a mixture of ethanol and propylene glycol (600 /xl; 1:1 v/v) and diluted with sterile saline (10 ml, 3.8% v/v). The radioactive product was sterilised by millipore filtration (0.22 /xm, Millex-GS) before intravenous injection.

Analysis The radioactive fraction was found to be radiochemically and chemically pure by two different tic systems; silica gel 60 F254 sheets (Merck): system A, C H C ^ M e O Ihc.Nt^OH (18:2:1.6:0.2 v/v), Rf = 0.81; system B, CHCl3 :EtQAc:c.NH40H (3:7:1 v/v), Rf = 0.27. [1:LC] Prazosin also had the same capacity factor as authentic prazosin both in normal phase hplc (condition as above) and reverse phase hplc {"/x-Bondapak C18", eluant, acetonitrile and phosphoric acid (25:75 v/v), flow 2.0 ml min”1}. Further validation of the radiosynthesis was obtained by performing the preparation using 13C-enriched carbon dioxide under identical conditions to the radioactive procedure and examining the purified product by 13C-NMR spectroscopy (^-decoupled,

267 22.5 MHz, EMSO). The spectrum exhibited a single peak at 158.6 ppm, in accord with the chemical shift assigned to the carboxamido-carbon in prazosin.

Radiochemical yield and specific activity [1XC]prazosin (4.1-6.1 GBq; 110-180 mCi) was prepared in 30-40% r.c.y from 11O 02 (44-55 GBq? 1.2-1.7 Ci) in 35 min from BOB. The specific activity was found to be in the range of 26-37 GBq//imol (0.7-1.0 Ci//imol) at BOS.

268 4.3.3 Description of % - and ^ P K M R Spectra of iY-Acvl-THTO Ocnpounds

4 .3 .3.1 nmyariscn of % ■ and ^ C - N M R spectra of N- c^clcxaropanecaTtxaTvl-^IHIO (30) at different NMR field strengths Comparison of the -*-H-NMR spectra of compound 30 at 80.9 MHz (Fig 4.20), 89.6 MHz (Figs 4.40a-b) and 250 MHz (Fig 4.26) resulted

in the following observations. The C-l- O ^ proton resonances were broad and featureless at two low field strengths (80.9 and 89.6 MHz) whereas they had resolved into two singlets at 250 MHz. This effect was comparable to the variable temperature study at 89.6 MHz where at 243 K (Fig 4.40e) and 273 K (Fig 4.40d) the C1-CH2 resonances were resolved. Similarly, the C10-CH proton of the cyclopropyl ring exhibited fine structure at 333 K (Fig 4.40b) and 363 K (Fig 4.40a) when observed at 89.6 MHz. However, it became broad and featureless at 250 MHz (Fig 4.26) and appeared much like the signal at 243 K (Fig 4.40e) at 89.6 MHz. The proton decoupled 13C-NMR spectra of compound 30 in CDCI3 at 22.5 MHz exhibited two resonances for the heterocyclic ring carbons as well as the Cga, C4a and C5 carbons of the aromatic ring. However, the 13C-NMR spectra of compound 30 at 62.9 MHz (Fig 4.34) displayed two resonances for each of the cyclopropyl ring carbons as well as those described for the low field strength case. Similarly, the 13C-NMR spectrum obtained at 62.9 MHz at ambient temperature (Fig 4.34) was comparable to the variable temperature spectrum obtained at 213 K (Fig 4.41b) at 22.5 MHz. The signal patterns observed in the % - and 13C-NMR spectra of compound 30 at different field strengths and temperatures suggested that an analogy can be drawn between high field strength and temperature since the effects observed at high field strength in both % - and 13C-NMR spectra were similar to those observed at low temperatures at lew field strength. The criteria for these observations were however distinctly different. lowering of temperature reduces the rate of interconversion between two

269 conformers whereas only the observation frequency is increased in the case of high field strength spectra at ambient temperature. Subsequently the and 13C-NMR spectra of most of the other N— acyl-THIQ derivatives were recorded at high field strengths (^H-NMR at 250 MHz and 13C-NMR at 62.9 MHz) although same additional information was acquire! from the low field strength spectra.

4.3.3.2 %-KMR spectra of ff-acvl-THIO ocmpounds at ambient temrerature The lew field %-NMR spectra of canpounds 56 and 57 at 89.6 MHz are shown in Figs 4.21 and 4.22. The high field %-NMR spectra of compounds 30, 53-55 and 57-60 at 250 MHz are shown in Figs 4.23- 4.30. The proton chemical shifts of canpounds 30, 53-55 and 57-60 at 250 MHz and for compound 56 at 89.6 MHz are given in Table 4.8. The -^H-NMR spectra of compound 53 {R = Me, Fig 4.23}, 54 {R = Et, Fig 4.24}, 55 (R = Pr, Fig 4.25}, 30 {R = c.Pr, Fig 4.26} and 57 {R = vinyl, Fig 4.27} exhibited two resolved resonances for each of the C1-CH2 and C3-CH2 protons at 250 MHz. Slight broadening and coalescing of these pairs of signals was observed going from alkyl to the acryloyl derivative. The C3-CH2 signals of the cyclopropyl derivative was the most affected. The signals for the C4-CH2 protons and the R substituent in canpounds 53-55 also displayed a doubling up effect but the component resonances were generally overlapped, although coupling constants for the C3-CH2 and C4-CH2 protons could be extracted. For the cyclopropyl derivative (30) a broad featureless doublet was observed, whereas for the acryloyl derivative (57), an unresolved and featureless singlet was observed for the C4-CH2 protons. The proton signals for the cyclopropyl ring were broad and featureless and the signals for the vinyl group were too complex to discern the doubling up effect. The aromatic protons of compounds 30, 53-55 and 57 were observed as singlets and appeared to be generally unaffected by the R substituent. The low field spectra of compounds 53-55 resembled the high field spectra except that the doubling up effect was absent for the

270 C4-CH2 and the R substituent protons. On the otherhand the low field spectra of ccarpound 57 (Fig 4.22) exhibited extremely broad and unresolved pair of signals for the and C3 protons. Compound 56 (R = c.Bu, Fig 4.21) was only analysed at low field and two resonances were observed for each of the Cj and C3 methylene protons. The C4 methylene protons exhibited a triplet whereas the cyclobutyl ring gave a broad complex multiplet. A singlet was observed for aromatic protons. In compound 58 {R = Fh, Fig 4.28), the signals for the C^, C3 and C4 methylene protons were extremely broad. Even so a pair of signals corresponding to the and C3 methylene protons were still observed. The C4-CH2 pair appeared to be near coalescence point. In contrast signals for aromatic protons in this compound were still sharp. Ihe low field spectra of this compound gave only unresolved and featureless signals for methylene protons of the heterocyclic ring. The ^H-NMR spectra of compound 59 (R = furanyl) at high field (Fig 4.29) and low field were similar. The proton signals did not appear to be doubled up e x c e p t that a broad singlet was observed for the Cj -CH.2 protons. Two triplets were observed for the C3 and C4 methylene protons. The aromatic and the furanyl ring protons also gave very sharp signals at both fields. In compound 60 {R = thiophenyl, Fig 4.30} all proton signals were resolved and very sharp at both high and low fields. No indication of doubling up of signals was present at either field.

271 5 4

I------1------1------1------1------1------1------1------1------1 9876543210 SHIFT(PPM)

Fig 4.20. 1H spectrum of N-cyclopropanecarbonyl-THIQ (30) at 80.9 MHz in CDCI3 at ambient temperature Fig 4.21.1H spectrum of N-cyclobutanecarbonyl-THIQ (56) at 89.6 MHz in CDC!3 at ambient temperature

Fig 4.22.1H spectrum of N-acryloyi-THlQ (57) at 89.6 MHz in toluene-d8 at ambient temperature

273 r

Rg 4.24.1H spectrum of N-propiorryl-THIQ (54) at 250 MHz in CDC13 at ambient temperature 274 -i—-1— ■ 1 1------■—1—.------1 I------1 I.i. ------1______i1______I______8 7 7 6 6 5 5 4 4 3 3 2 1 1 ppm ppm Fig 4.25.1H spectrum of N-butyryi-THIQ (55) at 250 MHz in CDCI3 at ambient temperature

Fig 4.26.1H spectrum of N-cyclopropanecartoonyl-THIQ (30) at 250 MHz in CDCI3 at ambient temperature 275 Fig 4.27. *H spectrum of N-acryloyl-THIQ (57) at 250 MHz in CDCI3 at ambient temperature

Fig 4.28.1H spectrum of N-benzoyl-THIQ (58) at 250 MHz in CDCI3 at ambient temperature

276 (

Fig 4.29 1H spectrum of N-{furan-2-carbonyl}TH!Q (59) at 250 MHz in CDC!3 at ambient temperature

Fig 4.30 1H spectrum of N-{thiophene-2-carbonyl}-THIQ (60) at 250 MHz in CDCI3 ambient temperature 277

(s)h 86 (60) . 7.40(m)v 2.95(t)h 7.1-7.25 (m) 7.1-7.25 3.92(t)h 4 7.07(m)v 7.46(m)v

5.8*e 3.97(t)h 6.48-6.5 (m)u 6.48-6.5 4.99(s)fc 7.51-7.52(m)u —— 2.85^ 2.96(t)h 3.6(s)c 'r 4.0(s)r 4.6(s)r 4.9(s)c 'r 7.44k 7.44k

(57) (58) (59) 3.87 (t)h 'b 3.87 6.28-6.71^ 7.44k 7.0-7.05(m)u 7.17k 7.2k 7.13-7.25(m) 4.70(s)h »x 3.75(t)c 'h'x 4.77(s)c 'h'x

° ° P 5.69-5.74P 6 6 6 . . . 2 2 2 - - - 6 6 6 . . . 3.78(f) 1 2.80(t)h 2.891 4.46(s)h 3.53(t)c 4.69(s)c 'h 1

Ccnpourd

1 1 to (30) (56)n 1.75-1.9 2.85(tp . 2.95(t)c,3 3.7-4.0(m) ^-'w 3.7-4.0(m) 4.73 (s)c 'k'w 4.73 4.86(s)h /w O.S-O.O111 0.9-1.3

3.81(f) 2.82(t)d (t)c 'd 2.87 3.68(t)c 5.9 e X.69(m)f 2.37 (t)9 2.37 2.37(t)9 7.6** 4.60(s) 4.71(s)c 1.70|m)c '*- 0.984 (t)C/ f (t)C/ 0.984 7.4* * 0.976(f)f

(t)c 'd 8 8 . 3.82(f) 2.83(t)d 2 5.9*e 4.60(s) 4.72(s)c 3.66(t)c 2.43 (q)f 2.43 2.42(q)c ':t 7.4** 1.19(t)c 'f 1.18(t)f chemical -^H-NMR sh ifts (ppm) of N-Acyl-THIQ compounds3 '^

4.8. (53) (54) (55) .95-7.35 (m).95-7.35 (m) 7.0-7.3 (m) 7.0-7.3 6.95-7.5k 7.11k 2.82(t)d 4.71(s)c 3.80(f) 5.9*® 2.89(t)c 'd 4.59 3.65(t)c 6 2.16(s)c 2.17(s) T a b le 1 3 4 1 0 1 1 1 2 13 5-8 CartxnNo

278 Footnotes of Table 4.8 a) Spectra obtained at 250 MHz in CDCI3 at ambient temperature unless otherwise noted b) In N-(COR) -THIQ compounds R as follows;

R = Me (53), Et (54), Pr (55), c.Pr (30), c.Bu (56), vinyl (57), Fh (58), furanyl (59), thiophenyl (60) c) Most intense signal of pair d) Signals partially overlapped e) Average value for coupling constant f) Signals overlapped but components resolved g) Signal components of two conformers just visible from each other h) Slightly broad i) Signals overlapped giving arise to a broad multiplet j) Partially resolved broad triplets k) Broad signal with a central prominent feature l) Signal broad and unresolved m) Two complex multiplets with range of 8 0.5-0.9 and 0.9-1.3 assigned to cyclopropyl ring Clla+j-, protons n) Spectra obtained at 89.6 MHz in CDCI3 at ambient temperature o) Complex multiplet in the range of 8 1.6-2.6 with prominent features at 8 1.9 and 2.3 p) Two doublets overlapped but components resolved q) Range containing three prominent features, doublet at 8 6.29, doublet at 8 6.36 and complex multiplet between 8 6.59-6.71 r) Signal extremely broad s) Very broad signal centred at 5 2.85 with shoulder at 6 3.0 t) Broad and components not resolved giving the appearance of a singlet u) Signals sharp and assignment by comparison with 2-furaldehyde and 2-acetylfuran100

279 V) Signals sharp and assignment by comparison with thiophene-2- carboxaldehyde,101 2-acetylthiophene101 and thiophene-2- methylester1^1

X) % spectrum in toluene-dg at 89.6 MHz at 303 K gave broad and unresolved signals; Ci“CH2 protons at 5 4.85c and 4.55 and C3-CH2 protons at 5 3.8 and 3.5C y) % spectrum in toluene-dg at 89.6 MHz at 303 K gave broad and unresolved signals; C1-CH2 protons at S 4.6C and 4.38 and C3- CH2 protons at 5 3.61 and 3.23c

* 3J(H3H 4) = Hz k k 3J(H10H1;l) = HZ k k k 3J(HXiHi2) = Hz

280 4.3.3.3 -^C-NMR spectra of ff-acvl-IHIO cxarpounds at ambient temperature The broad band proton decoupled 13C-NMR spectra of compounds 30, 53-55 and 57-60 at 62.9 MHz and compound 56 at 22.5 MHz are shown in Figs 4.31-4.39. The carbon chemical shifts are given in Table 4.9. The 13C-NMR spectra of compounds 53 {R = Me, Fig 4.31), 54 (R = Et, Fig 4.32), 55 {R = Pr, Fig 4.33), 30 {R = c.Pr, Fig 4.34), 56 {R = c.Bu, Fig 4.35} and 57 {R = vinyl, Fig 4.36} exhibited two signals for each of the C^, C3 and C4 carbons. For all of these compounds except 57, the carbon signals of the R substituent and the aromatic ring were also doubled up although in the case of 30 the aromatic signals were overlapped extensively. In the case of compound 57 both the aromatic and vinyl carbon signals were overlapped making it impossible to distinguish any doubling up effect. In contrast to the above compounds, 58 (R = Fh, Fig 4.37) displayed very broad signals for the Clf C3 and C4 carbons, although each one was resolved into two components. The phenyl ring signals were very sharp in comparison. In the case of compound 59 {R = furanyl, Fig 4.38) an extremely broad signal ranging over ca. 10 ppm was observed for the and C3 carbons, individual component resonances could not be established. The signal for the C4 carbon was also broad but much less so than the and C3 carbons, whereas sharp signals were observed for the furanyl and the aromatic ring carbons. Two broad signals just visible above the baseline were observed for the C]_ and C3 carbons of compound 60 {R = thiophenyl, Fig 4.39) whereas the C4 carbon signal was less broad. Again, the aromatic and the thiophenyl ring carbon signals were very sharp. In the simple aliphatic derivatives 53-55, signals corresponding to two conformers were observed for all the carbons indicating that the rate of interconversion was slow at ambient temperature. For the vinyl (57) and the cyclopropyl (30) derivatives some carbon signals were clearly doubled up but it was

281 difficult to distinguish whether all of the carton signals were exhibiting this effect, implying that the rate of interconversion was faster than for the alkyl derivatives. In the case of compound 58 (R = Fh) broad signals were observed for the nitrogen ring carbons indicating that the rate of interconversion was faster than both the alkyl and alicyclic derivatives. The heterocyclic derivatives 59 and 60 on the otherhand exhibited exceptionally broad resonances for the nitrogen ring carbons and individual conformer resonances were not observed. This implied that the rate of interconversion must be too fast on the NMR scale for component resonances to be resolved. The features observed in the 13C-NMR spectra of compounds 30, 53-58 were similar to those in the ^H-NMR spectra except that exceptionally broad resonances were observed for the furanyl and the thiophenyl derivatives (59) and (60).

282 ppm Fig 4.32. Proton decoupled 13C spectrum of N-propionyl-THIQ (54) at 62.9 MHz in CDCI3 at ambienttemperature at CDCI3 in MHz 62.9 4.32. N-propionyl-THIQat of spectrum (54) Fig decoupled Proton 13C

283 Fig 4.33. Proton decoupled 13C spectrum of N-butyryl-THIQ (55) at 62.9 MHz in CDCI3 at ambient temperature

ppm Fig 4.34. Proton decoupled 13C spectrum of N-cyclopropanecarbonyl-THIQ (30) at 62.9 MHz in CDCI3at ambient temperature 285 ppm atambient temperature 3 Fig 4.37. Protondecoupled spectrum 13C of N-benzoyl-THIQ(58) 62.9at MHzin CDCI Fig 4.36. Proton decoupled 13C spectrum of N-acryloyl-THIQ (57) at 62.9 MHz in CDCI3 at ambient temperature ambient at CDCI3 in MHz 62.9 at (57) N-acryloyl-THIQ of spectrum 4.36. Fig decoupled 13C Proton

286 ppm 10

at ambiemtat temperature 3 in CDCI3 at ambient temperature ambient at CDCI3 in in CDCI Fig 4.38. Fig Proton decoupledspectrum of N-{furan-2-carbonyl}-THIQ 13C (59) 62.9at MHz Fig 4.39. Proton decoupled spectrum13C of N-{Thiophene-2-carbonyl}-THIQ (60) at 62.9 MHz PPm

287 9 . 13C-NMR chemical sh ifts (ppm) of JV-Acyl-THI( 5 3 -5 5 )a ' b

No (5 3) (5 4 ) (5 5)

1 44.02°' 9 1 d 44.20c ' d '9 44.14°'d' 9 48.029 47.19d ' 9 47.36d ' 9

3 39.43^ 39.64d ' 9 39.58d '9 43.96°'9/d 43.06°'d '9 43.22°'d '9

4 28.48d 28.53d 28.52d 29.40°'d 29.49c ' d 29.58°'d

4i 134.02°'d'f 'h 134.13c ' d ' f 'h 134.08°'d'f 'h 135.02d ' f 135.45d ' f 135.12d' f

5 128.27c 'd 128.26c ' d 128.26°'d 128.87d 128.92d 128.90d

6 126.01e 126.04e 126.00e 126.48c ' e 126.47°'e 12 6.44°'e

7 126.31e 126.31e 126.68e 12 6.54°'e 12 6.53°'e 12 6.50°'e

8 12 6.57c ' e 126.64°'e 126.62°'e 126.85e 126.85e 126.82e

8< 132.55d ' f 13 2.7 0d ' f 132.72d ' f 133.46°'d'f 'h 133.68°'d'f 'h 133.63°'d'f 'h

9 169.34 172.70 171.88 1 69.43c 172.72c 171.99°

10 2 1 .63c 26.77° 35.52° 21.90 26.98 35.71

11 9.35 18.57° 9.33° 18.65

12 13.83 14.02

1 3 288 4.9 -^C-NMR chemical shifts (ppm) of N-Acyl-THIQ

No (56) (57) (58) (59)

1 4 4 .3 6 c ' d '9 4 4 .5 3 c ' d '9 4 5 .0 0 c ' d 'ln'9 4 6 .3 P 'r 4 6 .7 9 d '9 4 7 .4 7 d '9 4 9 .5 5 d 'm'9

3 3 9 .8 6 d '9 3 9 .9 9 d ,S 4 0 .2 7 ^ 'iri 4 3 .5 P 'r 4 2 .5 7 c ' d '9 4 3 .5 1 c ' d '9 4 4 .5 7 c '9,m

4 2 8 .47d 2 8 .4 2 d 2 7 .8 0 d 'm 2 9 .1 7 P 'V 2 9 .6 4 c ' d 2 9 .5 4 c ' d 2 9 .2 6 c ' d 'm

4a 134.06c'd'f 134.10c'd'f 'h 0 1 3 4 .62P 1 3 5 .1 5 d ' f 1 3 5 .01d , f

5 1 2 8 .4 0 c ' d 1 2 8 .3 0 c ' d n 1 2 8 .62P 1 2 8 .98d 1 2 8 .88d

6 1 2 5 .96e 1 n 1 2 6 .3 3 P 'V 1 2 6 .29e

7 1 2 6 .03e 1 n 1 2 6 .49P 1 2 6 .40e

8 1 2 6 .51e 1 n 1 2 6 .74P 1 2 6 .69e

8a 1 3 2 .90d ' f 1 3 2 .51d ' f 0 1 3 3 .0 9 133.63c'd'f 'h 133.37c'd'f 'h

9 1 7 3 .5 2 1 65.66 1 7 1 .9 7 1 5 9 .5 8 1 6 5 .81c

10 3 7 .5 6 ? '3 1 0 1 4 8 .1 6 s 37.683

11 25.083 1 n 1 1 6 .21f

12 1 7 .8 9 ? '3 n 1 1 1 .2 8 f 18.053

13 0 1 4 3 .8 3 s

289 Footnotes of Table 4.9

a) Spectra obtained at 62.9 MHz in CDCI3 and at ambient temperature unless otherwise noted. Multiplicity establed by DEFT unless otherwise noted b) In compounds N-(COR)-THIQ, R as follows;

R = Me (53), Et (54), Pr (55), c.Pr (30), c.Bu (56), vinyl (57), Fh (58), furanyl (59), thiophenyl (60)

c) Most intense component of pair

d) Assignment by analogy with THIQ (26)81'83'102 (see Table 4.15) and N-furan-2-carbonyl-THIQ (60)

e) Six aromatic resonances in the range 5 126.01-126.85 can be paired in six possible combinations of three pairs (other combinations ruled out on intensity considerations). One set of three pairs has been choosen at random but five alternative sets are equally possible. The small chemical shift variations of the possible pairs makes definitive assignment impossible

f) Other possible combinations of signals eliminated on the basis of intensity considerations

g) Assignment of pairing of signals confirmed by "carbon-proton 2D chemical shift correlation" study performed on compound 54

h) Alternative assignment

i) Overlapping signals

j) By analogy with data for cyclobutylcarboxamide.103

k) 1H decoupled spectra obtained only, multiplicity not established

l) Broad overlapping signals with peaks at S 126.04, 126.39, 126.61c, 126.90, 127.76c and 127.90 were assigned to C6, C7 , Cg, C10 and cll carbons. Assignment of individual resonances was impossible due to the proximity of the chemical shifts m) Signal very broad

n) Overlapping signals with peaks at S 126.24, 126.39, 126.67, 127.96, 128.22, 128.47, 129.05 were assigned to C5 , C5 , Cy, C8 , ciia+lib arid c12a+12t) carbons.d Assignment of individual resonances was impossible due to the proximity of the chemical shifts

290 0 ) Signals at S 132.59, 135.60 and 135.69 assigned to C4a, C8a/ C10 and c13 carbons. Individual resonances could not be assigned p) Assignment by analogy with compound (26)81'83 '102 q) Signal broad r) Signal extremely broad and overlapped, chemical shift measured from spectrum s) Assigned by comparison of intensity and chemical shift t) By analogy with data for 2-furaldehyde100'104 u) Signal not resolved at 62.9 MHz but resolved at 22.5 MHz v) Signals too broad to be observed therefore multiplicity by DEFT not established w) By analogy with data for thiophene-2-carboxaldehyde100'105 and thiophene-2 -carboxyl ic acid methyl aster100'105 x) Signal more intense probably due to overlap y) Signal at 128.81x assigned to and 05^ or C13w '11

291 NMR 4.3.3.4 Variable temperature -% 7spectra o f JV-cvclopropaiiecartxjnvl- THIQ (30) The % - spectra of compound 30 at 89.6 MHz in the temperature range 243-363 K are shown in Figs 4.40A-B. All proton signals at 363 K exhibited singlet behaviour, the cyclopropyl ring signals (2 0 .6-1 .8 ) were sharp with fine structure whereas the nitrogen ring C^, C3 and C4-CH2 proton signals were fairly broad. As the temperature was decreased from 363-243 K the Cy-CR.2 singlet (8 4.74, 363 K; Fig 4.40a) and the C3-CH2 triplet (5 3.72, 363 K; Fig 4.40a) both broadened and then decoalesced into two resonances for each of the methylene groups. Two singlets at 5 4.87 and S 4.55 (Fig 4.40e) and two triplets at 5 3.85 and 6 3.39 were observed for the and C3-CH2 protons respectively. Integration of signals at 273 and 243 K, demonstrated that the lower field component of C1-CH2 (6 4.87) was more intense whereas the higher field component of C3-CH2 (6 3.39) was more intense. The C4-CH2 triplet (5 2.70, 363 K; Fig 4.40a) did not change character in the 363-273 K range but became very broad and featureless at 243 K (Fig 4.40e).

The cyclopropyl C^q -CH multiplet (5 1.5-1.8 , 363 K; Fig 4.40a) started to lose its fine structure at 303 K (Fig 4.40c) whereas the clla+b methylene protons began to broaden and became featureless at 243 K (Fig 4.40e). The aromatic CH signals were also broadening and becoming featureless with decreasing temperature.

292 C) 303C) K ______ppm 1 1 A i ------_J at 89.6 MHz atdifferent temperatures 8 L^J ______I j ______1 ------1 ------H-NMR spectra ofN-cyclopropanecarbonyl-THIQ in toluene-d 1 b . a ______I------1 8 7 6 5 4 3 2 j Fig 4.40

293 4.3.3.5 Variable temperature ^C-NMR spectra of cwclopropanecarbcnvl-THIO (30) The proton decoupled 13C-NMR spectra at 22.5 MHz were acquired either in CDCI3 (302 and 213 K, Figs 4.41a & 4.41b) or in toluene-dg (302, 333 and 363 K, Figs 4.41c-e). The signals corresponding to only the C2, C3, C4 and the cyclopropyl ring carbons C1Q/ Clla+k are shown in Fig 4.41a-e. Multiplicity of signals was established by DEFT. At ambient temperature two pairs of signals were observed for the C]_ and C3 carbons both in CDC13 (Fig 4.41a) and toluene-d8 (Fig 4.41c). The two lower field signals (6 47.18 and 44.72) were assigned to the carbon whereas the two high field signals (8 43.28 and 40.0) corresponded to the C3 carbon (see Sec 4.2.3. for assignment). The and C3 carbon pair signals coalesced into two broad signals as the temperature was increased from 302 to 333 K (Fig 4.41c+d). They sharpened to give two singlets having an average chemical shift of the two signals observed for the individual paired resonances. A decrease in the temperature on the other hchd resulted in the sharpening of the paired resonances (Fig 4.41b). At ambient temperature partially resolved signals were observed for the C4 carbon in CDC13 (Fig 4.41a) whereas in toluene-dg they were less resolved (Fig 4.41c). An increase of temperature from 302-363 K resulted in the coalescence of these signals to give a sharp singlet at 363 K, whereas lowering of temperature gave resolved signals for the paired resonances. Two sharp signals were observed for the cyclopropyl ring carbons (<$ 11.46 and 7.45) at ambient temperature which remained unchanged with an increase of temperature but were resolved into two pairs with decreased temperature (213 K, Fig 4.41b). The aromatic carbons and the carbonyl carbon signals were also doubling up as the temperature was decreased from 302-213 K.

294 b) 213 K, in CDCI3 JUV.

a) 302 K, in CDCI3

■ I . I I. 60 30 20 10 0 ppm Fig 4.41 A.E Proton decoupled 13C spectra of N-cyclopropanecarbonyl-THIQ (30) either in CDCI3 ortoluene-d8 at 225 MHz 295 4 .3 .3 .6 Variable temperature % - N M R spectra of //-(thiochene-2- cartoiwll-THIQ (60) The variable temperature study of compound 60 was conducted over a range of 213-273 K at 250 MHz in CDCI3 . The proton chemical shifts at these temperatures are given in Table 4.10. The %-NMR spectra of compound 60 showed no indication of broadening or doubling up of signals at ambient temperature. The Cy- CH2 protons gave a singlet at 5 4.87 which was broad at 273 and broader still at the lower temperature 268 K. This signal decoalesced and resolved into two singlets (8 4.96 and 4.85) as the temperature was decreased from 263 to 233 K (Table 4.10). Sharpening of the two signals was observed with further decrease in tenperature to 213 K. The C3 (8 3.93) and C4 (8 2.95) methylene triplets were sharp between 273 and 268 K. As the temperature was decreased to 253 K, the individual triplets became broad and featureless. Further decrease in temperature resulted in the signals becoming more featured until two broad peaks each having two sharp central features (C3 8 3.95 and 3.97, C4 8 3.01 and 2.98, 213 K; Table 4.10) were observed. The thiophenyl ring protons gave rise to complex multiplets and elucidation of doubling up of signals was impossible although variation in the multiplet features was observed with decrease in temperature. The multiplet corresponding to the O q proton (5 7.35-7.45, 273 K, Table 4.10) broadened and decoalesced into two sets of multiplets (5 7.37-7.42 and 7.42-7.48, 213 K) as the temperature was decreased from 273-213 K. The C12 proton multiplet (6 6.95-7.13, 273 K) only began to broaden at 253 K and decoalesced with decreasing temperature into a complex set of multiplets. In contrast the C13 proton multiplet (<$ 7.45-7.60, 273 K) was virtually unaffected by the decrease in temperature although at lower temperature it was becoming less resolved. A singlet was observed for the aromatic protons between 273-243 K which became more featured as the temperature was decreased.

296 213 3.95n 3.97n 3.01n 2.98n 7.22 4.86(s) 4.99(s) 7.07-7.17 7.42-7.48 7.37-7.42 7.48-7.65 (m) 3.94n 3.96n 2.98n 2.99n 7.22 4.86(s)e ^h 7.26 4.98(s)h 7.08-7.16y 7.41-7.47t 7.37-7.41t 233 223 (m)z 3.94m 3.96m 2.99° 4.96(s) 4.85(s)e 7.24 2.97° 7.07-7.15^ 7.47-7.65 7.47-7.65 )9 s

(m)r 7.41-7.47s 3.93m 3.95ra 4.85(s)e '9 4.95( 2.97k 7.20 7.21 7.23 . 7.07-7.13x Temperature (K) (m)v (m)<3 253 243 3.931 4.94(s)^ 4.85(s)e '£ 2.97 7.22 7.20 6.92-7.13 6.96-7.07x 6.98-7.07u 6.95-7.08^ 6.85-7.07 7.35-7.45 7.33-7.47 7.35-7.41s (m)q (m)1 (m) (m) (m) 4.85(s)d 'e 2.96c 6.90-7.13 7.35-7.45 7.45-7.60 7.45-7.65 7.47-7.65 (m) (m) 3.93(b)3 3.94(b)k 2.96c 4.86(s)c 4.93(s)d 7.19 7.19 6.96-7.13 7.35-7.45 273 268 263 (m)P (m)P (m)u (m)P 3.93£ 4.87(s)b 2.95c 7.19 7.35-7.45 7.95-7.13 7.45-7.60 7.45-7.60 1 3 4 11 5-8 13 12 Table 4.10 Variable temperature 1H-NMR chemical shifts (ppm) of N-thiophene-2-carbonyl-THIQ (60)aTable 4.10N-thiophene-2-carbonyl-THIQ of (ppm) shifts temperature 1H-NMR Variable chemical Carbon No 297 Footnotes of Table 4.10 a) Spectra obtained at 250 MHz in CDCI3 b) Broad, c) Very broad d) Singlet pair partially resolved and very broad e) Most intense signal of pair f) Signal pair partially overlapped and fairly broad g) Singlet pair almost resolved and slightly breed h) Singlet pair resolved and fairly sharp i) Two multiplets partially resolved j) Triplet beginning to broaden k) Broad with minor features l) Signal very broad and featureless m) Signal pair overlapped giving arise to a broad base with two sharp features at centre n) Signal no resolved with two central sharp features o) Signal broad with minor features at 5 2.97 and 2.99 p) C^2 C13 protons very sharp but C-^]_ protons slightly broad q) Base of multiplet beginning to broaden r) Two multiplets partially resolved s) Two multiplets partially overlapped and fairly broad t) Two doublets sharpening and resolving u) Signal broader than at 273 K v) Presence of broad shoulder on main multiplet x) Main multiplet broad with features centred at 5 7.07 partially overlapped with a broad featureless signal centred at 5 7.02 y) Multiplets becoming more complex with signals sharpening and resolving z) Complex multiplets beginning to loose features

298 4.3.3.7 v ^tH piblp> temperature ^C-MMR spectra of N-

4 .4 2A —g • The 13C chemical shifts for these spectra are given in Table 4.11. Multiplicity of signals was established by DEFT. At ambient temperature (Fig 4.42a) the and C3 carbon signals (C! S 47.0, C3 S 43.8) were extremely broad and just visible above the noise. These signals coalesced into a very broad signal over a 10 ppm range at 283 K (Fig 4.42b) which resolved to give three broad signals (5 49.6, 45.47, 41.5) at 273 K (Fig 4.42c). The central signal (5 45.57) was the most intense signal due to the overlap of component resonances of the and C3 pairs. Sharpening of these signals was observed with decreasing tenperature with the most intense signal resolving into two partially overlapped components (5 45.30 and 45.21) at 223 K (Fig 4.42g). A broad signal was observed for the C4 carbon (5 28.91) at 302 K (Fig 4.42a). This signal broadened and then resolved into two signals with decrease of tenperature. Sharp signals (5 27.61 and 29.23) were observed at 223 K (Fig 4.42g). The lower field signal was the most intense. The thiophenyl and the aromatic ring carbon signals were generally overlapped except for the tertiary carbons (C4a, C8a and C10). However the two multiplets (5 126.1-128.7, 302 Fig 4.42a) observed for the CH carbons broadened and the signals coalesced as the tenperature was decreased from 302-253 K (Fig 4.42a- 4.42e). Further lowering of temperature resulted in the resolving and sharpening of these signals (Fig 4.42f). At 223 K (Fig 4.42g) fourteen sharp signals in the range 125.59-129.14 ppm (Table 4.11) were observed for the CH carbons. Cue to the close proximity of the chemical shifts it was impossible to assign them to individual carbons of the aromatic and thiophenyl rings. The three tertiary

carbons C4a, Cga and C^q (5 134.19, 132.71 and 137.31, 302 K) and the carbonyl Cg-carbon (S 163.57, 302 K) signals also broadened and then resolved into two resonances as the temperature was decreased from 302-223 K (Fig 4.42a-g).

299

ppm ppm Dom X C- & C, C3 c,&c3 C, C, — j - / 12Q 60 _i» _i» 120 eo r f 300 L \J 8a '8a 8a c '10 .L C4 L. c10 at 62.9 MHz in CDCI3 at different temperatures different at CDCI3 MHz 62.9 in at - S f Fig 4.42A.G (60) 4.42A.G Fig N-{thiophene-2-carbonyl}-THIQ of spectra 13C decoupled Proton ______Fig 4.42a)Fig T =303 K Fig 4.42b) 4.42b) Fig K =283 T Fig 4.42c) 4.42c) Fig K =273 T Cg Cg C10 J ° -i Fig 4.42d) T=263K

301 z o z

05 0* 031 OCI 0*1 ost 001 1 . . I |— i- " I"""" - | ' ■■■■■ | | — . |

03 Ludd OS 09 0 3 1 OCI 0*1 OSl osi - T " I ' I ------I ' I I ■

>1 =1 (izp'p By

b 6 < P

49 . 223 29.23b'k 27.61b 52b'k 45.21b 4 5 . 3 0 ^ 49 40. 163.12b 163.75b'k 132.36b,k 132.21b 126.65° 136. 126.59° 137.06k 126.28° 126.35° 125.59° 125.98° 128.88n 129.14n 134.21b 128.21n 128.83n 133.68b'k 126.47° 128.36n 128.74n

243 29.36°'k 27.76° 45.29°'k 40.76° 45.36°'k 49.55° 132.34 163.20 163.78k 132.51k 136.78k 126.81h 137.23 126.06h 125.65*1 129.34n 128.24n 128.89n 129.05n 133.76k 134.34 126.41h 128.42n 128.74n

253 29.52°'k 27.95° 49.72° 45.51°'3 40.95° . 40.95° 45.51°'j 164.07k'm 137.17k'm 137.44m 132.77 163.55m 128.999/1 126.87h 126.72h 125.96h 134.07k'm 128.529/1 126.87h 134.63m 128.729/1

263 94d'f'k 29.47d 28.17d'f 45.50d'3 41.0d 41.0d . 45.50d'3 49.69d 163.90° 132.78° 137.33° 126.81h 126.72h 126.47h 128.979/1 128.979/1 134.19° 126.81h 128.979/1 Temperature (K) Temperature 273 29.02d 45.47d'f'3 49.60d'f 45.47d'f'3 41.50d/f 163.56 137.19 132.62 128.729 126.60^ 126.54h 126.28h 128.459 126.6011 134.12 128.459 b 29 5i 283 28.90d 41. 41.5^ 163.58 137.26 128.669 132.67 126.38h 126. 126.17h 128.459 134.17 126.58h 128.459 302 28.91° 47.0d'f 43.8d'f 163.57 137.31 128.659 132.71 126.29*1 126.57h 128.449 126.15h 126.57h 134.19 128.449 a 8 6 8 9 7 5 4a 1 4 3 No Table 4.11 Variable temperature 13C-NMR chemical shifts (ppm) of N-{thiophene-2-carbonyl}-THIQ (60)a 10 1 1 12 13 Carbon

303 Footnotes of Table 4.11. a) Spectra obtained at 62.9 MHz in CDCI3 , multiplicity established by DEPT at 223 K b) Sharp, c)Slightly broad, d) Broad, e) E x t r e m e l y broad

f) Chemical shift measured from spectra g) Alternative assignment for C5 , and C13 carbons, signal assigned to more intense than C5 and C13 carbons h) Alternative assignment for C6, C7, C8 and C12 carbons, signal assigned to C12 mere intense than Cg_C8 carbons

i) Signal with a range of ca. 10 ppm, centre measured at 41.5 ppm from spectra j) Central feature of and C3 pairs overlapped k) Most intense signal of pair l) Broad and overlapped m) Signal pair partially overlapped and broad n) Combination of pairs chosen at random, alternative pairing equally possible. C5 carbon pair assigned by analogy with other N-acyl^IHIQ amides in Table 4.9 o) Pairing for Cg, C7, C8 and C^2 carbons chosen at random, alternative pairing equally possible

304 4.3.3.8 % - N M R spectra of ff-acyl-TBO ocBapounds at ambient temperature The proton spectra of compounds 29 {R =c.Pr}, 61 {R = Me}, 62 {R = Et}, 63 (R =Pr} and 64 {R = thiophenyl} were run in CDCI3 at 250 MHz and at ambient temperature. The chemical shifts and coupling constants are given in Table 4.12. The proton spectra of N-thiophene-2-carbonyl-THQ (64) is shown in Fig 4.43. For all compounds the expected chemical shift pattern was observed. All proton signals were sharp and no indication of broadening or doubling up of resonances was observed.

4.3.3.9 13C-NMR spectra of W-acyl-THO compounds at ambient temperature The proton decoupled 13C-NMR spectra of compounds 29, 61-64 were run in CDCI3 at 62.9 MHz and the 13C chemical shifts are given in Table 4.13. The 13C-NMR spectra of compound 61 (R = Me) and 64 (R = thiophenyl) are shown in Figs 4.45 and 4.46, respectively. Several interesting features were exhibited by compound 61. Very broad signals were observed for the C2 (5 43.22) and C8a (S 139.29) carbons whereas an extremely broad signal just visible above the noise level was exhibited by C4a (6 133.3; chemical shift estimated from spectrum). In contrast the other carbon signals were sharp and not doubled up. In the case of compounds 62 (R = Et) and (63) (R = Pr), C2 and C8a carbons were broad but less so than in compound (61), whereas C4a was still extremely broad. Again the other carbon signals were sharp. All of the carbons in compound 29 (R = c.Pr) and 64 (R = thiophenyl) exhibited sharp signals and only singlet behaviour was observed for all carbons.

4.3.3.10 %-NMR spectra of ff-aoetyl-THD (60} at low temperature The %-NMR spectra of compound 60 at 213 K in CDCI3 at 250 MHz is shown in Fig 4.47. The C2-CH2 and C3~CH2 protons gave two pairs of resonances with the lower field component of the C2-CH2 pair being the more intense and the higher field component of the C3-CH2 pair

305 at 62.9 MHz in CDCI3 at ambient temperature ambient at CDCI3 MHz 62.9 in at Fig 4.43 Proton decoupled spectrum 13C of N-{thiophene-2-carbonyl}-THQ (64)

306 at62.9 MHz in CDCI3 atambient temperature Fig 4.46 Proton decoupled 13C spectrum of N-{thiophene-2-carbonyl}-THQ (64)

307 308 8s ' t .6-7.0 (64) 6.61** 3.90 2.78 7.02-7.Is 2.01 7.15-7.22s 7.3-7.3 .6 6 * 6.70* 66 .9-7 . 1.96 2.74° 0.6-1.35r 0.6-1.35r 3.81 6.61** 6 6 ** 68 ^^kkkk . 6 1.5-1.83 0.9h 7.0-7.4 r-j r-j 3.78 3.79 1.95 1.96 6.95-7.37 1.16h 6.56* 6.62* 6.67** 2.71 2.71 2.51i 2.48h 7.41 7.52***

1 .29-j-'m 3.74 3.83m 2.7 3m 2.86 2 2.37 2.05-2.28n 4*4* (61) (61)e (62) (63) (29) 1.96 1.9-2.05m 3.79 6.57* 6.62 2.72 7.02-7.25 7.1-7.3 2.23f

(78)d 3.58k 6.90-7.10P'9 1.40^ 2.30k 8.9, 8.9, 6.7k '<3 8.08k 8.70k '1 h 2.90k 33 2 4 10 12 No 11 Table4.12. chemicalshifts (Km) N-acyl-THQof cxupounds 61-64,78)a_c (29, 13 5-83 Cartoon

309 Footnotes of Table 4.12 a) Spectra obtained at 250 MHz in CDCI3 at ambient temperature unless otherwise noted, IMS used as internal standard b) In conpounds N-(Q0R)-IHQ, R as follows;

R = H (78), Me (61), Et (62), Pr (63), c.Pr (29), thiophenyl (64) c) Assignment of all compounds by analogy with reference data for compounds 61 and 78 d) N-forrrryl-THQ (78) included for comparison65 e) Chemical shift data for compound 61 at 213 K f) Singlet, g) Doublet, h) Triplet, i) Quartet, j) Multiplet k) Toluene-dg used as solvent65 l) Centre of multiplet m) Most intense component of signal pair n) Multiplets partially overlapped o) Assignment confirmed by homonuclear decoupling experiment p) Chemical shift range obtained from Krivdin et al.68 Sample run in EMSO-d^ q) Chemical shift range of 8 6.9-7.1 refers to C5-C7 protons whereas 8 8.9 and 6.7 refers to the Cg proton r) Three sets of multiplets with ranges of 8 0.6-0.95, 0.98-1.1 (least intense) and 1.1-1.35 s) Assignment by analogy with thiophene-2-carboxaldehyde,100 thiophene-2-methylamide100 and thiophene-2 -methylester10 0 t) Doubled up doublet

* 3J(H2H 3) = Hz

** 3J(H3H4) = Hz

*** 3J(H10H h ) = Hz

**** 3J (H^H^) = Hz

310 T a b l e 4.13 . 13C-NMR chemical shifts (ppm) of tf-Acyl-THQ series (29, 61—64)a /b

Carbon No (61)c (62) (63) (29) (64)

2 43.22d'e 43.11°'^ 43.04c'i 43.15° 44.50°

3 24.09h 24.15c 24.25c 24.18° 24.24°

4 26.91 26.85c 26.87c 26.98° 26.70°

4a 133.39 133.19°'d '3 133.19c '9/j 133.33° 132.63°'°

5 128.44 128.47c 128.45c 128.45° 128.22°

6 125.17 125.12c 125.15c 124.89° 125.50°

7 126.09 126.02c 126.06c 125.96° 125.80°

8 124.62 124.63c 124.75c 124.67° 125.15°

8 a 139.29d'f 139.27c 139.39° 139.26° 139.31°'°

9 170.05 173.65 172.83 173.44 163.29

10 2 3 . 12h 27.88k 36.49k '1 13.60k 138.53° o 00 H o 11 19.30k '1 9 . 10k 'm 131.04n

12 13.84k '1 126.56n

13 129.72n

311 Footnotes of Table 4.13 a) Spectra obtained at 69.2 MHz in CDCI3 and at room temperature unless otherwise noted. Multiplicity of signals established by DEFT unless otherwise noted b) In N-(CQR)-THQ compounds, R as follows;

R = Me (61), Et (62), Pr (63), c.Pr (29), thiophenyl (64) c) Assignment by analogy with THQ (25)106 and N-acetyl-THQ d) Signal very broad e) Averaged value for chemical shift f) Signal just visible above noise g) Signal extremely broad, chemical shift measured from specturm h) Assignment opposite to literature confirmed by multiplicity established by DEPT i) Signal fairly broad j) Signal not broad compared to other carbon signals at 22.5 MHz k) Assignment based on chemical shift and multiplicity l) By analogy with literature values107 m) Cyclopropyl ring carbons Clla and C-^ equivalent and signal intense n) By analogy with data for thiophene-2-carboxaldehyde100'105 o) Assignment assisted by intensity

312 being the more intense. A large difference in the population ratio of the two conformers was observed (ca. 9:1). The C3-CH2 multiplet also showed signs of doubling up but the two sets of resonances were not resolved. In the case of the C10-CH3 group two sharp singlets were observed with the higher field component being the more intense. The aromatic protons exhibited a complex multiplet with fine structure so it was impossible to discern whether the signals were doubled up.

4.3.3.11 %-NMR spectra of N- ( -THIO compounds at ambient temperature The proton spectra of THIQ 26 and N-{ CH2R}-JTHIQ compounds where R = Me (65), Et (66), c.Pr (34), pro-2-ene (67), Eh (68) and furanyl (69) were run in CDCI3 at 250 MHz at ambient temperature. The chemical shifts and coupling constants are given in Table 4.14. For all compounds the expected chemical shift pattern was observed. In the case of the heterocyclic ring protons, a singlet was observed for the C1-CH2 and triplets for the C3-CH2 and C3-CH2 groups. A multiplet was observed for the aromatic ring protons. The individual R substituents displayed there corresponding signals. All signals were sharp and no indication of broadening or doubling up of signals was observed.

4.3.3.12 13C-NMR spectra of N- (CHoR) -IHIO compounds at ambient temperature The proton decoupled 13C-NMR spectra of N-{ O^Rj-THIQ compounds where R = H (79), Me (65), Et (66), C.Pr (34), pro-2-ene (67), Ph (68) and furanyl (69) were run in CDC13 62,9 MHz and the chemical shifts are given in Table 4.15. For all compounds sharp signals were obtained and no indication of broadening or doubling up of signals was observed.

313 4.3.3.13 -*H-NMR spectra of iV-ICHgRl-TIK) compounds at ambient temperature The l-H-NMR spectra of THQ (25) and N-{CH2R }-™ Q compounds where R = Et (70) and Pr (71) were run in CDCI3 at 250 MHz. The chemical shifts and coupling constants are given in Table 4.16. The heterocyclic ring C4-CH2 protons displayed a triplet whereas C3-CH2 protons exhibited a multiplet. However it was difficult to distinguish between the resonances exhibited by the C2-CH2 and C9-CH2 protons. For all compounds the signals were sharp and no indication of broadening or doubling up of signals was observed.

4.3.3.14 -^C-NMR spectra of iY-ICH^Rl-THO ocaroounds at ambient temperature The proton decoupled 13C-NMR spectra of compounds (25,70,71) were run in CDCI3 at 62.9 MHz and the chemical shifts are given in Table 4.17. For all compounds signals were sharp and no indication of broadening or doubling up of signals was observed.

314

(m) (m) (m) (m) 12 12 13 (m)° (m)° 6.33 7.38 7.2-7.5 7.2-7.5 4.95-5.25 11 (m) (t) 0.95 (d)° (m) 6.27 5.75-5.98 0.01-0.651 7.2-7.5

(m) 10 (m) (lTl) **• (lTl) 2.36 1.62 1.14 7.37**3 0.9-1.05 3

9 (s)n (s)n (d) 3.72 6.51 3.67 2.59m 'k 2.41 2.5i 7.72**3 2.5-2.99

(m)d (m)d (m)d (m)d (m)d (t) (m)e 7.0 5-8 6.9-7.2 6.9-7.2 6.93-7.2 6.95-7.25 6.9-7.4 6.94-7.24 Carbon No )1

3

3 4 (t)k (t)k . (t)k (t)k (t) * (t) 5.75*3 2.74 5.53*3 2.78 2.78 5.75*3 2.72 5.77 3 5.87 2.7? 3.09 5.95* 2.5-2.99 1 (s) (s) (s) (s)n (s)n (s) (s) 3.64 3.66 3.70 3.62 3.62 3.97 3.63 )h ) 66 68 (34) j (34) (67) ( ( (69) (65) f (65) (26)d Table 4.14. ^-H-NMR chemical shifts (ppm) of N-{CH2R}-THIQ compounds (26,34, 65-69)a c Compound

315 Footnotes of Table 4.14 a) Spectra obtained at 250 MHz in CDCI3 at ambient temperature unless otherwise noted, TMS used as internal standard b) Compound number and identity of R in N-{CH2R}-THIQ compounds as follows;

THIQ (26) and in N-{CH2R)-JIHIQ, R = Me (65), Et (66), c.Pr (34), pro-2-ene (67), Hi (68), furanyl (69) c) All chemical shifts assigned by comparison with THIQ, N- methyl-THIQ108 and multiplicity d) THIQ (26) included for reference. A broad signal with a chemical shift of S 1.98 was observed for NH protons. Shift confirmed by D2O exchange e) Complex multiplet with one major and one minor feature f) Spectra obtained at 89.56 MHz in CDC13 with TMS as internal standard g) Complex set of multiplets, impossible to assign individual methylene groups h) A carbon-proton 2D chemical shift correlation study performed

i) Chemical shifts assigned from carbon-proton 2D spectrum and triplets at 8 2.9 and 2.72 roofed towards each other j) Averaged values k) Assignment by analogy with compound (66)

l) Two sets of complex multiplets with chemical shift range of 5 0.1-0.3 and 0.45-0.65 assigned to Clla and C^^b protons m) Complex but basically a triplet n) Alternative assignment o) Assignment based on assuming a first-order spectrum and comparison with furan-2 -methanol10 0

* 3J(H3H 4) = Hz

** 3 J (%%()) = Hz

316 Table 4.15. 13C-NMR chemical shifts (ppn) of N-{CH.2^-)-rIHLQ (34, 65-69, 7 9 )a 'k

Carbon No (79) c (65) d (66) (34) (67) (68) (69)

1 47.92 56.86e 56.20^- 56.15e 56.02e 56.09e 'm 54.44e 'm

3 43.50 51.34e 50.951 50.87e 50.82e 50.60e 50.33e

4 28.88 29.84e 29.lO1 28.93e 29.00e 29.lle 28.93e

4af '

5f 129.08 127.30 128.59 128.56 128.57 128.63° 128.56 sf 125.51 126.24 125.49 125.44 125.50 125.52 125.49

7f 125.80 126.76 125.99 125.63 126.01 126.03 126.04

CO 126.01 127.29 126.56 126.54 126.52 126.55 126.51

8af '9 134.53 135.02 134.37 134.21 134.24 134.34 134.05

9 56.49f'9 60.461 63.52e 57.69e 62.76e 'm 53.39e 'm

10 13.05h 20.351 8.613 31.69*1 138.36^/h 151.73P

11 11.981 3.923'k 136.551 128.26n'° 108.67P

12 115.611 129.01n '° 110.03P

13 127.04n 142.13P

317 Footnotes for Table 4.15 a) Spectra obtained at 62.9 MHz in CDC13 and at ambient temperature unless otherwise noted. Multiplicity established by DEPT unless otherwise noted b) In compounds W-{CH2R}-THIQ where R as follows;

R = H (79), Me (65), Et (66), c.Pr (34), pro-2-ene (67), Fh (68), furanyl (69) c) All chemical shifts assigned by comparison with literature82 d) Spectra obtained at 22.5 MHz in CDCI3 and at ambient temperature. % decoupled spectra obtained only, multiplicity not established by DEFT e) Assignment by analogy with compound (66) where chemical shifts assigned by proton-carbon 2D correlation study f) Assignment by analogy with THIQ (26),82 N-methyl-THIQ82 and 6 ,7-dimethoxy-N-methyl-THIQ102 g) Assignment assisted by intensity h) Assignment based on chemical shift i) Unambiguous assignment of signals made from the carbon-proton chemical shift correlation study performed on this compound j) Assignment by analogy with diprenorphine109 and naltrexol109 k) Cyclopropyl ring carbons Clla and appear as equivalent and the signal was intense l) Assignment by analogy with 1-butene104 m) Alternative assignment n) Assignment by analogy with W-benzylmethylamine104 o) Alternative assignment p) Assignment by analogy with furan-2-methanol100

318 Table 4.16 1H-NMR chemical shifts (ppm) of tf-{CH2R}-THQ (29,70,71)a 'b 'c

C a r b o n No (25) (70) d (71)d

2 3 . 1 3 (t) 3 . 1 5 - 3 . 3 5 e 3 . 1 5 - 3 . 3 5 e 5 . 5 1 * f

3 1 . 8 2 (m) 6.41***- 6.34**r 6.33**r

4 2.67(t) 2.74(t) 2 . 7 3 (t)

5-8 6 . 2 0 - 7 . 1 0 9 6 . 4 0 - 7 . 1 5 9 6 . 3 7 - 7 . 1 5 9

9 3 . 1 5 - 3 . 3 5 e 3 . 1 5 - 3 . 3 5 e

10 1 . 1 6 (m) 1.48-1.65(m) 7 . 3 8 * * * f

11 0.93(t) 1.25-1.47(m) 7 . 2 1 * * * *

12 0 . 9 5 (t)

N H 3 . 60h a) Spectra obtained at 250 MHz in CDC1 3 at ambient temperature unless otherwise noted, TMS used as internal standard b) Compound number and identity of R in N-{CH 2 R)-THQ; THQ (25) and in //-{CH2R}-THQ, R = Et (70), Pr (71) c) Assignment by analogy with THQ (25)76• 79 d) Assignment by analogy with //-methyl-THQ,110 chemical shift and intergration e) Two triplets partially overlapped f) Average value calculated for coupling constant g) Complex multiplets with three main features h) Broad singlet

* 3J(H 2 H 3 ) = Hz ** 3J(H 3H 4 ) = Hz ** * 3J(H 10 H 1 1 ) = Hz

319 Table 4.17 13C-HMR ctenical shifts of compounds (25,70,71)a /b

Carbon No (25)c 'd (70) (71)

2 41.58 49.63e 49.47e

3 21.80 22.33e 22.29e

4 26.67 28.31e 28.25e

4a 120.89 122.30e'f 122.14e

5 129.08 129.32e 129.12e

6 116.38 115.37e 115.19e

7 126.32 127.25e 127.05e

8 113.79 110.65e 110.45e

8a 144.49 145.64e 'f 145.38e

9 53.419 51.24h

10 19.53f '9 28.43h

11 11.62f '9 20.48f 4-1 H 12 0 LD

a Spectra obtained at 62.9 MHz in CDC13 and at ambient temperature unless otherwise noted. Multiplicity established by DEPT unless otherwise noted

b Compound number and identity of R in N-{CH2R}-THQ; THQ (25) and in N-{CH2R}-THQ, R = Et (70), Pr (71)

c multiplicity not established

d Assignment by analogy with literature values,106 chemical shift and intensity

e Assignment by comparison with THQ (25)

f Assignment assisted by multiplicity and intensity g By comparison with literature values for R2NCH2CH2CH3104 h By comparison with literature values for R2NCH2CH2CH2CH3104

320

00 00 00

2 1 6 ppm . 4 00 . 4 50 - 5 00 - - _ 1 50 - . 3 00 _ _ 2 50 . 5 50 _ 3 50 ___ 6 6 ppm j

28 26 N-propionyl-THIQ (54) in CDCI3 at 303 K. ’H spectrum at 250 MHz and 13C at 62.9 MHz 62.9 at 13C and MHz 250 at K. 303 ’Hspectrum at CDCI3 in (54) N-propionyl-THIQ Fig 4.48. of density contour representation Fig and spectrum correlation shift chemical Carbon-proton 2D L ______54 52 _J

321 Table 4.18. Conformer population of N-acyl-HHQ series from ^H-NMR spectra.

Conformer Population^

C-|-CH2 C3-CH2 z (5) ~ E (5) Z (6) E (5) (%) (ppm) (%) (ppm) (%) (ppm) (%) (ppm)

(53)b 59 (4.71) 41 (4.49) 61 (3.65) 39 (3.8)

(54)b 59 (4.72) 41 (4.6) 60 (3.66) 40 (3.82)

(55)b 59 (4.71) 41 (4.6) 60 (3.68) 40 (3.81)

(30)b 59 (4.73) 41 (4.86) —— 61 (4.85)c 39 (4.55) 59 (3.5)c 41 (3.8)

(56)b 59 (4.69) 41 (4.46) — —

(57)b 62 (4.77) 38 (4.7) 61 (3.75) 39 (3.87) 60 (4.6)d 40 (4.38) 61 (3.23)d 39 (3.61)

(58)b 63 (4.9)e 37 (4.6) 55 (3.6) 45 (4.0)

(59)b f f f f

(60) <3 41 59 — —

a) Ratio of conformers determined by integrated signal intensities. Errors estimated as ± 5% b) CDCI3 as solvent, ambient temperature c) Toluene-dg as solvent at 273 K d) ^-NMR spectrum obtained in toluene-dg at 89.6 MHz. Broad overlapping signals observed for C1-CH2 protons. Error estimated as ± 10%. e) Signal pairs broad and not completely resolved therefore relatively large error {ca. 13% based on resolved signals of compound 53} present in the conformer ratios given f) At ambient temperature broad signal observed g) At ambient temperature a sharp singlet observed for the C-*—CH2 protons, at 223 K a resolved pair of signals observed

322 Table 4.19. Gonformer peculation of //-acyl-THIQ series from 13C-NMR spectra.

Compound Conformer Dooulation^

cl c3 z (S) E (S) Z (5) E (5) (%) (ppm) (%) (ppm) (%) (ppm) (%) (PPm)

(53)b 38 (48.02) 62 (44.02) 41 (39.43) 59 (43.96)

(54)b 41 (47.19) 59 (44.20) 41 (39.64) 59 (43.06)

(55)b 42 (47.36) 58 (44.14) 41 (39.58) 59 (43.22)

(30)b 42 (47.27) 58 (44.76) 42 (40.09) 58 (43.35)

(56)b 40 (46.79) 60 (44.36) 39 (39.86) 61 (42.57)

(57)b 38 (47.47) 62 (44.53) 37 (39.99) 63 (43.51)

(58)b 'c — —— —

(59)c — —— —

(60)e 34 (49.49) 66 (45.30) 32 (40.60) 68 (45.21)

a) Ratio of conformers determined by peak height measured from the spectrum b) Samples run in CDCI3 as solvent at ambient temperature c) Percentage of each conformer could not be determined due to signals overlapped but the population ratio appeared to follow the same pattern as for the other compounds d) Signals for and C3 extremely broad and overlapped. An estimation of individual conformer population could not be determined e) At ambient temperature signals for and C3 very broad and unresolved, at 243 K signal pairs resolved although still overlap of central signals (5 45.37 and 45.29)

323 4 .4 REFERENCES

1. Honkanen E. , Pippuri A., Kairisalo P., Nore P. , Karppanen H. and Paakkari I., J. Med. Chem. , 26., 1433 (1983).

2. Tamarkin L. , Baird C.J. and Almeida O.F.X., Science, 2 2 7 . 714 (1985).

3. Niles L.P. , Wong Y-W. , Mishra R.K. and Brown G.M. , Eur. J. Pharmacol., 55, 219 (1979).

4. M a r e n T . H . , Physiol. Rev., 47, 595 (1967).

5. Van Daele P.G.H., De Bruyn M.F.L., Boey J.M., Sanczuk S., Agten J.T.M. and Janssen P.A.J., Arzneim-Forsch. (Drug Res.), 26, 1521 (1976).

6. Foldes F.F., Med. Clin. North Am., 48, 421 (1964).

7. Sugiura M. , Torei S. and Narao T. , J. Chem. Soc. Perkin Trans. II, 907 (1983).

8. Dalton D.R., Ramey K.C., Grisler H.J.Jr., Lendvay L.J. and Abraham A., J . Am. Chem. Soc., 91, 6367 (1969).

9. Fraenkel G. , Cava M.P. and Dalton D.R. , J. Am. Chem. Soc., 8 9 . 329 (1967).

10. Potapov V.M., Dem'yanovich V.M., Krivdin L.B., Skvortosova T.V. and Bkhatti Z.I., J. Heter. Compd, 17, 486, (1981).

11. Kharasch M.S. and Reinmuth 0., "Grignard Reactions of Nonmetallic Substances", Prentice-Hall, Englewood Cliffs, N. Jersey, 1166 (1954).

12. Lindsell W.E., "Comprehensive Organometallic Chemistry", Ed. Wilkinson G., Stone F.G.A. and Abel E.W., Pergamon Press, Oxford, 1, Ch. 4, 155 (1982).

13. Ashby E.C., Bull. Soc. Chim. Fr. , 2133 (1972); Q. Rev. Chem. Soc., 21, 259 (1967).

14. W a k e f i e l d B.J., Organomet. Chem. Rev., 1, 131 (1966).

15. Schlenk W. and Schlenk W., Chem. Ber., 62, 920 (1929).

16. Parris G.E. and Ashby E.C., J . Am. Chem. Soc., 93., 1206 (1971).

17. Salinger R.M. and Mosher H.S., J . Am. Chem. Soc., 86, 1782 (1964).

18. Kirrmenn A., Hamelin R. and Hayes S., Bull. Soc. Chim. Fr. ,

324 1395 (1963).

19. Ashby E.C. and Becker W.E., J. Am. Chem. Soc. , .85., 118 (1 9 6 3 ).

20. Ashby E.C. and Smith M.B. , J. Am. Chem. Soc., SjS, 4363 (1964).

21. Sm ith M.B. and Becker W.E., Tetrahedron, 22, 3027 (1966); £3. 4215 (1967).

22. Evans D.F. and Khan M.S., J. Chem., Soc. A, 1643 (1967).

23. Evans D.F. and Fazakerley G.V. , J. Chem. Soc. Chem. Commun., 974 (1968).

24. Guggenberger L.J. and Rundle R.E. , J . Am. Chem. Soc., 90, 5375 (1968).

25. Stucky G. and Rundle R.E., J. Am. Chem. Soc. , 86, 4825 (1964).

26. Schleyer P. von R. , Pure Appl. Chem., 56., 151 (1984).

27. Wakefield B.J., "The Chemistry of Organometallic Compounds", Pergamon Press, N.Y., 3 (1974).

28. Nagren K. and Langstrom B. , J. Label. Compd Radiopharm. , 26., 83 (1989).

29. Normant H., C.R.Hebd. Seances Acad. Sci., 239. 1510 (1954).

30. March J., "Advanced Organic Chemistry", 3rdEd., John Wiley and Sons, Inc., 399 (1985).

31. Ashby E.C., Laemmle J . and Neumann H.M., J. Am. Chem. Soc., 94, 5421 (1972).

32. Sneeden R.P.A., "The C hem istry Of C a rb oxylic A cid s and E ste r s" , Ed. Patai S., Interscience Publishers, London, Ch. 4, 137 (1969) .

33. Wakefield B.J., "Comprehensive Organometallic Chemistry", Ed. Wilkinson G. , Stone F.G.A. and A bel E.W., Pergamon Press, O xford, 7, Ch. 44, 39 (1982).

34. Baldwin M. , Loudon A.G., Maccoll A., Haynes L.J. and Stuart K.L., J. Chem. Soc. C, 154 (1967).

35. Budzikiewicz H. , Djerassi C. and Williams D.H., "Interpretation of Mass Spectra of Organic Compounds", Holden-Day, Inc., San F r a n c is c o , Ch. 4, 63 (1964).

36. Loudon A.G. , Maccoll A. and Wong S.K., J. Chem. Soc. B, 1727 ( 1 9 7 0 ) .

325 37. Sample S.D., L ig h tn er D.A., Buchardt 0. and Djerassi C., J. Am. Chem. Soc., 997 (1967).

38. M achulla H-J. and Dutschka K. , J. Label. Compd Radiopharm., 1 6 . 287 (1978).

39. Le Bars D ., Luthra S.K., P ike V.W. and LuuDuc C. , Appl. Radiat. Isot., 38, 1073 (1987).

40. Le Bars D., Luthra S.K., P ik e V.W. and K irk K.L. , Appl. Radiat. I s o t., 39, 287 (1988).

41. Le Bars D. , Luthra S.K., P ike V.W. and Swenson E.R. , Appl. Radiat. Isot., 39, 671 (1988).

42. Gilman H., Mallory H.E. and Wright G.F., J. Am. Chem. Soc., 54, 733 (1932).

43. Shepard A.F., W inslow N.R. and Johnson J.R., J. Am. Chem. Soc., 52, 2083 (1930).

44. Oki M. , "Methods of Stereochemical Analysis: Application of Dynamic NMR Spectroscopy to Organic Chemistry", Ed. Marchand A.P., VCH Publishers, Inc., 4 (1985).

45. H a rris R.K., "Nuclear Magnetic Resonance Spectroscopy", Longman Group UK Ltd., Ch. 5, 119 (1986).

46. A k it t J.W., "NMR and Chemistry", 2nd Ed., Chapman and H a ll Ltd., London (1983).

47. Gutowsky H.S. and Holm C.H., J. Chem. Phys. , 23, 1363 (1955).

48. R ogers M.T. and Woodbrey J.C., J. Phys. Chem., 66., 540 (1962).

49. Challis B.C. and Challis J.A., "Comprehensive Organic Chemistry: The Synthesis and Reactions of Organic Compounds", Ed. Barton D. and Ollis W.D., Pergamon Press, Oxford, 2, Ch. 9.9, 957 (1979).

50. "Steric Effects in Organic Chemistry", Ed. Newman M.S., John Wiley and Sons, Inc., N.Y. (1956).

51. S tew a rt W.L. and Siddal T.H.III, Chem. Rev., 70, 517 (1970).

52. March J., "Advanced Organic Chemistry", 3rdEd., John Wiley and Sons, Inc., N.Y., Ch. 4, 82 (1985).

53. F in ar I.L., "Organic Chemistry", 6th Ed., Longman Group Ltd., London, 1, Ch. 19, 531 (1973).

54. Lambert J.B. and Featherman S.I., Chem. Rev., 75, 611 (1975)

326 55. Blackburne I.D. and Katritzky A.R., Account. Chem. Res., 8, 300 (1975).

56. Booth H., J. Chem. Soc., 1841 (1964).

57. Eyring H., Chem. Rev., 17, 65 (1935).

58. Glasstone S., Laidler K.J. and E yring H. , "The Theory o f Rate Process", McGraw-Hill, N,Y. (1 9 4 1 ).

59. Sandstrom J., "Dynamic NMR Spectrospy", Academic Press, London (1982).

60. Jaeschke A., Muensch H. , Schmid H.C., Friebolin H. and Mannschreck A., J. Mol. Spectros., 31, 14 (1969).

61. Shanan-Atidi H. and Bar-Eli K.H. , J. Phys. Chem., 74, 961 (1970) .

62. Pines A. and Robinovitz M., Tet. Lett., 3529 (1968).

63. Neumann R.C.Jr. and Jon es V. , J. Am. Chem. Soc., 90, 1970 (1968) .

64. Hirsch J.A., Augustine R.L., Koletar G. and Wolf H.G., J. Org. Chem., 40, 3547 (1975).

65. Combrisson S. and Roques B.P., Tetrahedron, 32, 1507 (1976).

66. Elguero J., Marzin C. and Peek M.E., Org. Magn. Reson. , 1_, 445 (1975) .

67. Begtrup M., Claramunt R.M. and Elguero J., J. Chem. Soc. Perkin Trans. II., 99 (1978).

68. K riv d in L.B., Torocheshnikov V.N., S ergeev N.M., Il'ina I.G. and Kazennova N.B., J. Heter. Compd, Vb, 849 (1980).

69. Anet F.A.L. and Bourn A.J., J. Am. Chem. Soc., 89, 760 (1967).

70. Lambert J.B. and Keske R.G., J. Am. Chem. Soc., 88., 620 (1966).

71. Lambert J.B., Keske R.G., C arhart R.E. and Jovanovich A.P., J. Am. Chem. Soc., 89, 3761 (1967).

72. Anet F.A.L. and Yavari I., J. Am. Chem. Soc., 99, 2794 (1977).

73. A net F.A.L., Yavari I., Ferguson I.J., Katritzky A.R., Moreno- Manas M. and Robinson M.J.T., J. Chem. Soc. Chem. Commun. , 399 ( 1 9 7 6 ) .

327 74. Lehn J.M. and Wagner J .} J. Chem. Soc. Chem. Commun. , 414 (1970).

75. S id d a ll T.H.III., S tew a rt A.E. and Knight F.D. , J. Phys. Chem., 74, 3580 (1970).

76. Nagarajan K. , Nair M.D. and Pillai P.M. , Tetrahedron, 23, 1683 ( 1 9 6 7 ) .

77. Monro A.M. and Sewell M.J., Tet. Lett., 595 (1969).

78. Nagarajan K. , Madhaven P. and Bhute R.S., Indian J. Chem., 848 (1969) .

79. Monro A.M. and Sewell M.J., J. Chem. Soc. B, 1227 (1971).

80. Garner G.V., Meth-Cohn 0. and Suschitzky H. , J. Chem. Soc. C, 1234 (1971).

81. Hughes D.W., H olland H.L. and MacLean D.B., Can. J. Chem., 54, 2252 (1976).

82. Adcock W. and Gupta B.D., J. Org. Chem., 41, 1498 (1976).

83. Singh S.P., Palmer S.S., S ten b erg V.I. and Farnum S.A., J. Heter. Chem., 15, 541 (1978) .

84. Jackman L.M. " Application of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry", Pergamon Press, London, 1959, p l 2 4 .

85. P ople J.A., Discuss. Faraday Soc., 34, 7 (1962).

86. Paulsen H. and Todt K. , Angrew. Chem. Int. Ed. Engl., 5., 899 (1966).

87. Paulsen H. and Todt K., Anal. Chem., 235. 30 (1968).

88. H atton J.V. and Richards R.E., Mol. Phys., 3, 253 (1960)

89. Kowalewski V.J. and De Kowalewski D.G., J. Chem. Phys., 32, 1272 (1960).

90. Anet A.L. and Born A.J.A., J. Am. Chem. Soc., 87., 5250 (1965) .

91. Whitteker A.G. , Moore D.W. and Siegal S., J. Phys. Chem., 68, 3431 (1964).

92. S id d a l T.H.III. and Stewart W.E., J. Chem Phys., 48, 2928 ( 1 9 6 8 ) .

328 93. Neumann R.C.Jr. and Young L.B. , J. Phys. Chem., 69, 1777 (1 9 6 5 ) .

94. Fritz H. , Hug P. , Sauter H. and Winkler T. , Org. Mag. Res., 9, 108 (1977).

95. McFarlane W., J. Chem. Soc. Chem. Commun., 418 (1970).

96. Jon es R.G. and Wilkins J.M., Org. Mag. Res., 11, 20 (1978).

97. Nagarajan K. and Nair M.D. , Tetrahedron, 2J3, 4493 (1967).

98. H atton J.V. and Richards R.E., Mol. Phys., 3, 253 (1960).

99. H atton J.V. and Richards R.E., Mol. Phys., 5., 139 (1962).

100. Dean F.M. and Sargent M.V., "Comprehensive Heterocyclic Chemistry", Ed. KatritzkyA.R. and Rees C.W., Pergamon Press, O xford, 4, Ch. 3.10, 531 (1984).

101. Kellogg R.M., "Comprehensive Heterocyclic Chemistry", Ed. K a tr itz k y A.R. and Rees C.W., Pergamon Press, Oxford, 4, Ch. 3.13, 713 (1984).

102. Hughes D.W. and MacLean D.M., "The A lk a lo id s" , Ed. Menske R.H.F. and Rodrigo R.G.A., Academic Press, Inc., 1£, 217 (1981).

103. U1 Hasan M., Org. Mag. Res., 14, 447 (1980).

104. Silverstein R.M., B a s s le r G.C. and Morrill T.C., "Spectrometric Identification of Organic Compounds", 4th Ed., John Wiley and Sons, Inc., Ch. 5, 249 (1981).

105. Takahashi K., Sone T. and Fujieda K., J. Phys. Chem., 74, 2765 (1970).

106. Fritz H. and Winkler T ., Helv. Chim. Acta, 59, 903 (1976).

107. W ehrli F.W. and Wirthlin T. , "Interpretation of Carbon-13 NMR Spectra", Heyden, London (1976).

108. Blagg J., Coote S.J., D avies S.G. and Mobbs B.E., J. Chem. Soc. Perkin Trans. I, 2257 (1986).

109. Carroll F.I., M oreland C.G., B rin e G.A. and Kepler J.A., J. Org. Chem., 6, 996 (1976).

110. Kametani T. and Kigasawa K. , Chem. Pharm. Bull., 14, 566 (1966) .

329 CHAPTER 5

NUCTEommc soBgiTrrrrcw wrm r18F1FEDCRIEE

330 5.1 IWCRODDCEECN Currently there is great interest in the study of neurological and psychiatric conditions (e.g. Parkinson's disease, Huntington's disease and schizophrenia) by PET. The measurement of receptor distribution and concentration within living brain (and other organs) can provide essential information on the relationship of perturbations in receptor systems to various disease states. Though both 1]-C and 18F have been used to label neurotransmitter receptor ligands, recent PET studies have shown the particular advantage of studying radioligand receptor interactions with 18F-labelled ligands. IXie to its longer half-life, 18F allows several hours of data acquisition and a longer time for non- specifically bound activity to clear and perhaps to give a high ratio of specific to non-specific binding in the neurotransmitter rich regions. The short half-life of 11C of course precludes studies of several hours. So far most 18F-labelled ligands have been prepared for the study of dopamine,1”4 estrogen5'6 and opiate receptors.7 18 F-Labe lied butyrophenone neuroleptics such as [18F]- haloperidol,8 '9 [ 18F] spiperone,8 '9 [18F]A/-methylspiperone10 and A/- ([18F]fluoroalkyl) -spiperone derivatives11'12 have been prepared for the localisation of dopamine (D2) receptors. The fluoroalkyl derivatives are easier to prepare than [18F] spiperone or [18F]iV- methylspiperone, have higher brain uptake and give similar or higher striatum to cerebellum uptake ratios.13 Fluorinated ligands for the estrogen receptor, such as 1 6 a - [ 18F] f luoroestradiol-17/3, 16,3- [ 18F] f luoroestradiol-17/3, (2R, 3S)-l- [ 18F] fluoro-2,3-5is-(4-hydroxyphenyl) pentane (1-[18F] fluoro- pentestrol) and (3R, 4S)-l-[18F]fluoro-3,4-bis-(4-hydroxyphenyl)- hexane (1- [ 18F] fluorohexestrol} have been compared in rats bearing mammary tumours.14 The most promising ligand is 1 6 a - ([ 18F] fluoro) -17/3-estradiol, which shows selective uptake into tissues rich in estrogen receptors such as the uterus, and into mammary tumours that are estrogen receptor-positive.

331 [ 18F] Fluorofentanol, 15 a fluoro-analogue of the opiate receptor agonist, fentanyl, and also 3-[18F]acetylcyclofoxy,7 a fluoro- analogue of the opiate receptor antagonist, naltrexone, have been synthesised to visualise opiate receptors. The uptake of [18F] fluorofentanol in mouse brain15 and of 3-[18F]acetylcyclofoxy in both rat and baboon brain7 match opiate receptor distribution and clearly reveal the basal ganglia and thalamus. Recently, enormous interest has developed in the characterisation of excitatory amino acid receptors. The finding nearly thirty years ago that D-glutamate exerted powerful excitatory action on neurons in the mammalian central nervous system (CNS) led to structure-activity studies to characterise this action.18 Preliminary studies inferred that Lr-glutamate and similar excitatory amino acids (EAA), such as L-aspartate and N-methy 1-D-aspartate (NMDA), act at the same glutamate receptor.18 Subsequent work utilising specific agonists,17 antagonists,17 electrophysiological observations18 and radioligand binding techniques19 has demonstrated that the action of EAA appears to be mediated by at least three receptor subtypes; these are now named according to the agonists that activate them, namely NMDA, kainate and quisqualate subtypes.20'23 The NMDA receptor has became one of the best characterized of all receptor types in the CNS.22 NMDA receptor activation results in the opening of ion channels in a receptor-ion channel complex (Fig 5.1). Opening of ion channels allows the influx of Na+ , Ca2+ and K* which initiate the neuronal response.24 However, excessive activation of NMDA receptors (excessive Ca2+ influx) leads to a cascade of cellular events which results in the death of most central neurons.25 Evidence is accumulating that brain damage associated with anoxia, stroke, hypoglycemia, epilepsy and perhaps neurodegenerative illnesses (e.g. Alzheimer’s and Huntington’s disease) is linked in part to the overstimulation of NMDA receptors.24

332 / T W/

— 'I

'RESTING' dosed

__ 4

Fig 5.1. Schematic diagram of NMDA receptor-associated ion channel Transmitter recognition site (T), Ion channel (C).

Studies in animals have shown that non-competitive antagonists (drugs that prevent the effect of an agonist by acting at a site different from that of the agonist on the same neuron) of NMDA. receptors can protect against EAA-induced (excitotoxic) neuronal loss.26 This finding may have important implications for the prevention and treatment of excitotoxic neuronal damage in man. The phencyclidine analogue, MK 801 ((+) 5-methyl-10, ll-dihydro-5tf-dibenzo [a, d ]cyclohepten-5,10-imine} (82; Fig 5.2), is the most potent non­ competitive antagonist of the NMDA receptor yet described.27

333 (82) ; n = 1, R = R’ = H {MK-801) (83) ; n = 1, R = F, R' = H {5-fluoromethyl analogue of MK 801) (84) ; n = 2, R = F, R' = H {5-p -fluoroethyl analogue of MK 801)

Fig 5.2. Structure of MK 801 and its fiuoroalkyl analogues.

It is believed that [3H]MK 801 binds at the level of the NMDA receptor-associated ion channel (Fig 5.1) and its binding appears to be entirely dependent upon activation of the NMDA. receptor by an agonist.26'28'29 For instance an agonist such as NMDA activates the receptor complex by combining with the "transmitter recognition site (T)" and this process opens the ion channel (C). Probably MK 801 binds to sites within this channel, thus preventing the passage of ions such as calcium. High affinity binding sites have been demonstrated in the mammalian brain, both in membranes27 and autoradiographically in brain sections.30 In addition it is known that MK 801 crosses the blood brain barrier readily.31 These properties suggested that MK 801 labelled with a positron-emitting radioisotope may have potential for the investigation of the NMDA receptor system in man by PET. The structure of MK 801 (82) does not lend itself to facile labelling with carbon-11. Thus the possibility of labelling MK 801 with fluorine-18 was considered. Lyle et al32 reported fluoride attack on cyclic sulphamates of MK 801 followed by acid hydrolysis as efficient routes to 5-fluoromethyl (83) and 5-/3 -fluoroethyl (84) analogues of MK 801. Furthermore these analogues of MK 801 retain

334 high affinity for the NMDA. receptor. The first part of this chapter reports on the evaluation of these cyclic sulphamates 85 and 86 as substrates for nucleophilic attack by [18F] fluoride at the n.c.a level and the preparation of the 5- [ 18F] fluoromethyl (89) and 5-/3- [18F]fiuoroethyl (90) analogues of MK 801 (Scheme 5.1) .

H+

(85) ; n = 1 {cyclic sulphamate (86) ; n = 2 {cyclic sulphamate} (89) ; n = 1 {5-[18F]fluoromethyl analogue} (90) ; n = 2 {5-p -[18F]fluoroethyl analogue} (87) ;n = 1 [18F]adduct / (88) ; n = 2 [18F]adduct 1SF

Scheme 5.1. Synthesis of 5-[18F]fluoromethyl and 5- p -[18F]fluoroethyl analogues of MK 801 from [18F]fluoride at the n.c.a level.

335 Mostly, 18F-chemistry can be conveniently divided into electrophilic and nucleophilic reactions (Ch. 1). Electrophilic reagents such as [18F] xenon difluoride,33 [18F]acetyl hypofluorite34 and dilute [18F]fluorine gas35 have all been used to prepare L-3,4- dihydroxy-6- [ 18F] f luorophenylalanine (L-6-[18F]f luoro-Dopa). All of these synthetic routes produce "carrier added" L-6-[18F]fluoro-Dopa in only 3-4% r.c.y at BOB. Nevertheless this radiotracer (L-6- [ 18F] fluoro-Dopa acts as a precursor to L-6- [ 18F] fluoro-dopamine) has been extensively used in PET studies to demonstrate the distribution of the neurotransmitter dopamine36 and the lack of dopamine in the striatum of patients with Parkinson's disease.37'38 L r-T h reo-3,4-dihydroxy-phenylserine (L-threo-DOPS) decarboxylates to nor-epinephrine (nor-adrenalin) in vivo.39'40 By analogy with the use of L-6-[18F]fluoro-Dopa for the study of the dopamine system, L- threo-DOPS labelled with 18F might be a useful radiotracer for the investigation of the nor-adrenergic system in man.41'42 Since the structures of L-Dopa (91) and L-threo-DOPS (92) are identical except in the side chain (Fig 5.3), the electrophilic reagents used for the preparation of L-6- [ 18F] fluoro-Dopa (93) can be considered for the preparation of L-6- [ 18F] fluoro-threo-DOPS (94).

Fig 5.3. Structure of L-DOPA and L-threo-DOPS

However these reagents would probably generate a mixture of "carrier added" fluorinated compounds (Scheme 5.2) and the r.c.y would probably be comparable to or less than that for to L-6-[18F] fluoro- Dopa.

336 RgHN COOR4 R5hn COOR, \HC / \HC / 4

R1 - r 5 = H or protecting group i) remove protecting groups ii) separate isomers by hpic L-6-[18F]fluoro-threo-DOPS Scheme 5.2. Preparation of L-6-[,8F|fluoro-threo-DOPS from (,sF]fIuorine

A recent paper has reported on the preparation of L-p- [ 18p] fluoro-phenylalanine freon n.c.a [18F] fluoride, using initially [18F]fluoro for nitro exchange in p-nitrobenzaldehyde.48 It was of interest to assess whether this approach could be extended to the preparation of L-6- [ 18F] fluoro-Dopa (93) or even L-6-[ 18F] fluoro- t h r e o-DOPS (94). A nucleophilic approach, such as this, potentially offers major advantages, such as the possibility to use high initial activities, regiospecificity of labelling and achievement of high specific activity. The second paid: of this chapter reports a preliminary investigation of same reactions which might serve as

337 labelling steps in the synthesis of L-6- [ 18F] fluoro-Dopa and L-6- [18F] fluoro-threo-DOPS from [18F] fluoride. These reactions would involve nucleophilic displacement of the activated nitro group of 2- nitro-4,5-dimethoxy-benzaldehyde (95) and of 2-nitro-4,5-dibenzyl- benzaldehyde (96) by [-^F] fluoride. Condensation of 2-[l8F]fluoro- 4,5-dimethoxy benzaldehyde (97) or 2-[18F]fluoro-4,5- dibenzylbenzaldehyde (98) with 2-phenyl-5-oxazolone in the presence of l,4-diazabicyclo[2.2.2]octane (DABCO) followed by hydrogenation, ring opening, deprotection and hplc purification might provide L-6- [18F]fluoro-Dopa (93). In the case of L-6-[18F]fluoro-threo-DOPS (94), reaction of (97) or (98) with glycine or a glycine synthon followed by deprotection and isomer purification might yield the required 18F-labelled product (Scheme 5.3).

338 CHO CHO

i) glycine and deprotect or ii) glycine synthon and deprotect

Mixture of D,L-6-[18F]fluoro-DOPA Mixture of D,L-threo- and D,L-erythro- 6-[18F]fluoro-DOPS

iii) chiral hplc purification iii) chiral hplc purification

COOH ,r COOH

6-[18F]fiuoro-L-DOPA 6-[18F]fluoro-L-threo-DOPS

(9 5) ; R = Me {2-nitro-4,5-dimethoxybenzaldehyde} (96) ; R = Bz {2-nitro-4,5-dibenzylbenzaldehyde} (97) ; R = Me { 2-[18F]fluoro-4,5-dimethoxybenzaldehyde} (98) ; R = Bz { 2-[18F]fluoro-4,5*dibenzylbenzaldehyhe}

Scheme 5.3. Proposal for the preparation of L-6-[18F]fluoro-DOPA and L-6-[18F]fluoro*threo-DOPS from[18F]fluoridealthen.c.a level. 339 5.2 DXSCDSSICN The exploitation of cyclic leaving groups in nucleophilic displacement reactions with [18F] fluoride has been limited. The first example was the synthesis of [ 18F] fluoroethanol.44 Glycol sulfite was reacted with cesium [18F] fluoride and the formed [18F]sulfinic acid derivative gave the desired product on aqueous hydrolysis. A more important application was the synthesis of 2- [ 18F] f luoro-2-deoxy-D-glucose (FDG) (QQ) from a cyclic sulphate derivative of mannopyranoside (PP) (Scheme 5.4). 4~* This overcame the limitations imposed by molecular fluorine in the synthesis of [18F]FDG (see Chapter 1).

(Et)4N 18F

H+

OH

Scheme 5.4. Fluorination and deprotection of 4,6-benzylidene-1- p-methyl- mannopyranoside-2,3-cyclic sulphate to give 2-[18F]fiuoro-2-deoxy-D-glucose

This approach led to the view that a carbohydrate containing a good leaving group axial at C-2, a protecting group (5 at C-l and a non­ participating protecting group at C-3, would yield isomerically pure [18F]FDG.46 A cyclic sulfate derivative has also been used in the preparation of 16a- [ 18F] fluoro-17/3-hydroxyestradiol in order to improve the specific activity of the product.47

340 The above studies indicate that sulphites and sulphates are suitable candidates for nucleophilic displacement reactions with

[18F j fluoride at the n.c.a level. The main advantages of these derivatives are their leaving group ability for [18F] fluoride attack and easy subsequent removal under mildly acidic conditions .44,45 Therefore, it was envisaged that the macroscale chemistry described by Lyle et al32 for the preparation of 5-fluoroalkyl analogues of the potent non-competitive NMDA antagonist MK 801 might be applicable to 18F-labelling at the n.c.a level. [18F] Fluoride was produced either by irradiation of H2160 with deuterons or of H2180 with protons. Recovered aqueous [18F] fluoride was converted into tetrabutylammonium [18F] fluoride by adding base (tetrabutylammonium hydroxide) then removing water by distillation. The fluoride salt was dried by azeotropic distillation with acetonitrile. This operation of activating the [18F] fluoride for nucleophilic displacement reactions requires careful manipulation, since the presence of even trace water (> 0.5 mmol) can completely solvate the fluoride ion, rendering it unreactive in displacement reactions.48 >49 The activated [ 18F] fluoride was then reacted with a cyclic sulphamate derivative of MK 801, either 85 or 86, in acetonitrile. The argument for choosing this solvent for aliphatic nucleophilic substitution was presented in Chapter 1. Reaction variables such as amount of substrate, temperature, fluorination time, hydrolysis time and reaction vessel were investigated. The results for the synthesis of the 5- [ 18F] fluoromethyl analogue of MK 801 (89) are presented in Table 5.1. It was found that as the amount of substrate 85 was reduced, the incorporation of [18F] fluoride decreased by almost 50% in both platinum and glass vessels. Additionally, it was observed that for all levels of substrate used the r.c.y were slightly lower (ca. 10%) in glass than in platinum. A experiment carried out in a rotary evaporator gave an extremely low r.c.y of the crude labelled product 89 (Table 5.1). However this result was from a single experiment and probably r.c.y's comparable to those obtained in glass vessels

341 could be obtained with further experimentation. Effects of reaction vessel material on the r.c.y obtained in nucleophilic displacements with [18F]fluoride have been noted by other investigators. loss of [ 18p]fluoride to glass surfaces has been reported50'51 although this was not observed here. The use of glassy carbon vessels51 and Vacutainers52 have been proposed to alleviate this problem. The reaction between tetrabutylammonium [18F] fluoride and cyclic sulphamate, either 85 or 86 occurred efficiently within 20 min in refluxing acetonitrile (353 K). The use of other bases for [18F]fluoride incorporation, such as, Kryptofix 2.2.2-potassium carbonate or Kryptofix 2.2.2-potassium carbonate/potassium oxalate gave extremely poor yield (< 2%) of the desired compound. Hydrolysis of the [18F]adduct (87) or (88) (Scheme 5.1) was efficiently achieved with dilute hydrochloric acid. Initially a reaction time of 10 min at 353 K was used. Subsequent experiments demonstrated that hydrolysis for 20 min was needed to achieve optimal yield. In early experiments the crude radioactive products 89 or 90 were extracted into chloroform, evaporated to dryness and injected onto a preparative hplc column for purification {Fig 5.4; an hplc chromatogram of compound 90}. The reaction mixture contained mainly one radioactive product (peak A) with the same retention time as the reference 5-/9-fluoroethyl analogue of MK 801 (peak B) and a number of by-products. Although the isolated product was radiochemically pure, there were traces of a number of stable impurities. To alleviate this problem the chloroform extract was passed through a reverse-phase Sep-pak. This procedure removed substantial amounts of by-products, thus enabling base-line hplc separation of "carrier" (peak B) associated with the 5-/9-[18F]fluoroethyl analogue (peak A; Fig 5.5). Each separated radioactive product, 89 or 90 was found to be radiochemically and chemically pure by both tic and hplc. Mass spectrometry confirmed the purity of the labelled products 89 and 90; the "carrier" in each [18F]fluoro compound exhibited a mass spectrum identical to that of the appropriate reference compound 83 or 84. Both fluoro analogues gave a peak at V[/Z = 220,

342 ___Jt* representing the loss of [M-(CH2)n-i(Scheme 5.5).

Scheme 5.5. Mass spectral fragmentation of fluoroalkyl analogues of MK 801

Mass spectrometry was also used to identify major by-products, the 5-chloromethyl analogue in the synthesis of the 5-[18F]fluoro- methyl analogue (89) and the 5-chloroethyl analogue (peak C, Fig 5.4) in the synthesis of the 5-/3- [ 18F] fluoroethyl analogue (90). These chloro analogues must have been formed during hydrochloric acid "work-up" in the radiosyntheses. In bio-medical applications of the [18F] fluoro analogues (89) or (90) these chloro compounds could possibly act as "pseudo carrier" by competing for the same NMDA receptor site. Thus their complete removal from the labelled compounds was vital and this was successfully achieved by a combination of Sep-pak treatment and preparative hplc. The amounts of "carrier" obtained in the 5- [ 18F] fluoromethyl (89) or 5-/3-[^8F] fluoroethyl (90) analogues (Table 5.3) correspond to specific activities in the range of 5.2-7.4 GBq//imol for the 5- [ 18F] f luoromethyl compound and 3.7-37 GBq/,umol for the 5 - f i- [18F]fluoroethyl analogue. "Carrier" (stable fluoroalkyl analogue) most probably originates from fluoride released into the target system and from traces of fluoride in the various reagents used. Yields of labelled products did not vary substantially with the amounts of carrier in the system. This is in marked contrast to some reactions of [18F] fluoride with substrates bearing a non-cyclic

343 group. For example in one route to 2-[^8F]FDG, where [^-8F]fluoride displaces triflyl it has been reported that the yield of 18F-labelled product decreases drastically as "carrier" increases.53 This phenomenon might be attributable to competing elimination reactions on the basis that with increasing concentration fluoride will express more basicity.54 In this study, the incorporation of [18F]fluoride into cyclic sulphamates, either 85 or 8 6 , was found to be highly reproducible and efficient (comparable to the reported macroscale chemistry) giving 5-[ 18F] f luoramethyl (89) or 5-/3- [ 18F] f luoroethyl (90) analogues in ca. 30% r.c.y (based on 18F used and at EOB) in 2.5 h preparation time. The specific activities (Table 5.3) of the fluoroalkyl analogues were consistent with published values on radiotracers produced from [ 18F] fluoride.10'52 The levels of "carrier" corresponding to these specific activities were sufficiently small to permit the labelled products to be evaluated for receptor binding studies in animals and in the case of the (5S,10R)-/3-[18F]fluoroethyl analogue (90), in man by PET.

Generally, nucleophilic substitution reactions on aromatic systems are only feasible when the position of substitution is sufficiently activated by an electron-withdrawing group such as nitro, cyano or carbonyl in an ortho or para position. In the case of exchange with [18F] fluoride strong leaving groups such as iodo, bromo, fluoro and nitro are required.55'56 These two requirements are met in the protected compounds, 2-nitro-4,5-dimethoxy- benzaldehyde (95) and 2-nitro-4,5-dibenzyl-benzaldehyde (96) which might serve as precursors to L-6- [ 18F] fluoro-Dopa (93) or L-6- [ 18F] fluoro- threo-DOPS (94) ( see Sec 5.1). Reactions of these compounds with [18F] fluoride were therefore attempted. For comparison nucleophilic substitution reactions with [ 18F] fluoride were also attempted on p-nitrobenzaldehyde (99) and o- nitrobenzaldehyde (100). For all the substrates concerned the choice of counter cation for the generation of reactive [18F] fluoride from aqueous

344 [18F] fluoride was investigated. All reactions were carried out in a platinum crucible and either EMSO or EMF was used to resolubilise the active [18F] fluoride for subsequent nucleophilic attack on the appropriate substrate. Preliminary results from these experiments are presented in Table 5.4. In p-nitrobenzaldehyde (93) and in o-nitrcbenzaldehyde (100) the extent of fluorodenitration did not vary extensively with counter cation. Yields of p - [ 18F] fluorobenzaldehyde (101) or o-[18F]fluoro benzaldehyde (102) were found to be in the range of 20-40% (based on 18F used). The results obtained for p-nitrobenzaldehyde are somewhat lower then those reported by Lemaire et al .43 These investigators used K+ 2 .2 .2-aminopolyether as the base for activating the [18F] fluoride. Attempts to reproduce these conditions resulted in negligible yield of the desired [18F]products. The incorporation of activated [18F] fluoride into 2-nitro-4,5- dimethoxybenzaldehyde (95) or 2-nitro-4,5-dibenzylbenzaldehyde (96) was lower than into o- or p-nitrobenzaldehyde for all cations used. Yields of the [18F]product 97 or 98 varied only slightly with counter cation or solvent. The highest yields (15% of 97; 23% of 98) were obtained with tetrabutylammonium [18F] fluoride and EMF as solvent. For all the experiments reported (Table 5.4), the r.c.y were based on the separation of radioactive product (organic phase) from [18F] fluoride (aqueous phase) by reverse phase Sep-pak treatment. TIC analysis of the radioactive products and subsequent autoradiography revealed that in the case of compound 97 and 98 an active impurity was also present in the organic phase. Thus, the actual yields of 97 and 98 are actually somewhat lower than quoted. Since the proposed preparation of L-6- [ 18F] fluoro-Dopa and L- 6- [ 18F] fluoro-threo-DOPS by the nucleophilic approach involves a multistep synthesis with isomer separation, the incorporation of [18F] fluoride at the first stage of the synthesis needs to be high (i.e > 50%) so that reasonable yields of the desired products can be obtained. Consequently, the above preliminary results require further optimisation in terms of counter cation, solvent, temperature, reaction vessel etc.

345 5.3 EXPERIMENTAL AND RESDIHS 5.3.1 r 18F1 Fluoride Production [1SF]Fluoride was produced by using either the 160(3He,p) 18F or 180 (p,n) 18F reaction. A stainless steel target containing either H2160 (3 ml) or H2180 (1.5 ml) was irradiated for either 45-60 min with 15 /iA beam of 53 MeV alpha particles or 30 min with a 20 /xA beam of 19 MeV protons. The aqueous solution was withdrawn remotely.

5.3.2 Preparation of Tetrabut^lammoniiirn f181 Fluoride The aqueous solution containing [18F] fluoride was transferred into a platinum crucible containing a teflon stirrer. A solution of tetrabutylammonium hydroxide (100 /xl, 4% v/v) was then added followed by acetonitrile (0.5 ml). The platinum crucible was placed in an oil bath (388 K) and the solution evaporated to dryness under a gentle stream of nitrogen. Drying was completed by further additions of dry acetonitrile (3 x 2 ml) evaporated under nitrogen.

5.3.3 Preparation of 5 -T-*-8F~lfluorranethvl (89) and 5 - 8 - r18Fl fluoroethvl (90) analogues of MK 801 To a dry tetrabutylammonium [18F] fluoride under nitrogen, cyclic sulphamate (5 mg) either (85) or (86) {gifts from Merck Sharp and Dohme} in acetonitrile (1.5 ml) was added and the platinum crucible was covered with a watch-glass. The reaction mixture was heated at reflux temperature (353 K) for 20 min. Dilute hydrochloric acid (1.5 ml, 3 M) was then added and the resulting mixture was further heated (353 K) for 25 min. The platinum crucible was removed from the oil bath and reaction mixture made alkaline (ca. pH 10) by the addition of dilute ammonia solution (3 M). The crude product was then extracted into chloroform (7 ml). A sample was chromatographed (Fig 5.4; see below for hplc conditions). The chloroform extract was passed through a pre-conditioned silica gel Sep-pak (Waters Associates, primed with CH2C12 , 10 ml). The Sep-pak was further eluted with dichloromethane (5 ml). Retained activity was recovered by elution with CH2Cl2:EtOH (5 ml, 9:1 v/v). The collected fraction was evaporated to dryness and the residue

346 taken up into hplc mobile phase. This was injected onto a silica gel column ("^-Porasil", 10 ura. particle size, 30 x 0.7 cm i.d.) eluted at 4.0 ml min-1 with dichloramethane containing 0.3% v/v of a mixture of ethanol, water and triethylamine (100:2:2 v/v). The eluate was monitored for radioactivity and UV absorbance at 280 nm. The radioactive fraction eluting at the same retention time as reference 5-fluoromethyl {(83); 19-20 min} or 5-£-fluoroethyl ((84); 22-24 min} analogue was collected. Figure 5.5 shews a typical hplc chromatogram of 5-/9-[18F]fluoroethyl analogue of MK 801 (90). Reaction variables such as the amount of cyclic sulphamate (85), temperature, fluorination time and reaction vessel were investigated and the results for 5- [ 18F] fluoromethyl analogue of MK 801 (89) are presented in Table 5.1.

Analysis i) TLC The collected radioactive fraction containing either (89) or (90) was analysed by three different tic systems using silica gel plates: system A, hexane and ethyl acetate (3:7 v/v); system B, dichloramethane containing 0.5% A (A = ethanol, water and triethylamine, 100:2:2 v/v); system C, chloroform, ethyl acetate and c.ammonia (3:7:1 v/v). Reference material was visualised by exposure to UV and radioactivity was detected by autoradiography. A single radioactive product which co-migrated with the corresponding reference 5-fluoromethyl (83) or 5-/3-fluoroethyl (84) analogue was observed on each tic system (Table 5.2).

347 ) ) ) ) 3 3 3 3

(CHCI (CHC13) (Sep-pak) (CHCI (CHCI (CHCI 6 2

5-/9-[ Temperature/Time Activity 343 (25) 383 (15) 383 338-343 (20) 348 343-365 (25) 359-368 (25) F] fluoride andF] this was used as the fluorinating agent 18 (85)amg K (min) K (min) % 6.2 2.1 5.2 352-368 (25) 368 5.0 6.2 Cyclic Sulphamate Fluorination fluorinationtime onthe yield of the 0 0 2.2 tetrabutylammonium [ Vessel Platinum Platinum Reaction Platinum Glass Glass Glass^ a) a) In each experimenttetrabutylammonium hydroxide mg) (4 was converted into c) c) Untreatedpyrex glass b) b) R.C.Ybased on labelledproduct collected frompreparative hplc column d) d) Complete preparation carried out in a rotary evaporator Table 5.1. Effectof reaction vessel, amountof cyclic sulphamate (85), tenperatureand

348 ABSORBANCE

Fig 5.4 Preparative hplc trace of the separation of the 5-p -[18F]ethyl analogue of MK 801 RADIOACTIVITY Time (min) Fig 5.5 Preparative hplc trace of the 5-p -[18F]ethyl analogue of MK 801 MK of -[18F]ethyl 5-panalogue of the trace hplc Preparative 5.5 Fig 60

40

20 o

ABSORBANCE Table 5.2. Rf of 5-[ 18F]fluorcmethyl and 5-£-[ 18F]fluoroethyl analogues o f MK 801

Analogue of MK 801 System A System B System C

*f Rf Rf

5-[18F]Fluoromethyl (89) 0.26 0.15 0.68

5-/3- [ 18F] Fluoroethyl (90) 0.38 0.43 0.64

i i ) HPLC Aliquots of the collected radioactive fractions containing (89) or (90) were also analysed using a normal phase hplc column ('V- Porasil", 10 /im particle size, 30 x 0.39 cm i.d.) eluted at 2.0 ml min-1 with dichloromethane containing 0.1% A (A = EtOH:H20:Et3N, 100:2:2: v/v). The eluant was monitored for radioactivity and UV absorbance (280 nm). For each analogue a single radioactive peak eluting at the same retention time as reference 5-fluoromethyl (9.8 min) or 5-/3-fluoroethyl (10.5 min) compound was detected. 5- Fluoromethyl (83) or 5-/3-fluoroethyl (84) compound was the only stable product detected. The appropriate hplc chromatogram was exploited to calculate the specific activity of the 5- [ 18F] f luoromethyl (89) and 5-/3-[18F]f luoroethyl (90) analogues (Table 5.3). iii) Mass spectrometry The collected radioactive fraction, containing (89) or (90), was evaporated and after decay, the residue was examined by mass spectrometry both by chemical ionisation and electron impact mode. The mass spectra were found to be identical to those of the corresponding reference compounds (83) or (84). Parent peaks were observed at M/Z 239 (M*, 100%} for the fluorcmethyl analogue (83) and at M/Z 253 (M*-, 100%} for the 5-/3-fluoroethyl analogue (84). In spectra for both compounds a characteristic peak at M/Z 220 was

351 observed. This was attributed to {[M-F]+ , 33%} for the fluoromethyl (83) and {[M-GH2F]+ , 100%} for the 5-0-fluoroethyl (84) analogue. Other major fragments that were observed in spectra for both compounds were M/Z 206, 204, 179 and 178.

Formulation for human Intravenous injection The appropriate fraction collected from the hplc column was evaporated to dryness and the labelled product solubilised by adding dilute acetic acid (700 n±, 0.1% v/v) and then diluting with normal saline for injection (7 ml, 0.9% NaCl v/v). Sterilisation was achieved by filtration through a sterile filter (0.22 fim pore size, Millex-GS).

Radiochemical yields and specific activities for the 5- f 18F~1 fluoromethvl and 5 -8 -[ ^8F1 fluoroethvl analogues of MK 801 The radiochemical yields and specific activities for the 5- [18F]fluoromethyl (89) and 5-0-*[18F]fluoroethyl (90) analogues of MK 801 obtained in various preparations are given in Table 5.3. Also included are the range of stable material observed in thirty preparations.

Table 5.3. Production parameters of [ 18F]fluoromethyl and 5-0-[ 18F] fluoroethyl analogues of MK 801

Analogue of R.C.Y Stable Specific Time for MK 801 material activity prep.

(%>a ng (nmol) GBq//xmola h

5- [ 18F] Fluoromethyl ca.30 2-5 (8-21) 5.2-7.4b 2.5 (89)

5-0-[ 18F] Fluoroethyl 25-35 2-36 (8-47) 3.7-37c 2.5 (90)

a) decay corrected to EOB b) starting activity of [18F]fluoride 111-260 MBq c) starting activity of [18F] fluoride 111-2673 MBq

352 5.3.4 f 18F1 Fluorination of p-Nitrobenzaldehvde (99) and o-Nitro- benzaldehvde (100) and its 4.5-Dimethoxv (95) and 4.5 - Dibenzvl (96) derivatives

F18piFluoride production [ 18p ] Fluoride was produced using the nuclear reaction 160(3He,p)18F in a stainless steel target containing water for injection (3 ml).

Preparation of f18FlKF/K 2.2.2 and reaction with 95, 96, 99 and 100 To a mixture of kryptofix 2.2.2 (24 mg, 64 //mol) and potassium carbonate (7 mg, 50.6 //mol) in a platinum crucible was added aqueous [18F]fluoride (0.5-3.0 ml) followed by acetonitrile (1.0 ml). The platinum crucible was then placed in a oil bath (288 K) and the solution evaporated to dryness under a slow stream of nitrogen. The residue was dried further by the subsequent additions and evaporation of dry acetonitrile (3 x 2 ml) . A solution of p-nitrobenzaldehyde {(99), 5 mg} or o- nitrobenzaldehyde ((100), 5 mg} or 2-nitro-4,5-dimethoxybenzaldehyde {(95), 5 mg} or 2-nitro-4,5-dibenzylbenzaldehyde {(96), 5 mg} in CMSO (1.5 ml) was then added to the [18F]KF/K 2.2.2 and the platinum crucible was covered with a watch glass. The reaction mixture was heated at 388 K (oil bath) for 20 min. The solution was then allowed to cool to ambient temperature and H20 for injection (10 ml) was added. The resulting mixture was passed through a pre-conditioned reverse phase Sep-pak column {primed with ethanol (5 ml) followed by a 10% aqueous solution of EMSO (10 ml)}. The Sep-pak was then washed with H20 (10 ml) and the retained activity was eluted with ethanol (5 ml). The results are presented in Table 5.4.

Preparation of f18F1PbF and reaction with 95. 96. 99 and 100 To rubidium carbonate (5 mg, 21.6 //mol) in a platinum crucible was added aqueous [18 F] fluoride (0.5-3.0 ml) followed by acetonitrile (1.0 ml). The solution was evaporated to dryness under nitrogen as described above. To the n.c.a Fb18F was added a solution

353 of {(95), 5 mg} or ((96), 5 mg) or {(99), 5 mg) or ((100), 5 mg} in EMSO (1.5 ml) and the reaction mixture was heated at 388 K for 20 min. In each case labelled product was isolated by Sep-pak treatment as described above. The r.c.y. are presented in Table 5.4.

Preparation of tetrabutvlammonium T18F1 fluoride and its reaction with 95, 96, 99 and 100 Tetrabutylammonium hydroxide (100 p i; 4% solution of Bu4N0H v/v) was added to the platinum crucible containing aqueous [18F] fluoride (0.5-3.0 ml) . Acetonitrile (1.0 ml) was then added and the solution was evaporated to dryness as described above. The substitution reaction of {(95), 5 mg) or {(96), 5 mg} or {(99), 5 mg} or ((100), 5 mg} was either carried out in EMSO (1.5 ml) or EMF (1.5 ml). Labelled product was isolated by Sep-pak treatment as described above. The results are presented in Table 5.4.

354

TLC?rf 0.67 0.67c+ inpurity 0.58c+ irtpurity Singleprod.c '^ Single prod.c 'd 8a - (%) 15b lla r.c.y 2-23b [18F]BU40H 9a 2a (%) r.c.y [18F]RbF

5a 4a (%) 34a 23a 21a 29a 39a r.c.y [18F]KF/K 2.2.2

(101) (97) (102) (98) 2_ [ 18 ] piuoro-4,5-dimeth- ] 18 [ 2_ 2-[18F]Fluoro-4,5-diben­-benzaldehydezyl o-[18F]Fluorobenzaldehyde oxy-benzaldehyde p-[18F]Fluorobenzaldehyde

(99) (100) (95) (96) Substrate Labelled Product [18F] fluoride was[18F] observed at the origin in systemA and B. andRf determinedby comparisonwith reference compounds; f) for all labelled products, a small amount of o-Nitrobenzaldehyde 2-Nitro-4,5-dimeth- zyl-benzaldehyde Table 5.4. Preliminaryresults fluoride of [18F] exchange inp~ ando-nitrofoenzaldehyde andderivatives 2-Nitro-4,5-diben- oxy-benzaldehyde p-Nitrobenzaldehyde a) a) reaction solvent EMSO; b) reaction solvent DMF; c) system A: silica gel plate, eluant- dichloromethane d) d) system B: silica gel plate, eluant- heptane and ethyl acetate e) ; (50:50 ticv/v) plates autoradiographed

355 5.4 REFERENCES

1. Wagner H.N.Jr., Burns H.D., Dannals R.F., Wong D.F., Langstrora B. , Duelfer T., Frost J.J., Ravert H.T., Links J.M., Rosenbloom S.H., Lukas S.E., Kramer A.V. and Kuhar M.J., Science, 221. 1264 (1983) .

2. Welch M.J., Kilbourn M.R., Mathias C.J., Mintun M.A. and Raichle M.E., L ife S ci., 33., 1687 (1983).

3. Arnett C.D., Shiue C-Y., Wolf A.P., Fowler J.S., Logan J. and Watanabe M . , J. Neurochem., 44, 835 (1985).

4. Arnett C.D., Fowler J.S., Wolf A.P., Shiue C-Y. and McPherson D.W., L ife S ci., 36, 1359 (1985).

5. Mathias C.J., Welch M.J., Katzanellanbogen J.A., Brodack J.W., Kilbourn M.R., Carlson K.E. and Kiesewetter D.O., N ucl. Med. B io l., 14, 15 (1987).

6. Landvatter S.W. , Kiesewetter D.O. , Kilbourn M.R. , Katzanellanbogen J.A. and Welch M.J., L ife S ci., ^3, 1933 (1983) .

7. Pert C.B., Danks J.A., Channing M.A., Eckelman W.C., Larson S.M., Bennett J.M., Burke T.R.Jr. and Rice K.C., FEBS., 1 7 7 . 281 (1984).

8. Kilbourn M.R., Welch M.J., Dence C.S., Tewson T.J., Saji H. and Maeda M. , Int. J. Appl. Radiat. Isot., 35, 591 (1984).

9. Shiue C-Y., Fowler J.S., Wolf A.P., Watanabe M. and Arnett C . D., J. Nucl. Med., 26, 181 (1985).

10. Shiue C-Y., Fowler J.S., Wolf A.P., McPherson D.W., Arnett C.D. a n d Z e c c a L. , J. Nucl. Med., 27., 226 (1986).

11. Kiesewetter D.O., Eckelman W.C., Cohen R.M., Finn R.D. and Larson S.M., Appl. Radiat. Isot., 37, 1181 (1986).

12. Barrio J.R., Satyamurthy N. , Keen R.E., Hawkins R.A., Bida G.T., Hoffman E.J., Huang S.C., Mazziotta J.C. and Phelps M.E., J. Nucl. Med., 27, 879 (1986).

13. Kilbourn M.R. and Welch M.J., Appl. Radiat. Isot., 3_7, 677 (1986).

14. Kiesewetter D.O., Kilbourn M.R., Landvatter S.N., Heiman D.F., Katzanellanbogen J.A. and Welch M.J., J. Nucl. Med., 25., 1212 ( 1 9 8 4 ) .

356 15. Hwang D-R., Falw A.L., Wolf A.P., MacGregor R.R., Fowler J.S. and Arnett C.D., J. Label. Compd Radiopharm., 23, 277 (1986).

16. Curtis D.R. and Watkins J.C., Pharmacol. Rev., 17, 347 (1965).

17. Watkins J.C. and Olverman H.J., Neurosciences, 10, 265 (1987).

18. Ascher P. and Nowak L., Neurosciences, 10, 284 (1987).

19. Cotman C.W., Monaghan D.T., Ottersen O.P. and Storm-Mathisen J., Neurosciences, 10, 273 (1987).

20. Watkins J.C. and Evans R.H., Ann. Rev. Pharmacol. Toxicol., 2 1 . 165 (1981).

21. Fagg G.A., Foster A.C. and Ganong A.H., Trends Pharmacol. Sci., 7, 357 (1986).

22. Mayer M.L. and Westbrook G.L., Prog. Neurobiol., 28, 197 ( 1 9 8 6 ) .

23. M c L e n n a n H., Prog. Neurobiol.,20> 251 (1983).

24. MacDermott A.B. and Dale N., Neurosciences, 10, 280 (1987).

25. Rothman S.M. and Olney J.W., Neurosciences, 10, 265 (1987).

26. Kemp J.A., Foster A.C. and Wong E.H.F., Neurosciences, 10, 294 (1987) .

27. Wong E.H.F., Kemp J.A., Priestley T., Knight A.R., Woodruff G.N. and Iversen L.L. , Proc. N atl. Acad. Sci. USA., 83., 7104 (1986).

28. Kemp J.A., Priestley T. and Woodruff G.N., Br. J. Pharmacol., 89, 535 (1986).

29. Largent B.L., Gundlach A.L. and Snyder S.H., J. Pharmacol.Exp. Ther., 238, 739 (1986).

30. Bowery N.G., Wong E.H.F. and Hudson A.L., Br. J. Pharmacol., 93, 944 (1988).

31. Patel S., Chapman A.G., Millan M.H. and Meldrum B.S., "Excitatory amino acids in Health and Disease", Ed. Loge D., John Wiley and Sons Ltd., 353 (1988).

32. Lyle T.A. , Magill C.A. and Pitzenberger S.M., J. Am. Chem. Soc., 109, 7890 (1987).

33. Firnau G., Chirakal R. , Sood S. and Garnett E.S., Can. J. Chem., 58, 1 4 4 9 (1980).

357 34. Chriakal R. , F i r n a u G. , C o u s e J . and Garnett E.S. , I n t . J. Appl. Radiat.I s o t . , 35. 651 (1984).

35. F i r n a u G. , C h i r a k a l R. and Garnett E.S ., J. Nucl. Med. ,25, 1228 (1984).

36. G a r n e t t E.S. , F i r n a u G and Nahmais C. , Nature, 305, 137 (1987) .

37. Gar n e t t E.S. , N a h m a i s C . and Firnau G . , Can. J. Neurol. S ci., 11, 174 (1984)

38. Garnett E.S. , J. Neurolog. Sci., 69., 223 (1985) .

39. Bartholini G., Constantinidis J., Puig M., Tissot R. and Pletscher A., J. Pharmacol. Exp. Ther., 193. 523 (1975).

40. Inagaki C. and Tanaka C., Biochem. Pharmacol., 27., 1081 (1978).

41. Suzuki T., Sakoda S., Ueji M., Kishimoto S., Hayashi A., Kondo T. and N a r a b a y a s h i H . , Neurology, 34, 1 4 4 6 (1984).

42. Y a m a m o t o M. , O g a w a N. a n d Uj ike H. , J. Neuro. Sci. 73.,, 39 (1986) .

43. Lemaire C., Guillaume L., Christiaens L., Palmer A.J. and Cantineau R . , Appl. Radiat. Isot., (1987).

44. Tewson T.J. and Welch M.J., J. Nucl. Med., 21, 559 (1980).

45. Tewson T.J., J. Nucl. Med., 24, 718 (1983).

46. Vora M.M., Boothe T.E., Finn R.D., Kothari P.J., Emran A.M. , Carroll S.T. and Gilson A.J., J. Label. Compd Radiopharm. 22., , 953 (1985).

47. Tewson T.J., J. Nucl. Med., 24, 52 (1983).

48. Ballinger J.R., Bowen B.M., Firnau G., Garnett E.S. and Teare F . W . , Int. J. Appl. Radiat. Isot., 35, 1125 (1984).

49. Cacace F. , Speranza M. and Wolf A.P., J. Fluorine Chem., 21, 145 (1982).

50. Gnade B.E., Schwaiger G.P., Liotta C.L. and Fink R.W. , In t. J. Appl. Radiat. Isot., 91 32, (1981).

51. Coenen H.H., Schuller M. and Stocklin G.L., J. Label. Compd Radiopharm., 1197 21, (1985).

52. Brodack J.W., Kilbourn M.R., Welch M.J. and Katzenellenbogen J.A., Int. J. Appl. Radiation. Isot.,.37, 217 (1986).

358 53. Hamacher K. , Coenen H.H. and Stocklin G. , J. Label. Compd Radiopharm. 2J3, , 1095 (1986) .

54. Clark J.H., Chem. Rev., 80, 429 (1980).

55. Shiue C-Y., Watanabe M. , Wolf A.P., Fowler J.S. and Salvadori P . , J. Label. Compd Radiopharm., 21, 533 (1984).

56. Attina M. , Cacace F. and Wolf A.P., J. Label. Compd Radiopharm., 20, 501 (1983).

359 APPENDIX

360 A P P E N D IX —1

Stock solutions5

Solution no. Nutrient Concentration g/1

1 k n o 3 200

2 MgS04 .7H20 100

3 k h 2p o 4 27

4 FeS04 .7H20 10 Na-citrate 20 Na2 .EDTA 20

5 Ca(N03)2 .4H20 2.36

Trace element solution

This solution is a mixture of: Trace element solution A (1000 ml) Trace element solution B ( 100 ml) Distilled water to a total volume of (2000 ml)

Trace element solution A

H 3BO3 2.86 g/1 MnCl2 .4H20 1.81 g/1 ZnS04 .7H20 0 .222g/l CuS04 .5H20 0.079g/l Dissolved in 0.05 M H2S04

Trace element solution B

(NH4) 6Mo ^024.4H20 27 mg/1 n h 4v o 3 22 mg/1 KCr (SOa ) ?.12H20 96 mg/1 NiS04 .6H20 45 mg/1 Co(NO3)2 .6H20 49 mg/1 NaVJ04 .2HpO 18 mg/1 TiCl3 33 mg/1 Dissolved in 0.05 M H2S04

361 Stock solutions^ (continued)

Culture Medium

Solution No. Volume (ml)

1 12.5

2 5

3 5

4 2

5 10

Trace element solution Distilled water to a total volume of 1000 ml.

a) The solutions were sterilised within 2-4 h of preparation in an autoclave at 393 K for 30 min. The bottles were covered with aluminium foil and stored in the cold room at 244-273 K.

362 APPENDIX-2

Nutrient Broth No. 2 Formula "Lab - Lemco" Beef extract 10 g/1 Peptone (Oxoid L37) 10 g/1 Sodium chloride 5 g/1 pH ca. 7.5

Preparation Granules of nutrient broth No.2 were dissolved in distilled water (1000 ml) and sterilised by autoclaving for 15 min at 100 kN m~2 .

363 APPENDIX-3

Nutrient Broth No. 2 Agar plates To a solution of nutrient broth NO.2 (6.25 g in 250 ml distilled water), Oxiod ionagar No.2 (4.5 g) was added. The mixture was sterilised by autoclaving for 15 min at 100 kN irf2. The solution was allowed to cool to 313-333 K and then transferred under aseptic conditions into petri dishes (ca.10 ml). These were allowed to set overnight and were stored at 237 K.

364 APPENDIX 4 Sequence of operations for the preparation of f^Cl Dinrenorphine and f11C1Buprenorphine (9) A schematic representation of the apparatus used is depicted in Fig 3.12. Vessels A, B and C were kept at 423 K. All FIFE lines were kept at 353 K.

Stage A: leak testing and flushing of apparatus.

Operation Valve Reagent n 2 Position Line (ml/min“l) 1) Flush cryogenic trap 2 + 2N on 15 2) Leak test vessel A 7 + 2N on 15 3) Leak test vessel B 7 off + 5N on 15 4) Prime "Sep-pak" with 1,4,4A + 2N on IV - THF 5) Seal "Sep-pak" 1,4,4A + 2N on - 6) Flush vessel A 1,7,10,4,4A + 2N on 15 7) Flush reagent lines 1,7,4,4A + 2N on I 15 to vessel A 8) Flush vessel B 1,4,4A,8 + 2N on 15 via distillation line 9) Flush lines to vessel B a) 1,4,4A,8,9 + 4N on II 15 + transfer both A and B b) 1,4,4A,10 + 5N on 15 to 448 K bath 1-2

Stage B: r -^^Cl carbonation and reduction.

10) Seal and trap 11002 2,7,8 ,10,1,4,4A + 5N on 1-2 11) Load Grignard reagent 2,7,8 ,10,1,4,4A + 5N on I 1-2 into vessel A 12) [ ] Carbonation 7,8,10,1,4,4A + 2N on 1-2 13) Addition of PDC and 7,8,10,1,4,4A 4- 2N on I 1-2 DTBP into vessel A

365 14) Removal of 6,7,8,1,4,4a + 2N on 5-10 at 338 K 15) Add NDRJ in THF 6 ,7,8,1,4,4A 2N on II 5-10 to vessel B 16) Distillation of [-^C] 6 off,8 on,7 off,l 15 acid chloride at 448 K 4,4A + 2 on 17) Addition of LiAlH4 8 ,1,4,4A + 2N on II 10 vessel B 18) Addition of MeOH to 8 ,1,4,4A, 11,9 + 4N on II 10 vessel B

Stage C: "Sep-pak" separation and purification.

19) Load onto "Sep-pak" 9 off,11 off,7 on, loff 15-20 4 + 4A off, 3 on, 8 off and 5N on 20) Elute "Sep-pak with 1,3 + 5N on IV 21) Removal of MeOH + THF 3,4,4A + IN on 15-20 at 338 K under red. pres, 22) Addition of hplc sol. 3,4,4A + IN on III 5 to vessel C 23) Load onto hplc loop 4,4A,5,12 + 3N on 24) Inject All off

366 APPENDIX 5

kBT k = k --- exp (-AG* /RT) Eq 4.1 h

In k = In (kg/h) + In T - AG*/RT

In k - In (kg/h) - In T = -AG*/kT

In (kg/h) + In (T/k) = AG*/kT

[In (kg/h) + In (T/k)] kT = AG*

[2.303 log (kg/h) + 2.303 log (T/k)] kT = AG*

[log (kg/h) + log (T/k)] 2.303 RT = AG*

h = 6.6261176 X 10*"34 J s k = 8.31441 J mol”1 IC1

[10.319 + log (T/k)] aT = AG* Eq 4.9

a = 1.914 x 10"2 (AG* is in kJ mol-1)

367