Synthesis and in vitro activity of 1-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-1H-1,2,4-triazol-5-amine and 4-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-4H-1,2,4-triazol-3-amine P2X7 antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.01.095

A novel series of aminotriazole-based P2X₇ antagonists was synthesized, and their structure-activity relationships (SAR) were investigated for activity at both human and rat P2X₇ receptors. Most compounds showed greater potency at th

Full text of DOI:10.1016/j.bmcl.2008.01.095

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2089–2092
Synthesis and in vitro activity of
1-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-1H-1,2,4-triazol-5-
amine and 4-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-4H-
1,2,4-triazol-3-amine P2X7 antagonists
Alan S. Florjancic,a,* Sridhar Peddi,a Arturo Perez-Medrano,a Biqin Li,b
Marian T. Namovic,a George Grayson,a Diana L. Donnelly-Roberts,a
Michael F. Jarvisa and William A. Carrolla,*
aAbbott Laboratories, Global Pharmaceutical Research and Development, 100 Abbott Park Road, Abbott Park, IL 60064, USA
bDepartment of Medicinal Chemistry, Abbott Bioresearch Center, 381 Plantation Street, Worcester, MA 01605, USA
Received 19 December 2007; revised 23 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—A novel series of aminotriazole-based P2X7 antagonists was synthesized, and their structure–activity relationships (SAR)
were investigated for activity at both human and rat P2X7 receptors. Most compounds showed greater potency at the human recep-
tor although several analogs were discovered with potent activity (pIC50 P 7.5) at both human and rat P2X7.
Ó 2008 Elsevier Ltd. All rights reserved.
The P2X7 receptor is an ATP-activated ion channel that
has received a notable amount of attention in recent
years from the academic and pharmaceutical sectors as
a potential therapeutic target.1–5 P2X7 is found on a
variety of cell types dealing with inflammation and im-
mune function,1 such as mast cells, macrophages, and
lymphocytes, and the receptor’s activation has been
found to be one of the most potent stimuli of IL-1b re-
lease. Receptor activation also drives the release of a
variety pro-inflammatory cytokines such as TNF-a as
well as giant cell formation and mast cell degranulation,
implicating it in a variety of potential disease states.
Although its presence on neurons has been debated,6,7
P2X7 is expressed in the central nervous system on
microglia and astrocytes.8 On glial cells, activation of
P2X7 has been found to mediate the release of gluta-
mate,9 which is involved in the neurotransmission of
painful sensory signals, potentially making P2X7 useful
in treating a variety of pain states. Studies with P2X7
KO mice showed a reduction in collagen-induced arthri-
tis severity,10 and offered protection to the KO animals
from inflammatory pain, and partial nerve ligation-in-
duced neuropathic pain.11
The potential therapeutic indications of inhibiting P2X7
have led to a recent proliferation in the number and
quality of small molecule antagonists in both the patent
and scientific literature.2–5 Agents from several disclosed
chemical classes have been found to possess activity in
rodent models of inflammatory and neuropathic pain.5
AZ9056, a P2X7 antagonist of undisclosed structure, is
reportedly under clinical investigation for rheumatoid
arthritis and inflammatory bowel disease.12
Recent reports from our laboratories have described the
in vitro and in vivo characterization of substituted aryl-
tetrazoles13 and aryltriazoles,14 exemplified, respectively,
by 1 and 2. Both of these P2X7 antagonists
N
N
N
N
N
Cl
N
Cl
N
Cl
Cl
N
1
2
Keywords: P2X7; Antagonist; Aminotriazole.
*
displayed activity in a rat model of neuropathic pain.
Interestingly, although attachment of the phenyl and
Corresponding authors. Tel.: +1 847 938 6041; fax: +1 847 935 5466
(W.A.C.); e-mail: william.a.carroll@abbott.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.095

2090
A. S. Florjancic et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2089–2092
R1
N
HN
35-42
benzyl groups on adjacent atoms was a requirement for
P2X7 activity, changing the positions of the nitrogen
atoms in the heterocyclic core with respect to the ap-
pended groups had little impact on activity. In an effort
to further explore the structure–activity relationships
around this pharmacophore, additional variations on
the linker between the heterocyclic core and the benzylic
appendage were investigated on two regioisomeric tria-
zoles. Surprisingly, it was found that the prototypical
methylene linker of 1 and 2 could be replaced with an
amino or aminomethyl linker with retention or improve-
ment in potency at both human and rat P2X7 receptors.
Cl
Cl
N
i-iii
Cl
NCS
N
Cl
N
R2
34
Scheme 2. Reagents and conditions: (i) RNH2, THF, rt, 1 h; (ii) NEt3,
HgCl2, NH2NH2, rt, 2–14 h; (iii) R1C(OEt)3, HCOOH, THF, reflux,
2 h, 20–35%.
formate under acidic conditions to afford ring closure,
giving the aminotriazole compounds 35–42 in 20–35%
yield over three steps.
Earlier work in our laboratories on triazole and tetrazole
cores established that the 2,3-dichlorophenyl substitution
pattern on the left-hand side enhanced the potency to-
ward P2X7. To quickly survey the aminotriazole systems,
we opted to hold the left-hand side 2,3-dichlorophenyl
moiety constant throughout our SAR studies.
In vitro P2X7 activity was assessed using the recombi-
nant rat and human receptors. Antagonist potencies
were determined by measuring the inhibition of Ca2+
flux with a fluorometric imaging plate reader (FLIPR)
using Fluo-4 as the dye and benzoylbenzoylATP
(BzATP) as the agonist.17
The target 5-amino-1-phenyl-1,2,4-triazoles 6–33 were
prepared using the synthetic route shown in Scheme
1. 2,3-Dichlorophenylhydrazine15 was reacted with
formamide at elevated temperature to provide the tri-
azole 4 which was in turn brominated under radical
conditions to give the key intermediate 5.16 Direct
reaction of 5 with anilines was unsuccessful under a
variety of conditions, however Buchwald–Hartwig
coupling conditions gave compounds 6–12 in low to
moderate yields, all unoptimized. Some intriguing pre-
liminary data led our main area of interest to be cen-
Previously, extensive SAR studies around the preferred
benzyl group of 2 revealed that substitution in the ortho
position gave rise to the most potent compounds. With
these results as a guide, compounds 6–12 were targeted
to ascertain if this same trend held true with an amino
linker (Table 1). Indeed, both the methyl (7), ethyl (8),
and methoxy (9) analogs proved more potent than the
unsubstituted compound 6. As the groups became larger
(10–12), however, the trend reversed leading to analogs
of lesser or equal potency to 6. Relative to compound 2,
with a methylene linker, the analogous 2-methyl com-
pound 7 displayed similar or improved potency at rat
and human P2X7. Interestingly, the corresponding ana-
log of 7 wherein the NH linker was replaced with oxygen
showed very weak potency.18
tered
on
compounds
which
contained
an
aminomethylene linker. We were able to generate a
medium-sized library of these compounds by treating
5 with a variety of commercial and custom benzyl
amines neat at 90 °C for 4–12 h to give compounds
13–33 in 10–48% yields.
Lengthening the linker by insertion of a methylene unit
gave rise to aminotriazole compounds 13–25. Although
the unsubstituted analog 13 displayed significantly im-
proved potency over 6, the previously observed trend
of enhanced potency with ortho-substitution was much
less prominent here or absent. Compounds 14, 17, 20
and 21, for example, possess essentially equivalent po-
tency to 13. The 2,3-disubstituted analogs 23 and 24
similarly did not improve upon 13. Only compound 22
shows an appreciable potency increase, which manifests
itself most noticeably at rP2X7. Surprisingly, the much
bulkier pyridin-3-yloxy compound 25 demonstrated
similar potency to the analogs with smaller groups in
the ortho position. In contrast to the decreasing potency
with larger groups seen with compounds 7–12, activity
seems to be independent of size with the extra atom in
the linker. This result gives some indication that with
the increased length of the linker, there is more tolerance
for varying the substitution at this position. Regarding
substitution in the meta and para positions, 15, 16 and
18, 19 were significantly less potent which is consistent
with previous observations.14
Synthesis of the regioisomeric 3-amino-4-phenyl-1,2,4-
triazoles 35–42 was accomplished utilizing a one-pot se-
quence (Scheme 2) starting from commercially available
2,3-dichloroisothiocyanate 34. Treatment with a benzyl
amine for approximately 1h in THF gave the intermedi-
ate thiourea, which, by addition of hydrazine in the
presence of base and HgCl2 for several hours, gave the
corresponding aminoguanidine. The crude compounds
were then heated to reflux in the presence of an ortho-
Cl
Cl
N
N
N
Cl
NHNH2
Cl
i
ii
3
4
Cl
N
N
Cl
N
N
N
N
Cl
Cl
iii or iv
Br
HN
n
R
5
6-33
Scheme 1. Reagents and conditions: (i) formamide, 120 °C, 16 h, 72%;
(ii) NBS, benzoyl peroxide, CCl4, reflux, 12 h, 35–42%; (iii) n = 0;
RNH2, Pd2(dba)3, XANTPHOS, NaOtBu, toluene, 100 °C, 12 h; (iv)
n = 1; RCH2NH2 (2–4 equiv), 90 °C, 4–12 h, 10–48%.
On the right-hand side of the pharmacophore, replace-
ment of phenyl with pyridine resulted in a loss of

A. S. Florjancic et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2089–2092
2091
Table 1. 5-Amino-1-(2,3,-dichlorophenyl)-1,2,4-triazole regioisomer:
N-phenyl and N-benzyl SAR
Table 2. 5-Amino-1-(2,3-dichlorophenyl)-1,2,4-triazole regioisomer:
N-pyridylmethyl SAR
Cl
N
N
Cl
N
N
N
N
Cl
R
Cl
HN
HN
N
n
R
a
hP2X7pIC50 rP2X7pIC50
a
Compound
n
R
b
b
Compound
R
hP2X7 pIC50
rP2X7 pIC50
1
2
—
—
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
—
—
H
6.91
7.11
6.44
7.75
7.34
6.99
5.73
6.21
6.51
6.68
5.41
6.62
6.54
5.69
4.96
5.26
5.31
6.68
6.70
5.99
5.15
6.15
5.86
4.98
6.31
6.54
7.40
6.87
6.20
6.80
26
27
28
29
30
31
32
33
H
6.93
<5
5.88
<5
Ha
Me
6
7.93
8.04
7.90
7.55
7.86
7.82
6.53
7.59
7.55
6.67
6.89
6.86
7
2-Me
2-Et
Azetidin-1-yl
8
Pyrrolidin-1-yl
Morpholin-4-yl
Pyridin-3-yloxy
Thiophen-3-yl
9
2-OMe
2-OEt
2-OCF3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2-Morpholin-4-yl 5.47
a 4-Pyridyl.
H
2-Me
7.32
7.68
6.81
6.32
7.24
6.54
5.79
7.34
7.31
7.70
7.51
7.17
b Values are means of 2–4 experiments. Standard deviations ranged
from 0.01 to 0.20 with an average of 0.11. Compounds tested at the
recombinant human and rat P2X7 receptors as described.17
3-Me
4-Me
2-OMe
3-OMe
4-OMe
2-OEt
no appreciable species differences and compound 38
was only 3-fold less potent at rat whereas its counter-
part 33 was 10-fold less potent at rP2X7 (Table 2).
The presence of a methyl group at R1 (cf. 38 and
39), had little effect on rP2X7 potency compared to
3-fold loss of activity at hP2X7. With the aim of
exploring potentially water-solubilizing functionality,
compounds 41 and 42 were synthesized.19 Both ana-
logs maintained moderate to potent activity at
hP2X7 demonstrating that additional hydrogen bond
donating and accepting groups may be installed in this
position.
2-SMe
2-SO2Me
2,3-Di-OMe
2,3-Di-Me
2-(Pyridin-3-yloxy) 7.33
a Values are means of 2–4 experiments. Standard deviations ranged
from 0.01 to 0.30 with an average of 0.14. Compounds tested at the
recombinant human and rat P2X7 receptors as described.17
potency (13 vs 26 and 27). Incorporation of a substi-
tuent in the 2-position of the 3-pyridyl group (28–33),
however, returned potency to the level observed with a
substituted phenyl. Compound 32, for example, is sim-
ilarly potent to 25 at rP2X7 and 3-fold more potent at
hP2X7. This group of structural analogs (28–33) fur-
ther underscores the range of tolerated substitutions
in the ortho position in this series, with amine (29–
31), arylether (25 and 32), and aryl (33) groups all
showing potent activity. Although many compounds
in this series display about 10-fold greater potency
at human over rat P2X7, the azetidine and pyrrolidine
analogs 29 and 30 show comparatively small species
difference (2- to 3-fold).
Overall, the wide variety of different functional groups
tolerated in the ortho (Tables 1–3) position suggests that
these moieties may not engage in specific receptor inter-
Table 3. 3-Amino-4-(2,3-dichlorophenyl)-1,2,4-triazole regioisomer:
N-pyridylmethyl SAR
R1
N
Cl
N
N
Cl
HN
N
R2
a
hP2X7pIC50 rP2X7 pIC50
a
Compound R1 R2
A smaller group of regioisomeric aminotriazoles 35–42
was also evaluated for activity at human and rat P2X7
(Table 3). This core afforded a synthetic handle which
allowed for limited probing of chemical space on the
aminotriazole at a position which was previously inac-
cessible (R1 group). In general, similar SAR trends
were observed in this regioisomeric series in that ortho
substitution (R2) substantially increased potency (ꢀ10-
fold), there was a high degree of tolerance for substi-
tution at R2 and compounds displayed similar potency
at hP2X7 to those in Table 2 (cf. 28, 30, 32, 33 vs 36–
38, and 40). Although the compounds in Table 3 gen-
erally showed greater potency for human over rat, the
species differences were less marked here for most ana-
logs. For example, compounds 37, 39, and 40 showed
35
36
37
38
39
40
H
H
H
H
H
Me
6.45
7.54
7.83
7.96
7.40
7.51
5.95
6.75
7.92
7.44
7.34
7.45
Pyrrolidin-1-yl
Thiophen-3-yl
Me Thiophen-3-yl
H
H
Pyridin-3-yloxy
N
N
NH2
41
42
7.49
7.17
NT
NT
O
H
N
N
a Values are means of 2–3 experiments. NT, not tested. Standard
deviations ranged from 0.02 to 0.20 with an average of 0.12. Com-
pounds tested at the recombinant human and rat P2X7 receptors as
described.17

2092
A. S. Florjancic et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2089–2092
8. Collo, G.; Neidhart, S.; Kawashima, E.; Kosco-Vibois,
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actions at hP2X7 beyond that afforded by relatively sim-
ple substituents such as methyl and methoxy. At rP2X7,
however, the two regioisomeric aminotriazoles appear
to diverge somewhat with regard to the preferred substi-
tutions in this part of the pharmacophore. A narrower
range of substituents in the 5-amino-1-phenyl-1,2,4-tria-
zole regioisomer displayed enhanced rP2X7 potency
compared to the 3-amino-4-phenyl-1,2,4-triazole regio-
isomer. Whereas in the former case amino substitution
(e.g., 29 and 30) met this criteria, in the latter case some-
what greater flexibility was observed with amino (37),
aryl (38) and arylether (40) groups all having enhanced
rP2X7 activity. The sulfone 22 also showed enhanced
rP2X7 potency, however this substitution was not inves-
tigated in both regioisomers. A generally more balanced
activity across species is clearly a desirable feature of this
subgroup of compounds that is shared by few other
P2X7 antagonists.14,20
12. Adis Data Information (2007). http://pipeline.dialog.com/
abb_lab/adrd.htm.
13. Nelson, D. W.; Gregg, R. J.; Kort, M. E.; Perez-Medrano,
A.; Voight, E. A.; Wang, Y.; Grayson, G.; Namovic, M.
T.; Donnelly-Roberts, D. L.; Niforatos, W.; Honore, P.;
Jarvis, M. F.; Faltynek, C. R.; Carroll, W. A. J. Med.
Chem. 2006, 49, 3659.
14. 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.
Bioorg. Med. Chem. Lett. 2007, 17, 4044.
15. Nagarajan, K.; Talwalker, P. K.; Kulkarni, C. L.; Ven-
kateswarlu, A.; Prabhu, S. S.; Nayak, G. V. Ind. J. Chem.
Sect. B 1984, 12, 1243.
16. X-Ray crystal analysis verified that the bromination
occurred exclusively at the 5-position of the triazole
ring.
17. Honore, P. M.; Donnelly-Roberts, D.; Namovic, M.;
Hsieh, G.; Zhu, C.; Mikusa, J.; Hernandez, G.; Zhong, C.;
Gauvin, D.; Chandran, P.; Harris, R.; Perez-Medrano, A.;
Carroll, W.; Marsh, K.; Sullivan, J.; Faltynek, C.; Jarvis,
M. F. J. Pharmacol. Exp. Ther. 2006, 319, 1376.
18. For 1-(2,3-dichlorophenyl)-5-(o-tolyloxy)-1H-1,2,4-tria-
zole: hP2X7 pIC50 = 5.8; rP2X7 pIC50 = 5.6.
In summary, a new series of potent and selective21
aminotriazole P2X7 antagonists has been discovered.
These studies have provided us with a wider under-
standing of the overall P2X7 pharmacophore by
expanding the diversity of linkers as well as substitu-
tions on the right hand aromatic group. The aminotri-
azole analogs described herein were found to possess
enhanced potency relative to earlier P2X7 triazoles.
Additionally, structural features were uncovered that
confer potent activity at both the human and rat
P2X7 receptors.
References and notes
19. Compounds 41 and 42 were prepared via the correspond-
ing Boc-protected homopiperazine intermediate that was
deprotected and alkylated with the appropriate alkylating
agent.
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