UNIVERSITY OF LJUBLJANA FACULTY OF PHARMACY JANJA ŠKRINJAR MASTER THESIS UNIFORM MASTER S STUDY PROGRAMME PHARMACY

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1 UIVERSITY F LJUBLJAA FACULTY F PARMACY JAJA ŠKRIJAR MASTER TESIS UIFRM MASTER S STUDY PRGRAMME PARMACY Ljubljana, 2015

2 UIVERSITY F LJUBLJAA FACULTY F PARMACY JAJA ŠKRIJAR DESIG AD SYTESIS F DIPEPTIDE L-[ 11 C]PE-L-PE- 2 AČRTVAJE I SITEZA DIPEPTIDA L-[ 11 C]PE-L-PE- 2 UIFRM MASTER S STUDY PRGRAMME PARMACY Ljubljana, 2015

3 Individual research work for the Master s Thesis was done at VU University Medical Center, Department of Radiology and uclear Medicine, Location Radionuclide Center, under supervision of assist. prof. Žiga Jakopin, PhD, co-supervision of prof. Albert D. Windhorst, PhD, and working co-supervision of Aleksandra Pekošak, MPharm. Measurements have been done at Vrije Universiteit Amsterdam (VU University Amsterdam) and Radionuclide center (VU University Medical Center). This individual research work was supported by the RADIMI Initial Training etwork (FP7-PEPLE-2012-IT). ACKWLEDGMETS First of all I would sincerely like to thank Aleksandra Pekošak, MPharm, for all the hours she invested in my project and all the support and knowledge she gave me. I would also like to thank assist. prof. Žiga Jakopin, PhD, for the support to do my thesis in the etherlands, to prof. Albert D. Windhorst, PhD, for the supervision over this project, to the whole Radionuclide center group for the great working environment and of course to my family for all the patience, love and support during my studies. STATEMET I declare that I have done the thesis independently under supervision of assist. prof. Žiga Jakopin, PhD, co-supervision of prof. A. D. Windhorst, PhD, and working co-supervision of Aleksandra Pekošak, MPharm. Amsterdam, 2015 Janja Škrinjar Chairman of committee: prof. Samo Kreft, PhD Member of committee: assist. prof. Pegi Ahlin Grabnar, PhD

4 CTETS ABSTRACT... V KEYWRDS... VI ABBREVIATIS... VI KLJUČE BESEDE... IX PVZETEK... IX 1. ITRDUCTI eurotransmitters Substance P Substance P europathic pain Positron emission tomography Radiopharmaceutical chemistry Carbon-11 chemistry Peptide coupling Asymmetric stereoselective phase-transfer synthesis WRK PLA REACTI SCEMES RGAIC CEMISTRY I

5 RADICEMISTRY MATERIALS AD METDS MATERIALS Reagents and solvents Laboratory equipment omenclature and molecule drawing Laboratory notebook Labeling programme METDS CRMATGRAPIC METDS Thin layer chromatography Column chromatography Mobile phases SPECTRSCPIC METDS uclear magnetic resonance Electrospray ionization-high resolution mass spectrometry RADICEMISTRY METDS Analytical isocratic high-performance liquid chromatography EXPERIMETAL PART RGAIC CEMISTRY II

6 SYTESIS F 2-((DIPEYLMETYLEE)AMI) ACETAMIDE (3) SYTESIS F (S)-2-((DIPEYLMETYLEE)AMI)-3- PEYLPRPAAMIDE (5) SYTESIS F (R)-2-((DIPEYLMETYLEE)AMI)-3- PEYLPRPAAMIDE (7) SYTESIS F 2-((DIPEYLMETYLEE)AMI)-3- PEYLPRPAAMIDE (9) [63] SYTESIS F (S)-TERT-BUTYL (2-((1-AMI-1-X-3- PEYLPRPA-2-YL)AMI)-2-XETYL)CARBAMATE (11) SYTESIS F (S)-2-((1-AMI-1-X-3-PEYLPRPA-2- YL)AMI)-2-XETAAMIIUM CLRIDE (12) SYTESIS F (S)-2-(2-((DIPEYLMETYLEE)AMI) ACETAMID)-3-PEYLPRPAAMIDE (13) SYTESIS F (S)--((S)-1-AMI-1-X-3-PEYLPRPA-2- YL)-2-((DIPEYLMETYLEE)AMI)-3-PEYLPRPAAMIDE (15) SYTESIS F TERT-BUTYL ((R)-1-(((S)-1-AMI-1-X-3- PEYLPRPA-2-YL)AMI)-1-X-3-PEYLPRPA-2- YL)CARBAMATE (17) SYTESIS F (R)-1-(((S)-1-AMI-1-X-3-PEYLPRPA-2- YL)AMI)-1-X-3-PEYLPRPA-2-AMIUM CLRIDE (18) SYTESIS F (R)--((S)-1-AMI-1-X-3-PEYLPRPA-2- YL)-2-((DIPEYLMETYLEE)AMI)-3-PEYLPRPAAMIDE (19) RADICEMISTRY SYTESIS F [ 11 C]BEZYL IDIDE [61] III

7 SYTESIS F RACEMIC -[ 11 C]Phe- 2 AD RACEMIC -[ 11 C]Phe- L-Phe SYTESIS F -L-[ 11 C]Phe- 2 AD -L-[ 11 C]Phe-L-Phe RESULTS AD DISCUSSI RGAIC CEMISTRY Phe L-Phe-L-Phe PLC AALYSIS RADICEMISTRY SYTESIS F Ph 2 C=-D,L-[ 11 C]Phe SYTESIS F Ph 2 C=-D,L-[ 11 C]Phe-L-Phe ASYMMETRIC SYTESIS F Ph 2 C=-L-[ 11 C]Phe ASYMMETRIC SYTESIS F Ph 2 C=-L-[ 11 C]Phe-L-Phe CCLUSIS REFERECES APPEDIX IV

8 ABSTRACT europathic pain is a disorder of peripheral nerves, causing pain and affecting large percentage of society, however no specific medicine indicated for its treatment has been developed yet. Biologically active fragment SP 1-7 as the major metabolite of neurotransmitter substance P (SP) has been discovered to induce antihyperalgesia in diabetic mice. Structure-activity relationship (SAR) study and truncation of this heptapeptide has resulted in dipeptide L-phenylalanine-L-phenylalanine amide (-L-Phe- L-Phe- 2 ) (K i =1.5 nm), as a small and high activity ligand for the SP 1-7 specific binding site. This lead compound can be used for development of new agents for treatment of neuropathic pain, due to its metabolic stability, high uptake, good permeability and affinity to SP 1-7 specific binding sites. Furthermore, labeled with a radionuclide it can be used with Positron Emission Tomography for the in vivo study of this peptide. The general aim of this research is to establish an asymmetric regioselective synthetic method for radiolabeling peptide -L-[ 11 C]Phe-L-Phe- 2 in a natural position, without changing the original structure. Therefore, an atom of the dipeptide needs to be substituted by a nuclide of the same element and carbon-11 seems to be best suited for this because of its most appropriate half-life. During our research we first focused on labeling unnatural amino acid phenylalanine amide (-Phe- 2 ) with carbon-11 to obtain optimal conditions for enantioselective synthesis with alkylation agent [ 11 C]benzyl iodide. Subsequently, obtained optimal conditions were transferred to label the desired dipeptide, -L-Phe-L- Phe- 2. In order to achieve asymmetric synthesis of the Schiff s base precursor and obtain the desired L,L-diastereomer, various phase-transfer catalysts have been studied. Both, -[ 11 C]Phe- 2 and -[ 11 C]Phe-L-Phe- 2 were labeled under mild conditions with high incorporation of the [ 11 C]benzyl group, however sufficient diastereomeric excess was not achieved. For further investigation we propose additional optimization of the radiolabeling, either with a different precursor or other phase-transfer catalysts, to accomplish sufficient diastereoselectivity and obtain the L,L-diastereomer. V

9 KEYWRDS Positron Emission Tomography (PET), Carbon-11, Radiolabeling, -L-[ 11 C]Phe-L-Phe- 2, [ 11 C]benzyl iodide, PET-tracer. ABBREVIATIS Arg: Arginine BnI: Benzyl iodide Boc: tert-butyloxycarbonyl BP: (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate DIPEA:,-Diisopropylethylamine DMF: Dimethylformamide DMS-d 6 : exadeuterodimethyl sulfoxide Cat. 1: -Allyl--(9-anthracenylmethyl)cinchonidinium bromide Cat. 2: (11bR)-( )-4,4-Dibutyl-4,5-dihydro-2,6-bis(3,4,5-trifluorophenyl)-3dinaphth[2,1-c:1,2 -e]azepinium bromide Cat. 3: (R,R)-3,4,5-Trifluorophenyl-AS bromide de: Diastereomeric excess ee: Enantiomeric excess eq: Equivalent ESI-RMS: Electrospray Ionization-igh Resolution Mass Spectrometry FDG: Fluorodeoxyglucose Gln: Glutamine VI

10 Gly: Glycine -L-Phe-L-Phe- 2 : L-Phenylalanine-L-phenylalanine amide -Phe- 2 : Phenylalanine amide e: exane PLC: igh-performance Liquid Chromatography Ki: Binding affinity Leu: Leucine blikovano: francoščina (Francija) Lys: Lysine Met: Methionine MP: Mobile phase MS: Mass Spectrometry K1R: eurokinin-1 receptor MR: uclear Magnetic Resonance PET: Positron Emission Tomography Phe: Phenylalanine PhMgBr: Phenylmagnesium bromide Pro: Proline PTC: Phase transfer catalyst Rf: Retention factor RT: Room temperature VII

11 SAR: Structure-activity relationship SP: Substance P SP 1-7 : Substance P 1-7 SPPS: Solid phase peptide synthesis T: Temperature TBAB: Tetrabutylammonium bromide TBAF: Tetrabutylammonium fluoride TBAS: Tetrabutylammonium hydrogen sulfate TF: Tetrahydrofuran TLC: Thin Layer Chromatography Tyr: Tyrosine δ: Chemical shift VIII

12 KLJUČE BESEDE Pozitronska emisijska tomografija (PET), ogljik-11, radiooznačevanje, -L-[ 11 C]Phe-L- Phe- 2, [ 11 C]benzil jodid, PET-radiofarmak. PVZETEK evropatska bolečina, ki je posledica okvare perifernega ali centralnega živčevja, dandanes prizadene velik odstotek populacije, a žal specifično zdravilo namenjeno zdravljenju te bolezni še ni bilo razvito. Dandanes se prizadetim bolečino lajša z zdravili, katerih indikacija so druge bolezni, npr. antiepileptiki, antidepresivi, kortikosteroidi, itd. Substanca P (SP), nevrotransmiter in nevromodulator v centralnem in perifernem živčnem sistemu, je znana po svojem sodelovanju pri vnetnih in nevropatskih bolečinah ter igra pomembno vlogo pri prenosu bolečinskih signalov od primarnih, aferentnih živčnih vlaken, v centralni živčni sistem in hrbtenjačo. SP 1-7, metabolit substance P, kaže v mnogih primerih ravno nasprotne učinke od izhodne molekule, in sicer blaži z SP povzročeni vnetni učinek, omili znake opioidne tolerance in odtegnitveni sindrom, v študijah na miškah obolelih za sladkorno boleznijo pa je pokazal sposobnost zmanjševanja preobčutljivosti na bolečino. SAR študije heptapeptida so pokazale, da je dipeptid L- fenilalanin-l-fenilalanin amide (-L-Phe-L-Phe- 2 ) potencialen ligand nevrotenzijskih receptorjev, z visoko afiniteto vezave (Ki 1.5 nm) na specifična vezavna mesta substance P 1-7, in lahko kot takšen velja za potencialno spojino vodnico za razvoj učinkovine na področju zdravljenja nevropatske bolečine. Vezavna mesta SP 1-7 in dipeptida so locirana v hrbtenjači miši in podgan ter v ventralnem tegmentalnem delu možganov podgan, kjer vezava posredno aktivira na nalokson občutljiv sigma receptor. Dipeptid, označen z radionuklidom, se nadalje lahko uporabi za preverjanje, ali se le-ta dejansko veže na želene predele možganov. Cilj raziskave je razvoj metode za enantioselektivno sintezo radioaktivno označenega dipeptida -L-[ 11 C]Phe-L-Phe- 2, brez spreminjanja originalne strukture. Za dosego letega moramo element dipeptida zamenjati z izotopom istega elementa. ajbolj primerna menjava v tem primeru je tako ogljik-11, katerega razpolovni čas znaša 20 min. aše raziskovalno delo je bilo sestavljeno iz treh medsebojno prepletenih področij; organske kemije, radiofarmacevtske kemije in PLC analize. Vsa tri področja smo najprej IX

13 združili na sami aminokislini fenilalanin amid (-Phe- 2 ), z namenom optimizacije pred nadaljnjim označevanjem želenega dipeptida. Sprva smo s klasično metodo sinteze peptidov v vodni fazi sintetizirali šest molekul, in sicer prekurzor (Ph 2 C=-Gly-L-Phe- 2 ) ter referenci (Ph 2 C=-L-Phe-L-Phe- 2, Ph 2 C=-D-Phe-L-Phe- 2 ) za dipeptid, ter po istem postopku v vodni fazi še prekurzor in referenci za nenaravno aminokislino. Sinteza molekul aminokisline ter vmesne stopnje dipeptida je potekla v enem koraku zaščite z benzofenon iminom ob prisotnosti ustreznega topila, medtem ko smo neželeni produkt in prekurzor dipeptida pridobili v tristopenjski sintezni poti. Prva stopnja, sklopitev aminokislin (coupling), je potekla preko aktivacije karboksilne skupine s pomočjo»coupling«reagenta (benzotriazol-1-iloksi)tris(dimetilamino)fosfonium heksafluorofosfat (BP) ob prisotnosti baze,-diizopropiletilamina (DIPEA). Sledila je odščita Boc skupine iz -terminalnega dela aminokisline, zadnji korak pa je bila že prej omenjena zaščita z benzofenon iminom. Reakcija zaščite je z največjim izkoristkom potekla v 2-propanolu, saj je zagotovil največjo topnost prekurzorja, nekoliko slabše v 1,2- dikloroetanu ob dodatku trietilamina oziroma v samem dikloroetanu, medtem ko v benzenu in diklorometanu prekurzor ni bil topen in posledično reakcija ni bila zmožna poteči. Vse sintetizirane komponente so bile analizirane z metodami TLC, MR, PLC in MS, s pomočjo analitične PLC pa je sledila še izdelava referenčnih kromatogramov prekurzorjev in referenc, tako za aminokislino kot tudi za dipeptid. amen referenčnih kromatogramov je bila sledljivost radiooznačevanja, predvsem preverjanje čistote, odstotka pretvorbe [ 11 C]benzil jodida in odstotek enantiomera oziroma diastereomera. Po uspešnem sintetiziranju referenc, prekurzorjev in pripravi PLC kromatogramov je sledilo radiooznačevanje z ogljikom-11. Prvi korak na področju radiokemije je bila sinteza [ 11 C]benzil jodida. Le-ta je bil uspešno sintetiziran iz [ 11 C]C 2 in Grignardovega reagenta, s čimer smo dobili [ 11 C]benzojsko kislino, ki smo jo ob dodatku LiAl 4 reducirali v [ 11 C]benzilni alkohol in ob dodatku 57 % I pretvorili v končni [ 11 C]benzil jodid. Produkt prve stopnje reakcije označevanja je bil pridobljen s 93 ± 2 % (n = 5) čistoto, v časovnem okviru 11 min, po postopku opisanem v literaturi [61], prikazanem na Shemi 3. adalje je [ 11 C]benzil jodid reagiral s prekurzorjem, Schiff-ovo bazo, ki smo ga predhodno deprotonirali z bazo z namenom aktivacije α-c-atoma na -terminalni strani in posledično selektivnim alkiliranjem omenjenega mesta. Z namenom optimizacije pogojev za enantioselektivno alkiliranje z [ 11 C]benzil jodidom je označevanje sprva potekalo na nenaravni aminokislini -Phe- 2, ti pogoji pa so bili nato preneseni na reakcije X

14 označevanja tarčnega dipeptida -L-Phe-L-Phe- 2. Tekom označevanja smo preizkušali različne pogoje; preverjali smo vpliv količine prekurzorja, potrebno množino in vrsto baze, preizkusili različni topili (toluen in diklorometan) kot topili za [ 11 C]benzil jodid, spreminjali temperaturo itd. Da bi selektivno alkilirali prekurzor Schiff-ovo bazo in tvorili želeni L,L-diastereomer, smo se poslužili dodatka različnih katalizatorjev faznega prehoda ter spremljali njihov doprinos k selektivnosti reakcije. Tako v primeru aminokisline kot tudi dipeptida je bila pretvorba [ 11 C]benzilne skupine uspešna, in sicer >90 % za - [ 11 C]Phe- 2 in >60 % za -[ 11 C]Phe-L-Phe- 2, kljub temu pa želen presežek L,Ldiastereomera ni bil dosežen. Razlog se verjetno skriva v dejstvu, da ima naš prekurzor amidirano C-terminalno stran, zato pretvorba v enolat, potreben za vsidranje katalizatorja, ni možna in stereoselektivnost v reakciji ni dosežena. Pri dipeptidu se je pojavil tudi neželeni stranski produkt, za katerega predvidevamo, da je posledica -alkiliranja. Zadnja stopnja pred končno tvorbo -L-[ 11 C]Phe-L-Phe- 2 je bila odščita benzofenon imina z acidolizo, ki je potekla kvantitativno. Za nestereoselektivno označevanje aminokisline smo sprva uporabili bazo tetrabutilamonijev fluorid (TBAF) (4-5 ekvivalentov), kjer smo tudi zasledili neželen stranski produkt, ki je verjetno posledica tvorbe kompleksa med TBA + in I -. [68] Pri optimalni temperaturi 45 C in ob uporabi diklorometana kot topila za [ 11 C]benzil jodid smo sintetizirali >70 % Ph 2 C=-D,L-[ 11 C]Phe- 2. ptimalne pogoje smo nato prenesli na reakcije z dipeptidom, kjer pa se je pojavil tudi neželeni produkt, predvidevamo, da je le-ta posledica -alkiliranja. Z uporabo 6-11 ekvivalentov baze TBAF smo dobili ugoden odstotek produkta, z ne previsokim deležem -alkilacije. Samo enantioselektivnost smo nadalje preverili s tremi različnimi katalizatorji prikazanimi na Sliki 1. Reakcijo smo ponovno sprva preizkusili na aminokislini. [ 11 C]benzil jodid smo raztopili v diklorometanu ali toluenu, in dodali presežek baze (optimalna količina je 205 ekvivalentov), kot je bilo ugotovljeno že izvedenih reakcijah za -L-[ 11 C]Phe. [6262] Tekom eksperimentalnega dela smo bazo TBAF nadomestili s Cs* 2, saj so se ob dodatku katalizatorjev faznega prenosa za najustreznejšo bazo izkazali hidroksidi, ob uporabi TBAF pa smo zasledili nezaželen stranski produkt. Temperaturo smo znižali na 0 C, saj nižja T pripomore k selektivnosti, oziroma večji pretvorbi v želeni L-enantiomer. Ph 2 C=-L-[ 11 C]Phe- 2 je bil pridobljen v visokem procentu ( 90 %), a kljub dodatku katalizatorja faznega prenosa presežka L-enantiomera nismo dosegli, temveč je bilo razmerje L-/D-enantiomer približno blikovano: nizozemščina (izozemska) Spremenjene kode polj blikovano: nizozemščina (izozemska) blikovano: nizozemščina (izozemska) XI

15 50%/50%, kakor v reakciji brez prisotnosti katalizatorja. ptimalne pogoje stereoselektivnega označevanja aminokisline smo prenesli na reakcije stereoselektivnega označevanja dipeptida s katalizatorji. Kot že v neselektivnem označevanju dipeptida, se tokrat ponovno pojavi stranski produkt. PLC je sicer pokazal uspešno vgradnjo [ 11 C]benzilne skupine v produkt Ph 2 C=-L-[ 11 C]Phe-L-Phe- 2, a zaradi pomanjkanja časa referenčnega kromatograma ločbe Ph 2 C=-L,L- in Ph 2 C=-D,L-Phe-L-Phe- 2 nismo uspeli pridobiti, zatorej spremljanje le-tega ni bilo možno. Tekom našega raziskovalnega dela smo uspešno z visokim procentom pretvorbe [ 11 C]benzil jodida sintetizirali -D,L-[ 11 C]Phe- 2 in -D,L-[ 11 C]Phe-L-Phe- 2. V nadaljnjih raziskovanjih na področju enantioselektivnosti s ciljem sinteze L,Ldiastereomera predlagamo dodatno optimizacijo označevanja, ki se nanaša tako na uporabo drugačnih, modificiranih prekurzorjev, npr. prekurzor s terc-butilnim estrom na C terminalni strani, kar bi ob dodatku baze omogočilo tvorbo potrebnega enolata, po končani enantioselektivni reakciji faznega prehoda, pa bi terc-butilni ester pretvorili v želen amid. Za prekurzor bi lahko izbrali ustrezen sekundarni, ali terciarni amid, kot tudi aplikacijo drugačnih katalizatorjev faznega prehoda, ki bi omogočili tvorbo ionskega para s prekurzorjem. b uspešno doseženi diastereoselektivnosti bi sledila izolacija diastereomera in njegova uporaba v avtoradiografiji. XII

16 1. ITRDUCTI 1.1. eurotransmitters eurotransmitters are small endogenous chemical messengers that transfer signals from one nerve cell, so called neuron, to another target neuron (Figure 1). So far, more than 100 neurotransmitters have been identified, however scientists still do not know the exact number of these messengers, which are essential for our life functioning. Most of the identified neurotransmitters are about the size of one amino acid, although they can also be as large as proteins or peptides. [ 1, 2 ] eurotransmitters are stored in synaptic or neurotransmitter vesicles in the sending neuron, from where the neurotransmitter molecules are released by an action potential into the synaptic cleft, a small gap between the sending and receiving neuron as shown in Figure 1. Duration of their stay in a synaptic cleft is highly important for synaptic transmission, therefore a short time passes before they are either taken back into the sending neuron by way of reuptake, degraded by enzymes or bind to the specific receptors in the membrane of the postsynaptic neuron. A short exposure (millisecond to microsecond) to the postsynaptic receptor of the neurotransmitter is already enough to induce a response on the receiving neuron either in an inhibitory or excitatory way. If the exposure lasted longer, the synapse would become refractory and a new neuronal signal would not occur. When the sum of excitatory signals caused by the depolarization is greater than that of inhibitory signals caused by hyperpolarization, the neuron will also create a new action potential, which will release neurotransmitters of its neuron terminal to another neuron. [ 3, 4, 5, 6 ] 1

17 Figure 1: Generic eurotransmitter System; Left: The connection between two neurons, Right: The synapse [ 7 ] Substance P Substance P (SP) is 11 amino acid residues containing neuropeptide transmitter, (-Arg- Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met- 2 ), with an amidated C-terminus, that belongs to the tachykinine family. SP acts as a neurotransmitter and neuromodulator in the central and peripheral nervous system and it is well-known for its involvement in inflammatory and neuropathic pain. [ 8, 9 ] SP not only alters the excitability of nociceptive neurons in the spinal horn, but also takes part in the regulation of important body functions, for instance mood disorders, nociception, stress, anxiety, respiratory rhythm, cell growth, diabetes, vasodilatation etc. Biosynthesis of SP occurs from polyprotein precursor preprotachykinin A, which is found in the central nervous system and the periphery. [ 8, 10, 11] SP is released from sensory nerves to spinal cord and brain, where it binds to the neurokinin-1 receptor (K1R), from tachykinin receptor sub-family, and often induces hyperalgesia. Together with K1R it forms acidified endosomes. Inside the endosome complex it breaks down into K1 and into several bioactive fragments, from which the most important one is -terminal metabolite substance P 1-7 (SP 1-7 ). Despite generally displaying lower affinity to other receptors, it also binds to neurokinin-2 (K2R) and neurokinin-3 (K3R). [12, 13] 2

18 SP plays an important role in transmission of pain signal from primary afferent nerve fibers into the central nerve system and spinal cord. Some researchers have attempted to develop K1R antagonists in order to treat pain, however research has resulted without significant success. Additional studies have provided valuable information that in mice, with disruption of SP function or lack expression of preprotachykinin A (PPT-A), response to painful stimuli was reduced, suggesting that SP indeed has an important role in pain perception and transmission. [ 8 ] Substance P 1-7 The -terminal biologically active fragment SP 1-7 (-Arg-Pro-Lys-Pro-Gln-Gln-Phe) is the most abundant metabolite of undecapeptide SP. Although this heptapeptide often displays similar biological effects as the parent compound, it also possesses opposite effects. Therefore, we can conclude that SP 1-7 acts as an endogenous modulator, as it antagonizes SP-induced actions. [ 9, 14] It has been shown that the heptapeptide has a specific binding site that differs from K1R, K2R and K3R. The mechanism of its action involves indirect activation of the naloxone-sensitive sigma receptor system. Since SP 1-7 is a non-active ligand for sigma-, tachykinin- or opioid receptors, the effects of SP 1-7 are generated through yet unidentified receptors in specific binding sites, which have been found in mouse and rat spinal cord and in the ventral tegmental area of the rat brain. [ 9, 15, 16, 17] SP 1-7 modulates some neural processes, for example learning and locomotor activity, it is involved in transmission of pain signal and modulates the function of immune response. In contrast to the parent compound, it also may reduce signs of opioid tolerance and withdrawal in animal models. Moreover, it attenuates the inflammatory effects exerted by SP. It was also observed that it induced antihyperalgesia in diabetic mice, which indicates a possible mode of regulation of neuropathic pain and consequently holds promise for drug development since no specific satisfactory treatment is yet available. [15, 17, 18, 19] Dipeptide -Phe-Phe- 2 Structure activity relationship (SAR) study of heptapeptide SP 1-7 revealed that first four - terminated amino acids are not necessary for binding to specific target, since affinity does not change significantly if those amino acids are substituted or removed. owever, binding affinity (Ki) increases following amidation of C-terminus resulting in a compound, binding with greater affinity than the native heptapeptide. This provides a valuable information that 3

19 the C-terminal part of SP 1-7, namely phenylalanine on the position 7, is pivotal for high binding affinity. Truncation of SP 1-7 (-Arg-Pro-Lys-Pro-Gln-Gln-Phe-) and also of endogenous µ-receptor agonist endomorphin-2 (-Tyr-Pro-Phe-Phe- 2 ), which also interacts with the SP 1-7 binding site with lower affinity, resulted in identification of dipeptide -Phe-Phe- 2, with the same binding affinity (Ki 1.5 nm) [19] as the endogenous heptapeptide SP 1-7. Therefore, the dipeptide was recognized as the lead compound in development of SP 1-7 mimetics and a promising molecule for the development of specific agents to treat neuropathic pain. [ 9, 20] SAR of some of the modified dipeptide analogues was already established, derived from the estimated Ki values of the synthesized analogues. Additionally, studies such as in vitro metabolism, in vitro permeability and uptake experiments were performed. In order to further improve in vitro pharmacokinetic properties of lead dipeptide -L-Phe-L-Phe- 2 (1a), different modifications of the peptide backbone and amino acids were introduced, as shown in Figure 2. Surprisingly, the lead compound 1a showed different Ki in two different studies, performed by the same group. Binding affinity obtained in the study published in 2013, was 8.4 ± 0.4 nm. [ 9 ] Firstly, methylation was done as the methyl group confers a higher metabolic stability, thus methylations of the dipeptide resulted in analogues 2a, 3a, 5a, 6a, with lower binding affinities, with the exception of analogue 4a, displaying the same binding affinity as the lead compound. Rigidization of the -terminal phenylalanine with a pyrrolidine analogue, also resulted in lower affinities, while the rigidization of the C-terminal phenylalanine resulted in 2 diastereomers 8a and 8b, with improved binding affinity and reduced binding affinity, respectively. owever, diastereomers were difficult to separate, therefore complete isolation of 8a from 8b was not possible. Both methylation and incorporation of pyrrolidine into the parent structure of dipeptide, increased metabolic stability and resulted in better intestinal permeability. Methylation of β-carbon on C-terminal side chain resulted in two-times lower binding affinity in case of diastereomer 9a and eight times lower in case of 9b. Elongation of the C-terminal phenylalanine side chain in compound 10a, and modification of the aromatic ring in C-terminus did not affect binding affinity. [ 9 ] 4

20 Compound Ki ± S. E. M ( n M) Compound Ki ± S. E. M ( n M) 2 1a ± a 2 7b 2 7a: ± b: ± a ± 3 2 8a 2 2 8b 2 8a: b: ± a ± a: ±0. 8 9b: ± a 5a 6a ± ± ± a 2 10a 2 11a 2 12a F 2 9b Figure 2: Binding Affinity of -Phe-Phe- 2 Analogues to the SP 1-7 Binding Site [ 9 ] 6. 2 ± ± ± 0. 2 The effect of the dipeptide, such as antihyperalgesia, antiallodynia and attenuation of nociception, occurs due to binding of dipeptide to the same specific binding target as SP 1-7. [21, 22] 1.2. europathic pain europathic pain is a complex, chronic pain state caused by damage or disease affecting nervous somatosensory system, from peripheral or central sources. ne or more nerve fibers can become damaged, dysfunctional, injured or with changed functions and can send 5

21 incorrect signals - pain messages- to the brain. europathic pain is often accompanied with hyperalgesia and allodynia, which appear as a result of neuropathic transformations in nerve system. It may have episodic or continuous components and is often described as stabbing, shooting, aching, tingling, electric burning sensation, like pin and needles [23, 24, 25, 26, 27] causing pain. It often looks like there is no special cause for neuropathic pain, but various conditions can affect nerves and consequently induce this kind of pain. Some of the causes are alcoholism, phantom pain following an amputation, cancer, pain following chemotherapy, trigeminal neuralgia, spine surgery, spinal cord injury, some strokes, toxins, radiation injury, multiple [24, 26, 27] sclerosis, diabetes, facial nerve problems, herpes zoster infection etc. Based on origin and mechanisms that cause pain, it is categorized into peripheral, central or mixed neuropathic pain. In case of peripheral neuropathic pain, damage of peripheral nerve causes development of sensitivity of nerve fiber, with spontaneous generation of action potentials. n the other hand, damage of the central nerve network first causes neurochemical and later anatomical changes in central nerve system, which become irreversible. Important cause of these plastic changes is deafferentation, the disconnection of normal afferent pathways on peripheral or central nerves. Common in chronic pain and as such in neuropathic pain is that it becomes autonomic, independent from trigger factors as a consequence of changes in nerves. Therefore, pain can also be spontaneous, especially in neuropathic pain, where normal sensory pathways from peripheral to central nerve system have been disconnected. As a result long-term changes occur in central and [26, 27, 28, 29] peripheral nerve system, which leads to reduced quality of life. europathic pain affects a large percentage of the population, around 8 % of European population, and is very difficult to treat as only % of people achieve some relief. There is no medicine developed for the purpose of neuropathic pain treatment yet, therefore medicines indicated for some other conditions are used to decrease pain intensity and improve quality of life in some patients. Pharmacological treatment includes use of certain anticonvulsants and antidepressants as favored, in some cases also antagonists of -methyl-d-aspartate (MDA) receptors, a + -channel blockers, corticosteroids and cannabinoids. Anticonvulsants are first-line drugs in treatment of diabetic neuropathy. pioid analgesic, also known as narcotics, are the most effective drugs to achieve pain relief, however are not recommended as first line treatments due to the risk of addiction 6

22 and are only used under medical supervision in some individuals. europathic pain does not respond to traditional painkillers and is not treatable with non-steroidal antiinflammatory drugs. Standard pain treatments often help only a minor fraction of people with neuropathic pain. Moreover, standard pain treatment can even be harmful for some patients and can lead to serious disability. Combined therapy represents an appropriate [24, 26, 27, 28] option for treatment to provide relief. Intensive research is in progress to improve efficiency of existing medications used for neuropathic pain treatment and to develop new drugs with specific indication for treatment of neuropathic pain. [28] 1.3. Positron emission tomography Positron emission tomography (PET) is an in vivo non-invasive molecular imaging technique, enabling to monitor fundamental biochemical and physiological processes in living organisms, using targeted radiopharmaceuticals. Clinical application in oncology, cardiology and neurology is of great value for early disease diagnosis and treatment monitoring. Moreover PET is applied in drug development, providing the information that is not available with other techniques. Detection of short-lived positron emitting radiopharmaceuticals enables three-dimensional image of biological processes in living organisms by monitoring the distribution and concentration of the decaying radioisotopes. PET is a sensitive, specific and selective imaging technique. [30, 31, 32] The PET radiopharmaceutical (or radiotracer) is inhaled or injected in the body, where it decays by positron emission (β + ). The positron particle is not detected directly, in fact it travels a short distance ( cm) in the body and loses its kinetic energy via collisions. After almost completely losing its kinetic energy, the positron collides with an electron and annihilates. As a result of the annihilation, two gamma photons are produced, as shown in Figure 3, under angle of 180, each with an energy of MeV. This type of radiation is called annihilation radiation and can be detected by circularly positioned scintillation detectors of a PET scanner. Consequently, from the collected data of many millions of individual annihilations, we can determine the time and location of the origin of detected photon/radiotracer, as a three-dimensional PET image. [31, 33, 34] 7

23 Figure 3: Principle of PET [35] Radiotracers used in PET are chemical compounds with one or more atoms replaced by a positron emitting radioisotope. Most common PET radionuclides are carbon-11, nitrogen- 13, oxygen-15 and fluorine-18. The first three mentioned nuclides are natural components of most important molecules in living organism, therefore labeling of biomolecules without interfering their function and structural modification is possible. Radiolabeling with radiometals (for instance 68 Ga, 99 Th or 89 Zr) is also an option, where nuclide has to be attached to the biomolecule via a chelate or/and linker, to form a strong complex. In these cases there is also a strong possibility that the radiolabeled molecule will change its original pharmacological characteristics. [31, 33, 36] Radiotracers are used to investigate metabolic processes in the body, to follow chemical and biological processes, and are very important in diagnosis of tumors and metastasis. Furthermore, they provide information on regional brain metabolism, blood flow and some specific neurochemical changes. The most widely used PET tracer is an analogue of glucose, [ 18 F]-2-fluoro-2-deoxyglucose ([ 18 F]FDG). It is taken up by high-glucose-using cells, for example brain, heart, kidney and tumor tissue, and is subsequently phosphorylated by hexokinase to [ 18 F]FDG-6-phosphate. Since this molecule lacks the hydroxyl group on position 2, further glycolysis is impossible and [ 18 F]FDG-6-phosphate is trapped in the cell and enables detection. Therefore, PET has an integral role in oncology, for the purpose of characterization and localization of many types of tumors. n the other hand, the drawback of [ 18 F]FDG is its unspecific uptake, in some cases making it impossible to distinguish tumor from inflamed tissue or from benign processes. Tumors 8

24 integrated in the high metabolic rate tissue with high background activity, such as brain, muscles and bladder, are also difficult to recognize. [30, 31, 33, 36] Many other PET radiotracers are used in the fields of neurology, one example is [ 18 F]fluorodopa, used in study of Parkinson s and Alzheimer disease and schizophrenia, providing information of neurotransmitter presynaptic distribution and metabolism. [37, 38] 1.4. Radiopharmaceutical chemistry Radiopharmaceutical chemistry is an important independent field in modern science, which plays tremendous role in nuclear medicine, especially as an integral part of diagnosis, therapy and drug development. For this purpose, radionuclides are created and radiotracers have overwhelmed the market, opening new possibilities for investigation of physiological processes in vivo. [39, 40] The production of most PET radionuclides requires a cyclotron, a particle accelerator for the production of nuclides that decay by β + emission as shown in Figure 4. A cyclotron is comprised of two D-shaped electrodes inside the vacuum chamber, between which high frequency alternating Figure 4: Scheme of cyclotron [41] voltage is applied. The perpendicular magnetic field enables particles to move in a circular way. When the velocity of accelerated particles is high enough, the beam is redirected onto the target system suitable for the production of required radioisotope, which is then transferred to the hot cell (Figure 5). To protect individuals from radioactive isotopes, further steps of synthesis are performed in hot cells. ot cells are closed chambers, protected with 6 cm of lead and a lead glass window, where radiolabeling is performed safely. The radiosynthesis is usually performed with computercontrolled robotic or automated systems. [42, 43, 44] 9

25 Figure 5: Research ot cells at Radionuclide center Amsterdam Contrary to traditional chemistry, radiochemistry deals with positron-emitting radionuclides with short half-lives, therefore total synthesis of radiopharmaceuticals should not take longer than three isotope half-lives ( rule of thumb ). For this reason, most PET facilities have cyclotrons, radiosynthesis laboratories and PET scanners under the same roof. A complicating factor of radiopharmaceutical chemistry is working with nanomolar, even picomolar amounts of radioisotopes produced in cyclotron. ormally, there is an excess of cold reagents, therefore kinetic of reactions are often pseudo-first order. The advantage of this type of kinetics is that even though cold reaction would take more hours or days, using PET radioisotopes the reaction is finished in few minutes. The goals in radiopharmaceutical chemistry are to make synthesis of radiotracers fast, highly efficient and on a small scale, to achieve products with high specific activity, defined as the amount of radioactivity per mole of labeled substance (GBq/μmol), which is an important parameter influencing the PET imaging outcome. igh specific activity means less unmodified drug, which consequently lowers the administered drug dose and decreases competitive binding of cold drug. [33, 34] Carbon-11 chemistry Carbon has 3 naturally occurring isotopes, 12 C and 13 C are stable, 14 C is a radioisotope. Carbon-11 is a positron emitting radioisotope and can be used for the labeling PET radiotracers. It is produced with a cyclotron by a nuclear reaction between protons and nitrogen-14 molecules, 14 (p,α) 11 C, in the gas phase. Its half-life is 20.3 minutes and it 10

26 decays to boron-11, which in % occurs due to positron emission, the rest of 0.21 % though due to electron capture. [45] In general, 11 C always binds covalently to the molecule we would like to label, often as [ 11 C]methyl group, attached to an amine, hydroxyl or carboxyl group. It is one of the most attractive and useful radioisotopes to work with, hot-for-cold substitution is not a problem, since the carbon-11 replaces a carbon-12 atom in a biologically active molecule without any significant effect on its biological properties. rganism is unable to distinguish between the original 12 C and the radiolabeled one as replacement, as both molecules will behave chemically and biologically the same. [45, 46, 47] The short half-life of carbon-11 makes the synthesis of 11 C-labeled tracers a special challenge. According to rule of thumb, with introduction of the radionuclide at the latest possible time point in reaction, the synthesis has to be shorter than 60 minutes. This can be achieved by using excess of reagents to achieve pseudo-first order kinetics, use of high precursor concentration in small volumes and use of sealed vessels for high reaction temperature. Consequently, hot chemistry synthesis takes less time (minutes) compared to traditional organic chemistry synthesis (hours). igh specific activity and high radiochemical purity at the end of the synthesis are required. The first parameter can be achieved by increasing the amount of radioactivity, higher amount of radioactive precursor, and preventing introduction of stable forms of carbon impurities, like avoiding C 2, which could still appear for example in the target gas, valves in the hot cells or as residue from solvents etc. The second parameter, namely high radiochemical purity, can be achieved by semi-preparative high-performance liquid chromatography (PLC) or solidphase extraction, to provide sterile, pyrogen-free radiopharmaceuticals, suitable for intravenous injections. [33] Besides the short half-life, another disadvantage is the fact that very small amounts of radioisotopes and radiolabeled intermediates are dealt with, typically on a picomolar to nanomolar scale. Such minute amounts of compounds are not common in traditional chemistry and even the smallest impurities in chemical for instance can disrupt the radiochemistry significantly. In addition, it is the underlying cause that specialized and miniaturized apparatus is needed for radiochemistry synthesis, in order to enable the labeling reactions on such a small scale. [32, 2 ] 11

27 Carbon-11 labeled reagents can sometimes be used directly as labeling agents in synthesis or can be converted into the molecule of interest. Conversion can be done on-line, to spare time and to achieve higher yield. Two major used carbon-11 labeled precursors used in almost all labeling syntheses are [ 11 C]C 2 and [ 11 C]C 4, both generated in situ in the target. Many carbon-11 radiopharmaceuticals are made from these two major building blocks. The most important secondary 11 C labeling agent for the alkylation of nucleophilic molecules is [ 11 C]methyl iodide, prepared from [ 11 C]C 2 by reduction with LiAl 4 and subsequent reaction with hydroiodic acid. ther important labeling agents are [ 11 C]phosgene synthesized from [ 11 C]C 4, [ 11 C]C, synthetized by reduction of [ 11 C]C 2 over zinc or molybdenum, and [ 11 C]benzyl iodide, which is successfully synthesized from [ 11 C]C 2 via Grignard reaction. [33, 45, 47] 1.5. Peptide coupling Peptides are synthesized by coupling the carboxylic group of one amino acid and amino group of another amino acid, in order to get a peptide bound. [48] Chemical synthesis enables the synthesis of natural proteins, which are difficult to obtain from bacteria, proteins constructed of D-amino acid, which are rarely present in nature, and also synthesis of modified and optimized amino acids. [49] Protein biosynthesis starts at the -terminus, which is contrary to chemical synthesis in which case it starts at the C-terminal side. [48] Chemical synthesis occurs, when activated carboxyl group of the incoming amino acid with protected -terminus is coupled with the amino group of another amino acid on the terminus of the growing peptide chain. Afterwards, deprotection of newly attached amino acid follows, thus revealing a new - terminal amine, to which a new amino acid can be coupled. This can be repeated as many times as necessary, in order to yield the desired peptide. [50] There are two possible ways of peptide synthesis, either solid-phase peptide synthesis (SPPS) or liquid-phase peptide synthesis. The latter used to be the classical method, but nowadays it is mostly replaced by SPPS, as a standard method. [48] Liquid phase peptide synthesis is more appropriate method for synthesis of short peptides, such as di-, tripeptides and C-terminally modified peptides [50], in which C-terminal amino acid is protected with different protecting group. Advantages of this method are that it is technically easier, raw materials are cheaper and synthesis is suitable for large-scale 12

28 production of peptides, for industrial purposes. [51] Downside of this approach is its long time and after each step the product has to be manually removed from the reaction solution. [48] n the other hand, in solid phase C-terminal amino acid is attached to a resin, with activated groups such as polystyrene or polyacrylamide, therefore resin works as a C- terminus protecting group. [48] SPPS is a rapid method, chemical properties of the synthesized protein can be controlled and there is no need of isolation intermediates. owever, disadvantage is its expensive raw material and the final product can contain up to 10 % of impurities. [48, 51] 1.6. Asymmetric stereoselective phase-transfer synthesis Phase-transfer catalysis is a firmly established synthetic organic chemistry technique that promotes reactions, bond formations in a heterogenous system, between two compounds located in different solvents that are immiscible. [52] Usually, the reactant in aqueous phase is insoluble in the organic phase, therefore phase-transfer catalyst (PTC) is added to transfer aqueous phase reactant into the organic phase, where reaction between both reagents occurs. [53] PTC enables both reactants, each originally dissolved in appropriate solvent, to move from one phase to another. [54] Addition of PTC can accelerate reactions, while this method is also known for its simple experimental procedures, reduction of excess reactants, using less expensive starting materials, higher yields and purities, milder and environmentally more safe reaction conditions and finally the possibility to use it in large scale production. [55, 56] Besides, chiral PTC is important and effective in reactions that demand high stereoselectivity. [57] There are several types of PTC, the most well-known are tetraalkylammonium PTCs, widely used in the industry because of their low price, or phosphonium salts. [58] For the ability to transfer reactants from aqueous solution, pairing cation (Q + ) needs to have a high lipophilicity, large ionic radius and has to be soluble in both phases. [56] The mechanism of PTC method is represented in Figure 6 which depicts an appropriate example reaction, namely the electrophilic alkylation of enolates, generated by the deprotonation of α-hydrogen of glycine derivatives, specifically the glycine Schiff s base. [56, 59] The added PTC (Q + X - ) dissolves in water and exchanges its anion with the anion of the reagent also dissolved in water phase. Afterwards, a newly formed ionic pair (Q + Y - ) is able to cross from water to organic phase, due to its high lipophilicity, explaining why the 13

29 catalyst is called phase transfer. In the organic phase the nucleophilic anion bound to PTC takes part in a nucleophilic substitution reaction with the reagent dissolved in the organic phase. Product (RX) is formed and the catalyst is transferred back to the aqueous phase, where the scheme is repeated. [58] Figure 6: Mechanism of phase-transfer catalysis [60] 14

30 2. WRK PLA The goal of this master s thesis is to label dipeptide -L-Phe-L-Phe- 2 with carbon-11, which displays a similar biological affinity as SP 1-7 neuropeptide and after radiolabeling it would represent a promising radiotracer to study with PET. [9, 19] To achieve this, the first step of radiolabeling will involve the synthesis of [ 11 C]benzyl iodide, according to the procedure established by Pekošak [61], as shown in Scheme 3. Subsequently, [ 11 C]benzyl iodide will react with the Schiff s base precursor under basic conditions, in order to activate the α-c-atom on the -terminus for α-alkylation (Scheme 4). Further, we will explore 3 different PTCs shown in Figure 7, in an attempt to find the most suitable one, which will enhance enantioselective synthesis to yield the L,L-diastereomer. Final step of the labeling will be the acidic deprotection of the benzophenone imine group, to yield -L- [ 11 C]Phe-L-Phe- 2 and the purification of the product on preparative PLC. In order to obtain a high radiopharmaceutical yield, the conversion of [ 11 C]benzyl group and diastereomeric excess (de) are most important and we will thoroughly examine different labeling conditions. Labeling will be performed with a highly useful radionuclide carbon-11, due to the fact that carbon is naturally present in all amino acids and peptides, therefore replacement of carbon-12 with carbon-11 during labeling will not cause any structural modification of the dipeptide. What is more, carbon-11 is poorly explored in peptide application, therefore this research will provide valuable contribution to the carbon-11 native peptides labeling. Prior to labeling we will synthesize Schiff s base dipeptide precursor (Ph 2 C=-Gly-L-Phe- 2 ) and cold references (Ph 2 C=-L-Phe-L-Phe- 2 and Ph 2 C=-D-Phe-L-Phe- 2 ), using standard organic liquid-phase peptide synthesis. To achieve successful and reliable synthesis of the desired dipeptide, we will first perform the radiolabeling of a model unnatural amino acid -Phe- 2 (Scheme 1). This labeling approach will later be implemented on the dipeptide. Therefore, also the glycine amide precursor and reference compounds will be synthesized using liquid-phase peptide synthesis. All required unnatural amino acids and dipeptides will be analyzed prior to labeling by TLC, MR, PLC and MS. In addition, reference chromatograms of precursors and references will be obtained on analytical PLC, for both amino acid amide and dipeptide. 15

31 After the successful synthesis of -L-[ 11 C]Phe-L-Phe- 2, it will be explored as a new PET radiotracer. Figure 7: PTC used in radiolabeling: -Allyl--(9-anthracenylmethyl)cinchonidinium bromide (Cat. 1), (11bR)-( )-4,4-Dibutyl-4,5-dihydro-2,6-bis(3,4,5-trifluorophenyl)-3dinaphth[2,1-c:1,2 -e]azepinium bromide (Cat. 2), (R,R)-3,4,5-Trifluorophenyl-AS bromide (Cat. 3) 16

32 2.1. REACTI SCEMES RGAIC CEMISTRY 2 R R 1 1,solvent 2 RT, overnight 2 Scheme 1: Model cold synthesis of precursor and reference compounds; (3) (2- ((diphenylmethylene)amino)acetamide): R 1 = -, (5) ((S)-2-((diphenylmethylene)amino)-3- phenylpropanamide): R 1 = -L-Phe, (7) ((R)-2-((diphenylmethylene)amino)-3- phenylpropanamide): R 1 = -D-Phe R 2 R DIPEA, BP 3 R DCM RT, overnight R 2 4M Cl/1,4-dioxane 4 h, RT R 2 R 3 2 RT, overnight,dce,tea + 3 R 2 R 3 2 Scheme 2: Model cold synthesis of dipeptide precursor and reference compounds; (13) ((S)-2-(2-((diphenylmethylene)amino)acetamido)-3-phenylpropanamide): R 2 = -, R 3 = -L- Phe, (15) ((S)--((S)-1-amino-1-oxo-3-phenylpropan-2-yl)-2-((diphenylmethylene)amino)- 3-phenylpropanamide): R 2 = -L-Phe, R 2 = -L-Phe, (19) ((R)--((S)-1-amino-1-oxo-3- phenylpropan-2-yl)-2-((diphenylmethylene)amino)-3-phenylpropanamide): R 2 = -D-Phe, R 3 = -L-Phe 17

33 RADICEMISTRY Synthesis of [ 11 C]benzyl iodide Prior to the synthesis of [ 11 C]phenylalanine amide and -[ 11 C]Phe-L-Phe- 2 we will have to synthesize [ 11 C]benzyl iodide ([ 11 C]BnI). The labeled benzyl iodide will be prepared using a one-pot procedure from [ 11 C]C 2 according to the procedure described by Pekošak. [61] [ 11 C]C 2 will be trapped in Grignard reagent (phenylmagnesium bromide) to obtain [ 11 C]benzoic acid, which will be reduced to [ 11 C]benzyl alcohol with LiAl 4 and iodinated with 57 % I to yield [ 11 C]BnI as depicted in Scheme 3. MgBr 11 C 11 C 2 11 C 2 I [ 11 C]C 2, TF LiAl 4, TF 35 C 130 C 57 % I 120 C [ 11 C]benzyl iodide Scheme 3: Synthesis of [ 11 C]benzyl iodide Synthesis of -[ 11 C]Phe- 2 and -[ 11 C]Phe-L-Phe- 2 Subsequently, [ 11 C]benzyl iodide will react with deprotonated Schiff's base precursor, Ph 2 C=-Gly- 2, after base treatment, and alkylation of [ 11 C]benzyl group will occur on α-carbon to yield racemic -[ 11 C]Phe- 2 or -L-[ 11 C]Phe- 2. If the precursor Ph 2 C=- Gly-L-Phe- 2 will be used, radiolabeling will yield in racemic -[ 11 C]Phe-L-Phe- 2 or -L-[ 11 C]Phe-L-Phe- 2 in presence of PTC. Final step will be the acidic deprotection of -terminus as summarized in Scheme 4. During our research following reaction conditions will be examined: - X µmol precursor Ph 2 C=-Gly- 2 or Ph 2 C=-Gly-L-Phe- 2 - Y eq base: Cs* 2, Cs (l), K (s), TBAF (s), TBAF (1 M in TF), TBAB (s), TBAS (s), Cs 2 C 3(s) - Solvent: DCM, toluene - Temperature: -10, 0, 25, 45 ºC - Phase transfer catalyst PTC: Cat. 1, Cat. 2, Cat. 3 18