Flow synthesis and biological studies of an analgesic adamantane derivative that inhibits P2X7-evoked glutamate release

Article abstract of DOI:10.1021/ml400079h

We report the biological evaluation of a class of adamantane derivatives, which were achieved via modified telescoped machine-assisted flow procedure. Among the series of compounds tested in this work, 5 demonstrated outstanding analgesic properties. This compound showed that its action was not mediated through direct interaction with opioid and/or cannabinoid receptors. Moreover, it did not display any significant anti-inflammatory properties. Experiments carried out on rat cerebrocortical purified synaptosomes indicated that 5 inhibits the P2X₇-evoked glutamate release, which may contribute to its antinociceptive properties. Nevertheless, further experiments are ongoing to characterize the pharmacological properties and mechanism of action of this molecule.

Full text of DOI:10.1021/ml400079h

Letter
pubs.acs.org/acsmedchemlett
Flow Synthesis and Biological Studies of an Analgesic Adamantane
Derivative That Inhibits P2X7‑Evoked Glutamate Release
Claudio Battilocchio,† Lucie Guetzoyan,† Chiara Cervetto,‡ Lorenzo Di Cesare Mannelli,§
Daniela Frattaroli,‡ Ian R. Baxendale,∥ Guido Maura,‡,⊥ Antonietta Rossi,# Lidia Sautebin,#
Mariangela Biava,¶ Carla Ghelardini,§ Manuela Marcoli,‡,⊥ and Steven V. Ley*,†
†Innovative Technology Centre, Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW, United Kingdom
‡Dipartimento di Farmacia, Sezione di Farmacologia e Tossicologia, Universita’di Genova, Viale Cembrano 4, 16148 Genova, Italy
§Dipartimento di Neuroscienze, Psicologia Area del Farmaco e Salute del Bambino, Universita’ di Firenze, Viale G. Pieraccini 6, 50139
Firenze, Italy
∥Department of Chemistry, Durham University, South Road, DH1 3LE Durham, United Kingdom
⊥Center of Excellence for Biomedical Research, Universita’ di Genova, Viale Benedetto XV 5, 16132 Genova, Italy
#Dipartimento di Farmacologia Sperimentale, Universita’ di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
¶Dipartmento di Chimica e Tecnologie del Farmaco, Sapienza Universita’ di Roma, P.le A. Moro 5, 00185 Rome, Italy
S
* Supporting Information
ABSTRACT: We report the biological evaluation of a class of adamantane derivatives, which
were achieved via modified telescoped machine-assisted flow procedure. Among the series of
compounds tested in this work, 5 demonstrated outstanding analgesic properties. This
compound showed that its action was not mediated through direct interaction with opioid and/
or cannabinoid receptors. Moreover, it did not display any significant anti-inflammatory
properties. Experiments carried out on rat cerebrocortical purified synaptosomes indicated that 5
inhibits the P2X7-evoked glutamate release, which may contribute to its antinociceptive
properties. Nevertheless, further experiments are ongoing to characterize the pharmacological
properties and mechanism of action of this molecule.
KEYWORDS: Adamantane derivatives, 2-aminoadamantane-2-carboxylic acid, analgesic agents, flow chemistry, purinergic receptors
mate.4,9−11 Additionally, activation of P2X7 can be equally
he ligand-gated ion channel superfamily of receptors is
Tintimately involved in the regulation of important responsible for the non-exocytotic release of glutamate through
the receptor-channel, in which the pore apoptotic state and
pannexin-1 recruitment are not required for the receptor
function and the glutamate efflux.1,4,12 This receptor seems to
modulate the death and survival of neurons and may play
different roles in the pathogenesis of disorders involving the
dysregulation of the glutamatergic transmission.2,3,13 Purinergic
signaling through P2X7 receptor seems to play primary roles in
neuroinflammation that underlie neurodegenerative diseases
including multiple sclerosis, Alzheimer’s and Huntington’s
diseases, or amyotrophic lateral sclerosis,14−19 and P2X7
inhibition has neuroprotective effects in animal models of
such diseases.19−21 A role for the P2X7 receptor in chronic pain
is suggested by findings in animal models and by the
effectiveness of P2X7 antagonists.22−28 Recent evidence
indicates that the receptor’s dilated pore conformation, linked
to downstream effects including the release of interleukin-1β
and ATP29−31 from microglia or macrophages, may be involved
in allodynia and pain hypersensitivity.28 These findings are
physiological functions. The alteration of these receptors has
been associated with a large range of diseases, including cardiac
disorders and perception of pain. The pivotal role associated
with these receptors is clearly linked to the interest of
researchers who are targeting these proteins for the develop-
ment of new medicines.
The purinergic P2X receptors are trimeric cell surface ion
channels gated by extracellular ATP. P2X3, P2X2/3, P2X4, and
P2X7 receptors have been associated with chronic pain and
arthritis, making these receptors potential targets for drug
discovery. Particularly interesting is the P2X7 class of receptors.
This ligand-gated ion channel is stimulated by extracellular
ATP and is present on the surface of hematopoietic lineage,
astrocytes, microglia, oligodendrocytes, and Schwann cells, as
well as on neurons (primarily glutamatergic neurons) in the
central and peripheral nervous system.1−6 P2X7 can switch from
a rapid-gating channel (selective for small cations) conforma-
tion to dilated pore conformations (permeable to molecules up
8
to 900 Da).7 The multiple permeation pathways of P2X7
appear to be linked to different modes of glutamate efflux.
Presynaptic influx of Ca2+ via P2X7 receptors (rapid-gating
channel) triggers the vesicular exocytotic release of gluta-
Received: February 25, 2013
Accepted: May 22, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
expected to strengthen interest in the P2X7 receptor and in the
different receptor conformations and permeation properties, as
a potential target in chronic pain.
Table 1. Mouse Abdominal Constriction Test (Acetic Acid
0.6%)
a
Several groups have reported P2X7 receptor antagonists with
a benzamide functionality; among them, AstraZeneca described
a class of adamantane containing benzamide molecules (1),
which showed potent antagonism toward P2X7 receptors.32
Abbott also reported an adamantane containing P2X7
antagonist (2) that exhibited efficacy against neuropathic pain
and inflammation in rat models.33 Noteworthy is that the
adamantyl motif is currently found in clinical use for the
treatment of neurogenerative diseases, as well as influenza, acne
vulgaris and type II diabetes. Further research is in progress for
the development of potential agents against iron overload
disease, cancer, malaria, and tuberculosis.34
We recently described the machine-assisted synthesis of an
unnatural amino acid (3) along with the generation of some
adamantane-containing molecules (4−8) (Figure 1).35 The
biological properties displayed by similar molecules prompted
us to investigate their antinociceptive properties.
Figure 1. Structures of P2X7 antagonists developed by AstraZeneca
(1) and Abbott (2); structures of 2-aminoadamantane-1-carboxylic
acid (3) and adamantane containing molecules tested in this work (4−
8).
The synthesis of a small collection of adamantane benzamide
compounds is briefly described in Scheme 1. The oxazoline (9)
can easily be obtained in 2 steps from commercially available 2-
adamantanone.35 Again using flow chemistry, compound 9 was
subjected to ozonolysis followed by reaction with different
nitrogen nucleophiles to afford 4−8 in good yields and purity
without the need for prior isolation of azalactone (10).
The analgesic profiles of the analogues are depicted in Table
1; BBG, a known antagonist of P2X7 receptors in the
nanomolar range,36 is also included for comparison purposes.
It has previously been reported that BBG exhibits therapeutic
effects in animal models for neurodegenerative diseases19,37,38
and reduces motor neuron damage in spinal cord injury or in
amyotrophic lateral sclerosis, by reducing microglia and/or
astrocyte activation and neutrophil infiltration.39−41 It also
blocks the P2X7 dilated pore formation, which is a key feature
a
b
All drugs were administrated per os 30 min before test. P < 0.05
c
versus vehicle-treated mice (control). P < 0.01 versus vehicle-treated
mice (control).
in chronic pain.28 More interestingly, biological studies
confirmed that the biological activity of BBG is mainly due to
its inhibitory activity on P2X7 receptors.19,41 However, it has
been shown that, at concentrations that may be reached after
repetitive dosage,19,39,41 BBG can also affect the functioning of
voltage-dependent Na+ channels,42 and therefore, this may play
a significant role in determining its biology.
Compounds 4 and 8 displayed no analgesic properties,
whereas 6 and 7 exhibited a moderate analgesic effect at higher
dosage (60 mg kg−1). Pleasingly, the antinociceptive effect of 5
was noticeable even at a low dose of 3 mg kg−1, and it also
Scheme 1. Telescoped Process for the Synthesis of 4−8: (a) Ozonolysis Step; (b) Amide Synthesis Step (Oxazolinone
Aminolysis)
B
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ACS Medicinal Chemistry Letters
Letter
displayed better analgesic profiles than BBG at the same
dosage. Further tests were then carried out to pinpoint the
molecular target(s) of 5.
We began by investigating whether the outstanding analgesic
profile of 5 could be induced via opioid and/or cannabinoid
receptors. As seen in Table 2, pretreatment with an opioid
removing any released compound and minimizing metabolism,
avoids receptor biophase and prevents indirect effects, enabling
nude receptors to be exposed. Under these experimental
conditions, only targets located on glutamatergic nerve
terminals (when monitoring glutamate release) are selectively
acted upon, allowing the pharmacological characterization of
release-regulating presynaptic receptors and allowing to assess if
a substance, added to the superfusion medium, could interfere
with the receptor activation.
Table 2. Lack of Effect by Opioid (Naxolone) and
Cannabinoid CB-1 (SR-141716A) Receptor Antagonists on
Analgesia Induced by 5 in the Mouse Abdominal
In this set of experiments, the rat cerebrocortical
synaptosomes were stimulated by BzATP, a preferential agonist
of P2X7 receptors,4,7 inducing an increase of aspartate efflux
(Chart 1a). By using this model of functional native rat P2X7
receptor, we found that, unlike BzATP, 5 did not activate the
receptor (Chart 1a), but behaved as an inhibitor of responses
mediated by P2X7 receptor activation (in our case, activation by
BzATP) in the micromolar range (IC50 value = 7.7 μM; Chart
1b). It has been previously reported that the activity of BzATP
stimulates both Ca2+-dependent and Ca2+-independent mech-
anisms of glutamate release.4,12,46 In our case, the BzATP-
evoked aspartate release was significantly reduced by
compound 5 (Chart 1c). Inhibitory effects were also detected
when BBG (P2X7 receptor antagonist)4,36,47 or A-43807924,25
(selective and competitive P2X7 receptor antagonist) were
administered, although their effects were more pronounced
than compound 5. We then turned our attention to whether
this inhibitory effect was mediated by extracellular levels of
Ca2+. While BBG and A-438079 could still exert some
inhibition under extra cellular Ca2+-free conditions, compound
5 was ineffective (Chart 1c). Moreover, its ineffectiveness on
the glutamate release in the presence of Rose Bengal48 (Chart
1c) confirmed that 5 only inhibited the glutamate release
dependent on the P2X7 function as a rapid gated Ca2+ channel,
while it did not affect the function of the receptor as a
permeation pathway for glutamate. Finally, the ineffectiveness
of 5, as well as of BBG, on the P2X7-independent stimulus (K+
depolarization, Chart 1d) represent a valid evidence that the
inhibition exerted by 5 (or BBG) on the BzATP-evoked
response may be related to a triggering of P2X7 receptor, rather
than to nonspecific effects.4
a
Constriction Test (Acetic Acid 0.6%)
no.
no. writhes
30 min
no. writhes
60 min
dose mg kg
treatment
control
mice
−1
12
12
8
30.4 2.9
33.1 2.2
b
b
5
10 p.o.
1 i.p.
16.5 2.3
28.3 3.2
33.7 3.6
15.1 1.9
17.8 2.5
19.3 3.1
31.9 2.5
32.6 2.4
20.7 2.7
Naxolone
SR-141716A
Naxolone + 5
SR-141716A + 5
8
1.5 i.p.
1.0 + 10
1.5 + 10
b
b
b
b
8
8
18.8 2.0
a
b
Naxolone and SR-141716A were administered 5 min before 5. P <
0.01 versus vehicle-treated mice (control).
receptor antagonist (Naxolone)43 or a cannabinoid 1 (CB-1)
receptor antagonist (SR-141716A)44 did not revert the
antinoception of 5, showing that its action was not mediated
through direct interaction with opioid or CB-1 receptors. We
then turned our attention to the effect of 5 on carrageenan
induced hyperalgesia and paw edema on rat models (Table 3).
Here again, as previously seen in Table 1, 5 and BBG displayed
similar activity although 5 was more effective at lower dosage.
Similar pharmacological profiles were also obtained, and both
compounds were cleared out after 90 min. A slight decrease of
the edema volume upon treatment after 30 min showed a low
anti-inflammatory activity; however, neither COX-1 nor COX-2
were inhibited by 5 (data not shown), highlighting the fact that
the analgesic activity is not associated with any anti-
inflammatory properties.
Finally, experiments were carried out on rat cerebrocortical
purified synaptosomes (Chart 1) in order to provide direct
evidence for interference with functioning native receptors. In
fact, isolated purified nerve terminals prepared from rodent
brain regions are helpful models allowing pharmacological
characterization of the receptors located on the plasma
membrane of the nerve terminals and revealing direct effects
of molecules at the receptors themselves. Indeed, release
monitoring from a superfused synaptosomal monolayer,45 by
Overall, these findings indicated that 5 behaves as an
inhibitor of Ca2+-dependent exocytotic [3H]D-aspartate efflux
and interferes with the rapid gating channel coupled to vesicular
release. The ability of 5 to distinguish among the P2X7 multiple
permeation pathways would merit further investigation.
On-going research toward the development of novel and
more efficient approaches for pain relief and treatment in
a
Table 3. Effect of 5 and BBG on Hyperalgesia and Paw Edema Induced by Carrageenan in the Rat Paw-Pressure Test
paw pressure (g)
after treatment
edema volume (mL)
pretreatment i.pl. treatment p.o. dose mg kg−1 before treatment
15 min
30 min
60 min
90 min
30 min
saline
control
control
5
61.2 2.5
36.1 3.3
34.2 2.4
32.5 3.2
35.7 3.1
35.5 2.1
30.9 1.3
33.1 1.8
65.6 3.4
32.8 2.9
39.1 3.5
44.2 3.2
48.4 3.4
38.7 2.5
46.3 1.8
47.2 2.3
58.9 3.8
30.6 3.2
40.8 3.7
52.7 3.8
53.9 4.6
43.1 2.2
50.8 2.5
49.6 2.8
61.4 4.1
31.8 2.2
35.6 2.8
43.4 3.9
48.3 4.2
38.5 2.9
45.6 1.8
43.8 2.2
63.6 4.0
34.8 3.1
36.9 3.3
38.2 2.5
33.6 3.0
37.1 1.9
40.2 2.1
36.2 2.4
1.44 0.05
2.71 0.08
2.53 0.05
2.06 0.08
2.10 0.09
2.63 0.07
2.17 0.08
2.21 0.08
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
carrageenan
3
b
c
c
b
c
c
c
̂
̂
5
10
20
10
40
60
c
5
b
̂
BBG
BBG
BBG
c
c
c
c
c
c
c
c
a
b
c
Carrageenan 100 μL, 1% i.pl. 2 h before test. P < 0.05. There were 5 rats per group. P < 0.01. There were 5 rats per group.
C
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ACS Medicinal Chemistry Letters
Letter
a
Chart 1. Action of 5 at Presynaptic P2X7 Receptors
a
(a) Ineffectiveness of 5 as a P2X7 receptor agonist. The preferential P2X7 agonist BzATP evoked [3H]D-aspartate efflux from superfused
cerebrocortical synaptosomes, while 5 was ineffective. (b) Inhibition of the response to BzATP by 5: log concentration−response relationship for 5
on the BzATP-evoked [3H]D-aspartate efflux. Effect of 100 μM BzATP alone (○) or in the presence of 5 (●). (c) Effect of 5, BBG, or the selective
P2X7 antagonist A-438079 on the BzATP-evoked [3H]D-aspartate efflux, in the presence or absence of extracellular Ca2+ or after preincubation with
the inhibitor of vesicular glutamate transporter Rose Bengal. (d) Ineffectiveness of 5 or BBG on the response to K+ depolarization. BzATP (or KCl)
was added for 120 s during superfusion; compound 5, BBG, or A-438079 was added 8 min before BzATP (or K+ depolarization). Ca2+-free EGTA
(0.5 mM)-containing medium was added 18 min before BzATP; Rose Bengal (0.5 μM) was preincubated (30 min at 37 °C) before and during
vesicle loading with [3H]D-aspartate. Data are mean SEM of 3−12 independent experiments in triplicate. *P < 0.05 vs BzATP alone; #P < 0.05 vs
BzATP in standard medium.
chronic pain is of considerable importance. Several classes of
drugs are already effective at controlling particular types of
pain; however, side-effects such as addictive issues or long-term
toxicity profiles hamper their use, especially during chronic
treatments.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and full characterization of compounds
4−8, material and methods for the mouse abdominal
constriction test, hyperalgesia and paw edema induced by
carrageenan in the rat paw-pressure test, and rat cerebrocortical
purified synaptosomes experiments. This material is available
free of charge via the Internet at http://pubs.acs.org.
Among the series of compounds tested in this work, 5
demonstrated outstanding in vivo analgesic properties in the
mouse abdominal constriction test. Further investigations
showed that its action was not mediated through direct
interaction with opioid and/or cannabinoid receptors. Com-
pound 5 also did not display any significant anti-inflammatory
properties. Experiments carried out on rat cerebrocortical
purified synaptosomes indicated that 5 behaved as an inhibitor
of P2X7-mediated responses. These preliminary findings also
point out that 5 would interfere only with Ca2+-dependent
exocytotic glutamate release. P2X7 receptors have been proven
to be a potential valid molecular targets for neuropathic pain
and neurodegenerative diseases;22−28,37 however, the in vitro
data suggest that an additional pathway may be involved. The
mechanistic features of 5 will be explored in more details in our
laboratories.
AUTHOR INFORMATION
Corresponding Author
*(S.V.L.) E-mail: svl1000@cam.ac.uk.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Pfizer Worldwide Research and Develop-
ment (to C.B.), EPSRC (to L.G., grant EP/F06968511), and
the BP 1702 endowment (to S.V.L.) for financial support.
ABBREVIATIONS
A-438079, 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)-methyl
pyridine; ATP, adenosine triphosphate; BBG, brilliant blue G;
■
D
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Letter
(19) Díaz-Hernan
Gom
́
dez, M.; Díez-Zaera, M.; San
ez-Villafuertes, R.; Canals, J. M.; Alberch, J.; Miras-Portugal, M.
́
chez-Nogueiro, J.;
BzATP, benzoyl benzoyl adenosine triphosphate; CB-1,
cannabinoid 1 receptor
́
T.; Lucas, J. J. Altered P2X7-receptor level and function in mouse
models of Huntington’s disease and therapeutic efficacy of antagonist
administration. FASEB J. 2009, 23, 1893−1906.
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