Cubyl amides: Novel P2X7 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.05.062

Polycyclic amides 2 and 5-9 were successfully synthesised and their lipophilicity profiles were evaluated using reverse-phase HPLC. All synthesised compounds possessed P2X₇R antagonistic properties when tested on rat spinal cord microglia cells. Extensive screening for binding to other neuroreceptor subtypes demonstrated their P2X₇ selectivity.

Full text of DOI:10.1016/j.bmcl.2008.05.062

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3720–3723
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cubyl amides: Novel P2X7 receptor antagonists
Hendra Gunosewoyo a, Jun Liu Guo b, Maxwell R. Bennett b, Mark J. Coster c, Michael Kassiou a,b,d,
*
a School of Chemistry, University of Sydney, NSW 2006, Australia
b Brain and Mind Research Institute, 100 Mallet Street, Camperdown, Sydney, NSW 2050, Australia
c Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Qld 4111, Australia
d Discipline of Medical Radiation Sciences, University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Polycyclic amides 2 and 5–9 were successfully synthesised and their lipophilicity profiles were evaluated
using reverse-phase HPLC. All synthesised compounds possessed P2X7R antagonistic properties when
tested on rat spinal cord microglia cells. Extensive screening for binding to other neuroreceptor subtypes
demonstrated their P2X7 selectivity.
Received 4 March 2008
Revised 13 May 2008
Accepted 15 May 2008
Available online 20 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
P2X7 antagonists
Polycyclic benzamides
Rational drug design
Microglia
The P2X7 receptor (P2X7R) is an unusual, non-desensitising cat-
ion-selective ion channel, directly gated by extracellular ATP. It is
ubiquitously found in a variety of cell types, most notably those
of haematopoietic origin, such as mast cells, macrophages and
lymphocytes, as well as brain glial cells, including microglia and
astrocytes.1 It possesses a 240-amino acid residue, cytoplasmic,
carboxy-terminal tail that is necessary for its bifunctionality as a
selective ion channel assembled from P2X7 subunits, or as a non-
selective pore. There are two hypotheses explaining P2X7-associ-
ated pore formation; in both scenarios brief activation of the
receptor with ATP leads to opening of the ion channel by confor-
mational changes within the P2X7 structure. In order to form the
membrane pore, the receptor complex recruits further subunits
to the original receptor complex. Alternatively, prolonged activa-
tion of the P2X7 receptor complex leads to activation of intracellu-
lar machineries that signal the opening of P2X7-independent pore
forming proteins such as pannexin 1 allowing entry of large (up to
molecular weights of 900 Da) cationic species.2
lagen antibody arthritis model.10 More strikingly, chronic inflam-
matory and neuropathic hypersensitivity are totally abolished in
P2X7-knockout mice despite the remaining normal nociceptive
processing.11 Furthermore, antagonism of the P2X7R on oligoden-
drocytes leads to failure of multiple sclerotic conditions to develop
in mouse EAE models of multiple sclerosis.12 In addition, linkage
studies show that one of the best logarithm of odds (LOD) scores
for bipolar disorder, clinical depression and anxiety disorders is
the P2X7 receptor.13,14 It could therefore be suggested that potent,
selective P2X7 antagonists may serve as novel all-purpose
analgesics.
To date, structure–activity relationship (SAR) studies of several
small, drug-like P2X7-active molecules have been reported and re-
cently reviewed (Fig. 1).15 Cyclic imide 1 and adamantyl benzam-
ide 2 were discovered by AstraZeneca through a high-throughput
Since the P2X7R-encoding cDNAs were isolated and character-
ised from three different mammalian sources (rats,2 mice3 and hu-
man4) about a decade ago, there has been increasing interest in
delineating different aspects of the P2X7R function, particularly
its role in microglia activation and neurodegenerative diseases,5,6
chronic pain modulation7 and the exact mechanism of the chan-
nel-to-pore transition.8,9 P2X7-knockout mice have been reported
to show reduced incidence and severity of arthritis in an anti-col-
* Corresponding author. Tel.: +612 9351 0849; fax; +612 9351 0852.
E-mail address: m.kassiou@usyd.edu.au (M. Kassiou).
Figure 1. Drug-like P2X7-active molecules.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.062

H. Gunosewoyo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3720–3723
3721
Table 1
as diagnostic probes when radiolabelled for use with in vivo
molecular imaging. We hypothesised that by replacing the ada-
mantane with a cubane moiety, these compounds could be ren-
dered suitable as in vivo radiotracers for positron emission
tomography (PET) whilst maintaining activity (Table 1). This would
allow for the first time to image P2X7R expression in disease and
disease progression. In addition they could also be used to assess
the efficacy of P2X7R therapy.
Cubyl amides and adamantyl amides
Although the original report by AstraZeneca mentioned that no
replacements were found for adamantane, we believed cubyl
amides 7–9 could offer unique possibilities for development of
P2X7R-active molecules due to their rigid molecular structure
and ability to orient substituents in three dimensions. As shown
in Table 1, the distance between the carbonyl carbon atom to the
furthest hydrogen atom in the adamantane series is ca 0.6 Å longer
than the cubane series. The dihedral angle about the amide C@O
and aromatic ring is very similar in both series, suggesting that
each of the cubyl amides adopts a very similar lowest energy con-
formation to the adamantane analogues. Moreover, systematic
alteration of their lipophilicity would allow easier access across
membranes or the blood–brain barrier. To the best of our knowl-
edge, there have only been a few studies that reported radiolabel-
ling of P2X7-active compounds.22,23 Thus far, no attempts have
been undertaken to radiolabel-specific P2X7R ligands for use in
PET imaging of the living brain. Herein, we report an efficient syn-
thesis of cubyl amides and their preliminary biological evaluation
at the P2X7R. Variations in the aromatic segment of the target com-
pounds are based primarily on maintaining strong binding to the
receptor whilst investigating potential sites for incorporation of a
fluorine-18 radiolabel (2 vs 5, 6 and 7 vs 8 and 9). Their preliminary
P2X7 functionality was tested based on the inhibition of dye uptake
using rat spinal cord microglia as P2X7 source. The binding affini-
ties towards other common neuroreceptors were also investigated.
Synthesis of novel N-cuban-1-ylmethyl-benzamides is illus-
trated in Scheme 1 (see Supplementary data for details). Commer-
cially available dimethyl 1,4-cubanedicarboxylate 10 was
converted to cubane-1-carboxylic acid 12 in 70% yield overall fol-
lowing a literature procedure utilising Barton decarboxylation as
the key step.24 Subsequent transformation of the acid to amine
13 followed by amide couplings afforded the desired amides 7–
9.25 To obtain more information on the optimal size of the ‘hydro-
phobic pocket’ at the P2X7R binding site, the adamantyl amides 2,
5, and 6 were also prepared in a similar fashion (Scheme 2). 2-
Chloro-5-methoxybenzoic acid 18 was readily prepared by diazoti-
sation of commercially available 5-amino-2-chlorobenzoic acid 16,
followed by treatment with excess MeI and K2CO3 and subsequent
Compound
\OCCCa (°)
ꢀ49.8
C(O)–H(poly) distanceb (Å)
ClogPc
2
7.392
5.40
5
6
7
8
ꢀ12.6
ꢀ24.4
ꢀ50.4
ꢀ12.3
ꢀ23.5
7.376
7.377
6.797
6.772
6.757
5.11
5.52
2.67
2.39
2.80
9
a
The dihedral angle about the amide C@O and aromatic ring obtained from
MacromodelTM
.
b
The distance between the carbonyl carbon atom to the furthest hydrogen atom
in the polycyclic moiety obtained from MacromodelTM
.
The ClogP values were obtained from ChemDraw 9TM. Surface areas of ada-
c
mantane = 156 Å2, cubane = 122 Å2 (MacromodelTM).
screening process of their compound library.16,17 More recently,
tetrazole 3 and triazole 4 have also been found to inhibit rat and
human P2X7R activity.18–21 These compounds are highly amenable
to structural modifications, indicating their potential as lead mole-
cules for further improving the potency and selectivity at P2X7R.
Of these the adamantyl benzamide 2 represents an intriguing
class of compound for development of P2X7 antagonists since the
adamantane could be replaced with other polycyclic moieties
and their influence on P2X7 receptor activity assessed. These mol-
ecules could potentially be used not only as therapeutics but also
Scheme 2. Reagents and conditions: (i) SOCl2, reflux, 3 h; (ii) aq NH3, THF, 0 °C–rt,
1 h; (iii) LiAlH4, THF, 0 °C to reflux, 16 h, 60% over three steps; (iv) aroyl chloride,
NEt3, MeCN, 0 °C–rt, 16 h.
Scheme 1. Reagents and conditions: (i) NaOH (1 equiv), MeOH, THF, rt, 16 h, 88%; (ii) (COCl)2, CH2Cl2, 30 min; (iii) sodium salt of N-hydroxypyridine-2-thione, hm, DMAP, t-
BuSH, benzene, reflux, 90 min; (iv) NaOH (1 equiv), MeOH, reflux, 1 h, 80% over three steps; (v) (COCl)2, CH2Cl2, rt, 45 min; (vi) NH3 (l), CH2Cl2, ꢀ78 °C, 30 min, 83% over two
steps; (vii) LiAlH4, THF, 0 °C to reflux, 16 h, 86%; (viii) aroyl chloride, NEt3, MeCN, 0 °C–rt, 16 h.

3722
H. Gunosewoyo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3720–3723
Table 2
Lipophilicity measurements for amides 2 and 5–9
Compound
Experimental logD
ChemDrawÒ logP
ChemDrawÒ ClogP
2
5
6
7
8
9
4.07 0.01
4.03 0.01
3.81 0.01
2.90 0.01
2.72 0.01
2.61 0.01
4.12
3.84
3.84
1.94
1.66
1.66
5.40
5.11
5.52
2.67
2.39
2.80
Scheme 3. Reagents and conditions: (i) NaNO2, H2SO4, 0–5 °C; (ii) hot H2O, reflux,
30 min; 70% over two steps; (iii) K2CO3, MeI, DMF, 40 °C, 20 h, 94%; (iv) LiOH, THF/
H2O, reflux, 6 h, 87%.
The P2X7 antagonistic properties of the polycyclic amides 2 and
5–9 were subsequently tested by measuring the decrease in the
fluorescent propidium iodide dye uptake in rat spinal cord microg-
lial cells stimulated by 20(30)-O-(4-benzoylbenzoyl)adenosine 50-
triphosphate (BzATP). To the best of our knowledge, this
demonstrates the first P2X7 functional studies performed on a
native, activated rat microglia system with all other purinergic
receptors intact. The cell types used for examining P2X7 function-
ality in the literature so far include: human THP-1 cells, HEK293
cells expressing either human or rat P2X7R, U373 cells expressing
human P2X7R and human 1321N1 astrocytoma cells devoid of
endogenous P2X function expressing either human or rat
P2X7R.4,16–19,28–30 As shown in Figure 2, all the six polycyclic
amides 2 and 5–9 exhibit a degree of antagonism on the rat
Scheme 4. Reagents and conditions: (i) BBr3, CH2Cl2, 0 °C–rt, 2 days, 84–100%.
hydrolysis of the resultant ester (Scheme 3). The 2-chloro-5-meth-
oxybenzamides 2 and 7 were also successfully demethylated using
boron tribromide to afford 19 and 20, potential compounds for 11
C
labelling (Scheme 4).
P2X7R at 1 lM concentration. Whilst IC50 values are yet to be
Since lipophilicity of the synthesised amides is central for their
use as brain imaging probes, we conducted logD (logP at pH7.4
determined, it is noteworthy that the P2X7R antagonistic proper-
ties are retained upon alteration of both the aromatic and polycy-
clic moieties in a wild-type P2X7R source.
)
measurements of these compounds. It is believed that the opti-
mum logP value for therapeutic CNS-active compounds is between
2 and 3.5.26 In this series, the logD values were evaluated using re-
verse-phase C-18 HPLC study by comparison of their average
retention times from three injections to that of reference com-
pounds with known logD values.27 All measurements were per-
formed on the same day to minimise day-to-day variations and
acetone was used to determine the void volume of the column
(1.72 min). The average retention time from three injections was
recorded for each of the selected six reference compounds and
the results are summarised in Table 2.
Extensive screening for binding to other common neurorecep-
tors was undertaken to ensure the specificity and selectivity of
amides 2 and 5–9 towards the P2X7R. Receptor-binding profiles
were generously provided by the National Institute of Mental
Health’s Psychoactive Drug Screening Program.31 The receptor pro-
filing studies indicated that none of the compounds 2 and 5–9
exhibited appreciable binding to the many neuroreceptor subtypes
assayed (see Supplementary information for details). In addition,
functional assays on P2Y1, P2Y2 and P2Y4 receptors expressed in
HEK293 cells using ATP as the agonist at a concentration of
An interesting observation in Table 2 is that whilst the ClogP
prediction for cubane derivatives 7–9 was quite accurate, it was
not the case for the adamantane derivatives 2 and 5–6. In contrast,
the logP prediction was excellent for adamantane derivatives but
not for the cubanes. Nevertheless, both ClogP prediction and the
measured logD from reverse-phase HPLC gave values greater than
4 for the adamantane derivatives, indicating that these compounds
are probably less suitable for crossing the blood–brain barrier. On
the other hand, the measured logD values of cubane derivatives 7–
9 were between 2 and 3. These values are considered to be within
the optimum range for potential PET radioligands (2 < logD < 3.5)
and therefore are suitable for brain imaging probes.26
4 mM and drug concentrations of 10 lM, 3 lM, 1 lM, 300 nM,
100 nM, 10 nM and 1 nM indicated that none of the compounds
2 and 5–9 had activity at the assayed metabotropic purinergic
receptors. The target structures and related compounds have been
judiciously chosen, taking into consideration the desired lipophil-
icity range and ability to radiolabel, and can be readily accessed
by concise synthetic routes from commercially available starting
materials, employing well-precedented and reliable synthetic
methods. The chemical synthesis and functional assays of related
compounds will be conducted in parallel as part of a continuous
and convergent assessment process to obtain derivatives with
optimum P2X7 activity (work in progress).
100
80
60
40
20
0
control
BzATP
BzATP + 2 BzATP + 5 BzATP + 6 BzATP + 7 BzATP + 8 BzATP + 9
Figure 2. P2X7 functional assay of amides 2 and 5–9. All experiments were performed using 150 lM BzATP and 1 lM of the antagonists. Average reading from four replicates
and standard errors are shown.

H. Gunosewoyo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3720–3723
3723
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Acknowledgment
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H.G. thanks the Commonwealth Scientific and Industrial Re-
search Organisation (CSIRO) for a Postgraduate Top Up Scholarship
and for the generous gift of dimethyl cubane-1,4-dicarboxylate.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.062.
20. Morytko, M. J.; Betschmann, P.; Woller, K.; Ericsson, A.; Chen, H.; Donnelly-
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