Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X7 receptor

Article abstract of DOI:10.1016/j.bmcl.2010.09.101

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X₇ antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X₇ in the etiology of pain is also presented.

Full text of DOI:10.1016/j.bmcl.2010.09.101

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6370–6374
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide
antagonists of the P2X7 receptor
Lee Abberley, Aude Bebius, Paul J. Beswick, Andy Billinton, Katharine L. Collis, David K. Dean, Elena Fonfria,
Robert J. Gleave, Stephen J. Medhurst, Anton D. Michel, Andrew P. Moses, Sadhana Patel, Shilina A. Roman,
⇑
Tiziana Scoccitti, Beverley Smith, Jon G. A. Steadman, Daryl S. Walter
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:
A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site
in this molecule. Compound 18 was subsequently identified as a potent P2X7 antagonist with very low
in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role
of P2X7 in the etiology of pain is also presented.
Received 13 August 2010
Revised 15 September 2010
Accepted 16 September 2010
Available online 22 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
P2X7
Antagonist
Pain
The P2X7 receptor is an ATP-gated ion-channel1–3 which con-
trols the activation and release of pro-inflammatory cytokines such
as interleukin-1b (IL-1b).4 Expression on cells in both the immune
and central nervous systems,5 together with preclinical in vivo
studies6 and reports detailing the activity of small molecule P2X7
antagonists in animal models of neuropathic pain7–10 directly
implicate the involvement of the P2X7 receptor in pain states.11
Consequently the potential for P2X7 antagonists to function as
novel human analgesics has resulted in significant interest in the
pharmaceutical sector.12 In our earlier publications13 we described
two series of compounds (exemplified by 1 and 2, Fig. 1) which
were shown to be active in animal models of pain.
N
Cl
O
HN
H
O
N
N
N
CF3
H
O
Cl
Cl
1
2
Figure 1. Compound 1: Human P2X7 pIC50 8.1; rat P2X7 pIC50 7.1; MW = 312;
LE = 0.55;14 LLE = 5.4;15 compound 2: human P2X7 pIC50 8.5; rat P2X7 pIC50 6.6; rat
MW = 334; LE = 0.53;14 LLE = 6.2.15
Table 1
Summary of the cross-species pharmacokinetic profile (n = 3)a of compound 2
Compound 2, in particular, was highlighted as having an excel-
lent preclinical physicochemical profile and a pharmacokinetic
profile in rats (see Table 1) which was suitable for progression to
clinical studies.13,16 However, the cross-species pharmacokinetic
profile,17 and particularly the short half-life in dog and monkey
raised concerns that the human half-life might also be relatively
short. Whilst the physicochemical profile of this compound sug-
gested that a delayed release formulation would be a viable ap-
proach to addressing this potential issue we also sought to
identify a backup molecule with an improved preclinical pharma-
cokinetic profile. One of the approaches we decided to adopt in or-
der to mitigate the risk of a short half-life in humans was to
Species
Rat
Dog
Monkey
CLb (mL/min/kg)
Vss (L/kg)
t1/2 (h)
9
14
0.9
0.7
69
17
1.0
1.5
65
1.1
0.9
57
Fpo (%)
a
Data generated from 1 mg/kg iv or 3 mg/kg po profiles.
explore modifications of compound 2 which, in the first instance,
were most likely to reduce the clearance rate in our preclinical spe-
cies. Initially we sought to achieve this in rats by blocking the sites
most prone to metabolic processes. Route of metabolism studies
suggested that hydroxylation and subsequent glucuronidation of
one of the carbon atoms in the pyrrolidinone ring (see 3 in
Fig. 2) was the most prevalent metabolic route and initial efforts
to block this pathway focused on substitution of the carbon atom
adjacent to the carbonyl group (which was predicted to be the
⇑
Corresponding author.
E-mail address: 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.2010.09.101

L. Abberley et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6370–6374
6371
The dimethylated analogue 4 (see Table 2) was 10-fold less po-
tent whilst difluorination gave 5 which was essentially equipotent
with 2. The intrinsic clearance in rat microsomes also increased
slightly, possibly indicative of a shift towards an alternative meta-
bolic route or an increase in metabolic rate. Replacing the carbon
atom with an oxygen 6 again led to a loss in activity whilst increas-
ing the ring size as in the morpholinone 7 was better tolerated and
low rat microsomal clearance was maintained. The most marked
effect, however, was observed when the carbon atom in the ring
was replaced with an N-methyl group. This led to a significant in-
crease in potency whilst also maintaining an encouraging in vitro
rat clearance profile. The remainder of this letter will focus on
the work done to exploit this latter observation.
OH
H
O
N
N
CF3
O
Cl
3
Figure 2. Proposed major metabolite of compound 2 in rats.
Table 2
Analogues of 2 in which the
a-carbon of the pyroglutamide ring is substituted or
replaced by a heteroatom
Compd
R
Human P2X7
Rat CLib mL/
min/g liver
a
pIC50
Compound 8 showed an increase in potency and maintained
many of the favourable properties of the pyroglutamide 2, includ-
ing the potential for good brain penetration (compound 8:
PSA = 52, c log D @ pH7.4 = 2.9, Pgp efflux ratio = 1.528), whilst
eliminating the metabolically labile carbon atom. Importantly the
nitrogen atom in this position provided a convenient synthetic
handle. We envisaged that appropriate substitution in this position
could lead to compounds with relatively low clearance and/or
higher volumes of distribution hence providing a complimentary
approach to increasing the in vivo half-life in rats. Compounds of
this general formula could be prepared21,22 according to the
method outlined in Scheme 1.
H
N
O
4
7.6
8.4
<0.5
0.7
N
F
CF3
O
Cl
F
H
N
O
O
5
N
CF3
CF3
O
O
Cl
Cl
O
N
H
N
6
7
7.8
8.4
—
Hofmann cyclisation23 of Cbz-protected asparagine followed by
tert-butyl esterification gave the key tert-butyl 2-oxo-N-(Cbz)-4-
imidazolidine carboxylate. The next step exploited an unusual
alkylative rearrangement,22 and then Cbz deprotection, and subse-
quent alkylation (or palladium-catalysed aryl halide cross-cou-
pling reaction) provided the desired tert-butyl 1,3-disubstituted
2-oxo-4-imidazolidine carboxylates. Subsequent ester deprotec-
tion and amide coupling then provided the target compounds in
good overall yields.
O
H
N
<0.5
O
O
N
CF3
O
Cl
N
H
N
8
9.2
<0.5
N
CF3
O
Cl
a
Data generated using an ethidium bromide release assay (Ref. 19), reporting an
A selection of the more interesting compounds prepared to
probe the effects of substitution on the ring nitrogen atom, is
shown in Table 3. We explored the effects of basic groups, of
varying strength, envisaging that a charged group could increase
the volume of distribution.25 At the same time we sought to ensure
that the overall physical properties of the compounds remained in
average value of n >3.
b
Microsomal clearance method described in Ref. 20.
most metabolically vulnerable position) or replacement of the car-
bon atom with a heteroatom.18
NH2
H
H
N
b
a
N
O
O
OH
O
N
O
OH
Cbz
N
O
N
O
Cbz
H
Cbz
O
Cbz
H
d
N
c
N
O
O
O
N
O
N
O
O
R
R
N
f
N
e
OH
O
O
O
N
N
O
O
R
N
H
g
N
R1
O
N
O
Scheme 1. Synthesis of 2-oxo-N-(phenylmethyl)-4-imidazolidine carboxamide analogues. Reagents and conditions: (a) bromine, aq NaOH;23 (b) t-BuOH, POCl3, pyridine,
CHCl3;24 (c) NaH, methyl iodide, DMF, 0 °C; (d) H2, Pd–C, MeOH; (e) NaH, alkyl halide, DMF, À10 °C; or aryl bromide, Pd2(dba)3, Cs2CO3, Xantphos™, 1,4-dioxane; (f) TFA,
DCM; (g) R1NH2, EDAC, HOBT, NEM, DCM.

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L. Abberley et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6370–6374
Table 3
Structure–activity data for N-substituted analogues of compound 8
R
N
H
O
N
N
CF3
O
Cl
a
Compd
R
PSA Å2
61
c log D @ pH 7.427
Human P2X7 pIC50
8.4
Rat CLib mL/min/g liver
0.6
Pgp efflux ratio28
2.1
9
H
2.2
HN
10
11
64
65
0.4
2.1
7.5
7.6
0.5
4.4
3.2
O
N
11.9
N
12
13
65
65
2.6
3.6
8.3
7.1
2.6
6.1
4.4
—
N
N
N
14
65
3.1
8.0
3.7
1.9
N
15
16
70
70
2.2
2.0
8.4
8.6
0.9
5.3
—
N
N
<0.5
a
Data generated using an ethidium bromide release assay (Ref. 19), reporting an average value of n >3.
Microsomal clearance method described in Ref. 20.
b
a desirable space particularly with respect to CNS penetration.26 In
addition, to ensure maximal CNS penetration, the propensity for
active efflux by P-glycoprotein (Pgp) was also monitored.28
increased levels of Pgp efflux. In general, only those analogues with
hydrogen (e.g., 9) or small alkyl (e.g., 8) substituents in this posi-
tion provided the appropriate mix of potency at the P2X7 receptor,
with low intrinsic clearance in liver microsomes, and relatively low
Pgp efflux.
Subsequently, and to explore the effects of the right-hand benz-
amide substitution pattern on potency and pharmacokinetics, a
two-dimensional array of NH and N-methyl substituted imidazoli-
dinecarboxamides was prepared and profiled. The choice of amides
prepared was largely based on the SAR previously derived from the
optimization of the pyroglutamide series (as exemplified by 2).
A subset of data obtained from the compounds prepared is listed
in Table 4.
Compound 9, the des-methyl analogue of 8, maintained a good
level of potency and in vitro clearance and Pgp efflux remained at
acceptable levels. The amino-substituted side-chains in 10 and 11
were less well tolerated by the P2X7 receptor and significant in-
creases in Pgp efflux ratios were also noted. The three isomeric
pyridines shown, 12, 13, and 14, retained a good level of potency
in all but the 2-isomer, but in all cases (and in step with increasing
lipophilicity) the in vitro clearance was significantly higher than in
the case of 8. The two imidazoles, 15 and 16, maintained good lev-
els of potency but the increased polar surface area resulted in
Table 4
P2X7 activity data for selected 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamides
R
N
H
O
N
N
R1
O
Rat CLib mL/min/g liver
Rat CLbc mL/min/kg
Rat t1/2c (h)
a
Compd
R
R1
Human P2X7 pIC50
8
9
17
18
19
20
Me
H
Me
H
Me
H
2-Cl-3-CF3-Phenylmethyl
2-Cl-3-CF3-Phenylmethyl
2,4-DiCl-phenylmethyl
2,4-DiCl-phenylmethyl
2,3-DiCl-phenylmethyl
2,3-DiCl-phenylmethyl
9.2
8.4
8.7
8.0
8.0
7.6
<0.5
0.6
<0.5
<0.5
<0.5
—
37
14
30
6
49
—
0.7
2.3
0.6
2.8
0.4
—
a
b
c
Data generated using an ethidium bromide release assay (Ref. 19), reporting an average value of n >3.
Microsomal clearance method described in Ref. 20.
As determined following a single iv infusion (1 mg/kg).

L. Abberley et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6370–6374
6373
A trend that emerged from this data was that whilst intrinsic
N
clearance in liver microsomes was low in all cases, the in vivo rat
clearance was generally higher in the N-Me examples (compare 8
with 9, and 17 with 18). This may hint at N-demethylation being
a route of metabolism in rats but no studies were conducted to
confirm this. Although the NH analogues were generally slightly
less potent, the improved pharmacokinetic profile (and low clear-
ance in particular) in rats singled these compounds out for further
study. Compound 18, a compound with high ligand efficiency
(Fig. 3), had very low in vivo clearance and displayed an oral bio-
availability of ꢀ100% in a rat iv/po crossover study (n = 3), and
was selected for progression to in vivo studies in animal models
of pain.17
Compound 18 is a selective antagonist of the P2X7 receptor,
showing no appreciable off-target activities (both in in-house
and CEREP29 selectivity panels). The rat receptor affinity of 18 is
relatively modest but we were confident that the selectivity for
the P2X7 receptor; and the high exposures obtained on oral dosing
in rats, together with the excellent solubility (0.78 mg/mL in
FeSSIF30), and low protein binding (65% in rat plasma) would allow
us to reliably evaluate the in vivo efficacy of this compound.
Indeed, compound 18 produced (Fig. 4) a highly significant
reversal of FCA-induced hypersensitivity in the knee joint model
of chronic inflammatory pain.31 An oral dose of 20 mg/kg produced
a maximal reversal of hypersensitivity which was statistically
indistinguishable from the effect obtained with the standard, cele-
coxib. Blood concentrations of 18 at 20 mg/kg, 1.0 h post-dose,
Cl
N
H
O
N
N
O
Cl
21
Figure 5. Compound 21: human P2X7 pIC50 8.0; rat P2X7 pIC50 7.2; MW = 393;
PSA = 65 Å2; Pgp efflux ratio = 4.9; Log D @ pH 7.4 = 2.2; rat in vivo clearance 17 mL/
min/kg; rat t1/2 = 2.3 h; rat Cmax (1 h) = 1073 ng/mL @ 3 mg/kg po.
Dosing period
Vehicle
100
75
50
25
0
20mg/kg
*
*
Celecoxib (30mg/kg)
*
*
*
0
5
10
13
14
15
16
17
Days post FCA (intra-articular)
Figure 6. Effect of 5 days of dosing a 20 mg/kg bid oral dose of 21 in the chronic
joint pain model in the rat. The effect of a 30 mg/kg bid oral dose of celecoxib is
included for comparison.
were 56.4
as 0.3:1.
lM and the terminal brain to blood ratio was measured
ratio was measured as <0.1:1. Protein binding of this compound
was relatively low (77% in rat plasma) so whilst the free concentra-
tions of 21 in the periphery are lower than those shown for 18 in a
similar study, the increased rat P2X7 activity of 21 relative to 18,
along with the data reported for compound 2 in our earlier paper,
would suggest that this compound should have been active in this
model if the efficacy was driven by peripheral concentrations
alone. A potential hypothesis to explain the differential activity
displayed by compounds 18 and 21 in the joint pain model could
be that the efficacy is driven by the free CNS concentrations in each
case and would lend support to the postulated role of P2X7 in the
release of IL-1b and/or glutamate in activated glia in the CNS.32
Finally, compound 18 was also profiled in dog and monkey and
pleasingly it was found that the modest improvements in half-life
in rats, relative to compound 2, translated to more substantial
improvements in the pharmacokinetics in these two additional
species (Table 5). In light of this data, compound 18 was selected
for further development as a potential analgesic agent.
We were interested in exploring the role of centrally expressed
P2X7 receptors in the etiology of pain and decided to also profile a
compound with low CNS penetration in our animal models. In con-
trast to the result shown above for compound 18, a compound with
a higher Pgp efflux ratio (see compound 21, Fig. 5) and poor brain
penetration (following oral dosing in rats) showed little or no effi-
cacy in the same rat pain model (see Fig. 6) even after 5 days of
dosing. Average blood concentrations of 21 at 20 mg/kg, 1.0 h
post-dose on day 1, were 16.8
l
M and the terminal brain to blood
H
N
Cl
H
N
O
N
O
Cl
18
In summary, our backup strategy sought to address the risk of
observing sub-optimal human pharmacokinetics with our first
candidate 2 by targeting the primary route of metabolism of this
molecule. This was achieved by replacing a metabolically labile
carbon atom with a nitrogen atom and compound 18 emerged as
the front runner from this new series. Compound 18 has a preclin-
ical pharmacokinetic profile which addresses the risks associated
with compound 2 and shows good efficacy in one of our key
in vivo rat pain models. In addition we have also described some
evidence which seems to support the role of central P2X7 in the
Figure 3. Compound 18: human P2X7 pIC50 8.0; rat P2X7 pIC50 6.4; MW = 302;
PSA = 61 Å2; LE = 0.58;14 LLE = 5.1;15 Pgp efflux ratio = 2.3; CYP450 inhibition pIC50
>25
lM at all isoforms tested (3A4, 2D6, 2C9, 1A2, 2C19); Log D @ pH 7.4 = 1.8.
Vehicle
Dose
13
14
15
16
20mg/kg
100
75
50
25
0
*
*
Celecoxib (30mg/kg)
*
*
*
Table 5
Summary of the cross-species pharmacokinetic profile (n = 3)a of compound 18
Species
Rat
Dog
Monkey
0
5
10 13
1 h
3 h
6 h
CLb (mL/min/kg)
Vss (L/kg)
t1/2 (h)
6
2
7
Days post FCA (intra-articular)
1.3
2.8
100
0.8
5.7
100
1.2
2.9
99
Figure 4. Effect of a 20 mg/kg oral dose of 18 in the chronic joint pain model in the
rat at 1, 3, and 6 h post-dose. The effect of a 30 mg/kg oral dose of celecoxib is
included for comparison.
Fpo (%)
a
Data generated from 1 mg/kg iv or 3 mg/kg po profiles.

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L. Abberley et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6370–6374
K.; Fonfria, E.; Gleave, R. J.; Lejeune, C. L.; Livermore, D. G.; Medhurst, S. J.;
Michel, A. D.; Moses, A. P.; Page, L.; Patel, S.; Roman, S. A.; Senger, S.; Slingsby,
B.; Steadman, J. G. A.; Stevens, A. J.; Walter, D. S. Bioorg. Med. Chem. Lett. 2010,
20, 5080.
processing of pain signals. Further details of related studies will be
published in due course.
14. Abad-Zapatero, C. Expert Opin. Drug Disc. 2007, 2, 469.
15. (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.
16. Details of clinical studies with compound 2 will be disclosed in a future
publication.
17. 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.
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