Synthesis and SAR development of novel P2X7 receptor antagonists for the treatment of pain: Part 1

Article abstract of DOI:10.1016/j.bmcl.2011.04.034

Structure-activity relationship (SAR) efforts around our initial lead compound 1 led to the identification of potent P2X₇ receptor antagonists with improved pharmacokinetic profiles. These compounds were potent and selective at the P2X₇ receptor in both human and rodent. Compound (entry 31) exhibited oral efficacy in the rat MIA and CCI pain models.

Full text of DOI:10.1016/j.bmcl.2011.04.034

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3805–3808
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR development of novel P2X7 receptor antagonists
for the treatment of pain: Part 1
Julius J. Matasi a, , Stephanie Brumfield a, Deen Tulshian a, Michael Czarnecki a, William Greenlee a,
⇑
Charles G. Garlisi a, Hongchen Qiu a, Kristine Devito a, Shu-Cheng Chen a, Youngliang Sun a,
Rosalia Bertorelli a, William Geiss b, Van-Duc Le b, Gregory S. Martin b, Samuel A. Vellekoop b,
James Haber b, Melissa L. Allard b
a Merck Research Laboratories, 2015 Galloping Hill Road, MS 2800, Kenilworth, NJ 07033-0539, USA
b Albany Molecular Research, Inc., 21 Corporate Circle, Albany, NY 12212-5098, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationship (SAR) efforts around our initial lead compound 1 led to the identification
of potent P2X7 receptor antagonists with improved pharmacokinetic profiles. These compounds were
potent and selective at the P2X7 receptor in both human and rodent. Compound (entry 31) exhibited oral
efficacy in the rat MIA and CCI pain models.
Received 21 January 2011
Revised 4 April 2011
Accepted 7 April 2011
Available online 24 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
P2X7
Purine derivatives
Inflammatory pain
Neuropathic pain
The P2X7 receptor is an adenosine triphosphate (ATP) activated
ligand-gated ion channel that is expressed mainly on immune cells
such as monocytes, macrophages, mast cells, lymphocytes and by
microglia and astrocytes in the central nervous system.1,2 The
expression of P2X7 on neurons is not yet completely established.3
Activation of the P2X7 with ATP leads to the opening of an ion
channel permeable to small ions (K+, Na+, Ca2+) and the formation
of a membrane pore with permeability for large molecules upon
continued exposure.4,5 This is accompanied by the downstream
activation of caspase-1 (ICE) leading to the processing and release
of mature forms of proinflammatory cytokines IL-1b and IL-18.6,7
Activation of P2X7 has also been shown to increase levels of other
exhibited similar potency for human and rat P2X7 in Ca2+ flux
assays16 (hIC50 = 248 nM; rIC50 = 233 nM). However potency in an
LPS/ATP-induced IL-1b human whole blood assay,17 was low
(IC50 = 3372 nM). The pharmacokinetic profile (PK), measured
using rapid rat model,18 was poor and not suitable for chronic
studies. Compound 1, was active in the rat mono-iodoacetate
(MIA)19 model of pain when administered orally at 100 mg/kg,
dose. Activity was also observed in the rat chronic constriction in-
jury (CCI)20 model of pain at the same dose, although via intraperi-
toneal (ip) route. This lead provided us with a starting point to
initiate SAR development. The Letter will describe the SAR that
led to the discovery of compounds with a better in vitro profile
and in vivo efficacy.
mediators of inflammation including MMPs, PGE2, and TNF-a,
although the mechanism for these effects is not as well stud-
ied.8–10 In addition, P2X7À/À mice were protected from both inflam-
matory and neuropathic pain.11,12 Recent publications of small
molecules also confirm that antagonism of P2X7 in vivo reduces
both inflammatory hyperalgesia and neuropathic pain.13–15 These
results support a role for this receptor in pain and neurodegenera-
tive diseases.
O
h P2X7 IC50 = 248 nM
r P2X7 IC50 = 233 nM
h WB (IL-1β) IC50 = 3372 nM
RR AUC = 1381 nM. hr, C6h = 0
hERG (VC, IC50) = 2300 nM
PDE5 (IC50) = 5200 nM
PDE11(IC50) = 140 nM
N
N
N
N
N
The screening of our internal compound library led to the
identification of compound 1 (Fig. 1) as initial lead. This lead
h-PXR Rif ratio @10μm=1.22
1
⇑
Corresponding author.
E-mail address: Julius.Matasi@merck.com (J.J. Matasi).
Figure 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.034

3806
J. J. Matasi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3805–3808
O
Cl
N
Cl
O
N
N
N
HN
N
N
N
N
d
a
b
c
N
N
N
N
Cl
N
N
H
Cl
Cl
N
2
Cl
3
4
F
F
F
F
F
F
O
N
O
N
O
N
N
N
e
N
N
N
f
g
N
N
7b, R = Aryl
R
HN
N
N
N
OH
N
F
F
5
6
F
7a, R= Br
F
F
F
Scheme 1. Reagents and conditions: (a) benzyl chloride, K2CO3, DMF, rt; (b) 0.2 N NaOH, reflux; (c) ethyl iodide, K2CO3, DMF, rt; (d) (R)-(À)-2-amino-3-methyl-1-butanol,
NMP, 120 °C; (e) thionyl chloride, DCM, rt; (f) N-bromosuccinamide, DCM, rt; (g) arylboronic acid, Pd(PPh3)4, Na2CO3, CH3CN/H2O, reflux.
O
O
O
N
N
N
N
HO
N
a , b
c
O
B
O
Br
Br
8
9
Scheme 2. Reagents and conditions: (a) oxalyl chloride, DCM, rt; (b) piperidine, DCM, 0 °C; (c) bis(pinacolato)diboran, PdCl2(dppf), KOAc, 1,4-dioxane, 80 °C.
The synthesis of these compounds was achieved from
commercially available 2,6-dichloropurine as shown in Scheme 1.
Alkylation with commercially available benzyl chlorides in the
presence of potassium carbonate in DMF provides 2 as the major
product. Hydrolysis of 2 using a 0.2 N sodium hydroxide solution
under reflux conditions gives compound 3. Compound 3 is then
converted to 4 as the major product using ethyl iodide in the pres-
ence of potassium carbonate. Compound 4 undergoes substitution
reactions with amino alcohols to give 5, followed by cyclization
using thionyl chloride to give compound 6. Compound 6 is then
brominated using NBS in dichloromethane to give the advanced
intermediate 7a. Suzuki coupling reaction with 7a provided us
with the desired targets.21
Most of the boronic acids/esters were purchased and used in the
Suzuki coupling reactions. The amino alcohols were either
purchased or prepared easily by treatment of commercially avail-
able amino acids with lithium aluminum hydride. Noncommercial
boronic acid derivatives were prepared as shown in Scheme 2.
4-Bromopyridine-2-carboxylic acid was treated with oxalyl
chloride and then followed by the piperidine to give compound
8. Compound 8 was then converted to the boronic acid ester 9
utilizing the typical conditions for carrying out this transformation.
All the boronic acid esters used in entries 17–20, Table 3 were sim-
ilarly prepared.
Our initial SAR determined that the isopropyl group was a suit-
able replacement for the spiroindane system of lead 1. It was also
established that C-2 benzyl group could be replaced with 4-flur-
ophenyl with similar potency as 1. Hence, majority of SAR reported
in this Letter was carried out by fixing the C-2 position with a
4-fluorophenyl group and the isopropyl group at the C-7 position
of the lead compound, 1. The scope of varying substituents on nitro-
gen at the 5-position was first explored. The results showed that the
ethyl and propyl groups were optimum at this position (Table 1, en-
tries 2 and 3). Hydrogen and the isopropyl group were not accept-
able replacements (entries 1 and 4). Although, compounds with
activity less than 200 nM in the Ca2+ flux assay were identified,
activity in the human whole blood assay remained unsatisfactory.
Entry 2, in Table 1 shows that by reducing the number of methylene
groups, a significant improvement in the PK was observed.
We next explored SAR at the N-1 position via modification of
the benzyl moiety (Table 2). The introduction of fluorine substitu-
ents on the benzyl ring improved P2X7 Ca2+ flux activity as well as
human whole blood activity (entries 6–10). The presence of elec-
tron donating groups at the 4-position of the benzyl ring were
not tolerated (entry 5). Cycloalkylmethyl derivatives were less po-
tent (entry 11) and the replacement of the phenyl with a pyridine
ring resulted in a dramatic loss in activity (entries 12 and 13).
We then turned to the C-7 position of the initial lead compound
(Table 3). This effort did identify compounds with similar potency
to the isopropyl derivative (entries 14, 15, and 17). However, there
was no additional improvement in the human whole blood po-
tency. The cyclopentyl, gem-dimethyl, and the spirocyclic com-
pounds were less potent (entries 16, 18–20), and substituted
phenyls were also not tolerated (entry 21).
These results led us to conduct further SAR modifications of the
C-2 position described in Table 4. Among the substituted phenyl
derivatives, only 4-cyano-3-fluorophenyl derivative (entry 26),
showed slight improvement in potency in human whole blood as-
say. Introduction of heteroaryls substituent’s at the C-2 position
led to a dramatic improvement in potencies in the human whole
blood assay (entries 31, 33, 36, and 38–40). The 2-trifluoro-
methyl-4-pyridyl derivative was less potent than the 5-pyridyl iso-
mer (entries 32 vs 31). The 2-piperidylpyridine derivative (entry
33) was equally potent in both Ca2+ flux and the human whole
blood assays. The other amino pyridines (entries 34 and 35) were
less active. The 2-substituted amide pyridines were very potent
in the human whole blood assay (entries 36, 38–40) but had poor
exposures in rat when evaluated using rapid rat PK assay. Efforts to
improve rat exposures of these amide derivatives were not
successful.

J. J. Matasi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3805–3808
3807
Table 1
SAR of the 5-position
Compound in entry 31 displayed best overall profile when eval-
uated for potency in whole blood assay and was found to be
equipotent in all three species (Fig. 2). This compound was then
evaluated for its efficacy in two pain models (MIA and CCI) in
rat. In the MIA pain model, it exhibited efficacy similar to naproxen
when given orally at a dose of 30 mg/kg. In the CCI model, it was
found to be significantly less active than gabapentin, with marginal
activity at 100 mg/kg when given orally. Cumulative in house
unpublished data suggest that a compound with predominantly
peripheral exposure tends to have more of an effect in the MIA
O
N
R
N
N
N
N
H
F
16
Entry
R
h-P2X7
IC50 (nM)
hwb IL-1b17
IC50 (nM)
RR AUC18
nM h
1
2
3
4
H
Et
1253.6
141
158
NT
2752
NT
NT
19,469
NT
Table 4
SAR modification of the C-2 position
Propyl
Isopropyl
470
NT
NT
O
N
N
R
N
N
N
Table 2
SAR of the benzyl group replacements
F
H
O
F
N
N
N
16
hwb IL-1b17
IC50 (nM)
RR AUC18
IC50 (nM)
F
Entry
R
h-P2X7
IC50 (nM)
N
N
R
6
4F-P
2F-Ph
29
439
25
64
40
295
NT
15,314
NT
H
22
23
24
25
26
27
4-CF3OPh
3-CF3OPh
4CN-Ph
4CN, 3F-Ph
3CN, 4F-Ph
223
279
223
163
NT
1154
6273
4318
5907
NT
16
Entry
R
h-P2X7
IC50 (nM)
hwb IL-1b17
IC50 (nM)
114
73
5
6
7
8
4MeO-Ph
3F,4FPh
2F,4FPh
4FPh
Inactive
29
35
52
36
NT
295
893
614
454
278
S
28
29
30
31
252
35
NT
NT
9
3F,5FPh
N
F
241
147
82
8706
1360
2685
10
3,4,5-TriF-Ph
24
N
11
114
NT
41
NC
12
13
2-Pyridyl
4-Pyridyl
3207
4065
NT
NT
N
50
F3C
N
32
132
NT
NT
F3C
N
33
34
35
64
114
NT
15
NT
NT
N
Table 3
SAR exploration of substituent’s at the C-7 position
N
207
1236
O
N
O
N
N
N
NT
F
H2N
N
N
N
O
O
R1
N
N
F
R2
36
37
38
N
N
42
43
82
92
NT
56
NT
29
64
F
Entry
R1
R2
h-P2X7
IC50 (nM)
hwb IL-1b17
IC50 (nM)
16
14
15
tButyl
H
H
41
52
614
248
O
N
N
16
H
111
NT
O
O
O
17
H
43
334
N
N
N
39
40
51
28
53
38
91
0
F
F
18
19
Me
Me
121
143
368
NT
(CH2)3
F
F
N
20
21
117
NT
NT
4-FPhenyl (RS)
1471

3808
J. J. Matasi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3805–3808
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O
N
8. Burnstock, G. Pharmacol. Rev. 2006, 58, 58.
N
N
N
h P2X7 IC50 = 50 nM
9. Samad, D. A.; Moore, K. A.; Sapirstein, A.; Billett, S.; Poole, S.; Allchorne, A.;
Bonventre, J. V.; Woolf, C. J. Nature (London, UK) 2001, 410, 471.
10. Wolf, C. J.; Allchorne, A.; Safieh-Garabedian, B.; Poole, S. Br. J. Pharmacol. 1997,
121, 417.
N
H
CF3
h WB (IL-1b) IC50 = 82 nM
r WB (IL-1b) IC50 = 17 nM
m WB (IL-1b) IC50 = 58 nM
RR AUC = 2685 nM. hr
C6h = 227 nM
N
11. 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.
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Medrano, A.; Carroll, W. A.; Marsh, K.; Sullivan, J.; Faltynek, C. R.; Jarvis, M. F. J.
Pharmacol. Exp. Ther. 2006, 319, 1376.
F
F
Figure 2.
model than in the CCI model. It did not have any affinity for either
CYP450 enzymes or PDE enzymes. The compound was also found to
be extremely selective for the P2X7 receptor when tested against
the other P2X receptor subtypes.
In conclusion, our efforts have identified potent P2X7 antago-
nists with significant improvement in human whole blood IL-1b
activity. The compound also displays oral efficacy similar to na-
proxen in rat MIA model and marginal activity in CCI model. Our
current efforts in this series are focused on improving oral efficacy
in neuropathic pain models. Results of these efforts will be re-
ported in future.
15. Carroll, W. A.; Kelvin, D. M.; Perez-Medrano, A.; Florjancic, A. S.; Wang, Y.;
Donnelly-Roberts, D. L.; Namovic, M. T.; Grayson, G.; Honore, P.; Jarvis, M. F.
Bioorg. Med. Chem. Lett. 2007, 17, 4044.
16. Cheewatrakoolpong, B.; Gilchrest, H.; Anthes, J. C.; Greenfeder, S. Biochem.
Biophys. Res. Commun 2005, 332, 17. Each compound was tested at least twice
and the average values are provided. For this assay, the average coefficient of
variance was 29%.
17. Compounds were assessed for their ability to inhibit ATP-mediated IL-1b
production in LPS-stimulated whole blood cultures. Compounds were added to
whole blood for 30 min followed by LPS for 3 h. ATP was added and IL-1b
concentrations were measured using commercially available ELISA kits. Each
compound was tested at least twice and the average values are provided. For
this assay, the average coefficient of variance was 37%.
18. Mei, H.; Korfmacher, W.; Morrison, R. AAPS J. 2006, 8, E493.
19. Bove, S. E.; Calcaterra, S. L.; Brooker, R. M.; Huber, C. M.; Guzman, R. E.; Juneau,
P. L.; Schrier, D. J.; Kilgore, K. S. Osteoarthritis Cartilage 2003, 11, 821.
20. (a) Bennett, G. J.; Xie, Y. K. Pain 1988, 33, 87; (b) Hunter, J. C.; Gogas, K. R.;
Hedley, L. R.; Jacobson, L. O.; Kassotakis, L.; Thompson, J.; Fontana, D. J. Eur. J.
Pharmacol. 1997, 324, 153.
Acknowledgments
The authors also acknowledge Emily Freeman, Geoffrey Giarmo,
Christian Holst and Lisa Peterson, all from Albany Molecular
Research Inc., for their contributions towards this work.
21. (a) An alternative synthesis of desired targets shown in Scheme
3 and
References and notes
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O
EtO2C
H2N
NC
EtO2C
N
N
NC
EtO2C
10
a
b, c
N
d, e
NOH
F
NH2
N
N
O
N
H
F
11
F
12
F
F
F
O
N
O
O
N
N
N
N
N
N
N
N
N
N
f
g
F
F
F
h
HN
N
N
Cl
OH
F
F
F
14
15
13
F
F
F
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Reagents and conditions: (a) Na2S2O4, NaHCO3, H2O; (b) ethyl 4-
fluorobenzimidate hydrochloride, CH3CN, reflux; (c) benzylamine, CHCl3,
reflux; (d) ethyl isocyanate, Et3N, CH3CN, reflux; (e) sodium methoxide,
MeOH, reflux; (f) POCl3, NH4Cl, reflux; (g) (R)-(À)-2-amino-3-methyl-1-
butanol, NMP, 120 °C; (h) thionyl chloride, DCM, rt.