UNIVERSITY OF LJUBLJANA FACULTY OF PHARMACY … · university of ljubljana . faculty of pharmacy . janja Škrinjar . design and synthesis of dipeptide l-[11c]phe-l-phe-nh 2. naČrtovanje

UNIVERSITY OF LJUBLJANA

FACULTY OF PHARMACY

JANJA ŠKRINJAR

MASTER THESIS

UNIFORM MASTER’S STUDY PROGRAMME PHARMACY

Ljubljana, 2015

UNIVERSITY OF LJUBLJANA

FACULTY OF PHARMACY

JANJA ŠKRINJAR

DESIGN AND SYNTHESIS OF DIPEPTIDE L-[11C]PHE-L-PHE-NH2

NAČRTOVANJE IN SINTEZA DIPEPTIDA L-[11C]PHE-L-PHE-NH2

UNIFORM MASTER’S STUDY PROGRAMME PHARMACY

Ljubljana, 2015

Individual research work for the Master’s Thesis was done at VU University Medical

Center, Department of Radiology and Nuclear 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 RADIOMI Initial Training Network

(FP7-PEOPLE-2012-ITN).

ACKNOWLEDGMENTS

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

Netherlands, 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.

STATEMENT

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

I

CONTENTS

ABSTRACT ......................................................................................................................... V

KEYWORDS ...................................................................................................................... VI

ABBREVIATIONS ............................................................................................................. VI

KLJUČNE BESEDE ........................................................................................................... IX

POVZETEK ........................................................................................................................ IX

1. INTRODUCTION ......................................................................................................... 1

1.1. Neurotransmitters .................................................................................................. 1

1.1.1. Substance P ...................................................................................................... 2

1.1.1.1. Substance P1-7 ........................................................................................... 3

1.2. Neuropathic pain.................................................................................................... 5

1.3. Positron emission tomography .............................................................................. 7

1.4. Radiopharmaceutical chemistry ............................................................................ 9

1.4.1. Carbon-11 chemistry ..................................................................................... 10

1.5. Peptide coupling .................................................................................................. 12

1.6. Asymmetric stereoselective phase-transfer synthesis ......................................... 13

2. WORK PLAN ............................................................................................................. 15

2.1. REACTION SCHEMES ..................................................................................... 17

2.1.1. ORGANIC CHEMISTRY ............................................................................. 17

II

2.1.2. RADIOCHEMISTRY ................................................................................... 18

3. MATERIALS AND METHODS ................................................................................ 20

3.1. MATERIALS ...................................................................................................... 20

Reagents and solvents .............................................................................................. 20

Laboratory equipment.............................................................................................. 21

Nomenclature and molecule drawing ...................................................................... 21

Laboratory notebook................................................................................................ 21

Labeling programme................................................................................................ 21

3.2. METHODS .......................................................................................................... 22

3.2.1. CHROMATOGRAPIC METHODS ............................................................. 22

Thin layer chromatography ..................................................................................... 22

Column chromatography ......................................................................................... 22

Mobile phases .......................................................................................................... 22

3.2.2. SPECTROSCOPIC METHODS ................................................................... 23

Nuclear magnetic resonance .................................................................................... 23

Electrospray ionization-high resolution mass spectrometry .................................... 24

3.2.3. RADIOCHEMISTRY METODS .................................................................. 24

Analytical isocratic high-performance liquid chromatography ............................... 24

4. EXPERIMENTAL PART ........................................................................................... 25

4.1. ORGANIC CHEMISTRY ................................................................................... 25

III

4.1.1. SYNTHESIS OF 2-((DIPHENYLMETHYLENE)AMINO) ACETAMIDE

(3) 25

4.1.2. SYNTHESIS OF (S)-2-((DIPHENYLMETHYLENE)AMINO)-3-

PHENYLPROPANAMIDE (5) ................................................................................... 26

4.1.3. SYNTHESIS OF (R)-2-((DIPHENYLMETHYLENE)AMINO)-3-

PHENYLPROPANAMIDE (7) ................................................................................... 27

4.1.4. SYNTHESIS OF 2-((DIPHENYLMETHYLENE)AMINO)-3-

PHENYLPROPANAMIDE (9) [63] ............................................................................. 28

4.1.5. SYNTHESIS OF (S)-TERT-BUTYL (2-((1-AMINO-1-OXO-3-

PHENYLPROPAN-2-YL)AMINO)-2-OXOETHYL)CARBAMATE (11) ............... 29

4.1.6. SYNTHESIS OF (S)-2-((1-AMINO-1-OXO-3-PHENYLPROPAN-2-

YL)AMINO)-2-OXOETHANAMINIUM CHLORIDE (12) ..................................... 30

4.1.7. SYNTHESIS OF (S)-2-(2-((DIPHENYLMETHYLENE)AMINO)

ACETAMIDO)-3-PHENYLPROPANAMIDE (13) ................................................... 31

4.1.8. SYNTHESIS OF (S)-N-((S)-1-AMINO-1-OXO-3-PHENYLPROPAN-2-

YL)-2-((DIPHENYLMETHYLENE)AMINO)-3-PHENYLPROPANAMIDE (15) . 33

4.1.9. SYNTHESIS OF TERT-BUTYL ((R)-1-(((S)-1-AMINO-1-OXO-3-

PHENYLPROPAN-2-YL)AMINO)-1-OXO-3-PHENYLPROPAN-2-

YL)CARBAMATE (17) .............................................................................................. 34

4.1.10. SYNTHESIS OF (R)-1-(((S)-1-AMINO-1-OXO-3-PHENYLPROPAN-2-

YL)AMINO)-1-OXO-3-PHENYLPROPAN-2-AMONIUM CHLORIDE (18) ........ 35

4.1.11. SYNTHESIS OF (R)-N-((S)-1-AMINO-1-OXO-3-PHENYLPROPAN-2-

YL)-2-((DIPHENYLMETHYLENE)AMINO)-3-PHENYLPROPANAMIDE (19) . 36

4.2. RADIOCHEMISTRY ......................................................................................... 38

4.2.1. SYNTHESIS OF [11C]BENZYL IODIDE [61] .............................................. 38

IV

4.2.2. SYNTHESIS OF RACEMIC H-[11C]Phe-NH2 AND RACEMIC H-[11C]Phe-

L-Phe-NH2 ................................................................................................................... 38

4.2.3. SYNTHESIS OF H-L-[11C]Phe-NH2 AND H-L-[11C]Phe-L-Phe-NH2 ........ 38

5. RESULTS AND DISCUSSION .................................................................................. 40

5.1. ORGANIC CHEMISTRY ................................................................................... 40

5.1.1. H-Phe-NH2 .................................................................................................... 40

5.1.2. H-L-Phe-L-Phe-NH2 ...................................................................................... 41

5.2. HPLC ANALYSIS .............................................................................................. 44

5.3. RADIOCHEMISTRY ......................................................................................... 44

5.3.1. SYNTHESIS OF Ph2C=N-D,L-[11C]Phe-NH2 .............................................. 44

5.3.2. SYNTHESIS OF Ph2C=N-D,L-[11C]Phe-L-Phe-NH2 ................................... 47

5.3.3. ASYMMETRIC SYNTHESIS OF Ph2C=N-L-[11C]Phe-NH2 ...................... 48

5.3.4. ASYMMETRIC SYNTHESIS OF Ph2C=N-L-[11C]Phe-L-Phe-NH2 ........... 51

6. CONCLUSIONS ......................................................................................................... 53

7. REFERENCES ............................................................................................................ 55

APPENDIX ......................................................................................................................... 61

V

ABSTRACT

Neuropathic 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 SP1-7 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 (H-L-Phe-

L-Phe-NH2) (Ki=1.5 nM), as a small and high activity ligand for the SP1-7 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 SP1-7 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 H-L-[11C]Phe-L-Phe-NH2 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 (H-Phe-NH2) with carbon-11 to obtain optimal conditions

for enantioselective synthesis with alkylation agent [11C]benzyl iodide. Subsequently,

obtained optimal conditions were transferred to label the desired dipeptide, H-L-Phe-L-

Phe-NH2. 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, H-[11C]Phe-NH2 and H-[11C]Phe-L-Phe-NH2 were labeled under mild conditions

with high incorporation of the [11C]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.

VI

KEYWORDS Positron Emission Tomography (PET), Carbon-11, Radiolabeling, H-L-[11C]Phe-L-Phe-

NH2, [11C]benzyl iodide, PET-tracer.

ABBREVIATIONS

Arg: Arginine

BnI: Benzyl iodide

Boc: tert-Butyloxycarbonyl

BOP: (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

DIPEA: N,N-Diisopropylethylamine

DMF: Dimethylformamide

DMSO-d6: Hexadeuterodimethyl sulfoxide

Cat. 1: O-Allyl-N-(9-anthracenylmethyl)cinchonidinium bromide

Cat. 2: (11bR)-(–)-4,4-Dibutyl-4,5-dihydro-2,6-bis(3,4,5-trifluorophenyl)-3H-

dinaphth[2,1-c:1′,2′-e]azepinium bromide

Cat. 3: (R,R)-3,4,5-Trifluorophenyl-NAS bromide

de: Diastereomeric excess

ee: Enantiomeric excess

eq: Equivalent

ESI-HRMS: Electrospray Ionization-High Resolution Mass Spectrometry

FDG: Fluorodeoxyglucose

Gln: Glutamine

VII

Gly: Glycine

H-L-Phe-L-Phe-NH2: L-Phenylalanine-L-phenylalanine amide

H-Phe-NH2: Phenylalanine amide

He: Hexane

HPLC: High-Performance Liquid Chromatography

Ki: Binding affinity

Leu: Leucine

Lys: Lysine

Met: Methionine

MP: Mobile phase

MS: Mass Spectrometry

NK1R: Neurokinin-1 receptor

NMR: Nuclear Magnetic Resonance

PET: Positron Emission Tomography

Phe: Phenylalanine

PhMgBr: Phenylmagnesium bromide

Pro: Proline

PTC: Phase transfer catalyst

Rf: Retention factor

RT: Room temperature

Oblikovano: francoščina (Francija)

VIII

SAR: Structure-activity relationship

SP: Substance P

SP 1-7: Substance P1-7

SPPS: Solid phase peptide synthesis

T: Temperature

TBAB: Tetrabutylammonium bromide

TBAF: Tetrabutylammonium fluoride

TBAHS: Tetrabutylammonium hydrogen sulfate

THF: Tetrahydrofuran

TLC: Thin Layer Chromatography

Tyr: Tyrosine

δ: Chemical shift

IX

KLJUČNE BESEDE

Pozitronska emisijska tomografija (PET), ogljik-11, radiooznačevanje, H-L-[11C]Phe-L-

Phe-NH2, [11C]benzil jodid, PET-radiofarmak.

POVZETEK

Nevropatska 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. SP1-7, 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 (H-L-Phe-L-Phe-NH2) potencialen ligand nevrotenzijskih

receptorjev, z visoko afiniteto vezave (Ki 1.5 nM) na specifična vezavna mesta substance

P1-7, in lahko kot takšen velja za potencialno spojino vodnico za razvoj učinkovine na

področju zdravljenja nevropatske bolečine. Vezavna mesta SP1-7 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 H-L-[11C]Phe-L-Phe-NH2, brez spreminjanja originalne strukture. Za dosego le-

tega moramo element dipeptida zamenjati z izotopom istega elementa. Najbolj primerna

menjava v tem primeru je tako ogljik-11, katerega razpolovni čas znaša 20 min.

Naše raziskovalno delo je bilo sestavljeno iz treh medsebojno prepletenih področij;

organske kemije, radiofarmacevtske kemije in HPLC analize. Vsa tri področja smo najprej

X

združili na sami aminokislini fenilalanin amid (H-Phe-NH2), z namenom optimizacije pred

nadaljnjim označevanjem želenega dipeptida. Sprva smo s klasično metodo sinteze

peptidov v vodni fazi sintetizirali šest molekul, in sicer prekurzor (Ph2C=N-Gly-L-Phe-

NH2) ter referenci (Ph2C=N-L-Phe-L-Phe-NH2, Ph2C=N-D-Phe-L-Phe-NH2) 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 (BOP) ob prisotnosti baze N,N-diizopropiletilamina (DIPEA). Sledila je

odščita Boc skupine iz N-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, NMR, HPLC in MS, s

pomočjo analitične HPLC pa je sledila še izdelava referenčnih kromatogramov

prekurzorjev in referenc, tako za aminokislino kot tudi za dipeptid. Namen referenčnih

kromatogramov je bila sledljivost radiooznačevanja, predvsem preverjanje čistote, odstotka

pretvorbe [11C]benzil jodida in odstotek enantiomera oziroma diastereomera. Po uspešnem

sintetiziranju referenc, prekurzorjev in pripravi HPLC kromatogramov je sledilo

radiooznačevanje z ogljikom-11. Prvi korak na področju radiokemije je bila sinteza

[11C]benzil jodida. Le-ta je bil uspešno sintetiziran iz [11C]CO2 in Grignardovega reagenta,

s čimer smo dobili [11C]benzojsko kislino, ki smo jo ob dodatku LiAlH4 reducirali v

[11C]benzilni alkohol in ob dodatku 57 % HI pretvorili v končni [11C]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. Nadalje je

[11C]benzil jodid reagiral s prekurzorjem, Schiff-ovo bazo, ki smo ga predhodno

deprotonirali z bazo z namenom aktivacije α-C-atoma na N-terminalni strani in posledično

selektivnim alkiliranjem omenjenega mesta. Z namenom optimizacije pogojev za

enantioselektivno alkiliranje z [11C]benzil jodidom je označevanje sprva potekalo na

nenaravni aminokislini H-Phe-NH2, ti pogoji pa so bili nato preneseni na reakcije

XI

označevanja tarčnega dipeptida H-L-Phe-L-Phe-NH2. 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 [11C]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 [11C]benzilne skupine uspešna, in sicer >90 % za H-

[11C]Phe-NH2 in >60 % za H-[11C]Phe-L-Phe-NH2, kljub temu pa želen presežek L,L-

diastereomera 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 N-alkiliranja. Zadnja

stopnja pred končno tvorbo H-L-[11C]Phe-L-Phe-NH2 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 [11C]benzil jodid

smo sintetizirali >70 % Ph2C=N-D,L-[11C]Phe-NH2. Optimalne pogoje smo nato prenesli

na reakcije z dipeptidom, kjer pa se je pojavil tudi neželeni produkt, predvidevamo, da je

le-ta posledica N-alkiliranja. Z uporabo 6 - 11 ekvivalentov baze TBAF smo dobili ugoden

odstotek produkta, z ne previsokim deležem N-alkilacije. Samo enantioselektivnost smo

nadalje preverili s tremi različnimi katalizatorji prikazanimi na Sliki 1. Reakcijo smo

ponovno sprva preizkusili na aminokislini. [11C]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 H-L-[11C]Phe. [6262] Tekom eksperimentalnega dela

smo bazo TBAF nadomestili s CsOH*H2O, 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. Ph2C=N-L-[11C]Phe-NH2 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

Oblikovano: nizozemščina(Nizozemska)

Spremenjene kode polj



XII

50%/50%, kakor v reakciji brez prisotnosti katalizatorja. Optimalne 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. HPLC je sicer pokazal uspešno vgradnjo

[11C]benzilne skupine v produkt Ph2C=N-L-[11C]Phe-L-Phe-NH2, a zaradi pomanjkanja

časa referenčnega kromatograma ločbe Ph2C=N-L,L- in Ph2C=N-D,L-Phe-L-Phe-NH2

nismo uspeli pridobiti, zatorej spremljanje le-tega ni bilo možno.

Tekom našega raziskovalnega dela smo uspešno z visokim procentom pretvorbe

[11C]benzil jodida sintetizirali H-D,L-[11C]Phe-NH2 in H-D,L-[11C]Phe-L-Phe-NH2. V

nadaljnjih raziskovanjih na področju enantioselektivnosti s ciljem sinteze L,L-

diastereomera 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. Ob uspešno doseženi diastereoselektivnosti bi sledila izolacija

diastereomera in njegova uporaba v avtoradiografiji.

1

1. INTRODUCTION

1.1. Neurotransmitters

Neurotransmitters 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]

Neurotransmitters 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]

2

Figure 1: Generic Neurotransmitter System; Left: The connection between two neurons,

Right: The synapse [7]

1.1.1. Substance P

Substance P (SP) is 11 amino acid residues containing neuropeptide transmitter, (H-Arg-

Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), 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 (NK1R), from

tachykinin receptor sub-family, and often induces hyperalgesia. Together with NK1R it

forms acidified endosomes. Inside the endosome complex it breaks down into NK1 and

into several bioactive fragments, from which the most important one is N-terminal

metabolite substance P1-7 (SP1-7). Despite generally displaying lower affinity to other

receptors, it also binds to neurokinin-2 (NK2R) and neurokinin-3 (NK3R). [12, 13]

3

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

NK1R 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]

1.1.1.1. Substance P1-7

The N-terminal biologically active fragment SP1-7 (H-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 SP1-7 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 NK1R, NK2R and NK3R. The mechanism of its

action involves indirect activation of the naloxone-sensitive sigma receptor system. Since

SP1-7 is a non-active ligand for sigma-, tachykinin- or opioid receptors, the effects of SP1-7

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] SP1-7 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]

1.1.1.2. Dipeptide H-Phe-Phe-NH2

Structure–activity relationship (SAR) study of heptapeptide SP1-7 revealed that first four N-

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. However, 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

4

the C-terminal part of SP1-7, namely phenylalanine on the position 7, is pivotal for high

binding affinity. Truncation of SP1-7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) and also of

endogenous µ-receptor agonist endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2), which also

interacts with the SP1-7 binding site with lower affinity, resulted in identification of

dipeptide H-Phe-Phe-NH2, with the same binding affinity (Ki 1.5 nM) [19] as the

endogenous heptapeptide SP1-7. Therefore, the dipeptide was recognized as the lead

compound in development of SP1-7 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 H-L-Phe-L-Phe-NH2

(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 N-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. However,

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]

5

H2NHN

ONH2

O

NH

HN

ONH2

O

H2NHN

ONH2

O

H2N N

ONH2

O

H2NHN

ONH2

O

H2NHN

ONH

O

HN

ONH2

O

NH

HN

ONH2

O

NH

H2NO

N

O NH2

H2NO

N

O NH2

H2N O

HN

O NH2

H2N O

HN

O NH2

H2N O

HN

O NH2

H2N O

HN

O NH2

H2N O

HN

O NH2

F

1a

2a

3a

4a

5a

6a

7a 7b

8a 8b

9a 9b

10a

11a

12a

8.4 ±

0.4

189 ±

3

70.4 ±

3.0

9.4 ±0.1

26.0 ±

1.2

136 ±

2

7a: 33.9 ±

2.57b: 50.5

± 1.5

8a: 2.28b: 93.1

± 0.2

9a: 17.5 ±0.8

9b: 68.0 ±

1.2

6.2 ±0.2

11.5 ±

0.1

3.3 ±

0.2

Ki ±

S.E.M (nM)

Ki ±

S.E.M (nM)

Compound Compound

Figure 2: Binding Affinity of H-Phe-Phe-NH2 Analogues to the SP1-7 Binding Site [9]

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 SP1-7. [21, 22]

1.2. Neuropathic pain Neuropathic pain is a complex, chronic pain state caused by damage or disease affecting

nervous somatosensory system, from peripheral or central sources. One or more nerve

fibers can become damaged, dysfunctional, injured or with changed functions and can send

6

incorrect signals - pain messages- to the brain. Neuropathic 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

causing pain. [23, 24, 25, 26, 27]

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

sclerosis, diabetes, facial nerve problems, herpes zoster infection etc. [24, 26, 27]

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. On 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

peripheral nerve system, which leads to reduced quality of life. [26, 27, 28, 29]

Neuropathic pain affects a large percentage of the population, around 8 % of European

population, and is very difficult to treat as only 40 – 60 % 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

N-methyl-D-aspartate (NMDA) receptors, Na+-channel blockers, corticosteroids and

cannabinoids. Anticonvulsants are first-line drugs in treatment of diabetic neuropathy.

Opioid 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

7

and are only used under medical supervision in some individuals. Neuropathic pain does

not respond to traditional painkillers and is not treatable with non-steroidal anti-

inflammatory 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

option for treatment to provide relief. [24, 26, 27, 28]

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 (0.5 - 2.0 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 180̊, each with an energy of 0.511 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]

8

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 68Ga, 99Th or 89Zr) 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, [18F]-2-fluoro-2-deoxyglucose ([18F]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 [18F]FDG-6-phosphate. Since this molecule lacks the

hydroxyl group on position 2, further glycolysis is impossible and [18F]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. On

the other hand, the drawback of [18F]FDG is its unspecific uptake, in some cases making it

impossible to distinguish tumor from inflamed tissue or from benign processes. Tumors

9

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 [18F]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

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. Hot 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 computer-

controlled robotic or automated systems. [42, 43, 44]

Figure 4: Scheme of cyclotron [41]

10

Figure 5: Research Hot 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. Normally, 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. High specific activity means less

“unmodified drug’, which consequently lowers the administered drug dose and decreases

competitive binding of “cold drug”. [33, 34]

1.4.1. Carbon-11 chemistry

Carbon has 3 naturally occurring isotopes, 12C and 13C are stable, 14C 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, 14N(p,α)11C, in the gas phase. Its half-life is 20.3 minutes and it

11

decays to boron-11, which in 99.79 % occurs due to positron emission, the rest of 0.21 %

though due to electron capture. [45]

In general, 11C always binds covalently to the molecule we would like to label, often as

[11C]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. Organism is unable to

distinguish between the original 12C 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 11C-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). High 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

CO2, 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 (HPLC) or solid-

phase 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]

12

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 [11C]CO2 and [11C]CH4, both generated in situ in the

target. Many carbon-11 radiopharmaceuticals are made from these two major building

blocks. The most important secondary 11C labeling agent for the alkylation of nucleophilic

molecules is [11C]methyl iodide, prepared from [11C]CO2 by reduction with LiAlH4 and

subsequent reaction with hydroiodic acid. Other important labeling agents are

[11C]phosgene synthesized from [11C]CH4, [11C]CO, synthetized by reduction of [11C]CO2

over zinc or molybdenum, and [11C]benzyl iodide, which is successfully synthesized from

[11C]CO2 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 N-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 N-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 N-

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

13

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] On 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.

However, 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

14

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]

15

2. WORK PLAN

The goal of this master’s thesis is to label dipeptide H-L-Phe-L-Phe-NH2 with carbon-11,

which displays a similar biological affinity as SP1-7 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 [11C]benzyl iodide, according to the

procedure established by Pekošak [61], as shown in Scheme 3. Subsequently, [11C]benzyl

iodide will react with the Schiff’s base precursor under basic conditions, in order to

activate the α-C-atom on the N-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 H-L-

[11C]Phe-L-Phe-NH2 and the purification of the product on preparative HPLC. In order to

obtain a high radiopharmaceutical yield, the conversion of [11C]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 (Ph2C=N-Gly-L-Phe-

NH2) and cold references (Ph2C=N-L-Phe-L-Phe-NH2 and Ph2C=N-D-Phe-L-Phe-NH2),

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 H-Phe-NH2 (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, NMR,

HPLC and MS. In addition, reference chromatograms of precursors and references will be

obtained on analytical HPLC, for both amino acid amide and dipeptide.

16

After the successful synthesis of H-L-[11C]Phe-L-Phe-NH2, it will be explored as a new

PET radiotracer.

Figure 7: PTC used in radiolabeling: O-Allyl-N-(9-anthracenylmethyl)cinchonidinium

bromide (Cat. 1), (11bR)-(–)-4,4-Dibutyl-4,5-dihydro-2,6-bis(3,4,5-trifluorophenyl)-3H-

dinaphth[2,1-c:1′,2′-e]azepinium bromide (Cat. 2), (R,R)-3,4,5-Trifluorophenyl-NAS

bromide (Cat. 3)

17

2.1. REACTION SCHEMES

2.1.1. ORGANIC CHEMISTRY

H2NNH2

O

NH

N

O

NH2

RT, overnight

,solventR1

R1

Scheme 1: Model “cold” synthesis of precursor and reference compounds; (3) (2-

((diphenylmethylene)amino)acetamide): R1= -H, (5) ((S)-2-((diphenylmethylene)amino)-3-

phenylpropanamide): R1= -L-Phe, (7) ((R)-2-((diphenylmethylene)amino)-3-

phenylpropanamide): R1= -D-Phe

H2NNH2

OO N

H

OOH

O

DIPEA, BOPDCM

+ NH

NH2

O

HN

OO

O

4M HCl/1,4-dioxane

NH

NH2

O

+H3NO

NH

NH

NH2

O

NO

RT, overnight

4 h, RT

RT, overnight

,DCE,TEA

R3 R3R2

R2

R2

R3R3

R2

Scheme 2: Model “cold” synthesis of dipeptide precursor and reference compounds; (13)

((S)-2-(2-((diphenylmethylene)amino)acetamido)-3-phenylpropanamide): R2= -H, R3= -L-

Phe, (15) ((S)-N-((S)-1-amino-1-oxo-3-phenylpropan-2-yl)-2-((diphenylmethylene)amino)-

3-phenylpropanamide): R2= -L-Phe, R2= -L-Phe, (19) ((R)-N-((S)-1-amino-1-oxo-3-

phenylpropan-2-yl)-2-((diphenylmethylene)amino)-3-phenylpropanamide): R2= -D-Phe,

R3= -L-Phe

18

2.1.2. RADIOCHEMISTRY

Synthesis of [11C]benzyl iodide

Prior to the synthesis of [11C]phenylalanine amide and H-[11C]Phe-L-Phe-NH2 we will

have to synthesize [11C]benzyl iodide ([11C]BnI). The labeled benzyl iodide will be

prepared using a one-pot procedure from [11C]CO2 according to the procedure described by

Pekošak. [61] [11C]CO2 will be trapped in Grignard reagent (phenylmagnesium bromide) to

obtain [11C]benzoic acid, which will be reduced to [11C]benzyl alcohol with LiAlH4 and

iodinated with 57 % HI to yield [11C]BnI as depicted in Scheme 3.

MgBr

[11C]CO2, THF LiAlH4,

THF

35 °C 130

°C

57 %

HI

120 °C

11COOH 11CH2OH 11CH2I

[11C]benzyl iodide

Scheme 3: Synthesis of [11C]benzyl iodide

Synthesis of H-[11C]Phe-NH2 and H-[11C]Phe-L-Phe-NH2

Subsequently, [11C]benzyl iodide will react with deprotonated Schiff's base precursor,

Ph2C=N-Gly-NH2, after base treatment, and alkylation of [11C]benzyl group will occur on

α-carbon to yield racemic H-[11C]Phe-NH2 or H-L-[11C]Phe-NH2. If the precursor Ph2C=N-

Gly-L-Phe-NH2 will be used, radiolabeling will yield in racemic H-[11C]Phe-L-Phe-NH2 or

H-L-[11C]Phe-L-Phe-NH2 in presence of PTC. Final step will be the acidic deprotection of

N-terminus as summarized in Scheme 4.

During our research following reaction conditions will be examined:

- X µmol precursor Ph2C=N-Gly-NH2 or Ph2C=N-Gly-L-Phe-NH2

- Y eq base: CsOH*H2O, CsOH(l), KOH(s), TBAF(s), TBAF (1 M in THF), TBAB(s),

TBAHS(s), Cs2CO3(s)

- Solvent: DCM, toluene

- Temperature: -10, 0, 25, 45 ºC

- Phase transfer catalyst PTC: Cat. 1, Cat. 2, Cat. 3

19

NNH2

O

11CH2I

[11C]benzyl iodide

No Cata lys

t or PTC

Reaction conditio

ns

NH2

O

11CH2

Ph2C=N

NNH

ON

NH

O

11CH2

No Catalyst or

PTC

Reaction conditi ons

NH2

O

NH2

O

Ph2C=N-D,L-[11C]Phe-NH2

or

Ph2C=N-L-[11C]Phe-NH2

Ph2C=N-D,L-[11C]Phe-L-Phe-NH2

or

Ph2C=N-L-[11C]Phe-L-Phe-NH2

37 %

HCl

120 °C, 2

min

120 °C, 2

min37

%

HCl

NH2

O

11CH2

H2N

H-D,L-[11C]Phe-NH2

or

H-L-[11C]Phe-NH2

H2NNH

O

11CH2

NH2

O

H-D,L-[11C]Phe-L-Phe-NH2

or

H-L-[11C]Phe-L-Phe-NH2

Scheme 4: Model “hot” synthesis of racemic- and H-L-[11C]Phe-NH2 or H-D,L-[11C]Phe-

L-Phe-NH2 and H-L-[11C]Phe-L-Phe-NH2

20

3. MATERIALS AND METHODS

3.1. MATERIALS

Reagents and solvents

Chemicals were supplied from different commercial sources: Sigma-Aldrich (Zwijndrecht,

the Netherlands), Fluorochem (Derbyshire, United Kingdom) and Bachem (Bubendorf,

Switzerland). Solvents used were obtained from Biosolve (Valkenswaard, the

Netherlands). Chemicals and solvents were used directly, without further purification,

unless stated otherwise.

The following chemicals were used to obtain desired products: (S)-1-(((S)-1-amino-1-oxo-

3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-aminium chloride (H-L-Phe-L-Phe-

NH2*HCl), benzophenone imine, (iodomethyl)benzene (BnI), benzyl bromide (BnBr),

N,N-Diisopropylethylamine (DIPEA), (Benzotriazol-1-yloxy)tris(dimethylamino)

phosphonium hexafluorophosphate (BOP), (S)-2-amino-3-phenylpropanamide (H-L-Phe-

NH2), 2-((tert-butoxycarbonyl)amino)acetic acid (Boc-Gly), (R)-2-((tert-butoxycarbonyl)

amino)-3-phenylpropanoic acid (Boc-D-Phe-OH), 2-aminoacetamide (H-Gly-NH2), 1 M

KHSO4, 10 % Na2CO3, MgSO4 and Na2SO4.

For radiolabeling, the following chemicals were used: LiAlH4 (1.0 M in THF),

phenylmagnesium bromide solution (PhMgBr) (1.0 M in THF), 57 % HI (aq), O-Allyl-N-(9-

anthracenylmethyl)cinchonidinium bromide (Cat. 1), (11bR)-(–)-4,4-Dibutyl-4,5-dihydro-

2,6-bis(3,4,5-trifluorophenyl)-3H-dinaphth[2,1-c:1′,2′-e]azepinium bromide (Cat. 2),

(R,R)-3,4,5-Trifluorophenyl-NAS bromide (Cat. 3), CsOH*H2O, KOH(s), TBAF(s), TBAF

(1.0 M in THF), TBAB, Cs2CO3, TBAHS, NaOH(s), MgSO4, K2CO3 and P2O5.

Solvents used to perform synthesis and isolation were: 1,2-dichloroethane (DCE),

triethylamine (TEA), n-hexane (He), ethylacetate (EtOAc), diethyl ether (Et2O), brine,

acetonitrile (MeCN), dichloromethane (DCM), methanol (MeOH), destilled water, acetone

(Me)2O, ethanol (EtOH), 1,4-dioxane, 4 M HCl in 1,4-dioxane, 37 % HCl, benzene,

hexadeuterodimethyl sulfoxide (DMSO-d6), dimethylformamide (DMF), toluene and 2-

propanol.

21

Laboratory equipment

− Ultrasonic bath: Ultrasonic Cleaner USC-THD, USC500THD (supply: 230 V, 50 Hz;

output: HF 45 kHz, 100 w, heater: 200 W), VWR International (Amsterdam, the

Netherlands)

− Rotary evaporator: Rotavapor® R II (Flawil, Switzerland)

− UV light: VL-215.LC (230 V, 50 Hz), Vilber Lourmat (Marne la Vallee, France)

− pH meter: CG 837, SCHOTT, BDL Czech Republic s.r.o. (Trutnov, Czech Republic)

− Vacuum oven: RVT360 (220 V, 50-60 Hz, 5 A, power 1.6 kV, temperature range 0°C to +

180°C), Heraeus

− Balance: MS303 S Precision Balance (Max = 320 g, d = 1 mg, 12 V, 0.84 A ), METTLER

TOLEDO (Greifensee, Switzerland), XP26DR Microbalance ( Max 5.1 g / 22 g, d = 0.002

mg/0.01 mg), METTLER TOLEDO (Greifensee, Switzerland)

− Dose calibrator: dose calibrator VDC – 304, Veenstra instuments (Joure, the Netherlands)

Nomenclature and molecule drawing

Molecules were drawn and named by computer software ChemBioDraw Ultra 12.0.2, from

CambridgeSoft Corporation (Massachusetts, USA).

Laboratory notebook

Laboratory notebook was recorded manually and on a computer software E-notebook

11.0.4.24, from CambridgeSoft Corporation (Massachusetts, USA).

Labeling programme

Labeling (helium flow, activity, temperature) was monitored by computer software

SpecView, version 2, build #767/32, from Spec View Corporation (Ridderkerk, the

Netherlands).


22

3.2. METHODS

3.2.1. CHROMATOGRAPIC METHODS

Thin layer chromatography

Reactions were monitored by thin layer chromatography (TLC), performed on pre-coated

silica 60 F254 aluminium plates, purchased from Merck (Darmstadt, Germany). As a mobile

phase, different solvents were used. Spots on chromatographic plates were detected using

ultraviolet (UV) light, at wavelength 254 nm. Subsequently, spots were visualized by two

spray reagents, ninhydrin, for detection of amines, and bromocresol green, for detection of

acids.

Column chromatography

Flash column chromatography was performed on Büchi (Flawil, Switzerland) Sepacore

system (comprising a C-620 control unit, a C-660 fraction collector, two C-601 gradient

pumps and a C-640 ultraviolet (UV) detector) equipped with Büchi Sepacore pre-packed

flash columns. If additional pre-column on Buchi was applied, silica gel 60 Å from Merck

(Darmstadt, Germany) was used. For mobile phase, following solvents and mobile phases

were used.

Chromatograms were acquired with Raytest GINA Star software, version 5.8 (Tilburg, the

Netherlands).

Mobile phases

Mobile phases (MP) used in thin layer chromatography and column chromatography:

Method 1: 0 – 10 min: 100 % He, 10 - 25 min: 0 – 20 % EtOAc, 25 – 32 min: 20 – 75 %

EtOAc, 32 min: 75 - 100 % EtOAc, 32 – 45 min: 100 % EtOAc

Method 2: 0 – 5 min: 100 % DCM, 5 - 15 min: 0 – 5 % MeOH, 15 – 30 min: 5 – 20 %

MeOH

Method 3: 0 – 10 min: 100 % He, 10 - 25 min: 0 – 50 % EtOAc, 25 – 35 min: 100 %

EtOAc

23

Method 4: 0 – 10 min: 100 % He + 1 % TEA, 10 - 35 min: 0 – 75 % EtOAc + 1 % TEA,

35 – 37 min: 75 % EtOAc + 1 % TEA


EtOAc, 35 – 45 min: 100 % EtOAc, 45 – 55 min: 100 % MeOH


EtOAc, 40 – 45 min: 95 % EtOAc

Method 7: 0 – 5 min: 100 % EtOAc, 5 - 15 min: 0 – 25 % He, 15 – 25 min: 25 - 100 % He,

25 – 30 min: 100 % He

MP 1: He/EtOAc: 1/1

MP 2: He/EtOAc: 1/2

MP 3: MeOH/DCM: 1/15

MP 4: He/EtOAc: 1/1 + 1% TEA

MP 5: He/EtOAc: 9/1

MP 6: He/EtOAc: 7/3

MP 7: He/EtOAc: 4/1

MP 8: MeOH/Na-formate: 7/3

MP 9: MeCN/H2O: 2/3

MP 10: MeOH/H20: 7/3

MP 11: MeOH/Na-formate: 4/1

MP 12: MeCN/ammonium formate: 1/2

MP 13: MeOH/0.1 HCO2NH4: 55/45

MP 14: He/2-propanol: 97/3

3.2.2. SPECTROSCOPIC METHODS

Nuclear magnetic resonance

1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance

250 (250.13 MHz for 1H) or a Bruker Avance 500 (500.23 MHz for 1H and 125.78 MHz

for 13C) with chemical shifts (δ) reported in parts per million (ppm) relative to the solvent

24

(CDCl3, 1H 7.26 ppm, 13C 77.16 ppm; D2O, 1H 4.79 ppm; DMSO, 1H 2.50 ppm, 13C 39.52

ppm) on the tetramethylsilane (TMS) scale. The obtained spectra were analyzed by the

program ACD/NMR Processor Academic Edition 12.01 from Advanced Chemistry

Development, Inc. (Bracknell, UK).

Electrospray ionization-high resolution mass spectrometry

Electrospray ionization-high resolution mass spectrometry (ESI-HRMS) was carried out

using a Bruker microTOF-Q instrument in a positive mode (capillary potential of 4500 V).

3.2.3. RADIOCHEMISTRY METODS

Radioactivity was quantified with a VDC-405 dose calibrator (Veenstra Instruments, Joure,

the Netherlands). All radiochemical reactions were carried out in homemade, remotely

controlled devices.

Analytical isocratic high-performance liquid chromatography

Analytical isocratic high-performance liquid chromatography (HPLC) was performed on a

Jasco (Easton, MD, USA) PU-2089 Plus station, with a Reprosil Chiral-AA column

(250*4.0 mm), Chiralcel OD-R (250*4.6 mm), Chiralcel OJ (250*4.6 mm) and Altima

C18 (250*4.6 mm, 5 u), with Jasco UV-2075 Plus UV detector (254 nm) and NaI

radioactivity detector (Raytest, Straubenhardt, Germany).

25

4. EXPERIMENTAL PART

4.1. ORGANIC CHEMISTRY

4.1.1. SYNTHESIS OF 2-((DIPHENYLMETHYLENE)AMINO)

ACETAMIDE (3)

Reaction:

H2NNH2

O NHN

NH2

O

DCE

1 3

+RT, overnight

2

Procedure:

To the solution of starting material H-Gly-NH2 (1000 mg, 13.50 mmol) (1), dissolved in 20

mL of DCE, 0.5 mL (12.15 mmol) of benzophenone imine (2), dissolved in 10 mL of

DCE, was added. Mixture was stirred overnight at room temperature (RT), monitored by

TLC, filtered and concentrated in vacuo. After addition of small amount of EtOAc and He,

precipitation occurred and crystals were filtered.

Results:

Appearance: white crystals

Yield: 6.310 % (203.0 mg)

Rf: 0.11 (MP 1)

HPLC purity: 96.15 % (MP 8)

1H-NMR (250.13 MHz, CDCl3): δ [ppm] = 7.62 – 7.31 (m, 10H, 10x Ar), 7.08 (s, 2H, -

CO-NH2), 3.91 (s, 2H, -CH2-CO-)

13C-NMR (125.78 MHz, CDCl3): δ [ppm] = 173.51 (-CO-NH2), 170.34 (-C=N-), 138.74

(Ar), 129.05 (Ar), 128.90 (Ar), 57.06 (=N-CH2-CO-)

26

HR-MS: 239.1216 [M+H+] (calculated for C15H14N2O: 238.11), 261.1033 [M+Na+]

(calculated: 261.10)

4.1.2. SYNTHESIS OF (S)-2-((DIPHENYLMETHYLENE)AMINO)-3-

PHENYLPROPANAMIDE (5)

Reaction:

NH

+ DCE, TEA

RT, overnightH2NNH2

ON

NH2

O4 52

Procedure:

To H-L-Phe-NH2 (200 mg, 1.22 mmol) (4), dissolved in 14 mL of DCE, 170 µl (1.22

mmol) TEA and 164 µl (0.974 mmol) (2), dissolved in 7 mL of DCE, was added at 40 °C

and stirred overnight at RT. Next day the reaction was monitored by TLC and after its

completion, the reaction mixture was concentrated in vacuo. Subsequently, flash column

chromatography was performed (Method 1) to obtain the pure product.

Results:

Appearance: white-yellow crystals

Yield: 13.0 % (52.0 mg)

Rf: 0.49 (MP 2)


1H-NMR (250.13 MHz, CDCl3): δ [ppm] = 7.53 – 6.93 (m, 15H, 15x Ar-H), 6.36 (s, 2H, -

CO-NH2), 4.14 – 3.99 (m, 1H, -CH-), 3.18 - 3.11 (m, 1H, -CH2-Ar), 3.07 – 2.93 (m, 1H, -

CH2-Ar)

27


(Ar), 137.85 (Ar-CH2-), 130.59 (Ar), 128.61 (Ar), 128.32 (Ar), 127.34 (Ar), 126.37 (Ar),

67.63 (-N-CH-), 41.52 (-CH2-Ar)



4.1.3. SYNTHESIS OF (R)-2-((DIPHENYLMETHYLENE)AMINO)-3-


Reaction:

H2NNH2

ONH N

NH2

O

2-propanol+RT, overnight

6 72

Procedure:

To H-D-Phe-NH2 (164 mg, 1.00 mmol) (6), dissolved in 20 mL of 2-propanol, 168 µl

(1.00 mmol) of (2) was added. The reaction was monitored by TLC and after its

completion, the reaction mixture was concentrated in vacuo. Afterwards, two sequential

flash column chromatographies with additional precolumn were performed (Method 2 and

Method 3) to obtain the pure product.

Results:


Yield: 29.2 % (96.0 mg)

Rf: 0.30 (MP 3)

HPLC purity: ˃99 % (MP 10)

28


CO-NH2), 4.13 – 4.01 (m, 1H, -CH-CO-), 3.18 – 3.11 (m, 1H, -CH2-Ar), 3.03 – 2.94 (m,

1H, -CH2-Ar)



126.44 (Ar), 67.79 (=N-CH-CO-), 41.57 (-CH2-Ar)



4.1.4. SYNTHESIS OF 2-((DIPHENYLMETHYLENE)AMINO)-3-

PHENYLPROPANAMIDE (9) [63]

Reaction:

NNH2

ON

NH2

O

DCM, TBAF, CsOH*H2O+RT, overnight

3 9

Br

8

Procedure:

To compound 3 (11 mg, 46 μmol), dissolved in 5 mL of DCM, 0.1 eq (0.78 mg, 4.6 µmol)

CsOH*H2O, dissolved in 1 mL of DCM, and 1 eq (55 µl, 0.055 mmol) of TBAF (in THF)

were added. Few minutes later, 1 eq (5.5 µl, 0.046 mmol) of benzyl bromide (8) was

added. Mixture was stirred overnight, monitored by TLC and later concentrated in vacuo.

Subsequently, flash column chromatography (silica gel was prewashed with 1 % TEA) was

performed (Method 4) to obtain the racemic product.

Results:


Yield: 66 % (10 mg)

29

Rf: 0.38 (MP 4)



CO-NH2), 4.12 – 4.06 (m, 1H, -CH-), 3.18 – 3.11 (m, 1H, -CH2-Ar), 3.04 – 2.92 (m, 1H, -

CH2-Ar)



67.91 (-N-CH-), 41.48 (-CH2-Ar)



4.1.5. SYNTHESIS OF (S)-TERT-BUTYL (2-((1-AMINO-1-OXO-3-

PHENYLPROPAN-2-YL)AMINO)-2-OXOETHYL)CARBAMATE (11)

Reaction:

ONH2

H2NO N

H

OOH

O

O

O

HN

NH

ONH2

O+

DCM, DIPEA, BOP

RT, overnight4 10 11

Procedure:

Boc-protected Gly (500 mg, 2.85 mmol) (10) was dissolved in 10 mL of DCM.

Subsequently, 1 eq (1.26 g, 2.85 mmol) of BOP dissolved in 10 mL of DCM and 2 eq

(0.997 mL, 5.71 mmol) of DIPEA were added simultaneously. After 10 min, H-L-Phe-NH2

(469 mg, 2.85 mmol) (4) dissolved in 10 mL of DCM, was added and the mixture was

stirred overnight. Next day reaction was monitored by TLC and after its completion the

solution was concentrated in vacuo. The solid residue was dissolved in 15 mL of EtOAc

and washed with 1 M KHSO4 (3x 15 mL), 10 % Na2CO3 (3 x 15 mL) and brine (1 x 15

mL). Organic phase was dried over MgSO4, the solvent was removed in vacuo and solid

was placed in vacuum oven at 40 °C and left to dry overnight.

30

Results:


Yield: 60.3 % (533 mg)

Rf: 0.66 (MP 3)


1H-NMR (500.23 MHz, CDCl3-): δ [ppm] = 7.85 (s, 1H, -CO-NH-), 7.24 - 7.14 (m, 5H,

Ar-H), 6.67 (s, 2H, -CO-NH2), 4.67 - 4.62 (m, 1H, -CH-CO-), 3.68 (s, 2H, -CH2-CO-),

3.12 - 3.10 (m, 1H, -CH2-Ar), 3.01 - 2.97 (m, 1H, -CH2-Ar), 1.30 (s, 9H, t-Bu)

13C-NMR (125.78 MHz, DMSO-d6): δ [ppm] = 173.24 (-CO-NH2), 169.50 (-CO-NH-),

156.23 (-O-CO-NH-), 138.40 (Ar-CH2-), 129.48 (Ar), 128.71 (Ar), 126.64 (Ar), 78.39

((CH3)3C), 54.20 (-NH-CH2-), 43.68 (-CH2-CO-), 39.40 (-CH-Ar), 28.97 ((CH3)3C)

HR-MS: 322.1726 [M+H+]( (calculated for C16H23N3O4: 321.17), [M+Na+] 344.1547


4.1.6. SYNTHESIS OF (S)-2-((1-AMINO-1-OXO-3-PHENYLPROPAN-2-

YL)AMINO)-2-OXOETHANAMINIUM CHLORIDE (12)

Reaction:

O

O

HN

NH

ONH2

O

-Cl+H3N

NH

ONH2

O

4 M

HCl/1,4-dioxane

11 12RT, 4

h

Procedure:

Boc protected intermediate (518 mg, 1.61 mmol) (11) was dissolved in 30 mL of 1,4-

dioxane followed by addition of 10 mL of 4 M HCl in 1,4-dioxane at 0 °C. Mixture was

stirred for 4 h, until TLC showed no presence of starting materials. Mixture was filtered,

31

and the solvent was removed in vacuo. Solid was placed in vacuum oven at 40 °C

overnight to obtain the dry product.

Results:


Yield: 72.3 % (300 mg)

Rf: 0.0 (MP 3)


1H-NMR (500.23 MHz, DMSO-d6): δ [ppm] = 8.16 (s, 3H, -NH3+

-), 7.95 (s, 1H, -CO-

NH-), 7.41 – 7.25 (m, 5H, 5x Ar-H), 7.22 – 7.18 (s, 1H, -CO-NH2), 4.49 – 4.45 (m, 1H , -

CH-CO-), 3.57 (s, 2H, -CH2-), 3.08 – 3.04 (m, 1H, -CH2-Ar), 2.81 - 2.76 (m, 1H, -CH2-Ar)


138.33 (Ar-CH2-), 129.74 (Ar), 128.55 (Ar), 126.74 (Ar), 55.77 (-CH-), 39.41 (-CH2-

NH3+Cl-), 38.21 (-CH2-Ar)

HR-MS: 222.1220 [M+H+] (calculated for C11H16N3O2+: 222.12), [M+Na+] 244.1033


4.1.7. SYNTHESIS OF (S)-2-(2-((DIPHENYLMETHYLENE)AMINO)

ACETAMIDO)-3-PHENYLPROPANAMIDE (13)

Reaction:

NHN

ONH2

O

-Cl+H3N

NH

ONH2

O NH

+ DCE, TEA

RT, overnight

12 132

Procedure:

32

H-Gly-L-Phe-NH2*HCl (300 mg, 1.02 mmol) (12) was dissolved in 30 mL of DCE and

placed in ultrasonic bath for 15 minutes at RT. Afterwards 143 µl (1.02 mmol) of TEA was

added dropwise, followed by the addition of 137 µl (0.819 mmol) (2) in 5 mL of DCE at

45 °C. After stirring the mixture overnight at RT, the reaction was monitored by TLC and

after its completion the mixture was filtered, as salt was expected as a by-product. Filtrate

was concentrated in vacuo, dissolved in 25 mL of diethyl ether and washed with 25 mL of

brine. Organic phase was collected, filtered, dried over MgSO4 and the solvent was

removed in vacuo. Subsequently, flash column chromatography with an additional

precolumn was performed (Method 5) to obtain the pure product.

Results:


Yield: 29.2 % (115 mg)

Rf: 0.13 (MP 6)


1H-NMR (250.13 MHz, CDCl3): δ [ppm] = 7.89 (d, 1H, J=5.0 Hz, -CO-NH-), 7.55 – 7.13

(m, 15H, 15x Ar-H), 7.02 – 6.95 (m, 2H, -CO-NH2), 4.71 (q, 1H, J=7.5 Hz, -CH-CO-),

3.87 (d, 2H, J=2.5 Hz, -CH2-CO-), 3.26 – 3.01 (m, 2H, -CH2-Ar)

13C-NMR (125.78 MHz, CDCl3): δ [ppm] = 172.93 (-CO-NH2), 171.16 (-CO-NH-),

170.76 (-C=N-), 138.42 (Ar), 136.56 (Ar-CH2-), 130.88 (Ar), 129.07 (Ar), 128.92 (Ar),

128.81 (Ar), 128.19 (Ar), 127.15 (Ar), 56.41 (-NH-CH2-CO-), 53.75 (=N-CH2-CO-), 37.54

(-CH2-Ar)

HR-MS: 386.1836 [M+H+] (calculated for C24H23N3O2: 385.18)

33

4.1.8. SYNTHESIS OF (S)-N-((S)-1-AMINO-1-OXO-3-PHENYLPROPAN-2-

YL)-2-((DIPHENYLMETHYLENE)AMINO)-3-


Reaction:

NH

NHN

ONH2

O

-Cl+H3N

HN

ONH2

O+

DCE, TEART, overnight

14 152

Procedure:

H-L-Phe-L-Phe-NH2*HCl (70 mg, 0.20 mmol) (14) was dissolved in 5 mL of DCE.

Afterwards 28 µl (0.20 mmol) of TEA was added dropwise, followed by addition of 34 µl

(0.20 mmol) (2), dissolved in 5 mL of DCE. The mixture was stirred overnight and

monitored by TLC. After filtration and concentration in vacuo, crude product was

dissolved in 25 mL of diethyl ether and washed with 25 mL of brine. Organic phase was

collected and dried over MgSO4, followed by the removal of solvent in vacuo.

Subsequently, flash column chromatography was performed (Method 6) to obtain the

desired product.

Results:


Yield: 52 % (50 mg)

Rf: 0.48 (MP 6)

HPLC purity: >99 % (MP 11)

1H-NMR (250.13 MHz, CDCl3): δ [ppm] = 7.45 – 7.08 (m, 20H, 20x Ar-H), 4.64 - 4.59

(m, 1H, -CH-CO-NH2), 4.09 – 4.05 (m, 1H, =N-CH-), 3.15 – 3.07 (m, 1H, -CH2-Ar), 2.95

– 2.89 (m, 2H, –CH2-Ar), 2.80 - 2.72 (m, 1H, –CH2-Ar)

34

13C-NMR (125.78 MHz, DMSO-d6): δ [ppm] = 172.83 (-CO-NH2), 170.97 (=N-CH-),

169.27 (-CO-NH-), 139.16 (Ar), 137.61 (Ar-CH2-; N-terminus), 135.75 (Ar-CH2-; C-

terminus), 130.17 (Ar), 129.85 (Ar), 128.92 (Ar), 128.61 (Ar), 127.53 (Ar), 126.75 (Ar),

68.26 (=N-CH-CO-), 53.05 (-NH-CH-CO-), 38.31 (-CH2-Ar; N-terminus), 38.16 (-CH2-

Ar; C-terminus)

HR-MS: 476.2321 [M+H+] (calculated for C31H29N3O2: 475.23), [M+Na+] 498.2140


4.1.9. SYNTHESIS OF TERT-BUTYL ((R)-1-(((S)-1-AMINO-1-OXO-3-

PHENYLPROPAN-2-YL)AMINO)-1-OXO-3-PHENYLPROPAN-2-

YL)CARBAMATE (17)

Reaction:

H2NNH2

O

O

NH

OH

OO

O

NH

HN

OO NH2

O

+DCM, DIPEA, BOP

RT, overnight

4 16 17

Procedure:

Boc-protected D-Phe-OH (300 mg, 1.13 mmol) (16) was dissolved in 13 mL of DCM and

1 mL of DMF. Subsequently, 1 eq (500 mg, 1.13 mmol) of BOP dissolved in 5 mL of

DCM and 2 eq (0.395 mL, 2.26 mmol) of DIPEA were added simultaneously. After 10

min of stirring at RT, H-L-Phe-NH2 (186 mg, 1.13 mmol) (4), dissolved in 5 mL of DCM

and 1 mL of DMF, was added and the mixture was stirred overnight and monitored by

TLC. After the completion of reaction, the solution was concentrated in vacuo. As a work-

up procedure, flash column chromatography with additional precolumn was performed

(Method 7) to afford the desired product.

Results:


35

Yield: 68.6 % (319 mg)

Rf: 0.30 (MP 7)


1H-NMR (500.23 MHz, DMSO-d6): δ [ppm] = 7.42 (s, 1H, -CO-NH-), 7.27 - 7.11 (m,

10H, 10x Ar-H), 6.83 (s, 2H, -CO-NH2), 4.47 – 4.42 (m, 1H, NH-CH-CO-), 4.15 – 4.12

(m, 1H, -CH-CO-), 3.08 – 3.04 (m, 1H, -CH2-Ar), 2.79 – 2.74 (m, 1H, -CH2-Ar), 2.65 –

2.62 (m, 1H, -CH2-Ar), 2.49 – 2.47 (m, 1H, -CH2-Ar), 1.29 (s, 9H, t-Bu)


155.72 (-O-CO-NH-), 138.45 (Ar), 129.69 (Ar), 128.48 (Ar), 126.73 (Ar), 79.40 (-CH-

(CH3)3), 54.08 (-CH-CH2-), 49.19 (-CH-CH2-), 37.59 (-CH2-Ar), 28.61 ((CH3)3C)

HR-MS: 412.2212 [M+H+] (calculated for C23H29N3O4: 411.22), 434.2025 [M+Na+]


4.1.10. SYNTHESIS OF (R)-1-(((S)-1-AMINO-1-OXO-3-PHENYLPROPAN-

2-YL)AMINO)-1-OXO-3-PHENYLPROPAN-2-AMONIUM CHLORIDE

(18)

Reaction:

-Cl+H3N

NH

ONH2

O

4 M

HCl/1,4-dioxane

O

NH

HN

OO NH2

O

17 18

RT, 3 h

Procedure:

Boc-protected D-Phe-L-Phe-NH2 (150 mg, 0.365 mmol) (17), dissolved in 10 mL of 1,4-

dioxane, was placed on ice bath and 4 mL of 4 M HCl in 1,4-dioxane was added. The

mixture was stirred until TLC showed complete disappearance of the starting material (3

h). Solvent was removed in vacuo, the residue was washed with DCM (2 x 5 mL), and left

to dry overnight at RT.

36

Results:


Yield: 87.0 % (99.0 mg)

Rf: 0.1 (MP 2)


1H-NMR (250.13 MHz, DMSO-d6): δ [ppm] = 8.12 (s, 3H, -NH3+), 7.35 - 7.20 (m, 10H,

10x Ar-H), 7.05 (s, 2H, -CO-NH2), 4.65 – 4.61 (m, 1H, NH-CH-CO-), 4.15 – 4.07 (m, 1H,

-CH-CO-), 3.15 – 3.08 (m, 1H, -CH2-Ar), 2.92 – 2.67 (m, 3H, -CH2-Ar)


138.18 (Ar), 129.98 (Ar), 129.79 (Ar), 128.90 (Ar), 128.60 (Ar), 66.82 (-CH-CH2-), 52.42

(-CH-CH2-), 37.80 (-CH2-Ar)



4.1.11. SYNTHESIS OF (R)-N-((S)-1-AMINO-1-OXO-3-PHENYLPROPAN-2-

YL)-2-((DIPHENYLMETHYLENE)AMINO)-3-


Reaction:

NHN

HN

ONH2

O-Cl+H3N

NH

ONH2

O

+ 2-propanol

18 19

RT, overnight

2

Procedure:

37

H-D-Phe-L-Phe-NH2*HCl (35 mg, 1.1 mmol) (18) was dissolved in 10 mL of 2-propanol.

Afterwards 15 µl (1.1 mmol) of benzophenone imine (2) was added. Mixture was stirred

overnight and monitored by TLC. After the completion of reaction, the mixture was

filtered and the filtrate was concentrated in vacuo. Crude product was precipitated from

He/EtOAc, and filtered afterwards. The obtained crystals were placed in a vacuum oven

and dried at 40 °C overnight.

Results:


Yield: 36 % (15 mg)

Rf: 0.13 (MP 1)


1H-NMR (250.13 MHz, CDCl3): δ [ppm] = 7.55 – 6.97 (m, 20H, 20x Ar-H), 4.71 - 4.63

(m, 1H, -CH-CO-NH2), 4.15 – 4.08 (m, 1H, =N-CH-), 3.19 – 3.00 (m, 4H, -CH2-Ar)

13C-NMR (125.78 MHz, CDCl3): δ [ppm] = 172.83 (-CO-NH2), 172.80 (=N-CH-), 169.10

(-CO-NH-), 139.14 (Ar), 136.99 (Ar-CH2- N-terminus), 136.22 (Ar-CH2- C-terminus),

128.70 (Ar), 128.37 (Ar), 128.13 (Ar), 127.04 (Ar), 69.25 (=N-CH-CO-), 56.14 (-NH-CH-

CO-), 37.14 (-CH2-Ar), 32.74 (-CH2-Ar)



38

4.2. RADIOCHEMISTRY

4.2.1. SYNTHESIS OF [11C]BENZYL IODIDE [61]

First stage of radiolabeling was a 3-step synthesis of [11C]benzyl iodide, as depicted in

Scheme 3. [11C]CO2 was transferred from loop by a steam of helium gas with flow rate 10

mL/min to a vessel containing 0.1 mL of PhMgBr (1 M in THF) at 35 °C. After maximum

of activity was trapped in the vessel, mixture was stirred with helium flow 10 mL/min for 1

min and an additional 1 min at higher helium flow (50 mL/min). Afterwards 0.1 mL

LiAlH4 (1 M in THF) was added at 35 °C (helium flow 10 mL/min) to reduce [11C]benzoic

acid to [11C]benzyl alcohol. Solvent was immediately evaporated at 130 °C. Next, 0.1 mL

of 57 % HI was added at 0 °C and left to react for 2 min at 120 °C. [11C]Benzyl iodide was

diluted with 2.5 mL of DCM or 3 mL of toluene and transferred by helium flow 100

mL/min through purification column (K2CO3/MgSO4 = 8/2) into a second reaction vessel

containing one KOH pellet (cca. 96 mg). [11C]benzyl iodide was obtained in 93 ± 2 % (n =

5) radiochemical purity in overall synthesis time of approximately 11 min (Appendix 1).

4.2.2. SYNTHESIS OF RACEMIC H-[11C]Phe-NH2 AND RACEMIC H-

[11C]Phe-L-Phe-NH2

Base, dissolved in 100 µl of DCM and 300 µl of DMSO, was added to the precursor, either

Ph2C=N-Gly-NH2 or Ph2C=N-Gly-L-Phe-NH2, prior the addition of [11C]benzyl iodide to

deprotonate the precursor, as depicted in Scheme 4. After addition of [11C]benzyl iodide, in

DCM, the mixture was stirred for 10 min with helium flow 10 mL/min at different T and

alkylation occurred on the α-C-position to yield racemic amino acid Ph2C=N-D,L-

[11C]Phe-NH2 and racemic dipeptide Ph2C=N-D,L-[11C]Phe-L-Phe-NH2. The exact

conditions used for alkylation are shown in Table I and Table II, respectively. Final

quantitative deprotection, with 0.2 mL of 37 % HCl at 120 °C for 2 min (helium flow 10

mL/min), resulted in H-D,L-[11C]Phe-NH2 and H-D,L-[11C]Phe-L-Phe-NH2.

4.2.3. SYNTHESIS OF H-L-[11C]Phe-NH2 AND H-L-[11C]Phe-L-Phe-NH2

Subsequently, [11C]benzyl iodide, in DCM or toluene, was added to the mixture of

precursor, either Ph2C=N-Gly-NH2 or Ph2C=N-Gly-L-Phe-NH2, and base, to deprotonate

precursor, as depicted in Scheme 4. Reaction mixture was stirred for 10 min, with helium

39

flow 10 mL/min, at different T and alkylation occurred on the α-C-position to yield

Ph2C=N-L-[11C]Phe-NH2 and Ph2C=N-L-[11C]Phe-L-Phe-NH2, with the aid of Cat. 1, Cat.

2 or Cat. 3 (Figure 7). The exact conditions used for alkylation are shown in Table III and

Table IV, respectively. Final quantitative deprotection, with 0.2 mL of 37 % HCl at 120 °C

for 2 min (helium flow 10 mL/min), resulted in H-L-[11C]Phe-NH2 and H-L-[11C]Phe-L-

Phe-NH2.

40

5. RESULTS AND DISCUSSION

Our research work consisted of three integrated fields: organic chemistry, radiochemistry

and HPLC analysis, which were all first performed on a model amino acid, to establish the

carbon-11 chemistry, prior to labeling of the desired dipeptide.

As radiochemical detection was performed on HPLC, with ultraviolet (UV) and

radioactivity detectors, standards of all compounds, namely the precursor, the wanted as

well as the unwanted products, for example both enantiomers (H-L-Phe-NH2 and H-D-Phe-

NH2), had to be synthesized using standard organic chemistry to identify the radiolabeling

products. During experimental work, precursor and reference compounds for model

unnatural amino acid H-Phe-NH2 (Scheme 1) and dipeptide H-L-Phe-L-Phe-NH2 (Scheme

2) have been successfully synthesized. In the next step, radiolabeling was performed under

different conditions, as further described, and quantified by analytical HPLC. Analysis

revealed that labeling with carbon-11 was achieved in high yields, however L-enantiomer

or L,L-diastereomer were not obtained.

5.1. ORGANIC CHEMISTRY

5.1.1. H-Phe-NH2

Precursor and both enantiomers (H-L-Phe-NH2 and H-D-Phe-NH2) were synthesized by

˝one-step˝ protection reaction with benzophenone imine (Scheme 1), following the

procedure by O´Donnell. [63] Diphenylmethylene, as a protecting group on N-terminus, has

already been successfully applied to asymmetric syntheses due to its ability to form

Schiff´s base, which was required to achieve α-alkylation with [11C]benzyl iodide. The

predicted selective α-monoalkylation, opposed to N-alkylation on C-terminus, should

occur due to a lower pKa on the α-C-atom which makes it more likely to dissociate. To

prove the above mentioned selectivity, a cold test reaction with more stable benzyl

bromide was performed to yield racemic the compound 9. [63] After labeling, the

diphenylmethylene protecting group could be quantitatively cleaved under acidic

conditions.

41

5.1.2. H-L-Phe-L-Phe-NH2

Precursor and reference compounds for dipeptide (Scheme 2) were synthesized using the

classical approach of liquid phase peptide synthesis. This method was chosen as it is a

convenient method for synthesis of short dipeptides and we wanted to avoid the influence

of resin for the labeling, used in solid-phase peptide synthesis.

Intermediate Ph2C=N-L-Phe-L-Phe-NH2 was synthesized from the commercially available

H-L-Phe-L-Phe-NH2*HCl, following the same protection reaction with benzophenone

imine in DCE and addition of TEA. [63] Furthermore, the syntheses of precursor (Ph2C=N-

Gly-L-Phe-NH2) and unwanted product (Ph2C=N-D-Phe-L-Phe-NH2) were achieved in

three steps (Scheme 2). Coupling of two amino acids was accomplished by liquid-phase

peptide synthesis in DCM, in presence of base and a coupling agent, DIPEA and BOP,

following the procedure by Ghalit. [65] DIPEA is a tertiary amine, used as a base (Hünig´s

base) to provide a slightly basic pH, required for peptide coupling. It is a weak nucleophile,

in which the central N-atom is surrounded by ethyl and two isopropyl groups, granting

space only for one proton to be accepted after deprotonation of carboxylic group, which

becomes activated. After deprotonation of the carboxylic group of Boc-protected amino

acid, BOP as an additional proton-accepting group leads to an increase of electrophilicity

of carboxyl group, by an in situ generation of an activated ester B, as depicted in Scheme

5. The ester is subsequently attacked by the amine group of the other amino acid, resulting

in the formation of peptide bond D via the hydrogen bonded transition state C. [66]

42

P+

N

N O

N

N

N

N

PF6-

O-

R1

O

P+ O N

NN

ON

N

O

R1

N

-PO(NMe2)3

N

N

N

O

O

R1

HN

R2

HN

N

N

H

N+

O

HR1

-O R2

N+

N

N

H

N

O

HR1

-O R2

R1 NH

O

R2

A

BC

D

+

N

N

N

HO

N+

N

N-O H

P+

N

N

O

N N

N

N

PF6-

BOP

Scheme 5: Reaction mechanism for BOP-mediated peptide couplings [66]

The next step was Boc deprotection (Scheme 6), accomplished under strong acidolytic

conditions (4 M HCl in 1,4-dioxane). Deprotection was fast and almost quantitative, more

importantly, the use of water was avoided, which could induce deprotection of

diphenylmethyleneamine, achieved in the last step.

43

OHN

R

O

H R

HN O+

O

H

R

HN O

O

H

NH2R

NH3ClRCl

Cl-

H Cl H Cl1 eq excess

+by-products: CO2(g)carbon dioxide1 eqt-butyl cation

1 eq

Scheme 6: Deprotection of tert-butyl carbamate in 4 M HCl in 1,4-dioxane

Last step, before obtaining the desired precursor and unwanted product, was the protection

reaction with benzophenone imine (shown in Scheme 7). [63] In order to optimize the

protection reaction, different solvents were examined. Reaction in DCM did not yield any

product, neither did protection in benzene with addition of 3Å molecular sieves. [67] This

can be explained by the fact that the dipeptide was bought, and it is commercially available

as a salt, therefore it was not properly dissolved in DCM and benzene. On the other hand,

in DCE [63] with addition of TEA and ultrasonic treatment, the product (3) was obtained in

6.3 % yield and in 13.0 % yield in case of compound (5). The highest yield of 29.2 % for

compound (7) was achieved in 2-propanol following the procedure described by Huang. [64] General explanation for the above listed yields is the low solubility of the precursor in

different solvents and an additional required step, namely the purification with flash

column chromatography. Due to the precursor’s polarity, it can better dissolve in DCE,

whereas the subsequent addition of TEA increases the lipophilicity of now deprotonated

precursor and improves solubility. As expected, the highest yield was obtained in 2-

propanol, as it is the most polar solvent out of those utilized. Moreover, we managed to

isolate our product with crystallization in 2-propanol.

NH

NHN

ONH2

O

-Cl+H3N

HN

ONH2

O+

RT, overnight

Ph2C=N-L-Phe-L-Phe-NH2L-Phe-L-Phe-NH2

solvent i. - v.

Scheme 7: Protection with benzophenone imine; Examination of different solvents: i.

DCM; ii. Benzene + molecular sieves; iii. DCE; iv. DCE + TEA; v. 2-propanol

44

5.2. HPLC ANALYSIS

Prior to labeling, reference chromatograms were prepared on analytical HPLC in order to

monitor radiolabeling, more precisely the radiopharmaceutical purity, conversion of

[11C]benzyl group and enantiomeric or diastereomeric excess. According to the

diastereoseparation of dipeptides by Schmidt in 2004 [68], separation of H-L-Phe-NH2 and

H-D-Phe-NH2 was examined on Reprosil Chiral-AA column (250*4.0 mm), with

macrocyclic antibiotic teicoplanin as chiral selector. Different ratios of water/MeOH

mixtures, with or without presence of TEA and TFA, were used, however no separation

was achieved. The reason for unsuccessful separation is believed to be amidated C-

terminus, since a free carboxylic group is essential for enantiorecognition and consequently

separation. [68] Subsequently, separation was achieved on normal phase Chiralcel OJ [69],

with He/2-propanol (Appendix 2).

5.3. RADIOCHEMISTRY

After synthesis of cold compounds and HPLC reference chromatograms, radiolabeling was

performed. First unnatural amino acid precursor Ph2C=N-Gly-NH2 without presence of

phase-transfer catalyst was labeled, to optimize the conditions. The obtained racemic H-

[11C]Phe-NH2 proved that labeling indeed took place selectively on α-carbon, without N-

alkylation.

5.3.1. SYNTHESIS OF Ph2C=N-D,L-[11C]Phe-NH2

The precursor Ph2C=N-Gly-NH2 was treated with strong base (TBAF) to deprotonate the

α-carbon and alkylated with [11C]benzyl iodide, dissolved in DCM. Afterwards reaction

mixture was stirred for 10 min, at different temperatures, with helium flow (10 mL/min).

During experimental work we also examined magnetic stirrers for mixing, but that did not

yield any product. Results in Table I show, that less than 2 eq of base is not sufficient for

the synthesis. Further, reaction under same conditions (Entry 8 and 12, Table I), but

different T revealed that optimal T is 45 °C, as previously stated by Pekošak. [62] Increasing

the base (Entry 9 – 12, Table I) improved the HPLC conversion to around 70 %, with

optimum around 4 – 5 eq of TBAF, however higher amounts (Entry 13 – 15, Table I)

45

though did not improve conversion. Furthermore, several other bases, TBAB(s), TBAHS(s)

and Cs2CO3(s) were used, at the optimal conditions from previous TBAF experiments,

however almost no significant conversion was achieved. Due to the poor solubility of

Cs2CO3 in DMSO, the base had to be dissolved in MeOH or DMF, which could also affect

the alkylation. In addition, we observed that all experiments using TBAF or TBAB(s)

resulted in unwanted peak on HPLC chromatogram. We assumed that the unwanted peak is

a result of the formation of a complex between tetrabutylammonium ions (TBA+) and

iodide ions (I-). [70] Reference chromatogram is presented in Appendix 3, moreover, an

example of labeling chromatogram (Entry 12, Table I) can also be found in Appendix 4.

46

Table I: Labeling of racemic H-[11C]Phe-NH2 with [11C]benzyl iodide in DCM; All HPLC

conversions are decay corrected.

Entry Precursor (µmol)

eq base Base Solvent* T (̊C)

HPLC conversion

(%) 1 12 1.1 TBAF (1M in THF) 300 µL DMSO 45 10.3

2 12 1.2 TBAF (1M in THF) 300 µL DMSO 25 9.3



5 14 1.9 TBAF (1M in THF)** 300 µL DMSO 45 3.9











16 21 6.0 TBAB(s) 300 µL DMSO 45 0.0

17 21 10.0 TBAB(s) 300 µL DMSO 45 0.0

18 11 4.6 Cs2CO3(s) 300 µL MeOH 45 0.0

19 10 5.0 Cs2CO3(s) 300 µL DMF 45 1.1

20 11 5.6 TBAHS(s) 300 µL DMSO 45 0.0

*Solvent: In general, 100µL of DCM was used with additional solvent included in the table. **Additional

base 0.9 eq CsOH*H2O was added.

47

5.3.2. SYNTHESIS OF Ph2C=N-D,L-[11C]Phe-L-Phe-NH2

Labeling of dipeptide started with less than 2 eq of base and was continued with optimal

conditions from the amino acid, as model reaction, with [11C]benzyl iodide dissolved in

DCM. Firstly, Entries 1 and 2 (Table II) confirmed that using less than 2 eq of base, the

activation indeed does not occur. Secondly, we proved the hypothesis that a sufficient

amount of base has to be added before the addition of labeling agent [11C]benzyl iodide to

activate the precursor. Thirdly, it was observed that the temperature does not play an

important role in reaction, since it gave approximately the same HPLC conversion either

25 °C or 45 °C (Entry 4 and 5, Table II). Contrary to what was proved for cold chemistry

and labeling of amino acid, we expected that the unwanted N-alkylation would occur as a

side reaction, whereas 6 - 11 eq of base is optimal to obtain good yield with not to high

percentage of N-alkylation. As already observed with phenylalanine amide labeling, an

unwanted peak appeared in all reaction mixtures, which is suspected to be a result of

formation of complex among TBA+ and I-. Last step of radiolabeling, deprotection under

strong acidic conditions (37 % HCl) was quantitative. The reference chromatogram is

presented in Appendix 5, moreover an example of labeling chromatogram (Entry 8, Table

II) can be also found in Appendix 6.

48

Table II: Labeling H-D,L-[11C]Phe-L-Phe-NH2; with [11C]benzyl iodide in DCM; All HPLC conversions are decay corrected.

Entry Precursor (µmol)

eq base

Base Solvent* T (°C)

HPLC conversion

(%)

Probably N-

alkylation (%)

1 14.8 1.1 TBAF (1M in THF) 300 µL DMSO

45 0.0 /


45 0.0 /


45 6.9 /


25 42.3 17.3


45 42.5 19.9


45 45.5 33.4


45 8.1 /


25 62.7 24.9

*Solvent: In general 100µL of DCM was used with additional solvent included in the table.

5.3.3. ASYMMETRIC SYNTHESIS OF Ph2C=N-L-[11C]Phe-NH2

As the asymmetric synthesis was the subject of interest of this research work,

enantioselective reaction was examined using 3 different phase-transfer catalysts, as shown

in Figure 1. [54] Reference chromatograms can be found in Appendix 3.

For this purpose, [11C]benzyl iodide was dissolved either in DCM or toluene, to monitor

the effect of the solvent. Despite previous labeling reactions, asymmetric synthesis in

presence of 10 % of PTC required an excess of base, approximately 205 eq, as previously

noticed by Pekošak for H-L-[11C]Phe. [62] As proven in standard asymmetric synthesis,

reactions with PTC are achieved with both high yield as well as ee in presence of a

hydroxide base (KOH or CsOH*H2O), therefore TBAF was replaced with CsOH*H2O.

Moreover, in order to obtain high ee, reactions were performed at lower T, which

contributes to selectivity and a higher conversion into the desired L-enantiomer. Obtained

results are shown in Table III and an example of chromatogram from labeling is presented

in Appendix 7.

Firstly, with Cat. 1 it was found that 205 eq of CsOH*H2O was the optimal amount of

base. More CsOH*H2O (250 eq), did not significantly increase the yield and different

bases KOH(s) or TBAF(s) did not yield the desired product. In the presence of TBAF the

49

unwanted side product was once again observed. Further, the temperature or solvent (DCM

or toluene) did not significantly affect the HPLC conversion with this catalyst. Moreover,

the use of CsOH in water solution, as presence of water could improve phase-transfer

reaction, yielded no product, therefore it was concluded that additional amount of water

does not improve the phase-transfer reaction.

Secondly, using the same conditions as for Cat. 1, Cat. 2 also showed no difference in

conversion between 0 °C or 25 °C, however higher conversion of [11C]benzyl group was

observed in DCM compared to toluene (Table III).

Thirdly, Cat. 3 was studied in the presence of 205 eq CsOH*H2O or TBAF(s) in

dichloromethane. TBAF was once again confirmed as inappropriate base, as unwanted

peak was observed which decreased the HPLC conversion. Further, the

tetrabutylammonium base did not improve the asymmetric synthesis.

Finally, reaction mixtures (Entry 9 to 17, Table III) were analyzed on a Chiralcel OJ

column in order to reveal enantiomeric excess (Appendix 8). All reactions with

CsOH*H2O as base gave excellent conversions, however instead of high amount of L-

enantiomer racemic mixture, approximately 50 % L- and 50 % H-D-Phe-NH2, was

observed. To study the influence of the catalyst on phase-transfer reaction, optimal

conditions were performed without presence of the catalyst (Entry 17, Table III), which

resulted with same excellent HPLC conversion and racemic mixture (46.1 % of L- and 53.9

% of Ph2C=N-D-[11C]Phe-NH2). Nevertheless, results from Table III show that the

reaction at lower temperature indeed favors the formation of the L-enantiomer compared to

25 °C. Lastly, it can be concluded that CsOH*H2O is the optimal base for the high

conversion, although the right PTC still has to be found.

50

Table III: Results for asymmetric synthesis of Ph2C=N-L-[11C]Phe-NH2; All HPLC conversions are decay-corrected.

Entry eq base

Base PTC T (°C)

solvent for BnI

HPLC conversion (%)

L-Phe-NH2 (%)

D-Phe-NH2 (%)

1 69 CsOH*H2O Cat. 1 25 Toluene 22.0 / / 2 144 CsOH*H2O Cat. 1 25 Toluene 27.0 / / 3 250 CsOH*H2O Cat. 1 25 Toluene 83.2 / / 4 205 CsOH*H2O Cat. 1 25 DCM 83.1 / / 5 205 CsOH*H2O Cat. 1 0 DCM 72.1 / / 6 205 CsOH solution Cat. 1 0 Toluene 0 / / 7 205 CsOH*H2O Cat. 2 25 Toluene 47.9 / / 8 205 CsOH*H2O Cat. 2 0 Toluene 28.1 / / 9 205 CsOH*H2O Cat. 1 25 Toluene 84.2 ± 8.7 (n=3) 47.2 52.8 10 205 CsOH*H2O Cat. 1 0 Toluene 74.1 ± 10.2 (n=3) 58.9 41.1 11 205 KOH(s) Cat. 1 0 DCM 0 0 0 12 205 TBAF(s) Cat. 1 0 DCM 64.5 58.7 41.3 13 205 CsOH*H2O Cat. 2 25 DCM 92.7 ± 4.2 (n=2) 45.7 54.3 14 205 CsOH*H2O Cat. 2 0 DCM 93.6 ± 4.0 (n=2) 54.2 45.8 15 205 TBAF(s) Cat. 3 0 DCM 57.5 68.5 31.5 16 205 CsOH*H2O Cat. 3 0 DCM 99.4 54.0 46.0 17 16.8 CsOH*H2O /* 0 DCM 98.6 46.1 53.9

* Without the aid of PTC.

Unsuccessful asymmetric synthesis can be explained by the fact that PTC has a rigid

structure, therefore the formation of ions can only succeed on sterically less hindered side

of the nitrogen. As depicted in Figure 8, where a similar catalyst is used, the C-terminus of

the precursor has a tert-butyl ester enolate, which is formed after base treatment and this

enables an optimal docking and consequently, an asymmetric induction. The formation of

the ion pair among (Z)-enolate imino ester and quaternary ammonium salt of the catalyst is

essential. Our precursor, however, has an amidated C-terminus, which probably does not

form the enolate and consequently it would not be possible to dock into the catalyst and to

direct a stereoselective alkylation. [71]

51

Figure 8: Attack of benzyl iodide (RX) on ion pair formed between catalyst and (Z)-

enolate of imino ester [72]

5.3.4. ASYMMETRIC SYNTHESIS OF Ph2C=N-L-[11C]Phe-L-Phe-NH2

Optimal conditions established on H-L-[11C]Phe-NH2 were transferred to dipeptide to

achieve the labeling and formation of H-L-[11C]Phe-L-Phe-NH2. During alkylation of the

precursor, with [11C]benzyl iodide dissolved in DCM, again two main products were

observed, the side product was probably the N-alkylated product. Comparing yields

obtained with Cat. 1 and Cat. 2, it was observed that temperature (-10 ⁰C or 0 ⁰C) does not

have significant influence on conversion of [11C]benzyl iodide. Thermodynamic reaction

of course should increase yield with increased temperature, but the difference is not

significant and because of preferred enantioselectivity, reactions were performed at lower

temperatures. Following the optimal results obtained for the catalyzed labeling of amino

acid amide, Cat. 3 was studied as the last one (Table IV, Entry 6 - 8). Using this catalyst, it

was confirmed that a minimum of 5 µmol of precursor is needed (Entry 6, Table IV). Same

conditions were repeated with higher amount of base, resulting in 33.2 % HPLC

conversion. Under the same conditions TBAF was studied and conversion was reduced

significantly. These optimal conditions were also performed without presence of a catalyst

(Entry 9, Table IV), showing the same excellent HPLC conversion. Unfortunately, due to

the short time of experimental work of this master’s thesis, diastereomers were not

separated on Chiralcel OJ column, therefore diastereomeric excess could not be studied.

52

The last step, deprotection under strong acidic conditions (37 % HCl at 120 ºC at 2 min)

was quantitative, 97.9 % ± 0.7 % SD (n = 2).

Table IV: Results for asymmetric synthesis of Ph2C=N-L-[11C]Phe-L-Phe-NH2; DCM is used as solvent for [11C]benzyl iodide; All HPLC conversions are decay corrected.

Entry Precursor

(µmol) eq base Base PTC

T (°C)

HPLC conversion (%)

Probably N-alkylation

(%)

1 13.2 205 CsOH*H2O Cat.1 0 35.9 55.2

2 11.7 205 CsOH*H2O Cat. 1 0 73.9 15.1

3 11.9 205 CsOH*H2O Cat. 1 -10 42.8 9.6

4 11.9 205 CsOH*H2O Cat. 2 0 45.4 32.2

5 12.7 205 CsOH*H2O Cat. 2 -10 37.2 8.6

6 3.6 140 CsOH*H2O Cat. 3 0 0 /

7 5.9 137 CsOH*H2O Cat.3 0 58.4 33.3

8 5.9 140 TBAF (1M in THF) Cat. 3 0 20.6 6.8

9 3.1 140 CsOH*H2O /* 0 35.1 13.9 * Without aid of PTC.

53

6. CONCLUSIONS

During this research 6 compounds have been successfully synthesized and analyzed prior

to radiolabeling. Precursor (3) and cold references (5, 7) for unnatural amino acid amide,

as model compounds, have been synthesized by a one-step protection reaction with

benzophenone imine. The same method was used for compound (15), precursor (13) and

unwanted dipeptide (19) which have been synthesized in a three-step reaction by liquid-

phase peptide synthesis. In protection reactions with benzophenone imine, 2-propanol

proved as the most appropriate solvent to obtain the highest yield, due to the good

solubility of starting material.

Prior to labeling, reference chromatograms were obtained for amino acid amide and the

dipeptide, with the enantiomeric separation achieved on a Chiralcel OJ column.

Subsequently, model reactions were done on a single amino acid to prove selectivity of the

α-alkylations on the Schiff’s base, however a subsequent labeling revealed that dipeptide

can also be a subject of N-alkylation, as the N-alkylated side product was obtained during

the alkylation with [11C]benzyl iodide.

[11C]benzyl iodide was obtained in high radiochemical purity. Successful radiolabeling of

racemic H-[11C]Phe-NH2 was achieved with high conversion (>90 %) of [11C]benzyl

group. Racemic dipeptide H-[11C]Phe-L-Phe-NH2 was obtained in moderate yield (>60 %),

with unwanted N-alkylation. Asymmetric synthesis, in the presence of a PTC, resulted in

excellent conversion (>90 %) of [11C]benzyl group. The last step of the reaction,

deprotection under strong acidic conditions, was quantitative as expected. From the

obtained HPLC chromatograms it can be concluded that the reactions in the presence of

PTC were not stereoselective and that the three studied phase-transfer catalysts did not

give stereoselective induction, therefore we did not obtain desired

enantiomeric/diastereomeric excess. For future research we propose esterification of C-

terminus of the precursor molecule, in order to achieve selectivity with PTC we used in

this research and afterwards transforming the ester moiety back to amidated C-terminus,

necessary for high binding affinity towards SP1-7 binding side. Another option could also

be the synthesis of PTC with the ability to form stable ion pair with precursor, activating α-

C-atom then attacked by benzyl iodide. When desired, H-L-[11C]Phe-L-Phe-NH2 would be

obtained in high yield and radiochemical purity, autoradiography would be performed to

54

verify if labeled dipeptide indeed had bound to the desired brain regions. If it did, the

dipeptide itself and its analogues, could open a new horizon in the development of first

specific medication for treatment of neuropathic pain, moreover they could be used as a

research tool for the identification of the SP1-7 target (receptor).

55

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61

APPENDIX

Appendix 1: Chromatogram of labeled [11C]benzyl iodide; Altima 250*4.6 mm,

MeOH/buffer (Na-formate): 70/30, 1 ml/min, 254 nm; Rt: 11.4 min [11C]benzyl iodide

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min

ACT

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min0,0

5,0

10,0

CPS *1000

03'28

04'20

05'13

06'05

09'10

11'35

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min

UV1

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min0

100

200

300

mV

03'21

04'27

05'50

07'11

62

Appendix 2: Reference chromatogram of Ph2C=N-L-Phe-NH2 and Ph2C=N-D-Phe-NH2;

Chiralcel OJ 250*4.6 mm, He/2-propanol: 97/3, 1 mL/min, 220 nm; Rt: 15.1 min Ph2C=N-

L-Phe-NH2, Rt: 19.1 min Ph2C=N-D-Phe-NH2

00"00 05'00 10'00 15'00 20'00 25'00 min

UV1

00"00 05'00 10'00 15'00 20'00 25'00 min

0

20

40

60

80

100

120

140

160

mV 11'56

15'08

19'11

63

Appendix 3: Reference chromatogram for Ph2C=N-D,L-Phe-NH2; Altima C18, 240*4.6

mm, MeOH/buffer (Na-formate): 70/30, 1 mL/min, 254 nm; Rt: 6.1 min Ph2C=N-Gly-

NH2, Rt: 11.1 min BnI, Rt: 15.3 min Ph2C=N-L-Phe-NH2

04'00 06'00 08'00 10'00 12'00 14'00 16'00 18'00 min

UV1

04'00 06'00 08'00 10'00 12'00 14'00 16'00 18'00 min

0

50

100

150

200

250

300

350

400 mV

06'10

09'00

11'06

15'25

64

Appendix 4: Chromatogram of labeled Ph2C=N-D,L-[11C]Phe-NH2 (Table I, Entry 12);

Altima C18, 250*4.6 mm, MeOH/buffer (Na-formate): 70/30, 1 mL/min, 254 nm; Rt: 10.3

min [11C]BnI, Rt: 15.3 min Ph2C=N-L-[11C]Phe-NH2. The identity of the product was

confirmed with analytical HPLC by co-injection of the product and non-labeled Ph2C=N-

L-Phe-NH2.

00"00 05'00 10'00 15'00 20'00 min

ACT

00"00 05'00 10'00 15'00 20'00 min0

100

200

300

CPS

07'41

10'27

15'27

23'33

00"00 05'00 10'00 15'00 20'00 min

UV1

00"00 05'00 10'00 15'00 20'00 min

0,00

0,20

0,40

0,60

0,80

1,00mV *1000

06'07

09'06

15'10

65

Appendix 5: Reference chromatogram for Ph2C=N-D,L-Phe-L-Phe-NH2; Altima C18,

250*4.6 mm, MeOH/buffer (Na-formate): 80/20, 1 mL/min, 254 nm; Rt: 5.2 min Ph2C=N-

Gly-L-Phe-NH2, Rt: 6.2 min BnI, Rt: 9.3 min Ph2C=N-L-Phe-L-Phe-NH2

00"00 01'00 02'00 03'00 04'00 05'00 06'00 07'00 08'00 09'00 min

UV1

00"00 01'00 02'00 03'00 04'00 05'00 06'00 07'00 08'00 09'00 min

0

50

100

150

200

250

300

350

400 mV

05'23

06'16

09'33

66

Appendix 6: Chromatogram of labeled H-D,L-[11C]Phe-L-Phe-NH2 (Table II, Entry 8); Altima C18, 250*4.6 mm, MeOH/buffer (Na-formate): 80/20, 1 mL/min, 254 nm; Rt: 6.5

min [11C]BnI, Rt: 10.4 min Ph2C=N-L-[11C]Phe-L-Phe-NH2

02'00 04'00 06'00 08'00 10'00 12'00 min

ACT

02'00 04'00 06'00 08'00 10'00 12'00 min0,00

0,50

1,00

1,50 CPS *1000

05'42

06'51

09'32

10'43

02'00 04'00 06'00 08'00 10'00 12'00 min

UV1

02'00 04'00 06'00 08'00 10'00 12'00 min0,00

0,20

0,40

0,60

0,80

1,00mV *1000

67

Appendix 7: Chromatogram of labeled Ph2C=N-L-[11C]Phe-NH2 (Table III, Entry 14);

Altima C18, 250*4.6 mm, MeOH/Na-formate: 70/30, 1 mL/min, 254 nm; Rt: 10.4 min

[11C]BnI, Rt: 15.3 min Ph2C=N-[11C]Phe-NH2. The identity of the product was confirmed

with analytical HPLC by co-injection of the product and non-labeled Ph2C=N-Phe-NH2.

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min

ACT

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min0,00

0,50

1,00

CPS *1000

07'46

10'40

15'32

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min

UV1

00"00 02'00 04'00 06'00 08'00 10'00 12'00 14'00 min0

100

200

300

mV

15'08

68

Appendix 8: Chromatogram of labeled Ph2C=N-L-[11C]Phe-NH2, showing enantiomeric

ratio (Table III, Entry 9); Chiralcel OJ column, 250*4.6 mm, He/2-propanol: 97/3, 1

mL/min, 220 nm; Rt: 15.0 min Ph2C=N-L-[11C]Phe-NH2, Rt: 19.2 min Ph2C=N-D-

[11C]Phe-NH2. The identity of the product was confirmed with analytical HPLC by co-

injection of the product and non-labeled Ph2C=N-L-Phe-NH2.

00"00 05'00 10'00 15'00 20'00 min

ACT

00"00 05'00 10'00 15'00 20'00 min0

200

400

600

800

CPS

07'39

11'38

15'03

19'23

00"00 05'00 10'00 15'00 20'00 min

UV1

00"00 05'00 10'00 15'00 20'00 min0,00

0,20

0,40

0,60

0,80

1,00mV *1000

02'22

03'48

11'45

14'44

22'56

Documents

UNIVERSITY OF LJUBLJANA FACULTY OF PHARMACY … · university of ljubljana . faculty of pharmacy . janja Škrinjar . design and synthesis of dipeptide l-[11c]phe-l-phe-nh 2. naČrtovanje