AU2013204848A1 - Labelling methods - Google Patents

Abstract

C:\NRPortbl\DCC\SXD\4914478_I.DOC-I2/04/2013 The invention provides a method for radiofluorination of biological vectors such as peptides comprising reaction of a compound of formula (II): or a salt thereof with a 5 source of (18F)-fluoride, to give a compound of formula (1): or a salt thereof. The method may be effected under mild reaction conditions and offers a more chemoselective labelling approach. Novel reagents for use in the radiofluoridation method, and uses of the resultant 18F-labelled vectors are also provided.

Description

C:\NRPortbl\DCC\SXD\4914478_l.DOC-12/04/2013 LABELLING METHODS This application is a divisional of Australian Patent Application No. 2008265184, the entire content of which is incorporated herein by reference 5 The present invention relates to methods and reagents for ( 18 F)-fluorination, particularly of biological vectors such as peptides. The resultant 1 8 F-labelled vectors are useful as radiopharmaceuticals, specifically for use in Positron Emission Tomography (PET). 10 The application of radiolabelled biological vectors for diagnostic imaging is gaining importance in nuclear medicine. Biologically active molecules which selectively interact with specific cell types are useful for the delivery of radioactivity to target tissues. For example, radiolabelled biological vectors have significant potential for the delivery of radionuclides to tumours, infarcts, and infected tissues for diagnostic 15 imaging, clinical research, and radiotherapy. 1 8 F, with its half-life of 110 minutes, is the positron-emitting nuclide of choice for many receptor imaging studies. Therefore, 1 8 F-labelled biological vectors have great clinical potential because of their utility in PET to quantitatively detect and characterise a wide variety of diseases. 20 One difficulty with certain 18 F-labelled biological vectors is that the existing 1 8 F-labelling agents are time-consuming to prepare. For example, efficient labelling of peptides and proteins with 18 F is mainly achieved by using suitable prosthetic groups. Several such prosthetic groups have been proposed in the literature, including N-succinimidyl-4( 1 8 F)fluorobenzoate, m-maleimido-N-(p-( 18 F)fluorobenzyl)-benzamide, 25 N-(p( 1 8 F)fluorophenyl) maleimide, and 4-( 1 8 F)fluorophenacylbromide. Many labelling methods using prosthetic groups give rise to multiple radiolabelled products. For example a peptide containing 3 lysine residues has three amine functions all equally reactive towards the labelled prosthetic group. This approach, often referred to as the "two-step" approach can also be time-consuming as the radiolabelled prothetic 30 group has to be prepared and then coupled to the biological vector in a second step. Therefore, there still exists a need for 1 8 F-labelling methodologies which allow rapid, chemoselective introduction of 18 F into biological vectors, particularly into peptides and proteins, under mild conditions to give 1 8 F-labelled products in high radiochemical yield - 1- WO 2008/155339 PCT/EP2008/057659 .nd purity. Additionally, there is a need for such methodologies whlch are amenable to automaton to facilitate preparation of rdiopharmcIceuticais in the clinical setting. Accordingly, the present invention orovidesa method for radiofluorination cornprisina 5 reaction of a compound of forrnula (II) VECTOR L LINKER -- CRYPTAN (II) or a salt thereof with a source of [F]-fluoride. to give a compound of formula () 10 VECTOR LINKER - - --- PAND -F (I) or a salt thereof, followed by the optional steps: (i) purification of the compoundi of formula () and/or (ii) formulation of the compound of formula )H. The present invention provides a more chemoselective aprooach to radiolaibeling where the exact site of introduction of the label is pre-selected during the synthesis of the precursor of formula [H). This methodology is therefore chemoselective and its 20 application is considered generic for o wide range of biological vectors. As used herein, the term "Vector" meons a biomolecule suitable for radiolabelling to form a radiopharmaceutical, such as a peptide, protein hormone, polysaccaride, oligonucleatide, antibody fragment, cell, bacteriurn, virus, r small drug-like molecule. In formulae (I) and (Il) and in other aspects of the invention unless specifically stated otherwise, particularly suitable Vectors are selected from peptides, proteins, and smalI drug-like molecules, and in one aspect of the invention are Vectors which do not need to cross the biood--broin barrier for their biological function. 30 Suitable peptides for use as a Vector in the invention include somatostatin analogues, such as octreotcide, bombesin, vasoactive intestinal peptide, chermotactic peptide WO 2008/155339 PCT/EP2008,,057659 analogues, cx-melnocyte stimulotng hormone, neurotenisin, ,,rg--C--.y-/Isp peptide, humfan pro-inSulin connecting peptidea, iNsuh lip teli on' -es, bi-,knm endotcui, anioali glutothioi-e calcitonin, Moirinin! and 1H, luteinizing ho~mune reheasng hormone, gostrins, cholecystachiniri, substance P', vasop ress-in, forraYl 5 norleucyileucv l-p ht i-/lal/-rIorleocyl-tyrosyl/- y sine,, Annecxin V ano, ,,,cagu- es, Vasoact~ve Protein-i. (yA P-i peptdes, ana caspase peptide substrates. Preferred peptides f-Or Use as a Vector in the invention are Arg-GlY-Asp peptide and its analogues, such as those described in DVO C,1/7 7415 and V'O 03/0064391, preferably a peptide comprising the frament: 1L C 0 H H I I ' 0 0 ---- 0 ( / HO HN H~ I ~ NH2 more preferably, the peptide of form-ula (A). -------------------

-

-

S 20 S 0

-

1 /0 0x HOH 30 H N H N 35 wh/erein W/ is ether -NJF 2 orI WO 2008/155339 PCT/EP20081057659 H N H, 'o 0 0 wheren a is on integer of from 1 to 10, preferably a is 1 1i0 In formulae (11) and (K, and in other aspects of the invention, the Linker is a C 5

-

0 hydrocarby! group optiona||y including 1 to 10 heterootoms such as oxygen or nitrogen, and may be chosen to provide good in vivo pharmacokinetics, such as favourable excretion characteristics. The term "hydrocarbyl group" means an organic is substituent consisting of carbon and hydrogen, such groups may include saturated, unsaturated, or aromatic porions. Suitable Linker groups include alkyl, alkenyl, alkyny chains, aromatic, polyaromatic, and heteroarcmatic rings (for example, triazoles), an polymers comprising ethyleneglycol, ornino acid, or carbohydrate subunits any of which may be optionally substituted for example with one or more ether, thiooether, 20 sulphonamide, or aide functionality. As used herein, the term "Cryptand" means a hi- or poly-cyclic rmultidentate ligcind for the fluoride anion. Suitable Cryptands for binding onions such as fluoride have been reviewed in JW. Steed, J.L. Atwood in Suprcmolecular Chemistry (vViley, New \York, 25 2000), pp198-249; Supromolecular Chemistry of Anions, Eds A Bianchi, K Bowmann james, E. Garcia-Espana (Wiley-VCH, New York, 1997t and PD. Beer, PA, Gale, Angew.Chem. 2001, 113, 502; Angew. Chem. Int. Ed. 2001, 0, 486. Suitable Cryptands used herein include those of formula (C): 30 R1 R3/ R4 R5 (C) R2 WO 2008/155339 PCT/EP2008/057659 wherein: 5 RI and R2 are independently selected from R3, R4 and RF are independent y s e ted rom: HN A NN NH HN N N HN +v N 10 Prei erired Cryptands usefud in may be selectedfrom: N NN NH HN HN-N N NH < N NH N, NHHNN N N H NN or mayir-l be- chosen to have1 desirable properties suchd- as a high binding Constant for friNhi stabilityof the fluoride nurd complex and high fluode selectivity over other anions. |n one aspect of the invention, the -Ciran bears a positive charge.

WO 2008/155339 PCT/EP2008/057659 In the compounds offormula (I) ard (), the Cryptand s attachedto a inker group. The point of attachment may be a nitrogen or carbon atom in the CryptIand. Thus the point of attachment to the Linker '" may be n group R1 or R2 L ,N N 5 or n R3, RA or RS: 4- H N HN HN L N N HN N HN N/ L se + L -* + N N N 4 N H H H N N N Ni, NH NH N NH H- H 4 L N L N N L 10 Suit'bek saits according to the 'inventior include (i) physiologicaly --cptable acd addition salts such as those derived from minerca acids, for example hydrochloric, hydrobrornic, phosphoric, metaphosphoric, nitric and sulphuric acids, arnd those derived from organic acids, for example tartaric, trifluoroacetic, citric. malic, a tic, 1 furnaric, benzoic, glycollic, gluconic, succinic, rnethanesulphonic, and parc toluenesulphonic acids; and (ii) physiologically acceptable base salts such ammonium salts, alkali metal salts for example those of sodium and potassium), alkaline earth metal salts (for example those of calcium and magnesium), salts with organic bases such as triethanolanine, N-methyl-D-glucamine, piperidine, pyridine, 20 piperazine, and rnorpholine, and salts with amino acids such as arginine and lysine. As used herein, the term "source of [mF]fluori de" means a reagent capable of delivering F]-flu ride in reactive form to the reaction mixture. ['Flfluoride is -6- WO 2008/155339 PCT/EP2008/057659 conveniently prepared in a cyclotron from 180-enriched water using the (p,n;-nuclear reaction, (Guillaume et a, App Radiat. sot. 42 (1991) 7/49-762) . For example, the source of [isF]-fluoride may be 'F]-fluoride in target water rm a cyclotror, or an [18F)-fluoride salt prepared from the target water such as: PF1-sodium fluoride, PF] 5 potassium fluoride, PF]-coesium fluoride, P 8 F]-tetraalkylammonium fluoride, 1 8

F

tetraolkylphosphonium fluoride in a suitable solvent such as acetonitrile, dimethylformamide, dimethysulphoxide, tetrahydrofura, dioxan, 1,2 dimethox~yethane, sulphone, M-methylpyroidinone or aqueous mixtures of any thereof. 10 The reaction of a compound of formulc (II) with a source of L 8 F]-fluoride may be effected at non-extreme temperature, such as 10 C to 50 C, and most preferably at ambient temperature and in a suitable solvent such as those listed hove as solvents for the "source of PF)-fluoride" or alternatively as a solid supported reaction as 15 described below. The ability to incorporate PFf-luoride into a biological vector at ambient temperature is a particular advantage of the invention as many biologica vectors are unstable at elevated temperatures. If the Cryptand in a compound of formula (II) does not have a fixed positive charge, the reaction with a source of SF fluoride is suitably performed at a pH of below 5, which is achieved by addition of acid 20 such as hydrochloric or sulphuric acid. Following preparation of a compound of formula W, a purification step (i) may be required wnch may compise, for example, removal of excess [F]-fluorid, removalof solvent, and/or separation from unreacted compound of formula (lH) Excess ' 8

F]

25 fluoride may be removed from a solution a the compound of furrnula (2 by conventional techniques such as ion-exchange chromatography (for example using BiO-RAD AG --X8 or Waters QMA) or solid-phase extraction (for example, using alumina. Excess solvents may be removed by conventional techniques such as evaporation at elevated temperature in vacuo or by passing a stream of inert gas (for 3 a example, nitrogen or argon) over the solution. Alternatively, the compound of formula (1) may be trapped on a solid-phase, for example a cartridge of reverse-phase absorbant for example a Csvs derivatized silica, whilst the unwanted excess reagents -7- WO 2008/155339 PCT/EP2008/057659 and by-products are eluted, and then the compound of formula 0) may be eluted from the solid-phase in purified form. Separation of a compound of formula ) from unreacted compound of formula (fl) may be effected bv conventional techniques, for example using solid-phase extraction on an anionic soli--phase (for example, a S macroporous sulphonated polystyrene resin) exploiting the reduced charge, and hence change in affinity caused by binding of 'IFj-fluoride to the compound of formula (l0 in one embodiment, the compounds of formulae (li) may be covalently bound vic the Vector to a solid support, such as polymer beads or coatings, for example, a trityl or 10 chlorotrityl resin. In this aspect, the excess reagents and by-products of the radio fluorination reaction may be separated from the polymer-bound product by washing. Cleavage f the compound of formula (W) from the solid support may be effected by conventional techniques of solid phase chemistry, for example as described in Florencio Zaraaoza Dorwald "Organic Synthesis on Solid Phase; Supports. Linker, 5 Reactions", VViley-VCH (2000) This approach may be particularly suitable for citomated production of the compounds of formula 0) i n which the Vector is a peptde or protein. Following preparation of a compound of formula ) or a salt thereof, it may be 20 appropriate to formulate it as a radiophrmaceutical, ready for administration to a subject. Such form; elation step Lii may comprise preparation of a aqueous solution or the compound af formula f cr a salt thereof by dissolving in sterile sotonic saline which may contain up to 10% of a suitable organic solvent such as ethanol, or a suitable buffered solution such as a phosphate buffer. Other additives such as 25 stabilizers, for example ascorbic ocid may be added to the formulation. Compounds of formula ill) may be prepared by reacting a compound of formula (lii: R LINKER- CRYPTAND (lii) 3c with a corripound of formula (IV): -8 - WO 2008/155339 PCT/EP2008/057659 VECTOR __---R (M wherein the Vector and Cryptand are as defined above, u nker'is a portion of the 1Unker as defined above, and RI and Rv are reactive groups capable of covalent bonding to 5 eachother so as to complete formation of the Linker. Suitably, one of R' and R is an amine and the other is a carboxylic ocic or an activated carboxylic ester, isocyanate or sothiocyanate such that the compounds of formulae (Ill) and (IV) may be joined by simple amine reaction. Suitable activated carboxAic esters include the N hydroxvsuccinimidyl and N-hydroxysulfosuccinimidvl esters: 0 0 0 J'""0 N N 10 0O Alternatively one o R and Rv may be a thiol and the other a group reactive towards a thiol, such as a maleiricle or an a-halocarbonyl. As would be cpoarent to the person skilled in the art it may also be desirable for the is Cryptand in the Compound of formula (111) to have protection groups on any exposed functional groups e.g. omino groups to prevent or reduce side-reactions during conversion t a CorTpound of formula l1). in these cases the protection group will be chosen from those commonly used for the functional group n question e.g tert butylcarbamate for an amine. Other suitable protecting groups may be found in 20 Protecting Groups in Organic Synthesis, Theodora W. Greene and Peter G. M. Wuts, published by John Wilev & Sons Inc. which further describes methods for incorporating and removing such protecting groups. Certain compounds of formula (I) may be prepared by reacting a compound of formula 25 (nli) wherein RI is either an amino or carboxylic acid group with a compound of formula (IV) wherein R"v is either a carboxylic acid or amine group respectively. In these cases a compound of formula (1I) nuy be coupled with ca compound of formula 'IV) optionally using in situ activating agents such as 2-lH-benzotriazole--yl-L13,3 -_9- WO 2008/155339 PCT/EP2008/057659 tetramethyluronium hexCIfluorophosphate (-- BTU) or N-[(dimethylanino)-1H-12,3 triazolo(4,5-bjpyridin-1-ylmethylene]-N-methylmethanamonium hexafluorophosphate N-oxiOde (HATUL. Standard conditions will be used e g. dimethylformrnide (DMF) solution and a base e.g. triethylamine or diisopropylethylanine Alternatively where Riv in the s compound of formula (IV) is a thiol group, this may be reacted v;ith a cornpound (Il) in which RJ' is a thiol reactive group such as o maleimide or an a-halocarbonyl. This reaction may be performed in a pH buffered solution or an organic solvent. The product compound having the formula (1) might be purified by preparative high performance liquid chromatography. 10 Compounds of formula (II) wherein the Vector is a peptide or protein may he prepared by standard methods of peptde synthesis, for example, soild-phose peptiae synthesis, for example, as described in Atherton, E and Sheoppoard, RC; "Sold Phase Synthesis"; !RL Press: Oxford, 1989. Incorporation of the Linker and Cryptand in a compound of 15 formula (l) may be achieved by reaction of the N or C-terminus of the peptide or with some other functional group contained within the peptide sequence, modification of which does not affect the binding characteristics of the Vector. The Compound of formula (Ill) as defined above, is preferably introduced by fon-nation of a stable amide bond formed by reaction of a peptide amine function (Rv) with ci compound of formula 20 (I) in which R is an activated acid or alternatively by reaction of a pepticle acid function (Riv) with a compound of formula (Il) in which R4 is an amine, and in either case the compound of formula III) may be introduced either during or following the peptide synthesis, for exam ple, solid-phase peptide synthesis. When either of Rill or Riv is an acid the reaction of compounds of formulae (HI) and I) may be effected using in 25 situ activating agents such as 2-(1H-benzotriazole-1-yl-1,1,3,3-tetramethyluroniunm hexafluorophosphate (HRTJU) or N-[(dimethyiamino)-1H-12,3-triaolo4,5-bpyridin-I ylmetvlenei-N-metylrnethanammonium hexafluorophosphate N-oxide (HATU). An embodiment of this particular aspect of the invention is shown in Scheme 1. -10- WO 2008/155339 PCT/EP2008/057659 NN b OC boc boo b O bc N N N PePdes N N NH, bo" boWc"l NN N N N N TFA PPv~~i N NH VFu d j-WSWPMo TFA = Trifluoroacetic acid Scheme 1 The Cryptands may be synthesised as described in US20040267009 Al, Bernard Dietrich, Jean-Marie Leh., Jean Guiihem and Claudine Pascard, [trehedron Letters, 1989, Vol 30, No. 31, pp 4125-4128, Paul H. Smith tai, J. Org Chem, 193, 58, 7939 7941, Jonathan W Steed et a, 2004, Joumal of the American Chemical Society, 126, 12395-12402, Bing-guang Zhang et al, Chem. Comm., 2004, 2206 2207. o The synthesis of a Compound of formula (I) may be achieved as described in the above references for the underivotized Cryptands with modifications to the starting materials or by subsequent chemistry, for example, by alkyiotion of a secondary amine group of the Cryptand as iustrated in the Examples below. Compounds of formula (ll) may also be prepared as shown in Schemes 2 to 5 in which L ard R"' are as defined 15 above for the Compound of formula (Ill. 2 OC WO 20081155339 PCT/EP2008,1057659 S c h emre 2 NHH'N1 HN' 0 11 NH H~ LBR / r', <<B /< NH;------- N H -------2N ~H] B rH ------ N H 1-,H11 < ScI-Im 3I1 H + NO N<H '"- L L " 01 -p theme Z. 2 N-, N H NHHrN H 3 NN. ) -12- WO 2008/155339 PCT/EP2008/1057659 0n o ' KNH H L L-S O [. ( - - : PG pr ot ect i ng gr OUp L-F" NH HN h , NI HN HN'4 As a further aspect of the invention, there is provided a compound of formnua (1) or a sait thereof, as defined above. These corrpods having utility as PET tracers. b Compounds of formula (1) in which the \/ector is a peptide suitably Ar-Gly-Asp peptide or its analogues are preferred, such as the peptides described in WO 01/77145 and WO 03/006491. Particularly preferred peoides in this aspect of the invention are those of formula (A) as defined above for the compounds of formula (I. 10 The compounds of formula (1) or a salt thereof may be administered to patients for PET imaging in amounts sufficient to yield the desired signal, typical radionuclide dosages of 0 01 to 100 mCi, preferably 0.1 to 50 mCi will normally be sufficient per 70kg bodyweight, though the exact dose will be dependent on the imaging method being performed and on the composition of the compound of formula (0 or salt thereof. The compounds of formula I0) or a salt thereof may therefore be formulated as a radiopharmaceutical for administration using physiologically acceptable carriers or excipients in a manner fully within the skill of the art. For example, a compound of forrrula (I) or a salt thereof, optionally with the addition of one or more 20 pharmaceutically acceptable excipients, rmnay be suspended or dissolved in an aqueous medium, with the resulting solution or suspension then being sterilized. Such rodiopharmaceuticals form a further aspect of the invention. Viewed from a further aspect the invention provides the a compound of formula (1) or a 25 salt thereof as defined above for use in medicine, more particularly in a method of in vivo imaging, suitably PET, said method involving administration of said compound to a WO 2008/155339 PCT/EP2008/057659 human or animal body and generation of an rage of at least part of said body. Vewed from a still further aspect the invention provides a method of generating an image of a human or animal body involving adminstering a radiopharmaceutical to s said body, e.g. into the vascular system and generating an image of at least a part of said body to which said radiopharmaceu tical has distributed using PET, wherein said radiopharmaceutical comprises a compound of formula (1) or a salt thereof as defined above. In a further aspect, there is provided a rnethod for in vivo imaging, suitablyPET imaging, of a body, preferably a human body, to which body a radiopharmaceutical 10 comrising a compound of formula (i) or a salt thereof as defined above has been preadministered , wherein the method comprises detecting the uptake of said radipharmaceutical by an in vivo imaging technique, suitably PET. In a further aspect, the present invention provides a compound of formula (II) or a salt is thereof as defined above, having use as a radiolabelling precursor. In another aspect, the present invention provides novel synthetic intermediates of formula (II ),useful for functionalising Vectors re-dy for radiofluoridation, for example by the methods described above. Accordingly, there is provided a compound of 2 o formula (ll) LINKER'H- CRYPTAND (Ill) wherein Rm is as defined above and is preferably selected from amine, carboxylic acid, activated corboxylic ester, isocyanate, isothiocyanate, thiol, moleimide, or o Nalocarbonyl, and the Linker' and Cryptand are as defined above. 25 Preferred compounds of formula (Ill) include: -14 - WO 2008/155339 PCT/EP2008,'f5'7659 / A N N 0. H H N / N t NHH N~ A&' 11 N'H N' N> N N H LR NH N < N~ -IN, i-IHN.NI / ~ N ' HI I .N i / N NH NH( LNR' INHzN Ni~~~ N ( N N /NHI_ whemb Lis a inker ' as dened abve and W". qs a teactive a -o as defie c-ibaVe, 5 cand is preferably selected flor amine, carboxylc acid, actvated carboxylic ester, sacyaaate, isothiocvainate, thiol, malriici or" Mhocrbanyl. Mo,-e preferred campounds of farmula OW OAK~d WO 2008/155339 PCT/EP2008/057659 L -R N N <NH HN HN- [NH HN. N N N N - NH HN HN NH HN. HN N N N N N L P N HN HNN1 NK N< N NH HN' HN< N N N<N N N KN N N N+ NN N N N N N,' N N, N N -,N N N - N N wherein Eis a Lnker as defied above, and R is a reactive group as defined above, and is preferably selected from amine, corboxylic acid, activated carboxylic ester, isocyanate, isothiocyanate, thiol, maleimide, or a-halocorbonyL in a further aspect of the invention, there is provided a compound of formula M: CRYPTAND F (V) or a sait thereof, wherein the Cryptand is as defined above, for use in medicine, for example as perusion imaging agents. Preferred compounds of formula (V) for this purpose comprise a preferred Cryptand as described above. For this use, the Compound of formula I) or a salt thereof is suitably -1 - WO 2008/155339 PCT/EP2008/057659 formulated as a radiopharmaceutical as described above for the Cornpounds of formla (IL In the altemative there s provided a method of imaging which comprises admrinistration to a subject of a detectable amount of a compound of formula M or a salt thereof as defined above, and imaging the subject using PET. Methods for perfusion iragin gusing PET are described in Swaiger, J. Nucl. Med. (1994) 693-8 and the references therein. 10 In some circumstances, it may be desirable to prepare a prosthetic group for rodilorilridCion o a Vector. Therefore, according to a further aspect of the invention there is provides a cormpound of formula MWK Rm- LNKER' CRYPTAND IF (VI) 15 wherein the Licker' and Cryptand and R' are cs defined for a compound of formula (lil) above. According to a further aspect of the inventior there is provided a kit for the preparation of a radioflurinated compound comprising a synthetic intermediate of formula (11), 20 and opuonally a compound of formula (IV) as defined cbove In use of the kit, the compound of formula (Ili, would be reacted with a compound of formula (IV), using methods described above to form the corresponding compound of formula (i) aid then reacted with a source of ' 18 F]-fluoride to farm a radiofluorinated 25 Vector of formula (I). Optionlly, the compound of formula (N may be purified end/or formulated as described above. The invention is i1ustrated by way of the following examples, in which these abbreviations are used: 3 C PriOH: isopropanol N triethylamine RT.: room temperature -17- WO 2008/155339 PCT/EP20081057659 MepH: methano! WI BOC (tertiary) hutc>xvcarhonyl TF tetrah Arauan HPILCI high performance liquid chromatagraphy [DCW dichloromethone LCOIS liquid chrom-otLography moass spectrometry 10 NI R nuLIcIear mcavgn et icr eEo nain cec TFA trifluoroacetic. oad Example I1: Synithesis of compound 4 15 ' q8 QK 3 RT r NaB N.H 'NI HN NH.IN, IN B r QLDMF / 1<2eoo NHN _NR NRl N~H iN H 4 Excomnle Iii) Sv thes of co un A IA 3-eck m und botRom 'ask equipe ar wth a mechanical stirmrwasO charcgad with -18 WO 2008/155339 PCT/EP2008/057659 16.7 mL of 98% tnpropylamine and 0,33L of 99% i-PrOH, and cooled to -- 78" C. in a dry ce-isopropoanol both. To this mature, solutions of 15.0 g 40% aqueous glyoxal (0.1103 maiole, diluted to 83 mL with isopropanol, and 10.0 g (0.0.683 moles) of 96% tris-(2 raminoethylamine (tren[ diluted to 83 mL. were simultaneoLsly added over a period of 5 2 hrs with Viorous stirring. (Initial concentrotion of glyoxal=1.24 M; initial concentration of tren=0. 82 M. Then the reaction mixture was allowed to warm up overnight and brieflywarmed up to 600 C. to ensure that the forrmnation of compound 2 was complete. It was cooled to room temperature while nitrogen gas was blown over its surface. The solvent was removed under vacuum and chloroform (250 mL) was added. The resulting 10 slurry was filtered through sand and concentrated under vacuum to give an orange solid (15.2 g, 43%). Examrote 1L) Synthesis of coround 2 Compound 1(4 g, 11.2 mmal) was dissolved in merhanol ((150 mi) and was cooled in an ce/water bath. Sodium borohydride (S g, 200 mmol) was added portion wise over 30 1s minutes The mixture was left to rise to room temperature with stiffing over 16 hours. The solution was concentrated to dryness under vacuum to give an off white solid. The solid was dissolved in water (100 mL and was heated to 60 0C for half on hour during which time an oily material formed in the mixture. THF (100 mL) was added and the organic layer was separated. The aqueous layer was extracted again with THF 100 20 mL). The combined extracts were filtered through a phase separator cartridge ond were concentrated to dryness under vacuum. The oily solids were re-dissolved inT (20 mL) and water (15 mLi was added. The solution was concentrated slowly until a white solid crystallized which was collected by filtration, washed with ice cold water and dried under high vacuum (1.6 g, 38%). 23 Example 1(iii) Synthesis of compound 3 Compound 2 (01 g, 0270 rnmol) was dissolved in dry DMF (5 mL) and potassium carbonate added (1 eq 0297 mmol, 0,041 g). The alkyl bromide (1.1 eq. 0.297 mmol, 81.7 mg) was added potion wise following the reaction by HPLC-mass spectrometry by taking approximately 0.1 mL volume front the reaction and diluting with 1:1 0.1% 30 formic acid in water:acetonitrile (10 mL. The reaction was stirred at room temperature for 16 hours. A further 025 equivalents of the alkyl bromide was added and the -.19 WO 2008/155339 PCT/EP2008/057659 reaction stirred for a further 16 hours. The reaction mixture was concentrated to dryness under vacuum. This was used in the next step without further purification. Example Ijivi Synthesis of compound 4 Crude compound 3 was dissoved in cry DMF (20 mL and pyridine (2 mUl was added 5 followed by ci-tert-butylcarbonate (I g, 458 rmmol, 17 eq.). The mixture was heated at 70 DC under nitrogen for 16 hours. The crude product was analysed by thin layer chromatography (slica gel plates elutng with 10% methanol/DCM) and by LCMS. Thin layer chromatography showed two major spots having Rf values of 0.2 and 0.5 and some minor spots. The mixture was purified by flash colurnn chromatography on silca 1o gel eluting with 100% petrol 40-60 to 100% ethyl acetate. The second rnajor peak was shown to be the desired penta-POC product by NMR and LCMS (50 rng). Example 2 Br N0N Ht HN HN DMF H HR HN NHHN HN NH HN N _N 1-5 Exam a 2 t Synth esisof compound 5 Compound 2 (01 g, 0.270 rmnol) was dissolved in dry DMF (2 ml (and a solution: of the alkyl bromide (11 eg, 0.297 mmol, 81.07 mg in dry DMF ( mU was added over 5 rninus. The soluton vs stirrel at roon temperature for 16 hours. The DMF was removed under reduced pressure and white slids dissolved in an miniurm volurne of 20 water/methanol (1:1). Preparative HPLC (Phenomenex luna C1O2 150x21.2, acetonitrile/water 5% to 70% over 10 minutes) gave a major peak having tr of 8-8.5 minutes which was freeze dried giving an white solid (15 mg). NMIR and LCMS confirmed the structure. -20- C.NRPortbl\DCC\SXD\4914478_l.DOC-12/04/2013 Example 2(ii) Fluoride binding studies with ( 1 9 F)-fluoride Compound 5 (1 mg) in water (0.1 mL) acidified to pH 1 with IN HCI and an aqueous solution of potassium fluoride (0.1-1 eq) was added at RT. The solutions were analysed by reversed phase HPLC (l%TFA/water, 1%TFA MeCN gradient on Luna C5 150x4 6 mm, 5 detecting at 254 nm). Example 2(iii) Fluoride radiolabelling of compound 5 with ( 1 8 F)-fluoride IM HCI (4.5pL, 4.5pmol) was added to compound 5 (0.1 mg, 180 nmol) in 50:50 methanol / water (0.2 mL). This acidified solution was added directly to a glass vial 10 containing ( 1 8 F)fluoride (98 MBq) in target water (0.05 mL) and left at room temperature for 20 minutes. The reaction was analayzed by reverse phase HPLC (solvent A = 0.1% TFA in water; Solvent B = 0.1% TFA in MeCN, Luna C5 150x4.6 mm, detecting at 254 nm; Gradient: 0 to 3 minutes (2% B), 3-10 minutes (2 to 70% B), 10 to 13 minutes (70% B); 13 to 16 minutes (70 to 2% B), 16 to 21 minutes (2% B); flow rate 15 lmL / minute ( 1 8 F)-5 has a retention time of 10.1 minutes. ( 1 8 F)-5 was purified using the same HPLC method with a decay corrected isolated yield of 64%. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", 20 will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from 25 it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. - 21 -

Claims (3)

1. A method for radiofluorination comprising reaction of a compound of formula (1i) VECTOR LINKED CRYPTAND (Il) or a salt thereof with a source of EF]-fluoride, to give a compound of formula (1): VECTOR LINKER CRYPTAND -F (I) 10 ---- ------------ or a salt thereof, followed by the optional steps: (i) purification of the compound of formula (i); and/or (ii) formulation of the coprnound of for mula (1). is

2. A method according to claim 1 wherein the Vector is a peptide, protein, hormone, polysaccaride, oligonucleotide, antibody fragment, cell bacterium, virus, or small drug like molecule. 20 3. A method according to claim I or 2 wherein the Vector is Arg-Gly-Asp peptide or en analogue thereof. 4 A method according to claim 3 wherein the Vector comprises the fragment: ------- /- - - ----------------------------- S \511 - e t' N -n HN HN H SNH HN HNN

25--5 -22-- WO 20081155339 PCT/EP2008,1057659 S. A m-ethod c-cordchnor to claims 4 wherein the Ve-ctor is of formul,11a (A), 0 0 //Ho H N N N 000 0 H N wheei v\4 i5 eihrCNL H N H 0 T-.N H 0/ R/ wherein: 40 PRi ondl R2 are independently selected from -23 - WO 2008/155339 PCT/EP2008/057659 N -N ( N N N N N and H R3 7 4 anthd accring toclaim 6sweethe rypadisslctdfo HN 'NH HN H N N NH HN N N I-I I7 H /H N NNH 4 NNN 'H hs de A tneo according to Claim 6 wherein the Crphod is celectad caem A A r /N' NH-N H NN NN, N N I) L.' N N H NJ HNIN N 3. A componund of formula W0 or 00or a salt theroof as dined iw any o[ ain r 1 to 7. 9. A rodiocharmaoceuticol formulation comprising a compound of form-ula (1) or a salt thr'maf as dekec-r in any of Claims i.to 7 and a physiologicaily acceptabe car-rier or exc i pient. 15 10. A compound of formula () or a salt thereof as defined in any of Claims ato 7 for use in medicine, more particularly in a method of in vivo imaging, suitably PET. -24 -- WO 2008/155339 PCT/EP2008/057659 11. A method of generating an image of a human or animal body involving administerinc a radiopharmaceuticol as defined in Claim 9 to said body, and aenerantia an irnage of asters a part of said body to which said radiophormaceutical " has distributed using PET. 12. A compound of formula (ll: R' LINKER' CRY PAND (lI) 10 wherein R' is a reactie group suitably selected from amine, carboxylic acid, activated carboxylic ester, isocyanate, isothiocyanate, thiol, maleimide, and a-halocarbonvl and the Linker is a portion of the Linker as defined in any of Claims 1 to 7 and the Cryptona' is as defined in any of Claims 1 o 7. 15 13. A compound of formula (V) CRYPTAND -'F (V) or a salt thereof wherein the Cryptand as defined in any of Claims 1to 7, for use in medicine, for example as perfusion imaging agents. 20 14. A method of imaging which comprises administration to a subject of a detectable amount of a compound of formula ) or a salt thereof as defined in Claim 13. and imaging the subject using PET. 15. A compound of formula M): 25 Ril LINKER '---YfA-- ---- F (VI) wherein Ril is a reactive group suitably selected from amine, carboxylic acid, activated carboxylic ester, socyanate. sothiocyanate, thiol, maleimide, and a-halocarbonyl; and the Linker' is a portion of the Lilker as defined i any of Clam-is l to 7 and the Cryptand 30 is as defined in any of Claims 1 to 7. -25-