Synthesis and structure-activity relationships of a series of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor

Article abstract of DOI:10.1016/j.bmcl.2010.03.096

High-throughput screening identified compound 1 as a potent P2X₇ receptor antagonist suitable for lead optimisation. Structure-activity relationships (SAR) of a series of (1H-pyrazol-4-yl)acetamides were investigated and compound 32 was identif

Full text of DOI:10.1016/j.bmcl.2010.03.096

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3161–3164
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of a series of (1H-pyrazol-4-yl)a-
cetamide antagonists of the P2X7 receptor
*
Laura J. Chambers , Alexander J. Stevens, Andrew P. Moses, Anton D. Michel, Daryl S. Walter,
David J. Davies, David G. Livermore, Elena Fonfria, Emmanuel H. Demont, Mythily Vimal, Pam J. Theobald,
Paul J. Beswick, Robert J. Gleave, Shilina A. Roman, Stefan Senger
Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening identified compound 1 as a potent P2X7 receptor antagonist suitable for lead
optimisation. Structure–activity relationships (SAR) of a series of (1H-pyrazol-4-yl)acetamides were
investigated and compound 32 was identified as a potent P2X7 antagonist with enhanced potency and
favourable physicochemical and pharmacokinetic properties.
Received 22 February 2010
Revised 25 March 2010
Accepted 25 March 2010
Available online 30 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
P2X7
P2X7 antagonist
Lead optimisation
The P2X7 receptor is an ATP-gated ion-channel which is ex-
pressed in cells of the hematopoietic lineage,1,2 (e.g., macrophages,
microglia, mast cells, and lymphocytes) and has been the subject of
considerable interest in the recent scientific and patent literature
as a target for potential therapeutic intervention.2–8 Activation of
P2X7 receptors has numerous effects including non-specific cation
flux;9 subsequent formation of a membrane pore, which can facil-
itate the transit of relatively large molecules;10 and activation and
release of pro-inflammatory cytokines such as interleukin-1b (IL-
1b).11 Furthermore, the P2X7 receptor is also expressed in the cen-
tral nervous system12 and has been shown to mediate glutamate
release in glial cells.13
The localisation of the P2X7 receptor to key cells of the immune
system, coupled with its ability to release important inflammatory
mediators from these cells and the emerging role of purinergic sig-
naling in the central nervous system,14 suggests a potential role for
P2X7 receptor antagonists in the treatment of a wide range of con-
ditions including inflammatory disease, pain and neurodegenera-
tive disorders. Recent preclinical in vivo studies have directly
implicated the P2X7 receptor in both inflammatory and neuro-
pathic pain15 and small molecule P2X7 antagonists have been dem-
onstrated to be efficacious in animal models of neuropathic
pain.16–19 Consequently, P2X7 antagonists are of interest as novel
therapeutic agents in a number of disease states. Two compounds
of undisclosed structure, AZD-9056 and CE-224535, have pro-
gressed to early proof-of-concept clinical trials in rheumatoid
arthritis patients.20,21
In order to identify novel P2X7 chemotypes, a high-throughput
screen (HTS) of the GSK compound collection was carried out. This
led to the discovery of compound 1 (Fig. 1), which exhibited a good
level of in vitro potency against both the human and rat ortho-
logues of P2X7 but displayed poor in vitro stability in microsomal
preparations.
The synthesis of this compound was achieved in four synthetic
steps as shown in Scheme 1. The first step involved alkylation of
acetylacetone to produce a functionalized 1,3-diketone. This was
followed by pyrazole formation with phenylhydrazine, ester
hydrolysis and amide coupling. Variation of the hydrazine or amine
components allowed rapid preparation of analogues with two
main points of diversity. Compound 1 demonstrated encouraging
levels of potency and low molecular weight; highlighted by the
promising ligand efficiency22 (LE) and ligand lipophilicity effi-
N
Cl
O
N
N
H
F
1
Figure 1. Compound 1, a P2X7 antagonist identified from HTS. Relevant data:
Human P2X7 pIC50 7.4; Rat P2X7 pIC50 7.0; Rat in vitro clearance = 46 mL/min/g
* Corresponding author. Tel.: +44 1279643070; fax: +44 1279643210.
E-mail address: laura.j.chambers@gsk.com (L.J. Chambers).
liver; cLog D
MW = 372; LE = 0.39; LLE = 4.0.
@ pH 7.4 = 3.4; Solubility in water = 110 lg/mL (1 h timepoint);
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.096

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L. J. Chambers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3161–3164
pound 9 illustrated that an aromatic ring was not necessarily re-
quired for activity. Overall, the changes made in this part of the
molecule suggested that the amide substituent was binding into
a relatively large pocket and that a moderate level of antagonism
could be obtained if this was filled appropriately. For instance,
O
O
O
O
a
OEt
O
the
a,
a0-dimethyl benzamide analogue (8) retained a good level
of inhibition. Regarding the benzylic group, the di-halo substitu-
tion patterns of compounds 5 and 6 appeared optimal, however
neither of these modifications offered a significant advantage when
compared to compound 1.
b
N
c, d
N
O
O
R1
N
In order to determine the importance of the amide group to
receptor binding, a number of alternative linkers were prepared25
(exemplified by compounds shown in Table 2). Methylation of
the amide nitrogen (compound 11) led to a marked loss of inhibi-
tion, which suggested that either the N–H was participating in an
important interaction, or that the secondary amide conformation
was preferred. Interestingly, the urea analogue (compound 14)
was tolerated, although potency was reduced somewhat compared
to compound 1. A range of heterocyclic amide isosteres (exempli-
fied by 15) also maintained a degree of activity, which suggested
that a hydrogen bond donor in this region of space is not an abso-
lute requirement. The H-bond acceptor and appropriate placement
of the amide group appeared to be the important factors in retain-
ing inhibitory activity in this series.
R1
N
R2
N
OEt
H
Scheme 1. Synthesis of pyrazolacetamide analogues. Reagents and conditions: (a)
ethyl bromoacetate, KOH, 1,4-dioxane, water, rt, ca. 16 h; (b) (i) R1NHNH2, AcOH,
EtOH, reflux, 2 h; or (ii) R1NHNH2ꢀHCl, NaOAc, EtOH, reflux, 2 h; (c) LiOH, H2O, THF,
50 °C, 2 h; (d) R2NH2, EDAC, HOBT, 4-ethylmorpholine, DCM, DMF, rt, ca 16 h.
Table 1
Structure–activity data for a range of 2-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)-
acetamides
N
O
N
R
To investigate the contribution of the pyrazole ring to receptor
binding, a number of heterocyclic replacements were prepared26
(Table 3). The results obtained for compounds 16 and 17 demon-
strated that removal of one of the methyl groups from the pyrazole
resulted in a reduction of potency so, where possible, methyl sub-
stituents were retained in analogous heterocycles. Most alternative
heterocycles were poorly tolerated (compounds 18–21), with com-
pound 22 being a notable exception.27 Although interpretation was
complicated by missing methyl groups in some instances, there
was a suggestion that one of the pyrazole/imidazole ring nitrogen
atoms participates in a binding interaction with the receptor, one
that the other ring systems are unable to access.
a
Compd
R
Human P2X7 pIC50
NH
2
<6.0
Cl
3
4
6.0
NH
O
<6.0
NH
NH
Cl
Table 2
5
6
7
7.1
Structure–activity data for a range of amide linker variations
N
Cl
Cl
N
R
X
Cl
Cl
7.6
NH
NH
a
Compd
X
R
Human P2X7 pIC50
<6.0
Cl
O
Cl
Cl
Cl
Cl
Cl
66.0b
N
11
F
F
H
N
8
9
6.9
NH
NH
12
13
14
15
<6.0
<6.0
6.6
O
6.8
H
N
O
10
<6.0
NH
F
N
O
a
Data generated using an ethidium bromide release assay (see Ref. 24), reporting
N
H
N
H
an average value of n >3.
b
This value is an average of four individual values: <6.0; 6.2; <6.0; 6.0.
F
O
ciency23 (LLE) values. This compound was therefore selected as a
promising starting point for further optimisation.
N N
5.9
Modifications of the amide substituent (R2 in Scheme 1) were
explored first and are summarized in Table 1. Although compounds
2–7 in Table 1 are benzamides, the moderate inhibition of com-
Cl
a
Data generated using an ethidium bromide release assay (see Ref. 24), reporting
an average value of n >3.

L. J. Chambers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3161–3164
3163
Table 3
Structure–activity data for a range of heterocycle variations
Table 4
Structure–activity data for a range of N-substituents
Cl
N
O
Cl
O
R N
X
N
N
H
H
F
F
a
Compd
X
Human P2X7 pIC50
a
Compd
23
R
Human P2X7 pIC50
Rat Clib mL/min/g liver
N
N
N
7.2
7.0
12.0
—
Cl
16
6.8
6.8
24
MeO
O
N
17
18
25
26
8.0
7.3
3.2
1.8
H2N
O
N
N
<6.0
N
N
O
O
27
28
6.5
6.9
>50
19
20
21
<6.0
<6.0
<6.0
N
S
N
20.0
N
N
29
30
6.5
6.7
5.7
2.4
N
HO
S
N
31
32
Me
H
6.8
7.7
3.5
0.58
22
7.3
a
Data generated using an ethidium bromide release assay (see Ref. 24), reporting
N
an average value of n >3.
b
Microsomal clearance method described in Ref. 29.
a
Data generated using an ethidium bromide release assay (see Ref. 24), reporting
an average value of n >3.
N
Cl
O
HN
N
As an orally bioavailable therapeutic agent was ultimately re-
quired, it was important to understand and address the high
in vitro clearance observed with early representatives of this series.
H
F
32
28
In silico profiling of compound (1) using the METASITE program
Figure 2. Lead compound from the optimisation of compound 1. Relevant data:
postulated the phenyl ring as a highly likely site of metabolism.
To test this hypothesis and to probe the SAR in this region of space,
ring substitutions and ring replacements were explored. As shown
in Table 4, most of these changes resulted in a good level of inhibi-
tion, and the binding site appeared to be tolerant of both lipophilic
and hydrophilic groups in this position. 4-Substitution of the phe-
nyl ring (compounds 23 and 25), or replacement by pyrimidine
(26) also successfully reduced the in vitro clearance. Incorporating
a hydrophilic group seemed to successfully lower the clearance;
however compound 23 is a notable exception, which may be ex-
plained by the 4-substituent blocking a potential site of oxidation.
Homologation to a benzyl group (27) led to a reduction of activity,
although the analogous pyridyl methyl group (28) did show a
slight reduction to the in vitro clearance.
Human P2X7 pIC50 7.7; Rat P2X7 pIC50 6.8; Rat in vitro clearance 0.58 mL/min/g;
cLog D @ pH 7.4 = 1.6; Solubility in water = 732 lg/mL (1 h timepoint); MW = 296;
LE = 0.53; LLE = 6.1.
Table 5
Summary of the pharmacokinetic profile of compound 32
Route of administration Dose Property
Measured value
IVa
1 mg/kg CLb (mL/min/kg)
T1/2 (h)
15
1.7
1.6
VDss (L/kg)
POb
3 mg/kg Tmax (h)
1.0
Cmax
(
lM)
2.92 0.33
AUC/dose (minꢀkg/L) 58 3.6
Fpo 90%
Small alkylated derivatives (compounds 29–31) retained a de-
gree of activity, and offered some significant metabolic advantages,
but removal of the N-substituent (compound 32) provided the
most significant improvements to both inhibition and metabolic
stability. Removing the pyrazole nitrogen substituent (R1 in
Scheme 1), which does not appear to make a significant interaction
with the human receptor, significantly lowered both the molecular
weight and the cLog D30 of the molecule (see Fig. 2). This can be
demonstrated by the improved ligand efficiency and high ligand
lipophilicity efficiency of compound 32. This modification also re-
sulted in the lowest level of in vitro metabolism observed in the
series to this point, as well as a marked improvement in solubility
when compared to 1. Compound 32 was selected for further pro-
a
Compound 32 was dissolved in 0.9% (w/v) saline containing 10% (w/v) HPB
(hydroxypropyl-b-cyclodextrin) and 2% (v/v) DMSO at a target concentration of
0.2 mg/mL. It was administered as a 1 h intravenous infusion to 1 rat to achieve a
target dose of 1 mg/kg.
b
Compound 32 was dissolved in 1% (w/v) methylcellulose in water at a concen-
tration of 0.6 mg/mL and dosed by oral gavage to 3 rats at a target dose of 3 mg/kg.
gression to determine whether the improved in vitro properties
translated to a desirable in vivo profile31 (Table 5).
Following intravenous administration (1 mg/kg), compound 32
had a low blood clearance (15 mL/min/kg, approximately 17% of li-
ver blood flow). The steady-state volume of distribution (VDss) was

3164
L. J. Chambers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3161–3164
17. Broom, D. C.; Matson, D. J.; Bradshaw, E.; Buck, M. E.; Meade, R.; Coombs, S.;
Matchett, M.; Ford, K. K.; Yu, W.; Yuan, J.; Sun, S. H.; Ochoa, R.; Krause, J. E.;
Wustrow, D. J.; Cortright, D. N. J. Pharmacol. Exp. Ther. 2008, 327, 620.
18. Perez-Medrano, A.; Donnelly-Roberts, D. L.; Honore, P.; Hsieh, G. C.; Namovic,
M. T.; Peddi, S.; Shuai, Q.; Wang, Y.; Faltynek, C. R.; Jarvis, M. F.; Carroll, W. A. J.
Med. Chem. 2009, 52, 3366.
19. Nelson, D. W.; Gregg, R. J.; Kort, M. E.; Perez-Medrano, A.; Voight, E. A.; Wang,
Y.; Grayson, G.; Namovic, M. T.; Donnelly-Roberts, D. L.; Niforatos, W.; Honore,
P.; Jarvis, M. F.; Faltynek, C. R.; Carroll, W. A. J. Med. Chem. 2006, 49, 3659.
20. McInnes, I. B.; Snell, N. J.; Perrett, J. H.; Parmar, H.; Wang, M. M.; Astbury, C.
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1.6 L/kg, indicative of good distribution into tissues, and a half-life
of 1.7 h was recorded. Compound 32 also exhibited good oral
absorption following a 3 mg/kg dose. Comparison of the intrave-
nous and oral studies resulted in an estimated oral bioavailability
of 90%.
In conclusion, it was demonstrated that the pyrazole N-substi-
tuent of the initial hit 1 offered the greatest opportunity to im-
prove the overall profile of this series. Compound 32 was
subsequently identified and shown to be a potent P2X7 antagonist
at both human and rat orthologues; with good oral absorption, low
in vivo clearance and an excellent physicochemical profile.
21. ClinicalTrial.gov Identifier: NCT00628095.
22. Abad-Zapatero, C. Expert Opin. Drug Disc. 2007, 2, 469–488.
23. (a) Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881; The same
concept was independently proposed by researchers at Pfizer and termed lipE:
(b) Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Monica Correia, A.; Owen, D. R.;
Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Bioorg. Med. Chem. Lett. 2009, 15,
4406.
24. Potencies are measured using a competition experiment using the natural
ligand ATP. ATP activation leads to formation of a large opening in the cell wall
and accumulation of ethidium bromide dye within the cell, levels of which are
measured using fluorescence. All test compounds had Imax values of 100%. Full
details of the assay protocol can be found in patent application WO 2007
141267 A1.
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31. 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.