Pharmacological evaluation of a novel series of urea, thiourea and guanidine derivatives as P2X7 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2017.04.005

We report on P2X₇ receptor antagonists based on a lead adamantly-cyanoguanidine-aryl moiety. We have investigated the importance of the central cyanoguanidine moiety by replacing it with urea, thiourea or guanidine moieties. We have also investigated the linker length between the central moiety and the aryl portion. All compounds were assessed for their inhibitory potency in a pore-formation dye uptake assay at the P2X₇ receptor. None of the compounds resulted in an improved potency illustrating the importance of the cyanoguanidine moiety in this chemotype.

Full text of DOI:10.1016/j.bmcl.2017.04.005

Accepted Manuscript
Pharmacological evaluation of a novel series of urea, thiourea and guanidine
derivatives as P2X7 receptor antagonists
Erick C.N. Wong, Tristan A. Reekie, Eryn L. Werry, James O'Brien-Brown,
Sarah L. Bowyer, Michael Kassiou
PII:
S0960-894X(17)30373-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.005
BMCL 24858
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 March 2017
29 March 2017
1 April 2017
Please cite this article as: Wong, E.C.N., Reekie, T.A., Werry, E.L., O'Brien-Brown, J., Bowyer, S.L., Kassiou, M.,
Pharmacological evaluation of a novel series of urea, thiourea and guanidine derivatives as P2X7 receptor
antagonists, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.005
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Pharmacological evaluation of a novel series of urea, thiourea and
guanidine derivatives as P2X7 receptor antagonists
Erick C. N. Wonga, #, Tristan A. Reekieb, #, Eryn L. Werryc, James O’Brien-Brownb, Sarah L.
Bowyerb and Michael Kassioub, ∗
aSchool of Medical Sciences, The University of Sydney, NSW 2006, Australia
bSchool of Chemistry, The University of Sydney, NSW 2006, Australia
cFaculty of Health Sciences, The University of Sydney, NSW 2006, Australia
# Joint first authors.
Michael Kassiou. Tel.: +612-9351-2745; e-mail: michael.kassiou@sydney.edu.au
Abstract - We report on P2X7 receptor antagonists based on a lead adamantly-
cyanoguanidine-aryl moiety. We have investigated the importance of the central
cyanoguanidine moiety by replacing it with urea, thiourea or guanidine moieties. We have
also investigated the linker length between the central moiety and the aryl portion. All
compounds were assessed for their inhibitory potency in a pore-formation dye uptake assay at
the P2X7 receptor. None of the compounds resulted in an improved potency illustrating the
importance of the cyanoguanidine moiety in this chemotype.
Keywords:P2X7R; inflammation; cyanoguanidine; urea; thiourea.
∗ Michael Kassiou. Tel.: +612-9351-2745; e-mail: michael.kassiou@sydney.edu.au

Adenosine 5’-triphosphate (ATP) is the endogenous ligand and physiological agonist of
the P2X7 purinoreceptor (P2X7R), a member of the P2X superfamily of trimeric ligand-gated
cation channels (P2X1-7).1-4 The P2X7R is predominantly and highly expressed on immune
cells of hematopoietic origin such as peripheral monocytes and macrophages, as well as their
centrally located counterparts, microglia.2,5 Transient P2X7R activation by low concentrations
of extracellular ATP permits the influx of calcium and sodium ions, and efflux of potassium
ions within milliseconds.1,4,6 However, psychological stress and insults to the CNS (including
neurodegenerative and ischemic related) incite enhanced release of ATP into the extracellular
environment.7-9 The prolonged activation of the P2X7R results in the immediate
rearrangement of the cell membrane and cytoskeleton,10,11 the formation of a large >900 Da
macro-pore, and eventual apoptosis.2,7 Prior to cell death, the proinflammatory cytokines IL-
1β and IL-18 are also processed and secreted,8,10,12-15 Significantly higher serum levels of IL-
1β, among other proinflammatory cytokines, have been reported in patients suffering from
Alzheimer’s disease (AD)16 and major depressive disorder (MDD),17 suggesting the
dysregulation of the inflammatory response may underpin these, and many other,
neurodegenerative (including Parkinson’s disease), and neuropsychiatric (including bipolar
disorder) conditions, as well as neuropathic pain.7,18-21 Mice lacking the P2X7R generated
macrophages that were incapable of releasing mature IL-1β in response to extracellular ATP,
or to its more stable and potent analogue, 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-
triphosphate tri(triethylammonium) salt (BzATP).22,23 Additionally, in several animal models
of depression, abolishing P2X7R expression offered an antidepressant effect.23 When
stimulated with the amyloid β protein (Aβ), a hallmark of AD pathology, lipopolysaccharide
(LPS)-primed microglia from wild type mice released ATP and large amounts of IL-1β,
whereas their P2X7R-deficient counterparts did not.24 Moreover the microglia derived from
these P2X7R-knockout mice were resistant to Aβ-induced plasma membrane
permeabilisation, potentially through their inability to form the large P2X7R pore.
Considering the significant influence the P2X7R has over the inflammatory response, and that
microglial P2X7R expression is upregulated in conditions such as AD and multiple sclerosis
(MS),1,25 P2X7R antagonism appears as a potential treatment strategy.
Initial P2X7R-focussed medicinal chemistry was directed at generating P2X7R antagonists
for the treatment of peripheral inflammatory diseases including rheumatoid arthritis and
Crohn’s disease, with a number of compounds having progressed to clinical trials.2,26
Adamantyl amide 1 was developed by AstraZeneca and showed promising pharmacokinetic

properties from in vivo studies in rats (Figure 1). Abbott Laboratories developed compounds
focused on the cyanoguanidine moiety. These compounds, with 2 and 3 being examples of
the more potent in the series, showed nanomolar potency, but a poorer pharmacokinetic
profile. We have recently reported an adamantyl-cyanoguanidine hybrid that combined
pharmacologically beneficial features from the Abbott cyanoguanidine derivatives with the
inclusion of an adamantane moiety for improved pharmacokinetics and enhanced blood-brain
barrier (BBB) penetration.27 The small cyanoguanidine hybrid 4a was reported to have
nanomolar potency at the P2X7R (IC50 = 100 nM). Potency could be improved by
lengthening the chain and incorporating a nitrogen atom into the aryl portion, particularly at
the 3-position to give 4b (IC50 = 69 nM). Further conclusions from this work showed that a
methylene linker between the cyanoguanidine and adamantyl moieties was essential for
potency, while either no linker (n = 0) or a methylene linker (n = 1) was acceptable for the
aryl portion.
Figure 1. P2X7R antagonists.
While there have been advancements in potency optimization and BBB permeability, many
currently disclosed compounds lack desirable characteristics for targeting central P2X7R,
being unable to permeate the BBB or offer insufficient target engagement for potential
therapeutic value.1 Therefore to increase the potential for translational clinical utility,
modifications were made to the cyanoguanidine moiety in an effort to optimize lipophilicity.
However, it is unknown whether the cyanoguanidine moiety was essential for potency or if
substitutions could be tolerated. In this article, we report urea, thiourea and guanidine
derivatives of this cyanoguanidine hybrid which explore the significance of the
cyanoguanidine linker moiety and the positioning of the phenyl group on P2X7R antagonist
potency.
The (adamantan-1-yl)methanamine hydrochloride (5) was obtained using previously
reported procedures28 and then reacted with the commercially available phenyl isocyanate (6)

to afford 7a in 72% yield (Scheme 1). Considering the range of analogues that we wanted to
develop, we sought adamantyl isocyanate as a key building block. However, we were
interested in a previous report of a methyl isocyanate equivalent derived from 1,1’-
carbonyldiimidazole (CDI) that was crystalline and stable.29 By using CDI and 5, compound
8 could be obtained as a white solid in 95% yield. This compound was stored for a year under
ambient conditions without any observed decomposition, making it a suitable isocyanate
equivalent. Reacting 8 with benzylamine or phenethylamine afforded 7b and 7c, respectively.
The same isocyanate equivalent 8 could be converted to the pyridyl containing ureas 9a-c,
using 2-, 3-, or 4-picolylamine respectively. The high yields obtained (73-81%) illustrate the
utility of 8 as a precursor for forming (adamantan-1-yl)methyl ureas.
The commercially available isothiocyanates 10a-b could be reacted with 5 to form the
thiourea derivatives 11a-b and further converted to the guanidine derivatives 12a-b.
Scheme 1. Synthesis of ureas, thioureas and guanidines. Reagents and conditions: a) Et3N
(1.0 equiv), CH2Cl2, RT, 18 h; b) 1,1’-carbonyldiimidazole (1.0 equiv), MeCN/DMF (3:1),
RT, 2 h; c) requisite amine (1.0 equiv), Et3N (1.0 equiv), CH2Cl2, RT, 18 h; d) Et3N (1.0
equiv), CH2Cl2, RT, 18 h; e) 1) MeI (10 equiv), MeCN, 40 ºC, 1 h; 2) NH4OH, RT, 3h.
The adamantyl urea, thiourea and guanidine compounds 7a-c, 9a-c, 11a-b and 12a-b, were
assayed for their ability to inhibit BzATP-induced P2X7R activity in human THP-1 cells.
BzATP is a stable and potent P2X7R agonist with the P2X7R being endogenously expressed
by THP-1 cells. The cellular uptake of the fluorescent dye, YO-PRO-1, was measured as an
indicator of pore formation, and therefore P2X7R activity. Experimental protocols are
detailed in the supplementary information. In addition to the newly synthesized compounds,
the previously reported cyanoguanidine 4a was also subjected to the same experiments.

The significance of the cyanoguanidine moiety to the nanomolar potency yielded by 4a in
previous reports27 was explored by replacing this with other functional groups, whilst
retaining the adamantyl and aryl portion of the molecule (Table 1). With the exception of
compound 11b, replacing the cyanoguanidine moiety with all three functional groups (urea,
thiourea, guanidine) decreased the lipophilicity (cLogP) value compared to 4a (Table 1).
Comparing variations in central functionality showed that substituting the cyanoguanidine
moeity (4a) with: (i) a guanidine (12a) proved detrimental to the molecule’s solubility, (ii) a
thiourea (11a) was poorly tolerated with an almost 24-fold decrease in potency and yielded
only low micromolar potency (2.4 µM), and (iii) a urea (7a) revealed an even greater 50-fold
reduction in potency (4.9 µM).
Our previous studies on the adamantyl cyanoguanidine compounds showed that altering
the linker length between the central moiety and aryl group impacted potency.27 Therefore,
the linker length between the phenyl group and the central functional group was investigated
in all the urea, thiourea, and guanidine derivatives to determine if this might result in
improved compound potency. Although extending the phenyl portion by one carbon from
the guanidine group 12b restored the molecule’s solubility, potency remained low (12b > 10
µM). Extending the phenyl group away from the urea group of 7a by one carbon only slightly
improved compound potency (7b = 4.54 µM), and by two carbons led to a further decrease in
potency (7c = 6.41 µM). These results mirror the results of the adamantyl cyanoguanidine
derivatives where a linker length of n = 0 or 1 was most tolerated and that extending the
linker further was detrimental. The thiourea derivatives showed a decrease in potency when
the linker was increased from n = 0 to 1 (11a = 2.42 µM, 11b = 4.24 µM, respectively) and
so further extensions were not investigated. Overall, increasing the distance between the
central functional group and the phenyl group did not produce higher potency compounds,
and may reflect a smaller binding pocket on the P2X7R where binding residues may favor
shorter molecules.30
Previous findings indicate the inclusion of a nitrogen atom in the aromatic ring of
adamantyl cyanoguanidines improved activity at the P2X7R.27 Investigating the role of
incorporating a nitrogen atom in the aromatic system was made on the urea moiety (n = 1)
giving the isomeric pyridyl derivatives 9a-c. Compound 9b displayed a slight increase in
potency (3.38 µM), while the analogues 9a and 9c were less potent than the phenyl analogue
7b (9a and 9c; 5.95 and 6.75 µM respectively). The improved potency of 9b is in agreement
with the observations of the cyanoguanidine derivative 4b that also showed improved

potency over its phenyl and pyridyl isomer analogues. The 3-pyridyl derivative 9b (n = 1)
positions the nitrogen atom in a similar location to that observed in the quinoline compounds
2 and 3. These combined results suggest that, as a hydrogen bond acceptor, the nitrogen atom
improves hydrogen bonding interactions with residues in the receptor’s ligand-binding site.
Table 1. IC50 values of compounds 4a, 7a-c, 9a-c, 11a-b and 12a-b derivatives against
hP2X7R using the YO-PRO-1 dye uptake functional assay in human THP-1 cells
Compound
Structure
cLogP a
IC50 mean SEM (µM) b
H
N
H
N
4a
4.6
0.10 0.01
N
O
NC
H
N
H
N
7a
7b
7c
9a
3.36
3.43
3.71
2.51
4.96 0.24
4.54 0.28
6.41 0.26
5.95 0.04
H
N
H
N
O
O
H
N
H
N
N
H
N
H
N
O
N
H
N
H
N
9b
2.09
2.09
4.5
3.38 0.14
6.75 0.17
2.42 0.12
4.24 0.14
O
O
S
N
H
N
H
N
9c
H
N
H
N
11a
11b
12a
H
N
H
N
4.71
4.18
S
H
N
H
N
b
–
NH
H
N
H
N
12b
4.39
>10,000
NH
a Calculated using ChemDraw Professional 15.
b Values are the mean of three to five experiments and uncertainty was determined by the standard error of the mean (SEM)
c
IC50 could not be determined due to compound insolubility
We have reported the synthesis of 10 adamantyl-containing compounds as well as the
adamantyl isocyanate analogue 8. However, the poor potency resulting from the chemical
modifications to the cyanoguanidine moiety, as revealed by the dye uptake functional assays,
render the compounds synthesized of unlikely therapeutic use. These results have clearly
shown the importance of the cyanoguanidine moiety for potent P2X7R inhibition in this class
of compounds, but also confirmed that the incorporation of a nitrogen atom in the aromatic
system, namely at the 3-position, can be used to improve potency. Based on these results, our

current work in this area will be to return the cyanoguanidine moiety and develop
functionality around that to improve potency.
Acknowledgments
Work performed was supported by the NHMRC and in part by the European Union’s
Seventh Framework Programme [FP7/2007-2013] INMiND (Grant agreement No.
HEALTH-F2-2011-278850).
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Supplementary Material
1
Full synthetic details, H and 13C NMR spectra and experimental protocols for biological
assays are listed in the supplementary material.

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Pharmacological evaluation of a novel series
of urea, thiourea and guanidine derivatives
as P2X7 receptor antagonists
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Erick C. N. Wong, Tristan A. Reekie, Eryn L. Werry, James O’Brien-Brown, Sarah L. Bowyer and Michael
Kassiou