Structure-activity relationship studies on N′-aryl carbohydrazide P2X7 antagonists

Article abstract of DOI:10.1021/jm701516f

N′-Aryl acyl hydrazides were identified as P2X₇ receptor antagonists. Structure-activity relationship (SAR) studies evaluated functional activity by monitoring calcium flux inhibition in cell lines expressing recombinant human and rat P2X₇ receptors. Selected analogs were assayed in vitro for their capacity to inhibit release of cytokine IL-1β. Compounds with potent antagonist function were evaluated in vivo using the zymosan-induced peritonitis model. A representative compound effectively attenuated mechanical allodynia in a rat model of neuropathic pain.

Full text of DOI:10.1021/jm701516f

3030
J. Med. Chem. 2008, 51, 3030–3034
Brief Articles
Structure-Activity Relationship Studies on N′-Aryl Carbohydrazide P2X7 Antagonists
Derek W. Nelson,*,† Kathy Sarris,‡ Douglas M. Kalvin,‡ Marian T. Namovic,† George Grayson,† Diana L. Donnelly-Roberts,†
Richard Harris,† Prisca Honore,† Michael F. Jarvis,† Connie R. Faltynek,† and William A. Carroll†
Abbott Laboratories, Neuroscience Research and AdVanced Technology, Global Pharmaceutical Research and
DeVelopment, Abbott Park, Illinois 60064-6101
ReceiVed December 5, 2007
N′-Aryl acyl hydrazides were identified as P2X7 receptor antagonists. Structure-activity relationship (SAR)
studies evaluated functional activity by monitoring calcium flux inhibition in cell lines expressing recombinant
human and rat P2X7 receptors. Selected analogs were assayed in vitro for their capacity to inhibit release
of cytokine IL-1ꢀ. Compounds with potent antagonist function were evaluated in vivo using the zymosan-
induced peritonitis model. A representative compound effectively attenuated mechanical allodynia in a rat
model of neuropathic pain.
Introduction
block the ATP binding site. Compound 1 inhibited Bz-ATP-
stimulated IL-1ꢀ release in human THP-1 cells and effectively
reversed mechanical allodynia in the Chung model of neuro-
pathic pain.
Recently, the physiology and pharmacology of the P2X family
of ligand-gated ion channels have been studied extensively.1–7
The ATP-sensitive, homomeric subtype P2X7 receptor has
received considerable attention because it is localized on cells
of hematopoietic origin, and recent data indicate a role in the
onset and persistence of certain pain states.8 Specifically, the
P2X7 receptor is expressed on macrophages and epidermal
Langerhans cells.9 In addition, P2X7 receptors are expressed
on microglia10 and astrocytes11 in the central nervous, but
expression on neurons remains uncertain.12 Activation of P2X7
by extracellular ATP leads to ion flux,13 caspase-1 activation,14
release of the cytokine IL-1ꢀ,15,16 p38 MAP kinase activation,17
and, ultimately, reversible cell membrane pore formation that
may lead to lysis and cell death.4 P2X7 receptor activation has
also been linked to glutamate release and inhibition of glutamate
uptake.18 The impact of P2X7 receptor activation on cytokines
and glutamate regulation contributes to the mechanistic rationale
for its role in the development and progression of several disease
states or conditions including inflammation,19 neurodegenera-
tion,20 and neuropathic pain.8,21 The phenotype of P2X7-
knockout mice, which showed reduction in symptom severity
in an arthritis model and resistance to the development of
inflammatory and neuropathic pain, is consistent with a sig-
nificant role of P2X7 in nociceptive signaling.22
Our program has identified other distinct structures that
function as P2X7 receptor antagonists. A high-throughput screen
(HTS) of the Abbott corporate compound library revealed the
terpene-derived acyl hydrazide 3 as a novel P2X7 antagonist,
as measured by the inhibition of calcium flux (hP2X7 pIC50
)
7.3, rP2X7 pIC50 ) 7.3). Herein, we describe the SAR studies
around analogs of 3 that identified key substitutions influencing
P2X7 functional potency for this pharmacophore. An example
from the new series of P2X7 receptor antagonists exhibited
efficacy in a behavioral model of neuropathic pain as well as a
model of induced peritonitis.
Chemistry
The acyl hydrazide core of structure 3 differs from other
reported pharmacophores that serve as P2X7 receptor antagonists
and presents new opportunities to probe the structural require-
ments for receptor activation.25 The SAR studies involved
manipulation of three different moieties within the acyl hy-
drazide pharmacophore: the aryl system bound to the terminal
hydrazide nitrogen, the groups substituted on the nitrogen atoms,
and the lipophilic acyl substituent. Synthesis of the acyl
hydrazide compounds was accomplished by three different
routes as shown in Scheme 1. Treatment of an aryl hydrazine
or the corresponding hydrochloride salt with an acyl chloride
in the presence of triethylamine generated the acyl hydrazides
(route 1). Alternately, the aryl hydrazines coupled with car-
boxylic acids using tetramethylbenzotriazole uronium tetrafluo-
roborate (TBTU) in the presence of triethyl amine (route 2).26
Selected libraries of acyl hydrazides were prepared by the high
throughput organic synthesis (HTOS) group using microwave-
Several different classes of P2X7 receptor antagonists have
been identified,8 but many of the early antagonist structures
lacked properties suitable for therapeutic use. Efforts from our
laboratories have focused on the development of small-molecule
P2X7 receptor antagonists for the treatment of pain. Two
structurally distinct classes of potent and selective P2X7 receptor
antagonists have been disclosed previously. The disubstituted
tetrazole 123 and cyanoguanidine-containing structure 224 rep-
resent selective, reversible P2X7 antagonists that competitively
* To whom correspondence should be addressed. Abbott Laboratories,
Neuroscience Research, R47W, AP10, 100 Abbott Park Road, Abbott Park,
Illinois 60064-6101. Tel.: (847)-937-9825. Fax: (847)-935-5466. E-mail:
derek.nelson@abbott.com.
†
Neuroscience Research.
Advanced Technology.
‡
10.1021/jm701516f CCC: $40.75
2008 American Chemical Society
Published on Web 04/26/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3031
Scheme 1a
Table 1. Functional Potency of Acyl Hydrazide P2X7 Antagonists with
Modified Acyl Groups
hP2X7 Ca2+
flux pIC50
rP2X7 Ca2+
flux pIC50
a,c
a,c
cmpd
R
4a
4b
4c
4d
4e
4f
n-hexyl
iso-pentyl
cyclohexyl
1-methylcyclohexyl
cyclohexylmethyl
cyclopentyl
1-phenylcyclopentyl
1-adamantyl
6.39 ( 0.05
6.83 ( 0.07
6.76 ( 0.03
7.15 ( 0.02
7.41 ( 0.04
7.47 ( 0.07b
6.88 ( 0.02
7.93 ( 0.01b
6.71 ( 0.06
6.47 ( 0.02
6.61 ( 0.17
6.83 ( 0.06
6.82 ( 0.01
7.31 ( 0.03b
6.82 ( 0.02
7.55 ( 0.09b
a Reagents and conditions: (a) NEt3, THF, rt; (b) TBTU, NEt3, MeCN,
rt or carbodiimide resin, HOBt, microwave; (c) NaNO2, aq. HCl; (d)
SnCl2/2H2O.
4g
4h
assisted coupling reactions between aryl hydrazine salts and
carboxylic acids using resin-bound carbodiimide reagents (route
3).27
a Number of determinations ) 2, unless otherwise indicated. b Number
of determinations g 3. c Standard error of measurement shown.
Several aryl hydrazines required for the present study were
commercially available, but brief syntheses of selected hydra-
zines were necessary. The quinoline and isoquinoline hydrazines
were prepared by literature methods involving generation of a
diazonium salt from the corresponding aminoquinoline deriva-
tive followed by reduction using tin(II) chloride.28 For the
chlorinated quinoline hydrazine required for the synthesis of
5h, the 5-amino-2-chloroquinoline precursor was prepared by
known procedures29 and transformed into the hydrazinoquinoline
as described above. The precursors of the lipophilic acyl
substituents were commercially available as either the acid
chlorides or the carboxylic acids.
spinal nerve ligation was performed 7-14 days prior to the
behavioral test. Mechanical allodynia was evaluated using von
Frey monofilaments. Rats were tested before compound admin-
istration. Rats with paw withdrawal threshold <5 g were kept
for compound testing. Rats were then tested again, 30 min
following intraperitoneal administration of the P2X7 antagonist.
Result and Discussion
The initial structure-activity relationship studies of the acyl
hydrazide compounds evaluated the spatial requirements for the
lipophilic acyl moiety. Compounds containing acyclic and cyclic
alkyl groups of varying size and flexibility indicated trends in
the functional potency as measured by the FLIPR calcium flux
assay (Table 1). Potency improved as larger, more rigid alkyl
groups were incorporated. Cycloalkyl groups containing rings
of 6-7 carbon atoms generated antagonists with comparable
potency at both the human and rat receptors. Two of the acyl
substitution patterns, specifically the benzylic groups with the
spirocyclopentane moiety in the benzylic position (4g) and the
adamantyl group (4h), afforded opportunities for rapid explora-
tion and optimization of the structural properties. In addition,
the rigid, three-dimensional adamantyl ring system distinguished
the acyl hydrazides from the structures of the other antagonists
under investigation in our laboratories.34
Following the survey of acyl substituents that could replace
the terpene-derived moiety in 3, attention focused on the aryl
portion of the pharmacophore. In the next cycle of SAR
investigations, the acyl moiety was maintained as the adamantyl
group while the aryl group was modified. The data are
summarized in Table 2. For N′-phenyl hydrazides, substitution
at the ortho position of the phenyl ring resulted in more potent
antagonists (5d, 5g, 5h). Compounds with simple aromatic
heterocycles bound to the terminal nitrogen of the hydrazide as
exemplified by the N′-pyridyl hydrazides (5j, 5k) afforded P2X7
receptor antagonists with only modest potency. However, the
N′-(5-isoquinolinyl) (5l) and N′-(5-quinolinyl)hydrazides (5m)
proved more potent. The quinolinyl hydrazide also represented
a core structure with more desirable physicochemical properties
and additional opportunities for SAR explorations.
Biology
In Vitro. Functional P2X7 receptor activity was measured
using two different methods: (1) inhibition of Ca2+ flux in cell
lines expressing recombinant human or rat P2X7 receptor, and
(2) inhibition of IL-1ꢀ release in differentiated THP-1 cells.
Inhibition of Ca2+ flux was measured with a Fluorometric
Imaging Plate Reader (FLIPR) using Fluo-4 as the calcium
sensing dye and benzoylbenzoylATP (BzATP) as the agonist.30
The FLIPR experiments utilized a 5 min pretreatment with the
antagonist. Previous work has demonstrated that increased
pretreatment times of 15, 30, and 60 min resulted in no change
in the measured IC50 values.23 Recombinant human and rat P2X7
were functionally expressed in stably transfected human 1321N1
astrocytoma cells devoid of endogenous P2X receptor function.
An assay for inhibition of IL-1ꢀ release was established in
human THP-1 cells that had been differentiated with LPS and
IFNγ then stimulated with BzATP for 30 min in the presence
of test compounds.31 Measurement of IL-1ꢀ release was
performed using commercially available ELISA kits.
In Vivo. The in vivo efficacy of the novel P2X7 antagonists
in the acyl hydrazide series was evaluated in two different
animal models. A model of zymosan-induced peritonitis deter-
mined compound-dependent modulation of cytokines including
interleukin-1 (IL-1ꢀ) in rats.32 In these experiments, acyl
hydrazides were administered intraperitoneally or subcutane-
ously 30 min prior to intraperitoneal administration of a saline
suspension of 2 mg zymosan. Four hours later, the animals were
euthanized by CO2 inhalation and the peritoneal cavities lavaged
(2 × 15 mL) with ice cold phosphate buffered saline (w/o Ca2+
and Mg2+) with 10 units heparin/mL. For cytokine determina-
tion, the samples were spun at 10000 × g in a refrigerated
microfuge (4 °C). The supernatants were removed and frozen
until IL-1ꢀ levels were determined by ELISA techniques.
Reversal of neuropathic pain was evaluated using the L5/L6
spinal nerve tight ligation (SNL) model.33 In these experiments,
The third iteration of SAR studies employed the N′-5-quinolyl
moiety in conjunction with a selection of cyclic acyl moieties.
Compounds 6a-e evaluated the impact of modifications to the
adamantyl ring system (Table 3). Contraction to the norada-
mantyl moiety (6a) caused a decrease in functional potency at
the human receptor that eliminated some of the species
discrepancy observed for adamantyl analog 5m. Insertion of a
methylene spacer between the adamantyl group and the carbonyl

3032 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Brief Articles
Table 4. Functional Potency of Acyl Hydrazide P2X7 Antagonists in
Table 2. Functional Potency of Acyl Hydrazide P2X7 Antagonists with
Modified Aryl Groups
the Inhibition of Human IL-1ꢀ Release In Vitro and Efficacy In Vivo in
the Zymosan Model
cmpd
(A-number)
hIL-1ꢀ release
zymosan (i.p.; %
a,b
pIC50
effect at 20 µmol/kg)a
5m
6a
6c
5g
4h
8.3 ( 0.2
6.8 ( 0.1
>9.0
63
55
49
63
66
hP2X7 Ca2+
flux pIC50
rP2X7 Ca2+
flux pIC50
a,c
a,c
cmpd
Ar
NTc
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5h
4-methoxyphenyl
2,5-dimethylphenyl
phenyl
2-fluorophenyl
3-fluororphenyl
4-fluorophenyl
2-chlorophenyl
2,3-dichlorophenyl
2,4-dichlorophenyl
2-pyridyl
6.01 ( 0.04
7.24 ( 0.06
6.96 ( 0.05b
7.40 ( 0.08b
6.49 ( 0.20
6.55 ( 0.19b
7.94 ( 0.07b
7.46 ( 0.05b
6.43 ( 0.27
6.66 ( 0.05b
6.08 ( 0.02
7.47 ( 0.07b
7.99 ( 0.05b
7.29 ( 0.14b
5.61 ( 0.03
6.87 ( 0.03
6.56 ( 0.07b
6.69 ( 0.01b
6.33 ( 0.10
5.97 ( 0.15b
7.47 ( 0.04b
7.29 ( 0.06b
>5
6.37 ( 0.02
6.01 ( 0.09
6.82 ( 0.08b
7.35 ( 0.06b
6.76 ( 0.01
NT
a Number of determinations ) 3, unless otherwise indicated. b Standard
error of measurement shown. c NT ) not tested.
3-pyridyl
5-isoquinolyl
5-quinolyl
2-chloro-5-quinolyl
a Number of determinations ) 2, unless otherwise indicated. b Number
of determinations g 3. c Standard error of measurement shown.
Table 3. Functional Potency of Quinoline Derived Acyl Hydrazide
P2X7 Antagonists
hP2X7 Ca2+
flux pIC50
7.56 ( 0.08b
7.24 ( 0.01
7.99 ( 0.04b
7.64 ( 0.17
rP2X7 Ca2+
flux pIC50
7.28 ( 0.08b
6.22 ( 0.02b
7.45 ( 0.06b
6.84 ( 0.07
a,c
a,c
cmpd
R1
3-noradamantyl
1-adamantylmethyl
3-chloro-1-adamantyl
3-ethyl-adamantyl
6a
6b
6c
6d
6e
6f
6g
6h
6i
3-hydroxy-1-adamantyl
6.56 ( 0.0.09 5.65 ( 0.07
Figure 1. Effects of 5m on mechanical allodynia observed in the SNL
model of neuropathic pain.
1-(4-methoxyphenyl)cyclohexyl 7.28 ( 0.04b
7.29 ( 0.10b
6.49 ( 0.0.07
6.59 ( 0.17b
6.76 ( 0.02b
1-(2-fluorophenyl)cyclohexyl
1-(3-fluorophenyl)cyclohexyl
1-(4-fluorophenyl)cyclohexyl
2,2,3,3-tetramethylcyclopropyl
1,2,2,3-tetramethylcyclopentyl
2-methyl-5-norbornen-2-yl
6.72 ( 0.06
6.85 ( 0.10b
7.15 ( 0.09b
Table 5. Pharmacokinetic Profile of 5m in Ratsa
Vꢀ
Clp
Cmax
AUC
6j
6k
6l
6.70 ( x0.11b 6.81 ( 0.04
-7.56 ( 0.06b 6.91 ( 0.02b
route (L/kg) (L/hr·kg) (µg/mL) Tmax (hr) (µg·hr/L) t1/2 (hr) F (%)
7.73 ( 0.12b
7.50 ( 0.08b
5.82 ( 0.13
IV
IP
0.90
1.7
0.94
0.39
0.40
0.60
0.44
0.50
42
6m 7,7-dimethyl-2-oxonorborn-1-yl 5.86 ( 0.03
a All animals administered doses of 5 µmol/kg in 10% DMSO/90%PEG.
a Number of determinations ) 2, unless otherwise indicated. b Number
of determinations g 3. c Standard error of measurement shown.
limit of the assay. Selected acyl hydrazides were also evaluated
using an in vivo model of zymosan-induced peritonitis in which
inhibition of production IL-1ꢀ was measured directly. Prophy-
lactic administration of selected acyl hydrazide P2X7 receptor
antagonists caused a substantial inhibition of IL-1ꢀ release.
Intraperitoneal administration of 20 mmol/kg of the acyl
hydrazides 5m, 5g, and 4h produced >60% reduction in IL-1ꢀ
release.
group in analog 6b decreased functional potency. Halogen and
alkyl substituents at the C(3) bridgehead of the adamanyl system
(6c, 6d) caused little change in antagonist potency, but the
hydroxyl group was not well tolerated (6e). Compounds 6f-i
evaluated the influence of electron-donating and electron-
withdrawing groups on the phenyl ring of the spirocyclic
benzylic groups. The fluoro group was tolerated at the ortho,
meta, and para positions, but the 4-methoxyphenyl substitution
pattern of 6f provided the greatest potency. Derivative 6f also
demonstrated comparable functional potency at both the human
and rat receptors. Alternate lipophilic acyl moieties were also
explored, and antagonist function was retained for a range of
differently substituted cycloalkyl and polycyclic hydrocarbon
groups (6j-6m).
Selected acyl hydrazide antagonists were evaluated for their
capacity to inhibit IL-1ꢀ release in vitro using human THP-1
cells (Table 4). The N′-quinoline acyl hydrazide 5m inhibited
IL-1ꢀ release efficiently, and the pIC(50) value for IL-1ꢀ
inhibition was comparable to the potency in the FLIPR assay.
Compound 6c was highly potent in the in vitro assay for
inhibition of IL-1ꢀ release, and the IC50 was below the detection
Previous work with other classes of P2X7 receptor antagonists
demonstrated a correlation between inhibition of IL-1ꢀ release
and efficacy in behavioral models of neuropathic pain,24
although a mechanistic rationale for this relationship was not
established. Behavioral effects of compound 5m were evaluated
in the spinal nerve ligation (SNL) model of neuropathic pain.
Intraperitoneal (ip) administration of 5m (30-300 µmol/kg)
resulted in a dose-related reversal of mechanical allodynia on
the injured (ipsilateral) side with an ED50 value of 92 µmol/kg
and a maximum response of 68% relative to the noninjured
(contralateral) side (Figure 1). The pharmacokinetic parameters
of 5m (Table 5) indicate plasma exposure levels consistent with
the observed behavioral effects.

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3033
(10) Ferrari, D.; Villalba, M.; Chiozzi, P.; Falzoni, S.; Ricciardi-Castagnoli,
P.; DiVirgilio, F. Mouse microglia cells express a plasma membrane
pore gated by extracellular ATP. J. Immunol. 1996, 156, 1531–1539.
(11) (a) Irnich, D.; Burgstahler, R.; Grafe, P. P2 nucleotide receptors in
peripheral nerve trunk. Drug DeV. Res. 2001, 52, 83–88. (b) Ballerini,
P.; Rathbone, M. P.; Di Iorio, P.; Renzetti, A.; Giuliani, P.;
D’Alimonte, I.; Trubiani, O.; Caciagli, F.; Ciccarelli, R. Rat astroglial
P2Z (P2X7) receptors regulate intracellular calcium and purine release.
NeuroReport 1996, 7, 2533–2537.
(12) (a) Deuchars, S. A.; Atkinson, L.; Brooke, R. E.; Musa, H.; Milligan,
C. J.; Batten, T. F. C.; Buckley, N. J.; Parson, S. H.; Deuchars, J.
Neuronal P2X7 receptors are targeted to presynaptic terminals in the
central and peripheral nervous system. J. Neurosci. 2001, 21, 7143–
7152. (b) Sim, J. A.; Young, M. T.; Sung, H.-Y.; North, R. A.;
Surprenant, A. Reanalysis of P2X7 receptor expression in rodent brain.
J. Neurosci. 2004, 24, 6307–6314. (c) Anderson, C. M.; Nedergaard,
M. Emerging challenges of assigning P2X(7) receptor function and
immunoreactivity in neurons. Trends Neurosci. 2006, 29, 257–262.
(13) Buisman, H. P.; Steinberg, T. H.; Fischbarg, J.; Silverstein, S. C.;
Vogelzang, S. A.; Ince, C.; Ypey, D. L.; Leijh, P. C. Extracellular
ATP induces a large nonselective conductance in macrophage plasma
membranes. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7988–7992.
(14) Kahlenberg, J. M.; Dubyak, G. W. Mechanisms of caspase-1 activation
by P2X7 receptor-mediated K+ release. Am. J. Cell Physiol. 2004,
286, C1100–C1108.
The above results demonstrate the potential of the acyl
hydrazide pharmacophore to reduce pain transmission and
cytokine production. The efficacy of 5m in the SNL model of
neuropathic pain (Figure 1) is consistent with previous reports
of antinociception using other structurally distinct P2X7 an-
tagonists.24 The mechanistic basis for the analgesic effects has
not been determined, but compound 5m was found to have no
significant interactions for concentrations up to 10 µM at other
P2 receptors (P2X3, P2X4, and P2Y2). In addition, a screen of
compound 5g against a variety of over 70 other receptors,
enzymes, and ion channels revealed no substantial interactions
using a concentration of 10 µM.
Conclusion
We have identified a novel series of acyl hydrazide P2X7
receptor antagonists. Examples of this new pharmacophore
exhibited functional potency using in vitro assays that monitor
distinct steps along the cascade initiated by P2X7 receptor
activation. The SAR studies conducted in this work revealed
preferred substitution patterns for the N′ and acyl termini of
the hydrazide structure. For the N′-aryl moiety, ortho-substituted
phenyl groups produced potent P2X7 receptor antagonists, as
did 5-substituted quinoline ring systems. Replacement of the
N-H bonds in the hydrazide was not tolerated. In the acyl
moiety, rigid polycyclic hydrocarbon ring systems exemplified
by the adamantyl group generated potent antagonist function.
Similarly, acyl groups derived from 1-phenylcyclohexanecar-
boxylic acid imparted a favorable balance of potency and
physicochemical properties. The primary in vitro FLIPR assay
utilized in this study revealed minor discrepancies between
functional potency at the recombinant human and rat receptors,
but certain substitution patterns minimized the differences.
Finally, compound 5m was found to possess antinociceptive
efficacy in a model of neuropathic pain, providing additional
evidence for the potential of the P2X7 receptor as a molecular
target for pain. Compound 5m also reduced the release of the
inflammatory cytokine IL-1ꢀ in a zymosan-induced model of
peritonitis demonstrating that P2X7 receptor antagonists can
inhibit inflammatory pathways.
(15) Perregaux, D.; Gabel, C. A. Interleukin-1 ꢀ maturation and release in
response to ATP and nigericin. Evidence that potassium depletion
mediated by these agents is a necessary and common feature of their
activity. J. Biol. Chem. 1994, 269, 15195–15203.
(16) Walev, I.; Klein, J.; Husmann, M.; Valeva, A.; Strauch, S.; Wittz, H.;
Weichel, O.; Bhakdi, S. Potassium inhibited processing of IL-1ꢀ in
human monocytes. EMBO J. 1995, 14, 1607–1614.
(17) Donnelly-Roberts, D. L.; Namovic, M.; Faltynek, C. R.; Jarvis, M. F.
Mitogen-activated protein kinase and caspase signaling pathways are
required for P2X7 receptor (P2X7R)-induced pore formation in human
THP-1 cells. J. Pharmacol. Exp. Ther. 2004, 308, 1053–1061.
(18) (a) Papp, L.; Vizi, E. S.; Sperla´gh, B. Lack of ATP-evoked GABA
and glutamate release in the hippocampus of P2X7 receptor-/-mice.
NeuroReport 2004, 15, 2387–2391. (b) Duan, S.; Anderson, C. M.;
Keung, E. C.; Chen, Y.; Chen, Y.; Swanson, R. A. P2X7 receptor-
mediated release of excitatory amino acids from astrocytes. J. Neurosci.
2003, 23, 1320–1328.
(19) (a) DiVirgilio, F.; Vishwanath, V.; Ferrari, D. On the role of the P2X7
receptor in the immune system. Handbook Exp. Pharmacol. 2001, 151,
355–374. (b) DiVirgilio, F.; Falzoni, S.; Mutini, C.; Sanz, J. M.;
Chiozzi, P. Purinergic P2X7 receptor: A pivotal role in inflammation
and immunomodulation. Drug. DeV. Res. 1998, 45, 207–213.
(20) (a) LeFeuvre, R. A.; Brough, D.; Touzani, O.; Rothwell, N. J. Role
of P2X7 receptors in ischemic and excitotoxic brain injury in vivo.
J. Cereb. Blood Flow Metab. 2003, 23, 381–384. (b) LeFeuvre, R.;
Brough, D.; Rothwell, N. Extracellular ATP and P2X7 receptors in
neurodegeneration. Eur. J. Pharmacol. 2002, 447, 261–269. (c) Rampe,
D.; Wang, L.; Ringheim, G. E. P2X7 receptor modulation of
ꢀ-amyloid- and LPS-induced cytokine secretion from human mac-
rophages and microglia. J. Neuroimmunol. 2004, 147, 56–61. (d)
Wang, X.; Arcuino, G.; Takano, T.; Lin, J.; Peng, W. G.; Wan, P.;
Li, P.; Xu, Q.; Liu, Q. S.; Goldman, S. A.; Nedergaard, M. P2X7
receptor inhibition improves recovery after spinal cord injury. Nature
Med. 2004, 10, 821–827.
Supporting Information Available: Synthetic procedures and
characterization data for intermediates and final products. Complete
descriptions of biological protocols. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(21) (a) Raghavendra, V.; DeLeo, J. A. The role of astrocytes and microglia
in persistent pain. AdV. Mol. Cell Biol. 2004, 31, 951–966. (b) Milligan,
E. D.; Maier, S. F.; Watkins, L. R. Review: Neuronal-glial interactions
in central sensitization. Semin. Pain Med. 2003, 1, 171–183.
(22) (a) Labasi, J. M.; Petrushova, N.; Donovan, C.; McCurdy, S.; Lira,
P.; Payette, M. M.; Brissette, W.; Wicks, J. R.; Audoly, L.; Gabel,
C. A. Absence of the P2X7 receptor alters leukocyte function and
attenuates an inflammatory response. J. Immunol. 2002, 168, 6436–
6445. (b) 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. Disruption of the P2X7 purinoceptor gene
abolishes chronic inflammatory and neuropathic pain. Pain 2005, 114,
386–396.
(23) (a) Nelson, D. W.; Gregg, R. J.; Kort, M. E.; Perez-Medrano, A.;
Voight, E. A.; Wang, Y.; Namovic, M. T.; Grayson, G.; Donnelly-
Roberts, D. L.; Niforatos, W.; Honore, P.; Jarvis, M. F.; Faltynek,
C. R.; Carroll, W. A. Structure-activity relationship studies on a series
of novel, substituted 1-benzyl-5-phenyltetrazole P2X7 antagonists.
J. Med. Chem. 2006, 49, 3659–3666. (b) Examples of triazole
derivatives of the tetrazole pharmacophore have also been disclosed:
Carroll, W. A.; Kalvin, D. M.; Perez-Medrano, A.; Florjancic, A. S.;
Wang, Y.; Donnelly-Roberts, D. L.; Namovic, M. T.; Grayson, G.;
Honore´, P.; Jarvis, M. F. Novel and potent 3-(2,3-dichlorophenyl)-4-
(1) North, R. A.; Barnard, E. A. Nucleotid receptors. Curr. Opin.
Neurobiol. 1997, 7, 346–357.
(2) Ralevic, V.; Burnstock, G. Receptors for purines and pyrimidines.
Pharmacol. ReV. 1998, 50, 413–492.
(3) Jacobsen, K. A.; Jarvis, M. F.; Williams, M. Purine and pyrimidine
(P2) receptors as drug targets. J. Med. Chem. 2002, 45, 4057–4093.
(4) North, R. A. Molecular physiology of P2X receptors. Physiol. ReV.
2002, 82, 1013–1067.
(5) Burnstock, G. Pathophysiology and therapeutic potential of purinergic
signaling. Pharmacol. ReV. 2006, 58, 58–78.
(6) Gever, J. R.; Cockayne, D. A.; Dillon, M. P.; Burnstock, G.; Ford,
A. P. D. W. Pharmacology of P2X channels. Eur. J. Physiol. 2006,
452, 513–537.
(7) Khakh, B. S.; North, R. A. P2X receptors as cell-surface ATP sensors
in health and disease. Nature 2006, 442, 527–532.
(8) (a) Donnelly-Roberts, D. L.; Jarvis, M. F. Discovery of P2X7 receptor
selective antagonists offers new insights into P2X7 receptor function
and indicates a role in chronic pain states. Br. J. Pharmacol. 2007,
151, 571–579. (b) Gunosewoyo, H.; Coster, M. J.; Kassiou, M.
Molecular probes for P2X7 receptor studies. Curr. Med. Chem. 2007,
14, 1505–1523.
(9) Surprenant, A.; Rassendren, F.; Kawashima, E.; North, R. A.; Buell,
G. The cytolytic P2Z receptor for extracellular ATP identified as a
P2X (P2X7) receptor. Science 1996, 272, 735–738.

3034 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Brief Articles
(benzyl)-4H-1,2,4-triazole P2X7 antagonists. Bioorg. Med. Chem. Lett.
2007, 17, 4044–4048. (c) Florjancic, A. S.; Peddi, S.; Perez-Medrano,
A.; Li, B.; Namovic, M. T.; Grayson, G.; Donnelly-Roberts, D. L.;
Jarvis, M. F.; Carroll, W. A. Synthesis and in vitro activity of 1-(2,3-
dichlorophenyl)-N-(pyridin-3-ylmethyl)-4H-1,2,4-triazol-3-amine P2X7
antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 2089–2092.
and 2-chloroquinolinearsonic acids. J. Am. Chem. Soc. 1938, 60, 2104–
2106.
(30) Bianchi, B. R.; Lynch, K. J.; Touma, E.; Niforatos, W.; Burgard, E. C.;
Alexander, K. M.; Park, H. S.; Yu, H.; Metzger, R.; Kowaluk, E.
Pharmacological characterization of recombinant human and rat P2X
receptor subtypes. Eur. J. Pharmacol. 1999, 376, 127–138.
(31) Donnelly-Roberts, D. L.; Namovic, M.; Faltynek, C. R.; Jarvis, M. F.
Mitogen-activated protein kinase and caspase signaling pathways are
required for P2X7 receptor (P2X7R)-induced pore formation in human
THP-1 cells. J. Pharmacol. Exp. Ther. 2004, 308, 1053–1061.
(32) (a) Perretti, M.; Solito, E.; Parente, L. Evidence that endogenous
interleukin-1 is involved in leukocyte migration in acute experimental
inflammation in rats and mice. Agents Actions 1992, 35, 71–78. (b)
Torok, K.; Nemeth, K.; Erdo, F.; Aranyi, P.; Szekely, J. I. Measure-
ment and drug induced modulation of interleukin-1 level during
zymosan peritonitis in mice. Inflammation Res. 1995, 44, 248–252.
(33) Jarvis, M. F.; Burgard, E. C.; McGaraughty, S.; Honore, P.; Lynch,
K.; Brennan, T. J.; Subieta, A.; van Biesen, T.; Cartmell, J.; Bianchi,
B.; Niforatos, W.; Kage, K.; Yu, H.; Mikusa, J.; Wismer, C. T.; Zhu,
C. Z.; Chu, K.; Lee, C.-H.; Stewart, A. O.; Polakowski, J.; Cox, B. F.;
Kowaluk, E.; Williams, M.; Sullivan, J.; Faltynek, C. A-317491, a
novel potent and selective nonnucleotide antagonist of P2X3 and
P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain
in the rat. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 17179–17184.
(34) (a) The adamantyl moiety has been employed in P2X7 antagonists
developed at other institutions. For an example, see: Baxter, A.; Bent,
J.; Bowers, K.; Braddock, M.; Brough, S.; Fagura, M.; Lawson, M.;
McInally, T.; Mortimer, M.; Robertson, M.; weaver, R.; Webborn, P.
Hit-to-lead studies: The discovery of potent adamantane amide P2X7
receptor antagonists. Bioorg. Med. Chem. Lett. 2003, 8, 4047–4050.
(b) Furber, M.; Alcaraz; L.; Bent, J. E.; Beyerbach, A.; Bowers, K.;
Braddock, M.; Caffrey, M. V.; Cladingboel, D.; Collington, J.; Donald,
D. K.; Fagura, M.; Ince, F.; Kinchin, E. C.; Laurent, C.; Lawson, M.;
Luker, T. J.; Mortimore, M. M. P.; Pimm; A. D.; Riley, R. J.; Roberts,
N.; Robertson, M.; Theaker, J.; Thorne, P. V.; Weaver, R.; Webborn,
P.; Willis, P. Discovery of potent and selective adamantane-based
small-molecule P2X7 receptor antagonists/interleukin-1 inhibitors.
J. Med. Chem. 2007, 50, 5882–5885.
(24) (a) Honore, P. M.; Donnelly-Roberts, D. L.; Namovic, M.; Hsieh, G.;
Zhu, C.; Mikusa, J.; Hernandez, G.; Zhong, C.; Gauvin, D.; Chandran,
P.; Harris, R.; Perez-Medrano, A.; Carroll, W. A.; Marsh, K.; Sullivan,
J.; Faltynek, C. R.; Jarvis, M. F. A-740003 (N-(1-{[(cyanoimino)(5-
quinolinylamino)methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxy-
phenyl)acetamide, a novel and selective P2X7 receptor antagonist dose-
dependently reduces neuropathic pain in the rat. J. Pharmacol. Exp.
Ther. 2006, 319, 1376–1385. (b) Perez-Medrano, A.; Donnelly-
Roberts, D. L.; Faltynek, C. R.; Honore, P.; Jarvis, M. F.; Peddi, S.;
Wang, Y.; Carroll, W. A. Discovery and biological evaluation of novel
cyanoguanidine P2X7 antagonists for the treatment of neuropathic pain.
J. Med. Chem. . submitted for publication. (c) Morytko, M. J.;
Betschmann, P.; Woller, K.; Ericsson, A.; Chen, H.; Donnelly-Roberts,
D. L.; Namovic, M. T.; Jarvis, M. F.; Carroll, W. A.; Rafferty, P. R.
Synthesis and in vitro activity of N’-cyano-4-(2-phenylacetyl-N-o-
tolylpiperazine-1-caboxamide P2X7 antagonists. Biorg. Med. Chem.
Lett 2008, 18, 2093–2096.
(25) Structures derived from adamantane amides were investigated inde-
pendently by AstraZeneca: Alcaraz, L.; Furber, M.; Nortimer, M.
Adamantane derivatives. WO 00/61569, 2000.
(26) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. New coupling
reagents in peptide chemistry. Tetrahedron Lett. 1989, 30, 1927–1930.
(27) The details of the procedure are available in the Supporting Informa-
tion.
(28) Dionne, G.; Humber, L. G.; Asselin, A.; McQuillan, J.; Treasurywala,
A. M. Ligand-receptor interaction via hydrogen-bond formation.
Synthesis and pharmacological evaluation of pyrrolo and pyrido
analogues of the cardiotonic agent 7-hydroxycyclindole. J. Med. Chem.
1986, 29, 1452–1457.
(29) (a) Deinet, A. J.; Lutz, R. E. Antimalarials 2-chloro-5-(3-diethylami-
nopropylamino)-quinoline. Useful preparations of 2-chloro-5- and
9-nitroquinolines. J. Am. Chem. Soc. 1946, 68, 1325–1326. (b) Capps,
J. D.; Hamilton, C. S. Syntheses in the quinoline series. I. 2-Hydroxy-
JM701516F