UNIVERSITÀ DI PISA

(1)

i

UNIVERSITÀ DI PISA

Doctoral School

“Science of Drug and Bioactive Substances”

XXIX cycle

“Development of New Small-Molecules Targeting

DNA Repair or Purinergic System for Therapeutic

or Diagnostic Applications”

Supervisor

Candidate

(2)

ii

PURINES AND PYRIMIDINES SIGNALING ... 1

Historical Perspectives ... 1

G-Protein Coupled Receptors (GPCRs) ... 3

Structure and Transduction Mechanism of P1 Receptors ... 4

Structure and Transduction Mechanism of P2Y Receptors ... 6

Ion Channels (P2X Receptors) ... 7

General Basis of Purinergic Transmission... 9

Source, Metabolism and Release ... 10

Biological Effects ... 12

ANTAGONISTS TOWARDS PURINERGIC SYSTEM ... 17

A1 Adenosine G-Protein Coupled Receptor Antagonists ... 17

A2A Adenosine G-Protein Coupled Receptor Antagonists ... 24

A2B Adenosine G-Protein Coupled Receptor Antagonists ... 32

A3 Adenosine G-Protein Coupled Receptor Antagonists ... 36

P2 Receptors Non-selective antagonists ... 43

P2X1 ATP-Ion Gated Channel Antagonists ... 45

P2Y1 G-Protein Coupled Receptor Antagonists ... 63

P2Y2 G-Protein Coupled Receptor Antagonists ... 64

P2Y4 G-Protein Coupled Receptor Antagonists ... 66

MOLECULAR IMAGING ... 77

PET and Radioligands ... 80

A2B PET RADIOTRACER ... 83

Chemistry ... 84

Binding Assays ... 86

(3)

iii

Stability Tests ... 88

Biodistribution Studies ... 88

AR mRNA and Protein Expression ... 91

P2X7 PET RADIOTRACER ... 93

EXPERIMENTAL SECTION ... 98

TUMOR: DEFINITION AND INTRODUCTION TO THE PATHOLOGY ... 107

Malignant and Benign Tumors: Their Characteristics and Differences ... 108

Pathogenesis of Tumors... 109

The Causes of Tumors ... 112

HOW ANTITUMOR DRUGS WORK ... 131

Heterocyclic Derivatives Possessing Intercalating Activity ... 134

TOPOISOMERASES ... 144

DNA Topoisomerase I ... 145

DNA Topoisomerase II ... 151

DNA TOPOISOMERASES AS TARGETS FOR ANTICANCER DRUGS ... 160

Topoisomerase I Inhibitors... 162

Topoisomerase II Inhibitors ... 168

Dual Topoisomerases I and II Inhibitors ... 172

Multiple Resistance Involves DNA Topoisomerases Inhibitors... 174

TYROSYL-DNA-PHOSPHODIESTERASES (TDP1 AND TDP2) ... 176

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) ... 176

Tyrosyl-DNA Phosphodiesterase 2 (TDP2) ... 180

TDP1 Inhibitors ... 183

TDP2 Inhibitors ... 192

DESIGN AND SYNTHESIS OF NEW NON-CAMPTHOTECIN DERIVATIVES ... 196

Biological Evaluation ... 204

DESIGN AND SYNTHESIS OF NEW DUAL TOPOISOMERASES I/II INHIBITORS ... 205

DESIGN AND SYNTHESIS OF NOVEL TDP-1 INHIBITORS ... 217

EXPERIMENTAL SECTION ... 230

(4)

1

PURINES AND PYRIMIDINES SIGNALING

Historical Perspectives

First studies about the potent actions of adenine compounds date back to 1929.1

Many years later, ATP was proposed as the transmitter responsible for non-adrenergic, non-cholinergic transmission in the gut and bladder, and the term purinergic was introduced by Burnstock in 1972.2_{Initially, there were some resistances in accepting it,}

since ATP was recognized first for its important intracellular roles in many biochemical processes, and its ubiquity and simplicity made it unlikely to be utilized as an extracellular messenger at a first impression, although powerful extracellular enzymes involved in its breakdown were known to be present. Implicit in the concept of purinergic neurotransmission was the existence of post-junctional purinergic receptors, and the potent actions of extracellular ATP on many different cell types implicated membrane receptors. Purinergic receptors were first defined in 19763 and distinguished two years later in P1 and P2.4 At about the same time, two subtypes of the P1 (adenosine) receptor were recognized,5,6 but it was not until 1985 that a proposal suggesting a pharmacological basis for distinguishing two types of P2 receptors (P2X and P2Y) was made.7

The first distinction between P1 and P2 was based on:

1. the relative potencies of adenosine and adenine nucleotides; 2. the sensitivity to antagonism by methylxanthines;

3. modulation of activity of adenylate cyclase.

In addition, the further P2 receptor classification was characterized by:

1. the rank order of potency: adenine nucleotides > adenosine; 2. insensitivity to antagonism by methylxanthine;

3. induction of prostaglandin synthesis.

In 1993, the first G protein-coupled P2 receptors were cloned,8,9_{and a year later}

two ion-gated receptors were cloned.10,11_{In 1994, on the basis of molecular structure and}

transduction mechanisms, it has been proposed that P2-purinoceptors should belong to two major families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G protein-coupled receptors.12

(5)

2

P3, P4, and P2YAp4A (or P2D) receptors have been also proposed, but evidence for

their existence is based solely on the distinct pharmacological profiles exhibited by some biological tissues.13

At present, we know that extracellular purines (adenosine 1, ADP 2, and ATP 3, Fig.1) and pyrimidines (UDP 4 and UTP 5, Fig.1) mediate diverse biological effects via the above mentioned two main families of purine receptors. Adenosine/P1 receptors have been further subdivided, according to convergent molecular, biochemical, and pharmacological evidences into four subtypes, A1, A2A, A2B, and A3, all coupled to G

proteins. Based on differences in molecular structure and signal transduction mechanisms, P2 receptors divide naturally into two families of ligand-gated ion channels and G protein-coupled receptors termed P2X and P2Y receptors, respectively; to date, seven mammalian P2X receptors (P2X1–7) and at least eight mammalian P2Y receptors

(P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14) have been cloned, characterized,

and accepted as valid members of the P2 receptor family.14

(6)

3

G-Protein Coupled Receptors (GPCRs)

Heterotrimeric G proteins are guanine-nucleotide regulatory protein complexes composed of α, and a dimer βγ subunits. They are responsible for transmitting signals from GPCRs to effectors, e.g. adenylyl cyclase (AC). Until now, 16 α, 5 β and 14 γ isoform have been reported.15

G proteins are divided into several subclasses with a specific activity profile:

1. Gs proteins stimulate adenylyl cyclase,

2. Gi proteins inhibit adenylyl cyclase and stimulate G protein-coupled

inwardly-rectifying potassium channels (GIRK), 3. G0 proteins stimulate K+ ion channels,

4. Gq/11 proteins activate phospholipase C (PLC),

5. G12 proteins activate Rho guanine-nucleotide exchange factors (GEFs),

6. olfactory G protein, Golf, stimulates adenylyl cyclase.

Interaction between a specific ligand and G protein-coupled receptor induces a conformational change in the receptor which begins to function as a guanine nucleotide

exchange factor (GEF) exchanging GDP for GTP on the Gα subunit. As a consequence,

according to the traditional view of heterotrimeric protein activation, this exchange causes the dissociation of the Gα subunit, bound to GTP, from the Gβγ dimer and the receptor. Recently, models suggesting molecular rearrangement, reorganization, and pre-complexing of effector molecules are beginning to be accepted. Both Gα-GTP and Gβγ can activate different signaling cascades (or second messenger pathways) and effector proteins, while the receptor is able to activate the next G protein. At the end of the cycle, Gα subunit hydrolyzes the attached GTP to GDP by its inherent enzymatic activity, being able again to re-associate with Gβγ. A group of proteins called Regulator of G protein

signaling (RGSs), act as GTPase-activating proteins (GAPs), specific for Gα subunits.

Their duty is to accelerate hydrolysis process of GTP to GDP, so that the transduced signal ends. Fig. 2

(7)

4

Figure 2. Activation cycle of G-protein by G-protein coupled receptors.

At this class of receptors belong both P1 and P2Y receptor subtypes.

Structure and Transduction Mechanism of P1 Receptors

The existences of two subtypes of P1 receptors was independently proposed.5,6 First proposal gained evidences from the observation of either increased stimulation or increased inhibition of cAMP formation by adenosine analogues compared to adenosine. The receptor subtypes mediating inhibition of cAMP formation were termed A1 subtypes, while the others, which mediated stimulation of cAMP formation, were named A2

subtypes.5 The other study reported the existence of two profiles for the relative effects of adenosine, N6_{-(phenyl isopropyl)adenosine and 5’-N-ethylcarboxamidoadenosine on}

adenylate cyclase activity. These receptor subtypes were termed Ra (activation of adenylate cyclase activity) and Ri (inhibition of adenylate cyclase activity), respectively.6

P1 receptors are also named adenosine receptors (referring to the endogenous ligand), while the subtypes are named the A1, A2A, A2B and A3 subtypes.16,17 Each of the

subtypes has been characterized by molecular cloning, agonist activity profile, antagonist activity profile, G protein-coupling and effector systems.18

The A1, A2A and A2B subtypes were initially discovered and classified in the

classical manner (i.e., by a study of agonist pharmacology). Evidence from recent cloning from a variety of mammals, including humans, sequencing and expression of each of these subtypes has provided structural and functional confirmation of their original classification as distinct adenosine receptor subtypes. In contrast, the A3 AR subtype was

discovered by molecular biology studies, then followed by classical pharmacological studies.18

(8)

5

Analysis of the amino acid sequences of the cloned receptors demonstrates that they all fit the seven transmembrane spanning domain structural motif, which is the model for all G protein-coupled receptors.19,20,21 This structural motif is characterized by seven domains, each composed by ~22-26 hydrophobic amino acids, which traverse the cell membrane. These domains possess alpha helical secondary structure. The extracellular domains of the receptor protein comprise the N-terminus, often containing one or more glycosylation sites, and three extracellular loops connecting the transmembrane domains (E I-III), while the cytoplasmic or intracellular domains comprise the C-terminus and similarly three intracellular loops (C I-III). Phosphorylation and palmitoylation sites, involved in regulation of receptor desensitization and internalization, are located on C-terminus domain. All ARs, with the exception of the A2A, contain a palmitoylation site

near the C-terminus. The A2A AR is the only subtype with an extraordinary long

C-terminus, 122 amino acids versus 36 amino acids in e.g. the A1 AR. Fig. 3

Figure 3. Structure of adenosine receptors

A1 and A3 receptor subtypes are coupled both to adenylyl cyclase inhibition,

leading to a reduction in cAMP levels, and to activation of phospholipase C, which in turn promotes IP3 production and calcium release from the intracellular stores. Moreover,

A1 can also promote potassium ion outflow from the cytosol, thanks to its coupling to a

yet-unknown G protein, leading to plasma membrane hyperpolarization. Conversely, the A2A and A2B subtypes are generally coupled to Gs members of the G-protein family,

(9)

6

Figure 4. Transduction mechanism of P1 receptors.22

Structure and Transduction Mechanism of P2Y Receptors

There are eight human P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11,

P2Y12, P2Y13, and P2Y14), but P2Y receptors are also found in many animal species,

indicating their development early in evolution.23 The nonconsecutive numbering of the mammalian P2Y receptors is due to the assignment of the P2Y nomenclature to species homologues or to proteins for which a function as receptor for extracellular nucleotides has not been confirmed.24_{All P2Y receptors belong to the δ-branch of class A GPCRs.}25,26

Crystal structures of the P2Y1 and P2Y12 receptors confirm typical features known for

GPCRs including seven hydrophobic transmembrane regions connected by three extracellular loops (E I-III) and three intracellular loops (C I-III)27,28,29 (Fig. 3). All P2Y receptors possess at their extracellular domains four cysteine residues, which, as shown for the P2Y1, P2Y2, and P2Y12 receptors, form two disulfide bridges: the first one between

the N-terminal domain and E-III, and the second bridge between E-I and E-II.30,31,32,33 The receptors of the P2Y receptor family show a relatively high diversity in the amino acid composition. P2Y receptors form homo- and heterodimers as known for other GPCRs,34,35 one example is a dimer composed of the P2Y1 receptor and the adenosine A1

receptor.36,37 There is also evidence for homodimerization of P2Y2, P2Y4, P2Y6 and

P2Y11 receptors.38,39,40 P2Y receptors are divided into two subfamilies on the basis of the

transduction mechanism.41,42 The receptors of the first subfamily (consisting of the P2Y1,

P2Y2, P2Y4, P2Y6 and P2Y11 receptors) couple via Gq-proteins to stimulate

phospholipase C followed by increase in inositol phosphates and mobilization of Ca2+ from intracellular stores. In addition, P2Y11 receptors couple to Gs proteins followed by

(10)

7

increased adenylate cyclase activity. In contrast, the P2Y12 receptor subfamily (P2Y12,

P2Y13, and P2Y14 receptors) signals primarily through activation of Gi proteins and

inhibition of adenylate cyclase activity or control of ion channel activity. (Fig. 5)

Figure 5. Transduction mechanism of P2Y receptors.22

Ion Channels (P2X Receptors)

Ion channels are specialized transmembrane proteins carry the ion currents. The term ionotropic refers to the ion-channel nature of the receptor and it is used to differentiate from metabotropic receptors, which are the previously discussed GPCRs. Ion channels are highly selective and discriminate not only between anions and cations, but even between different monovalent and divalent ions, for example, Na+, K+, and Ca2+. One crucial feature is their response to specific signals: at rest they are tightly closed and impermeable, but they are opened (“gated”) either by changes in the membrane potential or by certain ligands such as the neurotransmitters.43 According to this description, all ion channels are composed of two functional moieties (Fig. 6),

 a selectivity filter (S), which determines which types of ions may pass the membrane; and

 a gate (G), which specifies under which conditions the channel is opened.

Channels are subdivided into two major classes according to their gating trigger: the voltage-gated channels (VGCs) and the ligand-gated channels (LGCs).

(11)

8

Figure 6. Schematic representation of ligand-gated ion channels.43

Focusing on LGCs, it turned out that several of them, although different in function, have similar amino acid sequences. So LGCs can be classified into three superfamilies.

1. The superfamily of receptors that resemble the nicotinic acetylcholine receptors: glycine receptors (GlyR), GABAA receptors (GABAAR), nicotinic

acetylcholine receptors (nAChR), and some serotonin receptors (5-HT3R).

2. The superfamily of ionotropic glutamate receptors (GluR).

3. The ATP-gated purino receptors (P2X).

The members of the receptor superfamily 1 all have similar peptide loops formed by disulfide bridges, glycosylation patterns, distribution of proline, and (in one specific domain which forms the channel wall) Ser/Thr residues. All the receptors of this superfamily are characterized by four hydrophobic amino-acid sequences, which are long enough to span the plasma membrane. Accordingly, they sometimes are termed

four-transmembrane(4TM)-sequence receptors.

The members of the glutamate receptor superfamily presumably have three transmembrane sequences. A fourth hydrophobic sequence enters and leaves the plasma membrane from the cytoplasmic side, thereby forming a loop (reentrant loop), which is presumed to line the channel.

Members of the family ATP-gated ion channels P2X1–7 receptors show a subunit

topology of intracellular N and C termini that have consensus-binding motifs for protein kinases; two TM-spanning regions, the first (TM1) being involved with channel gating and the second (TM2) lining the ion pore; a large extracellular loop with 10 conserved

(12)

9

cysteine residues forming a series of disulphide bridges; a hydrophobic H5 region close to the pore vestibule, for possible receptor/channel modulation by cations; and an ATP-binding site, which might involve regions of the extracellular loop adjacent to TM1 and TM2. (Fig. 7)

Figure 7. P2X structure.

Structural features of P2X receptor and their function of rapid mediator (within 10 ms) to cations (Na+, K+ and Ca2+)44,45,46 are appropriate given their distribution on excitable cells (smooth muscle cells, neurons, and glial cells) and role as mediators of fast excitatory neurotransmission to ATP in both the central and peripheral nervous systems. This contrasts with the slower onset of response (less than 100 ms) to ATP acting at metabotropic P2Y receptors, which involves coupling to G proteins and second-messenger systems. Seven P2X receptor proteins (P2X1 to P2X7) have been cloned and

the ion channels formed from homomeric association of the subunits, when expressed in

Xenopus oocytes or in mammalian cells, have been functionally characterized and show

distinct pharmacological profiles. The P2X7 receptor is functionally unique among P2X

receptors in being able to act as a non-selective pore.

General Basis of Purinergic Transmission

Purines can be found together with classical neurotransmitters in various synapses of the central and peripheral nervous systems. They are also present with some releasable mediators in blood circulating cells, such as platelets. This means that their effects are a combination of modulation induced by the other transmitters with the independent activation of the purinergic receptors expressed in target tissues and organs. Within the

(13)

10

CNS and peripheral nervous system, they also regulate release of transmitters through presynaptic receptors.47_{Further complexity to the purinergic system is added by the}

following evidences:

 the neurotransmitter is not a single type of molecule, but is indeed a family of compounds, all biologically active (ATP, ADP and adenosine), resulting from stepwise hydrolysis of ATP (Fig. 8);

 the neurotransmitters are released not only by neurons and platelets but also by several other tissues, especially in metabolic emergencies.

Figure 8. Synthesis and metabolism of purines.22

Source, Metabolism and Release

In platelets, adenine nucleotides are stored as dense aggregates and released by exocytosis upon platelet activation. In the presynaptic terminals of the nervous system, ATP is stored in synaptic vesicles along with classical neurotransmitters (noradrenaline, acetylcholine, dopamine, serotonin, excitatory amino acids, and peptides). In presynaptic terminals of the peripheral nervous system, catecholamines and ATP are stored at a 4:1 molar ratio. In physiological conditions, only a calcium-dependent purine release from the presynaptic vesicular pool is observed. In emergency conditions (for example, hypoxia), during which alterations of plasma membrane permeability occur, purines are also released from their metabolic pool, not only by nervous cells, but also by endothelial, muscle, red blood cells and by other cell types damaged by the cytotoxic injury. In ischemic/hypoxic or traumatic areas, a huge increase in purines and pyrimidines

(14)

11

concentration has been detected, also due to degradation of nucleic acid released from dead cells.48

After release, ATP is a substrate of enzymes called ecto-ATPases (ectonucleotidases), which degrade it sequentially to ADP, AMP, and adenosine (Fig. 8). All these compounds are able to activate, with different efficacy, various purinergic receptors. Recently, pyrimidine di- or tri-phosphates (UDP and UTP), as well as UDP-glucose and UDP-galactose, have been discovered to be substrates of the same enzymes and able to activate the same G-protein-coupled receptors activated by ATP and ADP, suggesting an additional role for these molecules as intercellular mediators. To further support their biological role, UTP (which is particularly concentrated in circulating erythrocytes) has been shown to be significantly released in blood of patients undergoing heart failure. Although release and catabolism of uridine nucleotides are far less known, they seem to occur in a way similar to ATP, except for the apparent inactivity of UTP final product of catabolism, which is uridine. Both UTP and UDP are active on P2 purinergic receptors, even if with a response profile different from that of adenine nucleotides. Adenosine is degraded to the inactive metabolite inosine by the enzyme

exo-adenosine deaminase (ADA). The extra-cellular concentration of exo-adenosine is controlled

also by reuptake via specific transporters. In physiological conditions, reuptake is the most relevant mechanism to lower extracellular concentrations of adenosine, whereas its extracellular deamination becomes more relevant in pathological situations where adenosine concentrations are much higher.48

Following reuptake, adenosine can be either phosphorylated to AMP by an intracellular adenosine kinase, and converge again in the purine cellular pool, or be deaminated to inosine by an endo-ADA. Under physiological conditions, it is assumed that the majority of the reuptaken nucleoside is phosphorylated again to form AMP, whereas under pathological conditions, when intracellular concentrations of adenosine can be massive, deamination becomes the most relevant mechanism. This is due to the different affinity of adenosine for the adenosine kinase and the endo-ADA. Intracellular adenosine may also be produced through S-adenosylhomocysteine (SAH) hydrolysis and ATP hydrolysis. Whereas in physiological conditions adenosine seems to be mostly produced via SAH hydrolysis, in hypoxia and ischemia, the main metabolic pathway is represented by ATP hydrolysis.49

(15)

12

Finally, it has been demonstrated that tissue concentrations of ectonucleotidase enzymes increase substantially under particular physiological conditions or during damage repair. This suggests that nucleotides and nucleosides play a fundamental role in migration of embryonic stem cells, as well as in their terminal differentiation and recruitment of adult stem cells following traumatic events.48

Biological Effects

Cardiovascular system

In heart, ATP induces positive inotropism, which adds to that produced by β-adrenergic agonists. This is due to activation of P2X receptors and stimulation of calcium conductance. The negative inotropic and chronotropic effects of adenosine are much better characterized from a therapeutic point of view: the receptor involved is the A1

subtype that functionally antagonizes the positive inotropism and chronotropism produced by cathecolamines. In the atrium, the effects of adenosine are direct and are due to activation of K+ conductance; while in the ventricles, the effects are indirect and due to decrease in Ca2+ influx through L channels induced by β1-adrenoceptors.50

Recently, a protective role for adenosine has been described in ischemic heart preconditioning, a short (1-2 min) ischemia protecting myocardium from a subsequent and prolonged ischemia. This activity is mediated by A1 and A3 receptors through the

activation (and migration to the membrane) of protein kinase C and phosphorylation of a cellular substrate that protects from ischemic damage. The massive degranulation of residential cardiac immune cells during the short-lasting ischemia leads to a substantial decrease of releasable cytotoxic compounds during the second prolonged ischemia. Recent data suggest the involvement of A2A receptors, since through their activation by

adenosine there is an important anti-inflammatory process due to the reduced production of cytokines like TNFα and IL-6, which are present at high levels in patients with chronic cardiovascular diseases. Activation of A2A receptors could contribute to delay disease

progression and would offer new pharmacological tools for this disease.51

At vascular level, both ATP and adenosine are potent vasodilators. Adenosine acts directly on vascular receptors of the A2 subtypes on smooth muscle cells, which simulate

intracellular production of cAMP. On the other hand, the effect of ATP is indirect, as a consequence of the NO production resulting from activation of endothelial P2Y2,4

(16)

13

receptors. Adenosine is a potent platelet inhibitor, whereas ADP is a potent trigger of aggregation. The effect of adenosine is mediated by the A2 receptors that stimulate cAMP

production, whereas the effect of ADP is mediated by activation of P2Y12 receptor.52

CNS and Peripheral Nervous System

As already mentioned, purines colocalize with other neurotransmitters in the CNS. After release from presynaptic terminals, ATP generally induces rapid and transient excitatory responses mediated by P2X receptors. Through these receptors, ATP has an important role as excitatory transmitter and has been involved in phenomena like hippocampal LTP, which plays a key role in learning and memory. P2X receptors, especially P2X2/P2X3 types, are present on peripheral sensitive fibers and are responsible

for the transmission of pain stimuli to the spinal cord after being activated by ATP released by the surrounding cells as a consequence of trauma or inflammation. Sensitive fibers projecting to the spinal cord, in turn, release various excitatory neurotransmitters, such as glutamate, substance P, and again ATP, which contributes to central pain transmission via P2X2/P2X3 receptors expressed by nociceptive neurons. On the other

hand, adenosine exerts an extremely potent inhibition of excitatory neurotransmission and neurotransmitters exocytosis via the presynaptic A1 receptor subtype coupled to both

stimulation of K+_{and reduction of Ca}2+_{conductance. These effects are also enhanced by}

inhibitory postsynaptic A1 receptors that are present in all brain areas and antagonize the

excitatory effects induced by other transmitters like glutamate. A1 cerebral antagonism is

responsible for the stimulatory properties of methylxanthines such as caffeine, theophylline, and theobromine.50

A2A receptors are almost exclusively present in basal ganglia, where they

colocalize with D2 dopaminergic receptors on striatal interneurons containing GABA and

enkephalins. On these neurons, adenosine exerts inhibitory action through A2A activation,

whereas dopamine stimulates locomotor activity by interacting with D2 receptors. In

Parkinson’s disease, there is a progressive degeneration of dopaminergic neurons leading to bradykinesia and tremor; in rodent models of Parkinson’s disease, antagonists of A2A

receptors significantly alleviate symptoms by reducing adenosine inhibitory effect on motor activity. This mechanism also underlies the locomotor stimulation induced by methylxanthines at the same doses that induce stimulatory effects on the CNS. Experiments carried out on KO mice demonstrated a spontaneous rise in blood pressure and do not show any stimulation of locomotor activity after caffeine administration.

(17)

14

Moreover, A2A receptor KO animals show aggressiveness and hypoalgesia, suggesting

that A2A receptors have a role in the control of behavior and in mediating nociceptive

signals. An excessive activation of brain A2A receptors, especially those present in

microglia, seems to induce cell death and may contribute to neurodegeneration. This appears to be in contrast with the anti-inflammatory activity mediated by A2A receptors

on circulating cells and with the neuroprotective activity mediated by A1 receptors.

Recent experimental evidence also suggests a role for the A2A receptor in

neurodegenerative damage associated with ischemia and other chronic neurodegenerative disease of basal ganglia.47

Even if present at very low levels in the brain, the A3 receptor has been implicated

in regulation of cell survival. Depending on the pathophysiological conditions, activation of this receptor in neurons and astroglial cells induces either trophic and differentiating effects or necrosis and/or apoptosis, with possible implications in acute and chronic aging-associated diseases. The cytoprotection mediated by this receptor has interesting analogies with the myocardial protection and could be exploited to enhance brain resistance to neurodegeneration after trauma or ischemia.47

Respiratory System

Recently, it has been shown that at bronchial level, adenosine potentiates the release of histamine and proinflammatory and procontracting mediators from mastocytes and basophils by activating the A2B and A3 receptor subtypes. Selective A2B and A3

antagonists could therefore be useful in preventing (or reducing) bronchoconstriction caused by endogenous adenosine. Adenosine also inhibits acetylcholine release from vagal nerve terminals. Indeed, it has been reported that aerosol administration of an antisense oligonucleotide for A1 receptors in an animal model of allergic asthma

considerably reduces bronchoconstriction induced by subsequent administration of adenosine or allergens. This suggests a specific role of A1 receptors and opens up new

avenues for antisense therapy in asthma and related diseases.52

Nucleotide derivatives (especially UTP) have important effects on fluid and salt excretion in respiratory epithelia via P2Y2 receptors. Aerosol administration of these

compounds was initially found to normalize secretion of epithelia in patients affected by cystic fibrosis, suggesting new therapeutic perspectives for this serious disease.50

(18)

15 Other Systems

P1 and P2 receptors have been found in all organs and systems, even if their role, in some cases, is still unclear.

On bladder smooth muscle, adenosine acts as a potent relaxant via A2A receptors,52

whereas ATP induces rapid and transient contraction;50 this biphasic activity of purines has suggested that they may have a role in bladder emptying, with possible implications in the treatment of urinary incontinence.

In the gastrointestinal system, ATP controls hydrochloric acid secretion from gastric epithelial cells and exerts an inhibitory effect on intestinal motility; moreover, together with adenosine, it stimulates glycogenolysis and pancreatic secretion of insulin and glucagone.52

Adipocytes express A1 and A2 receptors that modulate lipolysis and glucose

oxidation; in line with this findings, high doses of methylxanthines induce weight loss.52

Purinoceptors are also expressed on spermatozoa, amniotic cells, chromaffin cells, and acinar cells in parotids. This suggests that purines might be involved also in exocrine and endocrine regulations and in reproduction.52

Adenosine Effects By-passing Receptor Activation

Studies about ADA deficiency syndrome, a disease characterized by lack of ADA enzyme, which is responsible for catabolism of adenosine, underlined how a high increase in concentration of this molecule and deoxyadenosine disrupts cellular energy equilibrium and activate death pathways. Some of the adenosine effects are directly induced by intracellular molecule involving well-defined intracellular pathways. Indeed, through these biochemical pathways, adenosine plays an important role in embryonic development, contributing to tissue remodeling by eliminating unnecessary structures and to the final configuration of the immune system by recognizing (and eliminating by apoptosis) potentially autoreactive clones.

In the following Table 1 are summarized the biological effects mediated by P1 and P2 receptors.

(19)

16

(20)

17

ANTAGONISTS TOWARDS PURINERGIC SYSTEM

Extracellular purines and pyrimidines have important and diverse effects on many biological processes including smooth muscle contraction, neurotransmission, exocrine and endocrine secretion, immune response, inflammation, platelet aggregation, pain, and modulation of cardiac function.13 Additional studies have shown the role of purines in emergency situations, such as cerebral and myocardial infarct, epileptic seizures, and infections where these molecules serve as danger signals family.14 In this view, during the years, a lot of efforts have been spent in order to discover and develop selective antagonists towards the numerous receptors involved in several physiopathological conditions. (Table 2)

A

1

Adenosine G-Protein Coupled Receptor Antagonists

A1 ARs are particularly expressed in the central nervous system (CNS), mostly in

the cerebral cortex, hippocampus, cerebellum, thalamus, brain stem and spinal cord. A1

ARs are also present in numerous peripheral tissues including vas deferens, testis, white adipose tissue, stomach, spleen, pituitary and adrenal gland, heart, aorta, liver, eye and bladder. In lungs, kidneys and small intestine low levels of this receptor has been found.15 A number of A1 AR antagonists has been studied as diuretic agents, for the treatment of

chronic lung diseases, for cardiac therapy and in dementia.53

Diuretic activity. Action of adenosine on A1 ARs exerts antidiuretic effects.

Consequently, A1 AR antagonists, blocking or reducing the adenosine action, may be

effective as diuretic agents. A1 AR antagonists showed to be potassium-sparing diuretics

with kidney-protecting properties, being particularly helpful in fluid retention disorders,

which are usually experienced by patients suffering from congestive heart failure. In these patients, A1 AR antagonists are able to increase both diuresis and glomerular filtration

rate, in contrast with the diuretic Furosemide which increases diuresis at the expense of glomerular filtration rate. Due to the capability of A1 AR antagonists to increase urine

formation without decrease renal blood flow, they are particularly useful to maintain glomerular filtration in patients having secondary edema to reduced cardiac function. Recent clinical trials confirmed their beneficial effects on renal function, even if high doses seem to cause the disappearance of renal benefits. Ongoing studies should be able

(21)

18

to demonstrate whether A1 AR antagonists can be used to improve renal function without

adversely affecting patients with heart failure.53

Action on heart. A1 AR antagonists are tested in the treatment of bradyarrhythmias

associated with inferior myocardial infarction, cardiac arrest and cardiac transplant rejection and could be useful in the treatment of chronic heart diseases, suppressing cardiac fibrosis and decreasing albuminuria, without effects on blood pressure.53

Antiasthma activity. As previously reported, adenosine plays a significant role in

lung diseases and conditions like hypoxia, stress, allergic stimulation, and exercise stimulate its endogenous production in many cells. Several in vitro and in vivo studies have been conducted on agonists and antagonists of the different receptor subtypes to evaluate their potential as novel antiasthma drugs. In particular, A1 AR antagonists could

represent useful drugs for the treatment of chronic lung diseases such as asthma, Chronic Obstructive Pulmonary Disease (COPD) and pulmonary fibrosis.53

Actions on CNS. The observation of the effects of caffeine, a classical non

selective adenosine antagonist, on the CNS, including an improvement of awareness and learning, encouraged the search of selective antagonists endowed with central activity. It has been reported that A1 AR blockade is involved in the discriminative-stimulus effects

of behaviorally relevant doses of caffeine. Nevertheless, other authors showed that the A2A AR is the main mediator of the behavioral stimulatory effect of caffeine. Selective

A1 AR antagonists induce cognition enhancement, leading to a general improvement in

memory performance. This may be useful in the treatment of dementia and anxiety disorders. Trevitt and colleagues reported a dose dependent improvement in locomotion in a model of Parkinson’s disease (PD) treated with the A1 AR antagonist CPT

(8-cyclopentyl-1,3-dimethylxanthine). These results suggest that A1 AR antagonism may

produce beneficial therapeutic effects, particularly at the beginning of treatment, even if the role of this receptor subtype in Parkinson’s disease (PD) is still unclear.53

Other activities. Adenosine is involved in the regulation of bone function and

recently it has been reported that A1 AR antagonists reduced bone loss, indicating their

potential use in the treatment of osteopoenia, osteoporosis and other bone diseases (e.g. Paget disease). Other potential therapeutic applications of A1 AR antagonists are

represented by the treatment of sepsis, in association with antibiotics and the treatment of hepatic ischemia-reperfusion injury. Initially, studies started from the synthesis and

(22)

19

evaluation of a multitude of xanthine derivatives, as the prototypic A1 AR antagonists

were teophylline and caffeine, which are part of xanthine family. However, also non-xanthine derivatives has been reported as potent and selective A1 antagonists, for

examples: adenine derivatives, pyrazolo[1,5-a]pyridines, tricyclic imidazolines. Both xanthine and non-xanthine derivatives are devoid of the sugar moiety that characterizes the majority of A1 AR agonists. In general, A1 AR antagonists are bicyclic or tricyclic

compounds, planar, aromatic or π-electron rich, nitrogen-containing heterocycles; hydrophobic substituents may enhance affinity, whereas hydrophilic substituents, which render many of the high-affinity antagonists quite insoluble in water, are usually not well tolerated.53

Xanthines derivatives

Alkylxanthines are the best-known class of compounds characterized as adenosine antagonists. This class of molecules has been deeply investigated in an effort to increase

potency and selectivity. Compounds more powerful than the prototypical caffeine have been identified, usually resulting from 1,3-dialkyl and 8-aryl or 8-cycloalkyl substitution. Unfortunately, additional activities unrelated to adenosine receptor blockade are triggered by these compounds, limiting success in preparing adenosine A1 selective agents as

therapeutic agents.54 Theophylline (6, 1,3-dimethylxanthine, Fig. 9) and caffeine (7, 1,3,7-trimethylxanthine, Fig. 9) are the classical non-selective xanthine antagonists of ARs that display micromolar affinity at all AR subtypes.

Theophylline was first extracted from tea leaves around 1888 and chemically identified and synthesized in 1896. There are many examples of potent A1 antagonists

that contain bulky lipophilic substitution at the 8-position of 1,3-dipropylxanthines. Doxophylline (8, 7-(1,3-dioxalan-2-ylmethyl)theophylline, Fig. 9) is a xanthine bronchodilator which differs from theophylline since it contains a dioxalane group at position 7. Several modifications on the xanthine core at the 1-, 3-, and 8-positions led to the discovery of 8-cyclopentyl-1,3-dipropyl xanthine (9, DPCPX, Fig. 9), showing a greater selectivity for rat A1 AR compared with the A2A AR and at the human (h) A1

compared with hA2A and hA2B ARs. Other substituted xanthines have been proposed as

A1 AR antagonists, in particular, 1,3-dipropyl-8-(3-noradamantyl)xanthine (10,

KW-3902, Fig. 9) and 1,3-dipropyl-8-(2-(5,6-epoxy)norbornyl)xanthine (11, BG-9719, Fig.

9). In this compound (also named Naxifylline), the xanthine ring and the epoxide are,

(23)

20

asymmetric center at C-2. While a small stereochemical effect on the affinity was present between the enantiomers at guinea pig and hA1 ARs, the R-isomer appeared to be less

potent than the S-isomer in the rat. Then, a series of xanthines substituted with norbornyl-lactones structurally related to BG-9719 (12, Fig. 9) was investigated. These derivatives, in which the xanthine occupies the exo position on the norbornyl ring system, showed high A1 binding affinity and selectivity over the closely related A2A AR. The lactones

possessed similar or higher in vivo activity to BG-9719 in the rat diuresis models.54

Figure 9. Xanthines derivatives A1 antagonists

Water-Soluble Xanthine Derivatives as A1 AR Antagonists

The highest affinity xanthine-based molecules pictured in Figure 9 lack appreciably polar substituents. As a consequence, solubility in water of these compounds is poor and it may limit their clinic application for intravenous administration in the treatment of acutely decompensate congestive heart failure patients. In the search for a selective A1 AR antagonist with greater aqueous solubility, a series of

1,3-substituted-8-cyclohexyl- and 8-bicyclo- [2.2.2]octylxanthines containing linear substitution patterns was investigated by Kiesman et al.55 Figure 10 Initial attention was on

(24)

8-cyclohexyl-21

trans-4-carboxylic acid xanthine derivatives published by Katsushima and co-workers,56

that showed low binding affinity at A1 AR. They expanded this series of compounds and

prepared the bicyclo-[2.2.2]octane derivatives by addition of a two-carbon bridge linking the 1- and 4-positions across the cyclohexane ring. Figure 10 Optimization of the bridgehead substituent led to propionic acid (13, BG-9928, Fig. 10),55 which retained high potency (dog A1, Ki 29 nM) and selectivity for the A1 AR (163-fold vs A2A AR; 24-fold

vs A2B AR; 1452 vs A3 AR) with improved oral efficacy in a rat diuresis model as well

as high oral bioavailability in rat, dog, and cynomolgus monkey. Another water-soluble molecule that has been described as a potential new oral drug for asthma is 3-[2-(4- aminophenyl)ethyl]-8-benzyl-7-{2-ethyl-(2-hydroxyethyl)amino]ethyl}-1-propyl-3,7-dihydropurine-2,6-dione] 14 labeled L-97-1. Figure 10 This compound is an adenosine A1 antagonist (Ki 580 nM) with at least 100-fold selectivity over A2A and A2B ARs.

Tricyclic imidazoline derivatives

These compounds are essentially derivatives of xanthines bearing an additional basic site, which significantly increase their water solubility compared to xanthines, without substantial loss in A1 binding affinity. In connection with the discovery of

BG-9928 and the tricyclic imidazoline derivatives reported in the literature, Vu et al.57 described the synthesis of compound 15, (Figure 10) the R-isomer of 7,8-dihydro-8-ethyl-2-(4-bicyclo[2.2.2]octan-1-ol)-4-propyl-1Himidazo-[2,1-i]purin-5(4H)-one, a potent A1

AR antagonist with good selectivity over the other three receptor subtypes. Imidazoline

15 is a potent competitive A1 AR antagonist, highly soluble in water (>100 mg/mL). In

addition, it has an oral bioavailability of 84% and an oral half-life of 3.8 h in rats. When orally administered in a rat diuresis model, compound 15 promoted sodium excretion (ED50 0.01 mg/kg). This level of efficacy is comparable to that of BG-9928. Additional

modifications of 15 also showed that the bridgehead hydroxyl group could be replaced with a propionic acid without a significant loss in binding affinity or in vivo activity.

(25)

22

Figure 10. Water-soluble A1 AR antagonists

Pyrazolo[1,5-a]pyridines

Another class of non-xanthine consists of derivatives possessing

pyrazolo[1,5-a]pyridine nucleus. One compound in this series, FK-453, (16, Fig. 11), synthesized by

Akahane et al. showed potent and selective adenosine A1 antagonist activity, so that it

was further investigated. Nucleus was modified different times such as constraining of acryloyl amide into a pyridazinone nucleus (compound 17, FK-838, Fig. 11) that produced a significant increase of potency and selectivity.58 FK-838 is an A1 selective

adenosine antagonist, as demonstrated in radioligand binding (IC50 A1 120 nM; IC50 A2

5900 nM) and functional assays, whose diuretic and natriuretic effects appear to be due to both its renal hemodynamic effects and a direct inhibition of proximal tubular Na+ reabsorption. The design process leading to the discovery of FR-166124 (18, 2-[2-[6-oxo-

3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1,6-dihydropyridazin-1-yl]-1-cyclohexenyl]acetic acid, Fig. 11) involved replacement of the butyric acid group of FK-838 with various cyclic acid groups at the N2_{of the pyridazinone ring in order to induce}

rigidity in the system and mimic the postulated conformation of FK-453. Cyclohexenyl acetic acid group was found to be especially effective. Compound 18 is reported as a more potent derivative with higher A2A/A1 selectivity and very high water solubility as the

(26)

23

Figure 11. Pyrazolopyridines

Adenine derivatives

The initial report of adenine derivatives showing adenosine antagonist activity only covered 1-methyl- and 9-methyladenine.60 It has been observed that adenine itself and 9-methyladenine showed specific competitive antagonism at low concentrations but exhibited nonspecific inhibitory activity at higher concentrations. Since N6-substitution of adenosine had been used to confer A1 AR agonist selectivity, it was clear that a

selective antagonist could potentially be obtained by replacing the ribose sugar with a methyl group to generate N6-substituted 9-methyladenines.61,62 Subsequent structure-activity work identified ((±)-N6-(endo-2-norbornyl)-9-methyladenine N-0861 (19, Fig.

12) as a lead compound, which has been undergoing development as a cardiovascular

agent, having negligible affinity (Ki > 10 000 nM) for alpha 1, alpha 2, and beta adrenoceptors, D1 and D2 dopamine receptors, and 5-HT2 serotonin receptors. N-0861

(19, Fig. 12) high affinity binding to A1 ARs in bovine caudate membranes has been

demonstrated by radioligand binding assay, showing a 600-fold selectivity for the A1 vs

the A2 subtype of ARs.63 N-0861 binds with lower affinity to A1 ARs in rat and guinea

pig cerebral cortex and to A1 ARs in atrial tissue from guinea pig and human heart.64

Substitution at the 8-position of adenine with isopropylmethyl-amine moiety gave the best results as observed for compound 20 (WRC-0571, Fig. 12), which is a highly potent and selective A1 AR antagonist with superior potency and aqueous solubility relative to

N-0861 (19, Fig. 12). In radioligand binding studies, it displayed high affinity for cloned

hA1 (Ki 1.7 nM) and much lower affinity for cloned hA2A and hA3 ARs (Ki 105 and 7940

nM, respectively). In functional studies it potently inhibited the A1-mediated negative

inotropic response to 5’-(N-ethyl-carboxamido)adenosine (NECA) in isolated guinea pig atria (pKb 8.41), whereas it was much less active against NECA-induced, A2B-mediated,

(27)

24

Figure 12. Adenine derivatives

Pyrrolopyrimidine derivative

In 2007, researchers from Solvay Pharmaceuticals65 described the pharmacological properties of a novel A1 adenosine receptor antagonist SL-V320 (21,

Fig. 13) and its protective effects on target organ damage in a rat model of CRF. They demonstrated that SLV320 is a selective A1 receptor antagonist both in vitro and in vivo.

Importantly, these investigations have revealed for the first time that blockade of an adenosine A1 receptor had protective effects in the heart and kidney, by attenuating

cardiac fibrosis and albuminuria in rats with 5/6 nephrectomy and that these protective effects occurred without changes in blood pressure. SLV320 is a selective and potent adenosine A1 antagonist (Ki = 1.0 nM) with a selectivity factor of at least 200 versus other

adenosine receptor subtypes and an even higher selectivity factor versus most other receptors.

Figure 13. Pyrrolopyrimidine derivative

A

2A

Adenosine G-Protein Coupled Receptor Antagonists

Within the brain A2A ARs are richly expressed in the striatum, nucleus accumbens,

and olfactory tubercle. A2A ARs are co-expressed with D2 dopamine receptors in the

(28)

25

antagonist effects in regulation of locomotor activity. Activation of A2A ARs in

striatopallidal neurons decreases the affinity of D2 receptors for dopamine, antagonizing

the effects of D2 receptors.66 The antithetical interaction between A2A and D2 receptors is

at the basis in using A2A antagonists as a novel therapeutic approach for the treatment of

Parkinson’s disease.66_{Actually, A}

2A ARs may have an important role in the

neurodegenerative process, as suggested by the neuroprotective effect showed after caffeine intake or A2A AR inactivation against dopaminergic neurodegeneration in a

neurotoxin model of Parkinson’s disease.67_{Concomitantly, two large prospective}

epidemiological studies have strongly associated caffeine consumption to a reduced risk of developing Parkinson’s disease.68,69 _{Last, the recent discovery that the A}

2A can form

functional heteromeric receptor complexes with other G protein-coupled receptors such as D2 and the mGlu5 receptors has also suggested new opportunities for the potential of

A2A antagonists in PD.70 Future development of bivalent ligands, being able to activate

D2 and block A2A ARs or antagonize both A2A and mGlu5 subtypes, would be a promising

strategy for the treatment of this neurodegenerative disease.71,72,73 In addition to the protection against striatal and nigral neuron loss by A2A antagonists, there are data also

supporting their protective role outside the basal ganglia.74_{Local injection of an A} 2A

antagonist prevents glutamate-dependent death of neurons in hippocampal cortex75_and

also reduced cortical damage in a variety of ischemic stroke models. In A2A knockout

(KO) mice transient focal ischemia caused less neuronal damage in comparison to their wildtype (WT) littermates.76 Therefore, it seems that tonic activation of A2A ARs may be

responsible for dangerous signal during injury, in contrast to the neuroprotective effects induced by endogenous A1 activation. A2A ARs contribution to the development of

ischemic brain injury have been suggested by selective inactivation or reconstitution of A2A ARs in bone-marrow cells.77 Complexity of involvement of A2A ARs in

neuroprotection is underlined also by the fact that this receptor subtype diminishes brain damage after excitotoxic and traumatic injury.78,79 A2A-mediated protection has been

reported against ischemia in the myocardia, kidney, and liver and in ischemia reperfusion injury in the spinal cord.80,81,82,83 High expression of A2A ARs has been found in platelets,

leukocytes, vascular smooth muscle, and endothelial cells with important implications in the regulation of inflammatory responses. It is now well established that stimulation of the A2A AR in immune cells induces anti-inflammatory effects, mostly due to its ability

to increase cAMP levels, which has strong immunosuppressive effects.84 Stimulation of A2A ARs inhibits neutrophil adherence to the endothelium, degranulation of activated

(29)

26

neutrophils and monocytes, plus superoxide anion generation. A2A ARs have been

recently defined as sensors and terminators of proinflammatory activities. The strongest evidence for the key role of A2A in inflammation derived by the elegant study of Ohta et

al.85 using mice deficient in A2A ARs. In this model the lack of A2A subtype led to

increased tissue inflammation and damage, thus suggesting a negative and nonredundant regulatory role for the A2A AR. This model has permitted to appreciate that adenosinergic

regulation of immune cells is fundamental in normal physiological control of inflammation in vivo in spite of the fact that other Gs protein-coupled receptors and cAMP

elevating ligands are present such as cathecolamines, prostaglandins, dopamine, and histamine.84Interestingly, the A2A AR has been demonstrated to be involved in promotion

of wound healing and angiogenesis in healing wounds.86,87 Moreover, it plays an active role in the pathogenesis of dermal fibrosis, suggesting a role for antagonists as novel therapeutic approach in the treatment and prevention of dermal fibrosis in diseases such as scleroderma.88

Styrylxanthines

1,3-Dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (22, KF17837, Fig. 14) was the first A2A AR antagonist in this chemical class of compounds.89 The

3-chlorostyrylcaffeine 23 (CSC, Fig. 14) was identified as being less potent than KF17837 but with an increased selectivity vs A1 AR subtype.90 Introduction of a propargyl moiety

at the 1-position in combination with the 8-styryl group in compound 24 (BS-DMPX, Fig.

14) increased affinity to the A2A AR with retention of selectivity.91

1,3-Diethyl-7-methyl-8-(3,4- dimethoxystyryl)-xanthine 25 (KW-6002, also named istradefylline, Fig. 14) is an 8-styrylxanthine with high affinity for the rat striatal A2A AR. Due to its high affinity and

selectivity, a radiolabeled derivative, [11C]-KW-6002 labeled at the aromatic O-methyl position, was developed to be used in pharmacological testing to trace the A2A ARs in

vivo.92,93 KW-6002 (compound 25, Fig. 14) was selected for phase III clinical trials for the treatment of Parkinson’s disease. However, in 2008 its approval was declined by the FDA, which expressed concern as to whether the results in clinical trials supported the clinical use of istradefylline and asking for more thorough clinical investigations. However, considering the positive clinical results obtained in PD patients, the company decided to perform a further trial with istradefylline and submitted another application for manufacturing and marketing approval on March 30, 2012, in Japan.94

(30)

27

Figure 14. Styrylxanthines A2A antagonists

9H-Purine derivatives

Minetti et al., on the basis of the molecular modeling of a number of potent AR antagonists, designed and synthesized a number of 2-alkyl-substituted purine derivatives as A2A AR antagonists.95 From them ST-1535 (2-n-butyl-9-methyl-8-

[1,2,3]triazol-2-yl-9H-purin-6-ylamine 26, Fig. 15), was the most interesting.

Parallel to this, in the same year, Pinna et al.96 presented results about the administration of the A2A receptor antagonist ANR-94, previously synthetized97 (27, Fig.

15). This molecule improved parkinsonian initiation movement deficits,

akinesia/hypokinesia, gait deficits, sensorimotor impairments and tremor even without the combined administration with L-dopa. ANR-94 also showed neuroprotective and anti-inflammatory effects offering a unique opportunity to counteract neurodegeneration, one of the most intriguing aspects of PD pathology.

(31)

28

Bicyclic derivatives

In the past years several researchers focused their attention on nitrogen bicyclic derivatives formed by 1,2,4-triazole nucleus fused to pyridine, pyrimidine or thiazole ring in which the nitrogen atom is shared by the two rings. In particular, ZM-241385 (28, Fig.

16) is one of the most potent A2A AR antagonists ever reported with favorable water

solubility. Although different substituents can be introduced in the bicyclic system, the antagonist activity increase when a 2-furanyl substituent is introduced at the carbon of the triazole nucleus and free or substituted aryl and amine groups are present on hexagonal ring95. Modification of the pharmacokinetic features of this type of derivatives can be achieved by the introduction of hydroxyl or amino substituent on the aryl group or various substituents on the position 5. In particular, derivative 29 (Fig. 16), showed great potency and selectivity for the A2A AR as compared with the A1 AR. Several isoster of

the triazolo-triazine nucleus have been synthesized; in particular some oxazolo-pyrimidines (30, Fig. 16) and triazolo-pyrazine (31, Fig. 16) showed good potency at the A2A AR and good selectivity versus A1 AR.95

Figure 16. Bi-heterocyclic derivatives

The non-xanthine compound Vipadenant (BIIB014/V2006) is a

triazolo[4,5-d]pyrimidine derivative (32, Fig. 17). Vipadenant displays high affinity for the A2A

receptor, with a Ki value of 1.3 nM, and selectivity for the A2A receptor subtype

(>50-fold vs A1 and A2B receptors and >1,000-fold vs A3 receptor). Although the results of

phase II clinical trials were promising, development of vipadenant was discontinued by Vernalis and Biogen in June 2010. The two companies have since decided to progress

(32)

29

preclinical studies of a next-generation compound, V81444 (structure not shown), which overcomes some toxicity concerns of previous compound and a phase 2 clinical trial has been planned to better evaluate its pharmacokinetics, safety and tolerability.98

Figure 17.

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines

9-Chloro-2-furan-2-yl-[1,2,4]triazolo[1,5-c]quinazolin-5-ylamine named CGS-15943 (33, Fig. 18) represented the first potent but poorly selective antagonist for the A2A

AR subtype.99 Bioisosteric replacement of the phenyl ring of CGS-15943 with an N7 -substituted pyrazole led to the first example of an adenosine antagonist displaying the pyrazolotriazolo-pyrimidine core named 8FBPTP (34, 8-(4-fluorobenzyl)-2-(2-furyl)-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin 5-amine, Fig. 18).100 Some structural features of this compound highlighted the essential requirements for the A2A affinity, i.e.,

the furyl moiety and the free amino group at the 5-position. Starting from these observations Baraldi et al.101 focused their interest on the pattern of substitution on the pyrazolo preserving the other structural elements. Several alkyl, aryl, and phenylalkyl substituents have been introduced at both the N7 and the N8 positions. The biological data derived from the molecules obtained indicated that the best radicals were phenylalkyl chains, and, among these, it was possible to discern the length of the spacer introduced between the phenyl ring and the pyrazolo nitrogen that was optimized in two or three carbon atoms. Two selected compounds of this family named SCH-58261 (35, 5-amino-7-(β-phenylethyl)2-(2-furyl)-pyrazolo[4,3-e][1,2,4]triazolo-[1,5-c]pyrimidine) and SCH-63390 (36, 5-amino-7-(3

phenylpropyl)2-(2-furyl)-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine)101,102 proved to be potent and selective A2A AR antagonists both in rat and

human models. Figure 18 It was also noted that the N7 derivatives were more selective for the A2A AR than the corresponding N8 derivatives. From the family of SCH

(33)

30

compounds, 5-amino-7-(3-(4-methoxyphenyl)propyl)-2-(2 furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH-442416, 37, Fig. 18) was selected for the development of a new positron emission tomography (PET) ligand, whose chemical structure allows an easy introduction of a methyl group by direct O-alkylation of the phenolic function with [11C]CH3I under alkaline conditions.103 The in vitro binding in the brain and

periphery, the good signal-to-noise ratio observed between 5 and 15 min after injection, and the low occurrence of radioactive metabolites all suggested that [11C]SCH-442416 was applicable as the first non-xanthine ligand suitable for the in vivo imaging of A2A

ARs using PET. In addition, the data obtained from the binding experiments showed a higher affinity of the title compound for hA2A vs rat ARs (0.048 vs 0.5 nM).103

(34)

31

Water-Soluble A2A Adenosine Receptor Antagonists

The major restriction of the tricyclic adenosine antagonists was the low solubility in aqueous media that limited the pharmacological screening. Starting from this limit Baraldi et al.101,104 reported a second generation of pyrazolotriazolopyrimidines bearing oxygenated substituents on the phenylalkyl chains at the 7-position (compounds 38-41). The most interesting compounds are depicted in Figure 19. Compound 38 displayed the best value of A2A AR affinity indicating that the 4-hydroxy group positively influenced

the receptor interaction but was not enough for reaching a good profile of water solubility. A watersoluble analogue of SCH-58261 (35), named SCH-BT2 (42), was prepared by introduction of a 4-methyl-piperazine-1-sulfonyl moiety at the para position of the phenyl ring. SCH-BT2 altered neither motor behavior nor produced postural asymmetry by itself. However, when infused concomitantly with levodopa (L-DOPA) (capable of inducing modest controlateral rotational behavior), SCH-BT2 significantly potentiated the number of contraversive rotations.105,106,107_{Recently, a novel series of 3-substituted}

8-furyl-[1,2,4]-triazolo[1,5-i]purin-5- amine analogs related to SCH-58261 (35) was reported as A2A AR antagonists.108

Figure 19. A2A water-soluble antagonists

Most of the N3-substituted aryl piperazine and piperidine analogs demonstrated in vivo A2A receptor binding affinity and A1 receptor selectivity profiles superior to those of

SCH-58261.Neustadt et al.109 reported the arylpiperazine derivatives of

(35)

32

compound 43 (SCH-420814, Predalenant, Fig. 19) displayed both superior in vitro and promising in vivo profiles, but it failed in 2013 in Phase 3 clinical trial.110_{In the same}

article, it is reported the successful Phase 2 clinical trial for benzothiazole derivative SYN-115 (Tozadenant, 44, Fig. 19), which is now in Phase 3 as announced by Biotie Therapies.

A

2B

Adenosine G-Protein Coupled Receptor Antagonists

A2B receptor is mainly expressed in the gastrointestinal tract, bladder, lung, and

on mast cells, but also in eye, adipose tissue, brain, kidney, liver, and other tissue. Apart from stimulation of adenylate cyclase through Gs proteins, A2B AR are associated to Ca2+

mobilization through Gq proteins and mitogen-activated protein kinase (MAPK) activation. Furthermore, in human mastocytoma-1 cells (HMC-1) adenosine can stimulate interleukin (IL)-8 secretion, in addition to IL-1b, IL-3, IL-4 and IL-13 secretion, all via the A2B AR.111 This enhances the release of inflammatory mediators in addition to

pro-inflammatory effects on airway smooth muscle cells, epithelial cells, and fibroblasts, confirming that A2B AR plays a key role in the inflammatory response associated with

asthma. First studies on the involvement of A2B AR in asthma-concerned selectivity of

enprofylline, a methylxanthine structurally related to theophylline. This anti-asthmatic drug was the most selective, though not potent A2B antagonist. Further studies showed

that A2B antagonists inhibited NECA (5'-N-ethylcarboxamidoadenosine)-induced

interleukin-8 secretion in HMC-1 and attenuated the release of IL-19 from human bronchial epithelial cells.111 Recently, it has been reported that the selective A2B

antagonist CVT-6883 attenuated the airway inflammation and fibrosis induced by inhaled AMP or allergens. In the human intestinal epithelial cells, adenosine was shown to stimulate via A2B AR activation an increase in cAMP levels that is responsible for Cl

secretion, which allows the natural movement of isotonic fluid into the lumen, but in abnormal conditions could cause a secretory diarrhea. Furthermore, a recent study demonstrated that the A2B AR blockade significantly down-regulates proinflammatory

cytokines and reduces the symptoms of colitis, indicating that A2B antagonists could be

an effective therapeutic strategy to treat colonic inflammation. Studies on the antinociceptive effects of some A2B antagonists supported the role of A2B AR in peripheral

pain signaling, with a synergistic effect between A2B-selective compounds and