Adenine-based acyclic nucleotides as novel P2X3 receptor ligands

Article abstract of DOI:10.1021/jm900131v

A new series of acyclic nucleotides based on the adenine skeleton and bearing in 9-position a phosphorylated four carbon chain has been synthesized. Various substituents were introduced in 2-position of the adenine core. The new compounds were evaluated o

Full text of DOI:10.1021/jm900131v

4596 J. Med. Chem. 2009, 52, 4596–4603
DOI: 10.1021/jm900131v
Adenine-Based Acyclic Nucleotides as Novel P2X3 Receptor Ligands
Rosaria Volpini,† Ram Chandra Mishra,† Dhuldeo D. Kachare,† Diego Dal Ben,† Catia Lambertucci,† Ippolito Antonini,†
Sauro Vittori,† Gabriella Marucci,† Elena Sokolova,‡ Andrea Nistri,‡ and Gloria Cristalli*,†
†Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy, and ‡Sector of Neurobiology and Italian
Institute of Technology Unit, International School for Advanced Studies (SISSA), Via Beirut 2/4, 34014 Trieste, Italy
Received February 3, 2009
A new series of acyclic nucleotides based on the adenine skeleton and bearing in 9-position a
phosphorylated four carbon chain has been synthesized. Various substituents were introduced in
2-position of the adenine core. The new compounds were evaluated on rat P2X3 receptors, using patch
clamp recording from HEK transfected cells and the full P2X3 agonist R,β-meATP as reference
compound. The results suggest that certain acyclic nucleotides, in particular compounds 28 and 29,
are endowed with modest partial agonism on P2X3 receptors. This is an interesting property that can
depress the function of P2X3 receptors, whose activation is believed to be involved in a number of
chronic pain conditions including neuropathic pain and migraine. In fact, the new acyclic nucleotides
are able to persistently block (by desensitization) P2X3 receptor activity after a brief, modest activation,
yet leaving the ability of sensory neurons to mediate responses to standard painful stimuli via a lower
level of signaling.
Introduction
In particular, they synthesized xanthine-triphosphate deriva-
tives, bearing an alkyl spacer instead of the ribose moiety, and
termed them “mini-nucleotides”.18 These compounds proved
to be useful selective agents for activation of P2X receptors.
On the other hand, 9-ethyladenine was used as the prototype
agent for a series of nonxanthine adenosine receptor antago-
nists. Previous studies from our group showed that many
derivatives of this scaffold are endowed with high binding
affinity toward P1 receptors and behave as antagonists.19-21
The first reported adenosine receptor antagonists are deriva-
tives of natural xanthines, caffeine or theophylline, whose
substitution at different positions led to highly potent and
selective P1 receptor antagonists.22
Starting from these observations, our search for new P2X3
receptor ligands led us to design and synthesize a series of
“acyclic nucleotides” based on the adenine skeleton and
bearing at the 9-position a functionalized four carbon chain,
which could mimic the ribose moiety of natural ligands and it
is suitable to be phosphorylated. Hence, various substituents
with different electronic and steric properties were introduced
in 2-position of adenine such as chlorine, iodine, amino,
phenylethoxy group, and hexynyl chain (general formula,
Figure 1).
P2X3 receptors, which belong to a large family of purinergic
receptors, are integral membrane proteins expressed by sen-
sory ganglion neurons to sense painful stimuli.1-3 Extensive
activation of such receptors is believed to be involved in a
numberofchronic pain conditions including neuropathicpain
and migraine.2 Because these states are notoriously resistant
to treatment, new molecules targeted to P2X3 receptors could
be useful to achieve better pain control.
P2X3 receptors are ligand-gated channels activated by
extracellular ATP to induce a rapid increase in membrane
permeability to mono- and divalent cations.4-7 One impor-
tant property of P2X3 receptors is the rapid transition to a
state of desensitization, which curtails their persistent signal-
ing in physiological conditions.8-10 P2X3 receptors regain
their ATP sensitivity relatively slowly via a multistep process
regulated by inflammatory substances.11-14 P2X3 receptor
desensitization is surprisingly agonist-specific,5 opening up
the possibility of blocking receptor activity with long-acting
agonists, ideally of low efficacy, to avoid pain stimulation.
Attaining this goal is made further feasible by the recent
observation that low concentrations of agonist can inactivate
a fraction of the receptors even in the absence of prior
activation.9,15 These data highlight the importance of identi-
fying ATP analogues that can shape P2X3 receptor function
by either desensitization or pharmacological antagonism.
Adenosine-based agonists and xanthine-based antagonists
bind adenosine purinergic A1 receptors with similar electro-
static properties.16,17 This observation led Fischer and co-
workers to design a set of modified xanthines (theobromine
and theophylline) in order to identify new purinergic ligands.
In most cases the mono-, di-, and triphosphate derivatives
were synthesized. The new compounds were evaluated for
their biological activity on rat P2X3 receptors by using patch
clamp recording from transfected human embryonic kidney
(HEK) cells.
Results and Discussion
Chemistry. Adenine based acyclic-nucleotides 14-29 were
synthesized using commercially available adenine (1), 2,6-
dichloropurine(2), and2-amino-6-chloropurine (3) asstarting
materials (Scheme 1). The introduction of a protected four
*To whom correspondence should be addressed. Phone: þ39-0737-
402252. Fax: þ39-0737-402295. E-mail: gloria.cristalli@unicam.it.
r
pubs.acs.org/jmc
Published on Web 07/16/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4597
and 1-hexyne, gave the desired 2-hexynyl derivative 13
(Scheme 2).
The adenine derivatives 7-10, 12, and 13 were phosphory-
lated by reacting them with phosphorus oxychloride in
trimethyl phosphate at room temperature, according to
Yoshikawa procedure.25 After purification on a diethylami-
noethyl resin (Sephadex DEAE A-25, HCO3- form) eluting
with a gradient of water and ammonium bicarbonate, the
corresponding monophosphates 14-19 were obtained in
good yield ranging from 35 to 78%. The syntheses of the
di- and triphosphate derivatives were carried out by a
modification of the Hoard-Ott method.26 Hence, the tri-n-
butylammonium salts of the monophosphate derivatives
14-19 were converted to phosphorimidazolates with 1,10-
carbonyldiimidazole. The phosphorimidazolates obtained
from compounds 14-17 were coupled with tri-n-butylam-
monium phosphate to give the corresponding diphosphate
derivatives 20-23.
Figure 1. Structure of R,β-meATP and general structure of synthe-
sized adenine “acyclic nucleotides”.
The triphosphates 24-29 were prepared by reacting the
phosphorimidazolates obtained from 14-19 with bis(tri-n-
butylammonium)pyrophosphate (Scheme 3).
carbon chain was performed by reacting 1-3 with 4-bromo-
butylacetate in the presence of potassium carbonate and dry
DMFa. Both substituted N-9 (4-6) and N-7 (4a-6a) isomers
were obtained in different ratios, depending on the used
starting purine derivative, and ranging from about 4:1 ratio
in the reaction with 1 to give 4 and 4a, to 5:1 ratio in the
reaction with compounds 2 and 3 to give 5, 5a and 6, 6a,
respectively.
Biological Data. The newly synthesized adenine acyclic
nucleotides were evaluated for their biological activity on rat
P2X3 receptors by using patch clamp recording from HEK-
transfected cells. The full agonist R,β-meATP was selected as
a reference compound because of its stability and high
selectivity for P2X3 receptors. Because we studied recombi-
nant receptors on unexcitable epithelial cells, which do not
constitutively synthesize and express them, we avoided inter-
ference by constitutive receptors and ion channels and
enhanced the reliability of our results by measuring ionic
currents exclusively caused by activation of P2X3 receptors
via a very rapid delivery system of the tested chemical agents.
Examples of inward currents induced by 2 s pulse applica-
tion of R,β-meATP (100 μM) or certain acyclic nucleotides
(100 μM) are shown in Figure 2. The inward current ob-
served in the presence of R,β-meATP possessed all the
characteristics typical of P2X3 receptor mediated responses,
namely rapid onset, large amplitude, and fast decay in the
continuous presence of this agonist (desensitization). While
the monophosphates 16, 18, and 19 lacked significant ago-
nist activity, the di- and triphosphate derivatives 22 and 26,
bearing an amino group in 2-position of the adenine core,
evoked responses smaller than those elicited by R,β-meATP
and accompanied by minimal desensitization during the 2 s
pulse application. The 2-iodo triphosphate derivative 28 did
evoke a rapid current of lower amplitude than the one
observed with R,β-meATP but still associated with rapid
desensitization, while the triphosphate 29, bearing a hexynyl
chain in 2-position, induced a small response with character-
istics intermediate between those of the triphosphates 26 and
28. Hence, the presence of at least two phosphate groups
seemed to be essential for receptor activation, while the
nature of the substituent present in 2-position of the adenine
moiety appeared to mostly influence the grade of receptor
activation and desensitization.
In all cases, the two isomers were isolated and character-
1
ized by their H NMR spectra, which showed significant
differences, and 1D NOE experiments. In the 1H NMR
spectra, the N-9 substituted compound 4 showed upfield
signals for H-8 and CH2-N compared to the corresponding
N-7 isomer 4a. The same differences were observed both
comparing 5 with 5a, and 6 with 6a (Table 1). These data are
in agreement with previous observations on N-9 and N-7
alkyl purine derivatives.19,23 Furthermore, the alkylation site
on the adenine derivatives 4 and 4a was confirmed by 1D
NOE experiments. In fact, after irradiation of the CH2-N in
compound 4, NOEs were observed for the H-2 and H-8 and
the vicinal CH2 (Table 2). The same experiment performed
on compound 4a showed NOE only for the H-8 and the
vicinal CH2, while no effect was observed for H-2 signal, so
unequivocally confirming 4 and 4a as N-9 and N-7 isomers,
respectively.
Reaction of N-9 isomers 4-6 with methanolic ammonia,
at 120 °C for 16 h, gave deprotection of the hydroxyl alkyl
chain and, in compounds 5 and 6, simultaneous substitution
of the chlorine atom in 6-position with an amino group to
obtain the desired derivatives 7-9, respectively.
The 2-phenethoxy derivative 10 was synthesized by treat-
ing 8 with phenethyl alcohol, in the presence of sodium
hydroxide (Scheme 1), while the synthesis of the 2-iodo and
2-hexynyl derivatives 12 and 13 was performed using 6 as
starting material. Hence, 6 was reacted in the Sandmeyer
conditions to obtain 11, which was then treated with metha-
nolic ammonia to give 12. Subsequent reaction of 12, using
Sonogashira coupling conditions21,24 with bis-triphenylpho-
sphine palladium dichloride, copper iodide, triethylamine,
The average P2X3 agonist activity of the tested acyclic
nucleotides in relation to R,β-meATP (taken as 100%;
dotted line) is summarized in Figure 3 (A; open bars). Most
of the tested monophosphate derivatives 14-19 were vir-
tually devoid of any agonist action. Among compounds
endowed with significant agonist activity, the triphosphates
24-29 were in general more active than the diphosphate 20-
23 and all of them behaved as much weaker agonists with
a Abbreviations: CDI, 1,10-carbonyldiimidazole; DEAE, diethylami-
noethyl; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
R,β-meATP, R,β-methyleneATP; HEK, human embryonic kidney;
HEPES, 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic sodium salt;
EGTA, glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid; MEM,
minimum essential medium; NOE, nuclear Overhauser effect.

4598 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Volpini et al.
Scheme 1. Synthesis of 2-Substituted 9-Hydroxybutyladenines 7-10a
a Reagents and conditions: (a) DMF, Br(CH2)4OCOCH3, rt, 16 h; (b) NH3/CH3OH, 120 °C, 16 h; (c) C6H5CH2CH2OH, NaOH, 80 °C, 4 h.
Table 1. 1H Chemical Shifts (ppm) of H-8 and CH2-N Signals of
Compounds 4-6 and 4a-6a in DMSO-d6
amino group is tolerated but the bulky phenethoxy substitu-
ent could not be allocated.
compd
H-8
CH2-N
We next evaluated whether continuously applied acyclic
nucleotides could modify subsequent responses to R,β-
meATP. To this end, we preapplied these compounds for
20 s prior to the pulse of R,β-meATP, as shown in the
examples of Figure 4 with 18 and 26. The monophosphate
18 did not produce an inward current (see Figure 4, top),
although it did depress the subsequent response to R,β-
meATP (on average, the response became 80% of control;
see B histograms in Figure 3), showing that 18 was a weak
antagonist. Similar results were obtained with the monopho-
sphates 16, 17, and 19 (Figure 3).
4
8.13
8.34
8.78
8.86
8.13
8.37
4.14
4.32
4.26
4.46
4.05
4.29
4a
5
5a
6
6a
Table 2. One-Dimensional NOE Data after Irradiation of CH2-N
Signal of Compounds 4 and 4a
compd
H-8
H-2
CH2CH2-N
Compound 26 produced an initial small current (25% of
the R,β-meATP response, indicating partial agonism;
Figure 3A) that fully desensitized after 2 s pulse applica-
tion and strongly decreased the subsequent response to R,β-
meATP: on average, the residual P2X3 receptor currents
after applying compound 26 were 17% of control (Figure 3B).
This pattern of effects was clearly observed also with tripho-
sphate derivatives like 24 and 25 that generated a 36 and 30%
response of the R,β-meATP maximum effect, yet they could
inhibit potently receptor function (R,β-meATP responses had
become 14 and 10%, respectively, of control). On the other
hand, compounds 28 and 29, which elicited the higher currents,
completely inhibited the subsequent response to R,β-meATP.
All these effects were reversible on washout. Hence, most
of the tested acyclic nucleotides could persistently block (by
desensitization) receptor activity after a brief, modest activa-
tion. In practice, these compounds behaved like weak partial
agonists.27 Full pharmacological demonstration of partial
agonism would require the construction of complete dose
response curves for each novel compound, and for validation
of the same maximum amplitude attained with 100 μM R,β-
meATP, a very large amount of the same compounds would
have been required, taking into account that they are less
potent than R,β-meATP and should be added by continuous
application. Notwithstanding this limitation, the early, low
amplitude receptor activation followed by desensitization
(see Figure 2) suggests that compounds endowed with activ-
ity like the one produced by 28 and 29 acted as partial
agonists on P2X3 receptors. Thus, they were clearly not
4
4a
yes
yes
yes
no
yes
yes
respect to R,β-meATP on P2X3 receptors. The nature of the
2-substitution modulates the receptor activation; hence, the
2-unsubstituted acyclic nucleotides di- and triphosphate 20
and 24 showed responses that were 32 and 36% of the
maximal effect elicited by R,β-meATP.
It is worthwhile to note that the diphosphate 20, lacking
substitution at the 2-position, is the only diphosphate deri-
vative endowed with notable activity, comparable to that of
the triphosphates. Considering the triphosphate derivatives
24-29, it is evident that the substitution in the 2-position of
lipophilic substituents like a chlorine and an iodine atom
or a hexynyl group (compounds 25, 28, and 29) was well
tolerated. In particular, the 2-iodo triphosphate 28 and
the 2-hexynyl triphosphate 29 showed responses of 60 and
38% of the maximal effect elicited by R,β-meATP, resulting
the most active compounds of the series. However, the
presence of a lipophilic sterically hindered phenethoxy group
was not tolerated, leading to compound 27, endowed with
very low activity. Also the presence of a hydrophilic amino
group produced a decrease of receptor activation ability
(compare 26 with 24). From these data, it is possible to
hypothesize that the new acyclic nucleotides interacted with a
receptor binding region so that the C-2 portion of the purine
scaffold was found in proximity of a small P2X3 receptor
hydrophobic pocket (capable of binding halogens or the
partially flexible hexynyl chain), where the small hydrophilic

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4599
Scheme 2. Synthesis of 2-Iodo and 2-Hexynyl-9-hydroxybutyladenines 12 and 13a
a Reagents and conditions: (a) C5H11ONO, CH2I2, 85 °C, 1 h; (b) NH3/CH3OH, 120 °C, 16 h; (c) 1-hexyne, (Ph3P)2PdCl2, Et3N, CuI, DMF, 90 °C, 3 h.
Scheme 3. Synthesis of 2-Substituted 9-Hydroxybutyladenine Mono- (14-19), Di- (20-23), and Triphosphates (24-29)a
a Reagents and conditions: (a) POCl3, (CH3O)3PO, rt, 3 h; (b) CDI, tri-n-butylammonium phosphate, DMF, rt, 16 h; (c) CDI, bis(tri-n-
butylammonium) pyrophosphate, DMF, rt, 16 h.
function yet leaving the ability of sensory neurons to mediate
responses via a lower level of signaling. This concept has
been applied, for instance, to the use of β-receptor partial
agonists to inhibit overactivity of β-receptors in hyperten-
sion and cardiac arrhythmias without excessive depression of
blood pressure and heart beating.29 Indeed, the very recent
study of the molecular mechanisms of partial agonism pre-
dicts that this phenomenon has implications for the design of
partial agonists for therapeutic use.28
Our experimental data from this relatively ample series of
acyclic nucleotides did not disclose any potent antagonist
because among the compounds devoid of significant agonist
activity at 100 μM concentration (15-19, Figure 3A). the
maximum degree of inhibition of R,β-meATP induced
responses was about 20% (Figure 3B). Nevertheless, the
Figure 2. Effects of acyclic nucleotides on P2X3 receptors ex-
pressed by HEK cells. Traces are sample records of inward currents
(obtained with patch clamp recording in the whole cell config-
general low ability to induce currents showed by the new
acyclic nucleotides may help to propose some hypothesis on
the molecular groups necessary to bind the receptor and to
uration) elicited by 2 s pulse application of the indicated com-
activate it partially or fully. In fact, the lack of an intact
ribose moiety in these molecules could be responsible of their
inability to fully activate the P2X3 receptor, being the sugar
moiety interactions necessary for full agonist activity.30
Of equal interest is the question of the mechanisms of
P2X3 receptor inactivation, which our present study can help
to address. A recent scheme of P2X3 receptor function based
on experimental investigations and kinetic modeling15 has
proposed that for equivalent degrees of receptor activation
and subsequent desensitization, recovery depends on the
chemical nature of the agonist that dissociates from the
bound receptor at different rates. The present study suggests
that the extent of receptor desensitization was mostly influ-
enced by the structure of the substituent present in 2-position
of the new acyclic nucleotides; however, our investigation
shows that the level of phosphorylation of the compound
also seemed to play a role. In fact, the 2-amino diphosphate
22 displayed weak agonist activity with minimal desensitiza-
tion, yielding square-shape inward currents in contrast with
the sharp transients induced by the full agonists, while the
pounds. The response evoked by R,β-meATP is taken as a standard
reference. Note that only 28 induces an effect partly similar in
amplitude and kinetics to the response to R,β-meATP. While 18 and
19 are virtually inactive, 22 and 26 produce current responses
without decay or with slow decay, respectively, suggestive of mini-
mal desensitization. All compounds are applied by fast superfusion
at 100 μM concentration.
typical antagonists in view of their observed ability to induce
receptor activation directly measured in terms of inward
current (Figure 3).
Partial agonism at ionotropic receptors has been recently
identified as due to a rapid conformation change (“flipping”)
of the receptor that occurs once the agonist is bound while
the channel still shut.28 This property applied to P2X3
receptors would be interesting to modulate their function
without generating full suppression of receptor signaling;
indeed, one might argue that a very strong antagonism of
P2X3 receptors can produce undesirable in vivo effects such
as lack of response to standard painful stimuli. Conversely,
administration of a partial agonist might depress receptor

4600 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Volpini et al.
Figure 4. Example of protocol to test the blocking action of acyclic
nucleotides on P2X3 receptor responses induced by R,β-meATP.
Application of 18 (100 μM; 20 s) does not evoke inward current and
partly inhibits the amplitude (84%) of subsequent response to R,β-
meATP (100 μM). Application of 26 (100 μM; 20 s) elicits a transient
inward current and largely depresses subsequent response to R,β-
meATP (12% residual response). All records are from the same cell.
activating P2X3 receptors is envisioned to be large,1,5 this
system would preserve sufficient sensitivity to respond to
natural stimuli. Future work is required to validate these
suggestions with an in vivo model of pain.
Experimental Section
Chemistry. Melting points were determined with a Buchi
apparatus and are uncorrected. UV spectra were collected on
a Varian Cary-1E UV-visible spectrophotometer. 1H NMR
spectra were obtained with Varian Mercury 400 MHz spectro-
meter; δ in ppm, J in Hz. All exchangeable protons were
confirmed by addition of D2O. 31P NMR spectra were recorded
at room temperature by using a Varian Mercury 400 MHz
spectrometer. Mass spectra were recorded on an HP 1100-
MSD series instrument. All measurements were performed
using electrospray ionization (ESI-MS) on a single quadrupole
analyzer. TLC were carried out on precoated TLC plates with
silica gel 60 F-254 (Merck). For column chromatography, silica
gel 60 (Merck) was used. For ion exchange chromatography,
Figure 3. Summary of agonist or antagonism activity of acyclic
nucleotides on P2X3 receptors. (A) Open bars show average inward
currents elicited by acyclic nucleotides applied for 2 s (100 μM) and
expressed as % of the response induced by R,β-meATP (100 μM),
n=7, P<0.05. (B) Filled bars show average inward currents indu-
ced by R,β-meATP (100 μM; 2 s) after preapplication of acyclic
nucleotides (100 μM) for 20 s; data are % values of control R,β-
meATP effects, n=7, P<0.05 for all acyclic nucleotides, except
15. For structures see Scheme 3.
chemically related triphosphate 26 elicited responses of
similar amplitude and characterized by more desensitization
(see Figure 2). Assuming that analogous current response
peaks indicated equivalent degree of receptor activation by
different compounds, it is likely that P2X3 receptors acti-
vated by 22 desensitized only very slowly because the dis-
sociation of such an agonist and, consequently, receptor
return to the resting state were intrinsically retarded due to
the chemical properties of this compound. Further studies
with 22 might, therefore, help to measure the time course of
receptor inactivation without the complication of interven-
ing desensitization, and to improve existing receptor models.
-
Sephadex DEAE A-25, HCO3 form was used. Elemental
analyses were determined on a Fisons model EA 1108 CHNS-O
model analyzer and are within (0.4% of theoretical values.
General Procedure for the Preparation of Compounds 4-6 and
4a-6a. To a solution of the suitable purine 1-3 (10 mmol) in dry
DMF (13.0 mL), K2CO3 (1.66 g, 12.0 mmol) was added. After
stirring for 5 min, 4-bromo-butylacetate (1.57 mL, 11.0 mmol)
was added and the reaction was left at rt for 16 h. Reactions
completion were checked by TLC eluted with CHCl3-CH3OH
(9:1): the N-9 isomers showed Rf values ranging from 0.2 to 0.6,
while N-7 isomers showed 0.1-0.5 Rf values, respectively. The
mixture was concentrated under vacuum and chromatographed
over flash silica gel column using the appropriate eluent to
obtain compounds 4-6 and 4a-6a as white solids.
9-Acetoxybutyladenine (4) and 7-Acetoxybutyladenine (4a).
Compounds 4 and 4a were obtained from 1 after chromatography
eluting with CHCl3-MeOH (98:2 to 95:5, v/v) as white solids, res-
pectively. 4: yield 54%, mp 176-178 °C. UV (EtOH) λmax: 205 nm
(ε 23335 dm3 mol-1 cm-1), 259 nm (ε 9404 dm3 mol-1 cm-1). 1H
NMR (DMSO-d6) δ 8.13 (s, 1H, H-8), 8.11 (s, 1H, H-2), 7.19 (brs,
2H, NH2), 4.14 (t, 2H, J = 7.0 Hz, NCH2), 3.98 (t, 2H, J = 6.6 Hz,
OCH2), 1.95 (s, 3H, CH3), 1.84 (m, 2H, NCH2CH2), 1.50 (m, 2H,
Conclusion
The present data suggest that certain acyclic nucleotides, in
particular compounds 28 and 29, obtained with de novo
synthesis and endowed with modest partial agonism on
P2X3 receptors are potentially useful drugs to regulate the
sensitivity of such receptors to painful stimuli. This goal may
be achieved by partially desensitizing the receptors so that
they wouldsignalless effectivelybut wouldstillretain acertain
degree of responsiveness to large concentrations of agonists.
Because the transient concentration of extracellular ATP

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4601
OCH2CH2). 4a: yield 15%, mp 135-137 °C. UV (EtOH) λmax
211 nm (ε13884 dm3 mol-1 cm-1),273nm(ε9175 dm3 mol-1 cm-1).
1H NMR (DMSO-d6) δ 8.34 (s, 1H, H-8), 7.87 (brs, 2H, NH2), 7.74
(s, 1H, H-2), 4.32 (t, 2H, J=7.0Hz,NCH2), 3.99 (t, 2H, J=6.4Hz,
OCH2), 1.95 (s, 3H, CH3), 1.91 (m, 2H, NCH2CH2), 1.54 (m, 2H,
OCH2CH2). Anal. (C11H15N5O2) C, H, N.
9-Acetoxybutyl-2,6-dichloropurine (5) and 7-Acetoxybutyl-
2,6-dichloropurine (5a). Compounds5 and 5a were obtained from
2 after chromatography eluting with CHCl3-MeOH (99:1, v/v)
:
9-Acetoxybutyl-6-chloro-2-iodopurine (11). To a solution of 6
(0.20 g, 0.71 mmol) indry DMF (3.0 mL), isoamylnitrite (0.30 mL)
and diiodomethane (0.94 mL) were added and the mixture was
heated at 85 °C for 1 h. After volatiles evaporation, the residue was
chromatographed on a flash silica gel column eluting with
CHCl3-MeOH (99:1, v/v) to get 11 as white solid. Yield 37%;
mp 127-129 °C. 1H NMR (DMSO-d6) δ 8.60 (s, 1H, H-8), 4.24 (t,
2H, J = 7.0 Hz, NCH2), 4.00 (t, 2H, J = 6.6 Hz, OCH2), 1.98 (s,
3H, CH3), 1.85 (m, 2H, NCH2CH2), 1.54 (m, 2H, OCH2CH2).
Anal. (C11H12ClIN4O2) C, H, N.
9-Hydroxybutyl-2-iodoadenine (12). Reaction of 11 gave 12 as
yellowish crystals. Yield 85%; mp 184-186 °C. 1H NMR
(DMSO-d6) δ 8.06 (s, 1H, H-8), 7.61 (brs, 2H, NH2), 4.43 (t,
1H, J = 5.2 Hz, OH), 4.06 (t, 2H, J = 7.2 Hz, NCH2), 3.37 (m,
2H, OCH2), 1.77 (m, 2H, NCH2CH2), 1.34 (m, 2H, OCH2CH2).
Anal. (C9H12IN5O) C, H, N.
2-Hex-1-ynyl-9-hydroxybutyladenine (13). To a solution of 12
(0.70 g, 2.10 mmol) in dry DMF (8.0 mL), bistriphenylpho-
sphine-palladium(II)chloride (31 mg, 0.043 mmol), copper io-
dide (30 mg, 0.16 mmol), triethylamine, and 1-hexyne (1.44 mL,
12.53 mmol (7.0 mL, 55.60 mmol) were added and the mixture
was heated at 90 °C for 3 h. The reaction mixture was filtered,
and after evaporation under vacuum of the filtrate, the residue
was chromatographed on a flash silica gel eluting with CHCl3-
MeOH (98:2 to 91:9, v/v) to give 13 as yellowish powder. Yield
78%; mp 150-152 °C. 1H NMR (DMSO-d6) δ 8.16 (s, 1H, H-8),
7.30 (brs, 2H, NH2), 4.44 (t, 1H, J = 4.2 Hz, OH), 4.09 (t, 2H,
J = 6.8 Hz, NCH2), 3.37 (m, 2H, OCH2), 2.38 (t, 2H, J = 6.8 Hz,
CH2CꢀC), 1.78 (m, 2H, NCH2CH2), 1.50 (m, 2H, OCH2CH2),
1.38 (m, 4H, CH2CH2CH3), 0.90 (t, 3H, J = 7.4 Hz, CH3). Anal.
(C15H21N5O) C, H, N.
as white solids. 5: yield 44%, mp 107-108 °C. UV (EtOH) λmax
:
213 nm (ε 21259 dm3 mol-1 cm-1), 273 nm (ε 9151 dm3 mol-1
cm-1). 1H NMR (DMSO-d6) δ 8.78 (s, 1H, H-8), 4.26 (t, 2H, J =
7.0 Hz, NCH2), 3.99 (t, 2H, J =6.6Hz, OCH2), 1.97 (s,3H, CH3),
1.87 (m, 2H, NCH2CH2), 1.55 (m, 2H, OCH2CH2). 5a: yield 8%,
mp 101-102 °C. UV (EtOH) λmax: 212 nm (ε 27450 dm3 mol-1
cm-1), 277 nm (ε 7194 dm3 mol-1 cm-1). 1H NMR (DMSO-d6): δ
8.86 (s, 1H, H-8), 4.46 (t, 2H, J = 7.0 Hz, NCH2), 3.99 (t, 2H, J =
6.6 Hz, OCH2), 1.97 (s, 3H, CH3), 1.84 (m, 2H, NCH2CH2), 1.58
(m, 2H, OCH2CH2). Anal. (C11H12Cl2N4O2) C, H, N.
9-Acetoxybutyl-2-chloroadenine (6) and 7-Acetoxybutyl-2-
chloroadenine (6a). Compounds 6 and 6a were obtained from
3 after chromatography eluting with CHCl3-MeOH (99:1, v/v)
as white solids. 6: yield 76%, mp 113-114 °C. UV (EtOH) λmax
:
202 nm (ε 3186 dm3 mol-1 cm-1), 222 nm (ε 15007 dm3 mol-1
cm-1), 246 nm (ε 3350 dm3 mol-1 cm-1), 309 nm (ε 3652 dm3
1
mol-1 cm-1). H NMR (DMSO-d6) δ 8.13 (s, 1H, H-8), 6.91
(brs, 2H, NH2), 4.05 (t, 2H, J = 7.0 Hz, NCH2), 3.97 (t, 2H, J =
6.6 Hz, OCH2), 1.96 (s, 3H, CH3), 1.80 (m, 2H, NCH2CH2), 1.51
(m, 2H, OCH2CH2). 6a: yield 15%, mp 163 °C dec. (EtOH) λmax
:
221 nm (ε 39800 dm3 mol-1 cm-1), 321 nm (ε 9154 dm3 mol-1
cm-1). 1H NMR (DMSO-d6): δ 8.37 (s, 1H, H-8), 6.62 (brs, 2H,
NH2), 4.29 (t, 2H, J = 7.0 Hz, NCH2), 3.98 (t, 2H, J = 6.6 Hz,
OCH2), 1.96 (s, 3H, CH3), 1.81 (m, 2H, NCH2CH2), 1.53 (m,
2H, OCH2CH2). Anal. (C11H14ClN5O2) C, H, N.
General Procedure for the Synthesis of Monophosphates 14-
19. Compounds 7-10, 12, or 13 (1.44 mmol) were in turn
dissolved in trimethyl phosphate (3.0 mL), and then 4 equiv of
POCl3 (539 μL, 5.78 mmol) were added at 0 °C. The solution was
stirred at rt for 3 h, and then H2O (3.0 mL) was added to the
reaction maintained at 0 °C and the solution neutralized by
adding triethylamine. The reactions were monitored by TLC,
using precoated TLC plates with silica gel 60 F-254 (Merck) and
i-C3H7OH-H2O-NH4OH (5.5:1.0:3.5) as mobile phase;
monophosphate derivatives showed 0.4-0.5 Rf values. The
nucleotides were purified by means of ionic exchange chroma-
General Procedure for the Preparation of Compounds 7-9 and
12. Compounds 4-6 or 11 (1.0 g, 4.01 mmol) were in turn added
in a steel vial containing saturated methanolic ammonia
(15 mL). The reaction was kept at 120 °C for 16 h unless
otherwise stated. After volatiles removing, compounds 7-9 or
12 were obtained by crystallization from methanol.
9-Hydroxybutyladenine (7). Reaction of 4 gave 7 white solid.
Yield 96%; mp 200-202 °C. 1H NMR (DMSO-d6) δ 8.12 (s, 1H,
H-8/H-2), 8.11 (s, 1H, H-8/H-2), 7.19 (brs, 2H, NH2), 4.44 (t,
1H, J = 5.2 Hz, OH), 4.12 (t, 2H, J = 7.2 Hz, NCH2), 3.38 (m,
2H, OCH2), 1.80 (m, 2H, NCH2CH2), 1.34 (m, 2H, OCH2CH2).
Anal. (C9H13N5O) C, H, N.
2-Chloro-9-hydroxybutyladenine (8). Reaction of 5, at 80 °C
for 16 h, gave 8 as white solid. Yield 90%; mp 188-190 °C. 1H
NMR (DMSO-d6) δ 8.15 (s, 1H, H-8), 7.73 (s, 2H, NH2) 4.44 (t,
1H, J = 7.0 Hz, OH), 4.09 (t, 2H, J = 7.0 Hz, NCH2), 3.38 (m,
2H, OCH2), 1.80 (m, 2H, NCH2CH2), 1.36 (m, 2H, OCH2CH2).
Anal. (C9H12ClN5O) C, H, N.
2-Amino-9-hydroxybutyladenine (9). Reaction of 6 gave 9 as
white crystals. Yield 89%; mp 191-193 °C. 1H NMR (DMSO-
d6) δ 7.67 (s, 1H, H-8), 6.61 (brs, 2H, NH2), 5.75 (brs, 2H, NH2),
4.44 (brs, 1H, OH), 3.92 (t, 2H, J = 7.0 Hz, NCH2), 3.37 (t, 2H,
J = 6.0 Hz, OCH2), 1.73 (m, 2H, NCH2CH2), 1.34 (m, 2H,
OCH2CH2). Anal. (C9H14N6O) C, H, N.
9-Hydroxybutyl-2-phenethoxyadenine (10). To compound 8
(0.50 g, 2.06 mmol), phenethyl alcohol (2.5 mL) and sodium
hydroxide (0.50 g) were added and the mixture was heated at
80 °C for 6 h. The solvent was removed under vacuum and the
residue chromatographed on a silica gel column eluting with
CHCl3-MeOH (95:5, v/v) to give 10 as white solid. Yield
79%; mp 113-115 °C. 1H NMR (DMSO-d6) δ 7.92 (s, 1H,
H-8), 7.32-7.18 (m, 7H, Ph-H and NH2), 4.39 (m, 3H, OH
and NCH2), 4.03 (t, 2H, J = 7.0 Hz, OCH2), 3.36 (m, 2H,
PhCH2CH2), 2.99 (t, 2H, J = 7.0 Hz, PhCH2), 1.79 (m, 2H,
NCH2CH2), 1.34 (m, 2H, OCH2CH2). Anal. (C17H21N5O2) C,
H, N.
-
tography on a Sephadex DEAE A-25 (Fluka) column (HCO3
form) equilibrated with H2O and eluted with a linear gradient of
H2O/0.5 M NH4HCO3.
9-(4-Monophosphate-butyl)adenine (14). Reaction of 7 gave 14
as white solid. Yield 68%. 1H NMR (D2O) δ 8.15 (s, 1H, H-2),
8.10 (s, 1H, H-8), 4.16 (t, 2H, J = 7.0 Hz, NCH2), 3.72 (m, 2H,
OCH2), 1.82 (m, 2H), 1.46 (m, 2H). 31P NMR (D2O) δ 1.07 (s).
Anal. (C9H17N6O4P) C, H, N.
General Procedure of Phosphorylation. Preparation of Bis(tri-
n-butylammonium) Pyrophosphate. Sodium pyrophosphate dec-
ahydrate 6.69 g (15 mmol) was dissolved in 150 mL of deionized
water. Excess of Dowex ion-exchange resin 20-50 mesh, proton
form, prewashed several times with water, was added to the
solution of sodium pyrophosphate, and the mixture was gently
stirred for 20 min. A mixture of 60 mL of EtOH and 7.14 mL of
tri-n-butylamine in a flask was placed in an ice-water bath, and
the pyrophosphate solution was filtered directly into the flask.
The resin was repeatedly washed with water until the filtrate was
no longer acidic. The solvent was then evaporated under
reduced pressure at 40 °C to give a thick, nearly colorless syrup.
This residue was treated twice with 100 mL of EtOH and then
evaporated. The residue was taken up in 40 mL of anhydrous
DMF and evaporated again. This residue was dissolved in
30 mL of anhydrous DMF, yielding 30 mL of a 0.5 M solution
of bis(tri-n-butylammonium) pyrophosphate in DMF. The
solution was stored, sealed, and cooled at 4 °C until use.31
Preparation of Tri-n-butylammonium Phosphate. The
same procedure was used as in case of the preparation of

4602 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Volpini et al.
bis(tri-n-butylammonium) pyrophosphate using sodium
monophosphate instead of sodium pyrophosphate.
examined for their intrinsic ability to generate an inward current
(agonist property) whose peak amplitude was compared with
the one elicited by the full agonist R,β-meATP. Furthermore, all
agonist compounds were also evaluated for their ability to
inhibit (after 20 s continuous application) the subsequent
response to R,β-meATP via the process of desensitization,
namely the depression of the subsequent R,β-meATP evoked
current after any early agonist activity of the novel drug.
Conversely, pure antagonism was defined as observed attenua-
tion of R,β-meATP induced responses in the absence of any
early inward current by the novel drug. We have previously
reported that preapplication (20 s) of selective antagonists and
agonists of P2X3 receptors fully blocks subsequent R,β-meATP-
induced currents.5 All chemical reagents, including enzymes for
cell culture, were from Sigma; culture mediums were obtained
from Gibco BRL.
General Procedure for the Synthesis of Diphosphates 20-23
and Triphosphates 24-29. To 0.15 mmol of monophosphates
14-19, in turn dissolved in dry DMF (1.0 mL), 36 μL of tri-n-
butylamine (28 mg, 0.15 mmol) were added. The solution was
stirred for 20 min at rt and then evaporated to dryness under
anhydrous conditions. After resuspension in dry DMF (1.4 mL),
N,N0-carbonyldiimidazole (122 mg, 0.75 mmol) was added and
the mixture was stirred at rt for 24 h. The reaction was quenched
by addition of dry CH3OH (49 μL, 38.5 mg 1.2 mmol) and stirred
for 30 min at rt. Then 6 mL (3 mmol) of a 0.5 M solution of tri-n-
butylammonium phosphate or bis(tri-n-butylammonium) pyro-
phosphate in DMF were added and the mixture was stirred at rt
for 16 h. The reactions were monitored by TLC, using precoated
TLC plates with silica gel 60 F-254 (Merck) and i-C3H7OH-
H2O-NH4OH (5.5:2.0:2.5) for the diphosphate derivatives or
i-C3H7OH-H2O-NH4OH (4.5:2.0:3.5) as mobile phase for
the triphosphate derivatives (0.4-0.5 Rf values), respectively.
The solvent was removed under vacuum and the residue, dis-
solved in H2O, was purified by means of ion exchange chroma-
tography to give the diphosphates 20-23 or the triphosphates
24-29, respectively.
9-(4-Diphosphate-butyl)adenine (20). Reaction of 14 gave 20
as white solid. Yield 15%, 1H NMR (D2O) δ 8.05 (s, 1H, H-2),
8.02 (s, 1H, H-8), 4.11 (t, 2H, J = 6.8 Hz, NCH2), 3.78 (m, 2H,
OCH2), 1.80 (m, 2H, CH2), 1.46 (m, 2H, CH2). 31P NMR (D2O)
δ -8.74 (d), -9.75 (m). ESI-MS: negative mode m/z 388.0 ([M -
2H þ Na]-), 366.0 ([M - H]-), 182.6 ([(M - 2H)/2]-). Anal.
(C9H24N8O7P2) C, H, N.
9-(4-Triphosphate-butyl)-adenine (24). Reaction of 14 gave 24
as white solid. Yield 22%, 1H NMR (D2O) δ 8.10 (s, 1H, H-2),
8.08 (s, 1H, H-8), 4.15 (t, 2H, J = 6.8 Hz, NCH2), 3.83 (m, 2H,
OCH2), 1.83 (m, 2H, CH2), 1.48 (m, 2H, CH2). 31P NMR (D2O)
δ-9.99 (m), -22.08 (m). ESI-MS: negative modem/z 446.0([M -
H]-), 222.4 ([(M - 2H)/2]-). Anal. (C9H28N9O10P3) C, H, N.
Biology. Cell Culture and Transfection. HEK 293T cells,
supplied by the in-house SISSA cell bank, were maintained in
culture in MEM-Glutamax medium (Invitrogen) supplemented
with 10% fetal calf serum (Sigma) and penicillin/streptomycin.
For each transfection experiment, 5 ꢁ 10-5 cells were plated and
transfected 24 h later with the calcium/phosphate method using
1 μg of high-quality purified plasmid DNA encoding for rat
P2X3 subunit (for details see reference; NCBI accession number:
CAA62594).32 Transfected cells were used 48 h later for further
experiments.
Electrophysiological Recording. Full details of the wholecell
patch clamp recording protocol can be found in previous
reports.9,31 In brief, HEK cells were continuously superfused
at fast rate (3 mL. min-1) with control solution containing (in
mM): NaCl 152, KCl 5, MgCl2 1, CaCl2 2, glucose 10, HEPES
10; pH was adjusted to 7.4 with NaOH. Patch pipettes had 3-
4 MΩ resistance when filled (in mM) with CsCl 130; HEPES 20;
MgCl2 1, magnesium ATP 3; EGTA 5; pH was adjusted to 7.2
with CsOH. Cells were voltage-clamped at -60 mV. Currents
were filtered at 1 kHz and acquired with pCLAMP 9.0 software
(Molecular Devices).
Chemicals and Their Application. Novel compounds were
applied via a rapid superfusion system (VC-6 with perfusion
fast step SF-77B, Warner Instruments) placed near the recorded
cell. Time for the solution exchange at cell membrane level was
20-30 ms. To screen the activity of the novel compounds, we
used, as a reference response, the fast inward current evoked by
the selective P2X3 full agonist R,β-methyleneATP (R,β-meATP)
usually applied for 2 s at 100 μM concentration that reliably
produces a maximal response.9,15,31 In each experiment, this
concentration of R,β-meATP was applied at least 3 times (at
5 min interval to avoid cumulative desensitization) to obtain an
average control response indicative of full activation of P2X3
receptors. All compounds (used at 100 μM concentration) were
Data Analysis. All data are presented as mean ( SEM (n=
number of cells) with statistical significance assessed with paired
t test (for parametric data) or Mann-Whitney rank sum test
(for nonparametric data). A value of p<0.05 was accepted as
indicative of statistically significant difference.
Acknowledgment. This work was supported by Fondo di
Ricerca di Ateneo (University of Camerino) and by grants
from the Italian Ministry of Research (FIRB 2003 and PRIN
2006) and from the Telethon Foundation (project GGP
07032).
Supporting Information Available: 1H NMR spectral data for
compounds 15-19, 21-23, and 25-29 and elemental analysis
for compounds 4-13, 4a-6a (analytical appendix I), and 14-29
(analytical appendix II). This material is available free of charge
via the Internet at http://pubs.acs.org.
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