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

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

GABA_Aand 5-HT₃receptors, 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 IC₅₀value 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-HT₃or GABA_Areceptors 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

−0.9 13

27

44

9

8

0.01

0.05

0.1

1

21

30

74

99

100

9

3

5

1

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

IC₅₀values (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

τ₁for both cases. Because τ₁expresses 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 τ₁depends 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.

τ_des1= 145 13 ms, τ_des2= 1.44 0.28 s, n = 7; GABA:

τ_des= 1.96 0.39 s, n = 10) indicating that they were mediated

by ionotropic receptors (5-HT₃and GABA_Areceptors, respec-

tively). Pre-application of 10 μM 4, 9 or 10 did not signifi-

cantly affected GABA_Aand 5-HT₃currents, 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 τ₁values 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-HT₃and GABA_Arecep-

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: τ_on= 65 9 ms, n = 7;

GABA: τ_on= 107 9 ms, n = 10) and decay (5-HT:

Fig. 8 Effect of compounds 4, 9 and 10 on GABA_Aand 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 IC₅₀in the nanomolar to sub-

micromolar range. The antagonisms of these new nucleotides

were selective at P2X3 receptors versus GABA_Aand 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⁻¹Å⁻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⁻¹Å⁻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-pK_ipredictor.

The latter tool allows estimating the pK_ifor 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 pK_ivalues 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 Na₂SO₄and evaporated to dryness. The resi-

due was purified by silica gel normal column chromatography

eluting with 4 % MeOH in CH₂Cl₂to 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 Na₂SO₄and 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 CH₂Cl₂to

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 NH₄HCO₃buffer.

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 (HCO₃form)

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=½AntÞ

;

ð2Þ

where [Ant] is the concentration of the antagonist, n_His the

Hill coefficient, IC₅₀is 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 EC₅₀and n_H, where

EC₅₀is 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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ð1Þ

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Article Doi

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

DOI: 10.1007/s11302-016-9539-y

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