Synthesis and in vitro activity of N′-cyano-4-(2-phenylacetyl)-N-o-tolylpiperazine-1-carboximidamide P2X7 antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.01.094

A novel series of cyanoguanidine-piperazine P2X₇ antagonists was designed based upon the structure of A-740003. Structure-activity relationship (SAR) studies focused on the piperazine moiety and the right hand side substitution. Compounds were assayed for activity at human and rat P2X₇ receptors and compound 29 was found to possess potent activity (IC₅₀ = 30-60 nM) at both species.

Full text of DOI:10.1016/j.bmcl.2008.01.094

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2093–2096
Synthesis and in vitro activity of N0-cyano-4-(2-phenylacetyl)-
N-o-tolylpiperazine-1-carboximidamide P2X7 antagonists
Michael J. Morytko,a Patrick Betschmann,a Kevin Woller,a Anna Ericsson,a
Haipeng Chen,a Diana L. Donnelly-Roberts,b Marian T. Namovic,b
Michael F. Jarvis,b William A. Carrollb,* and Paul Raffertya
aAbbott Bioresearch Center, Department of Medicinal Chemistry, 381 Plantation Street, Worcester, MA 01605, USA
bAbbott Laboratories, Global Pharmaceutical Research and Development, 100 Abbott Park Road, Abbott Park, IL 60064, USA
Received 19 December 2007; revised 21 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—A novel series of cyanoguanidine-piperazine P2X7 antagonists was designed based upon the structure of A-740003. Struc-
ture–activity relationship (SAR) studies focused on the piperazine moiety and the right hand side substitution. Compounds were
assayed for activity at human and rat P2X7 receptors and compound 29 was found to possess potent activity (IC50 = 30–60 nM)
at both species.
Ó 2008 Elsevier Ltd. All rights reserved.
The P2X7 receptor has drawn increasing attention in re-
cent years as a promising new therapeutic target for
small molecule drug research with potential applications
in the treatment of pain and inflammation.1–5 This
ligand-gated ion channel is primarily expressed on cells
of the immune system such as mast cells, macrophages,
lymphocytes, and glia. Acute activation of the P2X7
receptor by its endogenous ligand ATP gives rise to a
cascade of biochemical signaling events including revers-
ible channel opening, activation of caspases and proin-
structure, is reportedly under clinical investigation for
rheumatoid arthritis and inflammatory bowel disease.14
In the pain area, P2X7 KO mice displayed reduced sen-
sitivity to inflammatory or nerve insult15 in agreement
with the efficacy profile of P2X7 antagonists in models
of chronic inflammatory and neuropathic pain.8–10,12,16
These observations may be related to the involvement
of P2X7 in modulating either inflammatory cytokine
or glutamate release.5,17
flammatory
cytokine
(IL-1b,
IL-18,
TNF-a)
The potential therapeutic applications for a potent and
selective P2X7 antagonist has led to a flurry of recent re-
ports both in the patent and scientific literature describ-
ing a diverse array of small molecule ligands.2–5 The
cyanoguanidine P2X7 antagonists 1 (A-740003) and 2
provided an attractive starting point for the design of
additional novel agents due to their potent activity at
both rat and human receptors as well as their reported
selectivity over other P2X and P2Y receptors.9,16 An
early area of focus was to explore the potential to re-
place the unusual aminal unit present in 1 and 2 with
a piperazine moiety as shown. Described below are
structure–activity relationship studies that resulted in
the discovery of a novel series of cyanoguanidine-piper-
azine P2X7 antagonists around the general structure A.
release.1,5,6 Macrophages from P2X7 knock-out (KO)
mice show an impaired ability to release IL-1b in re-
sponse to stimulation with lipopolysaccaride (LPS)
and ATP both in vitro and in vivo.7 Small molecule
P2X7 antagonists have similarly been found to block
IL-1b and IL-18 release in vitro and/or in vivo.8–12 In
a model of collagen-induced arthritis, P2X7 KO mice
additionally showed a reduction in the incidence and
severity of arthritic symptoms compared to wild-type
controls.13 Collectively, these data suggest that P2X7
antagonists may offer a new approach to the treatment
of chronic inflammatory conditions such as rheumatoid
arthritis. AZ9056, a P2X7 antagonist of undisclosed
Compounds were prepared using the general synthetic
methods shown in Scheme 1; here illustrated for the syn-
thesis of target molecules V with the R1 group proximal
Keywords: P2X7; Antagonist; Cyanoguanidine; Piperazine.
*
Corresponding author. Tel.: +1 847 938 6041; fax: +1 847 935
5466; e-mail: william.a.carroll@abbott.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.094

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M. J. Morytko et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2093–2096
H
N
H
N
H
N
H
N
H
N
H
N
N
OCH3
OCH3
OCH3
OCH3
N
O
N
O
NC
NC
1 (A-740003)
2
O
N
R2
H
N
n
N
R1
N
NC
A
Table 1. SAR on the central piperazine ring
to the amide and distal to the cyanoguanidine. The
cyanoguanidine functionality is first constructed by cou-
pling intermediate II with intermediate III where bond
formation occurs predominantly at the less sterically
congested nitrogen. Subsequently, the intermediate IV
is reacted at the more hindered nitrogen to form the
amide bond using similar coupling conditions and giving
rise to final products V. To access analogs with the R1
group proximal to the cyanoguanidine and distal to
the amide, the sequence of coupling reactions was re-
versed making the amide first and the cyanoguanidine
second.
OCH3
OCH3
O
N
H
N
N
3
R1
2
N
NC
Compound
R1
rP2X7
IC50 (lM)a
hP2X7
IC50 (lM)
1
2
3
4
5
6
7
8
—
—
0.018
0.014
19
0.044
0.12
0.46
1.3
cis-3,5-Dimethyl
(R)-2-Isopropyl
(R)-2-Isobutyl
3-tert-Butyl
3-Phenyl
16
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.9
4.5
0.43
0.19
0.058
0.051
0.42
0.14
0.22
2-Phenyl
aValues are means of 2–3 experiments. Compounds tested at the
recombinant human and rat P2X7 receptors as described.9
Initial exploration around the general structure A began
with the evaluation of a variety of substituted pipera-
zines as shown in Table 1. The left and right hand aro-
matic substitutions selected for these investigations were
based upon those which provided potent activity for
compound 2. Although the early alkyl substituted ana-
logs 3–6 provided a moderate level of potency at
hP2X7, activity at the rat receptor was weaker and in
general potency fell short of that seen with 1 and 2.
Replacing the alkyl groups with phenyl in either the 2-
or 3-position (7, 8), however, resulted in a significant
improvement in potency at both rat and human recep-
tors. The potency at hP2X7 for 7 and 8 was comparable
to both 1 and 2.
With the promising activity of compound 7, additional
exploration was conducted on the right hand side
(RHS) in order to ascertain if further improvements in
activity could be attained by replacement of the 3,4-
dimethoxyphenyl with other aromatic groups. As shown
in Table 2, several heteroaryl replacements were found
to possess similar potency as 7 and 8, in particular the
pyridyl (12, 13), thiophene (14, 15), and isoxazole (18)
analogs. The pyrazole compounds 20 and 21 provided
potent activity at hP2X7 but were almost 10-fold weaker
at rP2X7. Some sensitivity to the attachment point of the
heteroaryl group was noted for the isomeric isoxazoles
18 and 19 with attachment at the 5-position of the isox-
azole giving ꢀ10-fold better potency than at the 3-posi-
tion. Other heteroaryls such as furan (16, 17) and indole
(23) were slightly weaker whereas the more basic imidaz-
ole (22) was considerably weaker. The phenyl (9),
4-chlorophenyl (10), and naphthyl (11) analogs also
displayed slightly attenuated activity compared to 7.
Interestingly, adamantyl substitution, which is a com-
mon feature of many P2X7 antagonists,3,4 was not found
to provide potent activity in this series (e.g., 24).
R1
Na+
H
N
S-
i
ii
NH
NCS
+
N
HN
NC
I
II
III
R1
R1
O
R2
NH
N
iii
H
N
H
N
N
N
N
N
NC
NC
IV
V
Scheme 1. Reagents: (i) NaHNCN, DMF; (ii) EDC, DMF; (iii) EDC,
DMF, R2CH2CO2H.

M. J. Morytko et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2093–2096
2095
Table 2. SAR on the right hand side aromatic group
as part of these early investigations, a limited foray in
this area did prove fruitful. Replacement of the o-tolyl
group with the 5-quinolinyl moiety as in A-740003 was
investigated. Gratifyingly, compound 29 proved to be
the most potent compound at rP2X7 from this series,
with an IC50 of 30 nM. This was an especially encourag-
ing result given the previous observations that direct
attachment of the right hand aromatic group was not
optimal for potency at the rat receptor. Compound 29
was also found to be selective for P2X7 over other P2
receptors, as evidenced by the lack of activity at
P2X2/3, P2X3, P2X4, and P2Y2 at 10 lM.
O
R2
N
H
N
N
N
NC
Compound R2
rP2X7
IC50 (lM)a IC50 (lM)
hP2X7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Phenyl
0.38
0.11
0.14
0.15
0.39
0.15
0.27
0.52
0.25
0.17
0.61
0.56
0.32
0.088
0.14
0.31
4-Chlorophenyl
2-Naphthyl
4-Pyridyl
In summary, a novel series of cyanoguanidine-pipera-
zines was discovered with potent and selective activity
at P2X7. It was found from these early studies that, with
suitable structural modifications, potency could be
achieved comparable to that of A-740003. Most analogs
displayed approximately 3- to 5-fold greater potency for
hP2X7 over rP2X7, however, compound 29 was highly
potent at both species. These findings indicate that sub-
stantial flexibility around the pharmacophore of 1 and
29 exists to incorporate structural changes with reten-
tion of potent P2X7 antagonism. Additional studies fur-
ther describing structural modifications around 7 and 29
will be the subject of future reports.
0.036
0.040
0.046
0.071
0.11
3-Pyridyl
2-Thienyl
3-Thienyl
2-Furanyl
3-Furanyl
0.22
0.046
0.41
3-Methylisoxazol-5-yl
5-Methylisoxazol-3-yl
5-Methyl-1H-pyrazol-1-yl
3-Methyl-1H-pyrazol-1-yl
1H-Imidazol-4-yl
0.060
0.040
1.0
14
1H-Indol-3-yl
1-Adamantyl
0.21
0.63
0.10
1.5
a Values are means of 2–3 experiments. Compounds tested at the
recombinant human and rat P2X7 receptors as described.9.
The influence of the length of the tether connecting the
piperazine with the right hand aromatic group was also
interrogated (Table 3, 25–28). Surprisingly, the length of
the tether had little effect on activity at hP2X7, with all
compounds displaying IC50s between 0.05 and 0.1 lM.
An expanded panel of directly attached aromatic amides
(n = 0) that overlapped most of the substitutions in
Table 2 was also investigated. As seen for 25, these addi-
tional directly attached amides displayed equivalent to
slightly reduced (2- to 3-fold) potency when compared
with their counterparts in Table 2 (data not shown).
At rP2X7 direct attachment of the aromatic group (25)
gave the weakest activity.
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replacement of o-tolyl with 5-quinolinyl
OCH3
O
N
OCH3
n
H
N
N
Ar
N
NC
Compound
Ar
n
rP2X7
IC50 (lM)a
hP2X7
IC50 (lM)
25
26
27
28
29
o-Tolyl
o-Tolyl
o-Tolyl
o-Tolyl
0
1
2
3
0
0.29
0.14
0.094
0.058
0.057
0.070
0.059
0.076
0.095
0.030
5-Quinolinyl
a Values are means of 2–3 experiments. Compounds tested at the
recombinant human and rat P2X7 receptors as described.9.

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