Structure-activity relationship studies on a series of novel, substituted 1-benzyl-5-phenyltetrazole P2X7 antagonists

Article abstract of DOI:10.1021/jm051202e

1-Benzyl-5-aryltetrazoles were discovered to be novel antagonists for the P2X? receptor. Structure-activity relationship (SAR) studies were conducted around both the benzyl and phenyl moieties. In addition, the importance of the regiochemical substitution

Full text of DOI:10.1021/jm051202e

J. Med. Chem. 2006, 49, 3659-3666
3659
Structure-Activity Relationship Studies on a Series of Novel, Substituted
1-Benzyl-5-phenyltetrazole P2X7 Antagonists
Derek W. Nelson,* Robert J. Gregg, Michael E. Kort, Arturo Perez-Medrano, Eric A. Voight, Ying Wang, George Grayson,
Marian T. Namovic, Diana L. Donnelly-Roberts, Wende Niforatos, Prisca Honore, Michael F. Jarvis, Connie R. Faltynek, and
William A. Carroll
Abbott Laboratories, Neuroscience Research, Global Pharmaceutical Research and DeVelopment, Abbott Park, Illinois 60064-6101
ReceiVed NoVember 29, 2005
1-Benzyl-5-aryltetrazoles were discovered to be novel antagonists for the P2X7 receptor. Structure-activity
relationship (SAR) studies were conducted around both the benzyl and phenyl moieties. In addition, the
importance of the regiochemical substitution on the tetrazole was examined. Compounds were evaluated
for activity to inhibit calcium flux in both human and rat recombinant P2X7 cell lines using fluorometric
imaging plate reader technology. Analogues were also assayed for their ability to inhibit IL-1â release and
to inhibit P2X7-mediated pore formation in human THP-1 cells. Compound 15d was advanced to efficacy
studies in a model of neuropathic pain where significant reversal of mechanical allodynia was observed at
doses that did not affect motor coordination.
Introduction
The P2X family of nucleotide receptors consists of nonspe-
cific, ligand-gated cation channels that participate in a variety
of physiological processes.1-4 The homomeric subtype P2X7,
formerly characterized as the P2Z receptor,5,6 possesses a large
extracellular loop, two transmembrane domains, a short intra-
cellular N-terminus, and a long intracellular C-terminus.7-9 P2X7
is expressed in the periphery on cells of the immune system
such as macrophages and epidermal Langerhans cells.6 The
receptor is also expressed in the central nervous system on
microglia10 and astrocytes,11 although the presence of P2X7 on
neurons remains controversial.12 Brief activation of P2X7 by
extracellular ATP initiates a complex sequence of events,
including ion influx,13 caspase-1 activation,14 release of the pro-
inflammatory cytokine IL-1â,15,16 and p38 MAP kinase activa-
tion.17 Prolonged activation results in reversible cell membrane
pore formation, leading to lysis and cell death.4 P2X7 receptor
activation has also been linked to glutamate release and
inhibition of glutamate uptake.18
Figure 1.
Despite the potential therapeutic applications, few druglike
Due to the presence of the P2X7 receptor on cells of the
immune system (macrophages, microglia, etc.) and the relation-
ship between P2X7 activation and cytokine or glutamate release,
this receptor may play an important role in the development
and progression of various disease states or conditions such as
chronic inflammation,19 neurodegeneration,20 and chronic pain.21
Particularly intriguing is the gathering body of literature linking
activated microglia and astrocytes to central sensitization and
the development and maintainence of neuropathic pain.21-25
More direct evidence for the role of P2X7 in the development
of inflammation and pain comes from experiments using P2X7-
deficient mice.26 In an anti-collagen arthritis model, knock-out
(KO) animals showed a substantial reduction in symptom
severity relative to wild-type controls.26a KO mice were also
protected from the development of both adjuvant-induced
inflammatory pain and partial nerve ligation induced neuropathic
pain.26b Recent in vivo pharmacological studies have also
pointed to the potential benefit of P2X7 antagonism in the
treatment of spinal cord injury20d and chronic pain.27
P2X7 antagonists have appeared in the literature. KN-62 (1)
(Figure 1) was the first small molecule antagonist to be
identified28 and it has been the starting point for several
extensive SAR studies.29 Two representative examples of novel
chemotypes that have recently been disclosed with potent in
vitro activity are compounds 230 and 3.31 From our own
laboratories, the cyanoguanidine-containing compound 4 (A-
759020) was found to have potent activity in vitro against P2X7
and was also active in in vivo pain models.32 A high-throughput
screen (HTS) of our corporate compound library revealed the
disubstituted tetrazole 5 (Figure 2) as a novel P2X7 antagonist
(pIC50 ) 6.9) in the recombinant human cell line. The low
molecular weight of compound 5 and potential for chemical
modification make it an attractive starting point for the
optimization of potency as well as physiochemical and phar-
macokinetic properties. We describe herein SAR studies around
5 that delineate the key substitutions influencing potency at P2X7
with this pharmacophore. In addition, we have identified
* Corresponding author. Tel: (847) 937-9825. Fax: (847) 935-5466.
E-mail: derek.nelson@abbott.com.
10.1021/jm051202e CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/23/2006

3660 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Nelson et al.
Table 1. Functional Potency of Tetrazole P2X7 Antagonists with
Modified Benzylic Groups
a
pIC50
Figure 2.
hP2X7
compd
R
Ca2+ fluxc
hYO-PROd
compound 15d as a representative example that exhibits efficacy
in a behavioral model of neuropathic pain.
15a
15b
15c
15d
15e
15f
15g
15h
15i
3
C6H5
7.4 ( 0.5
7.8 ( 0.5
6.3 ( 0.4b
6.9 ( 0.2
6.1 ( 0.1b
7.5 ( 0.2
4.9 ( 0.2b
7.3 ( 0.2
6.8 ( 0.3
7.2 ( 0.1
6.3 ( 0.1
6.9 ( 0.1
5.7 ( 0.1
6.7 ( 0.1
5.7 ( 0.1
6.7 ( 0.1
<5b
6.6 ( 0.1
6.1 ( 0.2
NT
2-CH3-C6H4
2-pyridyl
Chemistry
3-pyridyl
4-pyridyl
Initial SAR studies targeted the two phenyl rings of structure
5. Analogues were synthesized by one of the sequences shown
in Scheme 1. The tetrazole ring could be established by
cycloaddition of azide with benzonitrile derivatives (6).33
Subsequent benzylation of the 5-aryl N-H tetrazole (7) afforded
a separable mixture of regioisomeric tetrazoles containing the
desired 1,5-disubstituted product (8a) as the minor component
(Scheme 1, route 1).34 An alternate sequence for tetrazole
formation involved conversion of an amide (10) to only the
desired regioisomer (8a) using Mitsunobu conditions (Scheme
1, route 2).35 The Mitsunobu method worked with amides
derived from benzoic acids (9) but not with amides derived from
arylacetic acids (12). A general synthetic approach to tetrazoles
with reversed connectivity for the phenyl and benzyl moieties
involved conversion of the amide (12) to the corresponding
thioamide (13) followed by treatment with mercury(II) salts in
the presence of trimethylsilyl azide to form the tetrazole (14)
(Scheme 1, route 3).36
2-CH3-3-pyridyl
3-pyridinylmethyl
2,4-(CH3)2-5-thiazolyl
3,5-(CH3)2-4-isoxazolyl
-
a Number of determinations g 3 unless otherwise indicated. b Number
of determinations ) 2. c Standard deviation shown. d Standard error shown.
NT ) not tested.
been differentiated with LPS and IFNγ then stimulated with
BzATP for 30 min in the presence of test compounds.17
Measurement of IL-1â release was performed using com-
mercially available EIA kits. Finally, blockade of pore formation
by test compounds was assessed by the YO-PRO uptake assay
measuring the inhibition of cellular uptake of the impermeable
YO-PRO-1 diiodide dye using differentiated human THP-1 cells
stimulated with the agonist BzATP for 1 h.17
In Vivo. Efficacy in the reduction of neuropathic pain was
evaluated using the L5/L6 spinal nerve tight ligation (Chung)
model.38 In these experiments, spinal nerve ligation was
performed 7-14 days prior to assay. Tactile allodynia was
induced by application of a von Frey hair 30 min after
administration of the antagonist. Reduction in tactile allodynia
was measured by determination of the paw withdrawal threshold
and comparison to the contralateral paw. Potential motor
impairment in test animals subjected to compounds was
evaluated using the rotarod assay. Coordination was measured
using an accelerating rotorod apparatus with the time required
for the animal to fall from the rod being recorded.
Biology
In Vitro. Functional P2X7 activity was measured using three
different methods: (1) inhibition of Ca2+ flux in recombinant
human or rat cell lines, (2) inhibition of IL-1â release in human
THP-1 cells, and (3) blockade of pore formation in human
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.37 The FLIPR experiments utilized a 5-min pretreatment
with the antagonist. Increased pretreatment times of 15, 30, and
60 min resulted in no change in the measured IC50 values. The
recombinant human and rat P2X7 were functionally expressed
in stably transfected human 1321N1 astrocytoma cells devoid
of endogenous P2X receptor function. An inhibition assay of
IL-1â release was established in human THP-1 cells that had
Result and Discussion
Initial SAR studies focused on modification of the benzylic
moiety to incorporate substituted phenyl groups or heterocycles
(Table 1). Tetrazoles containing the benzyl group or a 2-sub-
Scheme 1a
a Conditions: (a) AlMe3, TMSN3, toluene, ∆; (b) ClCH2Ar′ or BrCH2Ar′, NEt3, CH3CN, rt; (c) SOCl2, DMF, toluene, ∆ then H2NCH2Ar′; (d) DIAD,
PPh3, TMSN3, THF, rt; (e) ClCOCH2Ar′, NEt3, THF; (f) Lawesson’s reagent, toluene, ∆; (g) Hg(OAc)2, TMSN3, NEt3, THF, 0 °C.

1-Benzyl-5-phenyltetrazole P2X7 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3661
Table 2. Functional Potency of Tetrazole P2X7 Antagonists with
Table 3. Functional Potency of Tetrazole P2X7 Antagonists with
Modified Phenyl Groups
Reversed Connectivity
a
pIC50
a
pIC50
hP2X7
compd
R1
R2
Ca2+ fluxc hYO-PROd
hP2X7
4.6 ( 0.4b
4.8 ( 0.1b
5.1 ( 0.3
4.8 ( 0.7b
5.4 ( 0.3
<5b
compd
R1
Ca2+ fluxc
hYO-PROd
16a
16b
16c
16d
16e
16f
16g
16h
16i
C6H5
2-Cl-C6H4
3-Cl-C6H4
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
NT
<5b
17a
17b
17c
17d
2,3-Cl2-C6H3
7.0 ( 0.2
7.6 ( 0.4
7.9 ( 0.1
7.7 ( 0.2
6.4 ( 0.1
NT
7.3 ( 0.1
6.8 ( 0.1
<5b
2,3-Cl2-4-F-C6H2
2-Cl-3-CF3-C6H3
2-Cl-3-CF3-4-F-C6H2
2,5-Cl2-C6H3
3,4-Cl2-C6H3
2,3-(CH3)2-C6H3
2,3-(OCH3)2-C6H3
4-pyridyl
5.1 ( 0.1
5.9 ( 0.2
<5b
2-CH3-C6H4 6.6 ( 0.4
2-Cl-C6H4
C6H5
C6H5
5.4 ( 0.2b
a Number of determinations g 3 unless otherwise indicated. b Number
of determinations ) 2. c Standard deviation shown. d Standard error shown.
NT ) not tested.
4.5 ( 0.42b NT
5-quinolyl
8-quinolyl
4.8 ( 0.2
4.6 ( 0.1b
<5b
<5b
16j
16k
16l
C6H5
2,3-Cl2-4-pyrrolidinyl-C6H2 C6H5
2-CF3-3-F-C6H3 3-pyridyl
NT
NT
6.8 ( 0.1
6.3 ( 0.4
6.4 ( 0.1
5.87 ( 0.1
Table 4. Functional Potency of Tetrazole P2X7 Antagonists against the
Rat P2X7 Receptor and in the Inhibition of Human IL-1â Release
6.2 ( 0.2b
7.9 ( 0.2
7.2 ( 0.4
7.9 ( 0.2
6.7 ( 0.3
16m 2-Cl-3-CF3-C6H3
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
16n
16o
16p
2-F-3-CF3-C6H3
2,3-Cl2-4-F-C6H2
2,3,4-Cl3-C6H2
a
a
pIC50
pIC50
rP2X7
compd Ca2+ fluxc
hIL-1â
release
rP2X7
compd Ca2+ fluxc
hIL-1â
release
a Number of determinations g 3 unless otherwise indicated. b Number
of determinations ) 2. c Standard deviation shown. d Standard error shown.
NT ) not tested.
15a
15b
15d
15e
15f
7.1 ( 0.4
7.2 ( 0.5
6.5 ( 0.2
5.5 ( 0.1
6.7 ( 0.2
5.6 ( 0.4
6.8 ( 0.3
6.4 ( 0.3
4.7 ( 0.7b
6.8 ( 0.3
15h
16m
16n
17b
17c
5.9 ( 0.2b NT
7.2 ( 0.1
7.1 ( 0.1
7.0 ( 0.2
7.3 ( 0.1
7.1 ( 0.2
6.3 ( 0.1
7.4 ( 0.4b
7.6 ( 0.1b
stituted benzylic group exhibited good potency (15a,b). The
limited solubility of such analogues in a suitable vehicle,
however, prevented a definitive assessment of their pharmaco-
kinetic properties, so attention was turned toward modifications
that could impart greater aqueous solubility. Incorporation of a
basic nitrogen into the benzylic moiety substantially decreased
antagonist potency for compounds 15c and 15e on the basis of
the calcium influx and pore formation assays. Compound 15d
containing the pyridine attached at the 3-position, however,
exhibited superior potency in both functional assays to 15c and
15e. Compound 15f, bearing a methyl group at the 2-position,
possessed 4-fold improved potency in the Ca2+ flux assay but
not in the pore formation assay. Elongation of the spacer
between the tetrazole and the pyridyl group significantly
decreased antagonist potency (15g). Heterocycles that main-
tained a nitrogen in an orientation similar to that of the 3-pyridyl
nitrogen (15h,i) afforded comparable potency in the calcium
influx assay to 15d, but potency in the pore formation assay
decreased. Finally, the 2,5-regioisomer 8b generated as the major
product in route 1 (Scheme 1) was found to be completely
inactive at P2X7 (data not shown).
a Number of determinations g 3 unless otherwise indicated. b Number
of determinations ) 2. c Standard deviation shown. NT ) not tested.
potency (16p). Although the favorable 2,3-dichloro substitution
pattern of analogues such as 15d is the same as that on the
aromatic ring of adamantylamide 3, it is unclear at present if
both of these molecules are binding in the same orientation with
respect to the 2,3-dichlorophenyl. The published SAR around
compound 3 certainly indicates a much greater latitude for
modification of the phenyl substitution with retention of P2X7
activity compared to the tetrazole analogues described here.31
Several tetrazoles in which the phenyl group was bound to
the tetrazole moiety at N(1) and the benzylic moiety at C(5)
were synthesized and evaluated in the in vitro assays (Table
3). The reversal of connectivity in the tetrazole pharmacophore
(17a-d vs 15d, 16m,o) had little effect on potency in the
calcium influx FLIPR assay. An improvement in potency in
the YO-PRO assay was observed for the reverse tetrazole
bearing the 2-chloro-3-trifluoromethylphenyl moiety (17c vs
16m).
After the SAR studies involving modification of the benzylic
group, optimization of the aromatic moiety bound directly to
the tetrazole core was undertaken (Table 2). It was found that
significant change to the substitution pattern of this region was
not well tolerated. For example, the unsubstituted and monochlo-
ro derivatives 16a-c were substantially less potent than
compound 15d. Likewise, both 2,5- and 3,4-dichloro variations
(16d,e) lost 30-100-fold in potency relative to 15d. Other 2,3-
disubstituted phenyl moieties with methyl or methoxy groups
decreased antagonist activity (16f,g). Heterocycles and amine
substituents also caused a substantial loss of antagonist potency
(16h-k). On the other hand, conservative modifications such
as selective replacement of the 3-chloro substituent with a
trifluoromethyl group generated more potent antagonists (16m,n).
Addition of halogens at C(4) of the phenyl ring to establish
trisubstituted benzene derivatives provided variable results, with
fluorine increasing potency in the calcium-flux assay but not
in the YO-PRO assay (16o) and chlorine having little effect on
Selected tetrazole antagonists were also evaluated at the
recombinant rat P2X7 receptor and for their ability to inhibit
IL-1â release in human THP-1 cells (Table 4). Generally, the
tetrazoles demonstrated greater potency (∼3-fold) at the human
P2X7 receptor compared to rat P2X7 in the calcium influx assay,
although the rank order potencies in the two assays were
qualitatively similar (Figure 3). Species differences can range
substantially higher, as indicated by the 20-fold difference in
potencies at rat and human P2X7 observed for compound 15h.
The compounds in Table 4 effectively inhibited IL-1â release
with potencies that were less than those in the Ca2+ flux assay
(Figure 4). Reduced potency was also observed in the pore
formation assay relative to Ca2+ flux (Figure 5), although the
magnitude of the effect was less than for the IL-1â assay
Differences in cell lines, times of exposure to the antagonist,
and the sequences of the signaling events measured in the three
different readouts of human P2X7 function may contribute to
the discrepancies, but the same rank order potencies were

3662 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Nelson et al.
As a representative early example from this series, compound
15d was evaluated for selectivity at other P2 receptors (P2X3,
P2X4, and P2Y2), where it was not found to have any significant
interactions up to 10 µM. Compound 15d was found to right-
shift the agonist dose-response curve for BzATP at the human
P2X7 receptor in the FLIPR assay (Figure 6). A Schild analysis
of these data generate a pA2 of 6.9 (slope ) 0.87 ( 0.14). The
FLIPR data used in the Schild analysis may not arise from a
system at equilibrium, but control experiments in which the time
of the FLIPR response after pretreatment with 15d was varied
from 3 to 60 min showed no shift in the dose response curve.
As a result, the Schild analysis shown in Figure 6 is consistent
with competitive antagonism by 15d. In electrophysiology
studies, 15d similarly inhibited BzATP-evoked current (pIC50
∼ 6.6) at the human P2X7 receptor (Figure 7). The BzATP-
induced currents were restored following washout of 15d. Given
its selectivity for P2X7, compound 15d represented an attractive
tool with which to probe the behavorial effects of this
pharmacology in the spinal nerve ligation (Chung) model of
neuropathic pain in rats. Intraperitoneal (ip) administration of
15d (10-300 µmol/kg) resulted in a dose-dependent reversal
of mechanical allodynia on the injured (left) side with an ED50
value of 76 µmol/kg and a maximal response of 78% relative
to the contralateral (noninjured) side (Figure 8). No behavorial
effect was observed on the contralateral side. Consistent with
these behavioral results, compound 15d showed 19% bioavail-
ability (ip) and a 1 h half-life in separate pharmacokinetic
studies.39 Evaluation of the effects of compound 15d on motor
coordination was also assessed using the rotarod assay. Up to
doses of 300 µmol/kg (ip), 15d failed to show a significant
impairment of motor function (data not shown).
Figure 3. Comparison of human and rat P2X7 potency. Slope ) 0.91;
R2 ) 0.70.
Conclusion
We have identified a novel tetrazole-based series of small
molecule P2X7 antagonists that shows potency comparable to
that of known P2X7 antagonists. The SAR studies conducted
on the phenyl and benzyl moieties established the preferred
substituents for P2X7 activity in both these regions. In general,
significant structural variation was not well tolerated on the
phenyl group. However, modification of the benzylic moiety,
particularly the incorporation of certain heterocycles, could
impart a favorable balance of potency and physiochemical
properties to allow for further in vivo evaluation. The three
different in vitro assays utilized in this study represent different
stages of the P2X7 signaling cascade, yet the rank order
potencies for compounds from this series were largely the same.
Some species differences in potency for rat versus human P2X7
were noted in this series, a potential limitation in selecting
analogues for in vivo evaluation. A Schild analysis using FLIPR
data to monitor calcium influx suggests competitive displace-
ment of agonist BzATP, which distinguishes 15d from non-
competitive antagonists such as KN62. Finally, compound 15d
was found to possess antinociceptive activity in a model of
neuropathic pain, providing additional evidence for the potential
of P2X7 as a novel target for pain.
Figure 4. Comparison of potency to inhibit Ca2+ flux and IL-1â
release. Slope ) 1.35; R2 ) 0.76.
Experimental Section
Figure 5. Comparison of potency to inhibit Ca2+ flux and pore
formation. Slope ) 0.84; R2 ) 0.84.
Chemistry. General. Proton NMR spectra were obtained on a
General Electric QE 300 or QZ 300 MHz instrument with chemical
shifts (δ) reported relative to tetramethylsilane as internal standard.
Mass spectral data were obtained using an electrospray (ESI)
technique or by direct chemical ionization (DCI) methods employ-
ing ammonia. Elemental analyses were performed by Robertson
Microlit Laboratories. Column chromatography was carried out on
silica gel (230-400 mesh). Thin-layer chromatography (TLC) was
observed in all three. Although the relationship between
inhibition of IL-1â release and potential antiinflammatory effects
of a P2X7 antagonist is evident, in the context of chronic or
neuropathic pain it is presently unclear which of the three in
vitro assays utilized in this study would be of greater relevance.

1-Benzyl-5-phenyltetrazole P2X7 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3663
Figure 6. (A) Effects of compound 15d to displace (parallel right-shift) the BzATP dose-response over the concentration range of 10-3000 nM.
(B) Schild analysis for compound 15d. Slope ) 0.87 ( 0.14, pA2 ) 6.88.
Figure 7. Concentration-dependent inhibition of BzATP-evoked current in hP2X7 stable 1321N1 cells by 15d. Representative current traces illustrating
inhibition of 100 µM BzATP-evoked current by 10, 100, and 1000 nM 15d. BzATP-evoked current amplitude after complete inhibition by antagonist
(1 µM) returned to >85% of control following approximately 2 min of antagonist washout.
and the mixture was stirred at room temperature for 24 h. The
reaction was monitored by TLC (silica gel/1:1 ethyl acetate:hexanes;
2,5-disubstituted product Rf ) 0.4, 1,5-disubstituted product Rf )
0.3). Brine (15 mL) was added to quench the reaction and the
mixture was transferred to a separatory funnel and then extracted
with ethyl acetate. The combined organic extracts were dried over
sodium sulfate, filtered, and concentrated by rotary evaporator. The
regioisomeric disubstituted tetrazoles were separated by flash
chromatography (TLC conditions) to give 540 mg (19%) of the
desired 1,5-disubstitued tetrazole as a white powder.
The hydrochloride salt was prepared by treatment of the free
base with excess etheric hydrogen chloride followed by removal
of the solvent/volatiles by rotary evaporator. The salt was recrystal-
lized from ethyl acetate/hexanes.
1-(2,6-Dichlorobenzyl)-5-(2,3-dichlorophenyl)-1H-tetrazole (5):
1H NMR (DMSO-d6) δ 7.94 (dd, J ) 8.1, 1.7 Hz, 1H), 7.90 (dd,
J ) 7.8, 1.7 Hz, 1H), 7.56 (t, J ) 8.0 Hz, 1H), 7.51-7.38 (m,
3H), 5.77 (s, 2H); MS (DCI/NH3) m/z 375 (M + H)+.
1-Benzyl-5-(2,3-Dichlorophenyl)-1H-tetrazole (15a): 1H NMR
(DMSO-d6) δ 7.90 (dd, J ) 7.7, 2.2 Hz, 1H), 7.60-7.50 (m, 2H),
7.31-7.23 (m, 3H), 7.08-7.00 (m, 2H), 5.56 (s, 2H); MS (DCI/
NH3) m/z 305 (M + H)+. Anal. (C14H10Cl2N4) C, H, N.
5-(2,3-Dichlorophenyl)-1-(2-methylbenzyl)-1H-tetrazole (15b):
1H NMR (DMSO-d6) δ 7.89 (dd, J ) 7.8, 1.7 Hz, 1H), 7.61 (dd,
1H, J ) 7.8, 1.7 Hz, 1H), 7.53 (t, J ) 7.8 Hz, 1H), 7.22-7.11 (m,
2H), 7.01 (td, J ) 7.5, 1.7 Hz, 1H), 6.75 (d, 1H, J ) 7.5 Hz, 1H),
5.6 (s, 2H), 2.08 (s, 3H); MS (DCI/NH3) m/z 319 (M + H)+. Anal.
(C15H12Cl2N4) C, H, N.
Figure 8. Effects of 15d on mechanical allodynia observed in the
Chung model of neuropathic pain.
performed using 250-mm silica gel 60 glass-backed plates with F254
as indicator. LC-MS analyses were performed on ThermoQuest
Navigator systems using 10-100% acetonitrile:10 mM ammonium
acetate gradient with MS data obtained using atmospheric pressure
chemical ionization (APCI) positive ionization over the range of
m/z from 170 to 1200.
Representative Procedure for Tetrazole Alkylation (Route
1, Step b). 3-[5-(2,3-Dichlorophenyl)tetrazol-1-ylmethyl]pyridine
(15d). To an oven-dried, round-bottomed flask under dry nitrogen
atmosphere were added 5-(2,3-dichloro-phenyl)-1H-tetrazole (2.0
g, 9.3 mmol) and 3-bromomethylpyridine hydrobromide (2.3 g, 9.3
mmol). The flask was sealed with a rubber septum. Anhydrous
acetonitrile (20 mL) was added via syringe to form a white slurry.
Triethylamine (3.2 mL, 2.3 g, 23 mmol) was added via syringe,
2-[5-(2,3-Dichlorophenyl)tetrazol-1-ylmethyl]pyridine hydro-
1
chloride (15c): H NMR (DMSO-d6) δ 8.45 (ddd, J ) 4.9, 1.5,
0.9 Hz, 1H), 7.89 (dd, J ) 8.3, 1.5 Hz, 1H), 7.81 (td, J ) 7.7, 1.8
Hz, 1H), 7.62 (dd, J ) 7.7, 1.5 Hz, 1H), 7.52 (t, J ) 7.8 Hz, 1H),
7.38-7.30 (m, 2H), 5.76 (s, 2H); MS (DCI/NH3) m/z 306 (M +
H)+. Anal. (C13H9Cl2N5‚HCl) C, H, N.
3-[5-(2,3-Dichlorophenyl)tetrazol-1-ylmethyl]pyridine hydro-
chloride (15d): 1H NMR (DMSO-d6) δ 8.70 (dd, J ) 5.1, 1.4 Hz,
1H), 8.57 (d, J ) 2.0 Hz, 1H), 7.99-7.92 (m, 2H), 7.72-7.56 (m,

3664 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Nelson et al.
3H), 5.74 (s, 2H); MS (DCI/NH3) m/z 306 (M + H)+. Anal.(C13H9-
Cl2N5‚HCl) C, H, N.
4-[5-(2,3-Dichlorophenyl)tetrazol-1-ylmethyl]pyridine hydro-
chloride (15e): 1H NMR (DMSO-d6) δ 8.66 (d, J ) 6.1 Hz, 2H),
7.94 (dd, J ) 8.1, 1.7 Hz, 1H), 7.66 (dd, 1H, J ) 7.8, 1.7 Hz, 1H),
7.57 (t, J ) 8.0 Hz, 1H), 7.37 (d, J ) 6.1 Hz, 2H), 5.80 (s, 2H).
Anal. (C13H9Cl2N5‚HCl) C, H, N.
1-Benzyl-5-(2,3-dichloro-4-pyrrolidin-1-yl-phenyl)-1H-tetra-
zole (16k): 1H NMR (CDCl3) δ 7.28-7.24 (m, 3H), 7.07-7.04
(m, 2H), 6.94 (d, J ) 8.8 Hz, 1H), 6.74 (d, J ) 8.8 Hz, 1H), 5.43
(s, 2H), 3.55-3.50 (m, 4H), 2.02-1.98 (m, 4H); MS (ESI+) m/z
374 (M + H)+. Anal. (C18H17Cl2N5) C, H, N.
3-[5-(3-Fluoro-2-trifluoromethylphenyl)tetrazol-1-ylmethyl]-
pyridine (16l): 1H NMR (CDCl3) δ 8.58 (d, J ) 3.7 Hz, 1H), 8.25
(br s, 1H), 7.69-7.63 (m, 1H) 7.56-7.46, m 2H), 7.31-7.26 (m,
1H), 6.97 (d, J ) 7.8 Hz, 1H), 5.41 (s, 2H); MS (ESI+) m/z 324
(M + H)+. Anal. (C14H9F4N5) C, H, N.
5-(2,3-Dichlorophenyl)-1-[(2-methylpyridin-3methyl)methyl]-
1
1H-tetrazole hydrochloride (15f): H NMR (DMSO-d6) δ 8.60
(dd, J ) 1.7, 5.3 Hz, 1H), 7.95 (dd, J ) 1.7, 7.8 Hz, 1H), 7.70-
7.67 (m, 1H), 7.70 (dd, J ) 1.7, 7.8 Hz, 1H), 7.59 (t, J ) 8.0 Hz,
1H), 7.49 (dd, J ) 5.4, 7.6 Hz, 1H), 5.77 (s, 2H), 2.47 (s, 3H); MS
(ESI+) m/z 321 (M + H)+. Anal. (C14H11Cl2N5‚HCl) C, H, N.
3-{2-[5-(2,3-Dichlorophenyl)tetrazol-1-yl]ethyl}pyridine hy-
drochloride (15g): 1H NMR (DMSO-d6) δ 8.70 (m, 2H), 8.22 (d,
J ) 8.5 Hz, 1H), 7.87 (dd, J ) 1.4, 7.1 Hz, 1H), 7.80 (m, 2H),
7.55 (t, J ) 7.8 Hz, 1H), 5.20 (t, J ) 6.8 Hz, 2H), 3.40 (t, J ) 6.8
Hz, 2H); MS (ESI+) m/z 321 (M + H)+. Anal. (C14H11Cl2N5‚HCl)
C, H, N.
3-[5-(2-Chloro-3-trifluoromethylphenyl)tetrazol-1-ylmethyl]-
pyridine hydrochloride (16m): 1H NMR (DMSO-d6) δ 8.66 (d, J
) 5.1 Hz, 1H), 8.53 (d, J ) 1.4 Hz, 1H), 8.17 (dd, J ) 7.8, 1.4
Hz, 1H), 7.89 (d, J ) 7.8 Hz, 1H), 7.80 (dd, J ) 7.8, 7.7 Hz, 1H),
7.61 (dd, J ) 8.1, 5.1 Hz, 1H), 5.74 (s, 2H), 5.70 (br s, 1H); MS
(ESI+) m/z 340 (M + H)+. Anal. (C14H9ClF4N5‚HCl) C, H, N.
3-[5-(2-Fluoro-3-trifluoromethylphenyl)tetrazol-1-ylmethyl]-
1
pyridine (16n): H NMR (CDCl3) δ 8.57 (br s, 1H), 8.39 (br s,
1H), 7.90-7.85 (m, 1H), 7.73-7.68 (m, 1H), 7.56 (d, J ) 7.8 Hz,
1H), 7.46-7.41 (m, 1H), 7.30 (br s, 1H), 5.61 (s, 2H); MS (ESI+)
m/z 324 (M + H)+. Anal. (C14H9F4N5) C, H, N.
5-(2,3-Dichlorophenyl)-1-(2,4-dimethylthiazol-5-ylmethyl)-1H-
1
tetrazole (15h): mp 174-175 °C; H NMR (DMSO-d6) δ 7.98
3-[5-(2,3,4-Trichlorophenyl)tetrazol-1-ylmethyl]pyridine (16p):
1H NMR (CDCl3) δ 8.59 (br s, 1H), 8.32 (br s, 1H), 7.54-7.50
(m, 2H), 7.30-7.26 (m, 1H), 7.10 (d, 1H), 5.48 (s, 2H); MS (ESI+)
m/z 340 (M + H)+. Anal. (C13H8Cl3N5) C, H, N.
(dd, J ) 3.1, 6.8 Hz, 1H), 7.63 (dd, J ) 4.5, 7.6 Hz, 1H), 7.61 (t,
J ) 7.5 Hz, 1H), 5.73 (s, 2H), 2.51 (s, 3H), 2.00 (s, 3H); MS (ESI+)
m/z 341 (M + H)+. Anal. (C13H11Cl2N5S) C, H, N.
5-(2,3-Dichlorophenyl)-1-(3,5-dimethylisoxazol-4-ylmethyl)-
1H-tetrazole (15i): mp 176-178 °C; 1H NMR (DMSO-d6) δ 7.96
(dd, J ) 1.7, 7.8 Hz, 1H), 7.70 (dd, J ) 1.7, 7.5 Hz, 1H), 7.62 (t,
J ) 7.8 Hz, 1H), 5.44 (s, 2H), 2.04 (s, 3H), 1.96 (s, 3H); MS (ESI+)
m/z 325 (M + H)+. Anal. (C13H11Cl2N5O) C, H, N.
Representative Procedure for Conversion of Amides to
Tetrazoles (Route 2, Step d). 3-[5-(2,3-Dichloro-4-fluorophenyl)-
tetrazol-1-ylmethyl]pyridine. To an oven-dried, N2-purged, 25-
mL, round-bottomed flask containing a magnetic stir bar were added
solid 2,3-dichloro-4-fluoro-N-pyridin-3-ylmethylbenzamide (299
mg, 1.00 mmol) and triphenylphosphine (525 mg, 2.00 mmol). The
flask was sealed with a septum and purged with N2 atmosphere.
Anhydrous THF (5 mL) was added via syringe to form a pale
yellow solution. Diisopropyl azodicarboxylate (387 µL, 2.00 mmol)
was added via syringe to form a light brown solution. Azidotri-
methylsilane (265 µL, 2.00 mmol) was added via syringe, and a
thick, white precipitate formed immediately. The reaction was
stirred at room temperature for 24 h, during which time the progress
of the reaction was monitored by TLC (silica gel, 40% isopropyl
alcohol, 60% hexanes; amide Rf ) 0.4, product Rf ) 0.3). Water
(10 mL) was added to quench the reaction and a brown solution
formed. The reaction was transferred to a 125-mL separatory funnel
and extracted with CH2Cl2 (3 × 10 mL). The combined organic
extracts were dried over MgSO4, filtered, and concentrated by rotary
evaporator to give a brown oil. The product was obtained by flash
chromatography using TLC conditions to give 46 mg (76%) of a
white powder.
3-(5-Phenyltetrazol-1-ylmethyl)pyridine (16a): 1H NMR
(DMSO-d6) δ 8.52 (dd, J ) 4.7, 1.4 Hz, 1H), 8.44 (d, J ) 2.4 Hz,
1H), 7.75-7.78 (m, 2H), 7.56-7.68 (m, 4H), 7.38 (dd, J ) 8.0,
4.9 Hz, 1H), 5.85 (s, 2H); MS (ESI+) m/z 237 (M + H)+. Anal.
(C13H11N5) C, H, N.
3-[5-(2-Chlorophenyl)tetrazol-1-ylmethyl]pyridine hydrochlo-
ride (16b): 1H NMR (DMSO-d6) δ 8.67 (dd, J ) 5.1, 1.4 Hz,
1H), 8.53 (d, J ) 2.0 Hz, 1H), 7.90 (dt, J ) 8.1, 1.7 Hz, 1H),
7.72-7.54 (m, 5H), 6.88 (br s, 1H, HCl), 5.71 (s, 2H); MS (DCI/
NH3) m/z 272 (M + H)+. Anal. (C13H10ClN5·HCl) C, H, N.
1
3-[5-(3-Chlorophenyl)tetrazol-1-ylmethyl]pyridine (16c): H
NMR (DMSO-d6) δ 8.82 (dd, J ) 4.8, 1.7 Hz, 1H), 8.44 (d, J )
2.0 Hz, 1H), 7.84 (dd, J ) 2.0, 1.8 Hz, 1H), 7.72-7.74 (m, 1H),
7.69-7.71 (m, 1H), 7.63 (d, J ) 7.8 Hz, 1H), 7.55-7.60 (m, 1H),
7.37 (dd, J ) 7.8, 4.7 Hz, 1H), 5.85 (s, 2H); MS (DCI/NH3) m/z
272 (M + H)+. Anal. (C13H10ClN5) C, H, N.
3-[5-(2,5-Dichlorophenyl)tetrazol-1-ylmethyl]pyridine
1
(16d): mp 200-201 °C; H NMR (DMSO-d6) δ 8.64 (dd, J )
1
5-(1-Benzyl-1H-tetrazol-5-yl)quinoline (16i): H NMR (DMSO-
1.7, 5.1 Hz, 1H), 8.51 (d, 1H, J ) 1.7 Hz, 1H), 7.89 (d, J ) 2.7
Hz, 1H), 7.82 (m, 1H), 7.79 (dd, J ) 2.7, 8.8 Hz, 1H), 7.74 (t, J
) 9.2 Hz, 1H), 7.58 (dd, J ) 5.1, 8.1 Hz, 1H), 5.71 (s, 2H); MS
(ESI+) m/z 307 (M + H)+. Anal. (C13H10Cl3N5) C, H, N.
3-[5-(3,4-Dichlorophenyl)tetrazol-1-ylmethyl]pyridine (16e):
d6) δ 8.98 (dd, J ) 4.2, 1.5 Hz, 1H), 8.28 (d, J ) 8.5 Hz, 1H),
7.96-7.84 (m, 3H), 7.52 (dd, J ) 8.6, 4.2 Hz, 1H), 7.20-7.15 (m,
3H), 6.97-6.93 (m, 2H), 5.63 (s, 2H); MS (ESI+) m/z 288 (M +
H)+. Anal. (C17H13N5) C, H, N.
1
8-(1-Benzyl-1H-tetrazol-5-yl)quinoline (16j): H NMR (DMSO-
1
mp 166-168 °C; H NMR (DMSO-d6) δ 8.53 (dd, J ) 1.7, 4.8
d6) δ 8.93 (dd, J ) 4.2, 1.9 Hz, 1H), 8.57 (dd, J ) 8.1, 1.7 Hz,
1H), 8.29 (dd, J ) 8.1, 1.4 Hz, 1H), 7.91 (dd, J ) 7.3, 1.5 Hz,
1H), 7.75 (dd, J ) 7.5, 7.4 Hz, 1H), 7.70 (dd, J ) 8.3, 4.2 Hz,
1H), 7.21-7.15 (m, 3H), 6.91-6.88 (m, 2H), 5.52 (s, 2H); LCMS
(ESI+) m/z 288 (M + H)+. Anal.(C17H13N5) C, H, N.
Hz, 1H), 8.45 (d, J ) 2.0 Hz, 1H), 8.05 (d, J ) 2.0 Hz, 1H), 7.88
(d, J ) 8.5 Hz, 1H), 7.75 (dd, J ) 2.0, 8.5 Hz, 1H), 7.58 (td, J )
1.7, 7.8 Hz, 1H), 7.38 (ddd, J ) 1.0, 4.7, 8.1 Hz, 1H), 5.84 (s,
2H); MS (ESI+) m/z 307 (M + H)+.
5-(2,3-Dimethylphenyl)-1-(2-methylbenzyl)-1H-tetrazole (16f):
1H NMR (CDCl3) δ 7.32 (d, J ) 7.4 Hz, 1H), 7.21-7.15 (m, 2H),
7.10 (d, J ) 7.4 Hz, 1H), 7.04-6.97 (m, 2H), 6.68 (d, J ) 7.8 Hz,
1H), 5.38 (s, 2H), 2.27 (s, 3H), 2.12 (s, 3H), 1.76 (s, 3H); MS
(ESI+) m/z 279 (M + H)+. Anal. (C17H18N4) C, H, N.
1-(2-Chlorobenzyl)-5-(2,3-dimethoxyphenyl)-1H-tetrazole (16g):
1H NMR (DMSO-d6) δ 7.46-7.16 (m, 6H), 7.02 (dd, J ) 7.5, 1.4
Hz, 1H), 5.59 (s, 2H), 3.88 (s, 3H), 3.60 (s, 3H); MS (DCI/NH3)
m/z 331 (M + H)+. Anal. (C16H15ClN4O2) C, H, N.
3-[5-(2,3-Dichloro-4-fluorophenyl)tetrazol-1-ylmethyl]pyri-
1
dine hydrochloride (16o): H NMR (DMSO-d6) δ 8.66(dd, J )
5.1, 1.4 Hz, 1H), 8.54 (d, J ) 1.7 Hz, 1H), 7.87 (d, J ) 7.8 Hz,
1H), 7.81-7.70 (m, 2H), 7.60 (dd, J ) 8.1, 5.1 Hz, 1H), 5.71 (s,
2H); MS (ESI+) m/z 324 (M + H)+. Anal. Calcd for C13H9Cl3FN5:
C, 43.30; H, 2.52; N, 19.42. Found: C, 42.66; H, 2.22; N, 19.06.
Representative Procedure for Conversion of Thioamides to
Tetrazoles (Route 3, Step g). 3-[1-(2,3-Dichlorophenyl)-1H-
tetrazol-5-ylmethyl]pyridine (17a). To an oven-dried, N2-purged,
25-mL, round-bottomed flask containing a magnetic stir bar was
added N-(2,3-dichlorophenyl)-2-pyridin-3-ylthioacetamide (208 mg,
0.700 mmol). Anhydrous tetrahydrofuran (7 mL) was added via
syringe to form a pale yellow solution. The flask was cooled to 0
4-(1-Benzyl-1H-tetrazol-5-yl)pyridine (16h): 1H NMR (DMSO-
d6) δ 8.76-8.78 (m, 2H), 7.97-7.99 (m, 2H), 7.36-7.45 (m, 5H),
6.05 (s, 2H); MS (ESI+) m/z 279 (M + H)+. Anal. (C17H18N4) C,
H, N.

1-Benzyl-5-phenyltetrazole P2X7 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3665
°C in an ice bath. Solid mercury(II) acetate (446 mg, 1.40 mmol)
was added to form a slurry. The flask was sealed with a septum
and purged with N2 atmosphere. Azidotrimethylsilane (806 mg, 921
mL, 7.00 mmol) was added via syringe. The yellow slurry was
stirred at 0 °C for 1 h. Saturated ammonium chloride solution (10
mL) was added and the mixture was transferred to a 125-mL
separatory funnel. The emulsion was extracted with dichlo-
romethane (3 × 10 mL). The combined organic extracts were dried
over sodium sulfate, filtered, and concentrated by rotary evaporator.
The product was purified by flash chromatography (silica gel/ethyl
acetate, product Rf ) 0.4) to give 203 mg (95%) of a white powder.
3-[1-(2,3-Dichlorophenyl)-1H-tetrazol-5-ylmethyl]pyridine (17a):
1H NMR (DMSO-d6) δ 8.43 (dd, J ) 4.7, 1.7 Hz, 1H), 8.27 (d, J
) 2.4 Hz, 1H), 7.99 (dd, J ) 8.1, 1.4 Hz, 1H), 7.83 (dd, J ) 8.0
1.5 Hz, 1H), 7.65 (dd, J ) 8.1, 8.0 Hz, 1H), 7.51-7.55 (m, 1H),
7.28 (dd, J ) 7.3, 5.3 Hz, 1H), 4.29 (s, 2H); MS (ESI+) m/z 306
(M + H)+. Anal.(C13H9Cl2N5) C, H, N.
3-[1-(2,3-Dichloro-4-fluorophenyl)-1H-tetrazol-5-ylmethyl]py-
ridine Hydrochloride (17b). The free base was prepared according
to the procedure for route 3, step g. The hydrochloride salt was
prepared by treatment of the free base with etheric HCl: 1H NMR
(DMSO-d6) δ 8.67 (d, J ) 3.7 Hz, 1H), 8.59 (d, J ) 1.7 Hz, 1H),
8.04 (d, J ) 8.1 Hz, 1H), 7.97 (dd, J ) 9.0, 5.3 Hz, 1H), 7.82 (dd,
J ) 8.8 Hz, 1H), 7.68 (dd, J ) 8.0, 5.3 Hz, 1H), 4.40 (s, 2H); MS
(ESI+) m/z 324 (M + H)+. Anal.(C13H9Cl3FN5) C, H, N.
3-[1-(2-Chloro-3-trifluoromethylphenyl)-1H-tetrazol-5-yl-
methyl]pyridine Hydrochloride (17c). The free base was prepared
according to the procedure for route 3, step g. The hydrochloride
salt was prepared by treatment of the free base with etheric HCl:
1H NMR (DMSO-d6) δ 8.63 (dd, J ) 5.4, 1.4 Hz, 1H), 8.53 (d, J
) 2.0 Hz, 1H), 8.23-8.19 (m, 2H), 7.97 (d, J ) 7.8 Hz, 1H), 7.88
(dd, J ) 7.8 Hz, 1H), 7.62 (dd, J ) 8.0, 5.3 Hz, 1H), 4.42 (s, 2H);
MS (ESI+) m/z 339.9 (M + H)+. Anal.(C14H10Cl2F3N5) C, H, N.
3-[1-(2-Chloro-4-fluoro-3-trifluoromethylphenyl)-1H-tetrazol-
5-ylmethyl]pyridine Hydrochloride (17d). The free base was
prepared according to the procedure for route 3, step g. The
hydrochloride salt was prepared by treatment of the free base with
etheric HCl: 1H NMR (DMSO-d6) δ 8.48 (dd, J ) 4.7, 1.4 Hz,
1H), 8.36 (d, J ) 2.0 Hz, 1H), 8.28 (dd, J ) 8.8, 5.1 Hz, 1H), 7.87
(dd, J ) 10.5, 10.0 Hz, 1H), 7.66 (d, J ) 7.8 Hz, 1H), 7.38 (dd, J
) 7.6, 4.9 Hz, 1H), 4.33 (s, 2H); MS (ESI+) m/z 358 (M + H)+.
Electrophysiology. Whole-cell patch-clamp recordings were
obtained from stably transfected hP2X7 1321N1 cells plated on
polyethylenimine-coated coverslips grown to approximately 50%
confluence. Currents were recorded using an Axopatch 200B
amplifier (Axon Instruments, Molecular Devices Corp., Union City,
CA) and digitized at 3 kHz for acquisition. The chamber containing
the cells was continuously perfused by an extracellular recording
solution (pH 7.4, 320 mosM) consisting of 147 mM NaCl, 2 mM
KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, and 13 mM
glucose. Patch electrodes were pulled from borosilicate glass and
fire-polished to 3-6 MΩ tip resistance. The intracellular solution
(pH 7.3, 300 mosM) contained 145 mM NaF, 10 mM EGTA, and
10 mM Hepes). All cells were voltage-clamped at -60 mV, and
series resistance was compensated 85-90%.
Cells were constantly perfused with extracellular solution at a
rate of 0.5 mL/min in the recording chamber. Agonist was delivered
to individual cells using a piezoelectric-driven rapid application
system (Burleigh Instruments, Fishers, NY). Extracellular solution
perfused the cell from one barrel of a glass θ tube positioned 100
µm away. Agonist solution perfused the other barrel, and was
applied by rapidly moving the solution interface across the cell.
Agonist applications were approximately 2 s in duration and were
given in approximately 1-min intervals. Antagonist was preapplied
using the extracellular solution barrel of the θ tube for at least 30
s before coapplication with agonist through the other barrel. Current
responses were acquired and analyzed using pClamp software
(Axon Instruments, Molecular Devices Corp., Union Cjity, CA).
Current amplitudes were measured at the end of the agonist
application pulse when a current plateau was achieved. All reagents
were purchased from Sigma Chemical (St Louis, MO).
Supporting Information Available: Elemental analysis data
and representative synthetic procedures and characterization data
for intermediates and immediate precursors to tetrazoles. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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