Abstract
P2 receptors are endowed with a number of biological effect, which are of extreme interest as
targets for new drugs. P2 receptor structure and functions are still not known in deep, and
new highly efficacious and selective ligands (both agonists and antagonists) are needed,
since they are at an early stage of development.
The scopes of our research has been the discovery and development of potent P2 ligands,
with the aim at designing selective pharmacological probes and potential drugs targeting P2
receptor subtypes. On this purpose, this thesis reports on two related topics:
-
the preparation and testing of 2-alkynyl ATP analogs as potential P2 agonists;
-
catalogues data mining for identification, purification and testing of polysulphonated
dyes as analogues of Reactive Blue 2, and preparation and testing of new RB-2 and suramin
analogues as potential P2 antagonists
As regard as the first topic, a new series of nucleotides with activity on human platelet
aggregation and adenylyl cyclase modulation through the interaction with P2Y1 and P2Y12
receptors has been prepared and well characterized. The di- and thriphosphates of 2hexynyladenosine (HEADP, 73, and HEATP, 75) are partial agonists at P2Y1 and agonists at
P2Y12 receptors while both the monophosphates (HEAMP, 71, and PEAMP, 72) and the diand thriphosphates of 2-phenyletynyladenosine (PEADP, 74, and PEATP, 76) mainly
antagonize the P2Y1 receptor. The surprisingly different behaviour of the diphosphates 73
and 74 (figure 18), which differ from structure point of view only for a portion of the alkynyl
chain, cannot be explained by different hydrolysis rates, but in terms of different size and
flexibility of the substituents in the position 2 of the purine base.
With regard to analogues of Reactive Blue 2 and suramin, with this work we have succeded
in the chromatographic separation of isomeric mixture of RB2. Furthermore, we have
prepared a number of RB2 and suramine analogues, which have been tested for their ability
of counteracting the toxic effect of hypoglicemia.
Some of tested compounds behave as protecting agents, being at least equiactive with RB2,
and being among the most active compounds of this category.
1
1. Introduction
Physiological evidence supporting the role of the adenine nucleotide, ATP (1, Figure 2), as a
neurotransmitter was first summarized in detail in 1972 [1], representing the basis of
Burnstock’s now seminal purinergic nerve hypothesis. This hypothesis was refined in 1978
with the delineation of distinct P1 (adenosine) and P2 (ATP) receptor classes [2]. However, it
is only in the past decade that definitive evidence for a family of discrete molecular targets
responsive to ATP and other nucleotides, the P2 purinergic receptor family, has been
obtained using molecular biological, pharmacological, and medicinal chemistry approaches
[3-11]. Thus, there is now a wealth of compelling data to support a role for extracellular
purine (ATP; ADP, 2) and pyrimidine (UTP, 5; UDP, 6) nucleotides acting as
neurotransmitter/neuromodulators to modulate the function of a diversity of mammalian cell
types and tissues under both normal and pathophysiological conditions [3,11-15]. The
concept that high-energy phosphate bond containing molecules, such as ATP, which play
such a key role in all aspects of cell function, might also function as extracellular chemical
messengers was initially met with considerable scepticism [16]. However, all molecules
mediating cellular communication involve energy-dependent processes that involve the
salvage of their breakdown products with the resynthesis of the active moiety. For ATP, this
involves adenosine reuptake and the subsequent phosphorylation of the nucleoside [17].
ATP can be constitutively released with steady-state basal extracellular concentrations in the
range of 3 nM [18]. Intracellular ATP release occurs by vascular release, cytolysis, and
activation of ABC (ATP-binding cassette) proteins [19]. The dynamic regulation of ATP
production by cells may also have provided a specific selective pressure for the evolutionary
progression from unicellular to multicellular organisms [20], a role that sustains the
functional importance of the purine nucleotide and its evolutionary conservation as an
energy source.
ATP functions as a pluripotent signalling molecule, eliciting direct effects on cell function via
its ability to activate the P2 receptor family and more indirectly via its conversion to ADP (2),
AMP (3) and adenosine (4) via a family of ectonucleotidases (E-NTPases) [21- 23], resulting in
a purinergic cascade (Figure 1) [11], the functional end product of which is adenosine, the
endogenous ligand for the P1 receptor family [3, 24]. There are also emerging data that
2
inorganic phosphate has the potential to function as a signalling molecule to induce gene
expression [25]. ATP and its analogues can also modulate the activity of other key ATP
binding proteins including the K-ATP ion channel family [26], neuronal nicotinic
acetylcholine receptors [27, 28], capsaicin-activated ion channels [29, 30], as well as ENTPases [31], ectoprotein kinases [32], acetylcholinesterase, and neuromuscular neuronal
nicotinic receptor expression [33],
thus making cellular responses to the nucleotide
potentially complex and multifunctional. The actions of the nucleotide are therefore
dependent on the enzymes and receptors modulated by ATP that are present in the
extracellular milieu.
Figure 1. Biochemical effects related to purimergic cascade
The expression of these targets can change during tissue development and as a result of
disease pathophysiology [33-35]. This type of neurotransmission has been termed “domain
specific” [36], with the extracellular availability of the neurotransmitter, the extent of its
metabolism, and the tissue/disease specific array of its potential recognition sites determining
the ultimate response of the target organ. In addition to this neurotransmitter role, ATP can
also effect intercellular information transfer via adenylate charge and can also modulate the
set-point of signal transduction pathways affecting responses to other neurotransmitters and
hormones [37]. P2 receptor ligands have therapeutic potential for, including ATP and UTP
(Figure 2) and receptor antagonists [13], in a variety of human disease states that include
cancer, chronic obstructive pulmonary disease (COPD), chronic bronchitis, asthma, bladder
3
and erectile dysfunction, reproduction, auditory and ocular function, pain, haemostasis
(platelet aggregation, Neutrogena and leukaemia, neurodegeneration, and immune system
function. An alternative, complementary approach to the discovery of novel drugs
interacting with the P2 receptor system is that of targeting the enzymes modulating ATP
(and UTP) metabolism. This approach is at a very early stage.
1.1. Evolution of the P2 Receptor Family
The initial characterization of P2 receptors [2] was empirically based on the rank order
agonist potency of a series of ATP analogues (Figure 3) to activate functional responses in a
variety of mammalian tissue preparations. While a number of distinct classes of P2
antagonists have been described (Figures 4-5), these are frequently lacking in both selectivity
for, and between, P2 receptors [38]. The effects of both P2 receptor agonists and antagonists
also
show
significant
tissue
and
species
dependency
that
further
complicates
pharmacological classification [38, 39].
NH2
PPP
O
O
P
P
P
OH
HN
N
N
O
PPPO
O
O
O
O
N
N
O
HO
O
N
O
OH
OH
1 (ATP)
5 (UTP)
HO
OH
b
d
OH
HO
d
b
O
NH2
PP
a
HO
N
N
O
O
P
P
N
N
N
O
PPO
O
O
O
HN
O
OH
OH
OH
HO
2 (ADP)
e
a
NH2
N
N
P
O
O
HO
NH2
N
N
O
3 (AMP)
OH
N
N
c
f
OH
HO
OH
HO
6 (UDP)
N
N
HO
O
HO
OH
4 (Adenosine)
Figure 2. Metabolic interconversion of naturally occurring purine and pyrimidine
nucleotides of relevance to extracellular action. Purine (adenosine derivatives, 1-4) and
pyrimidine (uridine derivatives, 5 and 6) nucleosides and nucleotide are shown. “P” refers to
4
a single phosphate group; thus, “PP” is diphosphate, etc.: (a) ectoapyrase, NTPDase1; (b)
ecto-ATPase, NTPDase2; (c) 5’-nucleotidase; (d) nucleoside diphophokinase; (e) AMP kinase;
(f) adenosine kinase.
In contrast, the discovery of the alkylxanthines, caffeine, and theophylline, as adenosine
receptor antagonists in the 1970s [40], provided a concrete basis for an extensive effort in P1
(adenosine) receptor medicinal chemistry, resulting in the identification of several clinical
drug candidates based on the xanthine pharmacophore some 20 years before the cloning of
this receptor family [41]. Furthermore, synthetic chemistry efforts in the area of nucleoside
triphosphates have been somewhat limited [42], with a systematic focus on developing
structure-activity relationships for the various P2 receptors and high-throughput screening
approaches to identifying novel pharmacophores being relatively recent [43, 44, 45]. Despite
the inherent limitations of P2 receptor characterization in the 1980s, the concept of ATPsensitive P2 receptors was expanded to encompass what were then designated as P2X and
P2Y receptors in 1985 [46], the P2X receptor being potently activated by the hydrolysisresistant ATP bioisosters (Figure 3A), α,β-methylene ATP (α,β-meATP, 10) and β,γmethylene-ATP (β,γ-meATP, 11). Accordingly, these ATP analogues were inactive at P2Y
receptors, with 2-methylthioATP (2-MeSATP, 13, Figure 3B) being the most potent agonist at
this P2 receptor subtype.
Pharmacological evidence for the existence of the P2T platelet ADP-sensitive receptor, a
pyrimidine (uracil nucleotide), P2U receptor, and the pore-forming mast cell P2Z receptor
was summarized in the following year. Unique pyrimidine-sensitive receptors, insensitive to
purines, were proposed in 1989 [15], and further evidence for their existence was derived in
the subsequent decade [47].
5
O
NH 2
NH 2
A
HS
O
O
O
P
P
P
OH
N
N
O
O
P
P
O
OH
HS
O
O
OH
O
OH
O
N
N
N
HO
P
P
P
OH
B
N
HO
H3CS
OH
HO
P
C
OH H2
O
OH
P
O
OH
OH
OH
HO
OH
OH
p-C6H5-C6H4CO ester at 2' or 3'
19. Bz-ATP
O
P
P
OH
O
O
P
OH
C
OH H2 OH
N
OH
H3CS
C6H5H2CHN
NH 2
N
N
N
N
O
N
N
N
N
PO
OH
HO
17.a R=H
17.b R=CH3 MRS2055
NH 2
N
N
O
OH
HO
N
N
PO
O
16.2-(4-aminophenylethylthio)ATP
N
N
N H C=HC( H C) S
2
2 4
N
PPP O
PPP O
O
HO
(H2C)2S
NH 2
N
O
NHR
N
N HN
2
N
N
O
OH
HO
12.beta,gamma-me-L-ATP
N
O
HO
N
PPP O
18.2-hexylthioATP,alfathio
O
O
OH
O
OH
15.2-hexylthioAMP
N
OH
N
NH 2
NH 2
O
N
N
O
O
OH
N
PO
N
N
N
N
N
14.2MeSADP
N
PPO
P
O
NH 2
S
P
N H C(H C) H CS
3
2 4 2
N
PPO
13.2MeSATP
S
O
N
H3CS
O
O
OH
O
O
N
N
NH 2
N
N
HO
O
NH 2
N
PPP O
O
11.beta,gamma-meATP HO
OH
HO
N
P
NH 2
N
N
N
NH 2
N
O
P
OH
NH 2
O
10.alfa,beta-meATP
O
P
8.ADPbetaS
N
O
C
OHH2 OH
O
O
OH
HO
OH
HO
N
O
HS
O
O
OH
NH 2
O
N
N
9.UTPgammaS
7.ATPgammaS
O
HN
N
N
N
H
PO
O
OH
20.3'-benzylamino
3'deoxy-ATP
OP
21.MRS 2255
PO
22.MRS 2268
Figure 3. (A) Structures of selected adenine nucleotides modified on the phosphate moiety
that have been investigated as P2 receptor agonists. (B) Structures of selected adenine
nucleotides modified on the base and ribose moieties that have been investigated as P2
receptor agonists. “P” refers to a single phosphate group; thus, “PP” is diphosphate, etc.
6
NH2
NH2
N
N
OH
OH
O
P
O
N
N
N
N
23a, Ap4A n=4
24, Ap5A n=5
25, Ap6A n=6
NH2
NH2
23b
HO
N
O
O
P
O
N
OH
n
NH3
OH
HO
OH
O
O
O
OH
O
N
N
O
O
O
N
N
N
N
P
O
OH
N
HN
OH
OH
HO
N
N
O
O
O
OH
2
N
O
OH
CHCl
OH
HO
N
P
O
OH
n
N
N
O
P
O
O
O
O
OH
N
N
N
O
N
N
OH
OH
OH
5
HO
OH
HN
O
26, Up3U n=3
27, Up4U n=4
28, Up5U n=5
29, Ip5I
Figure 4. Dinucleotides activating the putative P2D/P2YAp4A
In 1994, following from the seminal review of Dubyak [48] and the initial successes in the
cloning of P2 receptors, it was proposed a division of P2 receptors into two separate classes
based on both their structure and signal transduction properties [49]: the P2X (Table 1), a
family of ionotropic ligand gated ion channels (Logics); the P2Y (Table 2), a metabotropic,
heptahelical G-protein-coupled receptor (GPCR) family [50]. Other putative P2 receptors
including a UDP-glucose-sensitive GPCR (KIAA0001) cloned as an orphan receptor from
human brain [51], the putative P2D/P2YAp4A dinucleotide receptor responsive to dinucleotide
ligands (Figure 4) including Ap4A (23a), Ap5A (24), and Ap6A (25) [52], and a putative P3
receptor responsive to both nucleosides and nucleotides [53] have been reported. The existence
of these additional nucleotide-sensitive receptors has been based partly on their
pharmacological characterization, again with compounds that have inherent limitations in
their selectivity and potency. A P2X7 receptor paralog gene has been identified in the draft
human genome sequence [54]. It is important to note that many of the functional effects of
Ap4A and related urine and pyramiding polynucleotide’s may be ascribed to the activation of
other known P2 receptors [55], and it is possible that these molecules may have the potential
to act as depot sources for the generation of other nucleotides. As noted by Malevich and
7
Burnstock [3], in the absence of definitive molecular evidence for the existence of these other
P2 receptors, “receptor sub classification based on pharmacological criteria alone is no longer
tenable”. Thus the nomenclature system based on receptor structure has increasingly
supplanted the pharmacologically based historical system in the literature. For instance, many
of the receptors described previously as “P2U/P2U” are equivalent to the P2Y2 receptor while
the P2Z receptor has been cloned as the P2X7 receptor [71] and the elusive P2T as the P2Y12
receptor [56]. The use of structural homology alone is, however, an insufficient basis for
receptor classification. The recent discovery of the orphan GPCR, SP1999 [57] which is identical
to the cloned P2Y12 receptor [56, 58], illustrates that both structural and pharmacologically
relevant functions are necessary criteria for receptor classification. A novel sensory P2X
receptor has also been identified in Zebrafish [59].
1.2. ComplexitY of P2 Receptor Pharmacology
A variety of factors have tended to confound the pharmacological characterization of both
native and recombinant P2 receptors. These include the already mentioned early dependence
of P2 receptor classification on the rank order functional efficacy of ATP and UTP analogues
used as agonists and a lack of potent, selective, and bioavailable antagonists. In addition, the
intrinsic activity and metabolic stability of the various P2 agonists can also vary as a function
of the purity of the compounds used, the experimental protocol, the tissue(s) systems in which
their effects are assessed, and the species used [38, 39]. In addition to a lack of selectivity for the
different P2 receptors [38, 39], many P2 receptor antagonists reported to date have the potential
to interact with other biological targets. Suramin (30) (Figure 5), one of the most widely used
P2 receptor antagonists, antagonizes G proteins [60] and can inhibit proteases including HIV
reverse transcriptase. Furthermore, ATP can function as an agonist at the rat P2Y4 receptor but
as an antagonist (KB =700 nM) at the human receptor homologue [61]. Receptor reserve/
expression may also be a critical factor in defining pharmacophore efficacy, since ATP can
function as a partial agonist [62] or antagonist [63] at human P2Y1 receptors. Furthermore,
many commercially available preparations of P2 receptor ligands are chemically
heterogeneous or impure. For example, many of the early studies using Reactive Blue 2 (35) as
a P2 antagonist relied on samples that contained only 40-50% of the active entity with the
remaining components being unknown or uncharacterized. Similarly, many commercial
preparations of ATP can be contaminated with UTP. There are also marked differences in the
8
properties of native and expressed receptors both in terms of receptor expression number and
in regard to the associated proteins absent (or present) in either expression systems or the
natural milieu. These may provide markedly different data because of the consequent receptor
density, the propensity of the expressed receptor to interact with atypical protein partners, and
the nature of the reporter construct. At transfected P2X4 receptors, the anthelminthic ion
channel blocker, avermectin, noncompetitively modulates agonist responses. However, this
effect does not occur in vivo [64]. Also, in native cell types, evidence exists for more than one
P2 receptor subtype being expressed in a single cell. This is especially true for P2X receptors,
where the presence of unique, pharmacologically distinct, heteromers formed from the various
P2X receptor subunits in different permutations may be dynamically regulated [65-66]. This
further confounds data extrapolation from one system to another. Rapid agonist-induced
desensitization of P2-receptor-mediated responses would thus lead to the characterization of a
compound as an antagonist [3]. In the case of the quickly desensitizing P2X3 receptor
heteromer, agonist-mediated desensitization, resulting in functional receptor antagonism, can
occur in the absence of detectable receptor activation [67]. Even with bioisosteric replacements
of the labile phosphate groups of ATP, e.g., 10 and 11, these compounds can be readily
hydrolyzed by members of the ectonucleotidase/ E-NTPase family [23]. The activity of this
family of enzymes is highly tissue-dependent and varies according to the functional state of the
tissue [34, 35]. The net result of E-NTPase action is a reduction in the observed potency of ATP,
UTP, and their respective analogues as they are broken down to nucleoside 5’-diphosphates
(ADP, UDP) and to the nucleosides adenosine (from ATP) and uridine (from UTP) [23]. This
modification of agonist potency, which may vary from compound to compound, can then alter
the rank order activity of agonists, leading to a receptor characterization that is, in part,
dependent on the lability of the agonist rather than the intrinsic recognition properties of the
ligand for the receptor. This represents a generic problem when selective receptor antagonists
are not available. Nucleotidases can also be released in soluble form under physiological
conditions where they act to limit the effects of released ATP with distinct species differences
[68]. Since adenosine activates P1 receptors and purines can also modulate P2 receptor function
[69], the functional effects of these metabolic products become even more complex via the
context of the purinergic cascade [11]. Finally, nucleosides also undergo extracellular rephosphorylation to regenerate both the parent nucleotides and exchange phosphates between
purines and pyrimidines [21]. Several ATP analogues as well as putative P2 receptor
9
antagonists such as ARL 66096 (44) can inhibit the enzyme ectonucleoside triphosphate
diphosphohydrolyase (E-NTPDase; CD39 exists in two forms, one of which, E-NTPDase2, is
specific for ATP), leading to an augmentation of the effects of endogenous agonists, ATP,
and/or UTP. Thus, a compound, by inhibiting CD39 activity and increasing endogenous levels
of ATP, would appear to have agonist actions [70].
1.3. Radioligands
A lack of reliable radioligand binding assays for the characterization of P2 receptors has led to
additional confusion in the molecular characterization of P2 receptors.
While various bioisosteres of ATP (e.g., [3H]α,β-meATP, 10; [35S]ADPβS, 8; [35S]ATPγS, 7) have
been used to localize P2 receptors in various tissues [71], studies using transfected cells that
lack functional responses to P2 agonists show high levels of specific binding of [ 35S] ADPβS.
This binding actually decreases by 25% when a functional response is introduced via
transfection of the cells with cDNA for the P2X4 receptor subunit [72]. [33P]MRS 2179 (56, Figure
6), a high affinity P2Y1 receptor antagonist, has recently been developed as a receptor probe
[73]. Another critical issue in defining receptor function, not unique to P2 receptors, is that of
assigning significance to changes in receptor mRNA expression versus actual receptor protein
synthesis. While mRNA expression measured by Northern blot analysis may be altered as a
result of tissue or cell manipulation or in diseased tissues, such message lacks function at the
extracellular level, it is only when changes in receptor protein levels are detected at the cell
surface (Western blot) that definitive conclusions can be drawn regarding the physiological
and pathophysiological significance of such changes.
10
2. P2 Receptors
Fiftheen molecularly and functionally distinct mammalian P2 receptors have been generally
accepted up to today (Tables 1 and 2), the physiological function(s) of which, and their role in
tissue homeostasis and pathophysiology, are currently being elucidated using a variety of
pharmacological and genomic approaches including receptor antisense and receptor knockout
and knockin mice [3].
2.1. Relationship Between Native and Recombinant P2 Receptors
One of the main current challenges relating to P2 receptor research is the reconciliation of the
properties of recombinant receptors with those present in native tissues. Indeed, there are a
number of native P2 receptor phenotypes that, to date, do not have an exact molecular
correlate in recombinant studies of homo- or heteromeric P2X channels or P2Y receptors.
Clearly, the advances in studies on mouse knockouts of P2X [55, 57] and P2Y receptors [58, 63]
and the combination with behavioural and physiological paradigms will be a likely route for
future advance in relating the native phenotypes to the cloned P2 receptor subtypes, even if the
lack of subtype-selective ligands, especially potent and selective antagonists acts as a
considerable impediment to progress.
22..22 PP22X
XR
Reecceeppttoorrss
Members of the P2X receptor family are widely expressed in the central nervous system and in
the body periphery [1, 9, 23], where they play important physiological and pathophysiological
roles in a great variety of biological processes. These include muscle contraction, modulation of
the cardiovascular and respiratory system (P2X4), immunomodulation, inflammation and cell
death (P2X4,7), generation and transmission of nociceptive signals, fast synaptic transmission,
modulation of transmitter release and neuronal excitability (P2X2,4,6) [1, 2, 9, 23].
Functional P2X receptors are ATP-gated (ligand-gated) ion channels that mediate fast
excitatory neurotransmission in excitable tissues including neurons, glia, and smooth muscle
cells. ATP can elicit rapid responses (<10 ms) via these ion channels, esulting in selective equal
permeability to Na+, K+, and and significant permeability to Ca2+ cations [4, 9]. The membrane
depolarization resulting from the activation of P2X receptor multimers can lead to activation of
voltage-operated ion channels, L-type Ca2+ channels, and Ca2+-stimulated tyrosine kinases that
11
in turn activate MAP kinases (ERK1 and ERK2) that modulate transcriptional processing [74].
MAP kinase activation is also involved in P2Y1-receptor-mediated apotosis [75]. Seven P2X
receptor subunits with a two-transmembrane (2TM) motif structurally related to the amiloridesensitive epithelial Na+ channel of approximately amino acids in length with intracellular
termini and a cysteine-rich extracellular loop have been cloned from vertebrate tissues (human,
mouse and rat) and designated P2X1-7 .
The degree of sequence identity between these subunits ranges from 26% for P2X2 and P2X7 to
47% for P2X1 and P2X4 [3]. These subunits resemble proton-gated channels at a global but not
primary structure level [76] and form functional homomeric and heteromeric channels [68-69]
that currently available evidence suggests exist as stretched trimers (Figure 2) [77], contrasting
with the pentameric stoichiometry of other LGICs, e.g., the neuronal nicotinic receptor
superfamily [78]. Unlike the latter, very little is known regarding the nature of the ATP binding
site(s) on the proposed trimer, of distinct antagonist binding sites, of associated allosteric
binding sites, or of ancillary proteins necessary for native receptor function. P2X subunits, with
the exception of the P2X6 receptor [4], can form functional homomers that are activated by ATP
[68, 69]. The reason for the inability of P2X6 receptor subunits to form functional receptors is
unclear. Functional heteromers composed of P2X1/5, P2X2/3, P2X2/6, P2X2/6, and P2X1/2 subunits
have been described [68, 69, 79]. While there is convincing evidence for functional heteromers
from in vitro studies, in situ analysis studies of P2X receptors have resulted in the
identification of additional subunit combinations, e.g., a putative P2X2/5 heteromer, that are
pharmacologically distinct from those described above [80]. P2X receptors also appear to
undergo activation-dependent modifications that include cellular internalization for P2X1
receptors [69, 81, 82] or redistribution at synaptic junctions for the P2X2 receptor [83]. On the
basis of their kinetic parameters (table 1), P2X receptors have been divided into three groups
[3]. Group 1, which includes P2X1 and P2X3 homomers, is potently and rapidly activated by
agonists (0.01-0.1 s) and undergoes rapid inactivation (0.1-10 s) in the presence of prolonged
agonist activation. Group 2 includes P2X2, P2X2/3, P2X2/6, and P2X5 multimers that are rapidly
activated (0.1-1 s) and show slow inactivation/desensitization (10-100 s) profiles. Group 3 P2X
multimers include the P2X1/5, P2X4, P2X4/6, and P2X7 receptors. These show the same activation
kinetics as group 2 multimers but have both a fast and slow desensitization phase. In general,
those P2X receptors that undergo rapid desensitization are activated by α,β-meATP (10), 2MeSATP (13), and ATP (1). P2 receptors that undergo slower desensitization or show no
12
desensitization can be subdivided into two groups, those sensitive to α,β-meATP (P2X1/5, P2X4,
P2X4/6) and those that are only weakly sensitive to this agonist (P2X7).
In addition to functional homomeric channels (P2X1 – P2X5 and P2X7), P2X receptors may be
formed by hetero-oligomerization of different subunits (P2X1/5, P2X2/3, P2X2/6 and P2X4/6). P2X6
subunit does not function as homomultimer [13, 14, 24, 25]. It is not yet exactly known how the
properties of the different subunits influence the phenotype of the heteromeric channels.
However, the unique combination of properties that can be achieved through the formation of
heteromeric
P2X
receptors
confers
potentially
new
levels
of
physiological
and
pathophysiological control that still need to be elucidated. The heterogeneity of P2X receptors
may further be increased by alternative splicing [2, 14, 16]. To make matters more complex,
immunocytochemical studies suggest that cell populations in a variety of tissues may express
multiple subunit proteins [2]. In addition, time will tell whether P2X receptor subtypes also
show polymorphism of pharmacological importance. The overlapping expression of different
P2X subunits raises important questions about the subunit composition of the natively
assembled P2X receptors and their stoichiometry. Furthermore, the recent identification of
species differences in P2X receptor pharmacology is also important in the development of P2Xselective ligands and in the process of drug discovery [26, 27].
P2X1. The human, rat, and mouse P2X1 subunits (Table 1) were originally cloned from vas
deferens and urinary bladder. The functional P2X1 receptor can be rapidly activated by ATP
(pEC50 = 7.3) and its analogues (0.01-0.1 s) and undergoes rapid desensitization (0.1-10 s). The
rank order potency for agonist activation of recombinant P2X1 receptors was BzATP (19,
pEC50 = 8.8) » 2-Me-SATP (13) ≥ ATP > α,β-meATP (10) >> ADP. Agonists at this receptor are
potently and selectively blocked by Ip5I (29, pEC50 = 8.0) [55] as well as by other antagonists
including TNP-ATP (53), the suramin analogues, NF023 (31), NF279 (32), NF449 (33), and the
PPADS (42) analogues, MRS 2159 (45), and PPNDS (46) (Figure 5-6A). Messenger RNA for
the P2X1 receptor subunit is expressed in urinary bladder, smooth muscle layers of small
arteries and arterioles, vas deferens, lung and spleen, dorsal root, trigeminal and celiac
ganglia, spinal cord, and brain [84]. A P2X1 receptor has also been identified in platelets and
megakaryocytes. Deletion of the P2X1 receptor gene in male mice [85] results in a 90%
reduction in fertility, the consequence of a decrease in the amount of sperm in the ejaculate, a
reflection of a 60% reduction in the contraction sensitivity of the vas deferens to sympathetic
nerve stimulation and an abolition of the responsiveness to P2X receptor agonists.
13
P2X2.
The
human
rat
P2X2
subunit
was
initially
cloned
from
pancreas
and
pheochromocytoma PC12 cells [86]. At the recombinant P2X2 rec5.8) and ATPγS (7) are
equipotent as agonists, with α,β-meATP and‚ β,γ-meATP (11) being inactive. Suramin and
changes in P2Y1 receptor expression in dorsal root ganglion [97]. The N-type calcium channel
blocker, ῳ-conotoxin GVIA, is also an allosteric modulator of P2X3 receptor-mediated
responses in rat dorsal root ganglion neurons [98]. It is more potent (IC 50 = 21 nM) on P2X3
homomers than on the P2X2/3 heteromer (IC50 = 3.8 µM).
P2X3. The P2X3 subunit was originally cloned from rat dorsal root ganglion. The potency
order for agonist activation of the recombinant homomeric P2X3 receptor is BzATP (19)
(pEC50 = 7.1 >> 2-MeSATP > ATP ) = α,β-meATP. P2X3 receptor activation is potently blocked
by TNP-ATP (53) (pEC50 = 7.8). The P2X3 homomeric receptor undergoes rapid
desensitization (<100 ms). The P2X2/3 heteromeric receptor shares the pharmacological profile
(α,β-meATP sensitivity) of the homomeric P2X3 receptor and the slow desensitization
kinetics of the P2X2 homomer and potentially represents a naturally occurring form of the
receptor that is involved in pain perception [92]. Message for the P2X3 subunit was initially
reported to have a relatively restricted distribution compared to other P2X receptors being
expressed in a subset of sensory neurons including the trigeminal, dorsal root, and nodose
ganglia. It is largely absent from smooth muscle, sympathetic, and neurons. However, P2X3
receptor specific immunoreactivity in nucleus tractus solatarius and other brain regions has
been recently reported [93, 94] Mouse knockouts of the P2X3 receptor [95, 96] show a
complete absence of quickly desensitizing electrophysiological responses in dorsal root
ganglia, show no apparent compensatory alterations in the expression of other P2X subunits,
and show decreased nociceptive responses and bladder hyporeflexia [95], highlighting the
role of this receptor in sensory physiology. The incomplete reduction in nociceptive signaling
following the knockout of the P2X3 receptor is apparently due to a residual effect of ATP
acting via a P2Y1 receptor to modulate VR1 receptor function [30]. Axotomy also leads to
changes in P2Y1 receptor expression in dorsal root ganglion [97]. The N-type calcium channel
blocker, ῳ-conotoxin GVIA, is also an allosteric modulator of P2X3 receptor-mediated
responses in rat dorsal root ganglion neurons [98]. It is more potent (IC 50 = 21 nM) on P2X3
homomers than on the P2X2/3 heteromer (IC50 = 3.8 µM).
P2X4 The P2X4 subunit has been cloned from a variety of rat and human tissue sources [99].
P2X4 subunit message is found in brain, spinal cord, sensory ganglia, superior cervical
14
ganglion, lung, bronchial epithelium, bladder, thymus, salivary glands, testis, and vas
deferens. Expression of P2X4 subunits occurs at levels sometimes 100-fold greater than other
P2X subunits, particularly in regions of the cerebellum and hippocampus, where in the latter
brain region there is high colocalization with AMPA-sensitive glutamatergic receptors [100].
The recombinant P2X4 receptor shows greatest agonist sensitivity to BzATP (19) and ATP
(pEC50 = 6.3) and is insensitive to the P2 receptor antagonists suramin and PPADS [101].
Recombinant P2X4 receptor functional responses are potentiated by Zn 2+, by the channel
modulating macrolide anthelminthic, avermectin [64], and by the macrolide antibiotic
erythromycin, which, at concentrations that are clinically achievable (10 µM), can block the
effects of ATP on calcium influx in a human lung epithelial-like carcinoma cell line [102].
Alternatively, spliced forms of the P2X4 receptor subunit can form heteromers with wild-type
P2X4 subunits that are distinct from wild-type P2X4 homomers [101].
P2X5. The P2X5 subunit was initially cloned from rat celiac ganglion and shows an activation
profile of ATP > 2-MeSATP > ADP with α,β-meATP being inactive. It is relatively insensitive
to suramin and PPADS. Message for this P2 receptor subunit is present in the ventral horn of
the cervical spinal cord and in trigeminal and dorsal root ganglion neurons. The cloned chick
P2X8 receptor has a high sequence identity (59%) with the rat P2X5 receptor and may
represent the avian ortholog. As previously noted above, P2X5 subunits form functional
heteromers with P2X1 that show high sensitivity to ATP and TNP-ATP (53) [103].
P2X6. The P2X6 receptor subunit was originally cloned from a rat superior cervical ganglion
cDNA library. [149]. Its activation profile is ATP > 2-MeSATP > ADP, with α,β-meATP being
inactive. This receptor shows only partial inhibition by suramin or PPADS. Robust
transfection of functional P2X6 subunits has proven to be difficult. A p53-inducible P2
receptor designated P2XM, the function of which is altered in soft tissue tumors, has
sequence homology similar to that of the P2X6 subunit [104].
P2X7. The P2X7 subunit was cloned from rat and human brain and macrophages. It is
structurally different from other P2X receptor subunits in having a longer (240 amino acid)
intracellular C terminal. The P2X7 receptor was previously characterized as the P2Z receptor
[105], a cytolytic receptor present in cells of hematopoietic origin including mast cells,
macrophages, lymphocytes, erythrocytes, and fibroblasts. Brief activation (<10 s) of the P2X7
receptor results in a rapid, reversible membrane depolarization with Na+, K+, and Ca2+ influx
[105]. On prolonged exposure to ATP or other P2 agonists in the presence of low levels of
15
divalent cations, the P2X7 receptor converts to a nonselective pore that is permeable to small
molecules of molecular weight up to 900 Da, an event associated with cytotoxic effects, e.g.,
cell swelling, vacuolization, and necrotic and apoptotic cell death. All of the 240 amino acids
in the intracellular C terminal are required for induction of pore formation. The ability of P2
receptor agonists to induce pore formation was thought to be a unique property of the P2X 7
receptor, but other P2X subunit homomers, e.g., P2X2, P2X4 and P2X2/3 receptors, can also
form pores upon prolonged agonist application [106]. The potent cytotoxin maitotoxin,
derived from the dinoflagellate Gambierdiscus toxicus, induces a cell membrane pore that is
physiologically identical to the P2X7-induced pore, suggesting that this toxin may be a ligand
and/or a cofactor in P2X7-induced pore formation. The rank order agonist potency for
activation of the P2X7 receptor is BzATP (19 pEC50 = 5.3) » ATP, with 2-MeSATP, ATPγS (9),
and ADP being inactive. The human receptor has a lower sensitivity to agonists than the rat
receptor. Brilliant blue G (41, pEC50 = 5.3) is a potent noncompetitive antagonist of the P2X7
receptor [107] 2´,3´-dialdehyde ATP (54) (oxidized ATP, ox-ATP) is an irreversible inhibitor
of P2X7 receptors. In macrophages and lymphocytes, P2X7 receptor activation results in the
activation of phospholipase D and in human macrophages elicits the release of the
inflammatory cytokine IL-1‚ via activation of caspase-1 (IL-1‚ converting enzyme) [108, 109].
In addition to cells of hematopoietic origin, the P2X7 receptor is also found on hepatocytes
and acinar cells of the parotid and salivary glands. In macrophages, the P2X7 receptor is also
involved in the formation of multinucleated giant cells. A paralog of the P2X7 receptor with
80% homology to the designated P2X7 receptor has been identified in the draft sequence of
the human genome [44]. Proteomic analysis of the P2X7 receptor in HEK cells identified a
signaling complex comprised of 11 proteins that included laminin α-3, integrin ‚β2,β-actin,
supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4-kinase, and the
receptor protein tyrosine phosphatase-β (RPTP- β), the last of which may modulate P2X7
receptor function via control of its phosphorylation state. The C-terminal motif of the P2X7
receptor contains a conserved lipopolysaccharide (LPS) binding domain (amino acids 573590) that is structurally similar to the LPS binding site of the bactericidal/ permeability
increasing protein BPI [110]. Peptides derived from this latter motif bind LPS in vitro and
block the ability of LPS to activate ERK1 and ERK2 and degrade IkB-α in macrophages. A
P2X7 receptor knockout mouse shows a disruption in cytokine signaling cascades with
perturbation of ATP induced processing of pro-IL-1‚ by macrophages. In some patients with
16
B-chronic lymphocytic leukemia, the lymphocyte P2X7 receptor is non-functional [111].
Evaluation of single nucleotide polymorphisms (SNPs) for the P2X7 receptor in patients with
chronic lymphocytic leukaemia [112] showed SNPs at positions and that occurred with allele
frequencies of greater than 1%. Examination of the SNP at position, which is present on the
carboxy-terminal tail of the P2X7 receptor subunit, in normal subjects showed a Glu496, Ala
polymorphism associated with loss of function of the receptor.
17
18
O
NaO 3S
O
H
N
N
H
N
H
O
SO3Na
O
H
N
N
H
N
H
O
SO3Na
NaO 3S
30, SURAMINA
NaO 3S
SO3Na
NaO3S
SO3Na
SO3Na
NaO3S
SO3Na
O
NaO3S
HN
SO3Na
O
SO3Na
HN
NaO3S
O
O
SO3Na
NH
N
H
NH
HN
2
2
O
NaO3S
SO3Na
O
N
H
N
H
32, NF279
31, NF023
SO3Na
SO3Na
SO3Na
NaO3S
SO3Na
SO3Na
O
O
HN
NaO3S
O
NH
HN
NO 2
O2N
N
H
O
NaO3S
SO3Na
SO3Na
O
NH
N
H
NH
O
34, XAMR0721
SO3Na
33, NF449
SO3Na
NaO3S
N
N
OH
O
NH2
NH2
SO3Na
NH2
OH
N
N
SO3Na
NaO3S
O
R
39, Tripan blue
Cl
R'
SO3Na
N
R=
N
NaO 3S
N
H
N
N
H
NCS
H
C
C
H
35, R'= -SO3Na Reactive Blue 2 (m- and p- mixture)
36, R'=2 -SO3Na Cibacron Blue 3GA
SCN
SO3Na
40, DIDS
R=
R=
SO2
N+
NaO3S
37, Acid blue 129 38, UniblueA
O
N
H
N
SO3Na
41, Brialliant blue
19
Figure 5. Structures of selected polysulfonated
COH
O
HO
A
P
O
OH
OH
N
N
N
R1=
R1
SO3H
SO3H
HO3S
S
O
SO3H HO S
3
CO2H
3
CO2H
H
Cl
43, iso-PPADS
42, PPADS
NH
O
45,MRS 2519
44, MRS2160
SO3H
Cl
SO3H
O
PO3H
NO2
SO3H
46, PPNDS
49, SB9
N
H
SO3H
PO3H
47, MRS 2191
48, MRS 2257
NH(CH2)2SCH3
NH2
N
B
HO
O
O
O
P
P
P
O
O
C
OH OH OH
X2
N
N
N
N
S(CH2)2CH3
HO
O
O
O
O
P
P
P
N
N
O
O
C
OH OH OH
H2
N
S(CH2)2CF3
O
OH OH
OH OH
52, ARL 69931MX
50, X= Cl ARL66096
51, x= F ARL67085MX
NH2
NH2
N
N
NH2
N
N
O
HO
O
O
S
P
O
O
N
O
OH
N
O
OP
CHO
OP
54, oxidized-ATP
O
O
O
CHO
N
N
PPPO
PPPO
PO
N
N
N
N
N
NH2
N
N
N
55a, A3P5P
55b, A3P5PS
O
NO2
O2N
HN
NO2
53, TNP-ATP
R
PO
N
N
N
N
O
HN
HN
N
N
N
Cl
PO
N
O
N
N
N
N
PO
OP
56,R =H MRS 2179
57,R=Cl MRS 2216
PO
58, MRS 2286
PO
59.MRS 2279
Figure 6. (A) Pyridoxal phosphate derivatives. (B) Adenine nucleotide derivatives
20
N
N
SO2
O
SO2
SO2
N
N
O
N
N
N
60. (KN-62)
61. (KN-04)
NO 2
O
OH
P
O
O
O
O
O
N
H
62. Nicardipina
(CH2)2N(CH3)CH2Ph
N
N
N
C
O
SO2
HO
O
O
O
Et
P
O
N
63. MRS 2220
ONH4
ONH4
N
H
64. MRS 2154
Figure. 7 Non phosphorylated and non sulphonated derivatives that have been shown to act
as P2 antagonists.
21
2.3. P2Y Receptors
P2Y receptors are G-protein coupled receptors (GPCRs) with a typical hydrophobic
heptahelical transmembrane (7-TM) motif and are sensitive to activation by both purines
(ATP, ADP) and pyrimidines (UTP, 5, UDP, 6) [7, 10, 113]. Other receptors that are closest in
sequence homology to P2Y receptors include angiotensin and thrombin peptide receptors, an
observation that has led to speculation that there may be an as yet unidentified endogenous
peptide ligand for P2Y receptors.
The currently accepted members of this family are the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12,
P2Y13 and P2Y14 receptors (Table 2). They have been cloned from mammalian tissues (human,
mouse and rat). These subtypes (human) share an overall sequence identity ranging from
19% to 55% and their sizes range from 328 to 377 amino acids [1, 29-34]. Most of the P2Y
receptors produce their functional effects via G protein coupling (Gq/11) to activate
phoshoplipase C (PLC), forming IP3 and mobilizing intercellular calcium [10]. This in turn
leads to activation of other signaling pathways that include protein kinase C, PLA 2, calciumdependent K+ channels, nitric oxide synthase (NOS), voltage-operated calcium channels, and
MAP kinase pathways. Some P2Y receptors are also linked to inhibition of adenylate cyclase
activity [10]. Furthermore, the P2Y11 receptor is unique among P2Y receptors because it
couples to the stimulation of both phosphatidyl-inositol and adenylate cyclase signalling
pathways [33, 36, 37]. On the other hand, the activation of P2Y12 (P2T) receptors, present in
platelets and C6-2B glioma cells, results in the inhibition of adenylate cyclase via Gi/o [34, 48,
49]. On the basis of their structural similarities, P2Y receptors have been divided into two
distinct groups: group I consists of P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors; group II
consists of P2Y12 and P2Y13 receptors [114]. The UDP-glucose-sensitive GPCR KIAA0001 [42]
is a potential member of this second group and has been previsionally designated as the
P2Y14 receptor [114]. Despite emerging evidence [115] that functional dimerization of GPCRs
is a rule rather than an exception, there is currently little information regarding P2Y receptor
dimers, although there is evidence for P2Y1/ adenosine A1 receptor dimerization, the
resultant oligomer showing a distinct pharmacology with A1 agonists and antagonists
showing reduced affinity with a concomitant 400-fold increase in affinity for the P2 agonist
ADPβS (8) [116]. P2Y receptors can be characterized on the basis of responses to nucleotide
agonists and subtype-preferring antagonists (Table 2). Functional expression studies in
22
heterologous cell systems revealed three phenotypes of mammalian P2Y receptors: selective
purinoceptors (P2Y1, P2Y11 and P2Y12), selective pyrimidinoceptors (P2Y6) and receptors of
mixed selectivity (P2Y2 and P2Y4) [1, 29-32, 34, 36, 37].
Table .2. Mammalian P2Y receptors
P2Y1. The P2Y1 receptor was the first P2 receptor to be cloned [117]. The rank order of
agonist activation is 2- MeSATP ≥ ATP >> ADP, with α,β-meATP, β,γ-meATP, an UTP being
inactive. ADP is the most potent natural agonist. MRS 2279 (53) is the most potent P2Y1
antagonist yet reported [55] suramin (30) and Reactive Blue 2 (RB-2, 35) can also block the
effects of agonists at this receptor at micromolar concentrations. Activation of the P2Y 1
receptor results in either activation of PLC via the Gq coupling protein G11 or inhibition of
adenylate cyclase via Gi subunits that act independently [10]. P2Y1 receptor activation can
also directly modulate ion channel function, a G-protein mediated effect that is independent
of other second messenger systems. In rat cerebellar neurons, P2Y 1 receptor activation leads
to the opening of an outwardly rectifying K+ current via coupling of the βγ subunits of the G
23
protein to a K+ channel. P2Y1 receptor gene disrupted mice show altered platelet physiology
and a significantly reduced incidence of lethality from thrombosis [118].
P2Y2. The P2Y2 receptor (originally described as the P2U receptor) was first cloned from the
mouse neuroblastoma NG108-15 cell line [116] and subsequently from rodent and human
tissue ATP and UTP were equipotent in activating the receptor, with ADP, UDP, 2-MeSATP,
and α,β-meATP having weak to no activity. P2Y2 receptors are coupled to Gi and Gq proteins
that mediate phospholipid breakdown, IP3 formation, and calcium mobilization. In airway
epithelia, biliary epithelial cell lines, and avian exocrine salt gland cells, P2Y2 receptor
activation leads to opening of Ca2+-sensitive Cl-channels that are involved in epithelial fluid
secretion [119]. P2Y2 receptors negatively coupled to adenylate cyclase activation have been
reported as has P2Y2 activation of a member of the Kir 3.0 inward rectifier channel and
adenylyl cyclase via a cyclo-oxygenase (COX) dependent mechanism [120]. P2Y2 are
antagonized by suramin, but to a lesser extent than at the hP2Y1 receptor (about 28-fold),
whereas PPADS and MRS2279 are ineffective. The sensitivity of P2Y2 receptors to blockade
by suramin (30) and PPADS (42) has provided evidence for suramin-sensitive and-insensitive
responses and PPADS-sensitive and –insensitive responses suggestive of potential receptor
heterogeneity [121], although there is no molecular evidence for subtypes of the P2Y2
receptor. P2Y2 receptor knockout mice [122] show alterations in chloride transport function in
lung and other secretory tissues.
P2Y4. The P2Y4 receptor is a uridine-nucleotide specific receptor that was cloned from human
placenta and rat heart [123]. The human P2Y4 receptor has greater selectivity for UTP (full
agonist) over ATP and is insensitive to ADP and UDP. At the rat P2Y4 receptor, ATP
(competitive agonist) and UTP are equipotent, with ADP, ATPγS, 2-MeSATP, and Ap4A
(23a) acting as partial agonists. PPADS (42) has been reported as a weak antagonist for the
human P2Y4 receptor, with suramin (30) being inactive. The P2Y4 receptor has a relatively
restricted tissue expression being found only in placenta and pancreas with low levels in
lung and vascular smooth muscle [123]. There are currently no known selective P2Y4 receptor
antagonists. The P2Y2 and P2Y4 receptors can be distinguished with antagonists, i.e. suramin
blocks hP2Y2 and PPADS as well as reactive blue 2 block P2Y4 receptors.
P2Y6. The P2Y6 receptor was isolated from rat aortic smooth muscle, human placenta, and
human spleen [124]. The most potent agonist at the P2Y6 receptor is UDP with weak or no
effects seen with UTP, ATP, ADP, or 2-MeSATP. The P2Y6 receptor is linked to Gq
24
stimulating PLC with the formation of IP3 in monocytes, interleukin-8 [125], and is linked to
M-type potassium channels. Message for the P2Y6 subunit is widely distributed including
placenta, heart, lung, intestine, spleen, and airway and nasal epithelium [124]. There are
currently no selective antagonists for the P2Y6 receptor. The P2Y6 receptor is blocked by
Reactive Blue 2, PPADS and suramin with potency decreasing in that order.
P2Y11. The P2Y11 receptor was cloned from human placenta [126] and is also found on human
lymphocytes and canine kidney cells [127]. The P2Y11 receptor interacts with lymphocyte
adenosine A2A receptors in the developmental fate of B lymphocytes and is uniquely sensitive
to purine nucleotides with a rank order agonist potency of ATP > 2 MeS-ATP >>> ADP, with
UTP and UDP being inactive. The human receptor is also unique in that is coupled to both
adenylate cyclase and phosphoinositide pathways. The canine P2Y11 receptor in MadinDarby kidney cells is linked to an increase in short-circuit current (ISC)[127]. The P2Y11
subtype is highly sensitive to suramin and RB-2 antagonism, whereas PPADS is completely
inactive.
P2Y12. The P2Y12 receptor was cloned from rat and human cDNA libraries [56, 57, 58] and
represents the elusive ADP-sensitive P2 receptor on platelets previously termed P2T, P2YADP,
and P2YT. While previously thought to be a form of the P2Y1 receptor [57], the P2Y12 receptor
plays a discrete role in platelet shape change and aggregation. This confusion between the
P2Y1 and P2Y12 receptors may be explained by their combined role in platelet aggregation
[128]. ADP is a full agonist at the P2Y12 receptor with ATP and its bioisosteres, including ARL
67085 (51) and AR-C 69931MX (52) being functional antagonists that, like the
thienopyridines: ticlopidine and clopidigrel, are active in the clinic [129]. Ap4A (23a), a P2,
P3-monochloromethylene analogue, AppCHClppA (23b), and various phosphorothioate
analogues are competitive inhibitors of platelet aggregation. The P2Y12 receptor is present on
platelets and megakaryoblastic cell lines and is coupled to a Gi2 protein that inhibits
adenylate cyclise [72]. P2Y12 knockout mice in general have a normal phenotype but exhibit
prolonged bleeding time and reduced sensitivity to ADP, thrombin, and collagen.
P2Y13. The P2Y13 receptor is the most recent member of the P2Y family to be identified [114].
Initially cloned as an orphan GPCR alternatively termed GPR86210 and GPR94 the P2Y13
receptor has a high degree of homology compared to the P2Y12 receptor [114]. This receptor
has also been cloned as SP174 [130]. On the basis of its tissue distribution, the P2Y 13 receptor
has been implicated in immune system function, specifically T-cell maturation [114, 130].
25
P2Y14. This receptor, sensible to UDP- glucose, together with P2Y12 and P2Y13 may represent
a P2Y receptor subgroup for which transduction is exclusively through adenylate cyclase.
Other Putative P2 Receptors. As an addition to the list of receptors missasigned to the P2
receptor family already documented, the p2y3 receptor is a nucleotide-sensitive avian
receptor for which a mammalian homologue has not yet been described. The p2y5 receptor
was identified in activated chick T lymphocytes using radioligand approaches. No functional
or structural support for the existence of this exceptor has been reported. The P2Y 8 receptor
was cloned from Xenopus neural plate and was activated to an equal degree by ATP, UTP,
ITP, CTP, and GTP. Mammalian homologues of this receptor have yet to be identified. The
receptors cloned as P2Y9 and P2Y10 are not nucleotide receptors [113]. A novel cysteinyl
leukotriene receptor 1 (CysLT1) has been identified on human cordblood derived mast cells
(hMCs) and is functionally responsive to the cysteinyl leukotrienes LTC4 and LTD4 and also
to UDP. Interestingly, the effects of both the leukotrienes and UDP can be blocked by the
CysLT1 antagonist MK 571. This relationship between P2 receptors and CysLT receptors is
intriguing especially in light of the misidentification of the LTB4 receptor as the putative
P2Y7 receptor, suggesting perhaps that there is a close function relationship between the two
receptor families. A P2T-like receptor distinct from that on platelets that shows agonist
responses to 2-MeSATP has been identified in brain capillary epithelial cells. Some of the
missing numbers among the P2Y receptors are those cloned from non-mammalian sources.
The chicken p2y3 receptor likely represents the avian orthologue of the mammalian P2Y 6
receptor, whereas the turkey tp2y and the Xenopus p2y8 receptors appear to be similar to the
mammalian P2Y4 receptor [43, 44]. Other receptors have been mistakenly included in the P2Y
family. The p2y7 receptor is actually a leukotriene B4 receptor, whereas p2y5, p2y9 and
p2y10 receptors must be considered as orphan receptors [1, 29, 39, 48]. A wide variety of
tissues and peripheral as well as central cell types express at least one of the six cloned
mammalian P2Y receptor subtypes, where they mediate a broad range of physiological
responses. These effects are as diverse as platelet aggregation, stimulation of epithelial
chloride transport, granulocytic differentiation, regulation of vascular tone and modulation
of ion-channel activity and cell-to-cell signalling [1, 9, 29, 39, 45, 46, 48].
26
2.4. P2 Receptors Structure: P2X Receptors
P2X receptors are oligomeric proteins composed of more than one subunit per functional
receptor. Several approaches have been used to deduce the P2X receptor stoichiometry. Both
biochemical and electrophysiological analyses suggest [77] indicates that functional P2X
receptors have a trimeric motif. A model of the generalized P2X receptor subunit (Figure 8)
shows the topography of glycosylation sites and the cytoplasmic orientation of both amino
and carboxy termini. Features of the extracellular domain include 10 cysteine residues (-S),
that are highly conserved among different P2X subtypes and are thought to form disulfide
bridges. Four glycosylation sites have been identified on the rat P2X1 receptor. However, the
number and location of glycosylation sites differ within the different subunits of the P2X
family [131]. Numerous conserved positively charged residues (K and R) are present in the
extracellular loop of P2X receptors, four of which at positions 68, 70, 292, and 309 are
important for ATP binding to the human P2X1 receptor. The neutral residue I67 of the rat
P2X2 receptor defines a critical region of ATP binding. By use of the “SCAM” technique, in
which individual residues were replaced with cysteine and various alkylating reagents were
xamined for their ability to disrupt ligand recognition by reacting covalently with accessible
thiols, the residues within TM2 lining the pore of the rat P2X2 receptor were identified. The
results indicate that part of the pore of the P2X2 receptor is formed by the second
hydrophobic domain [20, 21], and also suggest that Val48 at the outer end of the first
hydrophobic segment takes part in the gating movement of channel opening [22, 51]. I328,
N333, and T336 were at the outer vestibule of the pore, while L338 and D349 lined the cation
channel. D349 was solvent-accessible only when ATP was applied. The use of chimeric
receptors has shown that the second transmembrane domain of P2X receptors is essential for
subunit assembly [38].
Chimeras of PX1 and P2X2 receptor subunits in concert with site-directed mutagenesis of the
N terminal of the first transmembrane domain demonstrated a critical role for this domain in
agonist effects [132], while calcium permeability of the P2X2 receptor was abolished by
replacing polar amino acid residues at Thr339 and Ser340 with tyrosine [133]. The P2X
receptor channels differ among themselves with respect to the rates of desensitization during
prolonged agonist stimulation. Site-directed mutagenesis studies suggest an important role
for amino acid residues in the C-terminus of rat P2X2 receptors in determining the rate of
27
desensitization. A protein kinase C site on the C terminus was responsible for slowing
desensitization of the P2X2 receptor [134]. In addition, other domains of P2X channels may
also contribute to desensitization [15].
Figure 8. Schematic representation of a P2X receptor. (A) An individual subunit showing the twotransmembrane (2TM) motif and amino acid residues that are implicated in ligand binding and
that maintain the conformation of the subunit. (B) Possible arrangement of subunits in a trimer,
based on the finding that intersubunit recognition is dependent on residues of the second TM
domain (II).
28
Figure 9. Schematic representation of a P2Y receptor showing the seven-transmembrane
(7TM) motif and amino acid residues that are implicated in ligand binding. The helices
shown are arranged in a roughly circular bundle, which is open in this diagram for
representational purposes. Model shows features of the hP2Y1 receptor important for
nucleotide binding both within the TMs (3, 6, and 7) and ELs (2 and 3), including three
positively charged residues (R128, K280, and R310) found to be important for ATP binding
(electrostatic interaction). Four Cys residues (-S), which are conserved among P2Y subtypes,
form disulfide bridges. The location (extracellular N-terminus) of the putative glycosylation
sites is conserved within the P2Y family. Shown also are residues within TM6 and -7, which
are modulatory for the activation of the hP2Y1 receptor. Molecular modeling has predicted
contact with the adenine and triphophate regions of ATP as shown. The 7TM bundle is
assumed to be closed (i.e., TM1 and TM7 are in contact), and EL2 is in proximity to the TM
ligand binding domain. Hydroxyl group on the intracellular C-teminal region represents
potential phosphorylation sites, for protein kinases that may participate in receptor
desensitization and internalization.
29
2.5. P2 Receptors Structure: P2Y Receptors
The structure of P2X receptors are not yet amenable to molecular modelling due to the lack
of protein template [135]. However, P2Y receptors have been successfully modelled using the
high resolution structure of bovine rhodopsin as a template [136, 137] by means of the
homology modelling technique.
P2Y receptors contain all of the typical features of the G-protein linked receptors (GPCRs),
including the seven hydrophobic transmembrane domains (TM), connected by three
extracellular (ELs) and three intracellular (ILs) loops. The great majority of the TM residues
are arranged in α-helical structures, while in EL2 an antiparallel β-sheet is present. Especially
in the regions with defined secondary structure, there is a great overall similarity between
P2Y and the templates, the only significant deviation being present in very flexible NT, EL1,
EL3, IL1, IL2, and IL3. As in the crystal structure of rhodopsin used as a template, the αhelical structure of TM3, TM4 and TM6 extend into the cytoplasm for all the P2Y subtypes.
At the cytoplasmic end of the TM7 in all subtypes, with the exception of P2Y 2 and P2Y11, the
protein folds at an angle of ̴ 90° to form a helical segment (H8) that runs parallel to the plane
of the cell membrane. Ligand affinity SAR, sequence analysis of cloned P2Y receptors, and
site-directed mutagenesis studies [138] have led to the refinement of computer-based models
for ligand binding to P2Y receptors. Recently structural insights have been gained using
molecular modelling based on a rhodopsin template in conjunction with mutagenesis to
suggest recognition elements important for nucleotide binding in TMs 3, 5, 6 and 7 and EL2.
Most of the mutagenesis work in the P2Y family has been carried out on the human P2Y1
receptor. To ascertain which residues of the human P2Y1 receptor were involved in ligand
recognition, individual residues of both the TMs (3, 5, 6, and 7) and ELs (2 and 3) were
mutated to alanine and various charged residues, and a cluster of positively charged lysine
and arginine residues near the exofacial side of TMs 3 and 7 and, to a lesser extent, TM6
putatively coordinated the phosphate moieties of nucleotide agonists and antagonists.
Agonists were inactive at R128A (TM3) and at R310A and S314A (TM7) mutant receptors and
had a markedly reduced potency at K280A (TM6) and Q307A (TM7) mutant receptors.
Positively charged residues of the human P2Y2 receptor (H262, R265, and R292 in TM6 and
TM7) were similarly found to be critical for activation, suggesting that residues on the
30
exofacial side of TM3 and TM7 were critical determinants of the ATP binding pocket. In
contrast, there was no change in the potency or efficacy of agonists in the S317A mutant
receptor, and alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant
receptors that exhibited a 7- to 18- fold reduction in potency compared to that observed with
the wild-type receptor. These residues thus appear to serve a less important modulatory role
in ligand binding to the P2Y1 receptor. Several charged residues in ELs 2 (E209) and 3 (R287)
were critical for receptor activation, suggesting that the role of the ELs in ligand recognition
was as important as that of the TMs. Moreover, energetically favorable “meta binding sites”
in the P2Y1 receptor have been defined, involving the critical residues of the ELs. At these
nucleotide docking sites that are postulated to lie distal to the principal TM site, a ligand may
bind en route to the principal TM binding site. Such secondary binding sites may then serve
to guide the ligand in its approach to the TM binding site and reduce the energy barrier to
ligand/ receptor complex formation. Two essential disulfide bridges in the extracellular
domains of the human P2Y1 receptor were also identified: one conserved among GPCRs and
another conserved between the N-terminal domain and EL3, characteristic of P2Y receptors.
Since changes in the potency of 2-MeSADP (14) and HT-AMP (15) paralleled the changes in
potency of 2-Me-SATP at the various mutant receptors, it appeared that the β- and γphosphates of the adenine nucleotides were less important than the α-phosphate in
ligand/P2Y1 receptor interactions. However, T221A and T222A mutant receptors exhibited
much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors,
respectively) versus di- or monophosphate potency, a result indicating a greater role of these
TM5 residues in γ-phosphate recognition. Taken together, the results suggest that the
adenosine and α-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the
exofacial side of P2Y receptors. Recently, a detailed model of antagonist binding to the P2Y1
receptor binding was presented. Ligand docking in the P2Y1 receptor model provided a
hypothesis for the coordination of ATP in the TM regions, consistent with site-directed
mutagenesis results, with a binding mode very similar to the one of the agonist. The
structural similarity between the potent nucleotide antagonist MRS 2179 (56) and nucleotide
agonists suggests that receptor activation resulting in a specific conformational change
depends on subtle differences between ligands.
31
3.1 Development of P2 Receptor Ligands
The identification of new pharmacophores that selectively and potently interact with (a) P2
receptors and (b) individual members of the P2X and P2Y families represents a major
challenge in medicinal chemistry.
Much of the historical data on P2 receptor ligands have been confounded by the use of a
limited repertoire of highly labile agonists, the majority related to ATP. Moreover,
moderately active antagonists with questionable purity, stability, and selectivity with
comparisons of such compounds being made between different tissue systems using
different physiological and pharmacological end points in different laboratories [45].
Evaluation of compounds in these systems is also confounded by different levels of
nucleotidase activity and by species differences in receptor pharmacology. This situation has,
to some extent, been simplified in recent years by the use of cell lines transfected with cDNA
for the various human and rodent P2 receptors. Even so, in many instances new compounds
have only been examined in limited assays, e.g., P2X or P2Y family only. This has led to the
description of compounds as being receptor-selective and then being used to define new
receptors/receptor systems before the compounds are found to be active at other P2
receptors. A case in point is that of BzATP; while it is the most potent of known purine
nucleotide analogues at the P2X7 receptor, it is far more potent at other P2 receptors [139]. Its
use as a selective ligand to delineate the involvement of P2X7 receptors in a given
system/tissue response can thus be misleading. Another complication is the degree to which
P2 receptor ligands behave as agonists, antagonists, or partial agonists. Depending on the
species, ATP can function as an agonist or an antagonist at the P2Y4 receptor [63]. Despite
these caveats, there has been considerable focus on modifying the parent nucleotides and the
various empirically identified antagonists, e.g., azo dyes, suramin, etc. It is only in the past 6
years, however, that molecular modeling and high-throughput screening approaches (HTS),
together with the systematic development of structure-activity relationships, have been used
in the identification and optimization of novel P2 receptor ligands. Anecdotally, HTS
approaches have yielded disappointing results in finding either agonist pharmacophores that
lack the tri- or dinucleotide motif of ATP, UTP, ADP, and UDP or antagonists that are active
in vivo. Modeling approaches remain at an early stage and are obviously more promising for
the better characterized P2Y GPCR family than for members of the P2X receptor family
32
where knowledge of subunit stoichiometry, agonist and antagonist recognition site
requirements, and the role of allosteric modulators are still in their infancy. Early studies to
derive SAR relationships for a variety of adenine nucleotide analogues interacting with P2X
and P2Y receptors used classical smooth muscle preparations, e.g., guinea pig taenia coli,
rabbit aorta, and rabbit mesenteric artery, to characterize P2Y receptor interactions, while
P2X receptor activity was measured in rabbit saphenous artery, guinea pig vas deferens, and
urinary bladder. Receptor heterogeneity and ligand instability confounded results from these
preparations. Nonetheless, they were used empirically to identify a large number of
compounds that have formed the basis of emerging medicinal chemistry efforts. With the
availability of cloned receptors (and sufficient resources), it is now possible to examine new
compounds at all members of P2X and P2Y receptor families under comparable conditions as
well as to evaluate compounds for effects on nucleotidase activity.
3.2. P2 Receptor agonists
Compounds that activate P2 receptors have distinct structural requirements from the
agonists active at adenosine (P1) receptors [43-45]. The structure-activity relationships for a
P2 receptor agonists are shown in figure 10. New ATP analogues containing modifications at
the triphosphate, ribose 2’or 3’, purine C2 or C8, or at the purine N6 position have been
synthesized [45,140].
Modification
of 1-position,
i.e. ,C-H is
tolerated
2-position thioethers, including
large groups, and 2-Cl enhance
P2Y1 affinity. Large groups
enhance stability
HO
Triphosphate required for P2Y2
receptor; antagonists at P2YT;
diphosphate preferred at P2Y1
and P2Y6,
-Thio is generally tolerated,
-methylene is tolerated at P2X.
NH2
O
O
P
P
P
O
N
N
O
OH
N6 -positionsmall alkyl
N
N
May be
CH2
O
O
O
OH
At 8 position are
possible, but
reduced potency
and efficacy
OH
HO
OH
Ribose-acylation
enhances affinity
for P2X1 and P2X3
receptors
Figure 10. Summary of structure-activity relationship for P2 receptor agonists.
33
Triphosphate Modifications (Figure 10). The 5’-di- and 5’-triphospate derivatives generally
diverge in activity at P2X. ADP elicits agonist action at P2X1 receptor but not at other P2X
subtypes. None of P2X receptors are not activated by AMP or adenosine, although both ringmodified adenosine monophosphates (e.g., hexylthioAMP, 15) can activate P2X1-P2X4
receptors to varying degrees [44]. Modification of the triphosphate group in the form of
replacement of the bridging oxygen atoms with methylene units or of the charged oxygen
atoms with sulphur has in some cases resulted in potent analogues that are resistant to
degradation by nucleotidases. α,β-MeATP (α,β-methylene adenosine 5’-triphosphate, 10) in
particular is highly potent at P2X receptors being selective for group 1 subtypes (EC50 = 1-10
μM), at which it causes rapid desensitization. α,β -MeATP also activates P2X2/3 heteromers
that are less susceptible to desensitization [90]. Another metabolically stable analogues are
β,γ-MeATP, 11 and the unnatural L-adenosine enantiomer β,γ-Me-L-ATP (L-adenyl-5’-( β,γmethylene)diphosphonate, L-AMP-PCP 12), which is a more potent agonist than ATP at the
P2X receptor in the guinea pig bladder [21], but is inactive in the guinea pig taenia coli P2Y
receptor.
The
ῳ-thionophospate
group
present
in
ATPγS
((adenosine
5’-O-(3-
thiotriphospate), 7) generally increase stability of the triphosphate group towards enzymatic
hydrolysis while maintaining potency at P2X1 receptor [141]. ATPγS activates all P2X, except
P2X7 receptor, in the concentration range of 3-16 µM.
These 2-thioether 5’-monophosphate derivatives are potent at P2Y1 receptors (see below). At
P2Y receptors, α,β -MeATP is weak or inactive. The thio substitution at the terminal
phosphate also provides enzymatic stability, leading to such analogues as ATPγS (adenosine
5’-O-(3-thiotriphosphate, 7), ADPγS (adenosine 5’-O-(2-thiodiphosphate, 8), and UTPγS
(uridine 5’-O-(3-thiotriphosphate, 9), a potent agonist at P2Y2 receptors. Compound 7 is a
potent agonist at various P2Y subtypes, but not P2X subtypes, and inhibited ecto-ATPase
competitively with micromolar affinity]. 9 Is a potent P2Y2 receptor agonist that is not readily
degraded by nucleotidases. The corresponding‚ γ-thiodiphosphate, UDP-γ-S, selectively
activates P2Y6 receptors.
a. Adenine Modifications. Pyrimidine-based nucleotides are generally weak P2X receptor
agonists, although CTP is active at P2X3 receptors and less so at P2X4 and P2X1/5 receptors.
UTP weakly activates P2X3 receptors. Substitution of the adenine ring, particularly at the 2position, is well tolerated. 2-MethylthioATP (2-Me-SATP, 13) is one of the most potent
34
agonists at P2Y and P2X receptors. It is thus typically more potent than ATP at P2X 1,2,3
receptors. At rat and human P2Y1 receptors, 5’-diphosphates (ADP and 2-MeSADP, 14) are
generally more potent than the corresponding 5’-triphosphates (ATP and 2-MeSATP, 13)
[142]. Long-chain 2-thioethers can enhance the potency (particularly at P2Y receptors) or
selectivity (particularly within the P2X class). A p-aminophenethylthio analogue (PAPETATP, 16) is the most potent agonist reported [143] of the rat P2X3 receptor (EC50 = 17nm). The
activity of 2-thioether derivatives of ATP at P2Y receptors varied somewhat, depending on
the distal structural features, and activity at P2X receptors varied to an even greater degree.
At rabbit saphenous artery P2X receptors, the thioethers were inactive but differing degrees
of activity were observed in the guinea pig vas deferens and bladder depending on distal
substituents in the 2-thioether moiety. The addition of a functionalized chain at the 2-position
allowed for truncation of the triphosphate group with retention of affinity, thus
circumventing one of the major complications in interpreting ATP pharmacological results,
e.g., the impact of ectonucleotidase action. While AMP was inactive at P2Y receptors, 2thioether analogues of AMP were full agonists at erythrocyte P2Y receptors although being
generally several orders of magnitude less potent than the corresponding 2-thioether
triphosphate analogue. For example, the 2-hexylthio ether of AMP (15), had an EC50 value of
59 nM in stimulating phospholipase C in turkey erythrocytes. The 2-hexenylthio ether of
AMP (17a), was 8-fold more potent than ATP itself but was 33-fold less potent than the
corresponding triphosphate. Thus, the long chain may act as a distal anchor of the ligand at
an accessory binding site on the receptor.
A further benefit of the presence of a long-chain thioether group at the 2-position was
increased stability of the triphosphate group at the 5’-position. It is likely that long chains,
although at a site on the molecule distal to the triphosphate group, interfere with the ATP
binding site of ectonucleotidases. Modifications of ATP other than 2-thioethers also resulted
in unexpected receptor selectivity with some analogues displaying selectivity or specificity at
P2X or P2Y receptors, suggesting the existence of possible subclasses. The potent agonist, N6methyl-ATP was selective for taenia coli P2Y receptors versus either vascular P2Y receptors or
P2X receptors. N6-Ethyl-ATP was approximately equipotent to ATP at taenia coli P2Y
receptors. N6 Modification may prove to be a general means of increasing P2Y selectivity,
since it was compatible with other modifications. A hybrid N6-methyl and 2-thioether ATP
derivative, N6-methyl-2-(5-hexenylthio)-ATP (17b), was a potent agonist at erythrocyte, taenia
35
coli, and C6 glioma cell P2Y receptors but was inactive at P2X receptors [144]. Large groups,
e.g., 2-phenylethyl, are not tolerated at the N6-position of P2Y1 receptor agonists.
b. Ribose Modifications. Modification of the ribose 2’-position and purine modifications of
ATP other than 2-thioethers can result in P2Y receptor selectivity. The ribose moiety of
agonists demonstrated most clearly for the P2Y1 receptor is amenable to extensive
modification. The weak agonist, 2’-deoxy-ATP (structure not shown) was selective for taenia
coli P2Y receptors versus either vascular P2Y receptors or P2X receptors. 3’-Deoxy-ATP is a
weak, but selective, P2X agonist, 3’-Benzylamino-3’-deoxy-ATP (20) had high potency and
selectivity for P2X receptors and was inactive at rabbit saphenous artery P2X receptors and at
all P2Y receptors. The potency of 20 at P2X receptors was approximately an order of
magnitude greater than that of α,β -MeATP. Expansion of the ribose ring resulted in the
anhydrohexitol derivative, MRS 2255 (21), which was monophosphorylated at two positions
on the ring, e.g, a bisphosphate that was a full agonist with an EC50 value of 3 μM, at the
turkey erythrocyte P2Y1 receptor [145]. P2 receptor nucleoside and nucleotide ligands
containing conformationally rigid ribose-like rings, based on carbocyclic rings were designed
using the methanocarba approach, e.g., fused cyclopropyl and cyclopentyl rings replacing
the ribose moiety. The position of fusion of the cyclopropane ring determined the
conformation of the ring, either Northern (N) or Southern (S). Rigid rings in the
methanocarba series have defined a preference or the (N) conformation of ribose at the P2Y 1
receptor. MRS 2268 (22), the (N)-methanocarba analogue of 2’-deoxyadenosine-3’,5’bisphosphate (antagonist; see below), was a potent P2Y1 agonist (EC50 = 155 nM), being 86fold more potent than the corresponding (S) isomer [145].
Dinucleotide Derivatives (Figure 4). Dinucleotides have both P2 receptor agonist and
antagonist activity (see below). The activity and selectivity of diadenosine polyphosphates
(ApnA, n = 4-6) 23-25 and mixed dinucleotide polyphosphates as agonists has been studied at
recombinant P2X receptors and depends on the number of phosphates [52]. At the rat P2X 1
receptor, Ap6A is a full agonist, while shorter homologues have increasingly diminished
potency and efficacy. Uracil dinucleotides 26-29 function as agonists at P2Y2 receptors [146].
36
3.3. P2 Receptor antagonists
Selective antagonists are preferable to agonist potency orders in pharmacologically defining
a receptor subtype. This is particularly relevant in the P2 receptor area given the complexities
in analyzing such agonist data (see above and ref. [77]). Although a great number of
compounds has been used to block P2X and P2Y receptor-mediated responses (Figure 5-6)
none is ideal. All compounds are limited in their usefulness in terms of their kinetics of
antagonism, receptor-affinity, subtype-selectivity or P2 receptor specificity. Their suspected
ability to be a substrate for ectonucleotidases or to inhibit these enzymes and thereby protect
ATP and other nucleotides from degradation also complicates their use [77]. In addition,
structural similarities between many well known P2 receptor antagonists (Figure 5-6) are
hardly apparent. Thus, pharmacophore geometries for antagonist binding sites are ill defined
[78]. In principle, any P2 receptor antagonist should be tested for its activity at ectonucleotidases, its P2 receptor specificity and its selectivity against all known subtypes of the
P2X and P2Y receptor family. In the face of this situation, a continuing need exists for agents
that overcome the deficiencies of prior P2 receptor antagonists.
Polysulphonates (Figure 5). A variety of aryl sulphonates, such as derivatives of the
antiparasitic drug suramin (e.g., 30-33) and derivatives of histochemical dyes, have been
described as P2 receptor antagonists. These contained anthraquinone (e.g., 35-38), arylazo
(e.g., 39), or triphenylmethane (e.g, 41) moieties.
Suramin Class. Suramin (30) is a weak antagonist at the P2Y2 receptor with a IC50 value of 48
μM [146], and at the P2Y13 receptor with a IC50 value of 2,3 μM [136]. A number of truncated
forms of suramin, e.g., 30-34, had P2 antagonist activity, with an higher potency and P2X
subtypes receptors (see below).
Anthraquinone Class. Reactive blue 2 ( RB-2, 35), among the most widely used P2 receptor
antagonists, is a mixture of m- and p-sulfonate isomers. RB-2 antagonize both P2X and P2Y
[147]. RB-2 inhibits P2X2 responses with an IC5O value of 0.36 μM [11]. Recently novel
analogues of RB-2 have been introduced, including compounds such us 37 [18]. RB-2 at the
concentration of 100 μm effectively blocks rat P2Y4 receptors, but only partially blocks human
P2Y4 receptors. ATP antagonizes the human but not rat P2Y4 receptors (see below).
37
c. Azo Dye Class. Trypan blue (39) had an IC50 value of 386 µM at P2X [148]. Related
analogues (structures not shown), reactive red 2 and acid red 33 (P2X, Kd = 0.42 μM) showed
an enhanced P2 antagonist potency compared to 33.
d. Triphenylmethane Dye Class. Coumassie brilliant blue G (41) is a potent antagonist at
P2X7 receptors with an IC50 value of approximately 400 Nm [149].
Pyridoxal Phosphate Derivatives (Figure 6A). Other structural classes of ATP antagonists
include derivatives of the coenzyme pyridoxal 5’-phosphate. The diazo-2’,4’-disulfonate
derivative of pyridoxal phosphate (PPADS, 42, Figure 6A) and the isomer 2’,5’-disulfonate
”isoPPADS” (43), were more potent and selective (10- to 20-fold) for P2X than P2Y. The
derivative PPNDS (46) is an highly potent antagonist of P2X1 receptor [17]. Phosphonate
analogues of PPADS were similar in potency to the phosphate derivatives, but phosphonate
linkage is more stable [19]. The most active analogue at P2X1 (IC50 = 5 nM) and P2X3 (IC50 = 22
nM) receptors was (42), being 14-fold and 10-fold more potent than PPADS itself. The diazo
linkage of PPADS was also replaced in analogues containing a carbon bridge, a modification
that maintained potency at P2X receptors and enhanced chemical stability. Thus, the PPADS
template can be altered at the pyridoxal and phenyl moieties to produce P2X1 and P2X3
receptor antagonists showing higher potency and a greater degree of reversibility than the
parent compound at these Group I P2X receptors.
Nucleotide Derivatives (Figure 6B). Nucleotide derivatives have long been used in various
modalities to block the effects of ATP. Such nucleotide antagonists are advantageous as they
display more favourable binding kinetic properties than the antagonists discussed
previously. The agonist α,β- MeATP (10) can block P2X receptor responses via rapid
desensitization of the receptor. Other nucleotide derivatives antagonize P2 receptor effects in
a more competitive manner. Trinitrophenyl-ATP (TNP-ATP, 53) and the corresponding diand monophosphate derivatives are nanomolar antagonists at P2X1, P2X3, and P2X2/3
(heteromeric) receptors [150]. Oxidized ATP (oATP, 54), proposed as an irreversible
antagonist for the P2X7 receptor in the mouse macrophage-like cell line. A number of
nucleotide derivatives, e.g., 50 - 52, have been developed as inhibitors of the platelet P2Y12
receptor. AR-C69931MX (52), which was in clinical trials as an antithrombotic agent. On the
contrary AR-C67085MX (51) has been shown to potently activate the P2Y12 receptor.
Both 2’- and 3’-deoxy modifications of A3P5P (55a) were well tolerated at P2Y1 receptors, and
the removal of the free hydroxyl group decreased agonist efficacy. The introduction of a 2-
38
chloro substituent resulted in 58, which was a selective antagonist at P2Y1 receptors, e.g.,
inactive at rat P2X1 receptors [150]. The N6-methyl modification resulting in the competitive
antagonist MRS 2179 (56) enhanced antagonistic potency of 2’-deoxyadenosine 3’,5’bisphosphate by 17-fold. The corresponding 2-chloro analogue MRS 2216 (57) was partial
agonists of intermediate potency. [33P] MRS 2179 and [3H] MRS 2279 have been introduced as
radioligands for P2Y1 receptors in platelets and other tissues. The N6-ethyl modification of
MRS 2179 was of intermediate potency as an antagonist, while the N6-propyl group
completely abolished both agonist and antagonist properties. Thus, the N6-binding region of
the P2Y1 receptor appears to contain a small hydrophobic pocket. For most applications 56
and its congeners are highly selective for P2Y1 receptor, with inactivity demonstrated at
P2Y2,4,6,11,12,13 and P2X2,3,4,7. Unlike 56, MRS 2216 (57) was inactive at Group I P2X receptors.
Nonhighly Charged Derivatives (Figure 7). The isoquinoline derivative KN-62 (60) is an
antagonist of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at micromolar
concentrations and a potent antagonist at P2X7 receptors at even lower concentrations. KN-04
(61) inhibits the human P2X7 receptor, but is inactive at CAM kinase II. The dihydropyridine
nicardipine (62), and related derivatives can antagonize and/or potentiate P2X receptor
esponses [151]. Thienopyridines, such as clopidogrel, have been developed as antagonists of
the ADP-induced aggregation of platelets, but the actual receptor antagonist is an active
metabolite of the clinically administered compound [152].
Miscellaneous Modulators of P2 Receptors (Figure 7). The potent activity of the ῳconotoxin GVIA [97], suggests hat other snail-derived peptides may function as P2X
antagonists, in line with their broad effects on other ion channels [153]. The macrolides,
avermectin and erythromycin [102] represent additional pharmacophores active at P2X
receptors that may bind to sites distinct from those recognizing nucleotides. Potentiators of
the action of ATP at P2X receptors have also been identified. Coumassie blue and the
pyridoxal phosphate derivative MRS 2220 (63) selectively enhance the effects of ATP at P2X1
receptors [154] and the charged dihydropyridine derivative MRS 2154 (64) selectively
enhances the effects of ATP at P2Y1, P2X1, and P2X2 receptors.
39
4.1. Biological Actions and Clinical Targets
ATP is a key component of every cell in the body and is ubiquitously available in the
extracellular medium as a neuromodulatory agent. Its effects on cell function are
multifactoral as a distinct ligand, as part of the purinergic cascade [11] and as a source of
cellular energy. Thus, modulation of P2-receptor-mediated ATP responsesmay be anticipated
to have profound effects oncellular and tissue function at both the cellular and intracellular
levels. However, since ATP appears to bea normal constituent of the extracellular
environment,it appears highly probable that functional alterationsin extracellular levels of
ATP and thus P2 receptor hypoorhyperfunction, or alterations in receptor numberassociated
with discrete disease states, will provide theopportunity for developing novel therapeutic
agents thatact via P2 receptors. In this context, the therapeuticareas currently of interest are
pulmonary (P2Y2/P2Y4, Phase III), thrombosis (P2Y12, Phase III), pain (P2X3, preclinical), and
bladder dysfunction (P2X3, preclinical). Furthermore, as detailed below, evidence is now
emerging that in some disorders, there are robust changes in P2 receptor message and/or
protein that will dictate tissue responses to extracellular nucleotide concentrations.
Cardiopulmonary Function. P2X1, P2X3, P2X4, P2Y2, P2Y4, and P2Y6 receptors are present in
human fetal heart
and mediate distinct effects from those of adenosine acting at P1
receptors. ATP is a mediator of vagal reflexes in the heart and lung and potently regulates
vascular tone causing either contraction or relaxation depending on receptor location.
Nucleotide-mediated vascular contraction occurs by a direct effect on smooth muscle cells
involving P2X receptor activation, while relaxation involves a P2Y-receptor-mediated,
endothelium-dependent mechanism [155]. ATP may also play a role in the development of
vascular disease, e.g., atherosclerosis and hypertension, via its trophic actions. In the
respiratory system ATP, acting via both P2X and P2Y receptors, maintains the patency of the
airspaces by modulating the release of phosphatidylcholine as a surfactant and by
stimulating mucus secretion, facilitating mucociliary clearance, regulating ciliary beat
frequency, and attenuating the inflammation associated with macrophage infiltration
following respiratory tract infection, allergen inhalation, and injury. ATP and UTP, acting via
P2Y2 receptors, stimulate chloride secretion in airway epithelium and mucin glycoprotein
release from epithelial goblet cells, enhancing mucociliary clearance. This represents a
potential treatment for cystic fibrosis (CF) and chronic bronchitis. In controlled clinical
40
studies UTP, used in preference to ATP as a P2Y2 receptor agonist because it does not form
cardiovascularly active metabolites such as adenosine, dose-dependently stimulated
mucociliary clearance and sputum expectoration in smokers, nonsmokers, and patients with
chronic bronchitis [156]. ATP may also have a direct role in asthma via its actions on
bronchial innervation. The nucleotide triggers a reflex bronchconstriction via activation of
P2X receptors on vagal C fibers, and both ATP and UTP can potentiate IgE-mediated mast
cell histamine release, effects involving P2Y receptors.
Hemostasis. ADP as already discussed above is a potent platelet recruiting factor, inducing
platelet aggregation. This involves a complex interplay among three distinct platelet P2
receptors: a P2Y1 receptor linked to phospholipase C pathways, the P2Y12 receptor linked to
adenylate cyclase inhibition and a P2X1 receptor. In P2Y1 knockout mice, which showed
increased bleeding time and resistance to thromboembolism, ADP was still able to inhibit
platelet adenylate cyclase activity, indicating the presence of a second ADP-responsive P2
receptor linked to adenylyl cyclise [118], while in P2Y12 knockout mice, there is prolonged
bleeding time and reduced sensitivity to ADP, thrombin, and collagen.
ATP is a competitive ADP antagonist at platelet P2Y receptors and stimulates production of
PGI2 and NO, which can also inhibit platelet aggregation and act as vasodilators. Exogenous
ATP can thus act to localize thrombus formation to areas of vascular damage, controlling the
relationship among hemostasis, thrombosis, and fibrinolysis. AR-C 69331-MX (52) is one of a
series of systemically active, synthetic P2Y12 receptor antagonists [129] that has a safer side
effect profile than aspirin and has superior antithrombotic properties compared to other
modulators of platelet activity, e.g., GPIIb/IIIa antagonists, which show a narrow margin of
safety [207]. An orally active P2Y12 receptor antagonist derived from 66 (Figure 7) is
reportedly entering Phase I trials as an antithrombotic agent. Both P2Y 11 and P2Y12 receptors
appear to play a role in hematopoesis.
ATP and Neuronal Excitability. The central and peipheral nervous systems contain both
P2X and P2Y receptors. ATP acts as fast transmitter in nervous tissue via activation of P2X
receptors, with other actions being mediated via P2Y receptors. ATP can produce its effects
directly via actions on the postsynaptic membrane as well as via an indirect action on
presynaptic P2 receptors to modulate the release of a variety of neurotransmitters including
acetylcholine, norepinephrine, dopamine, serotonin, glutamate and can enhance GABA
[157, 159], vasopressin, and oxytocin release [158]. In nervous tissue, given the co-release of
41
ATP with other transmitters [3], the effects of ATP on transmission can be amplified by
modulation of the effects of these other intercellular messengers. ATP also functions to
transmit information between neurons and glia [8]. P2X2, P2X4 and P2X6 receptors are
expressed in the prepiriform cortex [100], suggesting that a P2X receptor antagonist may
have potential as an antiepileptic [339]. These receptors are also highly expressed in
cerebellum and are localized with AMPA-sensitive glutamatergic neurons in hippocampus,
suggesting a role in the modulation of long-term potentiation [146].
Auditory and Visual Function. In the auditory system, ATP, acting via P2Y receptors,
depresses sound evoked gross compound action potentials in auditory nerves and the
distortion product otoacoustic emission, the latter a measure of the active process of the outer
hair cells. P2X and P2Y receptors are present in the vestibular system, and P2X 2 receptor
splice variants are present in the cochlea. In the rat, P2X splice variants (P2X2-1 and P2X2-3) are
found on the endolymphatic surface of the cochlear endothelium, an area associated with
sound transduction [160]. P2Y receptors are present in the marginal cells of the stria
vascularis, a tissue involved in regulating the ionic and electrical gradients of the cochlea.
While little is currently known regarding the pharmacology of hearing and vestibular
function, ATP may regulate fluid homeostasis, cochlear blood flow, hearing sensitivity, and
development. In the eye, ATP acting via both P2X and P2Y receptors modulates retinal
neurotransmission, affecting retinal blood flow and intraocular pressure ATP. In the ocular
mucosa, P2Y2 receptor activation increases salt, water, and mucus secretion and may thus
represent a potential treatment for dry eye disease [161]. In the retinal pigmented layer, P2Y 2
receptor activation can promote fluid absorption and may be effective in treating retinal
detachment. There is also emerging evidence that P2-receptor-dependent neurotransmission
may play a role in the olfactory and gustatory systems.
Pain. ATP is a cotransmitter with norepinephrine (NE) in sympathetic nerves, with
acetylcholine (ACh) in parasympathetic nerves supplying the bladder, and in nonadrenergic
and noncholinergic (NANC) inhibitory enteric nerves. The nucleotide has both excitatory
and sedative effects in the central nervous system (CNS), with both P2X and P2Y receptors
being widely distributed in the central and peripheral nervous systems [3]. A specific role for
ATP in pain signaling was indicated by seminal work showing that the nucleotide was
released from sensory nerves, that it produces fast excitatory potentials in dorsal root
ganglion neurons, and that it is a central mediator of primary afferent fiber conduction.
42
These actions appear to be physiologically relevant, since exogenous ATP enhances
hyperalgesia in a human blister base model and iontophoretic application of ATP to human
skin can elicit pain. The nucleotide is also a key mediator of neurogenic inflammation via its
actions on P2 receptors present on neutrophils, macrophages, monocytes, and microglia,
activation of which results in cytokine production and release. The potent P2X antagonist
TNP-ATP (53) attenuates the nociceptive effects of P2 receptor agonists following intrathecal
administration [162] and is antinociceptive given intradermally. These data suggest that
TNPATP can provide effective antinociception when this P2X receptor antagonist is
administreted directly to a relevant site of action. Consistent with these pharmacological
data, P2X3 knockout mice show a loss of rapidly desensitizing inward currents induced by
ATP in DRG neurons [94, 95] with evidence of a modest but nonsignificant increase in P2X 2
homomers [163] and a significantly reduced, but not elimination of, pain-related behaviors in
response to intraplantar ATP or formalin [94, 95]. ATP has also been implicated in the pain
associated with migraine by virtue of its effects on the neurovasculature . Coadministration
of ATP with nitric oxide (NO), the nucleotide probably acting as adenosine following
hydrolysis, mimics the effects of the inhalation anesthetic enflurane and reduces the amount
of inhalation anesthetic required for anesthesia. For visceral pain, a purinergic
mechanosensory transduction mechanism has evolved [92] where distention of tubes
including ureter, gut, salivary and bile ducts, and sacs such as the urinary and gall bladders
causes ATP release from the lining epithelial cells to act on P2X3 receptors located on the
subepithelial sensory nerve plexus to relay nociceptive signals to the CNS.
Trophic Actions. The viability and also the regeneration of nervous tissue are sustained by a
variety of endogenous polypeptide trophic factors. Neural injury increases growth factor
levels, e.g., fibroblast growth factor, epidermal growth factor, and platelet-derived growth
factor. ATP acts in combination with these growth factors to stimulate astrocyte proliferation
contributing to the process of reactive astrogliosis, a hypertrophic/ hyperplastic response that
is typically associated with nervous system trauma including stroke/ischemia, seizure
disorders, and neurodegenerative diseases, e.g., Alzheimer’s and Parkinson’s diseases. In
reactive astrogliosis, astrocytes undergo process elongation show an up-regulation of P2X
receptors. ATP and GTP can induce trophic factor (NGF, NT-3, FGF) synthesis in astrocytes
and neurons]. The effects of GTP are, however, inconsistent with any known P2 receptor.
NGF can up-regulate P2X2 receptor protein and induce neuritogenesis in PC12 cells. The
43
latter effect can be blocked by a number of putative P2 receptor antagonists, suggesting a
potential role of P2 receptors in NGF signal transduction processes. In vascular smooth
muscle cells, ATP can induce cell proliferation via modulation of the cell cycle, acting as a
“competence” factor in combination with other growth factors to facilitate tissue repair and
regeneration. The trophic effects of purines also extend to effects on immune cell function.
ATP can also induce cytolysis in macrophages infected with mycobacterium via P2X7receptor-mediated apoptotic and necrotic events [397]. While the novel antimicrobial activity
of ATP was initially thought to have potential utility in the treatment of tuberculosis, studies
in P2X7 receptor knockout mice showed that this receptor, while involved in bacterial killing,
was not essential for the antimicrobial effects of ATP [111]. The involvement of nucleotides in
developmental processes, where purinergic effects on tissue differentiation precede those of
adrenergic signaling, indicates that in many respects, purine-related effects on development
parallel the role of purines following tissue trauma, e.g., angiogenesis, cell proliferation, etc.
Bacterial Infection. Purinergic signaling mechanisms may be involved in the regulation of
bacterial growth. P2X4 receptors appear to be the site at which erythromycin can block the
effects of ATP on calcium influx, thus representing a potentially novel target to identify
compounds that suppress fluid secretion in chronic respiratory tract infections. Binding of P.
aeruginsa flagellin to a membrane glycolipid asialoGM1 (ASGM1) present on human HM3
epithelial cells promotes the autocrine release of ATP from host cells, activating, possibly, the
P2Y11 receptor, on the host cell to elicit a pathogenic defensive response.
Lower Urinary Tract Function. Urinary bladder function is regulated by sympathetic and
parasympathetic input with ATP mimicking the effects of parasympathetic nerve
stimulation, resulting in bladder contraction via activation of P2X receptors in the smooth
muscle of the urinary bladder detrusor muscle that is involved in bladder emptying.
Detrusor dysfunction results in urge urinary incontinence (UUI), a major health problem in
the aging female population. P2X receptors are also present in the bladder urothelium with
the P2X1 receptor being the predominant subtype in adult bladder. The P2X4 receptor shows
high levels of expression in the developing, but not adult bladder [164].
NO mediates the first stage of relaxation with ATP acting via P2 receptors to mediate the
second phase of the voiding response. Serosal ATP release occurs in rabbit because of the
hydrostatic pressure changes associated with bladder filling. Partial bladder outlet
obstruction in rabbit leads to an increase in purinergic and a decrease in cholinergic
44
components of nervemediated detrusir contaction a finding that may explain the poor
responses to anticholinergic therapy in patients with detrusor instability. In tissue from
patients with symptomatic outlet obstruction, P2X1 receptor expression was increased,
suggestive of an enhanced purinergic role in the unstable bladder resulting from outlet
obstruction [165]. In male rat genitalia, P2X1 and P2X2 seem to be involved in sperm transport
and ejaculation. In male P2X1 receptor knockout mice, fertility is reduced by approximately
90% with no effect on copulatory performance. While a P2X1 receptor antagonist may
conceptually represent a novel, nonhormonal male contraceptive, some additional attributes
will be necessary for it to be a reliable approach to birth control.
In the body of the penis, strong P2X1 with less P2X2 subunit immunoreactivity was present in
the smooth muscle of blood vessels and the corpus cavernosum, suggestive of a role in
erectile function. P2X receptors were present on Sertoli but not Leydig cells. ATP, acting via
germ cell P2X receptors, may therefore play a role in controlling germ cell maturation.
Hepatic Function, Diabetes, and Gastrointestinal Tract Function. There is a considerable
body of data on the role of purines in the control of gut function. Changes in liver cell
volume, e.g., in epithelium following exposure to insulin, and the uptake of amino and bile
acids increase ATP release [41], resulting in a change in extracellular concentration from
approximately 10 nM to greater than 300 nM. ATP then acts as an autocrine regulator to
modulate membrane chloride conductance and thus facilitates cell volume recovery, linking
the cellular hydration state to the P2-mediated pathways involved in cellular homeostasis.
ATP may also act as a paracrine mediator in hepatobiliary coupling, a process coordinating
the hepatocyte and ductular components of bile formation. Purines stimulate glycolysis in
isolated perfused rat liver via mechanisms involving both P1 and P2 receptor activation and
multiple signaling pathways resulting in an increase in hepatic glucose output. ATP also
stimulates pancreatic insulin release via a glucosedependent, P2Y-receptor-mediated
mechanism [166].
Bone Function. Both P2X and P2Y receptors are present on the two principle types of cell in
bone tissue, macrophage-derived osteoclasts and osteoblasts, that originate from
mesenchymal stem cells and are responsible for bone formation. ATP, released in response to
shear stress [21] functions as a mechanotransducer in skeletal tissue acting as an osteoblast
mitogen, potentiating the effects of growth factors on bone cells. ATP, but not adenosine, can
stimulate the formation of osteoclasts and their resorptive actions in vitro. P2 receptor
45
agonists may thus have potential in the treatment of osteoporosis, rheumatoid arthritis,
periodontitis, osteopenia, and inflammatory bone loss.
Cancer. While there is a considerable clinical data on the potential use of ATP as a treatment
for cancer and the cachexia associated with cancer], little progress has been made in
advancing the nucleotide to general use or more precisely in understanding its functional
role in attenuating metastasis progression. Given the proapoptotic effects of the nucleotide
acting via P2X7 receptors, it is likely that ATP effects on cancer growth may be cytokinedependent because the nucleotide modulates cytokine release [48]. An alternative
mechanism may be the ability of ATP to induce COX-2 expression [121], inhibitors of the
latter enzyme having been demonstrated to suppress colon carcinoma growth in controlled
clinical trials. P2Y2/P2Y4 receptors have been identified on human colorectal carcinoma cells
that may be associated with ATP-mediated control of cell proliferation control. In prostate
carcinoma cells, ATP and BzATP, but not UTP or adenosine, inhibit cell growth, effects
ascribed to P2X receptor activation. ATP has also been reported as inducing malignant tumor
growth in brain, nucleotide infusion eliciting in astrocyte proliferation, reactive astrogliosis,
and glioma formation. In stratified epithelium, P2X5 receptors are associated with
proliferating and differentiating cells, while P2X7 receptors label apoptotic cells. From these
findings, it has been suggested that selective P2X5 and P2X7 receptor agonists may have
potential in the treatment of skin disorders such as psoriasis, scleroderma, and basal cell
carcinoma and for restenosis following angioplasty.
Future Directions
In the past 5 years there has been an explosion in published studies characterizing P2
receptor function in a variety of tissue systems and disease states. This has been almost
exclusively driven by advances in the cloning, expression, and characterization of the P2
receptor family [3] that have provided the molecular tools necessary to begin the process of
understanding the role-(s) of ATP (and UTP) in disease pathophysiology. Many studies have
now been done showing differences in P2 receptor expression in development in diseased
tissues and from tissues derived from animal models of human disease. The recent flurry of
publications on changes in P2X receptors in various bladder disorders reflects the ease of
access to tissue from routine surgical procedures and cannot be easily duplicated for other
disease states, e.g., diabetes or neurodegenerative diseases involving apoptosis, where ATP
may play a key role. For the newcomer to the P2 area, the plethora of potential disease
46
targets described above, the role of ATP in energy-dependent processes within the cell, the
involvement of the nucleotide in the functional “yingyang” outcomes of P2-mediated
apoptotic
signalling
(e.g.,
increasing
apoptosis
to
treat
cancer
may
accelerate
neurodegenerative processes, while retarding apoptosis as a treatment for Alzheimer’s
disease may lead to metastasis formation), and the lack of progress in apparently promising
clinical studies for the use in ATP in cancer may be less suggestive of a viable approach to
drug discovery than an “energy priming” effect more akin to homeopathic medicine.
Nonethless, there are areas where ligands acting via P2 receptors are showing clinical
promise. In the field of antithrombotics, P2Y12 receptor antagonists represented by the ATP
bioisosteres AR-C 69931MX are superior in terms of safety to GPIIb/IIIa antagonists.
Similarly, in the respiratory area, P2Y2/P2Y4 agonists such as INS 365 (Up4U, 27), a direct
UTP/ATP mimic, has advanced to Phase II clinical testing for chronic bronchitis. Two other
areas of significant promise include pain and lower urinary tract dysfunction where
considerable evidence exists for a potential role for P2X3/P2X2/3 receptor antagonists. Finally,
the unique P2X7 receptor with a potential role in inflammation, cancer, and acute
neurodegenerative diseases such as stroke may, either via a directly acting ligand or an
allosteric modulator, e.g., KN-62-like (60), yield P2-based therapeutics. Medicinal chemistry
remains the key to advancing efforts in P2-receptor-based drug discovery. With only few
pharmacophores known that interact with the P2 receptor family, it will be imperative to
identify novel pharmacophores beyond suramin and the plethora of nucleotide-based
bioisosteres to identify nonnucleotide and “nonsuramin/PPADS, etc.” pharmacophores with
ADME properties consistent with “druggability”. This may require a more systematic
evaluation of compounds from natural sources, which have been a rich source of novel
pharmacophores for other receptor families. Together with an increased knowledge of the
properties of native receptors including P2X and P2Y oligomers and associated allosteric sites
that may have physiologically relevant effects on P2 receptor function, the choice of disease
states where robust information on the native human tissue is available, e.g., bladder, will do
much to convince the skeptic of the utility of P2-receptor-based therapeutics.
47
5. Aim of the research
On the basis of the above, taking into account that both agonists and antagonists for P2
receptors are at an early stage of development, and the fact that selective P2 ligands have the
potential for very promising drugs, the scopes of our research has been the discovery and
development of potent P2 ligands 2, with the aim at designing selective pharmacological
probes and potential drugs targeting P2 receptor subtypes. On this purpose, this thesis
reports on two related topics:
-
the preparation and testing of 2-alkynyl ATP analogs as potential P2 agonists;
-
catalogues data mining for identification, purification and testing of polysulphonated
dyes as analogues of Reactive Blue 2, and preparation and testing of new RB-2 and
suramin analogues as potential P2 antagonists
6. 2-Alkynyl-ATP analogues
Platelets possess mainly two P2 receptor subtypes, namely P2Y1 and P2Y12, whose combined
action is necessary for a full activation and aggregation response to stimulation by adenosine
diphosphate (ADP). P2Y1, coupled to the heterotrimeric GTP-binding protein Gq and
phospholipase C-β, is responsible for the mobilization of ionized calcium from internal stores
and mediates shape change and the initial wave of rapidly reversible platelet aggregation
induced by ADP. P2Y12, on the other hand, is negatively coupled to adenylyl cyclase through
Gi and mediates a progressive and sustained aggregation not preceded by shape change. The
latter receptor also plays an important role in the potentiation of platelet secretion induced by
several agonists, and its congenital deficiency results in a lifelong bleeding disorder [168, 169].
Moreover, there is an ionotropic receptor, P2X1, which is also involved in shape change, and
recent reports from our collaborators suggested a possible involvement in aggregation
(Figure 11).
Modulation of P2 receptors in platelets may be of paramount importance in regulating
platelet function, and, as a consequence, in controlling thrombotic diseases, which are the
most common cause of morbility and mortality in the Western World. As a matter of fact,
P2Y12 is the target of ticlopidine (65) and clopidogrel (66, figure 12-A), two platelet
aggregation inhibitors that are effective in the prevention and treatment of arterial
thrombosis [170]. The results of studies of experimental thrombosis in animals suggest that
48
antagonists of P2Y1 could also prove highly potent antithrombotic agents. Therefore, the
search for modified adenine nucleotides that could finely modulate the platelet P2 receptors
has been very active in the last few years.
Figure 11. Platelets P2 Receptors and aggregation
Cl
Cl
S
S
N
N
O
O
A)
65
66
NH 2
N
N
O
O
P
P
HO
O
OH
RS
R
N
N
O
O
O
N
N
N
N
O
EtHN
OH
HO
B)
NH 2
67
OH
HO
OH
68
Figure 12. Structures of Ticlopidine (65), Clopidrogel (66), 5’diphosphate of 2alkylthioadenosine (67), 2-alkynyl analogues of N-ethylcarboxamidoadenosine (NECA, 68).
49
6.1 SAR studies
Early structure-activity studies on the effects of ADP analogues on human platelets showed
that the introduction of substituents, such as alkylthio groups, in 2-position of the adenine
base enhanced potency (67, figure 12-B), while modifications of ribose moiety or diphosphate
chain had the opposite results [171, 172]. More recently, many papers reported that ribosemodified deoxyadenosine bisphosphate derivatives behaved as P2Y1 partial agonists or
antagonists [2, 173, 174], while the presence of constrained carbocyclic rings, as in
methanocarba analogues, led to compounds that proved to be agonists or antagonists,
depending on the presence of a bisphosphate group [2].
In the early nineties, we found that a series of 2-alkynyl derivatives of Nethylcarboxamidoadenosine (NECA) (68, figure 12-B) elicited potent inhibitory activity on
rabbit and human platelet aggregation induced by ADP, likely acting trough an A 2A
adenosine receptor subtype [175, 176]. In order to verify whether the introduction of such a
kind of side chains could generate nucleotides targeted to P2 platelet receptors, we
undertook the synthesis of mono-, di- and tri-phosphate derivatives of 2-hexynyladenosine
(HEAdo, 69) and 2-phenylethynyladenosine (PEAdo, 70). The selection of the substituent in
2-position was made on the observation that alkynyl and aralkynyl chains displayed quite
different activities at the platelet adenosine receptors [177-180].
6.2. Chemistry
New ligands for the P2 receptor subtypes were developed using our nucleoside library.
Nucleosides related to adenosine can be obtained using two different synthetic approaches, or
combinations of them. Convergent Synthesis: suitable bases are coupled to sugar derivatives.
Divergent Synthesis: commercially available nucleosides are modified to obtain new
compounds. Since many years our research group has developed extensive experience in the
convergent approach, coupling substituted purines and deazapurines with a variety of sugars.
A schematic representation of such synthetic work is shown in Figure 13.
As sugar we used ribose, 2- and 3-deoxyribose and 2,3-dideoxyribose derivatives; as far as
bases: 2,6-dichloropurine, 2-chloro-6-nitropurine, related deaza analogs, and a variety of 6amino derivatives. In figure 13 it is reported an example of divergent approach for the synthesis
of adenosine analogs.
50
O
O
HO
R2HN
O
HO
OH
OH
OH
O
R
HO
O
O
X
X
R1
OH
R2HN
OH
N
H
X
R= Cl or NO2
R1 = H or Cl
R2 = alkyl or cicloalkyl
x = N or CH
HO
O
O
O
R2HN
OH
OH
HO
O
Figure 13. Convergent Synthesis of nucleosides
O
N
HN
H2N
N
N
HO
N
N
3 steps
O
HO
R
Cl
I
N
OH
NH2-R
AcO
I
N
OAc
N
O
HO
O
AcO
N
N
N
R
NH
HO
NH
N
N
R'
H
R'
N
HO
OH
N
O
HO
OH
Figure 13. Divergent synthesis of nucleosides
As shown in Figure 13, the commercially available guanosine is converted in three steps into
the 6-chloro-2-iodo purine riboside, which is in turn treated with substituted amines and
then, under cross-coupling conditions, with the suitable terminal alkynes, to obtain 2,6disubstituted adenosines.
The 2-alkynyl-ATP and its analogues were obtained by commercially available guanosine,
according the divergent approach (Scheme 1). Guanosine and all the reagents were
purchased from Sigma-Aldrich (Italy). Guanosine was protected with acetic anhydride, using
51
triethylamine (NEt3) and dimethylaminopyridine. The protected derivative I was treated
with phosphorous oxychloride to obtain the substitution of the six position of the purine ring
with chlorine, and than with methylen iodide 2-penthyl nitrite in order to obtain the 6chloro-2-iodonucleoside (III). This derivative was treated with liquid ammonia (liq. NH 3),
which substituted selectively the chlorine atom in position 6, obtaining so the 2iodoadenosine (IV). The reason of this selective substitution can be explained by the higher
reactivity of the C-6 position in the purine ring toward aromatic nucleophilic substitution,
although iodine is a better leaving group with respect to chlorine. Finally, treating 2iodoadenosine with the appropriate terminal alkyne in cross-coupling conditions 2-HEAdo
(69) and 2-PHEAdo (70) were obtained.
The cross-coupling reactions is performed in presence of catalysts such as copper (I) iodide
(CuI) and triphenylphosphyne palladium chloride. The yields are ususally high (87-90%), but
while at room temperature the completion is achieved in many hours, with micro-wave the
reaction times become of few minutes, keeping the yields high. Hence, the novelty in this
application of synthetic divergent approach is the use of micro-wave for cross-coupling
reaction.
The synthesis of nucleoside diphosphates and triphosphates was carried out by a
modification
of
the
Hoard-Hot
method
[182],
through
the
activation
of
the
tributhylammonium salts of the monophosphate derivatives 71 and 72 with 1-1’carbonyldiimidazole (CDI) and the following treatment with tri-n-buthylammonium
pyrophosphate (scheme 2).
52
O
O
N
HN
N
N
H2N
O
HO
HO
Cl
N
HN
AC20
H2N
DMAP
AcO
OH
Guanosine
I
N
N
R'
HO
O
HO
H
O
HO
OH
N
N
I
OH
HO
R' = CH3(CH2)3
69
R' =
70
NH 3 liq
N
N
R'
OAc
AcO
III
NH 2
N
N
O
AcO
II
NH 2
N
N
I
CH2I2
OAc
AcO
N
N
C5H11ONO
O
AcO
OAc
N
N
H2N
POCl3
O
AcO
N
N
N
N
Cl
IV
Scheme 1. Synthesis of 2-exynyl (69) and 2-phenylethynyladenosine (70)
The synthesis of monophosphate derivatives 71 and 72 was achieved by reacting the
unprotected nucleosides 2-HEAdo (69) and 2-PEAdo (70), respectively, with phosphorous
oxychloride in trimethylphosphate at room temperature, following the Yoshikawa method
[181] (scheme 2).
53
NH 2
N
N
N
N
HO
NH 2
N
N
P
O
HO
O
POCl3
R'
R'
OH
HO
OH
N
O
O
OH
HO
(CH3O)3PO
N
R' = CH3(CH2)3
69
R' = CH3(CH2)3
71
R' =
70
R' =
72
O
Bu3NH+ H2PO4-
(Bu 3NH)2++ H2P2O7--
N
N
NH 2
NH 2
N
N
O
P
HO
O
OH
O
N
P
O
N
O
O
OH
R'
HO
P
OH
HO
N
N
O
O
O
P
P
N
OH
R'
O
O
OH
O
OH
N
HO
OH
R' = CH3(CH2)3
73
R' = CH3(CH2)3
75
R' =
74
R' =
76
Scheme 2. Synthesis of nucleotides.
54
6.3. Biological assays
Platelet shape change and aggregation induced by 10 microM ADP were studied in washed
human platelet.
Preparation of new ADP analogue suspensions was done in Tyrode solution containing
CaCl2 2 mM, MgCl2 1 mM, 1% glucose, 0.35% serum albumin and low concentrations of
apyrase, which was added to prevent P2 receptor desensitization by trace amounts of
contaminating ADP (released by platelets and red blood cells during the washing procedure)
in the suspending medium. Fibrinogen (40 mg/mL) was added to platelet suspensions to
support platelet aggregation. Two sets of experiments have been performed. Platelet
aggregometry (figure 14) was used to study the extent of platelet aggregation induced by
ADP, or the other synthesized compounds. Antagonistic studies, using the same conditions
but with the tested compounds added prior than ADP, were also performed, in order to
study if these compounds could modulate ADP effects.
Finally, platelet responses that are mediated by the interaction of ADP with P2Y 12 were
investigated by measuring the inhibition by ADP of the platelet adenylyl cyclase.
More in detail, prostaglandin (PGE1, 1 μM) was added to 1 mL of platelet suspension in the
presence or absence of 10 μM ADP and different concentrations of the test compounds. After
2 min incubation at 37°C, the reaction was stopped with 5% trichloroacetic acid, the platelet
cyclic AMP was extracted and then measured with a radioenzymatic technique.
The di- and triphophates of HEAdo (73 and 75 respectively) caused platelet shape change
and aggregation, with an EC50 ranging from 10 to 100 μM (Figure 14). The extent of
aggregation induced by 100 μM of these nucleotides is slightly lower (75 and 73, 80% and
70% respectively) than that elicited by 10 μM of ADP. The above results indicated that these
compounds activate both P2Y1 and P2Y12 receptors, but are not able to give 100% of the
response.
55
NH2
100 μM
N
-
N
O
O- O
O P O P O P O
O
OO-
N
N
O
%Aggregation
OH OH
100 μM
75
1 min
ADP 10 µM
NH 2
N
-O
100 μM
O- O
PO P O
O
O-
N
N
N
O
OH O H
73
1 min
1 min
Figure 14. Effects of ADP, 73 and 75 on human washed platelets
In figure 14 aggregation produced by 10 μM ADP was taken as 100%. Since it is clear that
pro-aggregatory nucleotides 73 and 75 induced a sub-maximal platelet aggregation, both
nucleotides were evaluated in washed platelets for their action on platelet aggregation
induced by 10 μM ADP. The results of this experiment are reported in Table 3 and showed
that the aggregation percentage induced by ADP decreased at the increase of the
concentration of both of them. In particular, at 100 μM concentration, HEADP and HEATP
partially antagonize the effect of ADP. Hence, compounds 73 and 75, promoting platelet
aggregation when given alone, and lowering the maximal aggregating potency of ADP, seem
to behave as partial agonists.
Aggregation percentage
Cp ()
1
10
100
73
98
99
83
92
90
64
75
Table 3. Effects of 73 and 75 on platelet aggregation induced by 10  ADP (taken as 100%).
On the contrary, the monophosphates of both 2-hexynyl and 2-phenylethynyladenosine
(HEAMP, 71 and PEAMP, 72) did neither induce platelet shape change nor aggregation
56
between 10 and 100 μM. But, surprisingly, neither the diphosphate (PEADP, 74) nor the
triphosphate (PEATP, 76) of the 2-phenylethynyladenosine elicited any effect (figure
15).
NH2
N
%
Aggregati
on
100
µM
N
N
O
-O P O
O-
N
O
OH OH
7
1
NH2
N
O
-O P O
O-
ADP 10
µM
N
O
OH OH
100
µM
N
N
72
NH2
N
-
1 min
O- O
O PO P O
O
O-
N
O
OH OH
100
µM
N
N
7
4
NH2
N
O
O- O
O PO P O P O
O
OO-
N
N
N
-
O
OH OH
100 µM
76
Figure 15. Effects of 71, 72, 74 and 76 on human washed platelets.
In order to verify the antagonistic behaviour of these nucleotides the second type of
experiments was carried out. The four compounds inhibited platelet aggregation induced by
10 μM ADP in a concentration-dependent manner. At a concentration of 800 μM, 71, 72, 74,
and 76 fully inhibited both platelet aggregation and shape change induced by 10 μM ADP. In
contrast, 2-phenylethynyladenosine diphosphate (74), did not inhibit the shape change and
inhibited the platelet aggregation by about 90%.
To check the interaction between the synthesized nucleotides and P2Y 12 subtype, the
modulation of the effect of ADP on the adenilate cyclase was evaluated (figure 17). The
activation of P2Y12 subtype by ADP yields the inhibition of this enzyme. After incubation of
the human platelets with ADP and the nucleotides, the platelet cyclic AMP was extracted and
then measured with a radioenzymatic technique.
57
At concentration of 100 μM none of the inhibitory nucleotides significantly antagonized the
inhibitory effect of 10 μM ADP on PGE1 induced platelet cAMP increase (red lines), despite
the fact that all of them brought about marked inhibition of platelet aggregation induced by
the same amount of ADP (pink lines). In particular, at the maximum concentration at which
the nucleotides were tested the inhibition of platelet aggregation was total or almost total for
74. At 800 μM the tested nucleotides partially antagonized ADP effect on cyclase; again, 74
resulted the less potent in the series.
These results clearly demonstrate that 71, 72, 74 and 76 inhibit mainly P2Y1 subtype and at
the maximum concentration of 800 m show even a partial inhibitory effect on P2Y12 subtype.
20
10 µM
100 µM
100
74
80
60
40
20
0
1 µM
10 µM
100 µM
800 µM
60
40
20
0
800 µM
% inhibition of AC
induced by ADP 10 µM
1 µM
72
80
1 µM
10 µM
100 µM
% aggregation
induced by ADP 10 µM
40
100
800 µM
100
76
80
60
40
20
0
1 µM
10 µM
100 µM
% aggregation
induced by ADP 10 µM
60
% inhibition of AC
induced by ADP 10 µM
80
0
% inhibition of AC
induced byADP 10 µM
71
% aggregation
induced byADP 10 µM
100
% aggregation
induced byADP 10 µM
% inhibition of AC
induced byADP 10 µM
74 Shows effect of minor entity.
800 µM
Figure 17. Effect of 71, 72, 74 and 76 on adenylate cyclase inhibition (red line) and platelet
aggregation (pink line) induced by ADP 10 µM.
6.4. Stability studies
Since the opposite behaviour of di- and threephosphates of 2-hexynyladenosine (73 and 75,
respectively; aggregation promoters) and of 2-phenylethynyladenosine (74 and 76,
respectively; aggregation inhibitors) and because generally di- and triphosphate are agonists,
while monophosphates are antagonists, experiments to check the stability of nucleotides in
Tyrode solution were set up, in order to exclude that different behaviour depended on a
difference in stability, i. e. a faster hydrolysis of the di- and threephosphates of 2-
58
phenylethynyladenosine to the relative monophosphate. Compounds 75 and 76 were
dissolved in a Tyrode solution and kept at room temperature (25 °C); the solution was
analyzed in a HPLC apparatus at 0, 5, 12, 24h and the area of peaks corresponding to three(starting compound), di- and monophosphate nucleotides were calculated. Results (Table 4)
clearly show that triphosphate nucleotides are moderately stable in solution at 25 °C, with a
rate of conversion to diphosphate, after 24h, of about 25% for compound 75 and 50 % for
compound 76. Monophosphate derivatives (whose presence would have accounted for
antagonistic activity) were formed only in negligible amount. The same experiments were
repeated at 37°C, yielding a moderate increase in degradation rate.
Time (h)
Cp
0
5
12
24
75
100
93.50
84.60
72.50
76
100
88.00
74.50
51.30
Table 4. Percentage of triphosphates in the pool of nucleotides in baffer solution at different
times.
To better check the di- to monophosphate hydrolysis, a similar experiment was performed
starting from 73 and 74. Table 5 shows that limited amount of diphosphate nucleotides was
converted to monophospates, accounting for a high stability of the former. This results clarify
that the different behavior of compounds 73 and 75 (aggregation promoter) and 74 and 76
(aggregation inhibitor) are not due to different stability in solution.
Time (h)
Cp
0
5
12
24
73
100
98.78
98.00
96.94
74
100
99.74
99.60
99.32
Table 5. Percentage of diphosphates in the pool of nucleotides in baffer solution at different
times.
59
6.5. Conclusions
A new series of nucleotides with activity on human platelet aggregation and adenylyl cyclase
modulation through the interaction with P2Y1 and P2Y12 receptors has been prepared and
well characterized. The di- and thriphosphates of 2- hexynyladenosine (HEADP, 73, and
HEATP, 75) are partial agonists at P2Y1 and agonists at P2Y12 receptors while both the
monophosphates (HEAMP, 71, and PEAMP, 72) and the di- and thriphosphates of 2phenyletynyladenosine (PEADP, 74, and PEATP, 76) mainly antagonize the P2Y1 receptor.
The surprisingly different behaviour of the diphosphates 73 and 74 (figure 18), which differ
from structure point of view only for a portion of the alkynyl chain, cannot be explained by
different hydrolysis rates, but in terms of different size and flexibility of the substituents in
the position 2 of the purine base.
Figure 18. 2-HEADP and 2-PEADP
60
74
75
73
Figure 19. 3D analytical chromatogram, showing the separation of nucleotides 73-75
61
7. Reactive Blue 2 and Suramin analogues
7.1. Reactive Blue 2
One of the few P2 antagonists known so far is the Reactive Blue 2. It belongs to the class of
anthraquinone-chlorotriazine reactive dyes, which were formerly used in textile industry and
later became important as ligands in affinity chromatography for purification of various
enzymes and protein [184, 185].
O
NH 2
O
s
A
B
C
O
HN
O
NH 2
O
O
A
B
O
C
O-O
O
O-
HN
D
O S
O
s
-
D
Cl
NH
N
E
N
N
F
H2N
O S
NH
O-O
O
S O-
N
E
N
H2N
Cl
N
F
O
S
O
77
O
O-
78
Figure 20. Structural isomers of RB-2
The structure of RB2 was claimed to be a ring F meta/para sulphonate mixture in the ratio of
65:35 (92 and 93). As commercial preparations, these dyes are of highly heterogeneous nature
and undefined in their actual chemical composition. For example, the Aldrich product is of
60% purity of a mixture about 2:1 of meta:para isomers. From the 1970s, these dyes have been
used as P2-receptor ligands, since they show a certain degree of antagonistic properties.
Reliable receptor research is warranted only with ligands of structural homogeneity and
unequivocal designation.
RB-2 was reported as a competitive P2Y antagonist at the
adenylate cyclase coupled P2Y receptors of C6 glioma cells, with a KB value of 25 nM, which
is at least a 50-fold higher affinity than those reported in other tissues [147]. In a recent study,
RB-2 and a series of compounds structurally related to this dye were investigated for their
ability to block different P2 receptor subtypes, P2Y in guinea pig taenia coli and P2X in rat vas
deferens [186]. Cybacron 3GA (36) (orto isomer analog of RB-2) is approximately 7-fold more
potent than RB-2 at P2X1-like receptors in rat vas deferens (IC50 = 9.6 μM, Kd =1.6 μM) but has
62
similar potency at P2Y1-like receptors in guinea pig taenia coli (Kd = 2.9 μM). The data
confirmed that P2 antagonists tend to be non-selective and to act with kinetics not purely
competitive. The structure-activity relationships for P2X and P2Y blockade in this series are
strikingly dissimilar [187]. In RB-2 and its isomers both the 1-amino- anthraquinone-2sulphonate core and the “side-chain” of the molecule are involved in P2X receptor binding;
P2Y affinity, in contrast, resides largely or totally in the anthraquinone core. The most
promising antagonists were Acid Blue 129 (37), which was shown to be P2Y versus P2X
selective, and Uniblue A (38), which was the most selective compound for P2X. Acid Blue 129
has modest antagonist activity at P2Y1-receptors in guinea pig taenia coli (IC50 = 3.0 M, Kd = 1.4
μM; RB-2 Kd = 3.4 μM) and shows greater than 71-fold selectivity versus P2X1-like receptors
in rat vas deferens (Kd > 100 μM; RB-2 IC50 = 30.4 μM and Kd = 11.4 μM) [186]. Uniblue A (38),
is a potent antagonist at P2X1-like receptors in rat vas deferens (IC50 = 7.5 μM, KD = 0.8 μM)
and shows greater than 125-fold selectivity versus P2Y1-like receptors in guinea pig taenia coli
(Kd > 100 μM) [186]. Both 37 and 38 presented few, if any, non-purinoceptor effects blocking
the respective P2-subtype. The limitation of these statements to the receptors tested must,
however, be emphasized [187]. For example, 38 appeared to be an antagonist at both P2 and
P1 (A1 adenosine) receptors in the rat superior cervical ganglion. In addition, Acid blue 129
and Acid blue 80 (79) show very poor selectivity between the native P2Y1 and P2Y2 receptors
in bovine aortic endothelial (BAE) cells. For this reason they are of limited use in the field of
P2Y receptor pharmacology. Furthermore, contrary to previous reports, acid blue 25 (80) is
not a BAE P2Y-selective antagonist [187]. These data demonstrate that it is not possible to
attribute antagonism at P2Y receptors to the affinity residues, which are largely or totally in
the 1-amino- anthraquinone-2-sulphonate core.
In these years the interest for RB-2 has been more than a pure research interest; in fact,
among the properties of such dye, it is worthy to remember the ability to prevent cellular
death induced by hypoglycaemia, hypoxia, mitochondrial dysfunction, so as glutamateevoked biological functions (excitotoxicity, neurotransmitter release, uptake of extracellular
Ca2+), or apoptosis, behaving as a neuroprotective agent [188].
On the above, our activities in this area has been the following:
i)
Individuate new compounds related to RB-2 to be tested as P2 receptor antagonists
in our cellular models in order to find structure-activity relationship, through the research of
63
compounds already available in data bases and catalogues, and syntheses of new
compounds.
ii)
Until now, in most of the biological assays, RB-2 has been tested as a mixture of m-
and p-sulphonate isomers (purity ̴ 97-98%). Recently scientific literature 147 reported that
it has been impossible to separate the two isomers, in spite of many different
chromatographic systems. Therefore, the two constituents contained in RB2 were obtained
through a time and money consuming stepwise synthesis.
Because optimization of chromatographic procedures would result in easily and
economically available P2 ligands, our research group has undertaken a project aiming at
chromatographic purification of anthraquinone-chlorotriazine reactive dyes and of the
organic dyes in general.
7.2. Suramin
Suramin (30) is a large, complex, naphthylsulphonated molecule. It is a highly polypharmic
ligand [146] that can act as an inhibitor of HIV reverse transcriptase as a competitive
antagonist at several P2 receptor subtypes and vasoactive intestinal peptide receptors, as an
inhibitor of G protein activity [60], and as an inhibitor of tyrosine phosphatase. However,
since suramin poorly penetrates cell membranes, these last actions may be relevant only in
broken cell preparations. Suramin and its derivatives were found to be P2X antagonists, and
suramin antagonism of P2 responses is readily reversible upon washout.
This antagonist action is not related to the clinical use of suramin. Dunn and Blakely [189]
were the first to show that suramin antagonizes P2X1 receptor mediated effects. Functional
antagonism by suramin has frequently been found to be non-competitive. The potency order
of 30 at P2X receptors is (IC50 in µM): P2X1, P2X5 › P2X2, P2X3 › P2X7 › P2X4, P2X6 (>500) [190,
191]. 30 Is a weak antagonist at the P2Y1,11,12 receptors (pA2 values 5.4-6.0) with similar
potencies, but it is very weak or inactive as antagonist at the uracil nucleotide P2Y receptors
(P2Y2,4,6). In particular it antagonizes P2Y2 receptor with an IC50 value of 48 µM [187]. Thus,
efforts have been made to identify the structural features required for P2 receptor
antagonism.
64
O
HN
SO3Na
O
O
HN
SO3Na
O
NH 2
SO3Na
79
HN
80
SO3Na
SO3Na
SO3Na
NaO3S
NaO3S
HN
O
O
O
N
H
H
N
N
H
NH
81
R
R
O
82
R
O
H
N
O
N
R
SO3Na
H
N
O
H
N
NH
O
NH
H
N
O
O
H
N
O
NH
HN
O
N
H
-
O
O
O
O
83
84
N
H
R
Figure. 21 RB-2 and suramin analogues
65
R
Suramin and its analogues have a dimeric structure, suggesting a possible bifunctional
interaction with P2 receptors. This seems even more likely considering the multimeric
structure of P2X ion channels and the possibility that ATP recognition sites may occur
between such subunits. Many of the truncated analogues of suramin also retain symmetry
suggestive of this type of bifunctional interaction. The structural parameters, which appear to
control the properties of P2 antagonism and ecto-nucleotidases inhibition, are:
- the molecular size of the compounds
- the position of the sulfonic acid residues in the naphthalene ring
- the nature and substitution pattern of the aromatic rings "1" or "2"
- the structure of the central urea or dicarboxylic acid diamide bridges [192].
Among the suramin analogs already known, a series of truncated forms results interesting:
NF023 (31), NF279 (32), NF449 (33) and XAMAR 0721 (34) (figure 5). NF023 is selective for
P2X1 (pA2 = 5.5 – 6.0) receptor versus P2X3 (pA2 = 4.45) and at P2Y receptors in different
tissues. NF023 inhibits ectonucleotidase activity but, unlike suramin, it has a high P2X 1selectivity vs ecto-nucleotidase inhibition. It is also noteworthy that the compound BSt101
(81, figure 19), in which three sulphonic groups were removed from one naphthyl ring of
NF023, displayed similar potency as NF023 at P2X1 receptors in rat vas deferens, P2Y
receptors in guinea-pig taenia coli and ecto-nucleotidases [166]. These results with BSt101 are
significant in that they illustrate that it is not necessary to have trisulphonic acid groups at
both ends of the molecule. NF279 (32), highly potent P2X1-selective antagonist, was
considerably (6-fold) more potent than NF023.The receptor selectivity profile of NF279 is :
P2X1 > P2X2 ≥ P2X3 ≥ P2X7 >> P2X4. This selectivity profile is clearly different from that of
suramin and NF023. At P2Y1 receptors NF279, NF023 and suramin present a reversible and
surmountable antagonism. The two analogues are less potent than suramin at P2Y1 and
inactive (up to 100 µM) at P2Y2,4,6 receptor. Like suramin and NF023, NF279 inhibited ectonucleotidase activity at the very high concentration of 300 µM. Formal exchange of one half
of NF279 molecule by a benzene residue gave the asymmetric urea 82, with reduced activity
at P2X1 by eight-fold and a loss of P2X1 vs P2Y1 selectivity. The compound NF449 (34) was
found to be the most potent P2 antagonist at P2X1 receptors present in rat vas deferens.
NF449 did not interact with α1A-adrenoceptors or histamine H1 and muscarinic M3 receptors.
Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the
subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 >> P2X3 > P2Y1
66
> P2Y2 > ectonucleotidases, make NF449 unique among the P2 receptor antagonists reported
to date. Any deletions or shifts of the sulfonic acid residues or exchange of the central urea
bridge by a bisamide of terephtalic acid (83) in NF449 led to decreased activity at P2X1 and a
loss of P2X1 selectivity. Nitro- and amine –precursors of the small and large ureas, truncated
form of the suramin,
do not display activities at any receptor except for the
naphthalenetrisulphonic acid derivatives of 84 which have antagonistic effects at the P2Y1
receptor. Compound (34) is selective for P2Y receptor and for P2 receptors versus
Ectonucleotidases [146].
On the above, in this thesis our attention has been focused at preparing more potent and/or
selective suramin derivatives, through simplification of its structure. Hence, we started
investigating on structure-activity relationships of suramin derivatives known as P2 receptor
antagonists in order to select lead structures.
67
7.3. Chemistry
7.3.1. Analysis
The search in data bases and chemical catalogues for polisulphonated RB-2 and Suramin
analogues, aiming at having more information on SAR of those compounds for their action
as P2 antagonist, resulted in the finding of a series of organic dyes (figure 21) which were
purchased from Sigma Aldrich (Italy): RB-2 (35, purity = 60%), Acid Blue 129 (37, purity =
25%), Cibacron Brilliant Red (85, purity = 50%), Reactive Blue 4 (86, purity = 35%), Remazol
Brilliant Blue R (87, purity = 45%), Acid Green 27 (88, purity = 60%), Bromaminic acid (89,
purity = 87.7%), Azocarmine B (ACB, 90, purity = 80 %), Sulphobromonaphtalein sodium
(SBNS, 91, purity = 90%), SPTBSA (92, purity = 85 % ), Reactive Black 5 (RB-5, 93, purity = 55
%), Brilliant Black BN (BBBN, 94, purity = 60%), Procion Red MX-5B (PRMX-5, 95, purity =
50%), Direct Red 80 (DR80, 96, purity = 25% ), Direct Red 81 (DR81, 97, purity = 50%), Direct
Red 23 (DR23, 98, purity=30%), Direct Red 75 (DR75, 99, purity = 75%). The series of the
Direct dyes, which structurally belongs to the category of azonaphatalensulphonic acid dyes,
are large and small ureas symmetrically and not symmetrically substituted. It seems that the
presence of the ureidic bridge is essential for selectivity toward P2X1 receptor on cell line
tested in previous works. Some of these dyes were chromatographically purified (37, 85-89)
before testing, and the structures of pure products confirmed by MS spectroscopy and/or by
nuclear magnetic resonance (NMR, 1H, and
C). The purity of the organic dyes after
13
chromatography was 97-99% and it was determined by HPLC. The analytical
chromatography was carried using the following system: Waters Binary Pump 1525, Waters
Spectrometer 2996 Diode Array Detector (DAD). The data were achieved through
proprietary Waters software Empower Millennium.
We paid particular attention to RB-2 purification, because a recent literature stated that no
chromatographic purification of commercial RB2 was possible, in spite of many different
attempts: the authors started a time consuming specific synthesis for the two isomers [147].
After a preliminary flash chromatography purification, the partially purified fraction
containing the two o- and m- isomers was subject to a HPLC method for the separation of the
two constituents of the blue mixture. The analytical apparatus used is the same described
above. On the basis of the analytical chromatography (fig. 23A and 23B) the semi-preparative
68
method was developed. For the separation, preparative system Waters Prepuce RCM was
used. The five fractions obtained with this last experiment were analyzed using the analytical
HPLC (fig. 23D).
C
A
D
B
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
Fraction 1
0.022
0.020
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
Figure 22. 3D analytical chromatogram, with
Fraction 2
0.030
0.028
0.026
0.024
0.022
0.020
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.035
0.000
Fraction 3
0.030
the separation of the two RB isomers (A). 2D
0.025
0.020
analytical chromatogram extracted at 625 nm
0.015
Fraction 4
0.010
0.005
(B); UV-VIS spectra of the two isomers (C);
0.000
analytical chromatograms of five fractions
0.025
collected from semiprep chromatography (D).
0.015
0.020
0.010
Fraction 5
0.005
0.000
7.50
8.00
8.50
9.00
9.50
10.00
Minutes
10.50
11.00
11.50
12.00
69
7.3.2. Synthesis
We set up also a program for the synthesis of a series of RB-2 and suramin analogues: a
number of naphatalensulphonic acid derivatives were synthesized. Analogues 100-110 show
variations in the length and in the constitution of the side chain, compared to the RB-2
structure. In the synthesis of these dyes the 1-amino-anthraquinone-2 sulphonate core was
maintained untouched, except for compounds 102-104. The structure of DESUR 1 (111) is
similar to that of XAMAR 0721 (34), suramin analogue already reported in literature. The
syntheses of compounds were all performed with modifications of previously published
methods [193, 194].
Structure of compounds was confirmed by MS spectroscopy. All synthesized compounds
were obtained as sodium salts. Thus, various anions (1–4 negative charges) with up to four
sodium ions could be identified in the mass spectra of all compounds. Purity of compounds
was demonstrated by thin layer chromatography (TLC) and a HPLC methods.
70
O
O
NaO3S
NH
NH 2
OH
O
HN
37 (see fig.5)
O
85
O
NH 2
N
N
SO3Na
NaO3S
HN
N
NaO3S
SO3Na
HN
O
SO3Na
SO2
HN
OSO3Na
NH
86
87
N
N
N
Cl
Cl
NH 2
SO3Na
O
NH
N=N
SO3Na
Cl
NaO3S
O
O
HN
NH 2
SO3Na
SO3-Na
SO3Na
O
HN
O
88
Br
89
SO3Na
Br
SO3-
N
O
Br
SO3Na
O
OH
Br
N
N
H
Br
SO3Na
NaO3S
OH
90
91
Figure 23. Commercial RB-2 and suramin analogues tested as P2 antagonists.
71
SO3H
O
O
S
N
O S
OH NH 2
O
O
N
OH
N
O
N
S O
OH
92
N N
N N
HO 3S
SO3H
93
Cl
O
OH HN
NaO3S
N N
N
N
N N
SO3H
S
O
Cl
N
NH
OH
NaO3S
N N
SO3Na
SO3Na
NaO3S
SO3Na
94
95
OH
O
N N
N
H
N N
SO3Na
SO3Na
96
N
O
OH
OH
N
N N
O
SO3Na
N
H
N
H
NaO3S
N
H
95
NH 2
SO3Na
N N
OH
SO3Na
N N
N N
SO3Na
SO3Na
O
N
H
NaO3S
2
HO
O
97
NH
96
2
Figure 24. Commercial RB-2 and suramin analogues tested as P2 antagonists.
72
O
O
NH 2
SO3Na
NH 2
SO3Na
Na 2CO3/Na2SO3 ; CuCl ; H2O
R-NH 2
O Br
O HN
R
time and temperature
of reaction R depended
sodium salt of
bromaminic acid
100-103
R
Cl
N
N
Cl
Na 2CO3 ; H2O/ acetone
N
HN
N
2 R-NH 2
Cl
N
N
NH
time and temperature
of reaction R depended
Cyanuric
chloride
Cl
R
104-107
O
NH 2
O
NH 2
SO3Na
SO3Na
O HN
O HN
SO3Na
Na 2CO3 ; H2O/ acetone
NH
Cl
N
NH
R-NH2
N
N
SO3Na
time and temperature
of reaction R depended
Cl
Cl
84
O
O
Br
N
R
108-109
NH 2
SO3Na
N
N
OH NH 2
SO3Na
NaBH4
OH Br
89
110
Figure 25. Synthetic procedures to obtained dyes
73
O
NH 2
O
NH 2
SO3Na
SO3Na
SO3Na
O HN
SO3Na
O HN
NH 2
100
O
101
NH 2
O
NH 2
SO3Na
SO3 Na
SO3K
O
NH
O HN
SO3Na
SO3Na
102
103
SO3Na
NH2
HN
N
NH
SO3Na
HN
N
N
Cl
SO3Na
HN
NaO3S
N
N
Cl
N
N
N
NH
N
HN
HN
NaO3S
SO3Na
SO3Na
NH2
104
105
106
74
O
O
NH2
NH2
SO3Na
SO3Na
OH
N
O HN
NH
SO3Na
O HN
N
N
OH
107
O
SO3Na
Cl
N
NH
N
N
NH
108
NH2
SO3Na
O HN
NaO3S
OH NH2
SO3Na
Cl
N
NH
N
N
NH
SO3Na
OH Br
SO3Na
110
SO3Na
NaO3S
SO3Na
109
NO2
KO3S
H
N
NO2
O
111
Figure 26. Synthetic RB-2 and Suramin analogues tested as P2 antagonists.
75
7.4. Biological assays
The commercial dyes, with different purity rate, were tested on Cerebellar granule neurones
in hypoglycaemic condition, in order to check their ability to give neuroprotection, through
the count of the intact nuclei [195-197].
7.4.1. Primary cell cultures
Cerebellar granule cultures from Wistar 8-day-old rat cerebellum were prepared as
previously described [195] and seeded (0.5 or 0.25 x 106 cells) on poly-L-lysine-coated 10- or
5-mm multi-well plastic dishes in Eagle’s basal medium (BME) (Gibco BRL, MI-Italy),
supplemented with 25 mM KCl, 2 mM glutamine, 0.1 mg/ml gentamycin, 10% heat
inactivated foetal calf serum (Gibco BRL, MI-Italy). At day 1 in vitro (DIV) cultures were
supplemented with 10 μM cytosine arabinoside and kept for 7-9 days, without replacing the
culture medium, until use.
7.4.2 Hypoglycaemia studies and evaluation of cell survival.
Hypoglycaemic conditions were obtained by maintaining the cells in Earle’s Balanced Salt
Solution (EBSS), a glucose-free buffer, enriched with 116 mM NaCl, 25 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, 26 mM NaHCO3, 0.6 mM NaH2PO4, pH 7.4, as described [196]. For
control conditions, EBSS was supplemented with 1 mg/ml glucose. Different compounds
were added as indicated. After treatment, cells were returned to the previously saved
culture medium and survival was evaluated by direct count of intact nuclei [197].
Figures 28 and 29 reports biological assay data. Cerebellar granule neurones at 8 DIV were
maintained under hypoglycaemic conditions for three hours, in the absence or presence of
our compounds (83-97). Cells were returned to the saved culture conditioned medium and
survival was assessed 20 hours later, as described in the methods section. The compounds
were used with different purity rate: RB-2 (37),99%; ACB (88), 80%; SBNS (89), 90%; SPTBSA
(90), 85%; RB-5 (91), 55%; BBBN (92), 60%; PRMX-5B (93), 50%; DR80 (94), 25%; DR81 (95),
50%; DR 23 (96), 30%. DR75 (97), 25%. They were all tested at 100µM. Data are expressed as
% of viable cells with respect to the Control condition (CTRL) maintained in the presence of 1
mg/ml glucose. Counts represent means ± SEM (n=4).
76
As shown in figure 28 and 29, which report biological data of some of the tested compounds,
there are three different behaviour: toxic, protective and almost inactive RB2 analogues. In
detail, in the first position of Figure 28, we can see the effect of hypoglycemia (violet bar),
which produces death of about 50% of the control cell, taken as 100% (light blue bar). RB2
(35) shows a very high protection from deadly effect of hypoglycemia; in fact, the violet bar is
very close in height to the light blue one, demonstrating a protection close to 100%.
Among tested compounds, 89 and, to some extent, 92 show a promising ability of protection.
In particular, compound 89 is as protective as RB2, hence it is one of the most active P2
antagonist of this class known so far.
Other derivatives, like 88, 91, and 95 seems not to have an evident role in this biological test.
For example, compound 95 is not toxic per se (light blue bar, of the same height as control),
while it gives a negligible amount of protection against hypoglycemia (violet bar similar to
that of control).
Finally, there are some derivatives, like 94, 97, and, to some extent, 90 and 93, which possess
from moderate to severe cytotoxicity. In particular, 94 shows a severe toxicity by itself (about
70% of cell die after administration of 100 microM of it) and possess a synergistic effect with
hypoglicemia, with almost no cells surviving to the combined actions.
140
CTRL
Viable cells (%)
120
Hypo
100
80
60
40
20
0
-
35
88
89
90
91
92
93
Figure 28. Biological data for compounds 88-93
77
120
CTRL
100
Hypo
80
60
40
20
0
-
35
94
95
96
97
Figure 29. Biological data of compounds 94-97
Table 5.
Table.6
78
7.4.3. Conclusions
With this work, we have succeded in the chromatographic separation of isomeric mixture of
RB2. Furthermore, we have prepared a number of RB2 analogues, which have been tested for
their ability of counteracting the toxic effect of hypoglicemia.
Some of tested compounds behave as protecting agents, being at least equiactive with RB2.
79
8. Experimental procedures
8.1. 2-Alkynyl ATP analogues
Melting points were determined with a Büchi apparatus and are uncorrected. 1H NMR
spectra were obtained with Varian VXR 300 MHz spectrometer;  in ppm, J in Hz. All
exchangeable protons were confirmed by addition of D2O. 31P NMR spectra were recorded at
room temperature using a Varian VXR 300 MHz spectrometer. Elemental analyses were
determined on a Fisons model EA 1108 analyzer and are within ± 0.4% of theoretical values.
Analytical HPLC for compounds 71-76 was performed on a on a Waters HPLC apparatus,
made of Waters 1525 Binary HPLC Pump (set at 1 ml/min) and Waters 2996 Photodiode
Array (set at 254 nm), using a Merck Lichrocart 125-4 HPLC Cartridge, packed with Lichro
Sphere® 100 RP 18 (5 μ). All analysis were performed in isocratic mode, using Methanol/
Na2H2PO4 0,5 M in water (30:70) as eluent. Injection volume of 20 μL.
8.1.1. General procedure for the synthesis of 2-alkynyladenosine derivatives
250 Mg (0.64 mmol) of IV (scheme 1) were dissolved in 2 ml of dry dimethylformamide
(dDMF). To the stirred solution under nitrogen stream, were added in the following order: 10
mg (0.014 mmol) triphenylphosphyne palladium chloride; 0,64 mg (0,003 mmol) of copper (I)
iodide; 2,64 ml of triethylamine (0,02 mmol) and the appropriate 1 - alkyne (6 equiv.) The
solution was irradiated in microwave. Exposure time changed with different compounds.
After completion, the mixture was evaporated and chromatographed on a silica gel column
in order to obtain the desired product. For each compound reaction conditions,
chromatography eluent, and yield are reported.
8.1.2. 2-Exynyladenosine (69, 2-HEAdo)
The reaction of IV (scheme 1) with 1-exyne (0,43 ml) was performed heating the reaction
mixture in microwave oven for three cycles of 4 minutes each setting the power at the level of
3. The product was purified on a Silica gel column chromatography using the following
eluent: cycloexane/ethyl acetate/methanol (60:22:18). The isolated product was crystallized
from ethanol and dried. Yield = 89%. Analytical data were in agreement with the previous
literature data. [347.37]. (C16H21N5O4) C, H, N
80
8.1.3. 2-Phenylethynyladenosine (70, 2-PEAdo)
The reaction of IV (scheme 1) with 1-phenylacetylene (0,473 ml) was performed heating the
reaction mixture in microwave oven for 2 minutes each setting the power at the level of 2.
The product was purified by Silica gel column chromatography with the following solvent
mixture: chloroform/methanol (90:10). The isolated product was crystallized from ethanol
and dried. Yield=84%. Analytical data are in agreement with the previous literature data.
[367.37]. (C18H17N5O4) C, H, N
8.1.4. General procedure for the synthesis of nucleoside-5’-monophosphates
To 0.56 mmol of the nucleoside, dissolved in 3.0 ml of trimethyl phosphate, 4 equivalents of
POCl3 (0,209 ml, 343 mg, 2.24 mmol) were added. The solution was stirred at room
temperature for 3 h. H2O (3 ml) was added and the solution was neutralized by dropwise
addition of triethylamine. The reaction mixture was purified by ion exchange
chromatography.
8.1.5. General procedure for the synthesis of nucleoside-5’-diphosphates and nucleoside-5’triphosphates
To 0.15 mmol of the nucleoside-5’-monophosphate, dissolved in 1 ml of dry DMF, were
added 0,036 ml of tri-n-butylamine (28 mg, 0.15 mmol). The solution was stirred for 20
minutes at room temperature and then evaporated to dryness under anhydrous conditions.
After suspension of residue in 1.4 ml of dry DMF, N-N'-carbonyldiimidazole (122 mg, 0.75
mmol) was added and the mixture was stirred for 3 h at room temperature. Methanol (0.049
ml, 38.5 mg, 1.2 mmol) was added and the mixture was stirred for 30 min at room
temperature. Then, 6 ml (3 mmol) of a 0.5 M solution of tri-n-butylammonium phosphate or
bis(tri-n-butylammonium) pyrophosphate in DMF were added (for the synthesis of the
diphosphate or the triphosphates derivatives). The mixture was stirred for 14 h at room
temperature. The solvent was removed in vacuo. The mixture, dissolved in H2O, was purified
by means of ion exchange chromatography.
8.1.5. Chromatographic procedures for monitoring and purification of phosphorilation
reactions
81
Reactions were monitored by TLC, using pre-coated TLC plates with silica gel 60 F-254
(Merck) and iPropanole-H2O-NH4OH (30%) (55/10/35) as mobile phase. The nucleotides were
purified by means of ionic exchange chromatography on a Sephadex® DEAE A-25 (Fluka)
column (HCO3- form) equilibrated with H2O and eluted with a linear gradient of H2O /0.5 M
NH4HCO3, which gives a gradual increase of PH and ionic strength.
8.1.6. 2-Hexynyladenosine-5’-monophosphate ammonium salt (2-HEAMP, 71)
From 169 mg (0,486 mmol) of the unprotected nucleoside 69 were obtained 78 mg (0.141
mmol) of 2-HEAMP ammonium salt. Yield = 29%. 1H NMR (DMSO-d6) d 0.93 (3H, t, CH3,
J=6.80 Hz), 1.51 (4H, m, CH2CH2CH3), 2.43 (2H, t, CH2CH2CH2CH3, J=6.86 Hz), 3.81 (2H, m,
H-5’), 4.05 (1H, m, H-4’), 4.23 (1H, m, H-3’), 4.58 (1H, t, H-2’, J=5.78 Hz), 5.89 (1 H, d, H-1',
J=6.22 Hz), 7.39 (1H, br s, NH2), 8.53 (1H, s, H-8). 1H NMR (D2O)  0.80 (3H, t, CH3, J=7.22
Hz), 1.34 (2H, m, CH2CH3), 1.47 (2H, m, CH2CH2CH3), 2.34 (2H, t, CH2CH2CH2CH3, J=6.75
Hz), 3.99 (2H, m, H-5’), 4.25 (1H, m, H-4’), 4.35 (1H, t, H-3’, J=4.20 Hz), 4.56 (1H, t, H-2’, J=5.28
Hz), 5.95 (1H, d, H-1', J=5.57 Hz), 8.34 (1H, s, H-8). 31P NMR (D2O)  0.91 (s) [ 444,38] Anal.(
C16H25 N6 O7P) C,H,N,P.
8.1.7. 2-Phenylethynyladenosine-5’-monophosphate ammonium salt (2-PEAMP, 72)
From 244 mg (0, 65 mmol) of the unprotected nucleoside 69 were obtained 252 mg (0. 54 mmol)
of 2-HEAMP ammonium salt. Yield = 83%. 1H NMR (DMSO-d6)  3.87 (2H, m, H-5’), 4.07 (1H,
m, H-4’), 4.23 (1H, m, H-3’), 4.64 (1H, m, H-2’), 5.96 (1 H, d, H-1', J=5.06 Hz), 7.52 (1H, br s,
NH2), 7.63-7.54 (5H, m, phenyl), 8.60 (1H, s, H-8). 1H NMR (D2O)  4.06 (2H, m, H-5’), 4.29 (1H,
m, H-4’), 4.36 (1H, m, H-3’), 4.49 (1H, m, H-2’), 5.87 (1H, d, H-1', J=4.38 Hz), 7.21-7.03 (5H, m,
phenyl), 8.23 (1H, s, H-8). 31P NMR (D2O)  1.04 (s). [464.37] Anal.(C18H21 N6 O7P) C,H,N,P.
8.1.8. 2-Hexynyladenosine-5’-diphosphate ammonium salt (2-HEADP, 73)
From 68 mg (0,15 mmol) of the tributhylammonium salt of the monophosphate derivative 71
were obtained 63 mg (0.11 mmol) of 2-HEADP ammonium salt. Yield =75%.1H NMR (D2O) 
0.77 (3H, t, CH3, J=6.97 Hz), 1.32 (2H, m, CH2CH3), 1.45 (2H, m, CH2CH2CH3), 2.32 (2H, t,
CH2CH2CH2CH3, J=6.58 Hz), 4.12 (2H, m, H-5’), 4.24 (1H, m, H-4’), 4.37 (1H, m, H-3’), 4.56
82
(1H, t, H-2’, J=5.28 Hz), 5.93 (1H, d, H-1', J=5.34 Hz), 8.37 (1H,s, H-8). 31P NMR (D2O)  -10.73
(d, P), -10.21 (d, P) [558.42]. Anal. (C16H32N8O10P2) C,H,N,P.
8.1.9. 2- Phenylethynyladenosine-5’-diphosphate ammonium salt (2-PEADP, 74)
From 67 mg (0, 14 mmol) of the tributhylammonium salt of the monophosphate derivative 71
were obtained 47 mg (0.08 mmol) of 2-PEADP ammonium salt. Yield = 57%. 1H NMR (D2O) 
4.20 (2H, m, H-5’), 4.35 (1H, m, H-4’), 4.48 (1H, m, H-3’), 4.63 (1H, m, H-2’), 6.00 (1H, d, H-1',
J=4.8 Hz), 7.54-7.09 (5H, m, phenyl), 8.44 (1H, s, H-8). 31P NMR (D2O)  -10.64 (s, P), -10.07
(s, P) P.M.: 578.41 Anal.(C18H28N8O10P2) C,H,N,P.
8.1.10. 2-Hexynyladenosine-5’-triphosphate ammonium salt (2-HEADP, 75)
From 67 mg (0,15 mmol) of the tributhylammonium salt of the monophosphate derivative 71
were obtained 55 mg (0.08 mmol) of 2-HEATP ammonium salt. Yield = 53%. 1H NMR (D2O)
 0.80 (3H, t, CH3, J=6.94 Hz), 1.34 (2H, m, CH2CH3), 1.47 (2H, m, CH2CH2CH3), 2.34 (2H, t,
CH2CH2CH2CH3, J=6.75 Hz), 4.12 (2H, m, H-5’), 4.26 (1H, m, H-4’), 4.47 (1H, m, H-3’), 4.58
(1H, t, H-2’, J=5.28 Hz), 5.95 (1H, d, H-1', J=6.07 Hz), 8.41 (1H, s, H-8). 31P NMR (D2O)  -21.70
(t, P), -10.66 (d, P), -7.28 (d, P). P.M.: 665.43 Anal. (C16H36N9O13P3) C,H,N,P.
8.1.11. 2- Phenylethynyladenosine-5’-triphosphate ammonium salt (2-PEATP, 76)
From 68 mg (0,15 mmol) of the tributhylammonium salt of the monophosphate derivative 71
were obtained 75 mg (0.08 mmol) of 2-PEATP ammonium salt. Yield = 58%. 1H NMR (D2O)
 0.80 (3H, t, CH3, J=6.94 Hz), 1.34 (2H, m, CH2CH3), 1.47 (2H, m, CH2CH2CH3), 2.34 (2H, t,
CH2CH2CH2CH3, J=6.75 Hz), 4.12 (2H, m, H-5’), 4.26 (1H, m, H-4’), 4.47 (1H, m, H-3’), 4.58
(1H, t, H-2’, J=5.28 Hz), 5.95 (1H, d, H-1', J=6.07 Hz), 8.41 (1H, s, H-8). 31P NMR (D2O)  -21.70
(t, P), -10.66 (d, P), -7.28 (d, P). P.M.: 665.43 Anal.( C16H36N9O13P3) C,H,N,P.
8.1.12. Preparation of tri-n-buthylammonium monophosphate
Reagens: Sodium diidrogenumphosphate monoidratus (NaH2PO4∙H2O; PM =137.99); resin
Dowex
50x8,
20-50
mesh;
tributhylamine
(PM=185.36;
d=
0.777;
bp=
88-90°C);
dimethylformide (DMF, PM=73.10; d= 0.95; bp= 152-154°C)
83
O-
nBu 3NH+
1,38 Mg di sodium diidrogenophosphate monohydrate (10
OH
P
O
mmol) were dissolved in 70 ml of water and put under stirring.
Dowex 50x8, 20-50 mesh, H+ form (21 g) was added and the
OH
suspension was stirred for 20 minutes more.
A mixture of ethanol (50 ml) and
tributhylamine (2,4 ml; 10 mmol) were put in an ice bath and the solution of orthophosphate
was filtered into the mixture. The resin on the filter was washed with water until the filtrated
showed to be neutral. The solution was stirred for 30 minutes in the ice bath. The residue was
evaporated (temperature below 35 °C) to dryness and co-evaporated three times with
ethanol. The residue was co-evaporated three times more with DMF. The residue is put in 20
ml of DMF, in order to obtain a solution 0.5 M of tri-n-buthylammonium monophosphate in
DMF. The solution was stored over molecular sieves at 4°C.
8.1.13. Preparation of tri-n-buthylammonium pyrophosphate
Reagent: Sodium pyrophosphate decahydrate (Na4P2O7∙10H2O; PM =446.06); Dowex resin
50x8, 20-50 mesh; tributhylamine (PM=185.36; d= 0.777; bp= 88-90°C); dimethylformide
(DMF, PM=73.10; d= 0.95; bp= 152-154°C).
nBu 3NH+
OP
O
3,34 Mg of sodium pyrophosphate decahydrate (7,5
OH
O
O
P
OH
O-
nBu 3NH+
mmol) are dissolved in 75 ml of water and put under
stirring. Dowex 50x8, 20-50 mesh, H+ form (21 g) was
added and the suspension was stirred for 20 minutes. A
mixture of ethanol (30 ml) and tributhylamine (3.57 ml; 15
mmol) were put in an ice bath and the solution of
pyrophosphate was filtered into the mixture. The resin on the filter was washed with water
until the filtrated showed to be neutral. The residue was evaporated (temperature no
superior to 35°C) and co-evaporated for three times with ethanol. The residue was coevaporated again for three times with 15 ml of DMF. The residue is put in 15 ml of DMF in
order to obtain a solution 0.5 M of tri-n-buthylammonium pyrophosphate in DMF. The
solution was stored over molecular sieves at 4°C.
8.1.14 .DEAE- Sephadex ion exchange resin
An ion exchange resin consists of an insoluble matrix to which charged groups are covalently
bound. The charged groups are associated with mobile counter-ions. These counter-ions can
84
be reversibly exchanged with other ions of the same charge without altering the matrix.
Sephadex DEAE ion exchangers are produced by introducing diethylamino ethyl (DEAE)
groups onto Sephadex, a cross-linked dextran matrix.
DEAE is a weak anionic exanchanger and its counter-ion is a chloride. The polydestrane
matrix is chemically unreactive. It is insoluble in water, where it swells, forming
stiff
spheres. The higher the number of cross-link the lower the swelling of spheres. Swelling
depends on PH, ionic strenght of the buffer, nature of the counter-ion. For the purification of
the phosphates either DEAE- Sephadex A-25 and A-50 can be used.
8.1.15. Preparation of the DEAE- Sephadex resin
Swelling of the resin: 10 gr of the resin were put into a beaker with 400-50o ml of deionizated
water (R>10 MΩ) for 24 hours at 4°C. After that the resin is swelled with 500 ml of NaHCO 3 1
M. The resin was kept for three days at 4°C, changing every day the supernatant buffer
solution with a fresh one.
8.2. RB-2 and Suramin
8.2.1. Purification methods
Dyes 37 and 85-89 were purchased from Signa-Aldrich. Flash chromatography was carried
out on Silica gel RP C18 (32x63 mm) and on Silica gel 60 (Sigma-Aldrich Italy). TLC was
performed on Silica gel aluminium sheets RP-18 F254s (E. Merck, Italy), and Silica gel 60 F254 (Fluka). The dye purity was determined by HPLC.
HPLC apparatus: Waters Binary Pump 1525, Waters Spectrometer 2996 Diode Array Detector
(DAD). The data were achieved through proprietary Waters software Empower Millennium.
All analysis were performed in isocratic mode, using different solvent mixture as eluent, and
a column Waters Nova-Pak C18 (3.9 x 150 mm). Injection volume of 20 μL.
8.2.1 Acid blue 129 (37, purity of commercial source=25%) was purified by normal flash
chromatography, with the eluent chloroform:methanol (85:15). The obtained dye purity was
99%, as determined by HPLC, elution in isocratic condition with MeOH:H 2O (50:50) and a
flow rate of 1 ml/min. [458.46] (C23H19N2O5SNa)MS : 435.0 ([M ]-).
85
8.2.2. Cibacron brilliant red (85, purity of commercial source =50%) was purified on a
preparative TLC (2 mm), using 2-propanol:ammonia (80:20) as eluent. The TLC run was
repeated three time. The product isolated has a characteristic scent of strawberry. The
obtained dye purity was 99%, as determined by HPLC, eluting in isocratic condition with
MeOH:H2O (14:86) and a flow rate of 0,7 ml/min. [992.20] (C32H19N6O14ClS4Na4) MS: 308.0
([M3- + Na+]=/3); 225.0 ([M4- ]/4)
8.2.3. Reactive blue 4 (86, purity of commercial source =35%). 1 Gr of the commercial
preparation
was
purified
by
flash
chromatography,
with
chloroform:ethyl
acetate:methanol:methanolic ammonia (25:65:8:2) as eluent. The obtained dye purity was
97%, as determined by HPLC, eluting in isocratic condition with MeOH:H 2O (30:70) and a
flow rate of 1 ml/min. [638.7] (C23H14N6O8Cl2S2Na2)MS: 637.7 ([M-H+]); 615.8 ([M-Na+]-); 307.4
([M-H+-Na+]/2).
8.2.4. Remazol brilliant blue R (87, purity of commercial source =45%), dissolved in water,
was purified by reverse phase (RP) flash chromatography, using the eluent methanol:water
(25:75). The obtained dye purity was 76%, as determined by HPLC, eluting in isocratic
condition with MeOH:H2O (20:80) and a flow rate of 1 ml/min. [626.55] (C22H16N2O11Cl2S3Na2)
MS: 603.0 ([M- + Na+]);
8.2.5. Acid Green 27 (88, purity of commercial source = 75%) dried on silica, was purified by
flash chromatography, with the eluent chloroform:methanol (80:20).The obtained dye purity
was 93%, as determined by HPLC, eluting in isocratic condition with MeOH:H 2O (50:50) and
a flow of 1 ml/min. [808.78] (C34H31N2O11S3Na3).
8.2.6. Bromaminic acid (89, purity of commercial source =87.7%), dried on silica, was purified
by flash chromatography, with chloroform:ethylacetate:methanol:methanolic ammonia as
eluent (40:50:9:1). The obtained dye purity was 92%, as determined by HPLC, eluting in
isocratic condition with MeOH/ H2O (20:80) and a flow rate of 1 ml/min. [404.2]
(C14H7NO5BrS3Na).
86
8.2.7. Reactive blue 2 (35, purity of commercial source =60%), dissolved in water, was
purified by reverse phase (RP) flash chromatography, using the eluent methanol:water
(35:75). The obtained dye purity was 99%, as determined by HPLC. The mixture contained
the two o- and m- isomers, whose ratio was determined with analytical HPLC. The mixture
was then separated by semipreparative HPLC
Analytical HPLC conditions for separation of RB2 isomers: Elution was performed using a
programmed sequence as follows: isocratic 10% MeOH for 6 minutes, 1030% MeOH with
gt = 4 min (gt = gradient time); isocratic 30% MeOH for 15 minutes; 3035% MeOH with gt =
1 min; isocratic 35% MeOH for 10 minutes.
Semi preparative HPLC conditions for separation of RB2 isomers: The elution with
methanol/water has been carried out with a programmed sequence as follow: isocratic 10%
MeOH for 5 minutes, gradient 1040% MeOH with gt = 25 min; isocratic 40% MeOH for 10
minutes.
Preparative system Waters Prepuce RCM was used, using three Prepack® 25 x 100 mm
cartridges, filled with the stationary phase Bondapak® C18 15-20 m 125 Å, obtaining a 25 x
300 mm column. After lyophilization, the blue isolated product was analyzed by 1H and 13C
NMR. Data were in agreement with the data in literature, confirming that the two isomers
had been separated.
8.3. Synthetic procedures
8.3.1.Chemicals
Bromaminic acid (89), 1,4-diaminobenzenesulphonic acid, cyanuric chloride (2,4,6-trichloro-s
-triazine), m- and p-aminobenzenesulphonic acids, 8-Amino-naphthalene-2-sulphonic acid,
3,5-dinitrobenzoic acid, 7-amino-naphtalen-1,3-disulphonic acid, 1-amino-3,6,8-trisulphonic
acid and all other reagents were purchased from Signa-Aldrich.
8.3.2. Analytical studies
H- and 13C NMR spectra were recorded on a Varian Gemini 200 and Varian Mercury 400
1
spectrometer; Chemical shift values  (in ppm) refers to the signal of DMSO in DMSO-d6,
with 2.49 for 1H-NMR and δTMS = δDMSO=39.7 for 13C-NMR.
H-NMR data are listed in the
1
following order: multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet), number of nuclei,
assignment. Flash chromatography was carried out on Silica gel RP C18 (32x63 mm) and on
87
Silica gel 60 (Sigma-Aldrich, Italy). TLC was performed on Silica gel aluminium sheets RP-18
F254s (E. Merck, Italy), and Silica gel 60 F-254 (Fluka). Elemental analyses were determined
on a Fisons model EA 1108 analyzer and are within ± 0.4% of theoretical values.
8.3.2.1.
1-Amino-4-(4-amino-3-sulphophenylamino)-9,10-dioxo-9,10-dihydranthracen-2-
sulphonic acid (100).
To a stirred solution of 500 mg of sodium carbonate and 400 mg of sodium sulphite in 50 ml
of
water
were
added
808.4
mg
(2
mmol)
of
1-amino-4-bromo-9,10-dioxo-9,10-
dihydroanthracene-2-sulphonic acid sodium salt (80) and 760 mg (4 mmol) of 1,4diaminosulphanilic acid. Then, 50 mg of copper (I) chloride were added and the mixture was
stirred at room temperature for 8 h, monitoring the formation of products by silica gel TLC,
using a mobile phase of chloroform/ethyl acetate/methanol (35:20:45).
After completion of the reaction the dark green solution was dried and the residue was
washed with methanol (5x40 ml). The combined filtrates were evaporated. The residue was
dried and chromatographed on a silica gel column in gradient conditions: chloroform:ethyl
acetate:methanol:metanolic
ammonia
eluent
(50:20:28:2→40:20:38:2).
From
the
chromatography four fractions were collected: 1) orange fraction; 2) yellow fraction; 3) blue
fraction; 4) green fraction (coming from a blue product plus a yellow product). The green
fraction was dried, triturated with acetonitrile and filtered. The red precipitate was removed,
while the blue-green filtrate (blue plus yellow spots) was purified using the chromatotrom
system with acetonytrile/acetone/water (70:29:1) as eluent. Collected fractions were put
together and dried under vacuum yielding 160 mg (0,34 mmol; yield 17%). 1H- and 13C- NMR
data were in agreement with literature data. [681.39] C23H12N6O8Cl2S2Na2
8.3.2.2. 1-Amino-4-(3-sulphophenylamino)-9,10-dioxides-9,10dihydroanthracene-2-sulphonic
acid (101).
To a stirred solution of 500 mg of sodium carbonate and 400 mg of sodium sulphite in 50 ml
of
water
were
added
808.4
mg
(2
mmol)
of
1-amino-4-bromo-9,10-dioxo-9,10-
dihydroanthracene-2-sulphonic acid sodium salt (89) and 692.76 mg (4 mmol) of 3aminobenzenesulphonic acid (metanilic acid). Then 50 mg of copper (I) chloride were added
88
and the mixture was stirred in oil bath at 130 °C for 8 h, monitoring the formation of
products by TLC, using a mobile phase of chloroform/ethyl acetate/methanol (50:20:30).
After completion of the reaction (about 8 h) the green mixture was dried, triturated with
acetonytrile and filtered over silica, washing with acetonitrile, for a total of 240 ml. The
orange filtrated was removed, while the violet residue was dried and chromatographed on a
silica gel column using an acetonytrile/acetone/water (70:29:1) eluent. From the
chromatography three main fractions: were collected 1) violet fraction (mixture of pink,
orange, and blue compounds); 2) blue fraction (pure compound); 3) green fraction (mixture
of blue, yellow, and red compounds). The blue residue (blue fraction) was dried under
vacuum. The chromatography was repeated on the violet and green fractions separately
using a chromatotrom system. From the violet fraction using acetonytrile as eluent the
orange impurity was removed. The green fraction was first crystallized from acetonytrile,
then the blue residue after evaporation of solvent was purified by chromatotron using
acetonytrile/acetone/water (70:29:1) as eluent. The blue residues were put togheter and the
solution was dried under vacuum, yielding in total 125 mg (0,25 mmol; yield 12,5 %).
C23H12N6O8Cl2S2Na2 MS: 495.0 ([M=+Na+]-), 473.0 ([M=+ H+]-),235.0 ([M]=/2).
8.3.2.3.
1-Amino-4-(8-amino-naphthalene-2-sulphonic
acid)-9,10-dioxyde-9,10
dihydroanthracene -2-sulphonic acid. (102)
To a stirred solution of 500 mg of sodium carbonate and 400 mg of sodium sulphite in 50 ml
of
water
were
added
808
mg
(2
mmol)
of
1-amino-4-bromo-9,10-dioxo-9,10-
dihydroanthracene- 2-sulphonic acid sodium salt (89) and 893 mg (4 mmol) of 8-Aminonaphthalene-2-sulphonic acid. Then, 50 mg of copper (I) chloride were added and the
mixture was stirred at room temperature for 24 h, monitoring the formation of product on
TLC using a mobile phase of acetonytrile/acetone/water (65:30:5) and the disappearance of
starting material with chloroform/ethyl acetate/methanol (40:30:30). After 24 h the brown
mixture was dried and chromatographed on a silica gel column using chloroform/ethyl
acetate/methanol (40:30:30) eluent. The combined fractions were filtered and evaporated. The
main faction was re-chromatographed by flash column chromatography on reversed phase
silica gel using a methanol/ammonium acetate 0,5 M (40:60) eluent. The combined fractions
were evaporated and freeze-dried. The blue residue was dried under vacuum yielding 189
mg (0, 34 mmol; yield 17%). [568.49] C24H14N2O8S2Na2 MS: 544.0 ([M]-), 206.7 ([M=]/2).
89
8.3.2.4. 1-Amino-4-(7-aminonaphtalen-3-disulphonic acid)-9,10 -dioxy-9,10-dihydroanthracene-2-sulphonic acid. (103)
To a stirred solution of 150 mg of sodium carbonate and 121 mg of sodium sulphite in 15 ml
of water were added 242.5 mg (0.6 mmol) of 1-amino-4-bromo-9,10-dioxo-9,10dihydroanthracene- 2-sulphonic acid sodium salt (89) and 404 mg (1,16 mmol) of 7-aminonaphtalen1,3-disulphonic acid. Then 15 mg of copper (I) chloride were added and the
mixture was stirred in oil bath at 90 °C for 5 h, monitoring the formation of product on TLC
using as mobile phase acetonytrile/acetone/water (70:29:1), while disappearance of starting
material with chloroform/ethyl acetate/methanol (40:30:30).
After 5 h the brown mixture was dried and chromatographed on a silica gel column using
chloroform/ethyl acetate/methanol (30:20:50) as eluent. The combined fractions were
evaporated. The fractions containing the blue spot were chromatographed again by flash
chromatography column on reverse phase silica gel using methanol/ammonium acetate 0,5
M (40:60) as eluent. The combined fractions were evaporated and lyophylized. The blue
residue was dried under vacuum yielding 43 mg (0, 12 mmol; 20%). [531.86]
(C15H12N7O6S2Na2) MS: 544.0 ([M—K+]-), 200.4 ([M3-]/3).
8.3.2.5. N-(4-Amino-3-sulfonyl-phenyl)-N'-(4-amino-2-sulfonyl-phenyl)-6-chloro[1,3,5]triazine-2,4-diamine sodium salt(104)
An ice-cooled solution of 35 mg (0.19 mmol) of cyanuric chloride in water (10 ml) and
acetone (10 ml) was added to a solution of 71.5 mg (3.8 mmol) of 2, 5-diaminosulphonic acid
in water (20 ml) at 0-5 °C. The resulting solution was stirred at 0-5 °C for 1 h while 2 M
sodium carbonate solution was added dropwise to keep the pH in the range 5-7. The reaction
was monitored by TLC using the following eluent: chloroform:methanol (60:40).
After 1 h the solvent was evaporated and the residue was purified by silica gel flash
chromatography column using chloroform:methanol (70:30) as eluent. The combined
fractions were filtered and evaporated. The product was dried under vacuum, yielding 29
mg (0,12 mmol; yield 64%). [487.90] (C23H12N6O8Cl2S2NaK) MS: 507.6 ([M-2H++Na+]-), 485.5
([M-H+] -), 242.4 ([M-2H+]=/2).
8.3.2.6. N,N',N''-Tri-m-sulfonylphenyl-[1,3,5]triazine-2,4,6-triamine sodium salt (105)
90
An ice-cooled solution of 35 mg (0.19 mmol) of cyanuric chloride in water (10 ml) and
acetone (10 ml) was added to a solution of 105 mg (0,95 mmol) of methanylic acid in water
(10 ml) at 0-5 °C. The resulting solution was stirred at 40-60 °C in oil bath for 2 h while 2 M
sodium carbonate solution was added dropwise to keep the pH in the range of 5-7. The
reaction was monitored by TLC using the eluent: chloroform/methanol/methanolic ammonia
(58:40:2). After 2 h the solvent was evaporated and the residue was purified by silica gel flash
chromatography column using chloroform/methanol/methanolic ammonia (68:30:2) as
eluent. The combined fractions were filtered and evaporated. The product was dried under
vacuum yielding 42 mg (0,07 mmol; yield 36%). [660.55] (C21H15N6O9S3Na3 ) MS: 637.0
([M=Na+]-), 307.0 ([M]=/2).
8.3.2.7. 6-Chloro-N,N'-di-p- sulfonylphenyl -[1,3,5]triazine-2,4-diamine sodium salt (106)
An ice-cooled solution of 35 mg (0.19 mmol) of cyanuric chloride in water (10 ml) and
acetone (10 ml) was added to a solution of 165 mg (0,95 mmol) of sulphanilic acid in water
(10 ml) at 0-5 °C. The resulting solution was stirred at 40-60 °C in oil bath for 2 h while 2 M
sodium carbonate solution was added dropwise to keep the pH in the range 5-7. The reaction
was monitored by TLC using a mobile phase of chloroform/methanol/methanolic ammonia
(58:40:2).
After 2 h the solvent was evaporated and the residue was purified by silica gel flash
chromatography column using chloroform:methanol:methanolic ammonia (68:30:2) as
eluent. The combined fractions were filtered and evaporated. The product was dried under
vacuum yielding 22 mg (0.05 mmol; yield 24%). [501] (C15H10N5O6ClS2Na2 ) MS: 227.4
([M]=/2).
8.3.2.8.
1-Amino-4-[4-(4,6-dihydroxy-[1,3,5]triazin-2-ylamino)-3-sulfo-phenylamino]-9,10-
dioxo-9,10-dihydro-anthracene-2-sulfonic acid sodium salt (107)
An ice-cooled solution of 49 mg (0.27 mmol) of cyanuric chloride in water (14,3 ml) and
acetone (14,3 ml) was added to a solution of 39 (0,27 mmol) of 100 in water (14.3 ml) at 0-5 °C.
The resulting solution was stirred at 0-5 °C for 1 h while 2 M sodium carbonate solution was
added dropwise to keep the pH in the range 5-7. The reaction was monitored on TLC using
acetonytrile/acetone/water (65:30:5) as eluent. The reaction was stirred at room temperature
91
overnight. The mixture was purified by flash chromatography column on silica gel using
acetonitrile/acetone/water (70:29:1) as eluent. The combined fractions were filtered and
evaporated. The blue residue was dried under vacuum, yielding 141 mg (0.22 mmol; yield =
81% ). [646.02] (C23H14N6O10S2Na2 ) MS: 623.0 ([M=-Na+]-), 299.0 ([M=-Na+]-/2).
8.3.2.9
1-Amino-4-{3-[4-chloro-6-(4-sulfo-phenylamino)-[1,3,5]triazin-2-ylamino]-4-sulfo-
phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulfonic acid sodium salt.(108)
An ice-cooled solution of 52, 56 mg (0.13 mmol) of sulphanilic acid in water (6,8 ml) and
acetone (6,8 ml) was added to a solution of 85 mg (0,13 mmol) of RB4 in water (6.8 ml) at 0-5
°C. The resulting solution was stirred at 0-5 °C for 1 h while 2 M sodium carbonate solution
was added dropwise to keep the pH in the range 5-7. The reaction was monitored by TLC
using acetonytrile/acetone/water (60:30:10) as eluent. The reaction was stirred at 120-130°C in
oil bath overnight. The crude was evaporated and purified by a silica gel chromatography
column using chloroform/ethyl acetate/methanol (15:50:35) as eluent. The combined fractions
were filtered and evaporated. The impure residue was chromatographed again on reverse
phase flash chromatography column using methanol/water (30:70) as eluent. The combined
fractions were evaporated and freeze-dried, yielding 29 mg (0,03 mmol; yield 23%). [840.11]
(C29H17N7O11ClS3Na3 ) MS:256.7 ([M]3-/3).
8.3.2.10.
8-{4-[5-(4-Amino-9,10-dioxo-3-sulfo-9,10-dihydro-anthracen-1-ylamino)-2-sulfo-
phenylamino]-6-chloro-[1,3,5]triazin-2-ylamino}-naphthalene-1,3,6-trisulfonic acid (109)
An ice-cooled solution of 52, 56 mg (0.13 mmol) of 1-aminonaphtalen-3,6,8-trisulphonic acid
in water (6,8 ml) and acetone (6,8 ml) was added to a solution of 85 mg (0,13 mmol) of RB4 in
water (6.8 ml) at 0-5 °C. The resulting solution was stirred at 0-5 °C for 1 h while 2 M sodium
carbonate solution was added dropwise to keep the pH in the range 5-7. The reaction was
monitored on TLC using acetonytrile/acetone/water (60:30:10) as eluent. The reaction was
stirred at 120-130°C in oil bath overnight. The mixture was evaporated and purified by a
silica gel chromatography column using chloroform/ethyl acetate/methanol (15:50:35) as
eluent. The fraction containing the blue product was chromatographed on reversed phase
chromatography flash column using methanol/water (30:70) as eluent. The combined
fractions were evaporated and freeze-dryed, yielding 60 mg (0,05 mmol; yield 38,4%).
[1094.26] (C33H17N7O17ClS5Na5 ) MS: 642.0 ([M=-Na+]-), 299.0 ([M=-Na+]-/2).
92
8.3.2.11. 1-Amino-4-bromo-9,10-dihydroxy-9,10-dihydro-anthracene-2-sulfonic acid sodium
salt (110)
1-Amino-4-brome-9,10-dioxide-9,10-dihydroanthracene-2-sulphonic acid sodium salt (89)
(512 mg; 1.27 mmol) was added of methanol (5,2 ml) and the resulting suspension was
stirred while cooling to 0-5°C. Solid sodium borohydride (190 mg; 5 mmol) was added in
small portions. Immediately after the addition of NaBH4 effervescence appeared and the
mixture from orange became green. The reaction was monitored by TLC using
chloroform/ethyl acetate/methanol/methanolic ammonia as eluent (25:50:24:1). After 1 h, the
mixture was dried under nitrogen stream and purified by silica gel flash chromatography
column, with chloroform/ethyl acetate/methanol/methanolic ammonia (30:50:18:2) as eluent.
The fractions were collected and evaporated. The residue was dried under vacuum, yielding
128 mg (0,31 mmol; yield 28%).[408.20] MS: 792.2 ([2M-+Na+]-), 487.5 ([M=+Na+ Br-]-), 385.6
([M]-).
8.3.2.12. 7-(3,5-Dinitro-benzoylamino)-naphthalene-1,3-disulfonic acid sodium salt (111)
3,5-Dinitrobenzoylchloride (80 mg, 0,38 mmol) was dissolved in toluene (0,5 ml). To the
stirred solution, 7-amino-naphtalen-1,3-disulfonic acid (100 mg, 0,29 mmol) dissolved in
water (1,1 ml) was slowly added and the pH was adjusted to 3.8. The reaction mixture was
kept at a constant pH of 3.8 by automatic addition of a 2 M Na2CO3 solution. After 30
minutes the mixture was neutralised to pH 7.0, the water was removed and the crude
product
was
purified
on
flash
chromatography
column
with
chloroform/methanol/methanolic ammonia (70:28:2) as eluent. The isolated product was
dried under vacuum, yielding 85 mg (0,15 mmol; 39%). [535.51], (C17H10N7O11S2K )MS: 210.9
([M-H+-K+]=/2).
93
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Index
1.Introduction
p.2
1.1.Evolution of the P2 Receptor Family
p.4
1.2. Complexities of P2 Receptor Pharmacology
p8
1.3. Radioligands
p.10
2. P2 Receptors
p.11
2.1. Relationship Between Native and Recombinant P2 Receptors
p.11
22..22.. PP22XX RReecceeppttoorrss
p.11
22..33.. P2Y Receptors
p.22
2.4.P2 Receptors Structure: P2X Receptors
p.27
2.5 P2 Receptors Structure: P2Y Receptors
p.30
3. Development of P2 Receptor Ligands
p.32
3.1. P2 Receptor agonists
p.33
3.2. P2 Receptor antagonists
p.37
4.Biological Actions and Clinical Targets
p.40
5. Aim of the research
p.48
6. 2-Alkynyl-ATP analogues
p.48
6.1. SAR studies
p.50
6.2. Chemistry
p.50
6.3. Biological assays
p.55
6.4.Stability studies
p.58
6.5.Conclusions
p.60
7. Reactive Blue 2 and Suramin analogues
p.61
7.1. Reactive Blue 2
p.61
7.2. Suramin
p.63
7.3. Chemistry
p.67
7.3.1. Analysis
p.65
7.3.2. Synthesis
p.69
8. Biology
p.75
8. Experimental procedures
p.80
10. References
p.94
113