Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor

Article abstract of DOI:10.1016/j.bmcl.2010.07.033

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X₇ receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X₇ antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.

Full text of DOI:10.1016/j.bmcl.2010.07.033

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5080–5084
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and structure–activity relationships of a series of pyroglutamic
acid amide antagonists of the P2X7 receptor
Muna H. Abdi, Paul J. Beswick, Andy Billinton, Laura J. Chambers, Andrew Charlton, Sue D. Collins,
Katharine L. Collis, David K. Dean, Elena Fonfria, Robert J. Gleave, Clarisse L. Lejeune, David G. Livermore,
Stephen J. Medhurst, Anton D. Michel, Andrew P. Moses, Lee Page, Sadhana Patel, Shilina A. Roman,
*
Stefan Senger, Brian Slingsby, Jon G. A. Steadman, Alexander J. Stevens, Daryl S. Walter
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 computational lead-hopping exercise identified compound 4 as a structurally distinct P2X7 receptor
antagonist. Structure–activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4
were investigated and compound 31 was identified as a potent P2X7 antagonist with excellent in vivo
activity in animal models of pain, and a profile suitable for progression to clinical studies.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 June 2010
Revised 6 July 2010
Accepted 8 July 2010
Available online 14 July 2010
Keywords:
P2X7
Antagonist
Pain
SAR
P2X7, an ATP-gated ion-channel,1–3 controls the activation and
release of pro-inflammatory cytokines such as interleukin-1b (IL-
1b).4 Antagonists of the P2X7 receptor can modulate such re-
sponses in P2X7-expressing cells in both the immune and central
nervous systems,5 suggesting a potential role for P2X7 receptor
antagonists in the treatment of a wide range of conditions. In par-
ticular, preclinical in vivo studies have directly implicated the P2X7
receptor in pain states6 and small molecule P2X7 antagonists have
been demonstrated to be efficacious in animal models of neuro-
pathic pain.7–10 Two compounds (AZD-9056 and CE-224535), have
progressed to early proof-of-concept clinical trials in rheumatoid
arthritis patients11,12 and whilst both of these compounds have
now been discontinued the question of whether a P2X7 antagonist,
with the appropriate physicochemical and pharmacokinetic pro-
file, could be therapeutically efficacious in treating human pain
states remains unanswered.
a potent antihyperalgesic agent in both the rat acute complete Fre-
und’s adjuvant (CFA) model of inflammatory pain19 and the knee
joint model of chronic inflammatory pain.20 However, compounds
of this class were subsequently found to be prone to time-depen-
dently inhibit the CYP3A421 isozyme and routes of metabolism
studies indicated oxidation of the methyl substituents as the most
likely cause.
Whilst this issue could be addressed (unpublished data) by
replacing the methyl groups with alternative substituents (e.g.,
N
Cl
O
N
N
H
F
1
O
Cl
N
In two earlier publications13 we described the optimization of a
series of (1H-pyrazol-4-yl)acetamides, derived from hit compound
Cl
O
N
HN
N
H
O
N
Cl
1
(Fig. 1) which had been identified via high-throughput
Cl
3
screening. From this series, compound 2, N-[(2,4-dichloro-
phenyl)methyl]-2-(3,5-dimethyl-1H-pyrazol-4-yl)acetamide, was
selected for evaluation in in vivo models of pain and shown to be
2
Cl
14
Figure 1. Compound 1: human P2X7 pIC50 7.4; rat P2X7 pIC50 7.0; rat in vitro
clearance15 = 46 mL/min/g liver; c log D16 at pH 7.4 = 3.4; MW = 372; LE = 0.3917
;
LLE18 = 4.0; compound 2: human P2X7 pIC50 8.1; Rat P2X7 pIC50 7.1; rat in vitro
clearance 1.3 mL/min/g; c log D at pH 7.4 = 2.7; MW = 312; LE = 0.55; LLE = 5.4;
compound 3: human P2X7 pIC50 6.9; rat P2X7 pIC50 6.5; rat in vitro clearance
>50 mL/min/g; c log D at pH 7.4 = 4.4; MW = 421; LE = 0.35; LLE = 2.5.
* Corresponding author.
E-mail addresses: daryl.s.walter@gsk.com, darylwmail-web@yahoo.co.uk (D.S.
Walter).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.033

M. H. Abdi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5080–5084
5081
CF3) this usually resulted in a significant loss of potency. In order to
O
address the potential developability risk presented by this observa-
tion we sought to identify alternative classes of P2X7 antagonists
which were free of this issue. Compound 3 (see Fig. 1) was also
identified as a P2X7 antagonist following our initial high-through-
put screening of the GSK compound collection and, whilst this
template was less attractive in general (higher molecular weight,
lower ligand efficiency (LE)), it presented the opportunity to iden-
tify common pharmacophoric features that are shared by different
P2X7 antagonist chemotypes. In order to identify these common
pharmacophoric features a simple overlay22 of compounds 1 and
3 was performed. As can be seen in Figure 2 (circled) the resulting
superposition of the two molecules indicated that the carbonyl
oxygens of the two (acyclic) amide groups as well as the pyrazole
nitrogen in 1 and the oxygen atom of the carbonyl in the pyridone
ring of 3, respectively, can interact with the same putative hydro-
gen-bond donor feature of the P2X7 receptor in each case.
O
N
H
O
c
O
O
O
O
O
O
a
b
O
O
N
R
O
H2N
N
H
R
O
O
d,e
H
N
R1
O
N
R
O
This observation led us to adopt the hypothesis that two suit-
ably positioned hydrogen-bond donor features are required (or at
least beneficial) when trying to achieve activity at the P2X7 recep-
tor in a low molecular weight template. Since only limited SAR was
available (particularly around compound 3) and we felt that we did
not have sufficient information to build a pharmacophore that
would be fit for virtual screening we decided to use fingerprint-
based similarity searching in order to identify additional
chemotypes of interest.23 After applying property filters (e.g.,
hydrogen-bond acceptor count P2, based on the overlay shown
in Fig. 2) hits of interest were selected by stepping through the
framework clusters and visually inspecting the members of the
individual clusters.24 A preference was given to molecules where
the bond count between the two hydrogen-bond acceptor atoms
was similar to that found in the query molecules (i.e., 5 in 1 and
6 in 3).
Scheme 1. Synthesis of pyroglutamic acid amide analogues. Reagents and condi-
tions: (a) aldehyde or ketone, AcOH, NaBH4, MeOH, 0 °C, ca. 1 h; (b) toluene, reflux,
Dean–Stark, ca. 16 h; (c) NaHMDS, alkyl halide, THF, À78 °C to room temperature,
ca. 1 h; or aryl bromide, Pd2(dba)3, Cs2CO3, Xantphos™, toluene, reflux, ca. 18 h (d)
2M NaOH (aq), MeOH, 0 °C to room temperature, ca. 4 h (e) R1NH2, EDAC, HOBT,
DCM/DMF (ꢀ3:1), rt, ca. 16 h.
weight and good ligand efficiency of 4 were particularly appealing.
Synthesis of the (S)-enantiomer of 4 (i.e., 4a) confirmed the activity
of the initial sample and we resolved to prepare additional ana-
logues to profile this template more thoroughly.
Analogues were, in the first instance, prepared using one of two
methods as shown in Scheme 1.27
Reductive alkylation of the dimethyl ester of glutamic acid fol-
lowed by thermal cyclisation gave the required N-substituted
methyl pyroglutamates. Alternatively these key intermediates
could be accessed by alkylation of methyl pyroglutamate with an
alkyl halide or arylation using Buchwald coupling conditions. Sub-
sequent saponification and amide coupling then provided the
N-substituted pyroglutamic acid amides in good overall yields.
Using the above methods we first prepared a number of
analogues which retained the 2-Cl, 4-F-benzylamide group and
explored the structure–activity relationships (SAR) around the N-
alkyl substituent in the five-membered ring (Table 1). Removing
the isopropyl group in 4a resulted in a complete loss of activity
(5) whereas replacement by methyl, ethyl and propyl gave
The most promising hit with a ligand efficiency = 0.4317 was
pyroglutamic amide 4 (see Fig. 3), one of only nine compounds se-
lected for screening, and ranked 595th amongst the 2000 top-
ranked hits from the 3-point pharmacophoric fingerprint similarity
search.26 Whilst the human potency was good, the potency at the
rat receptor was lower than preferred, but the low molecular
Table 1
Structure–activity data for pyroglutamic acid amide analogues of compound 4
F
H
O
N
N
R
O
Cl
Compd
R
Human P2X7
pIC50
Rat CLib
(mL/min/g) liver
Ligand
efficiency17
a
Figure 2. Overlay of compounds 1 (in orange) and 3.
5
6
7
8
9
10
11
12
13
H
Me
Et
n-Pr
2-Me-propyl
Benzyl
Cyclobutyl
Cyclopentyl
Phenyl
<6
—
—
<0.5
—
—
—
2.3
—
—
—
7.0
7.5
6.8
6.4
<6
7.6
6.3
6.1
0.50
0.52
0.45
0.40
—
0.47
0.38
0.35
F
F
H
N
H
N
O
O
N
N
O
Cl
O
Cl
4a
4
a
Data generated using an ethidium bromide release assay (Ref. 14), reporting an
Figure 3. Compound 4: human P2X7 pIC50 6.5; rat P2X7 pIC50 <5; c log D at pH
7.4 = 2.6; MW = 312; LE = 0.4317; LLE = 3.9.18 Compound 4a: human P2X7 pIC50 7.0;
rat P2X7 pIC50 5.8; rat in vivo clearance 31 mL/min/kg.
average value of n >3.
b
Microsomal clearance method described in Ref. 15.

5082
M. H. Abdi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5080–5084
Table 3
analogues (6, 7 and 8) with similar or slightly better potency in the
case of the ethyl group.
Structure–activity data for analogues of compound 4
a
Larger groups than this tended to have lower activity (e.g., 9)
and substitution with benzyl (10) was particularly detrimental to
activity. Small cycloalkyl groups such as cyclobutyl (11) were as
active as the ethyl analogue and this activity again dropped off
as the ring size was increased (e.g., 12) or if aromatic groups
(e.g., 13) were introduced.
Encouragingly, in the cases where data was generated, com-
pounds in this series demonstrated low levels of in vitro clearance
in rat microsomes (see Table 1) and showed very little propensity
to inhibit any of the CYP450 enzyme isoforms tested (i.e., 1A2, 2C9,
2C19, 2D6, and 3A4—data not shown). There was also no evidence
of time-dependent inhibition of the CYP450 3A4 enzyme thus
addressing the concerns with the pyrazole series exemplified by
compound 2.13
To explore the SAR of the right-hand benzamide we chose to use
the N-ethyl-substituted analogue 7 (the most ligand efficient ana-
logue prepared up to this point) as the basis for comparison of dif-
ferent amide substituents. A subset of the compounds prepared are
listed in Table 2. The cyclohexylmethyl 14 and simple benzyl 15
amides were essentially inactive. The adamantyl methyl group
16 (reported9 to be a preferred substituent in a number of other lit-
erature examples of P2X7 antagonists) was more active but not as
good as the original 2-Cl, 4-F-benzylamide. Lipophilic substitution
of the benzyl ring resulted in compounds with modest activity,
regardless of the position substituted (see Cl-substituted analogues
17, 18 and 19). A CF3 group in the 3-position 20 was, however,
more active than the 2-substituted analogue 21. More polar sub-
stituents (e.g., 2-OMe, 22) of the benzyl ring were not well
tolerated.
Compd
R
Human P2X7 pIC50
F
H
N
O
7
7.5
N
O
Cl
F
H
N
26
27
28
29
30
<6
N
O
Cl
F
H
N
7.0
<6
N
O
Cl
O
O
Cl
F
N
N
N
N
O
Cl
Cl
Cl
H
N
O
O
6.8
7.5
O
O
F
H
N
a
Data generated using an ethidium bromide release assay (Ref. 14), reporting an
average value of n >3.
to identify this series, acylating the ring nitrogen returned most
of the activity (see 27), possibly suggesting an H-bond with the
carbonyl via an interaction along a slightly different vector. The
amide NH also appeared to be important for receptor binding as
methylation led to inactive compounds (e.g., 28 cf. the analogous
NH amide, pIC50 = 7.9). Inverting the stereochemistry of compound
7 led to only a modest reduction in activity (29), whilst expanding
the five-membered ring to a six-membered ring (e.g., 30) was well
tolerated resulting in equipotent analogues.
Combination of the findings summarized above informed the
preparation of additional analogues and compound 31 (see
Fig. 4) subsequently emerged as a clear frontrunner with the best
overall property profile. This compound incorporates the favoured
2-Cl-3-CF3-benzylamide moiety and the methyl substituent on the
N atom of the lactam. Whilst the latter was not generally the most
potent substituent it tended to provide compounds with the best
balance of potency and pharmacokinetic properties.
Significant improvements in potency and ligand efficiency rela-
tive to the initial hit compound 4 were observed with disubstituted
benzylic amides in which both of the 2- and 3- or 4-positions were
substituted with
a lipophilic group. The 2,3- and 2,4-dic-
hlorobenzylamides, 23 and 24, were considerably more potent
than the monosubstituted analogues and the 2-Cl, 3-CF3-benzyla-
mide 25, in particular, was the most potent analogue prepared thus
far.
Analogues incorporating modifications to the pyrrolidinone ring
are listed in Table 3. When the ring-carbonyl oxygen in 7 was re-
moved, to give the corresponding proline derivative 26, most of
the activity was lost. Whilst this is not conclusive proof of an H-
bonding interaction as postulated in the initial hypothesis used
Table 2
Structure–activity data for a range of N-ethyl pyroglutamides
Compound 31 had excellent potency at the human receptor but
considerably lower affinity for the rat receptor (ꢀ100-fold lower);
low clearance in rat and human liver microsomes; no appreciable
inhibition of the CYP450 enzyme isoforms tested (as above) up to
H
O
N
N
R
O
concentrations of 100
lM; and no time-dependent inhibition of
Ligand efficiency17
Human P2X7 pIC50
R
a
Compd
any of these isoforms.21
14
15
16
17
18
19
20
21
22
23
24
25
Cyclohexylmethyl
Benzyl
<6
<6
—
—
0.44
0.45
0.44
0.45
0.43
0.38
Following intravenous administration (1 mg/kg) to rats, com-
pound 31 (see Table 4) had low blood clearance (9 mL/min/kg); a
Tricyclo[3.3.1.13,7]dec-1-ylmethyl
2-Cl-phenylmethyl
7.1
6.2
6.0
6.2
7.0
6.2
<6
7.9
8.2
8.8
3-Cl-phenylmethyl
4-Cl-phenylmethyl
3-CF3-phenylmethyl
2-CF3-phenylmethyl
2-OMe-phenylmethyl
2,3-DiCl-phenylmethyl
2,4-DiCl-phenylmethyl
2-Cl-3-CF3-phenylmethyl
H
O
N
N
CF3
—
O
Cl
0.54
0.56
0.53
31
Figure 4. Human P2X7 pIC50 8.5; rat P2X7 pIC50 6.5; rat and human in vitro
clearance <0.5 mL/min/g; CYP450 inhibition pIC50 >100 M at all isoforms tested;
log D at pH 7.4 = 1.9; MW = 334; LE = 0.5317; LLE = 6.2.18
a
Data generated using an ethidium bromide release assay (Ref. 14), reporting an
average value of n >3.
l

M. H. Abdi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5080–5084
5083
Table 4
Summary of the pharmacokinetic profile (n = 3) of compound 31
30
Dosing period
Vehicle (CCI)
Vehicle (sham)
20mg/kg
Route of administration Dose
Property
Measured value
20
10
0
IVa
1 mg/kg
CLb (mL/min/kg)
T1/2 (h)
9
Gabapentin (30mg/kg)
1.5
1.1
1.0
2.45
VDss (L/kg)
Tmax (h)
Cmax (lM)
POb
3 mg/
mg
AUC/dose (min kg/L) 73
Fpo 65%
0
20 24 26 28 30 32 34 36 38
Days (post-surgery)
a
Compound 31 was dissolved in 0.9% (w/v) saline containing 10% (w/v)
hydroxypropyl-b-cyclodextrins (HPB) and 2% (v/v) DMSO at a target concentration
of 0.2 mg/mL. It was administered as a 1 h intravenous infusion to achieve a target
dose of 1 mg/kg.
Figure 6. Effect of oral dose of 31 in the chronic constriction injury model in the rat
at oral doses of 20 mg/kg b.i.d. for 8 days. The effect of 8 days oral dosing of 30 mg/
kg b.i.d. gabapentin is included for comparison.
b
Compound 31 was dissolved in 1% (w/v) methylcellulose in water at a con-
centration of 0.6 mg/mL and dosed by oral gavage at a target dose of 3 mg/kg.
in vitro potency, selectivity, and pharmacokinetic properties, and
which do not time-dependently inhibit the CYP450 3A4 enzyme.
Compound 31 was shown to have good efficacy in both the chronic
joint pain model of inflammatory pain and the CCI model of neuro-
pathic pain. Compound 31 was thus identified as having clear po-
tential as a therapeutic agent for the treatment of pain and
addressed the developability concerns highlighted earlier for com-
pounds such as 2. Compound 31 was subsequently selected for
progression into early phase clinical studies. Details of these stud-
ies will be disclosed in a future publication.
steady-state volume of distribution of 1.1 L/kg; and a half-life of
1.5 h. Compound 31 was rapidly absorbed (Cmax achieved within
1 h) following a 3 mg/kg oral dose and had a bioavailability of 65%.
Compound 31 is a selective antagonist of the P2X7 receptor,
showing no appreciable off-target activities (>90 targets, both in-
house and CEREP28 selectivity panels). Whilst the rat receptor
affinity of 31 is relatively modest we were confident that the selec-
tivity for the P2X7 receptor along with the high exposures obtained
on oral dosing in rats, excellent solubility (0.97 mg/mL in FeSSIF29),
and low protein binding (60% in rat plasma) would allow us to reli-
ably evaluate the efficacy of this compound in animal models of
pain.30
Indeed, compound 31 produced (Fig. 5) a highly significant and
dose-related reversal of FCA-induced hypersensitivity in the knee
joint model of chronic inflammatory pain.20 Twice daily doses of
20 and 50 mg/kg produced a maximal reversal of hypersensitivity
which was not statistically different to that obtained with the stan-
dard, celecoxib. Average blood and brain concentrations of 31 at
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141267 A1.
Vehicle
5mg/kg
20mg/kg
50mg/kg
Dosing period
100
75
Celecoxib (30mg/kg)
50
25
0
0
5 10
13 14 15 16 17 18
Days post FCA (intra-articular)
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Figure 5. Effect of oral dose of 31 in the chronic joint pain model in the rat at oral
doses of 5, 20 and 50 mg/kg b.i.d. for 5 days. The effect of 5 days oral dosing of
30 mg/kg b.i.d. celecoxib is included for comparison.

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20. Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.;
Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.;
Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain 2006, 10, 537.
21. Time-dependent inhibition of CYP450s was assessed in the usual manner15
except that fluorescence was generated over 30 min (rather than 10 min) and
IC50s were determined after each 5 min time interval. An increase in IC50 of
greaterthantwo-tothreefoldwas consideredtobe significantand, depending on
the baseline degree of inhibition, to potentially represent a developability risk.
22. The overlay was generated using the software FLO (Thistlesoft, Colebrook,
CT06021, USA).
(basedonmolecular structure)24 andannotated with simple calculatedproperties
(e.g., molecular weight, hydrogen-bond donor and acceptor counts).
24. Harper, G.; Bravi, G. S.; Pickett, S. D.; Hussain, J.; Green, D. V. S. J. Chem. Inf.
Comput. Sci. 2004, 446, 2145.
25. Schneider, G.; Neidhart, W.; Giller, T.; Schmid, G. Angew. Chem., Int. Ed. 1999,
38, 2894.
26. Horvath, D. In Chemoinformatics Approaches to Virtual Screening; Varnek, A.,
Tropsha, A., Eds.; Royal Society of Chemistry: Cambridge, 2008; pp 44–72.
27. Also see WO 2008/003697A1 for additional synthetic details.
28. http://www.cerep.fr.
29. FeSSIF: Fed-state-simulated intestinal fluid. Galia, E.; Nicolaides, E.; Horter, D.;
Lobenberg, R.; Reppas, C.; Dressman, J. B. Pharm. Res. 1998, 15, 698.
30. 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.
31. Bennett, G. J.; Xie, Y. K. Pain 1988, 50, 355.
23. Searches were performed with either 1 or 3 as query molecule in combination
withreduced graph,24 topologicalpharmacophore,25 or3-pointpharmacophore26
fingerprints. Hits from the similarity searches were clustered using frameworks