Synthesis and biological activity of a series of tetrasubstituted- imidazoles as P2X7 antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.05.018

A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced with an imidazole. A number of potent antagonists were identified and structure-activity relationships (SAR) were investigated both with respect to activity at the P2X₇ receptor and in vitro metabolic stability. Compound 10 was identified as a potent P2X₇ antagonist with reduced in vitro metabolism and high solubility.

Full text of DOI:10.1016/j.bmcl.2010.05.018

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4951–4954
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of a series of tetrasubstituted-imidazoles
as P2X7 antagonists
*
Robert J. Gleave , Daryl S. Walter, Paul J. Beswick, Elena Fonfria, Anton D. Michel, Shilina A. Roman,
Sac-Pham Tang
Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced
with an imidazole. A number of potent antagonists were identified and structure–activity relationships
(SAR) were investigated both with respect to activity at the P2X7 receptor and in vitro metabolic stability.
Compound 10 was identified as a potent P2X7 antagonist with reduced in vitro metabolism and high
solubility.
Received 8 April 2010
Revised 6 May 2010
Accepted 9 May 2010
Available online 25 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
P2X7
Ion channel
Pain
The P2X7 receptor is a member of the P2X family of ligand gated
ion channels. Like other members, it is activated by ATP and can act
as a non-specific cation channel. It is however unique amongst this
family, in that its activation can also lead to the release of biolog-
ically active interleukin-1-b (IL-1b),1 and the up-regulation of other
proinflammatory mediators.2
in vitro metabolic stability in rat and human liver microsomes.
As part of the SAR studies around this template, it was decided
to replace the central pyrazole core. If tolerated, it was hoped this
would lead to an improvement in the overall developability profile
of this series.
Imidazole was selected as an isostere which could retain the
relative position of the ring substituents and the ability to act as
a hydrogen bond acceptor which was believed to be important
for P2X7 antagonism.7 This ring replacement would also lead to a
lower lipophilicity8 and solubility might also be gained through in-
creased basicity.
2-Aryl imidazoles, as shown in Figure 2, were selected for initial
investigation. A synthetic route was required which would enable
variation of each of the imidazole substituents. Literature prece-
dent identified keto-amides as imidazole precursors which would
enable facile variation of the nitrogen substituent.9 Incorporation
of an ester functionality, if tolerated in the cyclisation reaction,
would enable rapid optimization of the benzylic moiety.
P2X7 receptors are up-regulated and activated after inflamma-
tory insults and are located within cells of the immune system.3 This
localization, together with the established role for IL-1b in inflam-
matory cascades, suggests a key role in a number of disease states.
Recent data from experiments with knock-out mice has shown, for
example, a lack of the development of a pain phenotype in models
of inflammatory and neuropathic pain.4 A second study has shown
an improved score for P2X7 ꢀ/ꢀ mice when compared to wild-type
mice in a monoclonal antibody induced arthritis model.5 Recently a
number of novel antagonists have been disclosed, together with
encouraging in vivo data in animal models of inflammatory bowel
disease, rheumatoid arthritis, neuropathic and inflammatory pain.6
This evidence suggests the development of a potent antagonist
could provide an exciting approach to the treatment of a number of
inflammatory disorders. High throughput screening against the
P2X7 receptor lead to the discovery of a series of N-aryl-pyrazoles,
for example compound 1 (Fig. 1).7 This compound showed encour-
aging potency against both the human and rat channels, however
showed moderate aqueous solubility (0.11 mg/mL) and lacked
N
N
H
N
O
F
Cl
1
* Corresponding author. Tel.: +44 1920 421057.
E-mail addresses: robert.j.gleave@gsk.com, robert.gleave1@googlemail.com (R.J.
Gleave).
Figure 1. Lead identified through screening. P2X7 pIC50
clearance (Cli, mL/min/g liver) human >50, rat 46.
h 7.4, r 7.0; in vitro
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.018

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R. J. Gleave et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4951–4954
R1
Table 1
R1
P2X7 inhibition at human and rat receptors together with metabolic stability for
compounds 2–9
N
O
HN
H
N
N
N
N
Ar
O
O
R2
O
Ar
O
O
R3
R2
X
R2
R3
Human (rat)
P2X7 pIC50
Human (Rat) Clib
(mL/min/g liver)
c log Pc
a
Figure 2. Proposed target molecule and key intermediate.
Cl
Cl
N
H
2
3
Me
Et
7.4 (6.6)
7.0 (6.8)
9.5 (5.1)
16 (13)
3.7
The desired intermediates were prepared by a two step proce-
dure (Scheme 1). Coupling of glycine or alanine with an acid chloride
was carried out in good yield with transient protection of the acid
terminus using chlorotrimethylsilane (TMSCl). The intermediate
carboxylic acids were converted using 1,10-carbonyldiimidazole
(CDI) to the imidazolides, to which was added the magnesium eno-
late of mono-ethyl malonate. Upon addition, a decarboxylation led
to the desired cyclisation precursors in good to excellent yield.
Imidazole formation was successfully achieved by heating the
keto-amide, amine and acetic acid in xylenes using a Dean–Stark
apparatus in a typically moderate to good yield. Subsequent at-
tempts to replicate this reaction using microwave irradiation
proved unsuccessful. Ester cleavage using lithium hydroxide and
amide coupling led to formation of the desired analogues.
Initially the aryl and R1 substituents were held constant as phe-
nyl and methyl, respectively. Imidazole nitrogen substituents (R2)
were evaluated in combination with a small set of benzylic groups
which had been identified during SAR analysis around the original
pyrazole template and had shown to be well tolerated by the P2X7
receptor7 (Table 1).
F
N
H
4.3
F
Cl
N
H
4
5
Me
Et
7.3 (5.8)
7.3 (6.2)
16 (21)
4.0
4.6
Cl
N
H
>50 (43)
N
H
6
7
8
9
Me
Et
7.3 (5.5)
7.3 (6.3)
7.0 (5.3)
6.8 (5.4)
22 (43)
>50 (>50)
4.7 (2.0)
—
4.0
4.6
3.2
3.7
Cl
N
H
Cl
F
F
F
F
Encouragingly, the imidazole analogue 2 of the lead pyrazole 1
was shown to be equipotent against the human receptor with a
small drop-off observed in the rat assay. An increase in metabolic
stability was also observed with a reduction in human and rat
in vitro clearance upon replacement of the pyrazole core.
Each of the selected benzylic substituents showed similar hu-
man potency. A greater variation was observed with the rat ortho-
N
H
Me
Et
F
F
N
H
Compounds tested in an ethidium bromide release assay.10 Values represent a
a
mean with n P2.
b
In vitro microsomal stability.11
R1
R1
O
a
c
Daylight c log P.
OH
OH
H2N
Ar
N
H
O
O
logue, for example, 2,3,4-trifluorobenzyl (8, 9) was poorly tolerated
by the rat receptor, whereas 2-chloro-4-fluorobenzyl analogues (2,
3) had a much smaller drop-off in potency between species.
Increasing the size of the N-substituent had minimal effect on po-
tency and led to a decrease in metabolic stability. An emerging cor-
relation between lipophilicity and metabolic stability was
observed with the least lipophilic analogue 8 showing the greatest
stability in the microsomal preparation.
A potential metabolic liability within this series was thought to
be the unsubstituted phenyl ring and it was proposed that intro-
duction of a halogen substituent, such as fluorine, could block oxi-
dative metabolism. A series of compounds were synthesized, using
a method analogous to that shown in Scheme 1, with a 2,4-difluo-
rophenyl moiety (Table 2). The addition of fluorine yielded little
change in the affinity of analogues to P2X7 and generally showed
little benefit in terms of metabolic stability. An exception was com-
pound 10 which showed a good combination of potency at each
species and low to moderate metabolic turnover in both human
and rat.
b,c
O
O
R1
d
O
Ar
NH
N
O
O
R1
Ar
N
R2
O
e,f
R1
O
N
NH
R3
Ar
N
R2
A third set of analogues were prepared and evaluated in which
the potentially metabolically vulnerable R1 substituent at C4 was
replaced by hydrogen (Table 3). Removal of a methyl at this posi-
tion was detrimental to human potency in some cases, although
Scheme 1. (a) Reagents and conditions: (i) TMSCl, NEt3, DCM, 0 °C to rt, 1 h; (ii)
ArCOCl, 0 °C to rt, 18 h, 25–80%; (b) CDI, THF, rt 5 h; (c) EtO2CCH2CO2H, Mg(OEt)2, rt
18 h, 89–98%; (d) R2NH2, AcOH, xylenes, reflux, 2.5 h, 16–78%; (e) LiOH, THF, H2O,
0–5 °C, 4 h; (f) R3NH2, EDCI, HOBT, N-ethyl morpholine, DMF, rt, 18 h.

R. J. Gleave et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4951–4954
4953
Table 2
Table 4
P2X7 inhibition at human and rat receptors together with metabolic stability for
Solubility profile of compounds 1 and 10 (lg/mL, 1 h)
compounds 10–16
Aqueous media
pH
Solubility
F
1
10
N
Water
110
100
120
410
920
900
370
F
O
Fed state simulated intestinal fluid (FeSSIF)
Fasted state simulated intestinal fluid (FaSSIF)
Simulated gastric fluid
5
6.5
1.2
N
R3
R2
>1000
R2
R3
Human (rat)
P2X7 pIC50
Human (rat) Clib
(mL/min/g liver)
c log Pc
a
The hydrochloride salt of compound 10 was selected for further
evaluation of its developability profile since it showed the best
overall combination of potency and metabolic stability in both spe-
cies. The compound showed a moderate level of protein binding
(HSA 91% bound) and was not a potent competitive inhibitor of
Cl
Cl
N
H
10
11
Me
Et
7.4 (6.5)
7.0 (6.8)
3.0 (3.4)
20 (13)
4.0
F
CYP1A2, 2C9, 2C19, 2D6 and 3A4 with an IC50 of 5.5
lM at 3A4
N
H
4.6
and >10
l
M at each of the other isoforms.12 Solubility was assessed
F
in water and a number of simulated physiological fluids (Table
4).13 The data showed an improved profile compared with the pyr-
azole lead with high solubility in water, gastric fluid and simulated
fed state fluid. The compound showed moderate solubility in the
fasted state fluid, which may be a reflection of the fluid having a
pH value close to the measured pKa of compound 10 (6.47).
Compound 10 was also tested against 35 other ion channels,
receptors and enzymes. No significant off-target activity was ob-
served. Following on from this positive data the in vivo pharmaco-
kinetics of 10 were assessed in the rat. The compound was
administered orally at 3 mg/kg.14 Promisingly, oral exposure was
Cl
N
H
12
13
Me
Et
7.5 (6.2)
7.4 (6.4)
16 (16)
—
4.3
4.9
Cl
N
H
N
H
14
15
16
Et
7.3 (6.2)
7.2 (<5)
6.9 (6.0)
>50 (>50)
4.9
3.5
4.0
Cl
F
F
observed with
a Cmax of 0.43 lM ( 0.04), AUC (0–6 h)/dose
F
F
N
H
Me
Et
—
6.8 min kg/L ( 0.5) and a Tmax of 0.5 h indicating rapid absorption.
In summary, a combination of homologation and cyclisation
synthetic steps proved to be a versatile route to a number of tet-
ra-substituted imidazoles. This enabled a range of analogues to
be prepared for SAR studies. Replacement of the central core of
the pyrazole screening hit 1 with an imidazole was well tolerated.
Potency was successfully retained across a range of analogues at
both the human and rat receptor, whilst aqueous solubility was in-
creased. Several strategies were employed to block potential sites
of metabolism and to increase the in vitro metabolic stability.
Compound 10 was identified with an encouraging combination
of P2X7 inhibition and rat oral exposure and therefore with a suit-
able profile to take forward into in vivo PD studies.
F
F
N
H
18 (3.6)
a–c
See footnotes to Table 1 for details.
Table 3
P2X7 inhibition at human and rat receptors together with metabolic stability for
compounds 17–19
F
N
F
O
References and notes
N
R2
R3
1. Colomar, A.; Marty, V.; Médina, C.; Combe, C.; Parnet, P.; Amédée, T. J. Biol.
Chem. 2005, 278, 30732.
2. Hughes, J. P.; Hatcher, J. P.; Chessell, I. P. Purinergic Signalling 2007, 3, 163.
3. Collo, G.; Neidhart, S.; Kawashhima, E.; Kosco-Vilbois, M.; North, R. A.; Buell, G.
Neuropharmacology 1997, 36, 1277.
R2
R3
Human (rat)
P2X7 pIC50
Human (rat) Clib
(mL/min/g liver)
c log Pc
a
Cl
Cl
4. Chessell, I. P.; Hatcher, J. P.; Bountra, C.; Michel, A. D.; Hughes, J. P.; Green, P.;
Egerton, J.; Murfin, M.; Richardson, J.; Peck, W. L.; Grahames, C. B. A.; Casula, M.
A.; Yiangou, Y.; Birch, R.; Anand, P.; Buell, G. N. Pain 2005, 114, 386.
5. Labasi, J. M.; Petrushova, N.; Donovan, C.; McCurdy, S.; Lira, P.; Payette, M. M.;
Brissette, W.; Wicks, J. R.; Audoly, L.; Gabel, C. A. J. Immunol. 2002, 168, 6436.
6. Romagnoli, R.; Baraldi, P. G.; Cruz-Lopez, O.; Lopez-Cara, C.; Preti, D.; Borea, P.
A.; Gessi, S. Expert Opin. Ther. Targets 2008, 12, 647.
7. Chambers, L. J.; Stevens, A. J.; Moses, A. P.; Michel, A. D.; Walter, D. S.; Davies, D.
J.; Livermore, D. G.; Fonfria, E.; Demont, E. H.; Vimal, M.; Theobald, P. J.;
Beswick, P. J.; Gleave, R. J.; Roman, S. A.; Senger, S. Bioorg. Med. Chem. Lett. 2010.
doi:10.1016/j.bmcl.2010.03.096.
N
H
17
Me
6.7 (6.6)
2.7 (6.7)
3.8
F
N
H
18
19
Et
6.3 (6.4)
7.2 (6.2)
—
4.3
4.1
F
Cl
N
H
Me
10 (18)
8. c log D7.4 (ACD) compound 1 = 3.4, compound 2 = 2.2.
9. Gordon, T. D.; Singh, J.; Hansen, P. E.; Morgan, B. A. Tetrahedron Lett. 1993, 34,
1901.
a–c
See footnotes to Table 1 for details.
10. Ethidium bromide assay format described in patent application WO 2007
141267 A1. Briefly, the assay is carried out in HEK293 cells expressing
recombinant P2X7 receptors. ATP activation of the channel leads to an opening
of in cell wall and accumulation of ethidium bromide within the cell, levels of
which are determined using measurement of fluorescence. Inhibition of the
ATP response by a novel compound may be measured across a range of
when 2-methyl-3-chlorobenzyl is introduced (19) potency is main-
tained, indicating further evaluation of SAR may be warranted. The
R1 substituent seems unlikely to be a site of metabolism as its re-
moval has little effect on microsomal stability.
concentrations to generate an IC50
.
11. Clarke, S. E.; Jeffrey, P. Xenobiotica 2001, 31, 591.

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R. J. Gleave et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4951–4954
12. CYPEX assay using fluorescent substrates. Inhibition of CYP3A4 observed using
diethoxyflourescein as a substrate at 1 M.
13. Galia, E.; Nicolaides, E.; Horter, D.; Lobenberg, R.; Reppas, C.; Dressman, J. B.
Pharm. Res. 1998, 15, 698.
14. Compound 10 was dosed to three rats orally by gavage at a target dose of 3 mg/
kg. The compound was prepared, on the day of dosing, in 1% (w/v)
5 mL/kg. Serial blood samples were taken from each rat up to 6 h after dose
administration. Diluted blood samples were analysed for parent compound by
LC/MS/MS. All experiments were performed in accordance with the United
Kingdom Animals (Scientific Procedures) Act, 1986 under Project Licence as
well as under the review and approval of the GlaxoSmithKline Procedures
Review Panel. GlaxoSmithKline safety regulations were adhered to at all
times.
l
methylcellulose aq at
a concentration of 0.6 mg/mL and administered at