Academic year 2013-2014

Justine VAN OVERBERGE

First master of Pharmaceutical Care

Promoter

Prof. dr. apr. S. Van Calenbergh

Co-promoter

Prof. dr. R. Volpini

Commissioners

Prof. dr. apr. F. De Vos

dr. M. Risseeuw

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutics

Laboratory for Medicinal Chemistry

Master thesis performed at:

UNIVERSITA’ DEGLI STUDI DI

CAMERINO

SCHOOL OF PHARMACY

Department of Chemical Sciences

SYNTHESIS OF A PURINE ACYCLIC NUCLEOTIDE AS PARTIAL AGONIST

OF THE PURINERGIC P2X3 RECEPTOR

Academic year 2013-2014

Justine VAN OVERBERGE

First master of Pharmaceutical Care

Promoter

Prof. dr. apr. S. Van Calenbergh

Co-promoter

Prof. dr. R. Volpini

Commissioners

Prof. dr. apr. F. De Vos

dr. M. Risseeuw

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutics

Laboratory for Medicinal Chemistry

Master thesis performed at:

UNIVERSITA’ DEGLI STUDI DI

CAMERINO

SCHOOL OF PHARMACY

Department of Chemical Sciences

SYNTHESIS OF A PURINE ACYCLIC NUCLEOTIDE AS PARTIAL AGONIST

OF THE PURINERGIC P2X3 RECEPTOR

COPYRIGHT

"The author and the promoters give the authorization to consult and to copy parts of this

thesis for personal use only. Any other use is limited by the laws of copyright, especially

concerning the obligation to refer to the source whenever results from this thesis are cited."

3rd of June , 2014

Promoter Author

Prof. dr. apr. S. Van Calenbergh Justine Van Overberge

ABSTRACT

Adenosine-5’-triphosphate (ATP) is present in all living cells and can be released as a

result of several stimuli. It is the natural agonist of a family of cation-permeable ligand gated

ion channels, namely the P2X-receptors. The P2X-receptors are purinergic receptors which are

widely expressed in mammalian tissues and mediate a wide range of physiological functions.

ATP plays an important role as messenger in the central nervous system (CNS), hereby

triggering several processes, such as neurotransmission, hormone secretion, pain and

neuroinflammation. Because of these properties of extracellular ATP, the P2X-receptors are

currently receiving more attention.

The P2X3-receptors, mainly expressed on primary afferent nerves in the CNS, seem to

mediate the process of nociception, in particular the sensitization of chronic pain. This opens

interesting perspectives for future pain treatments. During the last few years, there has been

a lot of interest in developing compounds that block the P2X3-receptors. Moreover, acyclic

nucleotides, composed of an adenine moiety and substituted at position 9 with a

phosphorylated carbon chain showed modest partial agonism at the rat P2X3-receptors. This

partial agonistic activity opens opportunities for the treatment of chronic pain, because the

receptor is not fully suppressed and the defensive pain remains.

To further investigate the potential of this compound, in vivo results are required and

therefore the compound needs to be synthesized on bigger scale. Towards this goal, we

initially explored the same reaction conditions as in the previous (small scale) synthesis. Yet,

multiple difficulties were observed and in spite of several adjustments to the original reaction

procedure, the compound was obtained in a disappointing yield.

Consequently, in the future it will be necessary to improve the synthetic procedure in

order to obtain sufficient material for in vivo studies. Eventually, in vivo tests will be performed

in an available model for neuropathic pain. If favorable results are obtained, those tests can

be expanded to other models for pain. Moreover, since triphosphates are very likely to

degrade, more stable analogs may be developed towards future agents for the treatment of

chronic pain.

SAMENVATTING

Adenosine-5’-triphosphate (ATP) is aanwezig in alle levende cellen en kan vrijgelaten

worden als reactie op verscheidene stimuli. Het is de natuurlijke agonist van een familie van

cation-permeable ligand gated ion channels, namelijk de P2X-receptors. De P2X-receptors zijn

purinerge receptoren die tot expressie komen over het volledige lichaam en zodus een rol

spelen in vele fysiologische functies. Zo blijkt ATP belangrijk in het centraal zenuwstelsel als

trigger van verscheidene processen zoals neurotransmissie, hormoonsecretie, pijn en

neuroinflammatie. Wegens deze belangrijke rol van extracellulair ATP, krijgen de P2X-

receptors op dit moment dan ook heel wat aandacht.

De P2X3-receptors, die hoofdzakelijk tot expressie komen op de primaire afferente

neuronen in het centraal zenuwstelsel, lijken van belang in het proces van nociceptie, meer

bepaald in de sensitisatie van chronische pijn. Dit opent interessante perspectieven voor

toekomstige pijnbehandeling. De afgelopen jaren is de interesse voor de ontwikkeling van

moleculen die de P2X3-receptoren blokkeren dan ook gegroeid. Bovendien toonden

acyclische nucleotiden, gekenmerkt door een adenineskelet en een gefosforyleerde

koolstofketen op positie 9 van de purinering, een partieel agonistische activiteit tegenover

P2X3-receptors van de rat. Deze partieel agonistische activiteit biedt mogelijkheden in de

behandeling van chronische pijn omdat de receptor niet volledig onderdrukt wordt en de

beschermende pijn bijgevolg aanwezig blijft.

Om het potentieel van deze molecule verder te onderzoeken, zijn in vivo testen

vereist, wat betekent dat de synthese van de molecule moet opgeschaald worden. Om deze

opschaling te verwezenlijken, werden oorspronkelijk diezelfde reactiecondities gebruikt als bij

de voorgaande (kleine schaal) synthese. Er werden echter meerdere moeilijkheden

waargenomen en ondanks verscheidene aanpassingen aan de reactieprocedure, werd de

molecule uiteindelijk verkregen in een hoeveelheid die veel te laag was om de in vivo testen

op uit te voeren.

Bijgevolg zal het in de toekomst noodzakelijk zijn om een aangepaste reactie procedure

uit te voeren om zo de molecule in de gewenste hoeveelheid te krijgen om in vivo testen te

kunnen uitvoeren.

ACKNOWLEDGMENT

Writing this means the end of an unforgettable stay in Camerino, where I not only attended

an interesting research project, but also had the chance to meet great people who

contributed to lots of memories.

However, a number of people deserve my special thanks. Without their advice, tips,

knowledge and enthusiasm, I would never have been able to accomplish this thesis in a

positive way.

First of all, I would like to express my gratitude to Prof. dr. apr. Serge van Calenbergh for giving

me the opportunity to perform this research project in the medicinal chemistry unit of

Camerino and to proof-read my final report.

Secondly, I would like to thank Prof. dr. Rosaria Volpini for putting the laboratory at my

disposition and for giving me the chance to learn more about the fascinating field of purinergic

receptors.

Also many thanks to dr. Catia Lambertucci for her dedication and assistance during my stay.

A special thanks goes also to “Aji” Thomas, my mentor during this period. Besides the knowledge

he transmitted to me, he also supported me and encouraged me during every moment of this

research. His positive attitude and unlimited enthusiasm were really admirable. I want to wish

him a lot of luck in finishing his PhD research.

I’d also like to thank my labmates, Angela, Alice, Andrea and Giacomo for the nice atmosphere in

the lab. In addition, I also wish to thank my fellow Erasmus student, Gilles, for the support and his

contribution to great memories.

My final gratitude goes out to all the other people who stood by me during this period. I’m deeply

grateful for the unconditional support my mom has been giving me during my studies, but even

more important in every aspect of my life. Also Jan, my sister, my boyfriend and my close friends

deserve a warm thank you for their support.

TABLE OF CONTENTS

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

1.1 PURINOCEPTORS AND NUCLEOTIDES ......................................................................... 1

1.1.1. P1-receptors (adenosine receptors) .................................................................... 2

1.1.2. P2-receptors ......................................................................................................... 3

1.1.1.1 P2X-receptors ................................................................................................ 4

1.1.1.2 P2Y-receptors ................................................................................................ 6

1.2 P2X3-RECEPTOR ........................................................................................................... 7

1.2.1 The P2X3-receptor - agonists/antagonists ........................................................... 7

1.2.2 The P2X3 receptor and nociception ..................................................................... 8

1.2.2.1 The extracellular role of ATP ......................................................................... 8

1.2.2.2 P2X3-receptors in neural system .................................................................. 9

1.2.2.3 P2X3-receptors and pain transmission ....................................................... 10

1.2.2.4 The P2X3-receptor and the different pain conditions ................................ 11

1.2.3 Partial agonism and desensitization .................................................................. 13

1.2.4 New purinergic ligands: Adenine-based acyclic nucleotides ............................. 14

1.2.4.1 Binding pocket of P2X3-receptor ................................................................ 14

1.2.4.2 Synthesis of adenine-based acyclic ligands ................................................ 15

1.2.5 Instability of ATP and its variants ....................................................................... 16

2 OBJECTIVES .............................................................................................................. 17

3 MATERIAL AND METHODS ....................................................................................... 19

3.1 METHODS .................................................................................................................. 19

3.1.1 Nuclear magnetic resonance (NMR) .................................................................. 19

3.1.1.1 The principle of NMR .................................................................................. 19

3.1.1.2 NMR-spectrum ............................................................................................ 21

3.1.1.3 Instrumentation .......................................................................................... 21

3.1.2 Mass spectroscopy (MS)(44) ................................................................................ 21

3.1.2.1 The principle of MS ..................................................................................... 21

3.1.2.2 MS-spectrum ............................................................................................... 23

3.1.2.3 Instrumentation .......................................................................................... 23

3.2 MATERIALS ................................................................................................................. 23

4 CHEMISTRY .............................................................................................................. 25

4.1 REACTION SCHEME .................................................................................................... 25

4.2 SYNTHESIS OF 9-ACETOXYBUTYL-2-CHLOROADENINE AND 7-ACETOXYBUTYL -2-

CHLOROADENINE .................................................................................................................. 26

4.2.1 Reaction process ................................................................................................ 26

4.2.2 Detailed reaction mechanism ............................................................................ 26

4.2.3 Monitoring of the reaction ................................................................................. 27

4.3 SYNTHESIS OF 9-ACETOXYBUTYL-2-IODO-ADENINE.................................................. 27

4.3.1 Reaction process ................................................................................................ 27

4.3.2 Detailed reaction scheme .................................................................................. 28

4.3.3 Monitoring of the reaction ................................................................................. 30

4.4 SYNTHESIS OF 2-IODO-9-HYDROXYBUTYLADENINE .................................................. 31

4.4.1 Reaction process ................................................................................................ 31

4.4.2 Detailed reaction mechanism ............................................................................ 34

4.4.3 Monitoring of the reaction ................................................................................. 34

4.5 SYNTHESIS OF 2-IODO-9-(4-TRIPHOSPHATE-BUTYL)-ADENINE ................................. 34

4.5.1 Reaction process ................................................................................................ 34

4.5.2 Detailed reaction mechanism ............................................................................ 35

4.5.3 Monitoring of the reaction ................................................................................. 36

5 EXPERIMENTAL SECTION .......................................................................................... 37

5.1 SYNTHESIS OF 9-ACETOXYBUTYL-2-CHLOROADENINE .............................................. 37

5.2 SYNTHESIS OF 9-ACETOXYBUTYL-2-IODO-ADENINE.................................................. 38

5.3 SYNTHESIS OF 2-IODO-9-HYDROXYBUTYLADENINE .................................................. 39

5.4 SYNTHESIS OF 2-IODO-9-(4-TRIPHOSPHATE-BUTYL)-ADENINE ................................. 39

6 CONCLUSION AND FUTURE PROSPECTIVES ............................................................... 41

7 REFERENCES ............................................................................................................. 43

ABBREVIATIONS

ADP Adenosine-5’-diphosphate

Arg Arginine

ATP Adenosine-5’-triphosphate

cAMP Cyclic adenosine-5’-monophosphate

CGRP Calcitonin gene related peptide

CNS Central nervous system

cPLA2 Cytosolic phospholipase A2

CTP Cytidine-5’-triphosphate

ERK Extracellular receptor signal-induced kinase

ESI Electrospray ionization

GDNF Glial cell-derived neurotrophic factor

GPCRs G protein-coupled receptors

Hz Hertz

J Coupling constant

Lys Lysine

MALDI Matrix-Assisted Laser Desorption Ionization

MAPKs Mitogen-activated protein kinase

m/z Mass-to-charge ratio

NBS Nucleotide binding segment

NMR Nuclear magnetic resonance

PI3K Phosphoinositide 3-kinase

PPADS Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid

ppm Parts per million

PKC Protein kinase C

SAR Structure-activity-relationship

TM Transmembrane domain

UDP Uridine-5’-diphosphate

UTP Uridine-5’-triphosphate

1

1 INTRODUCTION

1.1 PURINOCEPTORS AND NUCLEOTIDES

Natural nucleotides consist of one or more phosphate groups, a pentose sugar, and a

base, in particular a heterocyclic base. There are different kinds of bases. Adenine and guanine

belong to the group of the purines, while cytosine, thymine, and uracil belong to the group of

the pyrimidines (Fig. 1.1). A nucleoside, on the other hand, is formed of just the heterocyclic

base bound to the sugar moiety without a phosphate group. Nucleotides are the monomer

building blocks of the nucleic acids. Important examples of nucleotides are adenosine-5’-

triphosphate (ATP), uridine-5’-triphosphate (UTP), adenosine-5’-diphosphate (ADP), and

uridine-5’-diphosphate (UDP). (http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html)

FIGURE 1.1: SRUCTURE OF THE DIFFERENT BASES: ADENINE, GUANINE, CYTOSINE, THYMINE, AND URACIL.

In 1929, the potent extracellular activities of purines, in particular adenosine and ATP

were first reported by Drury and Szent-Györgyi.(1) It soon became obvious that purinergic

signaling has an important effect in many physiological processes. Generally speaking, all cells

are able to release nucleotides, although the mechanisms of this extracellular discharge are

not yet fully clarified. In excitatory and secretory cells, it is believed that nucleotides are

packed in specialized granules (for example in synaptic vesicles) and in reaction of the right

stimulus they could be released in the extracellular spaces. For non-excitatory cells, the

mechanisms underlying the nucleotide release are more complex. In this type of tissues the

secretion of nucleotides can be induced for example by mechanical stimulation.(2)

The transmembrane receptors that interact with purines, were first proposed in 1971

by Burnstock and since that time there has been an enormous growth of interest. Purinergic

receptors, also known as purinoceptors, are found in many mammalian tissues. They are

activated by extracellular purine (and pyrimidine) nucleotides and adenosine.(1,3)

2

Due to their cellular regulatory function, the purinergic receptors are considered as

interesting pharmaceutical targets. Nowadays there is a division in 3 groups: P1 (adenosine)-

receptors, P2-receptors, and PO-receptors. About the latter group there is still little known.

Furthermore, the P1- and P2- families are divided in subclasses (Fig. 1.2).(4)

FIGURE 1.2: GENERAL CLASSIFICATION OF PURINERGIC RECEPTORS.

1.1.1. P1-receptors (adenosine receptors)

P1-receptors or adenosine-receptors form a subgroup of the G protein-coupled

receptors (GPCRs). These receptors are widely expressed in the body.(5) Four important

receptor subtypes are characterized: A1, A2A, A2B, and A3. The A1- and A3-receptors couple to

G-proteins of the Gi-family, while A2A- and A2B-receptors couple to Gs-type G-proteins. The A2B-

type is also coupled to the G-proteins of the Gq-family. Gi causes inhibition of the enzyme

adenylate cyclase activity, Gs produces stimulation of the enzyme adenylate cyclase activity,

and Gq modulates the action of phospholipase C. Other effector mechanisms are also

important for the activation of the adenosine-receptors, for example the activation of

Purinergic receptors

P1-receptors

A1

A2A

A2B

A3

P2-receptors

P2X

P2X1

P2X2

P2X3

P2X4

P2X5

P2X6

P2X7

P2Y

P2Y1

P2Y2

P2Y4

P2Y6

P2Y11

P2Y12

P2Y13

P2Y14

PO-receptors

3

phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs), and

extracellular receptor signal-induced kinase (ERK).(4–6)

The endogenous agonist of these receptors is adenosine, while inosine can act as a

partial agonist on the A1- and the A3-receptor.(5) Adenosine is produced intracellularly but due

to active transport there is always a finite amount of adenosine present in the extracellular

spaces, even under basal conditions. Therefore, the adenosine amount in the extracellular

spaces arises from intracellular adenosine or from the breakdown of the adenine nucleotides,

for example from ATP. The concentration of adenosine is low in the body fluid but this

concentration is sufficient to activate the receptors in tissues where these are highly

abundant. The levels of adenosine increase in stress and distress situations and help to

minimize the risk for adverse outcomes by increasing the energy supply and by decreasing

cellular work.(5–7)

P1-receptors can be selectively antagonized by low concentrations of methylxanthines,

such as caffeine and theophylline (Fig 1.3). Because of these properties of the adenosine-

receptor it is obvious that these receptors form interesting targets for drug development.

Nevertheless it is not easy to develop selective agonists or antagonists for P1-receptors.(5–7)

1.1.2. P2-receptors

Like P1-receptors, P2-receptors are membrane-bound receptors. In 1985, Burnstock

and Kennedy made a distinction between two major classes: the P2X- and P2Y-receptors. This

distinction was originally based on pharmacological profiles and tissue distributions. The P2X-

receptor was activated by stable analogs of ATP, namely α,β-methylene ATP and β,γ-

Adenosine Inosine Caffeine

FIGURE 1.1: STRUCTURE OF ADENOSINE (AGONIST OF P1-RECEPTORS), INOSINE (PARTIAL AGONIST OF P1-RECEPTORS) AND CAFFEINE (ANTAGONIST OF P1-RECEPTORS).

4

methylene ATP, while the P2Y-receptor was activated by 2-methylthio ATP. α,β-Methylene

ATP and β,γ-Methylene ATP seemed to be less potent agonists on the P2Y-receptor. This

classification into the two groups was also strengthened by the confirmation that the two

different groups of receptors were located on specific tissues. Later on, it was proven that the

heterogeneity in the tissues also could have been caused by different receptor subtypes or

small differences in the structure of the same receptor. That’s why the classification based on

localization of the receptor was no longer tenable. When also other P2-receptor subtypes

were discovered, also the classification based on pharmacological criteria was no longer

tenable. In 1994, it was suggested that the division into the two groups should be based on

the signal transduction mechanisms. It is ascertained that the P2X- and the P2Y-receptors

differ from each other in their receptor-mediated responses. P2X-receptors are ligand-gated

ion-channels for cations while P2Y-receptors are G protein-coupled receptors with 7

transmembrane regions.(1,4,8)

While the P1-receptors bind to adenosine, the P2-receptors mainly bind the purine

nucleotide ATP. ATP plays not only a key role in the cellular metabolism, it can also act as a

potent extracellular messenger by activating the P2-receptor. For example ATP is known to be

an important messenger molecule in the central nervous system (CNS) and in this way it plays

a role in neurotransmission, pain, hormone secretion, and neuroinflammation. ATP is present

in all cells and intracellular organelles, but also in the extracellular spaces of various tissues.

Furthermore, the pyrimidine nucleotide UTP and the dinucleotides ADP and UDP are agonists

of the P2-receptor as well.(9,10)

1.1.1.1 P2X-receptors

Seven genes are founded to code for the seven P2X-receptor subtypes (P2X1-7). These

seven subunits show a resemblance in 40-50% in amino acid sequence and each subunit is

around 400 amino acids in length (except for the P2X7 subunit which consists of 595 amino

acids).(11) Each subunit has two transmembrane domains (TM1 and TM2), separated by a large

extracellular loop consisting of 10 conserved cysteine residues.(12) The presence of only two

transmembrane segments allows to make a distinction between P2X-receptors and other

ligand-gated cation channels.(13) The N-and C-termini are located intracellularly and possess

binding motifs for protein kinases. Different homomeric or heteromeric ion channels can be

5

formed by these 7 subunits. There is biochemical and functional evidence that most likely

triplets of identical P2X-subunits are able to form homomeric ion channels (P2X1-7) and 3

other subunits which are not all identical are able to form heterotrimeric channels (for

example P2X1/5, P2X2/3, P2X4/6)(Fig.1.4).(14)(http://www.sigmaaldrich.com/technical-

documents/articles/biology/rbi-handbook/non-peptide-receptors-synthesis-and-metabolism/p2-receptors-p2x-ion-

channel-family.html)

FIGURE 1.1: STRUCTURE OF A P2X-RECEPTOR SUBUNIT, CONTAINING TWO TRANSMEMBRANE DOMAINS (TM). TM LINES

THE PORE OF THE CHANNEL. THE AMINO AND CARBOXYL TERMINI OF THE SUBUNITS ARE LOCATED INTRACELLULARLY.

THE SUBUNITS POSSESS A CONSERVED PROTEINEKINASE C (PKC) WHICH CAN BE PHOSPHORYLATED. THE EXTRACELLULAR

LOOP CONTAINS 10 CONSERVED CYSTEIN RESIDUS.(15)

P2X-receptors are ATP-gated ion channels for cations. When an agonist (for example

ATP) binds to the receptor, the conformation of the receptor changes and the ion channel

through which ions can pass opens or closes. Those ions (Na+, K+, and mostly Ca2+) are able to

modulate the cell function by depolarization. The P2X5-receptor is an exception because it

also shows a permeability for chloride ions.(10,13)

As mentioned before, all the subtypes of the P2X-receptor are activated by ATP.

Despite for a few exceptions ADP and AMP are not active. There are very few agonists which

are selective for one of the subtypes.(4)

6

P2X-receptors are widely distributed in excitable and non-excitable cells of

vertebrates. For example in neurons, glia, epithelia, endothelia, bone, muscle, and

hemopoietic tissues they can cause a functional response (Addendum 1). It is obvious that

these receptors play a major role in many physiological functions. They are important in

afferent signaling (including pain), regulation of renal blood flow, vascular endothelium, and

inflammatory responses.(14)

1.1.1.2 P2Y-receptors

P2Y-receptors are metabotropic receptors which belong to the family of G protein-

coupled receptors. So far, eight types of P2Y-receptors have been identified. P2Y1, P2Y2, P2Y4,

P2Y6 bind to Gq/11, activate phospholipase C, and, in this way, mobilize intracellular Ca2+. P2Y12,

P2Y13, P2Y14 bind to Gi, inhibit adenylate cyclase and subsequently reduce the levels of cAMP.

P2Y11 can bind to both Gq/11 and Gi. Eventually the cellular effects of binding with an agonist

arise by influencing different pathways such as inhibition of N-type voltage-gated Ca2+

channels in neurons and endocrine cell lines, activation of K+ channels in neurons, and

activation of Cl- channels in airway epithelium.(2)

The different kind of subtype receptors are distributed over a great variety of tissues.

The receptors are found in the neuronal system, in immune cells, in platelets, in the liver, in

the kidney and bladder, on the skin, in epithelial cells, and so on. In general, the P2Y-receptors

are involved in platelet aggregation, immune regulation, regulation of fluxes in airway

epithelia, and smooth muscle cell proliferation.(6)

P2Y-receptors are targets for many of the pharmaceutical drugs that are used in clinical

trials at the moment. One of the most famous drugs interacting with the P2Y-receptor is

Clopidrogel (Plavix®). It is a platelet aggregation inhibitor that blocks the P2Y12-receptor and

in this way reduces the risks on myocardial infarction, stroke, and mortality in patients with

cardiovascular diseases.(16)

7

1.2 P2X3-RECEPTOR

1.2.1 The P2X3-receptor - agonists/antagonists

As mentioned before, the P2X3 receptor belongs to the family of P2X-receptors. It is

an ionotropic receptor made up of 3 P2X3-subunits, which form functional homo-oligomeric

channels. Studies have shown the expression of the homomeric P2X3-receptor in dorsal roots

ganglia neurons, in myenteric neurons, in the heart, and in the cochlea. Since P2X3-receptors

are mainly found in primary afferent neurons, it is well-known that they play a role in various

neuropathic, inflammatory, and visceral pain conditions. There is also a P2X2/3-receptor that

has overlapping expression with the P2X3-receptor. This receptor consists of 2 units of P2X3-

monomer combined with one P2X2-monomer.(17,18)(http://www.sigmaaldrich.com/technical-

documents/articles/biology/rbi-handbook/non-peptide-receptors-synthesis-and-metabolism/p2-receptors-p2x-ion-

channel-family.html)

Next to the activation of the receptor by ATP, the P2X3-receptor can also be activated

by other nucleotides (analogs). By measuring the agonist-evoked currents on the human P2X3-

receptor, it was concluded that 2-methylthio ATP was the most potent agonist. ATP is slightly

less potent, followed by α,β-methylene ATP, CTP, and β,γ-methylene ATP with the same

effectiveness as ADP. Other compounds such as UTP, GTP, AMP, and adenosine showed no

significant responses on the P2X3-receptor (Fig. 1.5).(19)

8

Suramin and PPADS (Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid) were

the first identified P2X3-antagonists. Their poor pharmacokinetic properties, as well as the

lack of selectivity and potency make these compounds unattractive for further developments

as drugs. Different P2X3-antagonist are currently investigated for the treatments of chronic

pain. RO-3, which combines high affinity and selectivity for the P2X3-receptor represents an

important step towards the discovery of novel drug-like P2X-antagonists (Fig. 1.6).(19,20)

1.2.2 The P2X3 receptor and nociception

1.2.2.1 The extracellular role of ATP

ATP is found in all living cells and is most known because of its cellular function. It is an

important high-energy molecule because it is able to store and release the energy we need

for different metabolic processes. Next to this intracellular role, the extracellular activities of

ATP and adenosine on the heart and coronary bloods vessels were first reported in 1929. In

1970 it was suggested that ATP plays a role as neurotransmitter or co-transmitter.(10)

It was assumed for a long time that ATP is released from damaged or dying cells, but it

now seems that ATP is released from many cell types, in response to gentle mechanical

FIGURE 1.6: ANTAGONISTS OF THE P2X3-RECEPTOR.

FIGURE 1.5: AGONISTS OF THE P2X3-RECEPTOR

9

disturbance, hypoxia, and some agents. The combination of vesicular exocytosis and connexin

and/or pannexin 1 hemichannels appear to be responsible for the mechanism of ATP transport

from cells.(21)

Lastly, it should be noticed that the released ATP is broken down by ectonucleotidases.

Several enzyme families are involved in the breakdown of ATP (Fig. 1.7).(22)

FIGURE 1.7: SYNTHESIS, STORAGE, RELEASE AND INACTIVATION OF ATP. IN THIS EXAMPLE, THE RELEASED ATP ACTS ON

P2-RECEPTORS ON SMOOTH MUSCLE. ADENOSINE INTERACTS WITH THE P1-RECEPTOR. AFTER BREAKDOWN TO INOSINE,

IT IS REMOVED BY THE CIRCULATION. (22)

1.2.2.2 P2X3-receptors in neural system

The extracellularly released ATP is able to activate the purinergic receptors (P2-

receptors). As mentioned before, P2X-receptors are widely expressed in the CNS, generally

abundant on neurons, astrocytes, oligodendrocytes, and microglia.(10) More specific the P2X3-

receptors are localized dominantly in the subpopulation of small nociceptive sensory nerves

in trigeminal, nodose, and dorsal root ganglia. The majority of neurons that bear P2X3-

receptors belong to the GDNF (glial cell-derived neurotrophic factor)-sensitive population

which means that there is a gene present that is able to promote the survival and

10

differentiations of neurons. P2X3-receptors are co-localized with the vanilloid VR-1 receptor.

This co-expression secures the involvement of the P2X3-receptors in different states of

pain.(23)

1.2.2.3 P2X3-receptors and pain transmission

Due to the localization of the P2X-receptors in the CNS, many studies over the last few

years have shown some evidence for the involvement of P2X-receptors in central and

peripheral pain mechanisms. The P2X2-, P2X3-, P2X4-, P2X6- and P2X7-receptors are the main

P2X-receptors that seem to be involved in pain sensitization. The most important receptors in

pain sensitization are the P2X3-receptors.(14,24)

The homomeric P2X3-receptor is present in first-order sensory neurons or primary

afferent neurons, located on the dorsal side of the spinal column. These neurons conduct

sensory information from the peripheral sites to the dorsal horn of the spinal cord. The P2X3-

receptors which are present on the nerve endings at the peripheral site touch different tissues

such as the skin, the muscles, and joint. As mentioned before, ATP is released as a result of

different mechanisms such as tissue injury, visceral distention, inflammation, migraine or

sympathetic activation. By binding on the P2X3-receptors on the primary sensory neurons,

ATP causes the direct excitation of the nociceptive primary afferents (non-myelinated C-fibers

and myelinated Aδ-fibers). These neurons transmit signals to lamina II of the dorsal horn.

Second order neurons transmit the pain signals to the brain stem, thalamus, and other regions

in the brain. The brain processes these signals and ensures that pain is sensed (Fig. 1.8).(25,26)

However the relevance in acute tissue injury and inflammation is more limited, ATP

can also play a role in central mechanisms for nociception due to the presence of P2X-

receptors in the dorsal horn of the spinal cord and the brainstem. ATP, released in the spinal

cord, can stimulate presynaptic and/or postsynaptic P2X-receptors. The presynaptical

localization suggests that the binding of ATP can modulate the neurotransmitter release (for

example glutamate and substance P), while the postsynaptical localization indicates that ATP

can play a role in spinal nociceptive transmission. In contrast with the P2X3-receptors, which

are mainly involved in the peripheral mechanism, P2X2, P2X4, P2X6 – receptors and possibly

some other receptors are involved in this central mechanism.(23,27,28)

11

1.2.2.4 The P2X3-receptor and the different pain conditions

Studies with selective antagonists for the P2X3-receptor confirm the fact that the

P2X3-receptor is not only involved in acute pain sensation, but also in different chronic pain

conditions, such as chronic inflammatory pain and neuropathic pain.(28) In contrast to acute

pain, which is normally only present for a short period and acts as a warning signal, chronic

pain is a disease stage that has no identifiable cause and a poor prognosis due to the lack of

treatment possibilities. The discovery that the P2X3-receptor may be involved in this stage of

pain opens a lot of therapeutic perspectives.(23)

Inflammatory pain

Inflammation is a mechanism of self-protection of the body, through which cytokines,

growth factors, and inflammatory mediators are produced.

(http://www.medicalnewstoday.com/articles/248423.php) First of all, these mediators are able to directly

activate nociceptors by interacting with receptors on the nerve terminals. For example, ATP

can be released as a result of cell and tissue damage by mechanochemical stimulation during

the inflammation process and is hereby able to excite the nearby primary sensory nerves by

activation of the P2X3-receptors. Next to this, also indirect activation of the nerve terminals

FIGURE 1.8: THE SENSORY TRANSMISSION PATHWAY: ATP, RELEASED IN THE SKIN, ACTIVATES P2X3-RECEPTORS ON PRIMARY AFFERENT NEURONS. THIS SENSORY INFORMATION IS CONDUCTED TO THE SPINAL CORD OF THE DORSAL HORN. THEREAFTER, THE SIGNALS ARE TRANSMITTED BY THE SECOND ORDER NEURONS TO THE BRAIN.(26)

12

can take place. The inflammatory mediators are also able to indirectly sensitize the terminals

by reducing the transduction threshold. This sensitization is obtained by phosphorylating

some ligand-gated ion channels, such as the TTX-resistant sodium channels, the TRPV1-

receptor, and the P2X-receptors. Thus, different stimuli (for example neuropeptides,

arachidonic acid, and pH during pathologic conditions) are able to enhance the function of the

P2X-receptors by reducing their activation threshold. It is also known that these mediators

influence the neuronal expression of the nociceptors, including the P2X3-receptor, thereby

instating a spontaneous activity of sensory fibers (hyper-responsiveness). Inflammatory pain

usually disappears when the tissue is repaired.(23,29–31)

Neuropathic pain

Damage or disease in the neuronal system can evoke neuropathic pain. Different

investigators have reported that there is an up-regulation of P2X3-receptor and P2X2/3-

receptor expression or function in different models of neuropathic pain. Probably those P2X3-

receptors which are situated on primary afferent nerve terminals in inner lamina II and those

on the trigeminal brainstem sensory nuclei play a role in this state of pain. Normally, the

nociceptors are silent, but after peripheral nerve injury the neurons can become abnormally

sensitive and pathological spontaneous activity can be developed. Various changes in the

activity of somatosensory neurons can occur, such as increased excitability and sensitization.

In this way, the transmitted pain signals are altered and hyperalgesia and allodynia can

arise.(23)

However the underlying mechanisms of the involvement of the P2X3-receptor in

neuropathic pain remain relatively unknown, receptor-dependent cytosolic phospholipase A2

(cPLA2) seems to be an important mediator. P2X3-receptor stimulation after peripheral nerve

injury appears to induce the activity of cytosolic phospholipase A2 in primary afferent sensory

neurons. CPLA2 activation contributes to nerve injury-induced allodynia by releasing different

mediators such as arachidonic acid and lysophospholipids.

Different studies prove that the local administration of agonists show an increase in

nerve injury, resulting in hyperalgesia and allodynia. After the nerve injury, the administration

of selective or non-selective agonists demonstrated a decrease in nerve injury and the

associated neuropathic pain.(34–36)

13

Migraine pain

During the last few years evidence for the involvement of the P2X3-receptor in

migraine pain has grown. First of all, it is obvious that ATP, released in this pain condition,

activates P2X3-receptors. Moreover, the released neuropeptide calcitonin gene related

peptide (CGRP) seems to play a role in migraine as potent vasodilator and proinflammatory

agent. In an in vitro model CGRP seems to enhance the P2X3-receptor conductance by

accelerating the recovery of the receptor from desensitization.(23)

1.2.3 Partial agonism and desensitization

As previously mentioned, chronic pain is a state of pain which is very hard to treat.

Lately the P2X3-receptor has been examined as an interesting target to modulate pain. P2X3-

receptors antagonists are being investigated as analgesics in chronic pain situations.(18)

However, it is desirable that such agents don’t cause full suppression of the receptor because

that could produce unwanted side effects or a lack of response to standard painful stimuli.

This is the reason why partial agonism of the P2X3-receptor was suggested for pain

modulation. Partial agonists are able to activate the receptor but can’t cause a maximal

response.(35)

To understand the process of partial agonism on the P2X3-receptor, an important

characteristic of the receptor has to be clarified, namely desensitization. After a short period

of activation the receptor can lose its sensitivity very fast by structural and functional changes.

This conformational change occurs directly when an agonist binds to the receptor while the

channel is still shut.(35) There are indications that the desensitization is dependent on the

conformation of the ATP-pocket and on the structure of the transmembrane domains forming

the ion pore. Even very weak agonists which are not able to evoke a response, can cause

desensitization of the receptor (high affinity desensitization). The ATP-sensitivity recovers very

slowly by inflammatory substances via a multistep process. Studies have shown that the

velocity of the recovery process depends on the nature of the agonist and on the agonist

concentration.(36,37)

Partial agonists are able to block the receptor by desensitization, even in the absence

of prior activation. In this way they can manipulate the pain stimuli. The pain signal becomes

less strong but there is no complete block of the stimuli. This makes it possible that the

14

receptor still can respond to large concentrations of agonists and the defensive pain remains.

Consequently, partial agonism is very interesting in pain modulation.(25)

1.2.4 New purinergic ligands: Adenine-based acyclic nucleotides

1.2.4.1 Binding pocket of P2X3-receptor

Based on previous observations and new experiments, Thomas Riedel et al.

constructed a receptor model of the P2X3-receptor that may be helpful for the study of future

structure-activity relationships. With regard to the natural agonist (ATP), they suggest that the

P2X3-receptor has four possible nucleotide binding segments (NBS), which are located

pairwise at two neighboring subunits (Fig. 1.9). Those nucleotide binding sites consist of

groups of amino acids, rather than of individual amino acids. Lysine seems to be a central

amino acid that may be important in the binding of the purine, the ribose moiety, and the

phosphate chain of ATP. It is suggested that the negatively charged phosphate tail interacts

with positively charged lysine or arginine residues in the binding pocket of the receptor, while

the adenine ring of ATP seems to interact with aromatic phenylalanine residues.(38,39)

FIGURE 1.9: A a: PRESENTS THE EXTRACELLULAR LOOP OF THE HUMAN P2X3-RECEPTOR. 3 INDIVIDUAL SUBUNITS ARE

SEEN. A b: REPRESENTS THE SUPPOSED BINDING SITE AT THE INTERFACE OF TWO NEIGHBORING SUBUNITS,

CONTAINING FOUR NBS. B: NBS 1-2 AND NBS 3-4 ARE LOCATED AT OPPOSITE SITES OF A SINGLE SUBUNIT.(39)

15

1.2.4.2 Synthesis of adenine-based acyclic ligands

Initially, the synthesis of the adenine-based acyclic nucleotides was based on the

former synthesis of xanthine-triphosphate derivatives in which the ribose moiety of ATP was

replaced by an alkyl spacer. Those derivatives were capable of activating the P2X-receptors.

Xanthine has similar structural and electrostatic properties as adenine. As in the case of ATP,

the phosphate residues interact with the positively charged amino acid residues (Lys or Arg).

Also the heterocyclic planar skeleton is important in the binding . The analysis of the structure-

activity relationship (SAR) shows that the position of the N-alkyl phosphate (either on N1 or

N7) is not important for the activation of the receptor. It is suggested that the N1 and N7 alkyl

phosphate xanthines occupy the same conformational space in the binding site of the receptor

because of the flexibility of the alkyl chain. The optimal linker between the xanthine and the

phosphates seems to be ethylene. When the linker is longer than two methylenes it is possible

that the xanthine is shifted from its binding site. Further investigations suggested that the

xanthine N9 group serves as a H-bond acceptor. Steric factors at the C8 position possibly

contribute to inactivity of the compounds (Fig 1.10).(40)

Replacement of the adenosine sugar by 9-alkyl groups, like an ethyl, led to molecules

which show affinity for the P1-receptor and behave as antagonists. The activity can be

influenced by introducing alkynyl chains in the 2- or 8-position. 2-substituted analogues show

a higher affinity for the P1-receptor than 8-substituted analogs (Fig. 1.11).(41)

Both observations led to the development of acyclic nucleotides which are able to

partially activate the receptor. The structure is based on the adenine skeleton and bears a

phosphorylated four-carbon chain at the 9-position, which can mimic the ribose of the natural

FIGURE 1.10: XANTHINE-TRIPHOSPHATE DERIVATIVE WITH ALKYL SPACER ON THE N1 POSTION.

FIGURE 1.11: ADENINE DERIVATIVE WITH A 9-ETHYLGROUP AND AN ALKYNYLGROUP AT THE 2-(R2) OR 8-(R1) POSITION.

16

ligand ATP. In this case, the triphosphate group is present in a flexible chain and not in the

more rigid ribose moiety; probably this fact is responsible for the partial agonist behavior of

the acyclic nucleotides. At least 2 phosphate groups seemed to be essential for activation of

the receptor where the triphosphates showed the highest activity. All the phosphates behave

as much weaker P2X3 agonists than ATP. Many substituents were introduced at C2 such as

chloride, iodine, and amino. The nature of this group influenced the level of receptor

activation and desensitization.

The acyclic nucleotide, bearing a triphosphate group in 9-position and an iodine group

in 2-position showed the best partial agonistic activity at rat P2X3-receptors. (Fig. 1.12).(35)

1.2.5 Instability of ATP and its variants

In vivo ATP is released from both living and dying cells. It is well-known that the

released ATP can be enzymatically degraded by ectonucleotidases. There are several enzymes

which belong to the family of the ectonucleotidases. E-NTPD-ases (ecto-nucleoside-

triphosphate diphosphohydrolases) and E-NPP (ectonucleotide-

pyrophosphatase/phosphodiesterases) hydrolyse ATP into ADP and AMP, while ecto-5’-

nucleotidases are able to hydrolyse AMP into adenosine. The different enzymes have thus

another substrate specificity and product formation. When ATP is degraded, it can act as ADP

on P2Y1-, P2Y12-, and P2Y13-receptors or as adenosine on P1-receptors.(42,43)

Next to this enzymatic hydrolysis, also non-enzymatic hydrolysis can take place. The

phosphate groups are very sensitive to changes in temperature and pressure.

Due to this degradation, there is some evidence that the adenine-based acyclic

nucleotides are not very stable. Because of this instability, more stable analogs are desirable

as drugs acting on P2X3-receptors.

FIGURE 1.12: ADENINE-BASED ACYCLIC NUCLEOTIDES BEARING A PHOSPHORYLATED CARBON CHAIN IN 9-POSITION AND

AN IODINE GROUP IN 2-POSITION.

17

2 OBJECTIVES

In spite of continuous evolving science, the pharmaceutical field falls short to keep up

the increasing need for treatments for chronic pain. In chronic pain situations, the pain itself

has often become pathological. To treat this kind of pain, it is thus necessary to attack the

mechanisms that are leading to the abnormal pain sensitization. Therefore, the aim of this

treatment is to reduce the hyperalgesia leaving intact the normal defensive pain. This is in

contrast with the treatment for acute pain, where rapid action analgesia suppress the

perception of pain arising from tissue injury.

Over the last years, the purinergic receptors have gained a lot of interest as therapeutic

targets for chronic pain. ATP is present in all tissues and cells and seems to play an obvious

role in pathological conditions. The purinergic receptors are able to mediate the signal

functions of nucleotides, and in particular of ATP and hence play a role in the pain

sensitization. In this field, there has been a lot of focus on the P2X3-receptor, which affects

some chronic pain conditions, such as inflammatory pain, visceral pain, cancer pain, and

neuropathic pain.

In this thesis, the main focus of the research lies in the field of neuropathic pain, in

particular on P2X3-receptor modulators. From a series of analogues, the acyclic nucleotide,

composed of a 2-iodo-adenine that is substituted at position 9 with a phosphorylated carbon

chain emerged as the most potent partial agonist for the P2X3-receptor. The goal is to scale

up the synthesis of this compound in order to perform in vivo tests (Fig. 2.1).

FIGURE 2.1: ADENINE-BASED ACYCLIC NUCLEOTIDE WHICH SHOWS MODEST PARTIAL AGONISM TOWARDS THE P2X3-

RECEPTOR.

18

19

3 MATERIAL AND METHODS

3.1 METHODS

3.1.1 Nuclear magnetic resonance (NMR) (http://www.chem.ucla.edu/harding/notes/notes_14C_nmr02.pdf,http://teaching.shu.ac.uk/hwb/chemistry/tutorials/mols

pec/nmr1.htm)

3.1.1.1 The principle of NMR

Nuclear magnetic resonance (NMR) is a spectroscopic technique frequently used to

elucidate and confirm the unique structure of a compound by focusing on the molecular

network of an organic molecule. The most common NMR technique is H-NMR or proton

nuclear magnetic resonance.

The nucleus of an atom consists of protons and neutrons that have the property to

rotate around their axis. The proportion of protons and neutrons in that nucleus assigns a total

spin to the nucleus of the atom. Any nucleus possessing an odd number of protons or neutrons

or possessing an odd number of protons and neutrons, has a spin and thus contains a magnetic

moment. H-NMR focuses on the spinning property of the hydrogen atoms.

The spinning atom is able to generate a magnetic field. In the absence of an external

magnetic field (B0), the created magnetic moments (µ) will be orientated ad random.

Moreover, they have the same energy content. When an external magnetic field is applied,

the nuclei will rearrange themselves with or against the field of the external magnet. Those

two states have a different energy content wherein the parallel nucleus (the one that aligns

with the external magnetic field) has the lowest energy. The energy difference arises because

the positively charged nuclei generate their own magnetic field by moving. When an external

magnetic field is applied, a nucleus of spin I can only take 2I+1 orientations, with differences

from –I to +I. Hereby, I is the nuclear spin quantum number. For the H-nucleus, I is ½ and the

spin can be orientated in two states: parallel (+½) or antiparallel (-½). The energy difference

between the two states is dependent of the applied magnetic field (B0) and is called ΔE. The

greater the strength of the external magnetic field, the larger the difference between the two

energy states (Fig. 3.1).

20

Resonance is the process whereby the nuclei change to their opposite spin due to the

radiation they are submitted to. The nuclei (protons) absorb the energy of the applied

magnetic field, which forces them to rotate around their own spin axis. Absorption and

consequently, a flip of the spin will occur when the energy content of the electromagnetic

radiation is big enough to overcome the energy difference between the parallel and

antiparallel orientation. Since the energy difference between the parallel and antiparallel

state corresponds with a certain frequency, the electromagnetic radiation has to bear an equal

frequency.

However, this frequency is not equal for all the nuclei. An explanation for this should

be found in the fact that nuclear magnetic resonance of ‘naked’ protons doesn’t occur. The

nucleus of an atom is found in an environment (a cloud of electrons) that partly shields the

nucleus from the magnetic field. The greater the electron density around a nucleus, the more

shielded a hydrogen nucleus and the lower the field that a proton will experience. So nuclei

with a big shielding effect sense a smaller magnetic field because of the movement of the

electrons in the molecule that arouse little magnetic fields which counterwork the external

applied magnetic field. Thus, protons in an electron rich environment require a lower

frequency of resonance to change their spin. The resulting difference in frequencies of the

nuclei is very small and is therefore measured together with a reference compound,

tetramethylsilane (TMS). The protons of this molecule are more shielded than the protons of

most other molecules. Consequently, the resonance frequencies of the nuclei are reported as

in how far they are shifted from the reference compound (=chemical shift). When the chemical

shift (δ in ppm) has a lower value than zero, the signal is situated on the right of the signal of

TMS and the protons are more shielded than the protons in TMS. When it has a value higher

FIGURE 3.1: DIFFERENT ENERGY STATES OF A NUCLEUS UNDER THE INFLUENCE OF AN EXTERNAL MAGNETIC FIELD.

21

than zero, the signal lies on the left of the signal of the reference and the protons are less

shielded.

δ = 𝑆ℎ𝑖𝑓𝑡 𝑖𝑛 𝐻𝑧

𝑂𝑝𝑒𝑟𝑎𝑡𝑖𝑛𝑔 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑖𝑛 𝑀𝐻𝑧 δ: chemical shift

Moreover, a proton can feel the spin of the equivalent protons of the adjacent carbon

atom. This creates as it were a coupling between the protons of the neighboring nuclei and

this coupling provides a spin-spin split. This split signal can be explained by the N+1 rule. The

signal will split in N+1 peaks and N is the number of equivalent protons bounded to the

adjacent carbons. The distance between two peaks in a multiplet is the coupling constant or

J. This coupling constant is expressed in Hz and measures the influence of the nuclear spin of

the coupled protons on each other.

3.1.1.2 NMR-spectrum

A NMR-spectrum is a representation of the resonance frequencies of various protons

in function of their chemical environment. Four facts characterizing the spectrum are

important for the determination of the observed organic compound. First of all, the number

of peaks gives an indication of the number of various protons. Second, the chemical shift in

the spectrum gives an insight of the chemical environment. Signal splitting gives an idea of the

number of adjacent nuclei. And lastly, the area under the peak is in proportion with the

number of nuclei that belongs to this peak.

3.1.1.3 Instrumentation

For the recording of all the 1H-NMR spectra, a Varian Mercury 400 MHz spectrometer

was used. The samples were dissolved in DMSO-d6.

3.1.2 Mass spectroscopy (MS)(44)

3.1.2.1 The principle of MS

Likewise NMR, mass spectroscopy is a tool used for the qualitative and quantitative

identification of organic compounds. In a typical procedure of MS, a sample is ionized to

acquire positive or negative charges and some of the molecules, which are present in the

sample, fall apart in molecular ions and/or fragment ions. Eventually, those ions pass through

the mass analyzer and dependent on their m/z ratio they arrive at different parts of the

22

detector. This contact with the detector generates signals which are processed by the

computer.

There are different ways to produce the ions, but over the last few years the

electrospray ionization technique (ESI) has gained a lot of importance. All of the samples in

this thesis are submitted to this ionization technique. ESI is an atmospheric pressure ionization

technique and is seen as a ‘soft ionization’ procedure because the fragmentation is not that

clear. Concrete, in ESI, electrical energy is used to assist the transfer of ions from solution into

the gaseous phase, before they are subjected to mass spectrometric analysis. Firstly, the

prepared sample is pumped through a narrow, stainless steel capillary which is submitted to

a certain voltage and a dispersion of a fine spray of highly charged droplets is formed.

Subsequently, solvent evaporation diminishes the size of the charged particles. Thanks to this

evaporation, the charge density increases until the surface tension is no longer able to support

the charge. The repulsion forces cause the droplets to tear down in several smaller ones until

multiple charged molecules are formed. Eventually the charged sample ions are released from

the droplets and ejected into the mass analyzer (Fig. 3.2).

In order to perform the detection of the ions, a separation on m/z ratios by a mass

analyzer is necessary. Therefore a quadrupole is used. A quadrupole consists of four parallel

cylindrical rods whereby adjacent rods have opposite voltage. Also radio frequencies are

applied between each pair of rods. By alternating the potential of the bars, there arises an

electric field. Ions pass through the bars and the electric forces on the ions cause them to

oscillate. The positive bars are acting as high mass filters by eliminating the ions with a high

m/z value, while the negative bars are acting as low mass filters. Only the ions with a certain

FIGURE 3.2: MECHANISM OF ESI.

23

m/z ratio will pass through the oscillating electrical fields and reach the detectors for a given

ratio of voltages. The other ions are thrown out of their original path by colliding with one of

the rods. Finally, the ions are detected by an electron multiplier (Fig. 3.3).

3.1.2.2 MS-spectrum

After the ionization process, the separation based on the m/z ratios and the detection,

a MS-spectrum is generated. This spectrum gives an idea about the relative abundance as a

function of the mass-to-charge ratio. The amount, as well as the different kinds of the

produced molecular and fragment ions are specific for a certain molecule. That’s why MS is

an important technique used for the identification of a molecule.

3.1.2.3 Instrumentation

Mass spectra were recorded on an HP 1100-MSD series instrument. All measurements

were performed using electrospray ionization (ESI-MS) on a single quadrupole analyzer.

3.2 MATERIALS

Thin layer chromatography (TLC) was performed on precoated TLC plates with silica gel.

Visualization occurred by UV detection.

For column chromatography, silica gel 60 (Merck) was used and ion exchange

chromatography was performed, using Sephadex DEAE A-25, HCO3-.

The used reagents and solvents were purchased from Sigma-Aldrich (Steinheim,

Germany) or Alfa Aesar (Karlsruhe, Germany).

FIGURE 3.3: MECHANISM OF MS.

24

25

FIGURE 4.1: REACTION SCHEME.

4 CHEMISTRY

4.1 REACTION SCHEME

(a) 4-Bromobutylacetate, K2CO3, DMF, 24 h, RT, 68.66 %; (b) isoamyl nitrite, CH2I2, DMF, 4 h, 85 °C, 24.04 %; (c) NH3, 17 h, 120 °C, 68.26 %; (d) POCl3, bis(tri-n-butylammonium) pyrophosphate, triethylammonium bicarbonate buffer, (CH3O)3PO, 1.5 h, RT .

26

4.2 SYNTHESIS OF 9-ACETOXYBUTYL-2-CHLOROADENINE AND 7-ACETOXYBUTYL -2-

CHLOROADENINE

4.2.1 Reaction process

The first step of the synthesis of the adenine-based acyclic compounds consists of the

alkylation of 2-chloroadenine with 4-bromobutylacetate. The N9- (or N7)-atom of the

heterocyclic ring of 2-amino-6-chloropurine attacks the alpha-carbon of 4-bromobutylacetate

in a nucleophilic substitution reaction, more precisely a SN2 reaction. Hereby, the halogen is

responsible for an electron withdrawing effect in the haloalkane, which means that the carbon

obtains a partially positive charge (δ+) while the halogen becomes partially negatively charged

(δ-). The nucleophile (2-amino-6-chloropurine) is attracted by the positive charge of the

carbon atom of the halogen alkane. By this approach the electrons of the carbon will be

pushed even more in the direction of the halogen. The nucleophile moves until it is firmly

attached to the carbon and the leaving group is expelled. Dimethyl formamide (DMF) is used

as a polar aprotic solvent and facilitates the SN2-substitution.

4.2.2 Detailed reaction mechanism

Potassium carbonate is the base that abstracts a hydrogen atom of the N9- (or N7)-

atom and makes the heterocyclic ring negatively charged. The heterocyclic ring reacts as a

nucleophile with the positively charged carbon atom of 4-bromobutylacetate and the bromide

ion behaves as a good leaving group. Removing the proton from both the N7 (3) and N9-atom

(2) causes the formation of both isomers. The N9-isomer 2 is formed in excess because of the

negative inductive effect of the chloride ion that makes the N7-isomer a weaker nucleophile

(Fig. 4.2).

27

4.2.3 Monitoring of the reaction

The reaction completion was monitored by TLC. Two different spots, N7 as the lowest

one and N9 as the spot above were observed. The two isomers were isolated by flash silicagel

chromatography and the formation of compound 2 and 3 was confirmed by 1H-NMR.

4.3 SYNTHESIS OF 9-ACETOXYBUTYL-2-IODO-ADENINE

4.3.1 Reaction process

In the absence of simple and reliable methods for the synthesis of aryl iodides,

Sandmeyer conditions are used for the synthesis of 9-acetoxybutyl-2-iodo-adenine. The

Sandmeyer reaction is a method for replacing the amino group of a primary aromatic amine

with a number of different substituents. In general, it is used to synthesize aryl halides from

aryl diazonium salts. This means that first the diazonium ion salt is produced by reaction with

a nitrite. This salt can subsequently form an aryl halide by displacement with a nucleophile.

Generally the latter reaction is catalyzed by a copper(I) halide. A lot of different nucleophiles

can be used, such as halide anions, thiols, cyanide, etc.

(http://trutholic.wordpress.com/2011/11/27/sandmeyer-reaction/)

FIGURE 4.2: SYNTHESIS OF 9-ACETOXYBUTYL-2-CHLOROADENINE (2) AND 7-ACETOXYBUTYL -2-CHLOROADENINE (3).

28

Most variations of the Sandmeyer reaction consist of using different copper salts, but

also amyl nitrites can be used to halogenate the aromatic ring. Upon reaction of the amyl

nitrite with an aromatic amine in a halogenated solvent, an aromatic species, that is able to

abstract a halogen atom from the solvent, is formed.

(https://www.princeton.edu/~achaney/tmve/wiki100k/docs/Amyl_nitrite.html)

For the halogenation of 9-acetoxybutyl-2-chloro-adenine amyl nitrites are used as the

preferred diazotization agents. The compound is treated with isoamyl nitrite and forms the

diazonium ion after a couple of reallocations. Once the ion is formed, diiodomethane reacts

in a substitution reaction.

4.3.2 Detailed reaction scheme

The amino group of the primary aromatic amine reacts with isoamyl nitrite in an

addition-elimination reaction. After the nucleophilic addition, a temporary intermediate is

formed with a negatively charged oxygen and next the alkoxide acts as leaving group. After a

couple of rearrangements, driven by acid-base reactions with the alkoxide ion, water is

expelled to form a diazonium ion. What happens next, is not fully clarified. On the one hand,

a radical aromatic species can be formed via a single electron transfer. This radical is able to

abstract a halogen atom from the solvent (diiodomethane) to form the halogenated

compound. On the other hand an aromatic carbocation can be formed which is also able to

abstract a halogen atom from the solvent. Eventually, compound 4 is formed (Fig. 4.3).

29

FIGURE 4.3: SYNTHESIS OF 9-ACETOXYBUTYL-2-IODO-ADENINE (4), USING THE SANDMEYER CONDITIONS.

30

4.3.3 Monitoring of the reaction

The reaction completion was checked by TLC. For purification of the compound, flash

silicagel chromatography was used. After the elimination of the impurities, a dark violet color,

originating from diiodomethane, characterized the mixture. Normally diiodomethane can be

removed by sublimation but in spite of different attempts to remove the iodine (rotavapor,

oil pump and slight heating processes), a lot of impurities were still present.

That’s the reason why the purification process during scale-up was altered. The first

elution was performed with cyclohexane, which is a solvent that solutes iodine very well and

in this way removes it from the mixture. After the column, still a lot of impurities were present.

Another column was performed wherein cyclohexane was used again to wash away as much

as possible iodine and then the polarity was increased carefully. The compound eluted at a

ratio of 60:40 (CH2Cl2 - Cyclohexane) but in spite of the careful increase of polarity there were

still minimal impurities. Yet, a more pure and impure fraction were collected. The next

reaction was performed on these fractions, despite the presence of minimal impurities.

Because of difficulties encountered in the next step, it was decided to further purify

compound 4 until there were no impurities anymore (see below). Thus, a final step to obtain

a mixture free from impurities was crystallization from ethyl acetate. Ethyl acetate dissolves

the product moderately and in this manner crystals were formed. After those different

purification steps a pure product was obtained. Eventually the presence of compound 4 was

confirmed with 1H-NMR.

Because of the difficult purification process, the yield of the reaction was much lower

than expected. In the future it will therefore be necessary to adjust the purification process.

A bigger amount of 4 was necessary to perform the following reactions and therefore the

impure product, obtained after the second chromatography was also submitted to

crystallization with ethyl acetate. Against the expectations a pure product was obtained. This

purification step increased the yield of the reaction significantly.

31

4.4 SYNTHESIS OF 2-IODO-9-HYDROXYBUTYLADENINE

4.4.1 Reaction process

The 2-step synthesis of 2-iodo-9-hydroxybutyladenine was performed in a single pot.

The easiest and the first performed reaction is the deacetylation step. The acetate group is

removed by a nucleophilic acyl substitution reaction. Subsequently, the chloride group in the

6-position is replaced by an amino group in a nucleophilic aromatic substitution reaction.

Different attempts were carried out to obtain the desired compound. However, its formation

was always accompanied with the formation of unwanted side products e.g. 7 and 8 (Fig. 4.4).

FIGURE 4.4: COMPOUNDS FORMED AFTER DIFFERENT ATTEMPTS TO OBTAIN 2-IODO-9-HYDROXYBUTYLADENINE.

Attempt 1

First, a test amount of 9-acetoxybutyl-2-iodo-adenine was treated with a saturated

solution of ammonia in methanol at room temperature. Since TLC indicated that no product

was formed after 16 hours, an extra amount of saturated methanol was added. After 24 hours,

only a minor amount of the desired compound was formed and TLC showed the formation of

some side products. Since the deacetylation is seen as an easy and fast step, it was proposed

that one of the spots on the TLC was the compound with the deacetylated hydroxyl group 5.

The second side product was most likely the methoxy derivative 7. After separating the

different fractions with chromatography, the presumption on the presence of compound 7

was confirmed with 1H-NMR (Fig. 4.4). The end product 6 was obtained in a negligible amount.

32

Attempt 2

In a second attempt to obtain 6, a sealed tube fluxed with ammonia was used. The

mixture was heated at 80 °C for 18 hours and then it appeared the reaction was finished. The

end product 6 was formed but a lot of impurities were present. Most likely these impurities

arose because of the solvents that were used for dissolving the starting material, however

ethyl acetate was used to minimize the chance on formation of side products. Also the first

two attempts were carried out on less pure products of 4. Consequently, a lot of salts were

formed which caused a decrease of the yield of the reaction. These salts can also be a reason

for the difficult purification process. Flash silicagel chromatography was performed but the

isolated amount of the product was too low to use it in further reaction steps.

Attempt 3

The next attempt was carried out on a more pure fraction of 4 to evade the formation

of salts and thus of side products. Following literature, another reaction procedure was

proposed, namely a reaction temperature of 120 °C. Furthermore, there was no solvent used

to solve the crude. After 16 hours the reaction completion was checked by TLC and it seemed

that the deacetylated intermediate was still present. The sealed tube with the reaction

mixture was heated again overnight, but now by a lower temperature (80 °C). When the

reaction completion was checked, it seemed that also another product was formed. Probably

the mixture had been reacting too long and also the 2-position of the heterocyclic ring was

substituted with an aminogroup. After purification, two fractions were obtained. One with the

desired compound 6 and one containing the 2,6-diamino analogue 8. 6 was again present in a

negligible amount. In order to optimize the next attempt, a 1H-NMR was performed on the

latter fraction. This 1H-NMR confirmed the made presumption.

33

Attempt 4

In an attempt to exclude the formation of the diamino compound, the reaction was

now performed at room temperature. After two days the reaction completion was checked

but only the intermediate product 5 seemed present. Consequently, the temperature was

gradually increased (from 50°C till 120°C) and the progress of the reaction was checked

regularly. After two days of increasing the temperature, the intermediate compound was still

present. Because there seemed to be no change in the formation of the end product, a MS

was done to confirm the presence of the intermediate compound. This MS pointed out that 5

was indeed present, as well as the final product 6. This led us to proceed the reaction for 3

more hours at 120 °C. In spite of the close monitoring of the reaction, the compound 8 was

formed again. Consequently, the actual amount of end product 6 was again too low to perform

further reactions on.

Attempt 5

In the last attempt to obtain 2-iodo-9-hydroxybutyladenine the reaction was

performed at 120°C. This is because the previous attempts had shown that the actual product

6 was mainly formed at this temperature. After reacting for 17 hours at 120 °C, the product

was formed. Unfortunately, even after 7 hours, compound 8 started to arise. Because there

was still too much product in the deacetylated form, the reaction kept running until 5 wasn’t

present anymore. Finally, a mixture of 6 and 8 was obtained and was submitted to purification.

Conclusion

It can be concluded that it is very challenging to obtain a reaction mixture with mainly

6 as end product. Thus, it is necessary to regulate the conditions in such a way that a selective

replacement of the chloride group on the 6-position is obtained. At first, it seemed that the

reaction temperature of 120 °C was too high because particularly 8 was formed. That’s why in

the next attempt the temperature was increased gradually. However, when it appeared that

the desired compound was mainly formed at a temperature of 120°C instead of a lower

temperature, it was decided to carry out the last attempt at this temperature. Consequently,

to put the crude by a reaction temperature of 120°C for 16-17 hours seemed the best reaction

conditions, however the actual yield was not high as expected.

34

4.4.2 Detailed reaction mechanism

As mentioned before, two reactions take place in the same reaction pot. The

deacetylation step, which consists of a nucleophilic addition-elimination reaction, is the

fastest reaction. Ammonia attacks the carbonyl group and a temporary tetrahedral

intermediate with a negatively charged oxygen arises. This negative charge expels the final

product as a leaving group. Afterwards, ammonia interacts with the 6-position of the

heterocyclic ring in a nucleophilic aromatic substitution reaction. The chloride atom is now

expelled as leaving group (Fig. 4.5).

4.4.3 Monitoring of the reaction

During and after every attempt to obtain 6 the reaction completion was checked by

TLC. When the reaction was finished, the crude was purified with flash silicagel

chromatography. The presence of the compound was confirmed with 1H-NMR.

4.5 SYNTHESIS OF 2-IODO-9-(4-TRIPHOSPHATE-BUTYL)-ADENINE

4.5.1 Reaction process

The synthesis of compound 9 is based on the Yoskihawa reaction. This procedure

allows to phosphorylate unblocked nucleosides with phosphorus oxychloride (POCl3) in

trialkylphosphates leading predominantly to a nucleoside 5’-phosphorodichlorate. The latter

compound can then be transformed in nucleoside 5’-triphosphates using an excess of tri-(n-

butyl)-ammonium pyrophosphate in DMF. Finally, this step is followed by a neutral hydrolysis.

FIGURE 4.5: SYNTHESIS OF COMPOUND 5 AND 6.

35

In this reaction pathway, tiethylammonium bicarbonate buffer is used to open the cyclic

compound.(45)

4.5.2 Detailed reaction mechanism

Firstly, compound 6 is converted to an activated monophosphate. This happens in an

addition-elimination reaction. The nucleophile (compound 6) attacks with its hydroxylgroup

the partial positive charge of the phosphate group. The oxygen becomes negatively charged

during a short time and subsequently the double bond reforms by expelling a chloride atom.

In the next step, an addition-elimination reaction takes place again, now with tri-(n-butyl)-

ammonium pyrophosphate as the nucleophile. A chloride atom is expelled as leaving group.

Thereafter an intramolecular addition-elimination reaction takes place. The last chloride is

expelled and a cyclic intermediate is formed. Eventually, a triethylammonium bicarbonate

buffer is added to obtain hydrolysis of the cyclic compound and in this way, the triphosphate

is formed (Fig. 4.9).

FIGURE 4.6: SYNTHESIS OF 2-IODO-9-(4-TRIPHOSPHATE-BUTYL)-ADENINE (9).

36

4.5.3 Monitoring of the reaction

After adding POCl3, the reaction completion was checked on TLC. Subsequently, tri-(n-

butyl)-ammonium pyrophosphate and triethylammonium bicarbonate buffer were added and

the reaction completion was checked again.

Thereafter, the crude was submitted to purification. It is well-known that the

purification of phosphate compounds is not straightforward. First of all, it should be noticed

that phosphate compounds are very unstable. Changes in temperature or pH can cause a

hydrolysis of the triphosphates in diphosphates and/or monophosphates. This is why it is

necessary to be careful in the purification process. The crude is evaporated till dryness and

hereby the temperature has to be monitored closely. Because there is still DMF, originating

from the tri-(n-butyl)-ammonium pyrophosphate solution and trimethylphosphate present, it

is difficult to completely dry the crude. Since DMF has a boiling point of 153 °C, the crude dried

of the water content was directly charged into the ion exchange column.

In the first purification attempt, ion exchange chromatography was used. Herefore

resin (Sephadex DEAE A-25) was used as stationary phase and NH4HCO3- as mobile phase. To

remove the remaining solvents and possible other impurities the loaded column was washed

with water and thereafter a linear gradient of NH4HCO3- was applied. After the column, the

impurities were not fully removed and that’s why an ion exchange chromatography was

performed again. Instead of the application of a linear gradient of NH4HCO3-, the molarity and

thus the ion force was increased gradually. A pure compound was obtained by a constant

molarity of 0.25 M.

37

5 EXPERIMENTAL SECTION

5.1 SYNTHESIS OF 9-ACETOXYBUTYL-2-CHLOROADENINE

For the formation of 9-acetoxybutyl-2-chloroadenine 2,5 gram (14.7 mmol) 2-amino-

6-chloropurine was used. To the solution of this compound in dry DMF (19 ml) 4-

bromobutylacetate (2.4 ml, 21 mmol) and potassium carbonate (2.5 g, 18.1 mmol) were

added. The reaction mixture was stirred (under dry conditions) at room temperature (21°C)

for 16 hours. Eventually the reaction completion was checked by TLC, eluted with CH2Cl2 –

CH3OH (95:5). Because there was still starting material present, extra 4-bromobutylacetate

(0.6 ml, 4.2 mmol) was added and the mixture stirred for another 8 hours. Eventually the

reaction completion was checked again by TLC and it appeared that the reaction was finished.

On the TLC two different spots were visible. The N7 isomer showed a spot a bit lower than the

N9 isomer because of the difference in their polarity. The mixture was dried with the

evaporator under higher vacuum. Subsequently, to separate the final compound (N9) from its

isomer (N7) a flash silicagel column was used. Therefore the mixture was pre-adsorbed on

silica gel and subsequently purified. The desired compound 2 was eluting at CH2Cl2 – CH3OH

(98,5:1,5). At a ratio of CH2Cl2 – CH3OH (97:3), also the N7 isomer was eluting. The product was

collected in two different fractions: the pure and the impure fraction. After drying with the

rotavapor and the oil pump the yield of the reaction was calculated (68,66%). The presence of

compound 2 and 3 was confirmed with 1H-NMR.

1H-NMR (400 MHz, DMSO-d6): δ 8.13 (s, 1H, H-8), 6.90

(brs, 2H, NH2), 4.05 (t, J=7.2 Hz, 2H, NCH2), 3.98 (t, J=6.4

Hz, 2H, OCH2), 1.96 (s, 3H, CH3), 1.80 (m, 2H, NCH2CH2),

1.54 (m, 2H, OCH2CH2).

1H-NMR (400 MHz, DMSO-d6): δ 8.37 (s, 1H, H-8), 6.62

(brs, 2H, NH2), 4.29 (t, 2H, J=7.2 Hz, NCH2), 3.98 (t, 2H,

J=6.4 Hz, OCH2), 1.96 (s, 3H, CH3), 1.80 (m, 2H, NCH2CH2),

1.54 (m, 2H, OCH2CH2).

38

5.2 SYNTHESIS OF 9-ACETOXYBUTYL-2-IODO-ADENINE

2.871 gram of 2 was dissolved in DMF (30 ml). Thereafter, diiodomethane (13.5 ml,

167.5 mmol) and isoamyl nitrite (4.3 ml, 32 mmol) were added to this solution. The reaction

stirred for 3 hours in an anhydrous heating system (85°C). The reaction completion was

checked by TLC (CH2Cl2 – CH3OH (95:5)). Since there was still starting material present, 1.5 ml

isoamyl nitrite was added and the reaction kept stirring for one hour. Thereafter the reaction

completion was checked again and the reaction appeared to be finished. The volatiles were

removed and flash silicagel chromatography was performed. The first elution was performed

with 100% cyclohexane to remove as much iodine as possible. By increasing the polarity to

90:10 (CH2Cl2 - Cyclohexane), the product together with the impurities started to elute

unexpectedly. Thereafter another chromatography was done. This time the polarity was

increased very slowly. Again 100% cyclohexane was used to remove the iodine. Subsequently

the polarity of the used solvents was increased from 50:50 CH2Cl2 – Cyclohexane to 60:40

CH2Cl2 – Cyclohexane. In the 60:40 proportion, product started to elute. After the comparison

with a reference on TLC it appeared that the desired product 4 was eluting. The same polarity

of the solvent was maintained until all the product was eluted. However, the TLC showed still

a minimum of impurities. Thus, as final purification step crystallization was performed.

Therefore ethyl acetate was used. The crystals were separated from the other fraction by

filtration and now it seemed that a pure product was obtained. The crude was evaporated to

dryness and dried on the oil pump. Because of the presence of still a certain amount of iodine,

the crude was slightly heated in a warm water bad in purpose to obtain a faster elimination.

The yield of the reaction was calculated (9,36 %) and eventually the structure of the compound

was confirmed with 1H-NMR. Because of the extreme low yield of the reaction, a impure

fraction of 4, obtained after the second chromatography, was submitted to crystallization. In

spite of many impurities, a pure product was obtained. This process increased the yield till

24,04 %.

1H-NMR (400 MHz, DMSO-d6): δ 8.64 (s, 1H, H-8), 4.24 (t,

2H, J=7.2 Hz, NCH2), 3.99 (t, 2H, J=6.4 Hz, OCH2), 1.97 (s,

3H, CH3), 1.85 (m, 2H, NCH2CH2), 1.54 (m, 2H, OCH2CH2).

39

5.3 SYNTHESIS OF 2-IODO-9-HYDROXYBUTYLADENINE

A steel vial was cooled by submerging it into liquid nitrogen. Thereafter, N2 was fluxed

into the bomb during 10 minutes. Liquid ammonia was condensed in this bomb through a

system of mercury and potassium hydroxide. This condensing step was followed by adding 4

(0.496 g, 1.26 mmol) into the steel reaction bomb. The mixture was heated at 120°C for 17

hours. To monitor the reaction completion, the vessel was put in liquid nitrogen, the bomb

was opened and a TLC was ran.

When the reaction appeared to be finished, the ammonia was allowed to evaporate at

room temperature and the obtained crude was purified. Crystallization with a mixture of

methanol and ethylacetate was tried but it seemed that it was difficult to separate 6 from 8

via this way. Eventually, after pre-adsorption of the crude on silica gel, flash silicagel

chromatography was implemented. The compound (6) eluted at a ratio of CH2Cl2 - MeOH

(98:2). Two different fractions were collected, a fraction with 6, and a fraction with 6 and 8.

Subsequently, the yield of the reaction was calculated (68.25 %). The presence of the

compound was confirmed with 1H-NMR.

1H-NMR (400 MHz, DMSO-d6): δ 8.06 (s, 1H, H-8), 7.61 (brs,

2H, NH2), 4.44 (t, 1H, J=5.2 Hz, OH), 4.06 (t, 2H, J=7.2 Hz,

NCH2), 3.37 (m,2H, OCH2), 1.77 (m, 2H, NCH2CH2), 1.34 (m,

2H,OCH2CH2).

5.4 SYNTHESIS OF 2-IODO-9-(4-TRIPHOSPHATE-BUTYL)-ADENINE

For the synthesis of 9, a reaction with 100 mg of 6 was performed. The starting material

was dissolved in 6 ml of trimethylphosphate and thereafter the solution was cooled until zero

degrees for 10 minutes. 60 µl of phosphorus oxychloride was added and after 30 minutes the

reaction completion was checked on TLC (CH2Cl2-MeOH 90:10). Because the reaction was not

completely finished, another 60 µl of phosphorus oxychloride (POCl3) was added and the

mixture kept stirring for one hour. A 0.5 M solution of tri-(n-butyl)-ammonium pyrophosphate

was prepared by dissolving tetrasodiumpyrophosphate decahydrate (1.12 g, 2.50 mmol) in 75

ml of water and was left under stirring. To this solution an excess of the Dowex (50x8, 20-50

mesh, proton form; 6.75 ml of the resin (approximately 120 mequiv)) was added and the

40

mixture stirred for 20 more minutes. A mixture of ethanol (10 ml) and tributylamine (1.19 ml,

5 mmol) was taken in a 250 ml flask and cooled in an ice bath and the previous solution was

filtered directly in this flask. The resin was washed repeatedly with water until the filtrate

reached a neutral pH. The residue was evaporated at low temperature (< 35 oC) at high

vacuum and co-evaporated 3 times with ethanol. The residue was again co-evaporated 3 times

with anhydrous DMF. To the obtained residue, 5 ml of anhydrous DMF was added and it was

stored over dried molecular sieves at 0-4 oC. After confirming that the reaction was finished,

7 ml of this solution was added to the mixture. After reacting during 5 minutes, 25 ml of 1 M

triethylammonium bicarbonate was added to quench the reaction. This solution was prepared

by passing carbon dioxide gas into a 1.0 M aqueous solution of triethylamine at 5°C until the

pH of 7.9 was reached. The reaction completion was checked on TLC, eluted with isopropanol

– NH4OH – H2O (55:25:20) and the volatiles were removed as much as possible, hereby closely

monitoring the temperature. The first attempt to purify the crude was with ion exchange

chromatography. As stationary phase, resin was used. The Sephadex® A-25 DEAE

(diethylaminoethyl, weak anion exchanger) resin was left to swell in deionized H2O for 24 h at

4 °C. The supernatant H2O was frequently changed and then filtered over a filter paper without

suction. It was then placed in a 1 M NaHCO3 aqueous solution for 2 days at 4 °C, changing the

supernatant with a fresh solution and manually stirred from time to time. The resin was

filtered and washed with deionized H2O (and then with HCO3-). Finally, the resin was degassed

and ready to be used. After the sample was loaded, impurities and other neutrally charged

compounds (for example DMF and trimethyl phosphate) were removed from the column by

elution with water. Thereafter NH4HCO3- was used as mobile phase and a linear gradient (0 M

– 0.25 M) of NH4HCO3- was applied. TLC showed that the monophosphates eluted first,

followed by the triphosphates. Two fractions were obtained, both characterized with still a lot

of impurities. Therefore, another ion exchange chromatography was performed. The molarity

was kept at 0.25 M and the final compound was obtained.

1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H,

H-8), 4.07 (t, 2H, J = 7.0 Hz, NCH2), 3.85 (m,

2H, OCH2), 1.81 (m, 2H, CH2), 1.49 (m, 2H,

CH2).

31P (D20): δ 6.94 (m), -9.71 (d), -21.54 (t).

41

6 CONCLUSION AND FUTURE PROSPECTIVES

The aim of this thesis was to synthesize an adenine-based acyclic nucleotide (2-iodo-

9-(4-triphosphate-butyl)-adenine) which showed partial agonistic activity at the P2X3-

receptor. The goal was to obtain this compound on bigger scale in order to have a suitable

amount to perform in vivo studies.

In spite of many difficulties during the synthesis, eventually the final compound was

obtained. Unfortunately, the amount in which the triphosphate was isolated was too small to

perform the in vivo tests. The reason for the low overall yield has to be ascribed to the fact

that the yield of the individual reactions was not as high as expected.

In particular the purification of 9-acetoxybutyl-2-iodo-adenine significantly decreased

the yield, leading to necessary future adjustments of the purification process. Despite the fact

that different procedures were explored for the third reaction, we were not capable of

producing the expected amount of product. The presence of two halide atoms on the purine

ring made the selective replacement of the chloride atom in the 6-position very difficult. More

attempts to set up this problematic reaction are thus necessary. Also the purification process

of the final compound was very challenging and decreased the yield of the reaction with a

significant amount.

Future investigations should focus on obtaining the compound in an acceptable yield

in order to perform further tests on. If those in vivo tests contribute to a good result, further

investigations can focus on modifications of the compound in order to synthesize more stable

compounds.

42

43

7 REFERENCES

1. Abbracchio MP, Burnstock G. PURINOCEPTORS : ARE THERE FAMILIES OF P2X AND P2Y PURINOCEPTORS. 1994;7258(94).

2. Bronstein-sitton N. P2Y Receptors : Mediators of Extracellular Nucleotide Actions. 2006;(2):1–3.

3. Fredholm B, IJzerman A, Jacobson K, Klotz K, Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev. 2001 Dec;53(4):527–52.

4. Kügelgen I Von, Bonn D. Pharmacology of mammalian P2X- and P2Y-receptors. 2008;(3):59–61.

5. Fredholm BB. Adenosine receptors as drug targets. Exp Cell Res. Elsevier Inc.; 2010 May 1;316(8):1284–8.

6. Boyer L, Abbracchio MP, Burnstock G, Boeynaems J, Barnard EA, Kennedy C, et al. International Union of Pharmacology LVIII : Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology. 2006;58(3):281–341.

7. Burnstock G. Adenosine triphosphate as a neurotransmitter and neuromodulator. 1998.

8. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998 Sep;50(3):413–92.

9. Burnstock G, Williams M. P2 purinergic receptors: modulation of cell function and therapeutic potential. J Pharmacol Exp Ther. 2000 Dec;295(3):862–9.

10. Tu J, Wang L-P. Therapeutic potential of extracellular ATP and P2 receptors in nervous system diseases. Neurosci Bull. 2009 Mar;25(1):27–32.

11. Abbracchio M, Williams M. Purinergic and Pyrimidinergic signalling Volume 1. 2001.

12. North RA. Molecular physiology of P2X receptors. Physiol Rev. 2002 Oct;82(4):1013–67.

13. Xin L, Yanjuan X, Zhiyuan L. P2X Receptors as New Therapeutic Targets. 2002;13.

14. Surprenant A, North RA. Signaling at purinergic P2X receptors. Annu Rev Physiol. 2009 Jan;71:333–59.

15. Khakh BS. Molecular physiology of P2X receptors and atp signalling at synapses. Nat Rev Neurosci. 2001;2:165–74.

44

16. Weisman GA, Yu N, Liao Z, Gonzaléz F, Erb L, Seye C. P2 Receptors in Health and Disease. Biotechnol Genet Eng Rev. 2006;22:171–95.

17. Kaan TK, Yip PK, Patel S, Davies M, Marchand F, Cockayne D, et al. Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats. Brain. 2010 Sep;133(9):2549–64.

18. Cantin L-D, Bayrakdarian M, Buon C, Grazzini E, Hu Y-J, Labrecque J, et al. Discovery of P2X3 selective antagonists for the treatment of chronic pain. Bioorg Med Chem Lett. Elsevier Ltd; 2012 Apr 1;22(7):2565–71.

19. Garcia-Guzman M, Stuhmer W. Molecular characterization and pharmacological properties of the human P2X3 purinoceptor. 1997;59–66.

20. Lambertucci C, Sundukova M, Kachare DD, Panmand DS, Dal Ben D, Buccioni M, et al. Evaluation of adenine as scaffold for the development of novel P2X3 receptor antagonists. Eur J Med Chem. Elsevier Masson SAS; 2013 Jul;65:41–50.

21. Burnstock G. Introduction and perspective, historical note. Front Cell Neurosci. 2013 Jan;7:227.

22. Burnstock G. Historical review: ATP as a neurotransmitter. Trends Pharmacol Sci. 2006 Mar;27(3):166–76.

23. Wirkner K, Sperlagh B, Illes P. P2X3 receptor involvement in pain states. Mol Neurobiol. 2007 Oct;36(2):165–83.

24. Fabbretti E. ATP P2X3 receptors and neuronal sensitization. Front Cell Neurosci. 2013 Jan;7:236.

25. Ford AP. In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization. Purinergic Signal. 2012 Mar;8:3–26.

26. Nakatsuka T, Gu JG. P2X purinoceptors and sensory transmission. Pflugers Arch. 2006 Aug;452(5):598–607.

27. Chizh B, Illes P. P2X receptors and nociception. Pharmacol Rev. 2001 Dec;53(4):553–68.

28. Gum RJ, Wakefield B, Jarvis MF. P2X receptor antagonists for pain management: examination of binding and physicochemical properties. Purinergic Signal. 2012 Feb;8(Suppl 1):41–56.

29. Dai Y, Fukuoka T, Wang H, Yamanaka H, Obata K, Tokunaga A, et al. Contribution of sensitized P2X receptors in inflamed tissue to the mechanical hypersensitivity revealed by phosphorylated ERK in DRG neurons. Pain. 2004 Apr;108(3):258–66.

45

30. Di Virgilio F, Ferrari D, Idzko M, Panther E, Norgauer J, La Sala A, et al. Extracellular ATP, P2 receptors, and inflammation. Drug Dev Res. 2003 May 23;59(1):171–4.

31. Kidd BL, Urban LA. Mechanisms of inflammatory pain. 2001;87(1).

32. Burnstock G. Purinergic receptors and pain. Curr Pharm Des. 2009 Jan;15(15):1717–35.

33. Sharp CJ, Reeve AJ, Collins SD, Martindale JC, Summerfield SG, Sargent BS, et al. Investigation into the role of P2X(3)/P2X(2/3) receptors in neuropathic pain following chronic constriction injury in the rat: an electrophysiological study. Br J Pharmacol. 2006 Jul;148(6):845–52.

34. Tsuda M, Hasegawa S, Inoue K. P2X receptors-mediated cytosolic phospholipase A2 activation in primary afferent sensory neurons contributes to neuropathic pain. J Neurochem. 2007 Nov;103(4):1408–16.

35. Volpini R, Mishra RC, Kachare DD, Dal Ben D, Lambertucci C, Antonini I, et al. Adenine-based acyclic nucleotides as novel P2X3 receptor ligands. J Med Chem. 2009 Aug 13;52(15):4596–603.

36. Sokolova E, Skorinkin A, Fabbretti E, Masten L, Nistri A, Giniatullin R. Agonist-dependence of recovery from desensitization of P2X(3) receptors provides a novel and sensitive approach for their rapid up or downregulation. Br J Pharmacol. 2004 Mar;141(6):1048–58.

37. Giniatullin R, Nistri A. Desensitization properties of P2X3 receptors shaping pain signaling. Front Cell Neurosci. 2013 Jan;7:245.

38. Riedel T, Wiese S, Leichsenring A, Illes P. Effects of nucleotide analogs at the P2X3 receptor and its mutants identify the agonist binding pouch. Mol Pharmacol. 2012 Jul;82(1):80–9.

39. Bodnar M, Wang H, Riedel T, Hintze S, Kato E, Fallah G, et al. Amino acid residues constituting the agonist binding site of the human P2X3 receptor. J Biol Chem. 2011 Jan 28;286(4):2739–49.

40. Fischer B, Yefidoff R, Major DT, Rutman-Halili I, Shneyvays V, Zinman T, et al. Characterization of “mini-nucleotides” as P2X receptor agonists in rat cardiomyocyte cultures. An integrated synthetic, biochemical, and theoretical study. J Med Chem. 1999 Jul 15;42(14):2685–96.

41. Volpini R, Costanzi S, Lambertucci C, Vittori S, Martini C, Trincavelli ML, et al. 2- and 8-alkynyl-9-ethyladenines: Synthesis and biological activity at human and rat adenosine receptors. Purinergic Signal. 2005 Jul;1(2):173–81.

46

42. Joseph SM, Pifer MA, Przybylski RJ, Dubyak GR. Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation. Br J Pharmacol. 2004 Jul;142(6):1002–14.

43. Jacobson KA, Ivanov AA, de Castro S, Harden TK, Ko H. Development of selective agonists and antagonists of P2Y receptors. Purinergic Signal. 2009 Mar;5(1):75–89.

44. Ho CS, Lam CWK, Chan MHM, Cheung RCK, Law LK, Lit LCW, et al. Electrospray ionisation mass spectrometry: principles and clinical applications. Clin Biochem Rev. 2003 Jan;24(1):3–12.

45. Ludwig J. A New Route to Nucleoside 5’-triphosphates. Acta Biochim Biophys. 1981;16(3-4):131–3.

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm (9-04-2014)

http://trutholic.wordpress.com/2011/11/27/sandmeyer-reaction/ (15-04-2014)

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html (18-02-2014)

http://www.chem.ucla.edu/harding/notes/notes_14C_nmr02.pdf (9-04-2014)

http://www.medicalnewstoday.com/articles/248423.php (7-03-2014)

https://www.princeton.edu/~achaney/tmve/wiki100k/docs/Amyl_nitrite.html (15-04-2014)

http://www.sigmaaldrich.com/technical-documents/articles/biology/rbi-handbook/non-

peptide-receptors-synthesis-and-metabolism/p2-receptors-p2x-ion-channel-family.html (24-

02-2014)

8 ADDENDUM

Addendum 1

Overview of the location of principal P2X receptors expressed by excitable tissues and non-

neuronal cells.(21)