2′,3′-O-Substituted ATP derivatives as potent antagonists of purinergic P2X3 receptors and potential analgesic agents

Article abstract of DOI:10.1007/s11302-016-9539-y

Blocking membrane currents evoked by the activation of purinergic P2X3 receptors localized on nociceptive neurons represents a promising strategy for the development of agents useful for the treatment of chronic pain conditions. Among compounds endowed with such antagonistic action, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP analogue, whose inhibitory activity on P2X receptors has been previously reported. Based on the results of molecular modelling studies performed with homology models of the P2X3 receptor, novel adenosine nucleotide analogues bearing cycloalkyl or arylalkyl substituents replacing the trinitrophenyl moiety of TNP-ATP were designed and synthesized. These new compounds were functionally evaluated on native P2X3 receptors from mouse trigeminal ganglion (TG) sensory neurons using patch clamp recordings under voltage clamp configuration. Our data show that some of these molecules are potent (nanomolar range) and reversible inhibitors of P2X3 receptors, without any apparent effect on trigeminal GABA_A and 5-HT₃ receptors, whose membrane currents were unaffected by the tested compounds.

Full text of DOI:10.1007/s11302-016-9539-y

Purinergic Signalling
DOI 10.1007/s11302-016-9539-y
ORIGINAL ARTICLE
2′,3′-O-Substituted ATP derivatives as potent antagonists
of purinergic P2X3 receptors and potential analgesic agents
Diego Dal Ben1 & Anna Marchenkova2 & Ajiroghene Thomas1 & Catia Lambertucci1
Andrea Spinaci1 & Gabriella Marucci1 & Andrea Nistri2 & Rosaria Volpini1
&
Received: 22 April 2016 /Accepted: 19 September 2016
#
Springer Science+Business Media Dordrecht 2016
Abstract Blocking membrane currents evoked by the activa-
tion of purinergic P2X3 receptors localized on nociceptive
neurons represents a promising strategy for the development
of agents useful for the treatment of chronic pain conditions.
Among compounds endowed with such antagonistic action,
2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP an-
alogue, whose inhibitory activity on P2X receptors has been
previously reported. Based on the results of molecular model-
ling studies performed with homology models of the P2X3
receptor, novel adenosine nucleotide analogues bearing
cycloalkyl or arylalkyl substituents replacing the
trinitrophenyl moiety of TNP-ATP were designed and synthe-
sized. These new compounds were functionally evaluated on
native P2X3 receptors from mouse trigeminal ganglion (TG)
sensory neurons using patch clamp recordings under voltage
clamp configuration. Our data show that some of these mole-
cules are potent (nanomolar range) and reversible inhibitors of
P2X3 receptors, without any apparent effect on trigeminal
GABAA and 5-HT3 receptors, whose membrane currents were
unaffected by the tested compounds.
Introduction
The nucleotide adenosine-5′-triphosphate (ATP, Fig. 1), which
represents the main energy storage molecule in cells, is re-
leased from healthy or damaged tissues in high concentrations
(mM range) and acts also as a neuromodulator interacting with
specific membrane receptors belonging to the family of so-
called purinergic P2 receptors. This receptor family comprises
two different classes of membrane proteins named P2Y and
P2X receptors, which are distinguished on the base of their
structure and function. To date, eight subtypes of the P2Y
receptors, belonging to the family of G protein coupled recep-
tors (GPCRs), and seven subtypes (P2X1-7) of the P2X ion
channels, permeable to Na+, K+, Ca++ ions and small mole-
cules, have been cloned from humans [1–5]. P2X receptors
assemble as homo- or heterotrimers [6, 7] and share a common
structure, presenting intracellular N- and C-termini, two trans-
membrane domains (TM1 and TM2) and a large glycosylated
and cysteine-rich extracellular domain [8–10]. When ATP
binds to P2X receptors, a large conformational change causes
the opening of the transmembrane pore allowing the cation
flow and leading to the depolarization of the cell and genera-
tion of action potentials [11, 12]. After this activation process,
the P2X1 and P2X3 receptor subtypes undergo rapid desensi-
tization, while the P2X2, P2X4 and P2X7 subtypes desensi-
tize slowly [13]. Since P2X receptor activity is involved in a
number of physiological processes (regulation of cell cycle,
muscle cell contraction, platelet aggregation, nociception and
inflammation), they are considered as therapeutic targets for
several conditions including cancer and inflammatory, neuro-
logical, cardiovascular and endocrine diseases [3, 5, 14–26].
The P2X3 receptors were cloned in 1995 [27, 28], and they
are almost exclusively localized to nociceptive sensory gan-
glion neurons and nodose ganglion neurons [29, 30]. Different
chronic pain diseases are linked to the upregulation of these
.
.
Keywords Purinergic receptors P2X3 receptor antagonists
.
.
.
ATP derivatives Purinederivatives Patch clamp Molecular
modelling
Electronic supplementary material The online version of this article
(doi:10.1007/s11302-016-9539-y) contains supplementary material,
which is available to authorized users.
* Rosaria Volpini
rosaria.volpini@unicam.it
1
School of Pharmacy, Medicinal Chemistry Unit, University of
Camerino, via S. Agostino 1, 62032 Camerino, (MC), Italy
2
Neuroscience Department, International School for Advanced
Studies (SISSA), Via Bonomea 265, 34136 Trieste, Italy

Purinergic Signalling
Fig. 1 Ligands of the P2X3
receptor
receptors (i.e. neuropathic and inflammatory pain, headache
pain and migraine) [22, 31–39]. As a consequence, targeting
these receptors represents an innovative therapeutic strategy to
treat both neuropathic and inflammatory pain conditions [40,
41]. Recent papers described the rapid transition of the P2X3
receptors to a desensitization state followed by a relatively
slow recovery of sensitivity to ATP [13, 42]. Hence, long-
acting and low efficacy P2X3 agonists could be useful to
block nociceptive transmission via receptor inactivation.
Examples of such molecules are the so-called Bacyclic
nucleotides,^ which were obtained by modifying the sugar
moiety of ATP [43].
Another approach to obtain analgesic agents acting on
P2X3 receptors is by designing and synthesizing new antag-
onists. The first identified P2X3 antagonists were negatively
charged and/or high molecular weight arylpolysulfonate mol-
ecules [44], which showed to be unselective, as they were able
to antagonize also the metabotropic P2Y receptors [45].
Further studies led to the discovery of a potent and selective
P2X3 receptor antagonist, named A-317491 (Fig. 1), showing
nanomolar activity at both homomeric P2X3 and heteromeric
P2X2/3 receptors [46]. The most potent competitive antago-
nist of P2X3 receptors is the 2′,3′-O-(2,4,6-trinitrophenyl)-
ATP (TNP-ATP, Fig. 1) [47], with low nanomolar activity.
In fact, this molecule showed to inhibit ATP or α,β-meATP
evoked current at P2X3 receptors expressed by HEK293 cells
with an IC50 value of 2 nM [48]. This molecule was also able
to block (at 1 μM) by 91 % currents evoked by 100 μM α,β-
meATP at rat TG neurons [49] and to block currents evoked
by 30 μM α,β-meATP at mouse nodose neurons with an IC50
value of 11 nM [50]. Several P2X ligands were also discov-
ered through high-throughput screening (HTS) studies which
led to the discovery of a series of diaminopyrimidine deriva-
tives, like RO-3 and AF-353 (Fig. 1), acting as allosteric P2X3
antagonists [51–53]. Substitution of the diaminopyrimidine
scaffold with a diaminopurine moiety led to compounds that
maintained the antagonistic behaviour [54].
The screening efforts represented for several years the only
strategy to develop P2X ligands due to the lack of experimen-
tal structural data of P2X receptors. In 2009 and 2012, the
crystal structures of the zebrafish P2X4 receptor (zP2X4) in
the presence and absence of ATP were reported, providing
structural details of these membrane proteins [55, 56], useful
for the analysis of the ATP binding mode and the interpreta-
tion of mutagenesis data [12]. Furthermore, a comparative
analysis of the open (active state) and closed (inactive state)
receptor conformations provides a detailed view of the mech-
anism of P2X receptor activation.
We recently reported a molecular modelling analysis aimed
at developing 3D models of the human and rat P2X receptors
in both active and inactive state [57]. This study permitted the
analysis of the binding mode of the agonist ATP and the an-
tagonist TNP-ATP at the P2X3 receptor. The mechanism of
TNP-ATP antagonism, proposed on the basis of its ability to
prevent rearrangement of P2X subunits [56], was confirmed
by P2X3 modelling studies [57]. The comparison of the bind-
ing mode of ATP and TNP-ATP led to an interpretation of the
role of the 2′,3′-O-substituent in the antagonist molecule.

Purinergic Signalling
Starting from these results, we designed ATP analogues bear-
ing different substituents in the 2′,3′-O-position as new P2X3
antagonists. In the present study, we report the design, synthe-
sis and biological evaluation of three ATP derivatives present-
ing in the 2′,3′-O-position a cyclohexylidene or a benzylidene
moiety. The design was made with the aid of molecular
modelling using homology models of the human and mouse
P2X3 receptors that share 93.7 % identical residues and have
almost identical chemico-physical characteristics considering
the respective ATP binding cavities. In the design phase, we
decided to maintain the triphosphate chain of ATP even
though it is known to have low chemical and metabolic sta-
bility [58]. Our choice was made in order to make the new
molecules as similar as possible to the reference compound
TNP-ATP and hence to evaluate the impact of the different
2′,3′-substituent on the compound potency. Furthermore, the
corresponding monophosphate analogues of these compounds
were also synthesized, since it was observed that the mono- or
di-phosphate analogues of TNP-ATP were able to inhibit
P2X3 receptors as well [48]. The compounds were evaluated
for their biological activity on native P2X3 receptors of mouse
TG sensory neurons, using patch clamp technique to record
membrane currents. This method enabled a quantitative inves-
tigation of P2X3 agonist/antagonist interaction through the
use of dose-response curves in the presence of the reference
non-hydrolysable P2X3 agonist α,β-methyleneATP (α,β-
meATP; P2X3 EC50 = 1.8 μM [59]). This approach also
allowed us to study changes in receptor desensitization and
to test if the new compounds were able to modify membrane
currents evoked by activation of other membrane channels
like 5-HT3 or GABAA receptors that are expressed by TG
neurons [60–63].
Results
Molecular modelling design
We already reported the TNP-ATP docking conformation at
the inactive state human P2X3 binding site ([57] and Fig. 2).
In summary, the compound presents a similar binding mode to
the one of ATP, observable from X-ray data (zP2X4) and from
ATP-bound homology models of human [57] and mouse
(Supporting Material) P2X3 receptors developed using the
zP2X4 X-ray structure in its active state (PDB code: 4DW1;
2.8-Å resolution [56]) as a template. The phosphate groups of
the ligand are involved in a series of polar interactions with the
binding cavity residues, most of them being positively
charged residues (i.e. Lys63, Lys65, Ser275, Asn279,
Arg281 and Lys299; Fig. 2). The adenine moiety is inserted
between the side chains of Lys65, Phe174 and Ile215 and
provides H-bonding with Thr172 backbone and side chain
atoms. The 2′- and 3′-hydroxy groups of ATP are missing as
the two oxygen atoms are bound to the trinitrophenyl function,
which is inserted between two segments of adjacent P2X3
monomers and interacts with the residues located in its
Fig. 2 a, b Docking
conformation (schematic
description in panel b) of TNP-
ATP at the human P2X3 binding
site. Main residues for the ligand-
receptor interaction are indicated.
c, d Design of new ligands;
docking conformation of the
compound 4 (schematic
description in panel d)

Purinergic Signalling
proximity. At the same time, this group prevents the possible
approach of the two monomers observed during the activation
process of the receptor [56]. To verify that the role of the
trinitrophenyl group is mainly related to its steric hindrance
and not to other features given by its chemical structure (i.e.
interaction given by the nitro groups), our design process was
based on the substitution of the trinitrophenyl function with
other groups presenting comparable volume even if with dif-
ferent chemical properties. Hence, we designed ATP deriva-
tives bearing in the 2′,3′-O-position bulky groups like a
cyclohexylidene or a benzylidene function (2′,3′-O-
cyclohexylidene ATP and 2′,3′-O-benzylidene ATP, com-
pounds 4, 9 and 10, respectively, see Schemes 1 and 2). The
designed compounds were docked into the binding site of
homology models of the human and mouse P2X3, built using
the X-ray structure of the zP2X4 receptor in its apo (inactive)
state (PDB code: 4DW0; 2.9-Å resolution [56]) as a template.
Figure 2 reports a comparison of the simulated binding mode
of the TNP-ATP and compound 4 at the human P2X3 binding
site. In the Supporting Material, the binding mode of TNP-
ATP and the new triphosphate derivatives at the mouse P2X3
plus the docking conformations of compounds 9 and 10 at the
human and mouse receptors are reported. Docking results
present compound 4 inserted into the binding cavity in a very
similar way in respect to TNP-ATP, with the phosphate groups
positioned close to the cluster of positively charged residues in
the depth of the site. The adenine moiety is similarly posi-
tioned in respect to TNP-ATP (even if slightly reoriented
when compared to the same group of the latter compound),
while the cyclohexyl group is located within the interface of
the two subunits in a similar way of the trinitrophenyl function
of TNP-ATP. These data suggest that the new compound
could provide a similar steric hindrance of the reference mol-
ecule and hence could behave as obstacle for the receptor
activation mechanism in a similar way to TNP-ATP.
Analogous results were observed for the triphosphate deriva-
tives 9 and 10 (Supporting Material). The corresponding
monophosphate analogues (compounds 3, 7 and 8, respective-
ly) were synthesized as well, since it was observed that the
mono- or di-phosphate derivatives of TNP-ATP were able to
inhibit P2X3 receptors. In literature, we found that the com-
pound 3 was already claimed in a patent describing nucleoside
derivatives as P2X7 antagonists [64], although the synthesis
and the biological activity of this compound were not report-
ed. We also found that an analogue of the non-hydrolysable
P2X ligand β,γ-meATP, bearing a 2′,3′-O-benzylidene sub-
stituent and hence similar to the compounds 9 and 10 present-
ed in this work, was tested for its ability to induce contraction
at rabbit ear central artery showing a micromolar activity at
this tissue preparation [65].
Synthesis
New nucleotides 3 and 4 were prepared starting from the
suitable nucleoside 2. Reaction of commercially available
adenosine (1) with freshly distilled cyclohexanone in the pres-
ence of p-toluenesulfonic acid and 4 Å molecular sieves
(Scheme 1) yielded nucleoside 2 by using a modification of
a previously reported procedure [64] in which this compound
was not characterized. The purification of the reaction product
by silica gel chromatography and crystallization from
ethylacetate provided the intermediate compound 2 with
33 % yield.
Scheme 1 Reagents and
conditions: a, cyclohexanone, p-
toluenesulfonic acid, 4 Å
molecular sieves, 70 °C, 48 h; b, i.
phosphorus oxychloride, dry
trimethyl phosphate, 0 °C, 4 h; ii.
1 M TEAB, 0 °C to r. t., 15 min; c,
i. phosphorus oxychloride, dry
trimethyl phosphate, 0 °C, 4 h; ii.
bis-(tri-n-butylammonium)
pyrophosphate solution in dry
DMF, 0 °C, 10 min; iii. 1 M
TEAB, 0 °C to r. t., 15 min

Purinergic Signalling
[66]. Hence, these two epimers, compounds 5 and 6, were
separated by silica gel chromatography and characterized by
NOESY-NMR experiments; in fact, irradiation of the ketal CH
(H-1″ proton; Fig. 3) and measurements of its effects on the
ribose protons allowed to unequivocally attribute the molecule
configuration. By means of a 1D-NOESYexperiment (Fig. 3),
the irradiation of the signal at 6.02 ppm of compound 6, cor-
responding to the ketal CH (1″ position), induced a signal
increase of the peaks at 5.50, 5.08 and 7.56 corresponding to
the H-2′and H-3′ protons of the sugar moiety and the hydro-
gens in the orto-position of the phenyl ring, respectively.
Thus, we were able to ascribe the (R) configuration to the ketal
position of the dioxolane ring of compound 6, as the observed
interactions between the ketal proton and the hydrogens at the
2′ and 3′ positions of the sugar moiety was possible only when
the H-1″, H-2′ and H-3′ protons point toward the same side
with respect to the ribose plane. In the case of compound 5, a
NOESY-2D NMR experiment was performed due to the close
vicinity of the signals at 6.24 and 6.28 ppm, corresponding to
the H-1″ and H-1′, respectively, which did not permit a selec-
tive irradiation of the ketal CH proton. In the obtained spec-
trum, a clear interaction between the protons at 6.24 (H-1″),
4.28 (H-4′) and at 7.50 (2H-Ph) ppm was observed.
The signal at 4.28 corresponds to the ketal CH (1″-posi-
tion) of the dioxolane ring, while the other signals belong to
the H-4′ of the sugar moiety and to the protons in the orto-
position of the phenyl ring. In this case, we ascribed the (S)
configuration to the ketal chiral centre of compound 5. In fact,
only when the H-1″ and the H-4′ protons are on the same side,
it is possible to have proton magnetic field interaction.
Scheme 2 Reagents and conditions: a, benzaldehyde, p-toluenesulfonic
acid, 4 Å molecular sieves, 70 °C, 4 h. b, i. phosphorus oxychloride, dry
trimethyl phosphate, 0 °C, 4 h; ii. 1 M TEAB, 0 °C to r. t., 15 min; c, i.
phosphorus oxychloride, dry trimethyl phosphate, 0 °C, 4 h; ii. bis-(tri-n-
butylammonium) pyrophosphate solution in dry DMF, 0 °C, 10 min; iii.
1 M TEAB, 0 °C to r. t., 15 min
On the other hand, the reaction of adenosine with benzal-
dehyde, using the same reaction conditions described before,
supplied the suitable adenosine diastereomer derivatives 5 and
6 (Scheme 2). Although the synthesis of the 2′,3′-O-
benzylidene adenosine was previously reported, the reaction
was performed in different conditions with respect to this
work and the single epimers obtained were not characterized
The synthesis of desired nucleotides (compounds 3, 7 and
8) was performed by reaction of the suitable intermediates 2, 5
and 6 with phosphorus oxychloride in trimethyl phosphate at
0 °C and subsequent quenching with a cold triethylammonium
bicarbonate buffer solution. Compounds 3, 7 and 8 were ob-
tained as ammonium salts after ion exchange chromatography
on a Sephadex DEAE resin eluted with a solvent gradient of 0
Fig. 3 NOESY-1D NMR
characterization for the
configuration assignment of
epimers 6 and 5

Purinergic Signalling
to 0.2 M ammonium bicarbonate buffer (Schemes 1 and 2).
Previously, the diastereoisomeric mixture of 7 and 8 was ob-
tained by reaction of adenosine monophosphate with the benz-
aldehyde dimethylacetal, without a full characterization of the
separated diastereomers [67].
For the synthesis of the triphosphate derivatives 4, 9 and
10, after a first step reaction of the nucleosides 2, 5 or 6 with
phosphorus oxychloride in trimethylphosphate at 0 °C for 4 h,
a bis(tri-n-butylammonium) pyrophosphate solution in dry
DMF was added to the reaction mixtures. The reactions were
then quenched by adding slowly a cold triethylammonium
bicarbonate buffer solution and purified with ion exchange
chromatography on a Sephadex DEAE resin eluting with a
solvent gradient of 0 to 0.4 M ammonium bicarbonate buffer
(Schemes 1 and 2). All nucleotides were characterized by 1H-
NMR and 31P-NMR spectra, as usually performed.
Biological assays
Fig. 4 Effect of the application of compound 10 on currents evoked by
α,β-meATP at native mouse P2X3 receptors expressed by TG sensory
neurons. The current evoked by 10 μM α,β-meATP was taken as control
peak current and corresponds at a near-maximal response [37, 42]. After
5-min application of 10 (1 and 0.1 μM concentrations via fast
superperfusion), subsequent application of 10 μM α,β-meATP induced
smaller peak currents or no response at all. After 5-min washout, the
current amplitude was almost completely restored. Figure 4 shows
representative traces of the fast desensitizing inward currents evoked by
10 μM α,β-meATP and the effect of the synthesized compound 10
applied at 1 and 0.1 μM concentrations
Newly synthesized compounds 3, 4 and 7–10 were tested for
their biological activity at native mouse P2X3 receptors
expressed by TG sensory neurons in culture by using the patch
clamp recording technique as previously reported [37, 42, 43].
As a first approach, all compounds were applied for 5 min at
10 μM; none of them could elicit functional responses even at
concentration of 10 μM, showing lack of agonist properties.
However, as expected, they exhibited antagonistic activity on
P2X3 receptors, since their application inhibited subsequent
current responses evoked by 2 s pulses of 10 μM α,β-meATP
(Fig. 4) [37, 42, 43]. Such inhibition was reversible, as the
peak current amplitude fully or partially recovered after 5 min
wash out. Hence, the most potent compounds were further
evaluated also at lower concentrations up to 0.01 μM.
Table 1 shows the calculated percent inhibition of the
mouse P2X3-mediated currents elicited by tested compounds.
At the concentration of 10 μM, the monophosphate deriva-
tives 3, 7 and 8 showed no or low inhibition of P2X3 currents
with a percent change of −0.9 13 %, 27 9 % and 44 8 %,
respectively. These compounds were, therefore, not tested at
lower concentrations. The corresponding triphosphate deriva-
tives 4, 9 and 10 showed 100 % inhibition of the P2X3 cur-
rents at 10 μM concentration. Compounds 4 and 10 were able
to produce an almost complete inhibition (99 %) even at
1 μM. At sub-micromolar level, compounds 4 and 10 elicited
a dose-dependent inhibition (74 and 71 % at 0.5 μM, respec-
tively), while compound 9 was less potent (34 % inhibition at
the same concentration). Furthermore, compounds 4 and 10
behaved as moderate inhibitors also at nanomolar levels (30
and 43 % at 50 nM and 21 and 25 % at 10 nM concentrations,
respectively). Hence, these compounds show high potency
(although they are not efficacious as TNP-ATP, whose low
nanomolar activity is described within the Introduction).
Table 1 Summary of P2X3 antagonism of novel ATP derivatives
Compound
Concentration, μM
Inhibition of P2X3 current, %
3
7
8
4
10
10
10
−0.9 13
27
44
9
8
0.01
0.05
0.1
1
21
30
74
99
100
9
3
5
1
10
10
9
0.01
0.05
0.1
1
25 12
43 13
71
99
4
1
10
100
0.01
0.05
0.1
1
7
18
11
7
34 13
86
9
10
100
Effects of the novel compounds were investigated on P2X3 currents
evoked by 10 μM α,β-meATP; inhibition is expressed as % amplitude
of control α,β-meATP-evoked current; data for each compound concen-
tration are from 5 to 10 individual cells

Purinergic Signalling
Fig. 5 Dose-inhibition curves of P2X3-mediated currents for
compounds 4, 9 and 10 built by applying different concentrations of
antagonists using the same concentration of agonist (10 μM α,β-
meATP). Data points from n = 5–10 cells, fitted with Hill equation
(Eq. 2), asterisk indicates p < 0.05, two-sample t test
Dose response curves were built for the most potent com-
pounds (4, 9 and 10) as shown in Fig. 5, in which the log
compound concentration was plotted against the percent inhi-
bition of the α,β-meATP (10 μM) evoked current. Fitting the
data with the Hill equation (Eq. 2; see experimental details)
yielded for compound 4 a value of IC50 = 83 nM, for com-
pound 10 a value of IC50 = 81 nM, and for compound 9 an
IC50 = 211 nM (n = 5–10 cells). The difference of activity
between the two isomers 9 and 10 is not particularly high
(about 2.5-fold). Docking results show that the two com-
pounds could share the binding mode to the receptor cavity
and could give similar interaction with the protein residues.
On the other hand, the arrangement of the R-isomer 10 appears
more similar to the one of the reference compound TNP-ATP
with respect to the S-isomer 9 (see Supporting Material
Figure SI6). This data could explain the slightly higher poten-
cy of compound 10 compared to 9. It is noteworthy that, as
shown in Fig. 5, all three compounds produced full block of
the agonist responses: these observations together with the
absence of their direct stimulatory effect on P2X3 receptors
(as reported earlier) confirm these novel compounds to be full
antagonists.
To assess the mechanism of antagonism, we tested the abil-
ity of the two most potent compounds 4 and 10 to inhibit
P2X3 currents evoked by different concentrations of the ref-
erence agonist α,β-meATP. The potent non-competitive
P2X3 antagonist AF-353 [68] was also tested for comparison.
These antagonists were tested at concentrations close to their
IC50 values (0.05 μM for compounds 4 and 10; 0.01 μM for
AF-353) and applied for 5 min. The agonist α,β-meATP was
applied for 2 s at 1, 3, 10, 30 and 100 μM concentrations. The
dose-response curves are shown in Fig. 6. The extent of cur-
rent inhibition evoked by AF-353 did not depend on the α,β-
meATP concentration: this phenomenon is often referred to as
non-competitive antagonism. Conversely, the inhibitory
Fig. 6 Concentration-response curves for α,β-meATP-evoked P2X3
currents in control and with pre-application of compounds 4, 10 and
AF-353 (0.05 μM for 4 and 10; 0.01 μM for AF-353). The agonist was
applied at 1, 3, 10, 30 and 100 μM concentrations. Data for each point are
obtained from 5 to 11 cells, asterisk indicates p < 0.05, two-sample t test
potency of compounds 4 and 10 appeared to decrease with
increased agonist concentrations, suggesting a partially sur-
mountable antagonism exerted by these two molecules when
the agonist dose was raised (Fig. 6a). Figure 6b shows log
agonist concentration/response plots in control solution or in
the presence of each one of the three blockers. Since the
curves were clearly shifted rightwards and downwards by
compounds 4 or 10, it appears that neither drug could induce
competitive receptor antagonism that would have required
preservation of the agonist maximum response and parallel
shift of the dose response curve to the right. Thus, while the
inhibition evoked by AF-353 could be minimally reversed by
the P2X3 agonist, the antagonism by compounds 4 or 10 was
partially surmounted by the higher agonist concentrations,
suggesting a complex mode of pharmacological block in
which the non-competitive antagonism process was predom-
inant, yet not exclusive [69]. Nonetheless, in the presence of a
competitive antagonist, receptors are prone to fast desensiti-
zation (that is strongly dependent on agonist concentration)
and therefore might unexpectedly exhibit an apparent non-
competitive antagonism when high agonist doses are used

Purinergic Signalling
and/or the antagonist facilitates the development of desensiti-
zation. In such cases, the agonist maximum effect would be
decreased. We, therefore, explored if compounds 4 and 10
inhibited responses evoked by high agonist concentrations
might be explained by facilitated receptor desensitization. If
desensitization develops very rapidly, the response peak
would be curtailed, thus summating desensitization-induced
depression to any molecular antagonism by the novel com-
pounds. Thus, we analysed P2X3 currents in control and after
application of compound 10 with a two-exponential function
[59] that allowed calculating the desensitization time constant
τ1 for both cases. Because τ1 expresses the rate of current
decay, it actually quantifies the speed at which the activated
receptor enters into the desensitization state [59]. Since the
desensitization constant τ1 depends on the current amplitude
that reflects the number of active receptors [59], to obtain
suitable comparisons, the currents to be analysed should be
approximately of the same size. Thus, we used lower agonist
concentrations (5–10 μM) to elicit control currents and higher
α,β-meATP concentration (30 μM) after application of com-
pound 10 (0.03 μM), so that the evoked responses have com-
parable amplitudes. Figure 7 shows representative examples
of superimposed current traces in control and after compound
10 (0.03 μM), recorded from the same cell.
τdes1 = 145 13 ms, τdes2 = 1.44 0.28 s, n = 7; GABA:
τdes = 1.96 0.39 s, n = 10) indicating that they were mediated
by ionotropic receptors (5-HT3 and GABAA receptors, respec-
tively). Pre-application of 10 μM 4, 9 or 10 did not signifi-
cantly affected GABAA and 5-HT3 currents, indicating that
these compounds are selective for the P2X versus GABA
and 5-HT receptors at TG neurons (Figure 8).
Conclusion
The principal finding of the present investigation is the dem-
onstration of the highly potent and selective antagonism by
newly synthesized compounds on P2X3 receptors of trigem-
inal sensory neurons. Since these receptors are strongly
expressed by the vast majority of nociceptors in the trigeminal
ganglion without concomitant expression of other P2X recep-
tors [70, 71], this test system offers an advantageous opportu-
nity to explore new antagonists and their potential value in
dealing with trigeminal pain, an issue to be further studied
with in vivo preclinical experiments. Thus, this work repre-
sents a starting point for a structure-based design of P2X3
antagonists as a strategy for the development of novel
As seen from the bar graph of Fig. 7, mean τ1 values were
not significantly different between the experimental condi-
tions (p = 0.41681, paired t test), implicating that compound
10 did not affect the rate of agonist-induced desensitization.
P2X3 expressing sensory neurons such as DRG and TG are
also known to express ionotropic 5-HT3 and GABAA recep-
tors [60–63]. We used this property to evaluate the receptor
selectivity of the newly synthesized antagonists. As expected,
the majority of TG neurons responded to GABA (93 %) and
5-HT (63 %) applied at 10 μM concentration. All these re-
sponses had fast rise-time (5-HT: τon = 65 9 ms, n = 7;
GABA: τon = 107 9 ms, n = 10) and decay (5-HT:
Fig. 8 Effect of compounds 4, 9 and 10 on GABAA and 5-HT receptors.
a Diagram on the left summarizes mean normalized peak amplitude
values for currents evoked by 10 μM GABA (2 s pulses) in control and
after 5-min application of compounds 4, 10 or 9 (10 μM); n = 4, 6 and 5
for compounds 4, 10 and 9, respectively. Representative traces on the
right show GABA-mediated currents in control and after treatment with
compound 10, recorded from the same cell. b Diagram on the left shows
mean normalized peak amplitudes for currents evoked by 10 μM 5-HT
(2 s pulses) in control and after 5-min application of compounds 4, 10 or 9
(10 μM); n = 4. Examples of 5-HT-mediated current traces (recorded
from a single cell) in control and after compound 10 application are
presented on the right. p > 0.05 for each compound/receptor type,
paired t test
Fig. 7 Desensitization of P2X3 currents after application of compound
10. Superimposed current traces from the same cell in control and after
compound 10 (0.03 μM, 3 min) demonstrate no significant change in
current decay time. Control current is induced by 10 μM α,β-meATP,
current response after 10 treatment is evoked by 30 μM α,β-meATP.
Histograms on the right show values of desensitization time constant τ1
(mean S.E.); n cells = 7, p = 0.41681, paired t test

Purinergic Signalling
potential analgesic drugs. The rationale was the hypothesis
that the introduction of a bulky substituent in the 2′,3′-O-po-
sition of ATP, originally occupied by a 2,4,6-trinitrophenyl
moiety in TNP-ATP, may confer antagonist activity of the
obtained ATP derivatives at the P2X3 receptors. Hence, on
the basis of molecular modelling studies, we designed and
synthesized different mono- and triphosphate adenine nucle-
otides as TNP-ATP analogues in which the 2,4,6-
trinitrophenyl function was replaced with cycloalkyl or
arylalkyl groups. Patch-clamp recording experiments, per-
formed on mouse P2X3 receptors expressed by TG sensory
neurons, confirmed the original hypothesis demonstrating that
the new triphosphate derivatives behave as potent P2X3 re-
ceptor antagonists, with IC50 in the nanomolar to sub-
micromolar range. The antagonisms of these new nucleotides
were selective at P2X3 receptors versus GABAA and 5-HT3
receptors of the sensory TG neurons.
as a preliminary step. The alignment of the sequences of hu-
man and mouse P2X3 and zP2X4 can be found within the
Supporting Material. The boundaries identified from the used
X-ray crystal structure of zP2X4 receptor were applied for the
sequences of the human and mouse P2X3 receptors. The miss-
ing domains were built by the loop search method implement-
ed in MOE. Once the heavy atoms were modelled, all hydro-
gen atoms were added and the protein coordinates were then
minimized with MOE using the AMBER99 force field [74]
until the root mean square (RMS) gradient of the potential
energy was less than 0.05 kJ mol−1 Å−1. Reliability and qual-
ity of the model were checked using the Protein Geometry
Monitor application within MOE, which provides a variety
of stereochemical measurements for inspection of the struc-
tural quality in a given protein, like backbone bond lengths,
angles and dihedrals, Ramachandran φ-ψ dihedral plots, and
sidechain rotamer and non-bonded contact quality.
The future developments of the present work should deal
with two key points. First, it can be expected that, as triphos-
phate derivatives, the synthesized compounds 4, 9 and 10
might present low water and metabolic stability in physiolog-
ical condition, i.e. when analysed in vivo. Hence, further op-
timization of these compounds should improve the chemical
stability of the triphosphate chain. Second, the selectivity of
the synthesized derivatives versus the other P2X receptor sub-
types (not expressed by trigeminal neurons) will have to be
determined, to verify if systemic administration of these com-
pounds as analgesics might not be associated with pharmaco-
logical effects due to the activity on other members of the P2X
receptor family expressed in distinct body tissues.
Molecular docking analysis
The compound structures were docked into the binding site of
the P2X3 receptor models using the MOE Dock tool. This
method is divided into a number of stages. Conformational
Analysis of ligands: The algorithm generated conformations
from a single 3D conformation by conducting a systematic
search. In this way, all combinations of angles were created
for each ligand. Placement: A collection of poses was gener-
ated from the pool of ligand conformations using Triangle
Matcher placement method. Poses were generated by super-
position of ligand atom triplets and triplet points in the recep-
tor binding site. The receptor site points are alpha sphere cen-
tres which represent locations of tight packing. At each itera-
tion, a random conformation was selected, a random triplet of
ligand atoms and a random triplet of alpha sphere centres were
used to determine the pose. Scoring: Poses generated by the
placement methodology were scored using two available
methods implemented in MOE, the London dG scoring func-
tion which estimates the free energy of binding of the ligand
from a given pose, and Affinity dG scoring which estimates the
enthalpy contribution to the free energy of binding. The top 30
poses for each ligand were outputted to a MOE database.
Experimental section
Molecular modelling
All molecular modelling studies were performed on a Core i7
CPU (PIV 2.20 GHZ) PC workstation. Homology modelling,
energy minimization and docking studies were carried out
using Molecular Operating Environment (MOE, version
2012.10) suite [72]. All ligand structures were optimized
using RHF/AM1 semiempirical calculations and the software
package MOPAC [73] implemented in MOE was utilized for
these calculations.
Post docking analysis
The docking poses of each compound were then subjected to
AMBER99 force field energy minimization until the RMS
gradient of the potential energy was less than
0.05 kJ mol−1 Å−1. Receptor residues within 6 Å distance from
the ligand were left free to move, while the remaining receptor
coordinates were kept fixed. AMBER99 partial charges of
receptor and MOPAC output partial charges of ligands were
utilized. Once the compound-binding site energy minimiza-
tion was completed, receptor coordinates were fixed and a
Homology modelling of the human and mouse P2X3 receptor
Homology models of the human and mouse P2X3 receptor
were built using the X-ray structure of the zP2X4 receptor in
its apo (inactive) and ATP-bound (active) states (PDB code:
4DW0; 2.9-Å resolution and PDB code: 4DW1; 2.9-Å reso-
lution as templates [56], respectively). A multiple alignment
of the P2X receptor primary sequences was built within MOE

Purinergic Signalling
second energy minimization stage was performed, leaving
only compound atoms free to move. MMFF94 force field
[75–81] was applied. For each compound, the minimized
docking poses were then rescored using London dG and
Affinity dG scoring functions and the dock-pKi predictor.
The latter tool allows estimating the pKi for each ligand using
the Bscoring.svl^ script retrievable at the SVL exchange ser-
vice (Chemical Computing Group, Inc. SVL exchange:
http://svl.chemcomp.com). The algorithm is based on an
empirical scoring function consisting of a directional
hydrogen-bonding term, a directional hydrophobic interaction
term and an entropic term (ligand rotatable bonds immobilized
in binding). The obtained pKi values must be considered as
docking scores and not as a prediction of binding affinity. For
each compound, the three top-score docking poses according
to at least two out of three scoring functions were selected for
final ligand-target interaction analysis.
J = 2.8 Hz, J = 6.0 Hz, 1H, H-2′), 6.11 (d, J = 3.2 Hz, 1H,
H-1′), 7.36 (s, 2H, NH2), 8.14 (s, 1H, H-8), 8.33 ppm (s, 1H,
H-2). ESI-MS: positive mode m/z 348.0 [M + H]+, 370.0
[M + Na]+, 717.0 [2 M + Na]+; negative mode m/z 346.2
[M-H]−, 382.1 [M + Cl]−. Anal. Calcd. for C16H21N5O4: C,
55.32; H, 6.09; N, 20.16; found: C, 55.39; H, 6.12; N, 20.11.
2′,3′-O-(S)-Benzylideneadenosine (5) and 2′,3′-O-(R)-
benzylideneadenosine (6) Adenosine (1, 3 g, 11.23 mmol)
and p-toluenesulfonic acid (2.564 g, 13.48 mmol) were stirred
at 70 °C in freshly distilled benzaldehyde (53 mL) in the
presence of 4 Å molecular sieves. The reaction was stirred
for 4 h, cooled to room temperature, neutralized with
triethylamine and the molecular sieves were filtered. One hun-
dred millilitre of water was added and benzaldehyde was ex-
tracted with n-hexane (70 mL). The product was then extract-
ed from the water phase with ethylacetate (100 mL × 4), dried
over anhydrous Na2SO4 and evaporated to dryness. The resi-
due was purified by silica gel normal column chromatography
eluting with 4 % MeOH in CH2Cl2 to give 5 (672 mg,
1.89 mmol), 17 % yield, and 6 (1.484 g, 4.18 mmol), 37 %
yield, as a white powders after recrystallization from
ethylacetate. The configuration of the epimers was assigned
based on NOESY-NMR experiment data.
Chemistry
Melting points were determined with a Büchi apparatus and
1
were uncorrected. H-NMR and 31P-NMR spectra were ob-
tained with Varian Mercury 400 MHz spectrometer; δ in ppm,
J in Hz; all exchangeable protons were confirmed by addition
of D2O. Mass spectra were recorded on an HP 1100-MSD
series instrument. All measurements were performed using
electrospray ionization (ESI-MS) on a single quadrupole
analyser. TLC were carried out on pre-coated TLC plates with
silica gel 60 F-254 (Merck). For column chromatography,
silica gel 60 (Merck) was used. For ion exchange chromatog-
raphy, Sephadex DEAE A-25 resin, HCO3− form, was used.
Elemental analyses were determined on a Fisons model EA
1108 CHNS-O model analyser and are within 0.4 % of the-
oretical values.
1
5: m. p.: 217–219 °C; H-NMR (DMSO-d6, 400 MHz) δ
3.60 (m, 2H, H-5′), 4.28 (m, 1H, H-4′), 5.08 (m, 1H, H-3′),
5.17 (m, 1H, OH), 5.48 (m, 1H, H-2′), 6.24 (s, 1H, CH), 6.28
(d, J = 3.2 Hz, 1H, H-1′), 7.36 (s, 2H, NH2), 7.43 (m, 3H, Ph),
7.50 (m, 2H, Ph), 8.15 (br s, 1H, H-8), 8.37 ppm (s, 1H, H-2).
ESI-MS: positive mode m/z 355.9, 377.9, 732.9; negative
mode m/z 354.1, 390.0. Anal. Calcd. for C17H17N5O4: C,
57.46; H, 4.82; N, 19.71; found: C, 57.51; H, 4.89; N, 19.67.
6: m. p.: 229–231 °C ; 1H-NMR (DMSO-d6, 400 MHz) δ
3.54 (m, 2H, H-5′), 4.36 (m, 1H, H-4′), 5.08 (dd, J = 2.4 Hz,
J = 2.4 Hz, 1H, H-3′), 5.27 (t, J = 5.2 Hz, 1H, OH), 5.50 (dd,
J = 2.4 Hz, J = 2.4 Hz, 1H, H-2′), 6.02 (s, 1H, CH), 6.28 (d,
J = 2.8 Hz, 1H, H-1′), 7.37 (br s, 2H, NH2), 7.46 (m, 3H, Ph),
7.56 (m, 2H, Ph), 8.15 (s, 1H, H-8), 8.37 ppm (s, 1H, H-2).
ESI-MS: positive mode m/z 356.0, 377.9, 732.9; negative
mode m/z 354.1. Anal. Calcd. for C17H17N5O4: C, 57.46; H,
4.82; N, 19.71; found: C, 57.49; H, 4.85; N, 19.64.
2′,3′-O-Cyclohexylideneadenosine (2) Adenosine (1, 3 g,
11.23 mmol) and p-toluenesulfonic acid (2.564 g,
13.48 mmol) were stirred at 70 °C in freshly distilled cyclo-
hexanone (30 mL) in the presence of 4 Å molecular sieves.
The reaction was stirred for 48 h, cooled to room temperature,
neutralized with triethylamine, and the molecular sieves were
filtered. Water (100 mL) was added and cyclohexanone was
extracted with n-hexane (70 mL). The product was then ex-
tracted from the water phase with ethylacetate (100 mL × 4),
dried over anhydrous Na2SO4 and evaporated to dryness. The
residue was purified by silica gel flash column chromatogra-
phy eluting with a gradient of 0 to 1.5 % MeOH in CH2Cl2 to
give 2 (1.29 g, 3.70 mmol) with 33 % yield as a white powder
after recrystallization from ethylacetate. M. p.: 173–175 °C;
1H-NMR (DMSO-d6, 400 MHz) δ 1.35 (m, 2H, c-Hex), 1.47
(m, 2H, c-Hex), 1.57 (m, 4H, c-Hex), 1.76 (m, 2H, c-Hex),
3.52 (m, 2H, H-5′), 4.20 (m, 1H, H-4′), 4.95 (dd, J = 2.0 Hz,
J = 6.0 Hz, 1H, H-3′), 5.23 (t, J = 5.2 Hz, 1H, OH), 5.34 (dd,
General procedure for the synthesis of monophosphates 3, 7
and 8
POCl3 (52 μL, 0.56 mmol) was added dropwise in turn to a
solution of 2, 5 or 6 (50 mg, 0.14 mmol) in dry
trimethylphosphate (0.7 mL) placed in an ice bath. The reac-
tion was left to stir for 4 h, quenched by slowly adding 3 mL of
a cold 1 M TEAB solution in an ice bath and then stirred for
15 min at r. t. The reaction mixtures were evaporated to dry-
ness and co-evaporated with H2O (3 × 5 mL). The obtained
−
crude was purified over a Sephadex DEAE A-25 gel (HCO3
form) eluting with a solvent gradient of 0 to 0.2 M NH4HCO3

Purinergic Signalling
buffer. The appropriate fractions were collected, concentrated
under vacuum and co-evaporated several times with H2O to
yield pure monophosphates 3, 7 and 8 as white solids.
eluting with a solvent gradient of 0 to 0.4 M NH4HCO3 buffer.
The appropriate fractions were collected, concentrated under
vacuum, co-evaporated several times with H2O to yield pure
triphosphate derivatives 4, 9 and 10 as white powders.
Adenosine-2′,3′-cyclohexylidene-5′-monophosphate ketal
ammonium salt (3) The title compound was obtained from
2 with 93 % yield; 1H-NMR (D2O, 400 MHz) δ 1.26 (m, 2H,
c-Hex), 1.38 (m, 2H, c-Hex), 1.52 (m, 4H, c-Hex), 1.73 (m,
2H, c-Hex), 3.87 (m, 2H, H-5′), 4.47 (m, 1H, H-4′), 5.00 (dd,
J = 2.0 Hz, J = 2.0 Hz, 1H, H-3′), 5.23 (dd, J = 3.6 Hz,
J = 3.6 Hz, 1H, H-2′), 6.09 (d, J = 3.6 Hz, 1H, H-1′), 8.05
(s, 1H, H-8), 8.24 ppm (s, 1H, H-2). 31P-NMR (D2O,
162 MHz) δ 1.69 ppm. ESI-MS: negative mode m/z 426.0,
853.2 Anal. Calcd. for C16H25N6O7P: C, 43.25; H, 5.67; N,
18.91; found: C, 43.31; H, 5.77; N, 18.88.
Adenosine-2′,3′-cyclohexylidene-5′-triphosphate ketal
ammonium salt (4) The title compound was obtained from
2 with 25 % yield; 1H-NMR (D2O, 400 MHz) δ 1.29 (m, 2H,
c-Hex), 1.40 (m, 2H, c-Hex), 1.56 (m, 4H, c-Hex), 1.77 (m,
2H, c-Hex), 4.05 (m, 2H, H-5′), 4.55 (m, 1H, H-4′), 5.09 (m,
1H, H-3′), 5.27 (dd, J = 3.2 Hz, J = 3.6 Hz, 1H, H-2′), 6.13 (d,
J = 3.6 Hz, 1H, H-1′), 8.11 (s, 1H, H-8), 8.32 ppm (s, 1H,
H-2). 31P-NMR (D2O, 162 MHz) δ − 9.63, −10.65,
−22.06 ppm. ESI-MS: negative mode m/z 292.5, 586.1.
Anal. Calcd. for C16H36N9O13P3: C, 29.32; H, 5.54; N,
19.23; found: C, 29.38; H, 5.61; N, 19.18.
2′,3′-O-(S)-Benzylideneadenosine-5′-monophosphate am-
monium salt (7) The title compound was obtained from 5
with 63 % yield; 1H-NMR (D2O, 400 MHz) δ 3.94 (m, 2H,
H-5′), 4.46 (m, 1H, H-4′), 5.08 (dd, J = 3.6 Hz, J = 4.0 Hz, 1H,
H-3′), 5.33 (dd, J = 2.8 Hz, J = 3.2 Hz, 1H, H-2′), 6.08 (s, 1H,
CH), 6.17 (d, J = 3.6 Hz, 1H, H-1′), 7.29 (m, 3H, Ph), 7.36 (m,
2H, Ph), 8.01 (s, 1H, H-8), 8.18 ppm (s, 1H, H-2). 31P-NMR
(D2O, 162 MHz) δ 1.25 ppm. ESI-MS: negative mode m/z
433.9, 869.0. Anal. Calcd. for C17H21N6O7P: C, 45.14; H,
4.68; N, 18.58; found: C, 45.23; H, 4.72; N, 18.55.
2′,3′-O-(S)-Benzylideneadenosine-5′-triphosphate ammo-
nium salt (9) The title compound was obtained from 5 with
29 % yield; 1H-NMR (D2O, 400 MHz) δ 4.14 (m, 2H, H-5′),
4.65 (m, 1H, H-4′), 5.22 (m, 1H, H-3′), 5.42 (dd, J = 3.6 Hz,
J = 3.6 Hz, 1H, H-2′), 6.21 (s, 1H, CH), 6.29 (d, J = 4.0 Hz,
1H, H-1′), 7.37 (m, 3H, Ph), 7.47 (m, 2H, Ph), 8.12 (s, 1H,
H-8), 8.31 ppm (s, 1H, H-2). 31P-NMR (D2O, 162 MHz)
δ − 9.22, −10.53, −22.04 ppm. ESI-MS: negative mode m/z
296.5, 594.0. Anal. Calcd. for C17H32N9O13P3: C, 30.78; H,
4.86; N, 19.00; found: C, 30.84; H, 4.95; N, 18.92.
2′,3′-O-(R)-Benzylideneadenosine-5′-monophosphate am-
monium salt (8) The title compound was obtained from 6
with 23 % yield; 1H-NMR (D2O, 400 MHz) δ 3.94 (m, 2H,
H-5′), 4.63 (m, 1H, H-4′), 5.12 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H,
H-3′), 5.42 (dd, J = 2.8 Hz, J = 2.8 Hz, 1H, H-2′), 5.98 (s, 1H,
CH), 6.31 (d, J = 2.4 Hz, 1H, H-1′), 7.38 (m, 3H, Ph), 7.52 (m,
2H, Ph), 8.12 (s, 1H, H-8), 8.32 ppm (s, 1H, H-2). 31P-NMR
(D2O, 162 MHz) δ 0.98 ppm. ESI-MS: negative mode m/z
434.1. Anal. Calcd. for C17H21N6O7P: C, 45.14; H, 4.68; N,
18.58; found: C, 45.20; H, 4.75; N, 18.45.
2′,3′-O-(R)-Benzylideneadenosine-5′-triphosphate ammo-
nium salt (10) The title compound was obtained from 6 with
39 % yield:. 1H-NMR (D2O, 400 MHz) δ 4.04 (m, 1H, HCH-
5′), 4.10 (m, 1H, HCH − 5′), 4.65 (m, 1H, H-4′), 5.20 (m, 1H,
H-3′), 5.40 (dd, J = 3.2 Hz, J = 3.2 Hz, 1H, H-2′), 6.00 (s, 1H,
CH), 6.28 (d, J = 3.2 Hz, 1H, H-1′), 7.38 (m, 3H, Ph), 7.53 (m,
2H, Ph), 8.06 (s, 1H, H-8), 8.33 ppm (s, 1H, H-2). 31P-NMR
(D2O, 162 MHz) δ − 9.14, −10.66, −21.92 ppm. ESI-MS:
negative mode m/z 296.6, 593.9. Anal. Calcd. for
C17H32N9O13P3: C, 30.78; H, 4.86; N, 19.00; found: C,
30.84; H, 4.91; N, 18.96.
General procedure for the synthesis of triphosphates 4, 9
and 10
Biological assays
POCl3 (108 μL, 1.16 mmol) was added dropwise to a solution
of 2, 5, or 6 (100 mg, 0.29 mmol) in dry trimethylphosphate
(1.5 mL) placed in an ice bath. The reaction was left to stir for
4 h, after which 5.8 mL (2.90 mmol) of bis-(tri-n-
butylammonium) pyrophosphate solution in dry DMF was
added and stirred for 10 min. The reaction was quenched by
slowly adding 5 mL of a cold 1 M TEAB solution in an ice
bath and then stirred for 15 min at r. t. The mixture was ex-
tracted with tert-butylmethyl ether (3 × 15 mL), and the aque-
ous solution was evaporated and co-evaporated with H2O
(3 × 10 mL) to yield glassy oils. The obtained crude was
Trigeminal ganglia culture neurons
Cultures of TG sensory neurons were prepared from C57Bl/6
wild type mice (P10–14) as previously described [70]. In
brief, TG were rapidly excised and enzymatically dissociated
in F12 Medium (Invitrogen Corp, Milan, Italy) containing
0.25 mg/mL trypsin, 1 mg/mL collagenase, 0.2 mL DNAse
(Sigma, Milan, Italy) at 37 °C for 12 min. Cells were plated on
poly-L-lysine coated 35 mm Petri dishes on F12 medium sup-
plemented with 10 % foetal bovine serum and antibiotics and
incubated for 24 h (5 % CO2/95 % humidity, 37 °C).
−
purified over a Sephadex DEAE A-25 gel (HCO3 form)

Purinergic Signalling
Patch clamp recordings
For the most potent compounds, antagonist dose-inhibition
curves were constructed by applying for 5 min different con-
centrations of each test compound using the same maximal
concentration (10 μM) of α,β-meATP agonist. Data were
plotted and fitted with empirical Hill equation using Origin
6.0 (Microcal, Northampton, MA, USA):
Currents were recorded from small/medium size mouse TG
neurons (nociceptors that strongly express native P2X3 recep-
tors [70]) as previously described [70, 82] in whole cell volt-
age clamp configuration, at a holding potential of −65 mV
after correction for liquid junction potential. Cells were con-
tinuously superfused at room temperature with control solu-
tion containing (in mM): 152 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2,
10 glucose, 10 HEPES; pH 7.4 adjusted with NaOH. Patch
pipettes had a resistance of 3–4 MΩ when filled with follow-
ing solution (in mM): 140 KCl, 2 MgCl2, 0.5 CaCl2, 2 ATP-
Mg, 2 GTP-Li, 10 HEPES, 10 EGTA; pH 7.2 adjusted with
KOH (for recordings from TG neurons). After obtaining
whole cell configuration, cell slow capacitance was compen-
sated. Access resistance was never >10 MΩ and was routinely
compensated by at least 70 %. One kilohertz filtering was used
during recording; currents were acquired by means of a
DigiData 1200 interface and pClamp8.2 software (Molecular
Devices, Sunnyvale, CA, USA).
ꢀ
ꢁ
nH
%inhibition ¼ 100= 1 þ ðIC50=½AntŠÞ
;
ð2Þ
where [Ant] is the concentration of the antagonist, nH is the
Hill coefficient, IC50 is the concentration of antagonist re-
quired to block the maximal current by 50 %. Agonist
concentration-response curves in terms of normalized currents
versus log agonist concentration were fitted with the same Hill
equation, with corresponding parameters EC50 and nH, where
EC50 is the concentration of agonist required to produce the
half-maximal current. All data are presented as mean stan-
dard error of the mean (S.E.); n is the number of cells.
Statistical significance was evaluated with paired Student’s t
test (for parametric data) or Mann–Whitney rank-sum test (for
nonparametric data), as suggested by the software; p < 0.05
was considered statistically significant.
Drug application and data analysis
The receptor agonist α,β-meATP (Sigma) and test com-
pounds were applied by rapid solution changer system
(Rapid Solution Changer RSC-200; BioLogic Science
Instruments, Claix, France). Membrane currents were
analysed in terms of their peak amplitude and rise time
(10–90 % of the peak). Current decay due to receptor
desensitization during agonist application was fitted with
either a monoexponential or biexponential function using
pCLAMP Clampfit 9.2. Test compounds were kept at
+4 °C and dissolved to 10 mM in H2O before experiments
and finally diluted to necessary concentration in control
solution. For routine tests, the P2X3 selective agonist
α,β-meATP was applied at 10 μM concentration (2 s
pulse) at least 3 times (at 5 min interval to prevent cumu-
lative receptor desensitization) to obtain an average con-
trol response. Since activated P2X3 receptors desensitize
rapidly in the sustained presence of agonist, the inward
current transient is, of course, too short for the binding
equilibrium to be reached when agonist and antagonist are
co-applied. Thus, to assess potential receptor blocking ac-
tivity, each test compound was continuously pre-applied
for 5 min before the α,β-meATP application as previously
reported [42, 43]. Antagonist activity was quantified as
percent inhibition of the α,β-meATP-induced current:
Acknowledgment This work was supported by Fondo di Ricerca di
Ateneo (FPI000033 - University of Camerino) and the EU FP7 grant
EuroHeadPain (#602633).
Compliance with Ethical Standards As the corresponding authors of
the manuscript entitled “2′,3′-O-Substituted ATP derivatives as potent
antagonists of purinergic P2X3 receptors and potential analgesic agents”
I declare that:
All the authors have not any conflict of interest
Research involving Animals
All animal experiments were done in full accordance with the
European Union guidelines for animal use and the experimental protocols
were approved by the ethics committee of International School for
Advanced Studies (SISSA).
Conflict of Interest The authors declare that they have no conflict of
interest.
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%inhibition ¼ 100*ð1– I2=I1Þ;
ð1Þ
where I1 is the control peak current, I2 is the peak amplitude of
the current after test compound.

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