Synthesis, radiolabeling, and preliminary biological evaluation of [ 3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl] -4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7 receptor

Article abstract of DOI:10.1016/j.bmcl.2004.07.095

The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X₇ receptor, named [ ³H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl] -4-(o-tolyl)-piperazine (compound 13),

Full text of DOI:10.1016/j.bmcl.2004.07.095

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5709–5712
Synthesis, radiolabeling, and preliminary biological evaluation
of [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-
4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7
receptor
Romeo Romagnoli,a Pier Giovanni Baraldi,a,e, Maria Giovanna Pavani,
a
*
Mojgan Aghazadeh Tabrizi,a Allan R. Moorman,b Francesco Di Virgilio,c,e
Elena Cattabriga,d Cecilia Pancaldi,d Stefania Gessid and Pier Andrea Boread,e
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy
bKing Pharmaceuticals Research and Development Inc., 4000 CentreGreen Way, Cary, NC 27513, USA
c
`
Dipartimento di Medicina Diagnostica e Sperimentale, Sezione di Patologia Generale, Universita di Ferrara, 44100 Ferrara, Italy
d
`
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita di Ferrara, 44100 Ferrara, Italy
eInterdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara, 44100 Ferrara, Italy
Received 18 June 2004; accepted 22 July 2004
Available online 16 September 2004
Abstract—The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X7 recep-
tor, named [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine (compound 13), are reported. This
compound bound to human P2X7 receptors expressed in HEK transfected cells with KD and Bmax value of 3.46 0.1nM and
727 73fmol/mg of protein, respectively. The high affinity and facile labeling makes it a promising radioligand for a further char-
acterization of P2X7 receptor subtype.
Ó 2004 Elsevier Ltd. All rights reserved.
The P2X7 receptor (P2X7R) is an unusual nondesensitiz-
ing, cation selective, ion channel directly gated by extra-
cellular ATP, and a few pharmacological ATP analogs.1
This receptor, previously known as P2Z, was initially
described in lymphocytes and macrophages and thought
to be restricted to cells involved in inflammation and
immunity.2 However, we now know that, despite its
elective expression in immunocytes, P2X7 is also present
in cells not primarily participating in immunomodula-
tion.3 The structural feature that most characteristically
differentiates P2X7 from the other six members of the
P2X subfamily is the long (240aa) cytoplasmic carboxy-
terminal tail.4 It is understood that many of the typical
(and possibly exclusive) functional responses elicited by
P2X7 stimulation depend on this extended amino acid
stretch.5
tral neurons,3 lymphocytes7 epithelial cells,8 endothelial
cells,9 and fibroblasts.10 However, it is also well estab-
lished that mononuclear phagocytes (macrophages,
dendritic cells) express P2X7 to the highest level, and
these are the cells in which P2X7R functions have been
best characterized.2 There are few doubts that this recep-
tor is one of the most potent stimuli for the release of
N
O
S
O
O
N
N
S
R
O O
O
Expression of P2X7 message or protein has been
reported in cell types as different as macrophages6 cen-
N
N
1a, KN 62, R=H
1b, R=o-CH3
*
Corresponding author. Tel.: +39 0532 291293; fax: +39 0532
291296; e-mail: brlpgv@unife.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.095

5710
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5709–5712
OH
1500
1000
500
0
OH
OH
Total
Specific
Non-specific
Br
Br
Br
Br
a
b
N
O
N
O
N
O
Boc
Boc
Boc
HO
O
O
4
3
2
c
N
OH
Br
N
Br
N
O
S
O
0
5
10
15
20
25
30
O
[3H]Compound 13 free (nM)
Br
N
Br
N
Boc
250
d, e
KD = 3.46 0.1 nM
N
O
N
S
B
max = 727 73 fmol/mg of protein
200
150
100
50
O O
5
N
O
6
f
N
SO2
3H
0
O
N
0
100
200
300
400
500
600
700
3H
N
B
Figure 1. Saturation of 13 binding to human HEK P2X7 transfected
cells. In saturation experiments, 100lL of membrane homogenate
(80lg protein/assay) was incubated in duplicate with 10–12 different
concentrations of 13 in the range 0.3–30nM. Nonspecific binding,
defined as binding in the presence of 1lM of 1b, at the KD value for the
radioligand was 40% of total binding. Incubation time was 60min at
25°Cto allow equilibrium to be reached. Values are the means and
vertical lines SE mean of four separate experiments performed in
triplicate. In the lower panel the Schatchard plot of the same data is
shown. KD value was 3.46 0.1nM and Bmax value was 727 73fmol/
mg of protein.
N
S
O O
N
O
7
Scheme 1. Reagents and conditions: (a) Br2, C HCl2, rt, 30min; (b)
2
LiOH, THF, H2O, MeOH, rt, 1h; (c) o-tolyl piperazine, HOBt, EDC,
DMF, rt, 24h; (d) TFA, CH2Cl2, rt; (e) isoquinoline sulfonyl chloride,
TEA, CH2Cl2, rt, 18h; (f) tritium gas, 10% Pd/C, EtOH.
mature, biologically active, Interleukin-1b (IL-1b).11
Besides IL-1b, P2X7 also drives the release of other
key pro-inflammatory cytokines such as tumor necrosis
factor (TNF)-a and probably IL-6.12 These features
IC50 = 15nM), which results 3-fold more potent than
KN-62.14 Starting from this result, we focused our atten-
tion on the radiolabeling of 1b with tritium, which could
furnish a useful tool to investigate the receptor distribu-
tion and pharmacology. The incorporation of tritium
into the carbon framework of 1b is, however, not easily
performed.
make P2X7
receptor.
a new potential immunomodulatory
Until recently, the lack of potent and selective ligands
has hampered the development of radioligands to
devise useful receptor binding assays. Our group has
recently reported the identification and the functional
antagonistic properties of a novel series of KN-62-re-
lated compounds with general structure 1, focusing
our attention on the systematic modification of the phe-
nyl piperazine residue, by examining the effects due to
the insertion of different chemical functionalities on the
phenyl ring linked to the piperazine nitrogen.13 The
biological response investigated was the ATP-dependent
Ca2+ influx across the plasma membrane of human
monocytes. KN-62 (1a, IC50 = 51nM) is character-
ized by the presence of a phenyl-piperazine moiety. In
the series of synthesized compounds, which possess
different substituents on the phenyl ring, the presence
of a methyl group in the ortho position gives one of
the most potent compounds of the whole series (1b,
The first approach to the synthesis of the radiolabeled
form of 1b is shown in Scheme 1, and uses (L)-N-Boc-
N-methyl tyrosine methyl ester (2) as starting material.15
Compound 2 was dibrominated at the ortho positions to
the phenolic hydroxyl group to yield the derivative 3.
This intermediate was converted into the corresponding
carboxylic acid derivative 4 after saponification. The
subsequent condensation with o-tolyl piperazine fur-
nished the amide 5 in good yield. The protecting Boc-
group in 5 was conveniently removed by the use of TFA
and furnished the corresponding free amine, which was
then coupled with an excess of 5-isoquinolinesulfonyl
chloride16 to deliver the precursor 6. Unfortunately,
the dehalogenation with tritium gas in presence of 10%
Pd/C, did not furnish the final compound 7, affording

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5709–5712
5711
a, b
c
I
N
NH
10
N
NH
I
N
N Boc
8
9
2HCl
N
d
O
S
O
OH
O
e, f
O
N
O
N
N
Boc
N
N
O
N
11
I
S
O
I
12
N
g
N
O
S
O
O
O
N
N
O
N
S
O
3H
13
N
Scheme 2. Reagents and conditions: (a) (Boc)2O, TEA, DCM, 24h; (b) IPy2BF4, C HCl2, rt, 1h; (c) HCl/MeOH, rt, 2h; (d) Boc-N-methyl tyrosine,
2
HOBt, EDC, DMF, rt, 24h; (e) TFA, CH2Cl2, rt; (f) TEA, isoquinoline sulfonyl chloride, CH2Cl2, rt, 18h; (g) tritium gas, 10% Pd/C, EtOH.
instead a degradation product in which one of the iso-
quinolines was lost.
that determined by kinetic experiments. Kinetic studies
showed that at 25°C, compound 13 binding reached
equilibrium within 50min. Association and dissociation
were fitted to a one-component model significantly bet-
ter than a two-component model (P < 0.05). Calcula-
tions based on the kinetic data gave an observed
association constant (Kobs) of 0.046 0.002minÀ1 with
a corresponding association rate constant (k+1) of
0.007minÀ1 nMÀ1 in HEK 293 cells. Compound 13
binding was rapidly reversed by the addition of 1lM
An alternative approach was employed with the aim of
incorporating the label in a different position of com-
pound 1b in the last step of the synthesis (Scheme 2).
Commercially available o-tolyl piperazine 8 was con-
verted quantitatively into the corresponding N-Boc
derivative and then efficiently monoiodinated on the
17
aromatic ring using IPy2BF4 to furnish the corre-
sponding p-iodo-o-tolyl N-Boc piperazine 9. The subse-
quent N-Boc deprotection of 9, to yield 10, followed by
condensation with N-Boc-N-methyl tyrosine, gave the
amide 11. Removal of the Boc-group was performed
with TFA, followed by condensation with an excess of
5-isoquinolinesulfonyl chloride to provide the precursor
12.18 The final compound 13 ([3H]-1b) was obtained
by dehalogenation of 12 with tritium gas in the pres-
ence of 10% Pd/Cfollowed by HPLCpurification
in good yield (60%) and with a radiochemical purity
>95%.
1b, giving a dissociation rate constant (k ) of
À1
0.025 0.001minÀ1. From the rates of association and
dissociation, the equilibrium constant KD = kÀ1/k+1
was estimated to be 3.57nM.
Displacement studies of [3H]-1b (compound 13) with the
nonlabeled 1b led to an IC50 value of the same order of
magnitude as in the functional assay. In summary, based
upon these findings, the in vitro characterization of
compound 13 indicates that this new radioligand should
be considered a very useful tool to study the human
P2X7 receptor subtype.
Figure 1 shows a saturation curve of the binding of [3H]-
1b (compound 13) to the human P2X7 receptors
expressed in HEK 293 cells. The linearity of the Scat-
chard plot in the inset is indicative, in our experimental
conditions, of the presence of a single class of binding
sites with a Bmax value of 727 73fmol/mg of protein
and KD value of 3.46 0.1nM (n = 4), comparable to
Acknowledgements
The authors would like to thank King Pharmaceuti-
cals Research and Development, Inc. for financial
support and Amersham Biosciences for tritium gas

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R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5709–5712
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