synthesis of new constrained-‐ribose atp derivatives as antagonists

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 Master thesis performed at: GHENT UNIVERSITY UNIVERSITA’ DEGLI STUDI DI FACULTY OF PHARMACEUTICAL SCIENCES Department of Pharmaceutics Laboratory for Medicinal Chemistry CAMERINO SCHOOL OF PHARMACY Medicinal Chemistry Unit Academic year 2015-­‐2016 SYNTHESIS OF NEW CONSTRAINED-­‐RIBOSE ATP DERIVATIVES AS ANTAGONISTS OF P2X3 RECEPTORS Liesa TACK First Master of Drug Development Promoter Prof. dr. apr. S. Van Calenbergh Co-­‐promoter Prof. dr. C. Lambertucci Commissioners Prof. dr. apr. De Vos dr. M. Risseeuw Master thesis performed at: GHENT UNIVERSITY UNIVERSITA’ DEGLI STUDI DI FACULTY OF PHARMACEUTICAL SCIENCES Department of Pharmaceutics Laboratory for Medicinal Chemistry CAMERINO SCHOOL OF PHARMACY Medicinal Chemistry Unit Academic year 2015-­‐2016 SYNTHESIS OF NEW CONSTRAINED-­‐RIBOSE ATP DERIVATIVES AS ANTAGONISTS OF P2X3 RECEPTORS Liesa TACK First Master of Drug Development Promoter Prof. dr. apr. S. Van Calenbergh Co-­‐promoter Prof. dr. C. Lambertucci Commissioners Prof. dr. apr. De Vos dr. M. Risseeuw 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." May 30, 2016 Promoter Author
Prof. dr. S. Van Calenbergh Liesa Tack ABSTRACT An essential molecule present in the human body is adenosine triphosphate (ATP). It plays a crucial role in the cellular metabolism, especially in energy storage and transport. ATP is also involved in the synthesis of nucleic acids, is a neurotransmitter and it is the natural ligand of the purinergic P2 receptors. Purinergic receptors are divided into two big families: the P1 and the P2 receptor family. Those receptors are expressed in all mammalian cells having the biggest distribution in the central nervous system where they are located on nociceptive sensory neurons playing an important role in a lot of pain-­‐related conditions. The P2 receptors can be split into the P2X and the P2Y receptors. P2Y receptors are metabotropic, while P2X receptors are ionotropic. Of the latter exist seven subtypes with the focus of this study on the P2X3 receptor. Due to the involvement of the P2X3 receptor in case of acute pain, inflammatory pain, neuropathic pain, visceral pain, headache and migraine, cancer, and chronic pain, selective and potent ligands for this receptor are of enormous interest. A well known P2X3 receptor antagonist is trinitrophenyl-­‐ATP (TNP-­‐ATP). New P2X3 receptor antagonists are designed based on the structure of TNP-­‐ATP leading to the synthesis of 2’,3’-­‐O-­‐cyclohexylideneadenosine triphosphate. Aiming to obtain new P2X3 receptor antagonists, an analogous molecule with a minimal blockage of the 2’-­‐OH and 3’-­‐OH has been designed and synthesised. Also a stable analogue of 2’,3’-­‐O-­‐cyclohexylideneadenosine which can be achieved by replacing the oxygen atom at the α,β-­‐position by a methylene group was synthesised. Both compounds were synthesised and purified. For the synthesis of 2’,3’-­‐O-­‐
methyleneadenosinetriphosphate, alternative reaction conditions for removing the protection groups were found. For the work-­‐up of 2’,3’-­‐O-­‐cyclohexylideneadenosine α,β-­‐
methylene triphosphate, caution was needed to avoid alteration in chemical structure. SAMENVATTING Een essentiële molecule, aanwezig in het menselijk lichaam, is adenosine trifosfaat (ATP). Deze molecule speelt een cruciale rol in het metabolisme van de cel, voornamelijk bij energieopslag en energietransport. Naast deze functie is ATP ook betrokken bij de synthese van nucleïnezuren, is het een neurotransmitter en is het de natuurlijke ligand van de purinergische P2 receptoren. De purinergische receptoren zijn verdeeld in 2 grote families: de P1 en de P2 receptor familie. Ze worden in alle zoogdiercellen tot expressie gebracht, maar kennen de grootste distributie in het centraal zenuwstelsel waar ze gelokaliseerd zijn op nociceptieve, sensorische neuronen. Dit maakt dat ze van groot belang zijn in heel wat aan pijn gerelateerde condities. De P2 receptoren worden verder onderverdeeld in de P2X en de P2Y receptoren. De P2Y receptoren zijn metabotropische terwijl de P2X ionotropische receptoren zijn. Van de laatste bestaan er zeven subtypes met de focus van dit werk op de P2X3 receptor. Door de betrokkenheid van de P2X3 receptor in acute pijn, inflammatoire pijn, neuropathische pijn, viscerale pijn, hoofdpijn en migraine, kanker en chronische pijn is er veel interesse in selectieve en potente liganden voor deze receptor. Een gekende P2X3 receptor antagonist is trinitrophenyl-­‐ATP (TNP-­‐ATP). Nieuwe P2X3 receptor antagonisten zijn ontworpen, gebaseerd op de structuur van TNP-­‐ATP, aanleiding gevend tot de synthese van 2’,3’-­‐O-­‐cyclohexylideneadenosine trifosfaat. Met als doel nieuwe P2X3 receptor antagonisten te bekomen, wordt een analoog met een minimale blokkade van de 2’-­‐OH en 3’-­‐OH positie ontworpen en gesynthetiseerd. Een tweede uitdaging is de synthese van een stabiel analoog van 2’,3’-­‐O-­‐cyclohexylideneadenosine trifosfaat wat bereikt kan worden door het zuurstofatoom op de α,β-­‐positie te vervangen door een methyleengroep. Beide componenten werden gesynthetiseerd en opgezuiverd. Voor de synthese van de 2’,3’-­‐O-­‐methyleneadenosinetrifosfaat werden alternatieve reactiecondities voor het verwijderen van de protectiegroepen gevonden. Voor het opwerken van 2’,3’-­‐O-­‐
cyclohexylideneadenosine α,β-­‐methylenetrifosfaat was de nodige voorzichtigheid vereist om te vermijden dat er verandering optrad in chemische structuur. ACKNOWLEDGMENT First of all, I want to thank Prof. dr. apr. Serge Van Calenbergh, for giving me the possibility to write my thesis in the medicinal chemistry unit of Camerino. Off course, this would not have been possible without the support of his Italian counterpart Prof. dr. Rosaria Volpini, head off the medicinal chemistry unit of Camerino. Special thanks (or in Italian “grazie mille”) to Prof. dr. Catia Lambertucci who guided me throughout the whole period of my research in Italy up to the very last moment. “Aji”, as steady as a rock, has been my daily support within and outside the laboratory. Taking me to the airport, bringing me chocolates during a hard time, giving advice on my research, and keeping me calm whenever necessary. A friend forever! The lab experience would not have been the same if Alin, Michael, Ilenia, Andrea, Alessandra and Caterina would not have been around. Thanks for working together, laughing together and learning me Italian. Next, I like to thank all the Erasmus, international and Italian students I got to know during my stay in Camerino. Two special friends which I hope to see again are Sofia (I finally have a big sister) and Casey who played the violin with me every week. Last, but not least all my love goes to my parents, my sisters and my friends in Belgium. Not only during my stay in Italy, but already for a long time before they supported me in everything I did, gave me good advice and accepted me for who I am. TABLE OF CONTENTS 1. INTRODUCTION .............................................................................................................. 1 1.1. ADENOSINE-­‐5’-­‐TRIPHOSPHATE ...................................................................................... 1 1.2. THE PURINERGIC RECEPTORS ........................................................................................ 2 1.2.1. History ........................................................................................................... 3 1.2.2. Classification .................................................................................................. 3 1.2.3. The P2X receptor ............................................................................................ 5 1.2.3.1. Molecular physiology ............................................................................... 5 1.2.3.2. Kinetic properties .................................................................................... 6 1.2.3.3. Role in the human body ........................................................................... 6 1.2.3.4. Orthosteric agonists ................................................................................. 8 1.2.3.5. Orthosteric antagonists ........................................................................... 9 1.2.3.6. Allosteric modulation ............................................................................ 10 1.3. THE P2X3 RECEPTOR .................................................................................................... 11 1.3.1. Localisation .................................................................................................. 11 1.3.2. The P2X3 receptor and pathophysiology ...................................................... 11 1.3.3. Orthosteric agonists ..................................................................................... 13 1.3.4. Orthosteric antagonists ............................................................................... 14 1.3.5. Allosteric ligands ......................................................................................... 14 1.4. PERSPECTIVES .............................................................................................................. 14 2. OBJECTIVES .................................................................................................................. 16 3. MATERIALS AND METHODS .......................................................................................... 18 3.1. MATERIALS .................................................................................................................. 18 3.2. METHODS .................................................................................................................... 19 3.2.1. Liquid-­‐liquid ectraction (LLE) ........................................................................ 19 3.2.2. Column chromatography ............................................................................. 19 3.2.2.1. Silica flash column chromatography ..................................................... 20 3.2.2.2. Ion exchange chromatography ............................................................. 20 3.2.3. Thin Layer Chromatography (TLC) ................................................................ 21 3.2.4. Nuclear Magnetic Resonance Spectroscopy (NMR) ...................................... 21 3.2.4.1. Basic Principles ...................................................................................... 21 3.2.4.2. Acquiring the NMR spectrum ................................................................ 22 3.2.5. Massa Spectroscopy (MS) ............................................................................ 23 3.2.5.1. Basic principles ...................................................................................... 23 3.2.5.2. Instrumentation .................................................................................... 23 3.2.5.3. Electrospray ionisation – single quadrupole mass spectrometer ......... 23 3.2.6. Lyophylisation ............................................................................................. 24 4. CHEMISTRY ................................................................................................................... 25 4.1. 2’,3’-­‐O-­‐METHYLENEADENOSINE TRIPHOSPHATE ........................................................ 25 4.1.1. General reaction scheme ............................................................................. 25 4.1.2. Synthetic pathway B .................................................................................... 26 4.1.2.1. Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (2) ..... 27 4.1.2.2. Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)-­‐2’,3’-­‐O-­‐
methyleneadenosine (3) .................................................................................... 28 4.1.2.3. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) ....................................... 30 4.1.2.4. Alternative synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) ..................... 32 4.1.2.5. Conversion of by-­‐products .................................................................... 34 4.1.2.6. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5) ................. 34 4.2. 2’,3’-­‐O-­‐CYCLOHEXYLIDENEADENOSINE-­‐α,β-­‐METHYLENE TRIPHOSPHATE .................. 36 4.2.1. Synthesis of 2’,3’-­‐O-­‐cyclohexylideneadenosine-­‐α,β-­‐methylene triphosphate (9) ......................................................................................................................... 36 4.2.1.1. Reaction mechanism of compound 8 with methylenediphosphonic dichloride and then with tributylammonium phosphate ................................... 36 4.2.2. Work-­‐up of compound 9 .............................................................................. 37 5. EXPERIMENTAL SECTION .............................................................................................. 38 5.1 SYNTHESIS OF 2’,3’-­‐O-­‐METHYLENEADENOSINE TRIPHOSPHATE .................................. 38 5.1.1. Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (2) .............. 38 5.1.2. Synthesis of 2’,3’-­‐O-­‐methylene-­‐5’-­‐O,N6-­‐bis(4-­‐
methoxytriphenylmethyl)adenosine (3) ................................................................ 38 5.1.3. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) ............................................... 39 5.1.4. Alternative synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) ............................. 40 5.1.5. Conversion of by-­‐products ........................................................................... 40 5.1.5.1. Conversion of compound 6 .................................................................... 40 5.1.5.2. Conversion of compound 7 .................................................................... 40 5.1.6. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5) .......................... 40 5.1.6.1. Preparation of bis-­‐(tri-­‐n-­‐butylammonium)pyrophosphate in dry dimethylformamide (DMF) ................................................................................. 40 5.1.6.2. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5) .................. 41 5.2. SYNTHESIS OF 2’,3’-­‐O-­‐CYCLOHEXYLIDENEADENOSINE-­‐α,β-­‐METHYLENE TRIPHOSPHATE ................................................................................................................... 42 5.2.1. Effort 1 ......................................................................................................... 42 5.2.2. Effort 2 ......................................................................................................... 42 5.2.3. Effort 3 ......................................................................................................... 43 6. CONCLUSION ................................................................................................................ 44 7. LITERATURE .................................................................................................................. 45 LIST OF USED ABBREVIATIONS α,β-­‐MeATP α,β-­‐methylene adenosine-­‐5’-­‐triphosphate β,γ-­‐MeATP β,γ-­‐methylene adenosine-­‐5’-­‐triphosphate ADP Adenosine-­‐5’-­‐diphosphate AMP Adenosine-­‐5’-­‐monophosphate AR Adenosine receptors ATP Adenosine-­‐5’-­‐triphosphate BzATP 2’ & 3’-­‐O-­‐(4-­‐benzoyl-­‐benzoyl) adenosine-­‐5’-­‐triphosphate cAMP Cyclic adenosine-­‐5’-­‐monophosphate DCM Dichloromethane DMF Dimethylformamide DMSO-­‐D6 Dimethyl sulfoxide (deuterated) ESI Electrospray ionisation GPCR G-­‐protein coupled receptor I Spin LLE Liquid-­‐liquid extraction MS Mass spectroscopy m/z Mass-­‐to-­‐charge ratio NMR Nuclear magnetic resonance spectroscopy P Purinergic P2XR P2X receptors P2YR P2Y receptors PPADS Pyridoxalphosphate-­‐6-­‐azophenyl-­‐2’,4’-­‐disulphonic acid TEAB Triethylammonium bicarbonate TG Trigeminal ganglion TLC Thin layer chromatography TM Transmembrane TNP-­‐ATP Trinitrophenyl adenosine-­‐5’-­‐triphosphate UDP Uridine-­‐5’-­‐diphosphate UTP Uridine-­‐5’-­‐triphosphate 1. INTRODUCTION 1.1. ADENOSINE-­‐5’-­‐TRIPHOSPHATE Adenosine-­‐5’-­‐triphosphate (ATP) is a multifunctional molecule belonging to the chemical group of the nucleotides. It consists of three phosphate groups esterified to an adenosine at the 5’-­‐OH position, explaining its name: adenosine-­‐5’-­‐triphosphate (B, Figure 1.1). Adenosine exists of two parts: a nucleobase base (a nitrogen-­‐containing heterocyclic ring) and a ribose (a sugar with five carbons). In the case of ATP, the base is an adenine. NH 2
7
NH 2
6
N
5
N
N
1
N
8
N
9
4
2
N
O
5'
-O
O
1'
4'
O
γ
3
HO
P
O
O-
P
N
O
β
N
α
O
O-
P
O
O
O-
2'
3'
OH
OH
OH
A. Adenosine
OH
B. ATP
Figure 1.1: Chemical structure of adenosine (A) and ATP (B). This molecule can be considered as one of the most essential molecules found in the human body. It is present in every cell, where it executes its most important intracellular functions: energy storage and transport, a well established role in cellular metabolism. For this reason, ATP is called in biochemistry the “molecular currency” of intracellular energy transfer. It stores and transports chemical energy within cells. The energy carried by ATP, used to fulfil energy-­‐requiring reactions in the human body, is originating from the break-­‐down of nutrient molecules (e.g. oxidation of fat). The energy currency of life as it is called is referring to the cycle of producing, circulating, and break-­‐down of ATP. How does ATP ‘store’ this energy? Each phosphate group of ATP (B, Figure 1.1) is designated with a Greek letter. Starting from the phosphate closest to the ribose, we refer to these groups as alpha (α), beta (β), and gamma (γ). ATP stores its energy in the bonds between the phosphate groups. Especially, the bound between the β-­‐ and the γ-­‐ phosphate is very rich in chemical energy. 1 If this bound is broken and ATP is converted in adenosine-­‐5’-­‐diphosphate (ADP), this happens with an energy release of 12 kCal/mole. The breakdown or hydrolysis of ATP is catalysed by the enzyme, ATPase. ATP ADP + Pi Also the second phosphate group can be broken. This reaction converts ADP in adenosine-­‐5’-­‐
monophosphate (AMP) (1). ADP AMP + Pi A second intracellular function for ATP is its role in signal transduction pathways in which it provides the phosphate for protein-­‐kinase reactions (1). Besides these intracellular functions, ATP and its breakdown product adenosine exert several other functions. First of all, ATP plays an important role in the synthesis of nucleic acids, the building blocks of DNA and RNA. Secondly, ATP is a neurotransmitter playing a crucial role in intracellular communication and it has a role in biological processes such as muscle contraction, cardiac function, platelet function, vasodilatation, and liver glycogen metabolism. All these functions are mediated by purinergic P1 and P2 receptors, which are located on the surface of many cells: adenosine binds to the P1 receptors while ATP binds to the P2 receptors (2,3). 1.2. THE PURINERGIC RECEPTORS The purinergic receptor family is a large family of receptors expressed in all mammalian cells having the highest distribution in the nervous system (4,5). These receptors are proteins on the surface of the cells binding and responding to adenosine and to ATP. These two molecules are considered as the natural ligands of the purinergic receptors. Adenosine and ATP have purine-­‐type base moieties, explaining the name ‘purinergic receptors’. Today, we know those two molecules are not the only two molecules binding to the purinergic receptors (4). 2 The P2X3 receptor, the receptor highlighted in this work, is a member of the purinergic receptor family. It is mainly located on small nociceptive sensory neurons and plays an important role in primary afferent sensitization in many pain-­‐related conditions (5). 1.2.1. History The discovery, observation, and characterisation of the purinergic receptors was a long and slow process. In 1929, Drury and Szent-­‐Györgyi were the first to notice the effects of adenosine and its phosphorylated derivative AMP on the heart (sinus bradycardia). This observation was the first evidence of the existence of the purinergic receptors (6). In this period purinergic receptors were not yet known under this name. Successively, different physiological roles for adenosine were discovered. Adenosine was found to be a mediator of coronary vasodilation in response to myocardial hypoxia, and to stimulate the formation of cyclic AMP (cAMP) in the brain (4). AMP is also a general cell constituent with many functions, not being exclusive for the heart (6). In 1972, ATP was proposed as the transmitter responsible for non-­‐adrenergic transmission in the gut and bladder. At that time, Burnstock introduced the term ‘purinergic’ (7,8), stating that adenosine and its derivatives bind to a receptor called the adenosine or purinergic receptor. But the existence of this receptor only got accepted after saturable binding sites for radioactive adenosine analogues in the brain were demonstrated. In 1978, Burnstock divided the receptors which bind adenosine and ATP/ADP into two groups: P1 and P2 receptors, respectively. The P stands for purinergic receptor but later, it was discovered that some of the P2 receptors also bind pyrimidines like uridine triphosphate (UTP) and uridine diphosphate (UDP) (4,7). 1.2.2. Classification As mentioned above there is a distinction between P1 and P2 receptors. Adenosine is a cytoprotective modulator, activates P1 receptors family also known as the adenosine receptors (AR). These receptors are G protein-­‐coupled and can be divided in four subtypes: A1, A2A, A2B, and A3 (9). The sub-­‐classification is, among other things which will not be explained here, based on the ability of the receptor to activate or inhibit adenylyl cyclase. A1 and A3 receptors are usually coupled to inhibitory or Gi-­‐proteins, while A2A and A2B receptors 3 stimulate Gs-­‐proteins. The distinction between the A2A and A2B receptors is based on the difference in binding affinity of specific adenosinergic ligands (10). A1 receptors mediate the effect of adenosine on the heart and inhibit nerve cells. A2A receptors are stimulated in case of excessive tissue inflammation and have an anti-­‐
inflammatory effect. They also have anti-­‐fibrotic effects. A2B receptors are similar to A2A receptors but they are not completely identical. They are widespread through the body and promote vasodilatation which is also introduced during the anti-­‐inflammatory A2A-­‐effect. For the A3-­‐subtype conflicting effects are reported. This can be explained by the fact that different concentrations of agonist and different cell stages evoke different effects. It certainly is a key receptor in the stimulation and inhibition of cell growth (11). The P2 purinergic receptor family is a family of receptors which are activated by nucleotides. A nucleotide is a nucleoside (like adenosine) esterified with one, two or three phosphate groups (like ATP). The family is divided into two groups of receptors: the ionotropic1 P2X receptors and the metabotropic2 P2Y receptors (Figure 1.2). The P2Y-­‐receptors (P2YR) present eight sub-­‐types: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14. The native agonists for these receptors are ATP, ADP, UTP, UDP, and UDP-­‐glucose. These receptors are mainly present in the nervous system. They regulate the adaptation of the central nervous system to ischemia, tissue damage, inflammation, and chronic neurodegenerative diseases. P2YR ligands are tested to find their role in the treatment of neuropathic pain, cardiac arrhythmias, diabetes, thrombosis, Parkinson’s disease, rheumatoid arthritis, psoriasis, etc. (13,14). The P2X receptors (P2XR) are composed of three subunits. There are seven different subunits or monomers: P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, and P2X7. Each receptor exists of a functional combination of three monomers. The trimer represents an ion channel that is permeable to Na+, K+, and Ca2+. The activation of the P2X receptor will eventually lead to 1
An ionotropic receptor is a transmembrane molecule that can open or close a channel when a ligand binds the receptor. The channel allows smaller particles to travel in and/or out of the cell. In the case of ionotropic receptors these small particles are ions (12). 2
A metabotropic receptor is linked to so-­‐called G-­‐proteins. When a ligand binds the receptor of the G-­‐
protein, this gets activated, leading to the further activation of other molecules (the second messenger) (12). 4 depolarisation and excitation of cells. The native ligand of these receptors is ATP, although some subtypes are also sensitive to ADP. The receptors are expressed in all mammalian cells and are involved in various physiological and pathophysiological events, for example nerve transmission, pain sensation, and inflammation (3,5,13,15). Figure 1.2: Classification of the purinergic receptors (16). 1.2.3. The P2X receptor 1.2.3.1. Molecular physiology As mentioned in the section ‘classification’, the P2X receptors are a ligand-­‐gated ionotropic receptors, consisting of three monomers or subunits. The combination of 3 monomers is called a trimer. Each monomer has cytoplasmatic amino and carboxyl termini. The cytoplasmatic amino and carboxyl terminus are linked by a cysteine-­‐rich extracellular loop and two transmembrane domains (TM) (A, Figure 1.3). When the monomers are identical, we speak of a homotrimer. An example is the P2X3 receptor. This receptor has three identical subunits. If the monomers are not identical it is a heterotrimer. As example: P2X1/2. Not every combination of monomers gives a functional receptor. The P2X6 subtype does not exist as homotrimer (5). The binding cavity or the orthosteric binding site for ATP is located at the interface of two subunits, build up by five positively charged amino acid residues of both subunits (one 5 arginine and four lysines). The binding of ATP changes the conformation of the inactive receptor to a conformation that makes the receptor active (5,13). A B Figure 1.3: A P2X receptor monomer (A) (17), the architecture of the P2X receptor (B) (18). 1.2.3.2. Kinetic properties Based upon the kinetic properties of the P2X receptor, we can classify those receptors in three groups. The P2X1 and the P2X3 receptors belong to the first group. They are rapidly activated and undergo fast inactivation due to fast desensitization upon prolonged stimulation by agonists. Group two is characterised by inactivation due desensitization which is neither slow nor fast. Group two is the largest group containing P2X2, P2X2/3, P2X1/5, P2X4, and P2X4/6 receptors. Group three or the P2X7 receptors have a very slow or no inactivation at all due to slow or no desensitization (5,15). 1.2.3.3. Role in the human body ATP is the natural ligand of the P2X receptors. Binding of ATP to the P2X receptors activates the ion channel formed by the receptor. Activation can cause opening or closing of the ion channel to which they are linked. This changes the ion concentration in the cell and lead to intracellular effects. All P2X receptors are permeable to small monovalent cations. 6 Some are permeable for calcium or anions. P2X7 receptors can induce an increased permeability to larger organic cations (19). The P2X receptors are found in all mammalian tissues and are involved in a large variety of responses, such as platelet aggregation, cell death, contraction of the smooth muscle cells, etc. (3). However, they are especially involved in nerve transmission, pain sensation, and inflammation (13). The different P2X receptor subtypes will be briefly explained. P2X1 receptors The P2X1 receptor is particularly expressed on smooth muscles and in variety of hollow organs. There is an increased expression of this subtype in obstructed bladder making this subtype a potential target for the treatment of disorders of the bladder. The P2X1 receptor is also present on blood platelets where they have an important role in platelet physiology and haemostasis (5). P2X2 receptors P2X2 receptors are expressed in central and peripheral neurons. They are involved in the regulation of many central nervous system processes like memory and learning. At the periphery, they are involved in pain perception. In addition, these receptors also regulate other types of neuronal neurotransmitter-­‐gated channels (5). P2X3 receptors Both P2X3 and P2X2/3 receptors are present on small nociceptive sensory neurons and play a role in a variety of pain-­‐related diseases which will be discussed later (5). P2X4 receptors These receptors are widely distributed. We can find them on autonomic and sensory ganglia, arterial smooth muscles, osteoclasts, pancreas, kidney, liver, lung, heart, and human B lymphocytes but their functional role is still unclear (5). 7 P2X5 receptors We can find P2X5 receptors in the brain, cardiac muscle, eye, and spinal cord. They are also expressed at high levels in tissues related to the immune system. Also for this subtype of receptors the physiological and pathophysiological function is very unclear (5). P2X6 receptors These receptors always form functional heterotrimers with P2X2 or P2X4. We can find them in the central nervous system (5). P2X7 receptors This subtype of receptors is expressed on immune cells, within the central and peripheral nervous system, on osteoblasts and fibroblasts, and in epithelial cells. The receptors are involved in inflammatory and neuropathic pain (5). 1.2.3.4. Orthosteric agonists ATP and ADP, are well-­‐known natural ligands of the P2X receptors. ATP can bind with all different P2X receptors, but with different potency. The potency is expressed with the EC50, which ranges from 0.056 µM to 4 mM for ATP. ADP only weakly activates the P2X receptors, emphasising the importance of the triphosphate chain. Because ATP is prone to degradation, stable ATP analogues have been designed by replacing the oxygen between two phosphates by a substituted or not-­‐substituted methylene group, as in α,β-­‐methyleneATP (α,β-­‐MeATP) and β,γ-­‐methyleneATP (β,γ-­‐MeATP) (Figure 1.4). According to some studies these derivatives are not selective for a P2X receptor subtype and may even activate one or more P2Y receptors. Other sources state that α,β-­‐MeATP shows a preference for P2X1 and P2X3 and that β,γ-­‐MeATP is most potent at P2X1 (5,13,15). Other agonists are known but will not be further discussed here. Because of the fact that the orthosteric binding site is built up of positively charged amino acid residues, all potent agonists are negatively charged (13). 8 NH 2
N
O
-O
P
O-
O
Y
P
O-
N
O
X
P
O
N
N
O
O-
Figure 1.4: Chemical structure of ATP; X = Y = O (A), α,β-­‐MeATP; X = CH2, Y = O (B) and β,γ-­‐
MeATP; X = O, Y = CH2 (C). OH
OH
1.2.3.5. Orthosteric antagonists At the time of Burnstock’ discovery of purinergic transmission (1981), there were no useful antagonists. Apapim and ANAPP3 did inhibit some of the actions of ATP but not by binding on the P2X receptor (15). Today, we have numerous antagonists belonging to various chemical classes. Examples of antagonists are suramin (a arylpolysulfonate), pyridoxalphosphate-­‐6-­‐azophenyl-­‐2’,4’-­‐disulphonic acid (PPADS) and its isomer isoPPADS, and trinitrophenylATP (TNP-­‐ATP) with the latter being the most potent (Figure 1.5). Only PPADS and isoPPADS are selective for the P2X1 receptor, while the others are non-­‐selective (5). Most orthosteric antagonists are polar and will not penetrate in the central nervous system (13). 9 O
O
O
N
H
N
H
NH
HN
OH
O
NH O
S
OH
O
O
OH
O
O
OH
O
S
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O HN
HO
S
O
S
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S
A. Suramin
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O
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O
O
OH
O
OH
HO
-O
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O-
O
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O
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N
HO
OH
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O2N
S
NO 2
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B. PPADS
C. TNP-ATP
NO 2
Figure 1.5: P2X antagonists suramin (A), PPADS (B) and TNP-­‐ATP (C). 1.2.3.6. Allosteric modulation Many G-­‐protein coupled receptors (GPCRs) have an allosteric binding site, which can modify the binding and actions of molecules acting on the orthosteric binding site (20). The P2X function can be allosterically modulated by ions, steroids, and lipids. Also ethanol can have an influence. The development of positive and negative allosteric modulators can be a promising strategy in developing a treatment for diseases where the P2X receptors have an influence (13). 10 1.3. THE P2X3 RECEPTOR The focus of this thesis lies on the P2X3 receptor. The P2X3 receptor can exist as homotrimer or as heterotrimer with P2X2, P2X4 or P2X6. Due to the highly selective distribution of the P2X3 receptor within the nociceptive system, the receptor is involved in a diverse series of body functions (5). 1.3.1. Localisation The P2X3 receptor is mainly found on small nociceptive sensory neurons. Nociceptive neurons are neurons involved in pain perception. Three types of neuronal structures (relevant for pain) express selectively the P2X3 receptor at high levels: the trigeminal ganglion (TG), the dorsal root ganglion, and the nodose ganglion neurons. The binding of ATP to the P2X3 receptor present on these structures leads to depolarisation and activation of primary afferents. In order to achieve this, a certain concentration of ATP needs to be reached. At all times and in all cells ATP is presented in micromolar to millimolar concentrations, an amount that potentially can activate the P2X3 receptors on nociceptive neurons. But sometimes a higher concentration of ATP is needed to reach the threshold and activate the P2X3 receptor (21). 1.3.2. The P2X3 receptor and pathophysiology The activation of primary afferents, due to the binding of ATP on the P2X3 receptor is seen in a lot of different types of pain conditions and other pathophysiologic conditions. Here follows a brief summary of conditions in which the P2X3 receptor plays an important role. The administration of exogenous ATP or a P2X agonist gives rise to acute pain. Also the local administration of 2’-­‐ & 3’-­‐O-­‐(4-­‐benzoylbenzoyl)ATP (BzATP) on the skin gives pain due the binding on P2X3 receptors present on sensory neurons in the skin. This kind of pain will vanish after several minutes, even with continuous administration, due the rapid desensitization of the P2X3 receptor (21). Secondly, acute and chronic inflammatory pain is caused by a high concentration of extracellular ATP. Damaged cells lose their intracellular ATP, which can cause pain as a result of P2X receptor activation at the site of injury (21). 11 Damage or disease within the nervous system leads to neuropathic pain. First of all, neurons get abnormally sensitive to even low concentrations of ATP. Secondly, there is higher expression of P2X3 receptors in pain relevant neurons (21). Another kind of pain is visceral pain. The distension of visceral organs leads to the release of ATP from epithelial cells leading to an excess of ATP causing the pain sensation. An important example is the urinary bladder, where ATP is important in sensory transmission. The sensory transmission regulates the physiological action of the bladder filling and voiding. When there is an excess of ATP, the functional regulation of the bladder is disrupted and the patient will feel pain (21). Furthermore, the P2X3 receptor is involved in the neural dysfunction in trigeminal ganglia sensory neurons leading to migraine and headache pain (5,21). Cancer tissues express P2X and P2Y receptors, which are involved in tumour related pain (21). Finally, the P2X3 receptor plays a role in chronic cough. The upper and lower airways contain nodose ganglia which express P2X3 receptors. Stimulation, especially hypersensitisation of the receptors plays a key role in airway dysfunctions and their symptoms such as cough (22). 12 Figure 1.6: Pathophysiology of the P2X3 receptor (22). 1.3.3. Orthosteric agonists ATP as the natural ligand activates the P2X3 receptor at (sub)micromolar to millimolar concentrations. The agonist BzATP (Figure 1.7) is more potent than ATP. Other agonists of the P2X3 receptor are 2-­‐MeSATP, A317491, ATPγS, α,β-­‐MeATP, and β,γ-­‐MeATP. Ap4A is known as a partial agonist (5,13). NH 2
N
O
-O
P
O-
O
O
P
O-
O
O
P
N
N
O
O
OH
H
OH
OH
H
H
O
N
H
O
BzATP
Figure 1.7: Chemical structure of BzATP. 13 1.3.4. Orthosteric antagonists The first P2X receptor antagonists were suramin and its derivatives with IC50 values in the micromolar range. Later, a range of other antagonists were found. But it took a while before selective P2X3 receptors were discovered. A-­‐317491 (A, Figure 1.8) was the first potent and selective P2X3 receptor antagonist discovered. It is a non-­‐nucleotide small molecule with nanomolar affinity. Anyway, its ionic nature makes the molecule not suitable as drug because it will not penetrate in the central nervous system. (5,15,23). Today, there exist several new classes of molecules selective for the P2X3 receptor and the P2X2/3 receptor. Roche, the company who is the most active in the field of P2X3 receptor antagonists, has patents on a series of benzamides. Benzamides have a central benzamide ring and two aromatic or heteroaromatic rings in meta-­‐position. Another series of Roche are carboxamide derivatives such as example RO-­‐85 (B, Figure 1.8). A second important player in the field is AstraZeneca. Their most important compound is AZ-­‐2 (C, Figure 1.8). Also Renovis, Merck, Shionogi, and others have patents on several compounds (23,24). N
CO2H
S
N
CO2H
O
HN
HO 2C
NH
Cl
N
O
O
N
O
N
N
N
N
N
N
O
O
A. A-317491
C. AZ-2
B. RO-85
Figure 1.8: Chemical structure of A-­‐317491 (A), RO-­‐85 (B) and AZ-­‐2 (C). 1.3.5. Allosteric ligands A series of allosteric antagonists are known and patented by Roche, mostly derived from trimethoprim, such as RO-­‐3, RO-­‐4, and RO-­‐51 (13). They have a diaminopyrimidine ring and antagonise the P2X3 and the P2X2/3 receptors (23,24). 1.4. PERSPECTIVES A major challenge for researchers is to identify potent and selective ligands. Already two compounds have found their way to the clinic. The first compound who recently ended successful phase II clinical trials is AF-­‐219, a diaminopyrimidine analogue (structure not 14 available). It is an orally bioavailable P2X3 and P2X2/3 antagonist for the treatment of chronic cough and severe pain associated with interstitial cystitis/bladder pain syndrome (5,24). AF-­‐130 is the second molecule. It entered phase I trials on 8 December 2015. Afferent Pharmaceuticals is the company who launched the molecule. It is the leader in the development of P2X3 antagonists for the treatment of poorly managed and common neurogenic disorders. This molecule is evaluated in several conditions such as migraine, visceral pain, treatment-­‐resistant hypertension, and potentially in heart failure (25). 15 2. OBJECTIVES A lot of effort has been taken to elucidate the structure of the different P2X receptors. Crucial was the publication of the crystal structure of the zebrafish P2X4 receptor in the closed resting state in 2009, thereby making molecular modelling studies possible by using this crystal structure as template for homology modelling (26). In a following publication, a new ATP-­‐
bound open state P2X4 receptor crystal structure was obtained (27). These structural data make the understanding of the activation mechanism of the P2X4 receptors possible and permit the development of models for the other P2X receptors. These models are crucial for the structure-­‐based design of new ligands (5). Considerable efforts have been devoted to the design and identification of potent and selective ligands due to the potential therapeutic application of these compounds as drugs. However, for some receptor subtypes there is still a lack of such compounds. Since P2X receptors are promising targets in the treatment of inflammatory and chronic pain, rheumatoid arthritis, osteoarthritis, and urinary bladder dysfunctions, the development of new potent and selective molecules has been of big interest (5). The first objective of this study is designing a selective P2X3 receptor antagonist. In a previous work of the group where I performed this study, new P2X3 antagonists were designed based on the structure of TNP-­‐ATP, a known non-­‐selective P2X3 antagonist. This work obtained 2’,3’-­‐O-­‐cyclohexylideneadenosine triphosphate (A, Figure 2.1). This compound, together with others, was evaluated on native P2X3 receptors from mouse TG sensory neurons using patch clamp recording. It was able to inhibit current responses evoked by the full agonist α,β-­‐MeATP, behaving as a potent antagonist at P2X3 receptors with EC50 in the low nanomolar range (data not published). Hence, with the aim of obtaining new P2X3 antagonists, an analogous molecule with a minimal blockage of the 2’-­‐OH and 3’-­‐OH position is designed and synthesized (B, Figure 2.1). The synthesis and purification of this molecule is the first goal of this study. 16 NH 2
NH 2
N
N
OH
HO
OH
O
N
OH
O
OH
N
HO
O
P
P
P
O
O
O
OH
O
N
OH
O
N
O
P
P
P
O
O
O
O
O
O
N
N
O
O
O
B
A
Figure 2.1: Structure of 2’,3’-­‐O-­‐cyclohexylideneadenosine triphosphate (A), 2’,3’-­‐O-­‐
methyleneadenosine triphosphate (B). The second part of this study involves the design and synthesis of the stable analogue of compound C as a P2X3 receptor antagonist. In fact, this compound bearing a triphosphate chain in its chemical structure is unstable as they could be easily hydrolysed. Their stability can be increased by replacing one of the oxygen atoms in the triphosphate chain with a methylene group. Taking in mind that it is possible to introduce a methylene group preferentially at the α,β-­‐position (B, Figure 1.4) without losing potency of the compound obtained, the α,β-­‐methylene analogue of compound C is chosen for synthesis. Figure 2.2 shows the structure of compound C and the analogue to be synthesized (D, Figure 2.2). NH 2
N
OH
HO
OH
O
N
OH
O
NH 2
OH
N
O
P
P
P
O
O
O
N
N
HO
O
O
O
OH
O
H2
C
N
OH
N
N
O
P
P
P
O
O
O
O
O
O
D
C
Figure 2.2: Structure of 2’,3’-­‐O-­‐cyclohexylideneadenosine triphosphate (C), 2’,3’-­‐O-­‐ cyclohexylideneadenosine-­‐α,β-­‐methylene triphosphate (D). 17 3. MATERIALS AND METHODS 3.1. MATERIALS All solvents and reagents used, are purchased from Fluka Analytica (Steinheim, Germany), Sigma Aldrich (Steinheim, Germany), Alfa Aesar (Karlsruhe, Germany), Carlo Erba Reagenti (Val de Reuil, France), JT Baker (Deventer, Holland) or Analar Normapur (Leuven, Belgium). Silica gel column chromatography is performed with Silica Gel, high purity grade, pore size 60 Å, 230 – 400 mesh particle size, 40 – 63 µm purchased from Fluka Analytica (Steinheim, Germany). For anion exchange chromatography, a DEAE Sephadex A-­‐25 column is used. The matrix is a cross-­‐linked dextran matrix on which functional groups are attached by stable ether linkages (28). Thin Layer Chromatography (TLC) is performed using Silica Gel on aluminium foils with a pore diameter of 60 Å purchased from Fluka Analytical (Steinheim, Germany). They are cut to appropriate size before use. Visualisation is performed by UV-­‐detection at 254 nm and in an iodine chamber. Evaporation of solvents is executed with a Buchï Rotavapor and Buchï waterbath B-­‐480 (Switzerland), connected to a diaphragm vacuum pomp (Wertheim, Germany). All 1H-­‐NMR and 31P-­‐NMR spectra are recorded using a Varian Mercury 400 MHz spectrometer. Solvents used are DMSO-­‐d6 and D2O and all exchangeable protons were confirmed by the addition of D2O. All mass spectra are recorded using an HP 1100-­‐MSD series electrospray ionisation-­‐
single quadrupole mass spectrometer. Instrumentation used for lyophilisation is Labconco FreeZone® 1 Liter Benchtop-­‐Freeze Dry Systems-­‐Model 77400 Series (Kansas City, USA). 18 Melting points are determined with a Buchï Melting Point B450 apparatus (Switzerland) and are uncorrected. 3.2. METHODS 3.2.1. Liquid-­‐liquid extraction (LLE) Liquid-­‐liquid extraction (LLE) is an important separation technique. The basic principle of LLE is the different solubility of a compound in two immiscible solvents. Mostly there is an aqueous phase (water or a buffer) and an organic phase. The compound prefers to be in the phase in which it has the highest solubility. This can be expressed numerically by the partition coefficient: 𝑃 = 𝑐 (𝑜𝑟𝑔𝑎𝑛𝑖𝑐 𝑝ℎ𝑎𝑠𝑒)
𝑐 (𝑎𝑞𝑢𝑒𝑜𝑢𝑠 𝑝ℎ𝑎𝑠𝑒)
with P = partition coefficient; c = concentration (of the compound) (29,30). In case of a lipophilic compound, we prefer a high partition coefficient. In this way, our compound will be preferably present in the organic phase. For a hydrophilic compound, we prefer a low partition coefficient. Another condition to have a successful extraction is a difference in density between the two solvents/phases, so the two phases will form two separated layers. 3.2.2. Column chromatography Column chromatography is a second separation technique. On the one hand it uses a stationary phase which is a solid, an adsorbent or a gel. On the other hand, there is also a liquid mobile phase. The basic principle of column chromatography is the different affinity of compounds/impurities in a mixture to the stationary phase and mobile phase. The speed and the quality of separation depend on the characteristics of the stationary phase and the polarity of the mobile phase (31). An ideal compromise between the activity and polarity needs to be found depending on the characteristics of the compounds to be separated. Normally the mixture to be separated or purified is introduced on the top of the column. Different components of the mixture will run through the column with a different 19 speed. Those with a lower affinity for the stationary phase will move faster and will be eluted first. Those with a higher affinity will move slower and will be eluted later (32). There exist different types of column chromatography. The two types used for this study are silica flash column chromatography and ion exchange chromatography. 3.2.2.1. Silica flash column chromatography Silica flash column chromatography is typically used to purify mixtures. A good column chromatography needs a good preparation and choice of parameters. First of all, an appropriate solvent or mixture of solvents needs to be chosen. The choice of the solvent can be based on literature or can be determined based on the Rf-­‐value of the compound detected by TLC. An ideal solvent gives during detection of the compound on TLC a Rf-­‐value of 0.25 +/-­‐ 0.1. Also, the solvent system should provide the largest difference between the Rf-­‐value for the compound of interest and the Rf-­‐
Figure 3.1: TLC with Rf = a/b. value for the impurity. During elution the solvent or mixture of solvents can be changed gradually. This gradient elution makes it possible to separate the compound from other compounds/impurities present in the mixture. Secondly, a stationary phase needs to be chosen. We will use silica gel packed in a vertical column of glass. Finally, you need to determine the diameter of your column and the amount of silica needed (33). The rule used in the laboratory to determine the diameter is to take the square root of the amount of crude in mg giving the diameter in mm. 3.2.2.2. Ion exchange chromatography Ion exchange chromatography is a popular purification technique used for proteins and other charged molecules. A distinction is being made between cation and anion exchange chromatography. In cation exchange chromatography the negative charged stationary phase attracts positive charged molecules. Anion exchange chromatography is the opposite. The attraction is driven by ionic interaction. The strength of the interaction depends on the 20 number and the localisation of the charges on the molecule and on the stationary phase. When salts are present in the eluent, they too will interact with the stationary phase. This makes that there is no room for the molecules who have the weakest interaction with the stationary phase so they will elute first. Interactions which are stronger need a higher salt concentration before they start to elute. Generally, we are using a linear increasing salt concentration to elute all the components from the column (34). 3.2.3. Thin Layer Chromatography (TLC) Thin Layer Chromatography (TLC) can be used as well as an identifying technique as a semi quantitative method. TLC is based on its ability to separate different compounds in a mixture. Therefore, we need a stationary phase and a mobile phase. The stationary phase is the TLC plate. The mobile phase consists of a solvent or a solvent mixture. The process of TLC happens in a TLC chamber. This chamber maintains a uniform environment, prevents the evaporation of solvents, and keeps the system dust-­‐free. Another important part of the process is a filter paper, moistened in the mobile phase. It is placed inside the chamber and makes sure there is a uniform rise of the mobile phase (35). 3.2.4. Nuclear Magnetic Resonance Spectroscopy (NMR) 3.2.4.1. Basic Principles The main goal of NMR is to determine or to confirm the structure of an organic compound. A good interpretation of the spectrum needs a good understanding of the principles of NMR (36). A first principle considers the spin (I) of the nucleus (consists of neutrons and protons) of an isotope. Each nucleus has a characteristic spin. The two isotopes, to which our interest goes, are 1H (proton) and 31P (phosphorus). Both have a spin I = ½. A nucleus with I = ½ has 2 possible orientations or states, namely + ½ and – ½. These states have, in absence of an external magnetic field, an equal energy level. But when an external magnetic field is applied, both states have different energy levels. How big the difference is between the energy levels, depends on the strength of the external magnetic field. The energy difference is given as a frequency in MHz and will define which frequency of radiation is needed to excite a nucleus to a higher energy level or to bring the nucleus in resonance. 21 A second principle to be understood well is that a nucleus is a spinning charge so it will generate a magnetic field by itself (36,37). Next, you have the principle of spin-­‐spin coupling. For example, during the recording of a 1H-­‐
NMR spectrum, protons at a certain distance will interact with each other. They can alter the field that a proton experiences. Therefore, an altered field will be needed to bring a proton in resonance. This also splits the peak on a spectrum in a multiplet. The rule to determine the multiplicity of a multiplet is the number of equivalent protons in neighbouring atoms plus one. For example, considering the structure of ethanol: CH3CH2OH The peak of the methyl group will be a triplet because the neighbouring atom has two equivalent protons and two plus one makes three or a triplet (37). A last principle to be elucidated is the fact that nuclei of the same type of atom give resonance at different magnetic field values or different frequency values. You don’t expect this because they all have the same spin and generate the same magnetic field. The explanation is found with the electrons surrounding the nuclei in a compound. Electrons are negatively charged particles and will respond to the external magnetic field by generating a secondary field that opposes the external field. The secondary field of the electrons form a shield to the nucleus whereby a higher external magnetic field is needed to achieve resonance of the hydrogen/phosphorus atoms (36). 3.2.4.2. Acquiring the NMR spectrum NMR requires an accurate control of the settings. First of all, the spectrometer must be tuned to a specific nucleus. A sample is placed between the poles of the magnet and is spun. The radio frequency radiation of an appropriate energy is broadcasted into the sample and brings the nuclei to the higher energy level. The nuclei will go back to the lower energy level due emission of the absorbed radiation which gives a resonance signal which is monitored. The spectrum is acquired by varying the magnetic field or by varying the frequency of the radio frequency radiation (36). 22 3.2.5. Mass Spectroscopy (MS) 3.2.5.1. Basic principles Mass spectroscopy (MS) is an analytical technique mainly used to identify an unknown compound within a sample. It can also be used for quantification, elucidation of the structure and chemical properties of a compound or for confirmation of a compound (38). The basic principle of mass spectroscopy is to generate multiple ions from the studied compound, separate them according to the specific mass-­‐to-­‐charge ration (m/z) and to record the relative abundance of each type. The process requires conversion of the compound into gaseous ions, whether or not with fragmentation and detection of the different m/z ratios and their abundance. The result is a mass spectrum of your compound giving the ion abundance in function of the m/z ratio (38). A mass spectrum is the fingerprint of a compound. 3.2.5.2. Instrumentation A first component of a mass spectrometer is an ion source. This source converts your compound into gaseous ions, positively or negatively charged. Some sources will also fragmentise the compound. Secondly, an analyser separates ions with different m/z ratios. Finally, a detector system detects the ions and records their relative abundance. A computer program generates the mass spectrum. A lot of variations exist based on the above setting. A common variation is the following: an ion source produces ions which are separated according their m/z ratio in the analyser. One ion is selected and fragmentised. These fragments are analysed and identified within a second analyser (38,39). 3.2.5.3. Electrospray ionisation -­‐ single quadrupole mass spectrometer The ionization technique used for acquiring the spectra involves electrospray ionisation (ESI). From the sample (which needs to be a solution) an aerosol of charged droplets is generated making use of electrical energy. These droplets will shrink due evaporation of the solvent which makes that the charge density increases. Eventually, this will lead to a coulombic explosion producing smaller droplets. This process is repeated until individual sample ions are generated. ESI is a sensitive, robust and reliable tool (39,40). 23 Figure 3.2: Electrospray ionisation and quadrupole mass analyser (40). The mass analyser used is a single quadrupole. A single quadrupole exists out of four parallel rods which apply varying voltage and radiofrequency potential which makes that ions undergo complex trajectories depending on their m/z ratio. Only a certain ion will have a stable trajectories and will reach the detector (40). 3.2.6. Lyophilisation Lyophilisation is a technique allowing to preserve a product, it is a way of drying. The product is frozen rapidly, turning the water into ice without the formation of small ice crystals. Next, the product is heated under vacuum conditions. When you heat up water in a frozen state under vacuum conditions, the water changes immediately into the gaseous phase (= sublimation). The gaseous phase is removed immediately (41). 24 4. CHEMISTRY 4.1. 2’,3’-­‐O-­‐METHYLENEADENOSINE TRIPHOSPHATE 4.1.1. General reaction scheme In order to obtain the designed compound 5 (Figure 4.1) two possible synthetic pathways can be followed. Pathway A is a two-­‐step reaction, in which adenosine (1, Figure 4.1) is the starting material. In the first step, adenosine is blocked at the 2’,3’-­‐O-­‐position of the sugar moiety to obtain compound 4 (Figure 4.1). In the second step, the phosphorylation of the 5’-­‐OH group of compound 4 is achieved to yield compound 5. Pathway B is a multi-­‐step process. Again, adenosine is the starting material which requires protection of the 5’-­‐OH and 6-­‐NH2 groups, ribose blocking at the 2’,3’-­‐O-­‐position, removing of the protection groups of the 5’-­‐OH and 6-­‐NH2 groups, and phosphorylation of 5’-­‐OH to obtain compound 5. NH 2
NH 2
N
N
N
N
N
Pathway A
HO
N
N
O
N
HO
-O
O
O-
P
O
O
O-
P
N
N
N
O
O
O-
4NH4+
O
O
OH
1
O
P
O
O
OH
NH 2
N
O
O
5
4
Pathway B
O
O
HN
HN
N
N
O
N
N
N
N
O
O
N
O
O
O
O
O
OH
OH
N
2
3
Figure 4.1: General reaction scheme. Consulting procedures in literature, pathway A is possible by using formaldehyde. However, it is not possible to use the commercially available formaldehyde aqueous solution due to the fact that the presence of water prevents the formation of the product. In Figure 4.2 the reaction mechanism is showed in which water is released explaining why water should be avoided. On the other hand, gaseous formaldehyde is dangerous for health: it is corrosive, 25 toxic and induces respiratory sensitisation (42), hence the second synthetic approach has been used. HO
OH
O+
HO
H
H+
H
H
O
H
O+
O
H
HO
O
H
H
H
H
H
O+
H
H
H
- H 2O
- H+
O
O
H
H
H
O+
O
H
H
O+
HO
H
H
Figure 4.2: Reaction mechanism of Pathway A. 4.1.2. Synthetic pathway B Reaction pathway B is a multi-­‐step process. The synthesis starts with protection of the 5’-­‐OH and the 6-­‐NH2 groups of adenosine (1, Figure 4.3). The resulting compound 5’-­‐O,N6-­‐
bis(4-­‐methoxytriphenylmethyl)adenosine (2, Figure 4.3) is blocked on the ribose at the 2’,3’-­‐
O-­‐position, yielding 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)-­‐2’,3’-­‐O-­‐methyleneadenosine (3, Figure 4.3). From the latter, the protective groups at the 5’-­‐OH and 6-­‐NH2 positions are removed, resulting in 2’,3’-­‐O-­‐methyleneadenosine (4, Figure 4.3). Finally, the phosphorylation of the 5’-­‐OH group provides the desired compound 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5, Figure 4.3). Reaction conditions are derived from literature (43) or from existing in-­‐house procedures. 26 NH 2
NH 2
N
N
N
N
N
N
O
N
d
HO
HO
-O
O
O
1
P
O
O
P
O-
O
O
P
O-
N
N
N
O
O
O-
4NH4+
O
O
OH
OH
NH 2
N
N
O
O
5
4
a
c2 c1
O
O
HN
HN
N
N
O
N
N
N
O
b
N
O
N
O
O
O
OH
N
O
O
OH
2
3
a) Pyridine, 4-methoxytitrylchloride, 50 °C - 60 °C, 1 h
b) NaOH, CH 2Br 2, cetyltrimethylammonium bromide, CH 2Cl 2, H 2O, 70 °C, 24 h
c1) Acetic acid/water (80/20 V/V), 100 °C, 22 h
c2) Acetonnitrile, HCl (0.2 M), room temperature, 24 h
d) Ethyl acetate, POCl 3, bis(tri-n-butylammonium)pyrophosphate, TEAB, room temperature, 6 h
Figure 4.3: Synthetic reaction scheme to obtain compound 5. 4.1.2.1. Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (2) The first step of the synthesis is a nucleophilic substitution reaction of adenosine yielding 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (2, Figure 4.4). The reaction takes place at two positions: at the 5’-­‐OH and 6-­‐NH2 positions of adenosine securing the protection of these two moieties. Protection is needed so that the reagents of the next step of the synthesis only apply on the 2’-­‐OH and 3’-­‐OH positions of the ribose. O
NH 2
N
N
HN
N
N
N
N
HO
O
a
O
OH
O
O
OH
OH
OH
2
1
a) Pyridine, 4-methoxytitrylchloride, 50 °C - 60 °C, 1 h
Figure 4.4: Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (2). 27 N
N
The reaction is executed with 4-­‐methoxytritylchloride and pyridine to keep the reaction neutral. It is important that the reaction mixture doesn’t become acidic to avoid the removal of the protective groups. Pyridine catches the formed protons avoiding the reaction becomes acid. Reaction mechanism of 4-­‐methoxytritylchloride (Figure 4.5) The 5’-­‐OH position is an electron-­‐rich part of adenosine which attacks the electron-­‐
poor carbon atom of 4-­‐methoxytritylchloride. The chlorine atom is a good leaving group and can be eliminated easily. Next, the chloride will deprotonate the new formed molecule leading to the formation of hydrogen chloride which is scavenged by pyridine keeping the reaction mixture neutral. The same reaction mechanism applies also to the reaction with the amino group in 6 position. NH 2
NH 2
HCl
N
N
O
Cl
H
N
N
N
N
O
O
N
O
OH
N
N
O
O
+
OH
OH
OH
NH 2
N
N
H
Cl -
O
N+
N
N
O
O
+
OH
OH
Figure 4.5: Protection reaction mechanism of the 5’-­‐OH group of adenosine with 4-­‐
methoxytritylchloride. 4.1.2.2. Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)-­‐2’,3’-­‐O-­‐methyleneadenosine (3) The second step concerns the blocking of the H 3C
2’-­‐OH and 3’-­‐OH groups of the ribose moiety, yielding 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)-­‐
2’,3’-­‐O-­‐methyleneadenosine (3) obtained by reaction of the starting material compound 2 with 28 CH 3
N+
14
t-Bu
Br -
CH 3
Figure 4.6: Cetyltrimethylammonium bromide. dibromomethane (CH2Br2), sodiumhydroxide (NaOH) and cetyltrimethylammonium bromide (Figure 4.6). For the reaction scheme see Figure 4.7. The reaction is performed in a biphasic media of dichloromethane (CH2Cl2) and water (H2O) and cetyltrimethylammonium bromide acts as a phase transfer catalyst. O
O
HN
N
N
HN
N
N
N
N
O
N
N
O
CH 2Br 2, NaOH
O
O
OH
CH 3
N+
OH
14
t-Bu
2
O
O
H 3C
CH 3
Br -
(cat.)
O
O
3
CH 2Cl 2, H 2O, 70 °C, 24 h
Figure 4.7: Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)-­‐2’,3’-­‐O-­‐methyleneadenosine (3). Phase transfer catalyst mechanism Ionic reactions in biphasic systems consisting of water and an organic solvent are promoted by phase transfer catalysts. These catalysts (or amphiphilics) can be present in the two layers. The proposed mechanism is the phase-­‐extraction mechanism proposed by Starks and is presented in Figure 4.8 (44). Figure 4.8: Starks phase-­‐transfer reaction mechanism (44). 29 When we apply this general scheme specific on the reaction, the reaction mechanism is represented in Figure 4.9. Organic phase
O
O
HN
HN
N
N
N
N
N
N
H 3C
O
O
O
14
t-Bu
O
Br -
CH 2Br 2
compound 2
O
O
CH 3
N+
14
t-Bu
CH 3
CH 3
O
H 3C
N
O
CH 3
N+
N
O-
3
H 3C
2x
OH-
O-
CH 3
N+
14
t-Bu
CH 3
H 3C
H 3C
NaOH
Water phase
+
CH 3
N+
NaBr
14
t-Bu
CH 3
+
CH 3
N+
14
t-Bu
Br -
CH 3
OH-
Figure 4.9: Phase-­‐transfer catalyst mechanism. Reaction mechanism of CH2Br2/NaOH The ionisation of 2’-­‐OH and 3’-­‐OH groups of compound 2 is favoured by the intermediate formation of the ammonium hydroxide which is a stronger base in respect to NaOH. This makes of compound 2 a nucleophilic particle which will attack the electron-­‐poor carbon atom of CH2Br2 with the loss of one bromide. The second bromide leaves when the second nucleophilic position of compound 2 will attack the electron-­‐poor carbon atom of CH2Br2, yielding 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)-­‐2’,3’-­‐O-­‐methyleneadenosine (3). 4.1.2.3. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) After blocking of the 2’-­‐OH and 3’-­‐OH positions follows the removing of the protection groups at the 5’-­‐OH and N6 positions. The reaction is executed in acid conditions by using an aqueous solution of acetic acid. 30 O
HN
NH 2
N
N
N
N
N
N
N
O
N
HO
c1
O
O
O
O
O
O
O
4
3
c1) Acetic acid/water (80/20 V/V), 100 °C, 22 h
Figure 4.10: Synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4). Reaction mechanism of acetic acid in water Acetic acid is ionised by water. The released protons will protonate the 5’-­‐OH and N6 positions. The 4-­‐methoxytriphenylgroup is a good leaving group and will leave resulting in 2’,3’-­‐O-­‐methyleneadenosine (4, Figure 4.10). The reaction mechanism is visualised in Figure 4.11. O
O
O
HO
CH 3
H 2O
N
N
O
CH 3
H
H
N
N
N
N
O+
O
O
O
O
O
O
O
O
NH 2
O
N
N
HO
O
Figure 4.11: Reaction mechanism of acetic acid in water. 31 N
N
+2
O
N+
N
N
O
+ H+
-O
H
HN
O
While executing this reaction, some problems popped up. First of all, the reaction needed much more time in comparison with what was stated in literature. Secondly, the yield of the reaction was very low, around 20%, due to partial deprotection of the starting material and to the formation of by-­‐products (6 and 7, respectively, Figure 4.12). O
HN
N
N
NH 2
N
N
N
N
HO
N
N
O
O
O
O
O
O
O
O
7
6
Figure 4.12: By-­‐product 1 (6), by-­‐product 2 (7). 4.1.2.4. Alternative synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) To avoid formation of side products and to obtain the complete removal of the protection groups of the starting material a different procedure was used. El-­‐Kattan, Y et al. used different conditions: hydrogenchloride (HCl) (0.2 M) in acetonitrile, which gives again acid reaction conditions (Figure 4.13) but in this case the reaction is performed at room temperature. The yield of this reaction is 70 % and there is no formation of by-­‐products (45). 32 O
HN
NH 2
N
N
N
N
N
N
O
N
N
HO
O
c2
O
O
O
O
O
O
4
3
c2) Acetonitrile, HCl (0.2 M), room temperature, 24 h.
Figure 4.13: Alternative synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4). Reaction mechanism of HCl in acetonitrile HCl is ionised by acetonitrile. The released protons will protonate the 5’-­‐OH and N6 positions. The 4-­‐methoxytriphenylgroup is a good leaving group and will leave resulting in 2’,3’-­‐O-­‐methyleneadenosine (4, Figure 4.13). The reaction mechanism is visualised in Figure 4.14. O
O
H
HN
N
N
H + + Cl -
HCl
H
N
N
H
N
N
N
N
O+
O
O
H 3C C N
O
O
O
O
O
O
O
NH 2
O
N
N
HO
O
Figure 4.14: Reaction mechanism of HCl in acetonitrile. 33 N
N
+2
O
N+
O
4.1.2.5. Conversion of by-­‐products In order to recover more final product, also the conversion of side products formed in the deprotection procedure with acetic acid has been performed. Conversion of compound 6 Compound 6 is converted to 2’,3’-­‐O-­‐methyleneadenosine (4), following the same reaction conditions and mechanism as the alternative synthesis of 2’,3’-­‐O-­‐
methyleneadenosine (4) with HCl (0.2 M) in acetonitrile (45). Conversion of compound 7 To de-­‐acetylate compound 7, it has been treated with ammonia (NH3) and methanol (MeOH). Figure 4.15 gives the reaction mechanism. NH 2
NH 2
N
N
N
O
O
MeOH
O
O
N
N
N
N
N
NH 2
N
N
O
N
N
O
-O
HO
O
O
O
O
O
O
H
N
H
H
H
N+
H
H
Figure 4.15: Reaction mechanism of NH3 in MeOH. 4.1.2.6. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5) The last step to obtain the final compound concerns phosphorylation of the 5’-­‐OH position of compound 4 (Figure 4.16). It is a two-­‐step-­‐reaction in which first the monophosphorylation is attempted with POCl3. In the second step the triphosphate is formed by reaction, of the intermediate, with bis(tri-­‐n-­‐butylammonium)pyrophosphate. Reaction conditions are based on reference 46 (46). 34 NH 2
NH 2
N
N
O
N
O
O
N
N
-O
HO
P
d
O
O
N
N
O
P
O-
O
P
O-
N
O
O
O-
4NH4+
O
O
O
5
4
d) Ethyl acetate, POCl 3, bis(tri-n-butylammonium)pyrophosphate, triethylammonium bicarbonate, room temperature, 6 h.
Figure 4.16: Synthesis of 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5). Reaction mechanism POCl3/bis(tri-­‐n-­‐butylammonium)pyrophosphate In a first step, the dichlorophosphoryl derivative of compound 4 is formed. The reaction of this intermediate with pyrophosphate gave the formation of a cyclic intermediate which is split into the triphosphate (Figure 4.17). HCl formed is neutralised with triethylammonium bicarbonate (TEAB) added at the end of the reaction. It is also important to maintain anhydrous conditions during the reaction to avoid the conversion of the reactive phosphorous trichloride to the unreactive phosphoric acid. NH 2
NH 2
N
H
N
H
N
N
N
N
N
Cl
N
N
Cl
P
O
O
O
O
H+
O
O
O
bis(tri-n-butylammonium)pyrophosphate
O
Me
Cl
N
O
Cl -
O
N
O
P
O
O
N
Cl
O+
Cl
O
NH 2
P
Me
Cl
O
Cl
NH +
HO
NH 2
NH 2
N
N
HO
N
O-
OO
N
OO
O
P
P
P
O
O
O
O
N
N
O
P
-O
2
N
O
N
O
O
P
O
P
O
O
HO
O
H
4NH4+
O
O
O
O
Figure 4.17: Reaction mechanism of POCl3/bis(tri-­‐n-­‐butylammonium)pyrophosphate. 35 P
O
O-
Me
O
P
O-
O
H
4.2. 2’,3’-­‐O-­‐CYCLOHEXYLIDENEADENOSINE-­‐α,β-­‐METHYLENE TRIPHOSPHATE 4.2.1. Synthesis of 2’,3’-­‐O-­‐cyclohexylideneadenosine-­‐α,β-­‐methylene triphosphate (9) In order to obtain compound 9, a two-­‐step reaction, like the one described previously, is required starting from 2’,3’-­‐O-­‐cyclohexylideneadenosine (Figure 4.18). First, compound 8 is reacted with methylenediphosphonic dichloride in PO(OCH3)3 as solvent. Once the reaction is complete, by the disappearance of the starting material, the tributylammonium phosphate (nBu3NHPO4H2) is added together with tributylamine (nBu3N) to form 2’,3’-­‐O-­‐
cyclohexylideneadenosine-­‐α,β-­‐methylene triphosphate (9) after work up of the reaction with an aqueous solution of TEAB. TEAB is used to neutralise the protons formed during the reaction (Figure 4.19). It is important to maintain anhydrous conditions during the reaction to avoid the conversion of the reactive methylenediphosphonic dichloride to in the unreactive methylenediphosphonic acid. Reaction conditions and work-­‐up of the reaction are based on existing in-­‐house procedures. NH 2
N
N
NH 2
N
N
O-
N
HO
-O
e
O
O
OO
H2
C
P
P
O
O
O
4Et3NH +
8
N
O
P
O
N
O-
N
O
O
O
9
e) PO(OCH 3) 3, methylenediphosphonic dichloride, nBu 3N, nBu 3NHPO 4H 2, TEAB, room temperature, 4.5 h
Figure 4.18: Synthesis of 2’,3’-­‐O-­‐cyclohexylideneadenosine-­‐α,β-­‐methylene triphosphate (9). 4.2.1.1. Reaction mechanism of compound 8 with methylenediphosphonic dichloride and then with tributylammonium phosphate The 5’-­‐OH position of compound 8 will attack the electron-­‐poor phosphor. Chlorine is a good leaving group and will be removed from the molecule. An analogue mechanism will happen with nBu3NHPO4H2 yielding compound 9. 36 NH 2
NH 2
N
N
O
O
P
P
OH
OH
N
Cl
Cl
N
N
O
O
P
P
OH
OH
N
N
Cl
HO
O
N
O
O
O
O
O
H 3C
O
P
O
O
CH 3
O
O
CH 3
nBu 3N
NH 2
NH 2
N
N
O-O
OO
H2
C
N
O-
P
P
O
O
O
Cl
O
TEAB
O
O
P
P
OH
OH
N
O
O
O
N+
N
O
O
H
4Et3NH +
O
N
O
P
N
N
-O
P
O
OH
OH
Figure 4.19: Reaction mechanism of compound 8 with methylenediphosphonic dichloride and then with nBu3NHPO4H2. 4.2.2. Work-­‐up of compound 9 Previously, the reaction to obtain compound 9 was done by removing the solvent, trimethylphosphate, by extraction with tert-­‐butylmethyl ether. This procedure led to the formation of side products after ion-­‐exchange chromatography that were not present before. From TLC, it became clear that there was no problem during the synthesis of the compound but the work-­‐up of the reaction mixture led to the formation of those side products. Hence, no work up of the reaction was performed and the reaction mixture was directly brought on the column to purify the product. After purification 1H-­‐NMR, 31P-­‐NMR and mass spectroscopy confirmed the presence of the desired molecule. 37 5. EXPERIMENTAL SECTION 5.1. SYNTHESIS OF 2’,3’-­‐O-­‐METHYLENEADENOSINE TRIPHOSPHATE 5.1.1. Synthesis of 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (2) A three-­‐neck round bottom flask is fluxed with N2. To the flask, adenosine (2.000 g, 7.480 mmol), pyridine (15 mL) and 4-­‐methoxytritylchloride (6.929 g, 22.44 mmol) is added. The flask is left to react at 50 °C to 60 °C during one hour. The flask is cooled down into an ice bath. NaHCO3 is added until a pH of 7 to 8 is reached. A liquid-­‐liquid-­‐extraction is executed with dichloromethane (DCM) (3 x 100 mL) and water (100 mL). The organic phase is dried over sodium sulphate, filtered and the solvent is removed by evaporation. Flash column chromatography is performed on the remaining crude (100 % DCM to 97/3 DCM/MeOH). TLC showed the product was not clean, so a second flash column is executed (100 % DCM to 97/3 DCM/MeOH, gradient of 0.5 %), yielding compound 2 (2.135 g, 2.630 mmol, yield of 35.16 %). O
M.P.: 144-­‐164 °C. 1H-­‐NMR (DMSO-­‐d6, 400 MHz) δ 3.17 (d, J=4.8 Hz, 2H, H-­‐5’), 3.69 (s, HN
N
N
O
3H, OCH3), 3.70 (s, 3H, OCH3), 4.03 (q, J=4.4 N
Hz, 1H, H-­‐4’), 4.26 (q, J=5.2 Hz, 1H, H-­‐3’), 4.70 N
(q, J=4.8 Hz, 1H, H-­‐2’), 5.20 (d, J=6.0 Hz, 1H, O
O
OH), 5.51 (d, J=5.6 Hz, 1H, OH), 5.89 (d, J=4.8 OH
OH
Hz, 1H, H-­‐1’), 6.81-­‐6.85 (m, 4H, Ph), 7.17-­‐
2
7.32 (m, 24H, Ph), 7.82 (s, 1H, H-­‐8), 8.33 ppm (s, 1H, H-­‐2). ESI-­‐MS: positive mode m/z 812.2, 834.1. 5.1.2. Synthesis of 2’,3’-­‐O-­‐methylene-­‐5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (3) To a round bottom flask, containing 5’-­‐O,N6-­‐bis(4-­‐methoxytriphenylmethyl)adenosine (2.135 g, 2.630 mmol), DCM (21.04 mL), cetyltrimethylammonium bromide (0.0890 g, 0.2446 mmol) and CH2Br2 (5.5 mL) is added. In a beaker, NaOH (7.889 g, 197.3 mmol) is dissolved in water. This solution is added dropwise to the reaction flask. The flask is left to react under reflux for 24 hours at 70 °C. Liquid-­‐liquid extraction is performed with DCM (3 x 100 mL) and water (100 mL). The organic phase is dried with sodium sulphate, filtered and the solvent is removed by evaporation. Flash column chromatography is performed on the remaining crude 38 (100 % DCM to 99.5/0.5 DCM/MeOH, gradient of 0.25 %). The product is crystallised with ethanol and ethyl acetate, yielding compound 3 (1.781 g, 2.163 mmol, yield of 80.24 %). O
MHz) δ 3.00-­‐3.03 (m, 1H, H-­‐5’), 3.18-­‐3.22 (m, HN
N
1H, H-­‐5’’), 3.68 (s, 3H, OCH3), 3.70 (s, 3H, N
N
O
OCH3), 4.29-­‐4.31 (m, 1H, 4’), 4.88 (dd, J=6.4 N
O
Hz, J=6.0 Hz, 1H, H-­‐3’), 5.10 (s, 1H HCH), 5.12 O
(s, 1H HCH), 5.28 (dd, J=6.0 Hz, J=5.6 Hz, 1H, O
O
M.P.: 126-­‐146 °C. 1H-­‐NMR (DMSO-­‐d6, 400 3
H-­‐2’), 6.22 (d, J=2.4 Hz, 1H, 1’), 6.79 (d, J=8.8 Hz, 2H, Ph), 6.83 (d, J=8.8 Hz, 2H, Ph), 7.11-­‐7.28 (m, 24H, Ph), 7.37 (s, 1H, NH), 7.73 (s, 1H, H-­‐
8), 8.39 ppm (s, 1H, H-­‐2). 5.1.3. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) To a round bottom flask, containing 2’,3’-­‐O-­‐methylene-­‐5’-­‐O,N6-­‐bis(4-­‐
methoxytriphenylmethyl)adenosine (1.304 g, 1.5826 mmol), acetic acid (20.5 mL) and water (5.1 mL) is added. The flask is left to react under reflux for 22 hours at 100 °C. The reaction is cooled down to room temperature before putting the flask in an ice bath. The reaction is neutralised to pH 7 with NaHCO3. Liquid-­‐liquid extraction is performed with ethyl acetate (3 x 100 mL) and water (100 mL). The organic phase is dried with sodium sulphate, filtered and the solvents are removed by evaporation. On the remaining crude flash column chromatography is performed (100 % DCM to 97/3 DCM/MeOH), yielding three products: compound 4 (0.103 g, 0.3688 mmol, yield of 23.30 %), compound 6 (0.216 g) and compound 7 (0.039 g). NH 2
N
N
HO
O
4
N
N
(m, 2H, H-­‐5’), 4.14 (q, J=3.6 Hz, 1H, 4’), 4.88 (dd, J=6.4 Hz, J=6.4 Hz, 1H, H-­‐3’), 5.12 (s, 1H HCH), 5.15 (s, 1H HCH), 5.16 (t, J=5.6 Hz, 1H, OH), 5.29 (dd, J=6.4 Hz, J=6.4 Hz, 1H, H-­‐2’), 6.11 (d, J=3.2 Hz, 1H, 1’), 7.36 (br s, 2H, NH2), 8.14 (s, 1H, H-­‐8), 8.33 ppm (s, O
O
M.P.: 212-­‐214 °C. 1H-­‐NMR (DMSO-­‐d6, 400 MHz) δ 3.49-­‐3.59 1H, H-­‐2). ESI-­‐MS: positive mode m/z 279.9, 301.9; negative mode m/z 278.1, 314.0. 39 5.1.4. Alternative synthesis of 2’,3’-­‐O-­‐methyleneadenosine (4) To a round bottom flask, containing 2’,3’-­‐O-­‐methylene-­‐5’-­‐O,N6-­‐bis(4-­‐
methoxytriphenylmethyl)adenosine (0.530 g, 0.6432 mmol), acetonitrile (6.4 mL) and 0.2 M HCl (3.2 mL) is added. The reaction is left to stir at room temperature for 24 hours. The reaction is neutralised with Et3N to a pH of 6. Liquid-­‐liquid extraction is performed with ethyl acetate (3 x 100 mL) and water (100 mL). The organic phase is dried with sodium sulphate, filtered and the solvent is removed by evaporation. Flash column chromatography is performed on the remaining crude (100 % DCM to 97/3 DCM/MeOH). The product is crystallised with DCM, yielding compound 4 (0.127 g, 0.4548 mmol, yield of 70.71 %). 5.1.5. Conversion of by-­‐products 5.1.5.1. Conversion of compound 6 To the round bottom flask, containing compound 6 (0.216 g, 0.3916 mmol), acetonitrile (3.9 mL) and 0.2 M HCl (3.9 mL) is added. The reaction is left to stir at room temperature for 24 hours. The reaction is neutralised with Et3 to until a pH of 6 is reached. Liquid-­‐liquid extraction is performed with ethyl acetate (3 x 100 mL) and water (100 mL). The organic phase is dried with sodium sulphate, filtered and the solvent is removed by evaporation. The product is crystallised with MeOH, yielding compound 4 and is directly added to the vial containing compound 4. 5.1.5.2. Conversion of compound 7 To the round bottom flask, containing compound 7 (0.039 g, 0.0280 mmol) NH3CH3OH (10 mL) is added. The reaction is left to stir overnight. The solvent is removed from the reaction by evaporation. Further work-­‐up of the reaction is needed, but is never performed. 5.1.6. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5) 5.1.6.1. Preparation of bis-­‐(tri-­‐n-­‐butylammonium)pyrophosphate in dry dimethylformamide (DMF) In a beaker sodium pyrophosphate decahydrate (Na4P2O7.10H2O) (6.692 g, 15 mmol) is dissolved in water (100 mL) and is stirred at room temperature until a clear solution is attained. The solution is passed through a column of activated Dowex 50WX-­‐8 200 mesh, H+ form. Next, the column is washed with deionized water until the pH is neutral. The eluate is 40 collected in a 250 mL flask containing tributylamine (7.14 mL) and EtOH (75 mL) and is stirred in an ice bath. Solvents are removed by evaporation. The remaining product is dissolved in EtOH and the solvent is removed by evaporation. This process is repeated three times with dry DMF, resulting in a thick oil which is dissolved in dry DMF. This is stored in the fridge over activated molecular sieves. 5.1.6.2. Synthesis of 2’,3’-­‐O-­‐methyleneadenosine triphosphate (5) A round bottom flask, containing 2’,3’-­‐O-­‐methyleneadenosine (100 mg, 0.3581 mmol) is fluxed with N2. Ethyl acetate (1.9 mL) and POCl3 (0.1 mL) is added in dry conditions. The reaction is left to stir at room temperature for 6 hours. TLC has to confirm this first step of the reaction is finished. To the reaction mixture bis(tri-­‐n-­‐butylammonium)pyrophosphate (7.2 mL) is added in dry conditions and is left to react for 10 minutes. The flask is placed in nitrogen atmosphere and cold 1 M TEAB (6.2 mL) is added and is left to react for 15 minutes at room temperature. Liquid-­‐liquid extraction is performed with tert-­‐butyl ether (3 x 15 mL) and water (50 mL). The water phase is dried by evaporation and the resulting product is purified with ion exchange chromatography (0 M, from 0.10 M NH4HCO3 to 0.30 M NH4HCO3). Using TLC, the fractions containing the final compound are evaporated, gathered together in three different flasks and co-­‐evaporated until constant weight. The three flasks are sent for lyophilisation and a sample of each for NMR. As the final compound is not yet pure a new ion exchange column chromatography was executed (0 M, from 0.10 M NH4HCO3 to 0.30 M NH4HCO3). Using TLC, two fractions with the final compound are evaporated, co-­‐evaporated and sent for lyophilisation, yielding compound 5 (3 mg, 0.0051 mmol, 1.42 %). Because of the low yield, all dirty fractions are gathered together and a new ion exchange column chromatography is performed (0 M, from 0.10 M NH4HCO3 to 0.30 M NH4HCO3). Fractions, evaluated with TLC, which contains compound 5 are evaporated, co-­‐evaporated and send for lyophilisation, yielding an extra 34 mg of final compound (37 mg, 0.063 mmol, 17.60 %). 41 1
NH 2
N
O
-O
P
O-
O
O
P
O
O
O-
P
N
N
N
O
4NH4+
H-­‐5’), 4.49-­‐4.51 (m, 1H, H-­‐4’), 4.97-­‐5.00 (m, 1H, H-­‐3’), 5.07 (s, 1H HCH), 5.15 (s, 1H HCH), 5.18-­‐5.21 (m, 1H, H-­‐2’), 6.12 (d, J=3.6 Hz, 1H, O
O-
H-­‐NMR (D2O, 400 MHz) δ 4.07-­‐4.13 (m, 2H, H-­‐1’), 8.10 (s, 1H, H-­‐8), 8.30 ppm (s, 1H, H-­‐2). O
31
O
5
P NMR (D2O, 162 MHz) δ -­‐8.30, -­‐10.55, -­‐
21.43 ppm. ESI-­‐MS: negative mode m/z 257.0, 517.8. 5.2. SYNTHESIS OF 2’,3’-­‐O-­‐CYLCOHEXYLIDENEADENOSINE-­‐α,β-­‐METHYLENE TRIPHOSPHATE 5.2.1. Effort 1 A round bottom flask is fluxed with N2 and placed in an ice bath. Compound 8 (100 mg, 0.2870 mmol), PO(OCH3)3 (4.3 mL) and methylenediphosphonic dichloride (144 mg, 0.5758 mmol) is added in dry conditions. The ice bath is removed and the reaction is left to react at room temperature for 4.5 hours. In dry conditions and in the presence of an ice bath, nBu3N (1.6 mL) and nBu3NHPO4H2 (2.9 mL) is added and left to stir for 30 minutes. The reaction is quenched with TEAB (10.7 mL) in dry conditions during 15 minutes. The ice bath is removed and the reaction is left for 1 hour to come at room temperature. When TLC confirms the final compound is present, liquid-­‐liquid extraction is performed with tert-­‐butylmethyl ether (3 x 100 mL) and water (100 mL). TLC is performed and shows an altered compound, so the reaction is interrupted. 5.2.1. Effort 2 A round bottom flask is fluxed with N2 and placed in an ice bath. Compound 8 (100 mg, 0.2870 mmol), PO(OCH3)3 (4.3 mL) and methylenediphosphonic dichloride (144 mg, 0.5758 mmol) is added in dry conditions. The ice batch is removed and the reaction is left to react at room temperature for 4.5 hours. In dry conditions and in the presence of an ice bath, nBu3N (1.6 mL) and nBu3NHPO4H2 (2.9 mL) is added and left to stir for 30 minutes. The reaction is quenched with TEAB (10.7 mL) in dry conditions during 15 minutes. The ice bath is removed and the reaction is left for 1 hour to come at room temperature. When TLC confirms the final compound is present, the reaction mixture is dried using a vacuum pomp. TLC is performed and shows an altered compound, so the reaction is interrupted. 42 5.2.1. Effort 3 A round bottom flask is fluxed with N2 and placed in an ice bath. Compound 8 (100 mg, 0.2870 mmol), PO(OCH3)3 (4.3 mL) and methylenediphosphonic dichloride (144 mg, 0.5758 mmol) is added in dry conditions. The ice batch is removed and the reaction is left to react at room temperature for 4.5 hours. In dry conditions and in the presence of an ice bath, nBu3N (1.6 mL) and nBu3NHPO4H2 (2.9 mL) is added and left to stir for 30 minutes. The reaction is quenched with TEAB (10.7 mL) in dry conditions during 15 minutes. The ice bath is removed and the reaction is left for 1 hour to come at room temperature. When TLC confirms the final compound is present, the reaction mixture is brought on the column and ion exchange chromatography is performed (0 M, from 0.10 M NH4HCO3 to 0.30 M NH4HCO3). TLC is performed to detect in which fractions the final compound is present. These fractions are evaporated, co-­‐evaporated, transferred to a smaller flask and sent for lyophilisation. Performed NMR and mass show the presence of the final compound with still some impurities present. Another ion exchange chromatography should be performed, but by reason of ending the internship this will be executed by someone else. 1
NH 2
N
O-
O-
-O
O
H2
C
P
P
O
O
O
O
O
c-­‐Hex), 1.37-­‐1.41 (m, 2H, c-­‐Hex), 1.48-­‐1.59 (m, 4H, c-­‐Hex), 1.72-­‐1.75 (m, 2H, c-­‐Hex), N
O
P
4Et3NH +
N
O-
N
H-­‐NMR (D2O, 400 MHz) δ 1.21-­‐1.31 (m, 2H, 2.17 (app t, J=20 Hz, 2H, CH2), 3.99 (m, 2H, H-­‐5’), 4.56 (m, 1H, H-­‐4’), 5.08 (m, 1H, H-­‐3’), 5.30 (m, 1H, H-­‐2’), 6.15 (d, J=3.2 Hz, 1H, H-­‐
O
1’), 8.20 (s, 1H, H-­‐8), 8.44 ppm (s, 1H, H-­‐2). 31
9
ppm. ESI-­‐MS: negative mode m/z 291.5, 584.0. P NMR (D2O, 162 MHz) δ 18.26, 8.51, -­‐9.62 43 6. CONCLUSION The first aim of this study was to synthesise a selective P2X3 receptor antagonist with a minimal constrain at the 2’-­‐OH and 3’-­‐OH positions of the sugar moiety. Two possible pathways were presented based on literature and in-­‐house knowledge and procedures to produce the suitable intermediate nucleoside. Among the possible synthetic approach, the synthesis has been performed by using a four step synthetic pathway which include the protection of 5’-­‐OH and 6-­‐NH2 groups of adenosine as methoxytriphenylmethyl derivative. This compound reacted with dibromomethane in a phase transfer catalyst reaction to obtain the 2’,3’-­‐O-­‐methylene derivative which after deprotection with HCl/CH3CN gave the appropriate nucleoside 4. Nucleoside 4 was then phosphorylated in a two step phosphorylation reaction which produced the desired nucleotide 5. Second aim was to synthesise a more stable P2X3 receptor antagonist stabilising the phosphate chain of the triphosphate compound 8 previously synthesised. This higher chemical and enzymatic stability is obtained by replacing the triphosphate chain with a α,β-­‐methylene phonate chain. Problems encountered in the synthesis of this compound during the work up were solved and the desired compound 9 was obtained as confirmed by 1H NMR, 31P NMR and mass analysis. Both molecules are synthesised and purified successful. They will be sent to the University of Trieste (Italy) to evaluate their activity on P2X3 receptors by biological assays. 44 7. LITERATURE 1. New World Encyclopedia contributors. Adenosine triphosphate [Internet]. [Place unknown]: New World Encyclopedia; 15 Feb 2016 [cited 2016 April 04]. Available from: http://www.newworldencyclopedia.org/p/index.php?title=Adenosine_triphosphate&oldid=
994007 2. Agteresch HJ, Dagnelie PC, van den Berg JWO, Wilson JHP Adenosine Triphosphate: Established and Potential Clinical Applications. Drugs. August 1999; 58 (2): 211-­‐232. 3. Kaczmarek-­‐Hájek K, Lörinczi E, Hausmann R, Nicke A. Molecular and functional properties of P2X receptors: recent progress and presisting chanllenges. Purinergic Signalling. 2012; 8: 375-­‐417. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360091/ 4. JM L. Purinergic Receptors. In: Siegel GJ, Agranoff BW, Albers RW, et al., editors. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. [Internet]. 6th edition. Philadelphia: Lippincott-­‐Raven; 1999. Available from: http://www.ncbi.nlm.nih.gov/books/NBK27952/ 5. Lambertucci C, Dal Ben D, Buccioni M, Marucci G, Thomas A, Volpini R. Medicinal Chemistry of P2X Receptors: Agonists and Othosteric Antagonists. Current Medicinal Chemistry. 2015; 22: 915-­‐928. 6. Drury A, Szent-­‐Györgyi A. 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