From tyrosine to glycine: Synthesis and biological activity of potent antagonists of the purinergic P2X7 receptor

Article abstract of DOI:10.1021/jm070443e

The characterization of the native and recombinant P2X₇ receptor continues to be hindered by the lack of specific and subtype-selective antagonists with a "druglike" profile. However, a tyrosine derivative named KN-62 exhibits selective P2X

Full text of DOI:10.1021/jm070443e

3706
J. Med. Chem. 2007, 50, 3706-3715
From Tyrosine to Glycine: Synthesis and Biological Activity of Potent Antagonists of the
Purinergic P2X7 Receptor
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Maria Dora Carrion,† Carlota Lopez Cara,† Delia Preti,† Olga Cruz-Lopez,†
Mojgan Aghazadeh Tabrizi,† Allan R. Moorman,§ Stefania Gessi,‡ Eleonora Fogli,‡ Valeria Sacchetto,‡ and Pier Andrea Borea‡
Dipartimento di Scienze Farmaceutiche, UniVersita` di Ferrara, Ferrara, Italy, Research and DeVelopment, King Pharmaceuticals Inc., 4000
CentreGreen Way, Suite 300, Cary, North Carolina, and Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia,
UniVersita` di Ferrara, Ferrara, Italy
ReceiVed April 14, 2007
The characterization of the native and recombinant P2X7 receptor continues to be hindered by the lack of
specific and subtype-selective antagonists with a “druglike” profile. However, a tyrosine derivative named
KN-62 exhibits selective P2X7 receptor-blocking properties. As a molecular simplification of KN-62, the
present study was designed to evaluate the functional antagonistic properties of a novel series of glycine
derivatives characterized by the presence of different phenyl-substituted piperazine moieties. Antagonistic
activity of these glycine derivatives was tested on HEK293 cells transfected with the human P2X7 receptor.
The most potent P2X7 receptor antagonist identified in this study (compound 4g) contains an o-fluorine
substituent on the phenylpiperazine moiety and had an IC50 of 12.1 nM. The biological responses investigated
were ATP-dependent Ca2+ influx across the plasma membrane and ethidium bromide uptake.
Introduction
subtype.7 Opening of the P2X7 pore allows plasma membrane
fluxes of ions as well as of hydrophilic molecules with molecular
masses up to 900 Da.8-10
The P2X7 receptor is an ATP-sensitive, ligand-gated ion
channel that, like other members of the P2X family, mediates
a nonselective cation conductance when stimulated with an
appropriate ligand.1 Currently, seven different P2X receptor
subtypes have been molecularly defined and the P2X7 receptor
seems to be the most divergent member of this family in terms
of molecular structure, pharmacology, and function.1 The
structural features that more typically differentiate P2X7 from
the other members of the P2X subfamily are its size (595 amino
acids), its long cytoplasmic carboxyterminal tail (242 residues),2
and its peculiar ability to form a nonselective plasma membrane
pore of 3-5 nm size when exposed to prolonged or repeated
agonist stimulation.3,4 The “large pore” properties of this
receptor are usually verified by uptake of fluorescent dyes such
as ethidium and YoPro1a (4-[(3-methyl-2-(3H)-benzoxazolyliden-
e)methyl]-1-[3-(triethylammonio)propyl] diiodide).1 The mech-
anisms underlying the process of channel dilation were originally
thought to depend on the C-terminus region of the P2X7
receptor.2 However, emerging evidence indicates that distinct
proteins, activated by P2X7 receptors, may play a key role in
the dye uptake signal.5,6 Recent work has identified panx1 as
the large pore pathway activated by P2X7 receptors and as an
upstream molecule essential for the activation of the inflam-
matory cascade triggered by activation of this purinergic
Since their discovery in immune cells, P2X7 receptors have
been proposed as mediators of inflammation.11 In particular,
P2X7 receptors are found in most immune cells of the periphery
and the brain, where their activation leads to multiple down-
stream events such as cell permeabilization, apoptosis, and/or
cytokine release.12-14 In the case of prolonged stimulation, cell
death occurs through necrotic or apoptotic pathways.8,15,16
Interestingly, P2X7 receptors are rapidly up-regulated and
activated after inflammatory insults.17
However, despite its selective expression in immunocytes,
P2X7 is also present in cells not primarily participating in
immunomodulation, such as presynaptic terminals in the central
and peripheral nervous system18 and human epidermal Langer-
hans cells.19
On glial cells, the P2X7 receptor has been shown to mediate
release of glutamate, which is known to be involved in the
neurotransmission of painful sensory signals.20 Up-regulation
of the P2X7 receptor, most likely on activated microglia, was
reported in association with ischemic damage and necrosis by
occlusion of rat middle cerebral artery.21 Recent studies indicate
a role of the P2X7 receptor in the generation of superoxide in
microglia around â-amyloid plaques in a transgenic model for
Alzheimer’s disease22a and in multiple sclerosis lesions from
22b
autopsy brain sections.
* To whom correspondence should be addressed. For R.R.: phone, 39-
(0)532-291290; fax, 39-(0)532-291296; e-mail, rmr@unife.it. For P.G.B.:
phone, 39-(0)532-291293; fax, 39-(0)532-291296; e-mail, baraldi@unife.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
§ King Pharmaceuticals Inc.
These features make desirable P2X7 antagonists that can be
efficiently used in preventing, treating, or ameliorating a variety
of pain states (e.g., chronic inflammatory pain and neuropathic
pain), inflammatory processes, and neurodegenerative conditions
(e.g., stroke and Alzheimer’s disease).
‡ Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farma-
cologia, Universita` di Ferrara.
a Abbreviations: Boc, tert-butyloxycarbonyl; Cbz, benzyloxycarbonyl;
EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride; HEK,
human embryonic kidney; HOBt, 1-hydroxybenzotriazole; KN-62, 1-[N,O-
bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; Pd/C,
palladium on activated charcoal; TFA, trifluoroacetic acid; YOPRO-1,
quinolinium, 4-[(3-methyl-2-(3H)-benzoxazolylidene)methyl]-1-[3-(triethy-
lammonio)propyl] diiodide. These abbreviations and symbols are used in
addition to those by the IUPAC-IUB Commission on Biochemical
Nomenclature (J. Biol. Chem. 1985, 260, 14-42).
Among these, a tyrosine derivative named KN-62 (1-(N,O-
bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl)-4-phenylpip-
erazine, compound 1a, Chart 1) has been considered for several
years to be one of the most potent antagonists for the human
P2X7 receptors with an IC50 of 51 nM.23-25
In connection with the attempt to improve the activity and
to study the structure-activity relationship of KN-62 inhibition
10.1021/jm070443e CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/23/2007

Antagonists of the Purinergic P2X7 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3707
Scheme 1a
Chart 1. Chemical Structures of L-Tyrosine (1a and 1b),
L-Phenylalanine (2), L-Alanine (3), and Glycine (4) Derivatives
on the P2X7 receptor, our group has prepared a novel series of
KN-62-related compounds with general structure 1 and de-
scribed their functional antagonistic properties, focusing our
attention on the systematic modification of the phenylpiperazine
residue.26 In this series, the p-fluorophenyl derivative was the
most potent compound (1b, IC50 ) 6.3 nM), found to be 8-fold
more potent than KN-62. The potency of this latter derivative
was maintained by removing the methyl group linked to the
nitrogen on the R-position of the tyrosine moiety. This was
confirmed by the preparation of another series of tyrosine
derivatives synthesized by Jacobson’s group.27
a Reagents: (a) arylpiperazine, EDCl, HOBt, DMF, room temp, 18 h;
(b) TFA, CH2Cl2, room temp; (c) 10% Pd/C, EtOH, or TFA, CH2Cl2, room
temp; (d) ArSO2Cl, TEA, CH2Cl2, room temp.
different arylsulfonyl moieties, corresponding to quinoline-5-
and quinoline-8-sulfonyl functions, and with the naphthalene-
1-sulfonyl group. Finally, we prepared a small series of
homologues of glycine (n ) 2) corresponding to the â-alanine
derivatives 4c, 4d, 4ab, and 4ae.
Chemistry
Tyrosine derivatives with general formula 1 do not represent
ideal candidates for the discovery of oral drugs because of their
high molecular weight (>700 Da), high lipophilicity (clogP >
6), and the presence of metabolically labile sulfonate groups.
However, the lack of actvity of the L-phenylalaninylsulfonamido
derivative 2 suggests the importance of sulfonate for the P2X7
antagonism. Because of our efforts to synthesize new P2X7
receptor antagonists with druglike properties, by removing the
phenyl sulfonate ring to yield the L-alanine derivative 3, the
activity was dramatically reduced. Surprisingly, the replacement
of the amino acid L-tyrosine with glycine allowed us to
synthesize a new series of compounds with general formula 4,
active at nanomolar concentration and with acceptable molecular
weight. In particular, compound 4a was equipotent with KN-
62, although 10-fold less potent than compound 1b.
Compound 4a then became the starting point for the synthesis
of a series of derivatives with general structure 4, in which four
different positions of the aminoacid glycine were systematically
modified in the structure. A large series of compounds were
synthesized in order to study the effects of the insertion of
electron-donating and electron-withdrawing groups on the
phenyl ring linked to the piperazine nitrogen. These modifica-
tions served to alter the electronic, steric, and lipo/hydrophilic
features of the aromatic portion joined to the piperazine. By
synthesis of a small series of sarcosine derivatives (R2 ) Me;
4b, 4h, and 4ak), we were able to evaluated the importance of
the presence of a methyl on the R-nitrogen of glycine.
Compounds with general structure 4 were prepared using
N-Boc-glycine (5a), N-Cbz-glycine (5b), N-Boc-sarcosine (5c),
and N-Cbz-â-alanine (5d) as starting materials, following the
synthetic sequence outlined in Scheme 1. These amino-protected
precursors (5a-d) were converted to activated 1-hydroxy-1,2,3-
benzotriazole (HOBt) esters with HOBt and 1-(3-dimethylami-
nopropyl)-3-ethylcarbodimide hydrochloride (EDC) and coupled
with the appropriate commercially available N-arylpiperazines28
to give amides with general formula 6 in good yields. The
protecting tert-butyloxycarbonyl (Boc) groups were conveniently
removed using trifluoroacetic acid (TFA), while the benzyloxy-
carbonyl functions (Cbz or Z) were cleaved off using a catalytic
hydrogenation with 10% palladium on activated charcoal (Pd/
C), furnishing the corresponding free amines. These were then
coupled with an excess of arylsulfonyl chloride29 in CH2Cl2 in
the presence of triethylamine to furnish the respective sulfona-
mides 4a-ap in acceptable yields. Compound 4ac was obtained
by a reductive hydrogenation of 4aa in the presence of 10%
Pd/C.
The structures of the synthesized compounds 4a-ap and the
yields of the syntheses are presented in Table 1.
Results and Discussion
The potency of derivatives with general formula 4 in the
inhibition of ATP-stimulated Ca2+ influx and ethidium bromide
uptake in HEK293 cells transfected with the human P2X7
receptor (HEK293-P2X7) was investigated. Figure 1 shows
typical traces of ATP-dependent calcium increase in HEK 293-
We also evaluated the antagonistic effect of the replacement
of the N-isoquinoline-5-sulfonyl group in the compound with

3708 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Table 1. Physical and Synthetic Data of Novel Glycine Analogues
Romagnoli et al.
compd
Ar
R1
R3
n
m
mp, °C
yield,a %
formulab
anal.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
4ai
4aj
4ak
4al
4am
4an
4ao
4ap
p-F-C6H4
p-F-C6H4
p-F-C6H4
p-F-C6H4
p-F-C6H4CH2
m-F-C6H4
o-F-C6H4
H
Me
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
A
A
A
D
A
A
A
A
A
A
A
A
B
C
D
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
D
A
D
A
A
A
D
A
D
A
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
71-73
62
64
58
65
61
51
69
63
55
55
57
48
54
53
73
58
54
58
52
58
63
52
54
54
49
67
68
52
77
44
43
60
52
52
61
62
52
57
54
46
51
57
C21H21N4O3SF
C22H23N4O3SF
C22H23N4O3SF
C22H22N3O3SF
C22H23N4O3SF
C21H21N4O3SF
C21H21N4O3SF
C22H23N4O3SF
C21H20N4O3SF2
C21H20N4O3SF2
C21H20N4O3SF2
C21H20N4O3SF2
C21H21N4O3SF
C21H21N4O3SF
C22H22N3O3SF
C21H21N4O3SCl
C22H23N4O3SCl
C21H21N4O3SBr
C21H21N4O3SI
C22H24N4O3S
C23H26N4O3S
C22H24N4O3S
C23H24N4O4S
C22H21N5O3S
C22H21N4O3SF3
C22H24N4O4S
C21H21N5O5SF
C22H23N5O5S
C21H23N5O3S
C21H22N4O3S
C22H24N4O3S
C25H24N4O3S
C22H23N3O3S
C19H20N6O3S
C26H25N3O3S
C22H24N4O3S
C23H26N4O3S
C23H26N4O3S
C23H25N3O3S
C23H26N4O3S
C24H27N3O3S
C24H28N4O3S
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
58-60
73-75
63-65
50-51
68-70
67-69
o-F-C6H4
51-52
o,p-2F-C6H3
m,p-2F-C6H3
o,m-2F-C6H3
o,m′-2F-C6H3
o-F-C6H4
o-F-C6H4
o-F-C6H4
p-Cl-C6H4
p-Cl-C6H4CH2
p-Br-C6H4
p-I-C6H4
p-CH3-C6H4
p-CH3-C6H4CH2
o-CH3-C6H4
p-CH3CO-C6H4
p-CN-C6H4
p-CF3-C6H4
p-OCH3-C6H4
p-NO2-C6H4
p-NO2-C6H4
p-NH2-C6H4
C6H5
C6H5
1-naphthyl
C6H5
2-pyrimidinyl
1-naphthyl
C6H5CH2
186-187
73-75
202-204
215-217
62-63
52-53
68-70
70-71
68-70
103-15
202-205
73-75
146-148
153-155
153-155
52-53
88-90
76-78
170-172
72-73
93-95
63-65
178-180
203-205
58-60
185-187
86-88
202-204
53-55
C6H5CH2
C6H5CH2
C6H5CH2
C6H5(CH2)2
C6H5(CH2)2
C6H5(CH2)3
58-60
52-53
53-55
50-52
72-74
a Yield of synthesized compounds after purification by column chromatography. b All compounds were analyzed for C, H, N. Analytical results were
within 0.4% of the theoretical values.
P2X7 cells in the absence and in the presence of 4g (see below).
Results of experiments similar to the one shown in Figure 1,
performed with a large number of compounds synthesized in
our laboratory, are summarized in Table 2. Compound 4g was
the most active P2X7 receptor antagonist identified in this series,
and several compounds (i.e., 4w, 4aa, 4aj, and 4an) showed
an antagonistic activity more potent than KN-62. Figure 2
displays the inhibition curves of the four most potent compounds
(4g, 4aa, 4aj, and 4an).
Compounds 4g and 4aa were also evaluated for selectivity
at other P2 receptors (P2X1, P2X2, and P2X4), where they were
not found to have significant interactions up to 10 µM.
Initial SAR studies of 4a-ap focused on examining of the
effects on P2X7 inhibition induced by changes in the phenylpip-
erazine portion. As shown in Table 1, the replacement of the
fluoro group with a hydrogen (4ad) gave an inactive compound.
Also, the replacement of the phenyl ring with a pyrimidine
(compound 4ah) decreased the activity dramatically. Some
activity was regained with a more bulky 1-naphthyl group
(compound 4af), which was 1.5-fold less potent then KN-62.
The compounds that possessed different substituents at the
para position of the phenyl ring showed variable potencies. The
derivative 4aa, characterized by the presence of a nitro group,
was the most potent compound of the series, being 3-fold more
potent than KN-62. In the series of para-halogenated analogues,
only the p-fluoro derivative 4a was active. The increase of the
size of the halogen atom from chlorine (4p) to bromine (4r)
and finally to iodine (4s) caused a loss of potency. Starting from
compound 4a, the placement of the fluoro group in the meta
position (compound 4f) diminished the activity, while the ortho
position substitution (compound 4g) was beneficial for the
activity. This latter compound was 5-fold more potent than KN-
62.
Compounds 4a and 4g represented the starting point for the
synthesis of a large series of derivatives in which we introduced
two fluorine atoms in different positions on the phenyl ring. In
contrast to the o- and p-fluorophenyl substitutions, none of the
difluoro substituted analogues (4i-l) yielded encouraging levels
of activity. The dramatic reduction of activity due to the insertion
of a second fluoro group in the phenyl ring could be attributed

Antagonists of the Purinergic P2X7 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3709
Figure 2. Effect of P2X7 antagonists 4g, 4aa, 4aj, and 4an on
intracellular calcium increase induced by 1 mM ATP. P2X7-transfected
HEK 293 cells were incubated in a fluorimeter cuvette with standard
saline solution. The P2X7 antagonists were added 10 min before ATP.
Experimental points are averages of five determinations.
Figure 1. Typical traces that visualize stimulation of cytoplasmic Ca2+
increase in P2X7-transfected HEK 293 cells by 1 mM ATP in the
absence and in the presence of compound 4g. Cells were resuspended
in the fluorometer cuvette in the saline solution described in Chemical
Materials and Methods at a concentration of 106 cells/mL. The P2X7
antagonist was added at a concentration of 100 nM 10 min prior to
ATP.
receptor interaction can be improved with the presence of
chemical groups in the phenyl ring.
To investigate the importance of the distance between the
piperazine basic nitrogen and the phenyl ring by insertion of
one, two, and three methylene units, the corresponding com-
pounds 4aj, 4an, and 4ap were synthesized and tested.
Table 2. Activities of Synthesized Compounds with General Formula 6
on the Calcium Influx in HEK 293-P2X7 Cellsa
The biological data suggested that the optimal chain length
was one or two carbon atoms, which is presumably important
for the relative position of the phenyl ring in space. In fact, a
methylene or an ethylene spacer (compounds 4aj and 4an)
improved the activity, making it 1.5-fold higher than that of
the reference compound KN-62. However, extending the chain
to three carbon atoms (4ap) was clearly detrimental to the
antagonist potency. Cycle enlargement from piperazine (4aj)
to homopiperazine (4al) led to an almost 30-fold decrease in
activity.
compd
IC50 ( SE (nM)
compd
IC50 ( SE (nM)
KN-62
4a
4b
4c
4d
4e
51.1 ( 1.126
60.0 ( 5.2
>1000
4v
4w
4x
4y
>1000
40.2 ( 5.1
>1000
>1000
>1000
>1000
>1000
4z
>1000
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
4ai
4aj
4ak
4al
4am
4an
4ao
4ap
21.1 ( 2.0
>1000
4f
>1000
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
12.1 ( 3.0
>1000
>1000
>1000
>1000
800 ( 10
90.2 ( 5.1
>1000
>1000
A loss of potency was also observed when a substituent such
as fluoro, chloro, or methyl (compounds 4e, 4q and 4u,
respectively) was introduced in the para aryl position of the
benzyl group.
>1000
>1000
>1000
>1000
35.1 ( 3.0
100 ( 11
900 ( 7
>1000
40.0 ( 3.1
>1000
>1000
>1000
>1000
>1000
By synthesizing three compounds (4b, 4h, and 4ak), we were
able to evaluate the biological effect of the insertion of a NR-
methyl group on the glycine skeleton. While NR-methyl
analogues 4b and 4h were inactive with respect to the parent
compounds 4a and 4g, compound 4ak was only 3-fold less
active than the corresponding analogue 4aj.
>1000
>1000
>1000
4s
4t
4u
>1000
130 ( 10
>1000
a IC50 is the 50% inhibitory concentration representing the mean from
dose response curves of at least five experiments. All experiments were
repeated five times.
In the compounds 4b, 4aa, and 4ad, the replacement of the
glycine segment with its homologue, corresponding to â-alanine
derivatives 4c, 4ab, and 4ae, resulted in inactive compounds.
to steric and electronic factors, which may have prevented the
interaction of the difluorophenyl residue with the receptor.
The P2X7 receptor-binding capacity of the nitrogen of the
isoquinoline-5-sulfonyl moiety was studied by replacing this
function with a 1-naphthyl group to obtain the corresponding
derivatives 4d, 4o, 4ag, 4ai, 4am, and 4ao. Unfortunately, these
compounds were not able to maintain the antagonist activity of
the parent compounds 4c, 4g, 4ad, 4af, 4aj, and 4an. We found
that not only the presence but also the position of the nitrogen
atom on the arylsulfonyl moiety is important for the antagonist
activity. In fact, in compound 4g the inhibitory effect was lost
when the isoquinoline-5-sulfonyl moiety was replaced with its
two isomers corresponding to quinoline-5- and quinoline-8-
sulfonyl moieties (derivatives 4m and 4n, respectively).
Replacing the p-fluoro with a p-methyl group (compound 4t)
resulted in a 10-fold drop in potency. When the methyl group
was moved from the para to the ortho position (compound 4v),
the activity was completely lost.
A potency increase was observed with compounds 4aa and
4w, which contain nitro and acetyl groups in the para position
of the phenyl ring. These compounds were 3- and 1.5-fold more
active than KN-62, respectively. The addition of both electron-
withdrawing groups, such as nitrile (4x) and trifluoromethyl (4y),
and electron-donating groups, such as methoxy (4z) and amino
(4ac), was deleterious to the activity. These results support the
suggestion that the phenylpiperazine moiety interacts with the
active site of the receptor and indicate that the compound/
The effect of the four most potent compounds was also
assessed on ethidium bromide uptake induced by 1 mM ATP,
a typical P2X7 response. Figure 3 shows that the novel

3710 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Romagnoli et al.
N-Z-glycine (5b), Boc-sarcosine (5c), and Z-â-alanine (5d) (1
mmol) in dry DMF (5 mL) cooled at 0 °C was added EDC (211
mg, 1.1 mmol, 1.1 equiv), HOBt (1.1 mmol). and the suitable
N-substituted piperazine (1.1 mmol). This mixture was stirred for
17 h and then concentrated in vacuo. The residue was dissolved in
dichloromethane (10 mL), washed with water (5 mL), and then
washed with brine (5 mL). The organic layer was dried and
concentrated in vacuo. The residue, purified by column chroma-
tography, gave 6a-ag.
Benzyl Ester of {2-[4-(4-Fluorophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6a). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 3-7 v/v. The product was obtained
1
as a white solid (yield 76%), mp 118-120 °C. H NMR (CDCl3)
δ: 3.10 (m, 4H), 3.59 (t, J ) 5.2 Hz, 2H), 3.82 (t, J ) 5.2 Hz,
2H), 4.07 (d, J ) 4.2 Hz, 2H), 5.13 (s, 2H), 5.92 (bs, 1H), 7.00
(m, 4H), 7.37 (m, 5H).
Figure 3. Effect of P2X7 antagonists 4g, 4aa, 4aj, and 4an on ethidium
bromide uptake. P2X7-transfected HEK 293 cells were incubated in a
fluorimeter cuvette in the saline solution described in Chemical
Materials and Methods. The P2X7 antagonists were added 10 min
before ATP. Uptake inhibition is expressed as a percentage of maximal
bromide (EB) uptake rate (arbitrary fluorescence units per min)
measured in the absence of inhibitor. Experimental points are averages
of five determinations.
tert-Butyl Ester of {2-[4-(4-Fluorophenyl)piperazin-1-yl]-2-
oxoethyl}methylcarbamic Acid (6b). Eluent for the column
chromatography: EtOAc-petroleum ether, 4-6 v/v. The product
1
was obtained as a colorless oil (yield 77%). H NMR (CDCl3) δ:
1.40 (m, 9H), 2.87 (s, 3H), 3.02 (m, 4H), 3.57 (t, J ) 5.0 Hz, 2H),
3.72 (t, J ) 5.0 Hz, 2H), 4.04 (s, 2H), 6.90 (m, 2H), 7.39 (m, 1H),
7.72 (m, 1H).
Benzyl Ester of {3-[4-(4-Fluorophenyl)piperazin-1-yl]-3-
oxopropyl}carbamic Acid (6c). Eluent for the column chroma-
tography: EtOAc-petroleum ether, 7-3 v/v. The product was
compounds 4g, 4aa, 4aj, and 4an were able to reduce the agonist
effect with the same order of potency obtained in the calcium
studies.
1
obtained as an orange solid, mp 95-97 °C (yield 88%). H NMR
In conclusion, we have discovered a new series of phenylpip-
erazine derivatives that are potent P2X7 antagonists, in which
the original L-tyrosine moiety and the metabolically fragile
sulfonate group of KN-62 and its derivatives were replaced with
glycine. In the series of phenylpiperazine derivatives, the nature
and the position of substituents on the phenyl ring seemed to
exert an important influence on the biological activity. In fact,
only a few substituents (p- and o-F, p-Me, p-COCH3, and
p-NO2) were tolerated for the maintenance of antagonism, and
the isoquinolinesulfonyl moiety was essential for the activity.
Also. a dramatic reduction in potency was observed with the
methylation of the nitrogen atom in the glycine moiety. No clear
structure-activity relationship can be observed based on data
from antagonistic activity, but we have identified several
compounds, in particular 4g and 4aa, that appear to be more
potent than KN-62 and that may be selected for further
development.
(CDCl3) δ: 2.51 (t, J ) 5.6 Hz, 2H), 3.02 (m, 4H), 3.52 (m, 4H),
3.72 (t, J ) 5.6 Hz, 2H), 5.02 (s, 2H), 5.49 (bs, 1H), 6.86 (m, 4H),
7.19 (m, 5H).
tert-Butyl Ester of {2-[4-(4-Fluorobenzyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6d). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 9-1 v/v. The product was obtained
1
as a yellow oil (yield 68%). H NMR (CDCl3) δ: 1.42 (s, 9H),
2.48 (m, 4H), 3.40 (t, J ) 5.2 Hz, 2H), 3.54 (s, 2H), 3.64 (t, J )
5.2 Hz, 2H), 3.93 (d, J ) 4.4 Hz, 2H), 5.50 (s, 1H), 6.93 (t, J )
8.6 Hz, 2H), 7.22 (d, J ) 8.6 Hz, 1H), 7.26 (d, J ) 8.6 Hz, 1H).
Benzyl Ester of {2-[4-(3-Fluorophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6e). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 4-6 v/v. The product was obtained
1
as a white solid (yield 74%), mp 125-126 °C. H NMR (CDCl3)
δ: 3.12 (m, 4H), 3.50 (t, J ) 4.8 Hz, 2H), 3.73 (t, J ) 4.8 Hz,
2H), 4.01 (d, J ) 4.2 Hz, 2H), 5.07 (s, 2H), 5.72 (bs, 1H), 6.54
(m, 4H), 7.37 (m, 5H).
Benzyl Ester of {2-[4-(2-Fluorophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6f). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 4-6 v/v. The product was obtained
Experimental Section
1
as a white solid (yield 78%), mp 108-109 °C. H NMR (CDCl3)
Chemical Materials and Methods. General Procedure. All
reactions were carried out under in inert atmosphere of dry nitrogen
unless otherwise described. Standard hypodermic syringe (glass/
metal Luer) techniques were applied for transferring dry solvents.
Starting materials were purchased and used without any purification.
Reaction courses and product mixtures were routinely monitored
by TLC on silica gel (precoated F254 Macherey-Nagel plates) and
visualized with aqueous KMnO4. Melting points (mp) were
determined on a Buchi-Tottoli apparatus and are uncorrected.
Optical rotations were measured on a Perkin-Elmer 241 polarimeter.
Chemical shifts (δ) are given in ppm upfield from tetramethylsilane.
All products reported showed 1H NMR spectra in agreement with
the assigned structures. 1H NMR was determined in CDCl3 solution
with a Bruker AC200 spectrometer. Microanalytical analyses were
conducted by the Mycroanalytical Laboratory of the Chemistry
Department of the University of Ferrara. Organic solutions were
dried over anhydrous Na2SO4. Methanol was distilled from
magnesium turnings, dioxan was distilled from calcium hydride,
and dry DMF was distilled from calcium chloride. All three were
stored over molecular sieves (3 Å). In high-pressure hydrogenation
experiments, a Parr shaker on a high-pressure autoclave was used.
General Procedure A for the Synthesis of Compounds with
General Formula 6a-ag. To a solution of N-Boc-glycine (5a),
δ: 3.08 (m, 4H), 3.58 (t, J ) 5.2 Hz, 2H), 3.82 (t, J ) 5.2 Hz,
2H), 4.09 (d, J ) 4.4 Hz, 2H), 5.13 (s, 2H), 5.92 (bs, 1H), 6.87
(m, 4H), 7.37 (m, 5H).
tert-Butyl Ester of {2-[4-(2-Fluorophenyl)piperazin-1-yl]-2-
oxoethyl}methylcarbamic Acid (6g). Eluent for the column
chromatography: EtOAc-petroleum ether, 1-1 v/v. The product
1
was obtained as a colorless oil (yield 65%). H NMR (CDCl3) δ:
1.47 (m, 9H), 2.94 (s, 3H), 3.08 (t, J ) 5.2 Hz, 4H), 3.62 (t, J )
5.2 Hz, 2H), 3.79 (t, J ) 5.2 Hz, 2H), 4.09 (s, 2H), 7.07 (m, 4H).
Benzyl Ester of {2-[4-(2,4-Difluorophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6h). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 1-1 v/v. The product was obtained
1
as a white solid (yield 71%), mp 112-113 °C. H NMR (CDCl3)
δ: 3.02 (m, 4H), 3.57 (t, J ) 5.0 Hz, 2H), 3.80 (t, J ) 5.0 Hz,
2H), 4.08 (d, J ) 4.4 Hz, 2H), 5.13 (s, 2H), 5.80 (bs, 1H), 6.83
(m, 3H), 7.33 (m, 5H).
Benzyl Ester of {2-[4-(3,4-Difluorophenyl)piperazin-1-yl]-2-
oxoethyl}methylcarbamic Acid (6i). Eluent for the column
chromatography: EtOAc-petroleum ether, 6-4 v/v. The product
1
was obtained as a white solid (yield 72%), mp 115-116 °C. H
NMR (CDCl3) δ: 3.07 (m, 4H), 3.52 (t, J ) 5.0 Hz, 2H), 3.72 (t,
J ) 5.0 Hz, 2H), 4.02 (d, J ) 4.4 Hz, 2H), 5.08 (s, 2H), 5.75 (bs,

Antagonists of the Purinergic P2X7 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3711
1H), 6.59 (m, 2H), 7.04 (q, J ) 9.8 Hz, 1H), 7.33 (m, 3H), 7.50
(m, 1H), 7.85 (m, 1H).
5.4 Hz, 2H), 4.01 (d, J ) 3.8 Hz, 2H), 5.44 (bs, 1H), 6.90 (d, J )
9.0 Hz, 2H), 7.90 (d, J ) 9.0 Hz, 2H).
tert-Butyl Ester of {2-[4-(4-Cyanophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6t). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 6-4 v/v. The product was obtained
tert-Butyl Ester of {2-[4-(2,3-Difluorophenyl)piperazin-1-yl]-
2-oxoethyl}methylcarbamic Acid (6j). Eluent for the column
chromatography: EtOAc-petroleum ether, 6-4 v/v. The product
1
1
as a white solid (yield 78%), mp 110-112 °C. H NMR (CDCl3)
was obtained as an oil (yield 96%). H NMR (CDCl3) δ: 1.45 (s,
δ: 1.45 (s, 9H), 3.36 (m, 4H), 3.55 (t, J ) 5.6 Hz, 2H), 3.76 (t, J
) 5.6 Hz, 2H), 4.00 (d, J ) 4.4 Hz, 2H), 5.62 (s, 1H), 6.85 (d, J
) 9.0 Hz, 2H), 7.51 (d, J ) 9.0 Hz, 2H).
9H), 3.08 (m, 4H), 3.56 (t, J ) 5.2 Hz, 2H), 3.80 (t, J ) 5.2 Hz,
2H), 4.02 (d, J ) 4.4 Hz, 2H), 5.62 (bs, 1H), 6.72 (m, 1H), 6.84
(m, 1H), 7.02 (m, 1H).
Benzyl Ester of {2-[4-(4-Trifluoromethylphenyl)piperazin-1-
yl]-2-oxoethyl}carbamic Acid (6u). Eluent for the column chro-
matography: EtOAc-petroleum ether, 4-6 v/v. The product was
tert-Butyl Ester of {2-[4-(2,5-Difluorophenyl)piperazin-1-yl]-
2-oxoethyl}methylcarbamic Acid (6k). Eluent for the column
chromatography: EtOAc-petroleum ether, 7-3 v/v. The product
1
1
obtained as a white solid (yield 87%), mp 155-157 °C. H NMR
was obtained as a white solid (yield 87%). H NMR (CDCl3) δ:
(CDCl3) δ: 3.27 (m, 4H), 3.59 (t, J ) 4.8 Hz, 2H), 3.80 (t, J )
4.8 Hz, 2H), 4.07 (d, J ) 3.8 Hz, 2H), 5.13 (s, 2H), 5.83 (bs, 1H),
6.94 (d, J ) 8.6 Hz, 2H), 7.37 (m, 5H), 7.51 (d, J ) 8.6 Hz, 2H).
tert-Butyl Ester of {2-[4-(4-Methoxyphenyl)piperazin-1-yl]-
2-oxoethyl}carbamic Acid (6v). Eluent for the column chroma-
tography: EtOAc-petroleum ether, 6-4 v/v. The product was
1.45 (s, 9H), 3.08 (m, 4H), 3.56 (t, J ) 5.2 Hz, 2H), 3.78 (t, J )
5.2 Hz, 2H), 4.00 (d, J ) 4.4 Hz, 2H), 5.62 (bs, 1H), 6.61 (m,
2H), 7.02 (m, 1H).
Benzyl Ester of {2-[4-(4-Chlorophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6l). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 7-3 v/v. The product was obtained
1
obtained as a pink solid (yield 72%), mp 82-84 °C. H NMR
1
as a white solid (yield 74%), mp 145-146 °C. H NMR (CDCl3)
(CDCl3) δ: 1.35 (s, 9H), 2.97 (m, 4H), 3.45 (t, J ) 5.2 Hz, 2H),
3.63 (t, J ) 5.2 Hz, 2H), 3.68 (s, 3H), 3.92 (d, J ) 4.2 Hz, 2H),
5.42 (s, 1H), 6.77 (m, 4H).
tert-Butyl Ester of {2-[4-(4-Nitrophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6w). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 3-7 v/v. The product was obtained
as a yellow solid (yield 75%), mp 125-127 °C. 1H NMR (CDCl3)
δ: 1.45 (s, 9H), 3.48 (m, 4H), 3.59 (t, J ) 5.4 Hz, 2H), 3.82 (t, J
) 5.4 Hz, 2H), 4.03 (d, J ) 4.2 Hz, 2H), 5.50 (s, 1H), 6.84 (d, J
) 7.4 Hz, 2H), 8.17 (d, J ) 7.4 Hz, 2H).
tert-Butyl Ester of {3-[4-(4-Nitrophenyl)piperazin-1-yl]-3-
oxopropyl}carbamic Acid (6x). Eluent for the column chroma-
tography: EtOAc-petroleum ether, 4-6 v/v. The product was
obtained as a yellow solid (yield 95%), mp 160-162 °C. 1H NMR
(CDCl3) δ: 1.42 (s, 9H), 2.56 (t, J ) 5.6 Hz, 2H), 3.46 (m, 6H),
3.62 (t, J ) 5.6 Hz, 2H), 3.78 (t, J ) 5.6 Hz, 2H), 5.32 (s, 1H),
6.82 (d, J ) 9.4 Hz, 2H), 8.12 (d, J ) 9.4 Hz, 2H).
δ: 3.16 (m, 4H), 3.62 (t, J ) 5.2 Hz, 2H), 3.84 (t, J ) 5.2 Hz,
2H), 4.07 (d, J ) 4.4 Hz, 2H), 5.13 (s, 2H), 5.77 (bs, 1H), 6.93 (d,
J ) 9.0 Hz, 2H), 7.26 (m, 5H), 7.38 (d, J ) 9.0 Hz, 2H).
Benzyl Ester of {2-[4-(4-Chlorobenzyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6m). Eluent for the column chromatog-
raphy: EtOAc. The product was obtained as a white solid (yield
1
68%), mp 93-95 °C. H NMR (CDCl3) δ: 2.39 (m, 4H), 3.34 (t,
J ) 5.0 Hz, 2H), 3.544 (s, 2H), 3.59 (t, J ) 5.0 Hz, 2H), 3.95 (d,
J ) 4.0 Hz, 2H), 5.06 (s, 2H), 5.74 (bs, 1H), 7.27 (m, 9H).
tert-Butyl Ester of {2-[4-(4-Bromophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6n). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 4-6 v/v. The product was obtained
1
as a white solid (yield 65%), mp 110-111 °C. H NMR (CDCl3)
δ: 1.45 (m, 9H), 314 (m, 4H), 3.58 (t, J ) 5.0 Hz, 2H), 3.82 (t, J
) 5.0 Hz, 2H), 4.01 (d, J ) 4.4 Hz, 2H), 5.51 (bs, 1H), 6.83 (d, J
) 8.6 Hz, 2H), 7.38 (d, J ) 8.6 Hz, 2H),.
tert-Butyl Ester of {2-[4-(4-Iodophenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6o). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 4-6 v/v. The product was obtained
Benzyl Ester of [2-(4-Phenylpiperazin-1-yl)-2-oxoethyl]car-
bamic Acid (6y). Eluent for the column chromatography: EtOAc-
petroleum ether, 4-6 v/v. The product was obtained as a white
1
as a white solid (yield 78%), mp 110-112 °C. H NMR (CDCl3)
1
solid (yield 71%), mp 121-123 °C. H NMR (CDCl3) δ: 3.20
δ: 1.38 (m, 9H), 3.09 (m, 4H), 3.48 (t, J ) 5.4 Hz, 2H), 3.74 (t,
J ) 5.4 Hz, 2H), 3.93 (d, J ) 4.4 Hz, 2H), 5.43 (bs, 1H), 6.63 (d,
J ) 7.4 Hz, 2H), 7.49 (d, J ) 7.4 Hz, 2H).
(m, 4H), 3.63 (m, 2H), 3.84 (m, 2H), 4.09 (d, J ) 4.4 Hz, 2H),
5.13 (s, 2H), 5.94 (bs, 1H), 6.87 (m, 3H), 7.37 (m, 7H).
Benzyl Ester of [3-Oxo-3-(4-phenylpiperazin-1-yl)propyl]-
carbamic Acid (6z). Eluent for the column chromatography:
EtOAc-petroleum ether, 1-1 v/v. The product was obtained as a
tert-Butyl Ester of [2-Oxo-2-(4-p-tolylpiperazin-1-yl)ethyl]-
carbamic Acid (6p). Eluent for the column chromatography:
EtOAc-petroleum ether, 1-1 v/v. The product was obtained as a
1
pink solid (yield 82%), mp 94-96 °C. H NMR (CDCl3) δ: 2.05
1
white solid (yield 69%), mp 118-120 °C. H NMR (CDCl3) δ:
(m, 2H), 2.56 (m, 2H), 3.16 (m, 4H), 3.52 (m, 2H), 3.74 (m, 2H),
5.07 (s, 2H), 5.62 (bs, 1H), 6.89 (m, 3H), 7.33 (m, 7H).
1.36 (s, 9H), 2.18 (s, 3H), 3.03 (m, 4H), 3.45 (t, J ) 4.8 Hz, 2H),
3.69 (t, J ) 4.8 Hz, 2H), 3.90 (d, J ) 4.4 Hz, 2H), 5.42 (s, 1H),
6.75 (d, J ) 7.8 Hz, 2H), 6.98 (d, J ) 7.8 Hz, 2H).
Benzyl Ester of [2-(4-Phenylpiperazin-1-yl)-2-oxoethyl]car-
bamic Acid (6aa). Eluent for the column chromatography: EtOAc-
petroleum ether, 4-6 v/v. The product was obtained as a white
Benzyl Ester of {2-[4-(4-Methylbenzyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6q). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 7-3 v/v. The product was obtained
1
solid (yield 71%), mp 121-123 °C. H NMR (CDCl3) δ: 3.20
(m, 4H), 3.63 (m, 2H), 3.84 (m, 2H), 4.09 (d, J ) 4.4 Hz, 2H),
1
as a colorless oil (yield 53%). H NMR (CDCl3) δ: 2.27 (s, 3H),
5.13 (s, 2H), 5.94 (bs, 1H), 6.87 (m, 3H), 7.37 (m, 7H).
2.36 (m, 4H), 3.31 (t, J ) 4.8 Hz, 2H), 3.42 (s, 2H), 3.56 (t, J )
4.8 Hz, 2H), 3.93 (d, J ) 4.2 Hz, 2H), 5.05 (s, 2H), 5.73 (bs, 1H),
7.08 (d, J ) 8.2 Hz, 2H), 7.12 (d, J ) 8.2 Hz, 2H), 7.30 (m, 5H).
Benzyl Ester of [2-Oxo-2-(4-o-tolylpiperazin-1-yl)ethyl]car-
bamic Acid (6r). Eluent for the column chromatography: EtOAc-
petroleum ether, 1-1 v/v. The product was obtained as a white
solid (yield 73%), mp 134-136 °C. 1H NMR (CDCl3) δ: 2.33 (s,
3H), 2.92 (m, 4H), 3.55 (t, J ) 4.8 Hz, 2H), 3.79 (t, J ) 4.8 Hz,
2H), 4.09 (d, J ) 4.4 Hz, 2H), 5.42 (s, 2H), 5.83 (bs, 1H), 6.98 (d,
J ) 7.8 Hz, 1H), 7.03 (d, J ) 7.8 Hz, 1H), 7.14 (t, J ) 7.4 Hz,
1H), 7.37 (m, 7H).
Benzyl Ester of [2-Oxo-2-(4-pyrimidin-2-yl-piperazin-1-yl)-
ethyl]carbamic Acid (6ab). Eluent for the column chromatogra-
phy: EtOAc-petroleum ether, 7-3 v/v. The product was obtained
1
as a white solid (yield 95%), mp 103-105 °C. H NMR (CDCl3)
δ: 3.45 (t, J ) 4.8 Hz, 2H), 3.73 (t, J ) 4.8 Hz, 2H), 3.86 (m,
4H), 4.00 (d, J ) 4.4 Hz, 2H), 5.07 (s, 2H), 5.75 (bs, 1H), 6.55 (t,
J ) 4.8 Hz, 1H), 7.29 (m, 5H), 8.33 (d, J ) 5.0 Hz, 2H),.
Benzyl Ester of [2-(4-Benzylpiperazin-1-yl)-2-oxoethyl]car-
bamic Acid (6ac). Eluent for the column chromatography: EtOAc-
petroleum ether, 8-2 v/v. The product was obtained as a colorless
oil (yield 78%). 1H NMR (CDCl3) δ: 2.34 (m, 2H), 3.58 (m, 2H),
3.62 (t, J ) 4.8 Hz, 2H), 3.76 (t, J ) 4.8 Hz, 2H), 3.84 (s, 2H),
4.06 (d, J ) 4.2 Hz, 2H), 5.12 (s, 2H), 5.80 (bs, 1H), 7.35 (m,
10H).
tert-Butyl Ester of {2-[4-(4-Acetylphenyl)piperazin-1-yl]-2-
oxoethyl}carbamic Acid (6s). Eluent for the column chromatog-
raphy: EtOAc-petroleum ether, 6-4 v/v. The product was obtained
1
as a colorless oil (yield 76%). H NMR (CDCl3) δ: 1.45 (s, 9H),
tert-Butyl Ester of [2-(4-Benzylpiperazin-1-yl)-2-oxoethyl]-
methylcarbamic (6ad). Eluent for the column chromatography:
2.53 (s, 3H), 3.39 (m, 4H), 3.57 (t, J ) 5.4 Hz, 2H), 3.80 (t, J )

3712 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Romagnoli et al.
EtOAc. The product was obtained as a colorless oil (yield 68%).
1H NMR (CDCl3) δ: 1.44 (s, 9H), 2.43 (m, 4H), 2.90 (s, 3H),
3.50 (m, 2H), 3.54 (m, 2H), 3.62 (m, 2H), 4.03 (s, 2H), 7.31 (m,
5H).
Benzyl Ester of [2-(4-Benzyl[1,4]diazepan-1-yl)-2-oxoethyl]-
carbamic Acid (6ae). Eluent for the column chromatography:
EtOAc-MeOH, 9.5-0.5 v/v. The product was obtained as a
colorless oil (yield 62%). 1H NMR (CDCl3) δ: 1.84 (m, 2H), 2.59
(m, 4H), 3.40 (t, J ) 6.0 Hz, 2H), 3.56 (s, 2H), 3.60 (t, J ) 6.0
Hz, 2H), 3.94 (t, J ) 4.4 Hz, 2H), 5.06 (s, 2H), 5.80 (bs, 1H), 7.26
(m, 10H).
Benzyl Ester of [2-Oxo-2-(4-phenethylpiperazin-1-yl)ethyl]-
carbamic Acid (6af). Eluent for the column chromatography:
EtOAc. The product was obtained as a colorless oil (yield 60%).
1H NMR (CDCl3) δ: 2.54 (m, 4H), 2.64 (m, 2H), 2.82 (m, 2H),
3.44 (m, 2H), 3.70 (m, 2H), 4.02 (s, 2H), 5.12 (s, 2H), 5.90 (bs,
1H), 7.21 (m, 3H), 7.31 (m, 4H), 7.36 (m, 3H).
Benzyl Ester of {2-Oxo-2-[4-(3-phenylpropyl)piperazin-1-yl]-
ethyl}carbamic Acid (6ag). Eluent for the column chromatogra-
phy: EtOAc-MeOH, 9-1 v/v. The product was obtained as a
colorless oil (yield 79%). 1H NMR (CDCl3) δ: 2.60 (m, 4H), 3.25
(d, J ) 6.2 Hz, 2H), 3.45 (d, J ) 4.6 Hz, 2H), 3.70 (t, J ) 4.6 Hz,
2H), 4.02 (d, J ) 4.2 Hz, 2H), 5.11 (s, 2H), 5.80 (bs, 1H), 6.20
(m, 1H), 6.54 (d, J ) 15.8 Hz, 1H), 7.33 (m, 10H).
General Procedure for Removing the Boc Protecting Group.
The Boc derivative (1.5 mmol) was stirred at room temperature in
a mixture of TFA/CH2Cl2 (1:1, 5 mL) for 3 h. The volatiles were
removed in vacuo, and the residue was diluted with 5% aqueous
NaHCO3 (5 mL). The aqueous mixture was extracted with CH2Cl2
(3 × 5 mL), and the combined organic extracts were dried and
concentrated in vacuo. The residue obtained was used for the next
reaction without any purification.
General Procedure for Removing the Benzyloxycarbonyl (Z)
Protecting Group. A solution of Z-derivative (1 mmol) in 10 mL
of a EtOH was hydrogenated over 75 mg of 10% Pd/C at 50 psi
for 3 h. The catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure, giving a residue that was used
without purification for the next step.
General Procedure for the Synthesis of Compounds 4a-ap.
To a solution of the appropriate amine (0.5 mmol) in dry DCM (5
mL) was added at 0 °C Et3N (70 µL, 0.5 mmol, 1 equiv) and the
corresponding arylsulfonyl chloride (1 mmol, 2 equiv). The reaction
mixture was allowed to slowly warm up to room temperature and
then stirred for 12 h. After the end of this time, a saturated aqueous
NaHCO3 solution (2 mL) was added to the reaction mixture. After
the layers were separated, the organic layer was dried and
concentrated in vacuo. The residue, after purification for column
chromatography, furnished the appropriate product.
Isoquinolinesulfon-5-yl-{2-[4-(4-fluorophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4a). Eluent for the column chromatography:
EtOAc. The product was obtained as a white solid. 1H NMR
(CDCl3) δ: 2.98 (m, 4H), 3.39 (t, J ) 4.8 Hz, 2H), 3.65 (t, J )
4.8 Hz, 2H), 3.82 (d, J ) 4.0 Hz, 2H), 6.07 (bs, 1H), 6.82 (m,
2H), 6.96 (m, 2H), 7.69 (t, J ) 8.0 Hz, 1H), 8.20 (d, J ) 8.4 Hz,
1H), 8.43 (m, 2H), 8.72 (d, J ) 6.0 Hz, 1H), 9.35 (s, 1H). Anal.
(C21H21N4O3SF) C, H, N.
8.4 Hz, 1H), 8.42 (d, J ) 7.6, 1H), 8.49 (d, J ) 7.6, 1H), 8.71 (d,
J ) 6.4 Hz, 1H), 9.36 (s, 1H). Anal. (C22H23N4O3SF) C, H, N.
Naphthalenesulfon-1-yl-{2-[4-(4-fluorophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4d). Eluent for the column chromatography:
EtOAc-petroleum ether, 3-7 v/v. The product was obtained as a
1
white solid. H NMR (CDCl3) δ: 2.99 (m, 4H), 3.39 (t, J ) 4.4
Hz, 2H), 3.65 (t, J ) 4.42 Hz, 2H), 3.79 (d, J ) 4.4 Hz, 2H), 6.01
(bs, 1H), 6.86 (t, J ) 8.8 Hz, 2H), 6.99 (t, J ) 8.8 Hz, 2H), 7.54
(t, J ) 8.8 Hz, 1H), 7.62 (t, J ) 8.8 Hz, 1H), 7.69 (t, J ) 8.8 Hz,
1H), 7.93 (d, J ) 8.8 Hz, 1H), 8.07 (d, J ) 8.8 Hz, 1H), 8.26 (d,
J ) 8.8 Hz, 1H), 8.71 (d, J ) 8.0 Hz, 1H). Anal. (C22H22N3O3SF)
C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-fluorobenzyl)piperazin-1-yl]-
2-oxoethyl}amide (4e). Eluent for the column chromatography:
EtOAc. The product was obtained as a white solid. 1H NMR
(CDCl3) δ: 2.32 (m, 4H), 3.24 (t, J ) 4.6 Hz, 2H), 3.45 (s, 2H),
3.49 (t, J ) 4.6 Hz, 2H), 3.75 (s, 2H), 6.05 (s, 1H), 6.98 (t, J )
8.6 Hz, 2H), 7.19 (d, J ) 8.6 Hz, 1H), 7.26 (d, J ) 8.6 Hz, 1H),
7.68 (t, J ) 8.0 Hz, 1H), 8.20 (d, J ) 8.0 Hz, 1H), 8.43 (d, J ) 7.4
Hz, 2H), 8.72 (d, J ) 6.0 Hz, 1H), 9.35 (s, 1H). Anal. (C22H23N4O3-
SF) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(3-fluorophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4f). Eluent for the column chromatography:
EtOAc-MeOH, 9.8-0.2 v/v. The product was obtained as a white
solid. 1H NMR (CDCl3) δ: 3.01 (m, 4H), 3.34 (t, J ) 5.0 Hz, 2H),
3.58 (t, J ) 5.0 Hz, 2H), 3.75 (d, J ) 4.2 Hz, 2H), 6.03 (bs, 1H),
6.44 (m, 2H), 7.07 (t, J ) 8.0 Hz, 1H), 7.19 (s, 1H), 7.63 (t, J )
7.8 Hz, 1H), 8.14 (d, J ) 8.2 Hz, 1H), 8.38 (d, J ) 6.0 Hz, 2H),
8.67 (d, J ) 6.2 Hz, 1H), 9.29 (s, 1H). Anal. (C21H21N4O3SF) C,
H, N.
Isoquinolinesulfon-5-yl-{2-[4-(2-fluorophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4g). Eluent for the column chromatography:
EtOAc-petroleum ether, 8-2 v/v. The product was obtained as a
white solid. 1H NMR (CDCl3) δ: 2.97 (t, J ) 5.2 Hz, 2H), 3.02 (t,
J ) 5.2 Hz, 2H), 3.51 (t, J ) 5.2 Hz, 2H), 3.64 (t, J ) 5.2 Hz,
2H), 3.90 (d, J ) 4.0 Hz, 2H), 6.15 (bs, 1H), 6.89 (t, J ) 7.6 Hz,
1H), 7.02 (m, 3H), 7.57 (m, 2H), 7.65 (t, J ) 7.2 Hz, 1H), 8.08 (d,
J ) 7.2 Hz, 1H), 8.43 (d, J ) 7.2 Hz, 1H), 9.12 (s, 1H). Anal.
(C21H21N4O3SF) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(2-fluorophenyl)piperazin-1-yl]-
2-oxoethyl}methylamide (4h). Eluent for the column chromatog-
raphy: EtOAc-MeOH, 9.5-0.5 v/v. The product was obtained as
1
a white solid. H NMR (CDCl3) δ: 2.88 (s, 3H), 2.93 (t, J ) 4.8
Hz, 2H), 3.04 (t, J ) 4.8 Hz, 2H), 3.63 (m, 4H), 4.12 (s, 2H), 6.99
(m, 4H), 7.66 (t, J ) 7.8 Hz, 1H), 8.15 (d, J ) 8.4 Hz, 1H), 8.43
(m, 2H), 8.61 (d, J ) 6.2 Hz, 1H), 9.28 (s, 1H). Anal. (C22H23N4O3-
SF) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(2,4-difluorophenyl)piperazin-
1-yl]-2-oxoethyl}amide (4i). Eluent for the column chromatogra-
phy: EtOAc. The product was obtained as a white solid. 1H NMR
(CDCl3) δ: 2.87 (m, 4H), 3.33 (t, J ) 5.4 Hz, 2H), 3.74 (t, J )
5.4 Hz, 2H), 3.77 (d, J ) 3.8 Hz, 2H), 6.01 (bs, 1H), 6.76 (m,
3H), 7.64 (t, J ) 7.6 Hz, 1H), 8.14 (d, J ) 8.4 Hz, 1H), 8.39 (t, J
) 7.6 Hz, 2H), 8.65 (d, J ) 6.0 Hz, 1H), 9.30 (s, 1H). Anal.
(C21H20N4O3SF2) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(3,4-difluorophenyl)piperazin-
1-yl]-2-oxoethyl}amide (4j). Eluent for the column chromatogra-
phy: EtOAc-MeOH, 9.8-0.2 v/v. The product was obtained as a
Isoquinolinesulfon-5-yl-{2-[4-(4-fluorophenyl)piperazin-1-yl]-
2-oxoethyl}methylamide (4b). Eluent for the column chromatog-
raphy: EtOAc-MeOH, 9.5-0.5 v/v. The product was obtained as
1
white solid. H NMR (CDCl3) δ: 2.95 (m, 4H), 3.33 (t, J ) 5.0
1
Hz, 2H), 3.58 (t, J ) 5.0 Hz, 2H), 3.77 (d, J ) 3.8 Hz, 2H), 5.96
(bs, 1H), 6.60 (m, 1H), 6.90 (q, J ) 9.0 Hz, 1H), 7.00 (s, 1H),
7.64 (t, J ) 7.4 Hz, 1H), 8.17 (d, J ) 8.0 Hz, 1H), 8.42 (t, J ) 6.8
Hz, 2H), 8.66 (d, J ) 6.4 Hz, 1H), 9.32 (s, 1H). Anal. (C21H20N4O3-
SF2) C, H, N.
a white solid. H NMR (CDCl3) δ: 2.87 (s, 3H), 2.97 (t, J ) 4.6
Hz, 2H), 3.05 (t, J ) 4.6 Hz, 2H), 3.61 (m, 4H), 4.10 (s, 2H), 6.84
(m, 4H), 7.65 (t, J ) 7.8 Hz, 1H), 8.15 (d, J ) 8.0 Hz, 1H), 8.40
(d, J ) 7.6, 2H), 8.45 (d, J ) 7.6, 2H), 8.61 (d, J ) 6.2 Hz, 1H),
9.28 (s, 1H). Anal. (C22H23N4O3SF) C, H, N.
Isoquinolinesulfon-5-yl-{3-[4-(4-fluorophenyl)piperazin-1-yl]-
3-oxopropyl}amide (4c). Eluent for the column chromatography:
EtOAc-MeOH, 9.75-0.25 v/v. The product was obtained as a
Isoquinolinesulfon-5-yl-{2-[4-(2,3-difluorophenyl)piperazin-
1-yl]-2-oxoethyl}amide (4k). Eluent for the column chromatog-
raphy: EtOAc. The product was obtained as a white solid. 1H NMR
(CDCl3) δ: 3.02 (m, 4H), 3.41 (t, J ) 5.2 Hz, 2H), 3.68 (t, J )
5.2 Hz, 2H), 3.84 (d, J ) 4.0 Hz, 2H), 6.05 (bs, 1H), 6.65 (t, J )
6.8 Hz, 1H), 6.82 (q, J ) 8.6 Hz, 1H), 6.96 (m, 1H), 7.74 (t, J )
1
white solid. H NMR (CDCl3) δ: 2.53 (t, J ) 5.6 Hz, 2H), 3.04
(m, 4H), 3.12 (m, 2H), 3.44 (m, 2H), 3.72 (t, J ) 5.6 Hz, 2H),
6.06 (bs, 1H), 6.92 (m, 4H), 7.70 (t, J ) 7.4 Hz, 1H), 8.22 (d, J )

Antagonists of the Purinergic P2X7 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3713
7.6 Hz, 1H), 8.26 (d, J ) 7.6 Hz, 1H), 8.50 (d, J ) 7.6 Hz, 1H),
8.58 (m, 1H), 8.72 (m, 1H), 9.44 (s, 1H). Anal. (C21H20N4O3SF2)
C, H, N.
8.8 Hz, 2H), 7.72 (t, J ) 7.8 Hz, 1H), 8.00 (t, J ) 5.4 Hz, 1H),
8.22 (d, J ) 8.4 Hz, 1H), 8.32 (d, J ) 8.4 Hz, 1H), 8.46 (d, J )
6.2 Hz, 1H), 8.58 (d, J ) 6.2 Hz, 1H), 9.32 (s, 1H). Anal.
(C21H21N4O3SI) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(2,5-difluorophenyl)piperazin-
1-yl]-2-oxoethyl}amide (4l). Eluent for the column chromatogra-
phy: EtOAc. The product was obtained as a white solid. 1H NMR
(CDCl3) δ: 2.90 (m, 4H), 3.43 (t, J ) 5.4 Hz, 2H), 3.61 (t, J )
5.4 Hz, 2H), 3.76 (d, J ) 4.0 Hz, 2H), 5.97 (bs, 1H), 6.49 (m,
1H), 6.96 (m, 2H), 7.67 (t, J ) 7.8 Hz, 1H), 8.18 (d, J ) 8.2 Hz,
1H), 8.38 (d, J ) 7.6 Hz, 1H), 8.48 (d, J ) 7.6 Hz, 1H), 8.68 (d,
J ) 6.0 Hz,1H), 9.33 (s, 1H). Anal. (C21H20N4O3SF2) C, H, N.
Quinolinesulfon-5-yl-{2-[4-(2-fluorophenyl)piperazin-1-yl]-2-
oxoethyl}amide (4m). Eluent for the column chromatography:
EtOAc-petroleum ether, 6-4 v/v. The product was obtained as a
white solid. 1H NMR (CDCl3) δ: 2.96 (t, J ) 5.2 Hz, 2H), 3.04 (t,
J ) 5.2 Hz, 2H), 3.50 (t, J ) 5.2 Hz, 2H), 3.68 (t, J ) 5.2 Hz,
2H), 3.90 (d, J ) 4.0 Hz, 2H), 6.89 (t, J ) 7.6 Hz, 1H), 7.02 (m,
3H), 7.20 (bs, 1H), 7.58 (dd, J ) 4.0 and 8.4 Hz, 1H), 7.65 (t, J )
8.4 Hz, 1H), 8.08 (d, J ) 8.0 Hz, 1H), 8.26 (d, J ) 8.4 Hz, 1H),
8.43 (d, J ) 7.2 Hz, 1H), 9.12 (m, 1H). Anal. (C21H21N4O3SF) C,
H, N.
Quinolinesulfon-8-yl-{2-[4-(2-fluorophenyl)piperazin-1-yl]-2-
oxoethyl}amide (4n). Eluent for the column chromatography:
EtOAc-petroleum ether, 6-4 v/v. The product was obtained as a
white solid. 1H NMR (CDCl3) δ: 2.95 (t, J ) 5.2 Hz, 2H), 3.01 (t,
J ) 5.2 Hz, 2H), 3.48 (t, J ) 5.2 Hz, 2H), 3.63 (t, J ) 5.2 Hz,
2H), 3.90 (d, J ) 4.0 Hz, 2H), 6.89 (t, J ) 8.0 Hz, 1H), 7.02 (m,
2H), 7.20 (t, J ) 7.8 Hz, 1H), 7.26 (d, J ) 7.8 Hz, 1H), 7.55 (t, J
) 8.4 Hz, 1H), 7.64 (t, J ) 8.0 Hz, 1H), 8.06 (d, J ) 8.4 Hz, 1H),
8.26 (d, J ) 8.0 Hz, 1H), 8.423 (d, J ) 7.2 Hz, 1H), 9.12 (m, 1H).
Anal. (C21H21N4O3SF) C, H, N.
Isoquinolinesulfon-5-yl-[2-oxo-2-(4-p-tolylpiperazin-1-yl)ethy-
l]amide (4t). Eluent for the column chromatography: EtOAc-
petroleum ether, 7-3 v/v. The product was obtained as a white
1
solid. H NMR (CDCl3) δ: 2.25 (s, 3H), 3.01 (m, 4H), 3.38 (t, J
) 5.2 Hz, 2H), 3.64 (t, J ) 5.2 Hz, 2H), 3.83 (d, J ) 4.0 Hz, 2H),
6.17 (bs, 1H), 6.76 (d, J ) 7.8 Hz, 2H), 7.06 (d, J ) 7.8 Hz, 2H),
7.67 (t, J ) 7.8 Hz, 1H), 8.21 (d, J ) 8.0 Hz, 1H), 8.44 (d, J ) 7.8
Hz, 2H), 8.70 (d, J ) 6.0 Hz, 1H), 9.34 (s, 1H). Anal. (C22H24N4O3S)
C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-methylbenzyl)piperazin-1-yl]-
2-oxoethyl}amide (4u). Eluent for the column chromatography:
EtOAc-MeOH, 9.5-0.5 v/v. The product was obtained as a white
1
solid. H NMR (CDCl3) δ: 2.32 (s, 3H), 2.42 (m, 4H), 3.19 (t, J
) 4.8 Hz, 2H), 3.46 (t, J ) 4.8 Hz, 2H), 3.48 (s, 2H), 3.74 (d, J
) 3.6 Hz, 2H), 6.10 (s, 1H), 7.12 (m, 4H), 7.67 (t, J ) 7.8 Hz,
1H), 8.19 (d, J ) 8.2 Hz, 1H), 8.42 (d, J ) 8.2 Hz, 2H), 8.70 (d,
J ) 6.2 Hz, 1H), 9.34 (s, 1H). Anal. (C23H26N4O3S) C, H, N.
Isoquinolinesulfon-5-yl-[2-oxo-2-(4-o-tolylpiperazin-1-yl)ethy-
l]amide (4v). Eluent for the column chromatography: EtOAc-
petroleum ether, 7-3 v/v. The product was obtained as a yellow
1
solid. H NMR (CDCl3) δ: 2.17 (s, 3H), 2.84 (m, 4H), 3.40 (t, J
) 5.2 Hz, 2H), 3.62 (t, J ) 5.2 Hz, 2H), 3.88 (d, J ) 4.2 Hz, 2H),
6.20 (bs, 1H), 6.84 (d, J ) 7.8 Hz, 1H), 6.96 (d, J ) 7.8 Hz, 1H),
7.22(m, 2H), 7.67 (t, J ) 7.6 Hz, 1H), 8.22 (d, J ) 7.8 Hz, 1H),
8.46 (d, J ) 7.8 Hz, 2H), 8.70 (d, J ) 6.0 Hz, 1H), 9.52 (s, 1H).
Anal. (C22H24N4O3S) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-acetylphenyl)piperazin-1-yl]-
2-oxoethyl}amide (4w). Eluent for the column chromatography:
EtOAc. The product was obtained as a white solid. 1H NMR
(CDCl3) δ: 2.34 (s, 3H), 3.12 (m, 4H), 3.39 (t, J ) 5.4 Hz, 2H),
3.72 (t, J ) 5.4 Hz, 2H), 3.90 (d, J ) 4.2 Hz, 2H), 6.09 (bs, 1H),
6.76 (d, J ) 8.6 Hz, 2H), 7.64 (d, J ) 8.6 Hz, 2H), 7.67 (t, J ) 7.8
Hz, 1H), 8.21 (d, J ) 7.8 Hz, 1H), 8.42 (d, J ) 5.8 Hz, 2H), 8.67
(d, J ) 5.8 Hz, 1H), 9.25 (s, 1H). Anal. (C23H24N4O4S) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-cyanophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4x). Eluent for the column chromatography:
EtOAc-petroleum ether, 6-4 v/v. The product was obtained as a
Naphthalenesulfon-1-yl-{2-[4-(2-fluorophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4o). Eluent for the column chromatography:
EtOAc-petroleum ether, 4-6 v/v. The product was obtained as a
1
white solid. H NMR (CDCl3) δ: 2.96 (m, 4H), 3.38 (t, J ) 5.2
Hz, 2H), 3.65 (t, J ) 5.2 Hz, 2H), 3.79 (d, J ) 4.0 Hz, 2H), 6.01
(t, J ) 4.0 Hz, 1H), 6.86 (t, J ) 8.4 Hz, 1H), 7.02 (m, 3H), 7.54
(t, J ) 8.0 Hz, 1H), 7.62 (t, J ) 8.0 Hz, 1H), 7.72 (t, J ) 8.0 Hz,
1H), 7.92 (d, J ) 8.0 Hz, 1H), 8.07 (d, J ) 8.0 Hz, 1H), 8.26 (d,
J ) 8.0 Hz, 1H), 8.70 (d, J ) 8.0 Hz, 1H). Anal. (C22H22N3O3SF)
C, H, N.
1
Isoquinolinesulfon-5-yl-{2-[4-(4-chlorophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4p). Eluent for the column chromatography:
EtOAc-MeOH, 9.9-0.1 v/v. The product was obtained as a white
solid. 1H NMR (CDCl3) δ: 3.09 (m, 4H), 3.42 (t, J ) 5.2 Hz, 2H),
3.64 (t, J ) 5.2 Hz, 2H), 3.82 (d, J ) 4.0 Hz, 2H), 6.07 (bs, 1H),
6.89 (d, J ) 6.2 Hz, 2H), 7.24 (d, J ) 6.2 Hz, 2H), 7.69 (t, J ) 8.0
Hz, 1H), 8.22 (d, J ) 8.0 Hz, 1H), 8.45 (m, 2H), 8.72 (d, J ) 6.0
Hz, 1H), 9.36 (s, 1H). Anal. (C21H21N4O3SCl) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-chlorobenzyl)piperazin-1-yl]-
2-oxoethyl}amide (4q). Eluent for the column chromatography:
EtOAc-MeOH, 9.5-0.5 v/v. The product was obtained as a white
solid. 1H NMR (CDCl3) δ: 2.27 (m, 4H), 3.16 (t, J ) 4.6 Hz, 2H),
3.37 (s, 2H), 3.42 (t, J ) 4.6 Hz, 2H), 3.68 (d, J ) 4.0 Hz, 2H),
6.00 (s, 1H), 7.19 (m, 4H), 7.62 (t, J ) 7.8 Hz, 1H), 8.14 (d, J )
8.2 Hz, 1H), 8.37 (d, J ) 8.2 Hz, 2H), 8.64 (d, J ) 6.0 Hz, 1H),
9.29 (s, 1H). Anal. (C22H23N4O3SCl) C, H, N.
white solid. H NMR (CDCl3) δ: 3.27 (m, 4H), 3.43 (t, J ) 5.2
Hz, 2H), 3.66 (t, J ) 5.2 Hz, 2H), 3.82 (d, J ) 4.2 Hz, 2H), 6.08
(bs, 1H), 6.80 (d, J ) 9.0 Hz, 2H), 7.49 (d, J ) 9.0 Hz, 2H), 7.68
(t, J ) 7.8 Hz, 1H), 8.21 (d, J ) 8.4 Hz, 1H), 8.40 (d, J ) 5.2 Hz,
1H), 8.44 (d, J ) 5.2 Hz, 1H), 8.71 (d, J ) 5.2 Hz, 1H), 9.34 (s,
1H). Anal. (C22H21N5O3S) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-trifluoromethylphenyl)piper-
azin-1-yl]-2-oxoethyl}amide (4y). Eluent for the column chroma-
1
tography: EtOAc. The product was obtained as a white solid. H
NMR (CDCl3) δ: 3.17 (m, 4H), 3.39 (t, J ) 4.6 Hz, 2H), 3.62 (t,
J ) 4.6 Hz, 2H), 3.76 (d, J ) 4.4 Hz, 2H), 5.93 (bs, 1H), 6.81 (d,
J ) 8.6 Hz, 2H), 7.42 (d, J ) 8.6 Hz, 2H), 7.64 (t, J ) 8.2 Hz,
1H), 8.15 (d, J ) 8.2 Hz, 1H), 8.37 (t, J ) 6.8 Hz, 1H), 8.42 (t, J
) 6.8 Hz, 1H), 8.66 (d, J ) 6.0 Hz, 1H), 9.31 (s, 1H). Anal.
(C22H21N4O3SF3) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-methoxyphenyl)piperazin-1-
yl]-2-oxoethyl}amide (4z). Eluent for the column chromatogra-
phy: EtOAc-petroleum ether, 8-2 v/v. The product was obtained
Isoquinolinesulfon-5-yl-{2-[4-(4-bromophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4r). Eluent for the column chromatography:
1
1
EtOAc. The product was obtained as a yellow solid. H NMR
as a white solid. H NMR (CDCl3) δ: 3.95 (m, 4H), 3.38 (t, J )
(CDCl3) δ: 3.04 (m, 4H), 3.11 (t, J ) 5.0 Hz, 2H), 3.34 (t, J )
5.0 Hz, 2H), 3.91 (d, J ) 4.0 Hz, 2H), 6.07 (bs, 1H), 6.88 (d, J )
8.6 Hz, 2H), 7.37 (d, J ) 8.6 Hz, 2H), 7.80 (t, J ) 7.6 Hz, 1H),
8.32 (d, J ) 7.60 Hz, 1H), 8.48 (m, 1H), 8.51 (d, J ) 6.0 Hz, 1H),
8.66 (d, J ) 6.0 Hz, 1H), 9.47 (s, 1H). Anal. (C21H21N4O3SBr) C,
H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-iodophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4s). Eluent for the column chromatography:
EtOAc-petroleum ether, 9-1 v/v. The product was obtained as a
brown solid. 1H NMR (DMSO-d6) δ: 2.99 (m, 4H), 3.40 (m, 4H),
3.80 (d, J ) 5.4 Hz, 2H), 6.64 (d, J ) 8.8 Hz, 2H), 7.44 (d, J )
5.2 Hz, 2H), 3.63 (t, J ) 5.2 Hz, 2H), 3.75 (s, 3H), 3.82 (d, J )
4.2 Hz, 2H), 6.09 (bs, 1H), 6.83 (m, 4H), 7.68 (t, J ) 8.0 Hz, 1H),
8.21 (d, J ) 8.2 Hz, 1H), 8.40 (d, J ) 5.6 Hz, 1H), 8.47 (d, J )
5.6 Hz, 1H), 8.72 (d, J ) 6.2 Hz, 1H), 9.35 (s, 1H). Anal.
(C22H24N4O4S) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-nitrophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4aa). Eluent for the column chromatography:
EtOAc-MeOH, 9.5-0.5 v/v. The product was obtained as a yellow
solid. 1H NMR (CDCl3) δ: 3.39 (m, 6H), 3.69 (t, J ) 5.2 Hz, 2H),
3.83 (d, J ) 4.4 Hz, 2H), 5.96 (d, J ) 4.4 Hz, 1H), 6.78 (d, J )
7.4 Hz, 2H), 7.69 (t, J ) 7.4 Hz, 1H), 8.11 (d, J ) 7.4 Hz, 2H),

3714 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Romagnoli et al.
1
8.22 (d, J ) 8.2 Hz, 1H), 8.42 (m, 2H), 8.73 (d, J ) 6.2 Hz, 1H),
9.36 (s, 1H). Anal. (C21H21N5O5SF) C, H, N.
MeOH, 9-1 v/v. The product was obtained as a yellow solid. H
NMR (CDCl3) δ: 2.30 (m, 4H), 3.18 (t, J ) 4.8 Hz, 2H), 3.50 (m,
4H), 3.70 (s, 2H), 5.99 (bs, 1H), 7.02 (m, 5H), 7.64 (t, J ) 7.8 Hz,
1H), 8.12 (d, J ) 7.8 Hz, 1H), 8.40 (d, J ) 7.8 Hz, 1H), 8.44 (d,
J ) 7.8 Hz, 1H), 8.68 (d, J ) 7.8 Hz, 1H), 9.30 (s, 1H). Anal.
(C22H24N4O3S) C, H, N.
Isoquinolinesulfon-5-yl-{3-[4-(4-nitrophenyl)piperazin-1-yl]-
3-oxopropyl}amide (4ab). Eluent for the column chromatogra-
phy: EtOAc. The product was obtained as a yellow solid. 1H NMR
(CDCl3) δ: 2.57 (t, J ) 5.4 Hz, 2H), 3.27 (q, J ) 5.2 Hz, 2H),
3.40 (m, 4H), 3.49 (t, J ) 5.0 Hz, 2H), 3.72 (d, J ) 5.0 Hz, 2H),
5.96 (d, J ) 4.4 Hz,, 1H), 6.80 (d, J ) 9.2 Hz, 2H), 7.70 (t, J )
7.8 Hz, 1H), 8.18 (m, 3H), 8.45 (t, J ) 8.6 Hz, 2H), 8.73 (d, J )
6.2 Hz, 1H), 9.40 (s, 1H). Anal. (C22H23N5O5S) C, H, N.
Isoquinolinesulfon-5-yl-{2-[4-(4-aminophenyl)piperazin-1-yl]-
2-oxoethyl}amide (4ac). 4ac was recrystallized from ethyl ether.
Isoquinolinesulfon-5-yl-[2-(4-benzylpiperazin-1-yl)-2-oxoeth-
yl]methylamide (4ak). Eluent for the column chromatography:
EtOAc-MeOH, 9-1 v/v. The product was obtained as a white
1
solid. H NMR (CDCl3) δ: 2.39 (t, J ) 5.2 Hz, 2H), 2.46 (t, J )
5.2 Hz, 2H), 2.92 (s, 3H), 3.50 (m, 6H), 4.12 (s, 2H), 7.32 (m,
5H), 7.69 (t, J ) 7.8 Hz, 1H), 8.20 (d, J ) 8.0 Hz, 1H), 8.48 (d,
J ) 6.2 Hz, 2H), 8.67 (d, J ) 6.2 Hz, 1H), 9.32 (s, 1H). Anal.
(C23H26N4O3S) C, H, N.
1
The product was obtained as a yellow solid. H NMR (CDCl3) δ:
2.88 (m, 4H), 3.04 (m, 2H), 3.36 (t, J ) 5.2 Hz, 2H), 3.62 (t, J )
5.2 Hz, 2H), 3.80 (d, J ) 4.4 Hz, 2H), 6.02 (bs, 1H), 6.57 (d, J )
8.2 Hz, 2H), 6.76 (t, J ) 8.2 Hz, 1H), 7.68 (t, J ) 8.2 Hz, 1H),
8.18 (d, J ) 7.4 Hz, 2H), 8.44 (t, J ) 6.2 Hz, 2H), 8.72 (d, J ) 5.8
Hz, 1H), 9.34 (s, 1H). Anal. (C21H23N5O3S) C, H, N.
Isoquinolinesulfon-5-yl-[2-(4-benzyl[1,4]diazepan-1-yl)-2-oxo-
ethyl]amide (4al). Eluent for the column chromatography: EtOAc-
1
MeOH, 8-2 v/v. The product was obtained as a white solid. H
NMR (CDCl3) δ: 1.75 (m, 2H), 2.52 (m, 4H), 3.24 (t, J ) 7.0 Hz,
2H), 3.54 (m, 4H), 3.75 (d, J ) 6.0 Hz, 2H), 6.09 (bs, 1H), 7.25
(m, 5H), 7.64 (t, J ) 7.6 Hz, 1H), 8.17 (d, J ) 7.6 Hz, 1H), 8.42
(d, J ) 7.6 Hz, 1H), 8.44 (d, J ) 7.6 Hz, 1H), 8.68 (d, J ) 7.6 Hz,
1H), 9.33 (s, 1H). Anal. (C23H26N4O3S) C, H, N.
Isoquinolinesulfon-5-yl-[2-(4-phenylpiperazin-1-yl)-2-oxoet-
hyl]amide (4ad). Eluent for the column chromatography: EtOAc-
MeOH, 9.8-0.2 v/v. The product was obtained as a white solid.
1H NMR (CDCl3) δ: 3.05 (m, 4H), 3.42 (t, J ) 4.8 Hz, 2H), 3.63
(t, J ) 4.8 Hz, 2H), 3.82 (d, J ) 3.6 Hz, 2H), 6.11 (bs, 1H), 6.88
(m, 3H), 7.24 (m, 2H), 7.67 (t, J ) 7.8 Hz, 1H), 8.20 (d, J ) 8.2
Hz, 1H), 8.43 (m, 2H), 8.69 (d, J ) 8.2 Hz, 1H), 9.34 (d, J ) 8.0
Hz, 1H). Anal. (C21H22N4O3S) C, H, N.
Isoquinolinesulfon-5-yl-[3-oxo-3-(4-phenylpiperazin-1-yl)pro-
pyl]amide (4ae). Eluent for the column chromatography: EtOAc-
MeOH, 9.75-0.25 v/v. The product was obtained as a white solid.
1H NMR (CDCl3) δ: 2.52 (t, J ) 5.8 Hz, 2H), 3.09 (m, 4H), 3.26
(m, 2H), 3.49 (t, J ) 4.6 Hz, 2H), 3.69 (t, J ) 4.6 Hz, 2H), 6.02
(bs, 1H), 6.94 (m, 3H), 7.29 (t, J ) 7.8 Hz, 2H), 7.72 (t, J ) 7.8
Hz, 1H), 8.24 (d, J ) 8.2 Hz, 1H), 8.46 (m, 2H), 8.74 (d, J ) 6.2
Hz, 1H), 9.36 (m, 1H). Anal. (C22H24N4O3S) C, H, N.
Isoquinolinesulfon-5-yl-[2-(4-naphthalen-1-yl-piperazin-1-yl)-
2-oxoethyl]amide (4af). Eluent for the column chromatography:
EtOAc-petroleum ether, 8-2 v/v. The product was obtained as a
white solid. 1H NMR (CDCl3) δ: 3.04 (m, 4H), 3.48 (m, 4H), 3.87
(d, J ) 3.8 Hz, 2H), 6.08 (bs, 1H), 6.99 (d, J ) 6.8 Hz, 1H), 7.48
(m, 2H), 7.56 (t, J ) 8.2 Hz, 1H), 7.74 (t, J ) 8.2 Hz, 1H), 7.82
(m, 3H), 8.20 (d, J ) 8.2 Hz, 1H), 8.44 (t, J ) 7.0 Hz, 2H), 8.73
(d, J ) 6.2 Hz, 1H), 9.36 (s, 1H). Anal. (C25H24N4O3S) C, H, N.
Naphthalenesulfon-1-yl-[2-(4-phenylpiperazin-1-yl)-2-oxoet-
hyl]amide (4ag). Eluent for the column chromatography: EtOAc-
petroleum ether, 4-6 v/v. The product was obtained as a white
solid. 1H NMR (CDCl3) δ: 3.07 (m, 4H), 3.38 (t, J ) 4.8 Hz, 2H),
3.66 (t, J ) 4.8 Hz, 2H), 3.80 (d, J ) 4.4 Hz, 2H), 5.97 (bs, 1H),
6.88 (m, 3H), 7.27 (m, 2H), 7.54 (t, J ) 8.0 Hz, 1H), 7.63 (t, J )
8.0 Hz, 1H), 7.70 (t, J ) 8.0 Hz, 1H), 7.95 (d, J ) 8.0 Hz, 1H),
8.08 (d, J ) 8.0 Hz, 1H), 8.25 (d, J ) 8.0 Hz, 1H), 8.71 (d, J )
8.0 Hz, 1H). Anal. (C22H23N3O3S) C, H, N.
Isoquinolinesulfon-5-yl-[2-oxo-2-(4-pyrimidin-2-ylpiperazin-
1-yl)ethyl]amide (4ah). Eluent for the column chromatography:
EtOAc-MeOH, 9-1. The product was obtained as a yellow solid.
1H NMR (CDCl3) δ: 3.30 (t, J ) 4.8 Hz, 2H), 3.57 (t, J ) 4.8 Hz,
2H), 3.82 (m, 4H), 3.84 (d, J ) 4.0 Hz, 2H), 6.06 (t, J ) 4.4 Hz,
1H), 6.55 (t, J ) 4.6 Hz, 1H), 7.71 (t, J ) 8.2 Hz, 1H), 8.22 (d, J
) 8.4 Hz, 1H), 8.30 (d, J ) 5.0 Hz, 2H), 8.45 (t, J ) 5.6 Hz, 1H),
8.51 (d, J ) 5.6 Hz, 1H), 8.72 (d, J ) 4.4 Hz, 1H), 9.36 (s, 1H).
Anal. (C19H20N6O3S) C, H, N.
Naphthalenesulfon-1-yl-[2-(4-benzylpiperazin-1-yl)-2-oxoet-
hyl]amide (4am). Eluent for the column chromatography: EtOAc-
petroleum ether, 6-4 v/v. The product was obtained as a white
solid. 1H NMR (CDCl3) δ: 2.32 (m, 4H), 3.20 (t, J ) 5.2 Hz, 2H),
3.48 (m, 4H), 3.73 (s, 2H), 5.99 (bs, 1H), 7.29 (m, 5H), 7.52 (t, J
) 8.4 Hz, 1H), 7.62 (t, J ) 8.4 Hz, 1H), 7.69 (t, J ) 8.4 Hz, 1H),
7.94 (t, J ) 8.4 Hz, 1H), 8.07 (t, J ) 8.4 Hz, 1H), 8.24 (t, J ) 8.4
Hz, 1H), 8.70 (t, J ) 8.4 Hz, 1H). Anal. (C23H25N3O3S) C, H, N.
Isoquinolinesulfon-5-yl-[2-oxo-2-(4-phenethylpiperazin-1-yl)-
ethyl]amide (4an). Eluent for the column chromatography: EtOAc-
1
MeOH, 9-1 v/v. The product was obtained as a white solid. H
NMR (CDCl3) δ: 2.41 (m, 4H), 2.58 (t, J ) 5.0 Hz, 2H), 2.71 (t,
J ) 5.0 Hz, 2H), 3.26 (t, J ) 4.8 Hz, 2H), 3.76 (t, J ) 4.8 Hz,
2H), 3.77 (d, J ) 4.4 Hz, 2H), 6.1 (bs, 1H), 7.21 (m, 5H), 7.67 (t,
J ) 7.8 Hz, 1H), 8.19 (d, J ) 8.2 Hz, 1H), 8.42 (d, J ) 6.2 Hz,
2H), 8.69 (d, J ) 6.2 Hz, 1H), 9.34 (s, 1H). Anal. (C23H26N4O3S)
C, H, N.
Naphthalenesulfon-1-yl-[2-oxo-2-(4-phenethylpiperazin-1-yl)-
ethyl]amide (4ao). Eluent for the column chromatography: EtOAc-
petroleum ether, 8-2 v/v. The product was obtained as a white
solid. 1H NMR (CDCl3) δ: 2.42 (m, 4H), 2.56 (t, J ) 8.4 Hz, 2H),
2.73 (t, J ) 8.4 Hz, 2H), 3.24 (t, J ) 4.4 Hz, 2H), 3.53 (s, 2H),
3.73 (d, J ) 3.6 Hz, 2H), 5.98 (bs, 1H), 7.18 (m, 3H), 7.26 (m,
2H), 7.53 (t, J ) 8.0 Hz, 1H), 7.61 (t, J ) 8.0 Hz, 1H), 7.68 (t, J
) 8.0 Hz, 1H), 7.94 (t, J ) 8.0 Hz, 1H), 8.07 (t, J ) 8.0 Hz, 1H),
8.23 (t, J ) 8.0 Hz, 1H), 8.69 (t, J ) 8.0 Hz, 1H). Anal.
(C24H27N3O3S) C, H, N.
Isoquinolinesulfon-5-yl-{2-oxo-2-[4-(3-phenylpropyl)piperazin-
1-yl]amide (4ap). Eluent for the column chromatography: EtOAc-
1
MeOH, 9-1 v/v. The product was obtained as a white solid. H
NMR (CDCl3) δ: 1.76 (m, 2H), 2.32 (m, 6H), 2.57 (t, J ) 7.6 Hz,
2H), 3.22 (t, J ) 5.2 Hz, 2H), 3.49 (t, J ) 5.2 Hz, 2H), 3.75 (d, J
) 3.4 Hz, 2H), 6.06 (bs, 1H), 7.19 (m, 5H), 7.67 (t, J ) 7.8 Hz,
1H), 8.19 (d, J ) 8.4 Hz, 1H), 8.39 (d, J ) 6.0 Hz, 1H), 8.42 (d,
J ) 7.8 Hz, 1H), 8.71 (d, J ) 6.2 Hz, 1H), 9.34 (s, 1H). Anal.
(C24H28N4O3S) C, H, N.
Biological Materials and Methods. Cell Cultures. HEK293
cells were cultured in a 1:1 DMEM/F-12 medium (Lonza, Verviers,
Belgium) containing 15% heat inactivated FCS (Lonza, Verviers,
Belgium), 100 U/mL penicillin, and 100 µg/mL streptomycin. HEK
293-P2X7 cells were kindly provided by Prof. A. Surprenant from
Department of Biomedical Science, University of Sheffield, U.K.
Stable clones were cultured in the same medium containing G418
sulfate (Geneticin) (Sigma Aldrich, Milan, Italy) at a concentration
of 0.2 mg/mL.
Naphthalenesulfon-1-yl-[2-(4-naphthalen-1-ylpiperazin-1-yl)-
2-oxoethyl]amide (4ai). Eluent for the column chromatography:
EtOAc-petroleum ether, 4-6 v/v. The product was obtained as a
white solid. 1H NMR (CDCl3) δ: 3.14 (m, 4H), 3.49 (m, 2H), 3.83
(m, 2H), 4.11 (m, 2H), 6.04 (bs, 1H), 7.00 (d, J ) 8.8 Hz, 1H),
7.12 (d, J ) 8.8 Hz, 1H), 7.38 (m, 3H), 7.57 (m, 2H), 7.72 (t, J )
8.8 Hz, 1H), 7.94 (d, J ) 8.8 Hz, 1H), 8.08 (m, 2H), 8.12 (d J )
8.8 Hz, 1H), 8.29 (d, J ) 8.8 Hz, 1H), 8.74 (d, J ) 8.4 Hz, 1H).
Anal. (C26H25N3O3S) C, H, N.
Ca2+ Measurements. Changes in [Ca2+]i were measured with
the fluorescent indicator Fura-2/AM (Calbiochem Inalco, Milan,
Italy) as previously described.30 Briefly, HEK 293-P2X7 cells were
loaded with 4 µM Fura-2/AM in a saline solution (125 mM NaCl,
Isoquinolinesulfon-5-yl-[2-(4-benzylpiperazin-1-yl)-2-oxoethy-
l]amide (4aj). Eluent for the column chromatography: EtOAc-

Antagonists of the Purinergic P2X7 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3715
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5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM glucose, 5
mM NaHCO3, 1 mM CaCl2, 20 mM HEPES, pH 7.4) for 30 min
at 37 °C in the presence of 250 µM sulfinpyrazone. Cells were
then centrifuged at 1000g for 10 min to remove the extracellular
dye and resuspended in saline solution. Ca2+ traces were obtained
by using a LS50, Perkin-Elmer (Norwalk, CT) spectrofluorimeter
at an excitation wavelength of 340 and 380 nm and emission
wavelength of 505 nm. Measurements were performed in samples
that were thermostatically controlled (37 °C) and continuously
stirred in cuvettes. After a stable baseline had been established,
ATP was added and emitted light recorded. All experiments were
performed five times.
Ethidium Uptake. ATP-dependent increase in plasma membrane
permeability was measured using the extracellular fluorescent tracer
ethidium bromide (Sigma Aldrich, Milan, Italy) as previously
described.26 Cells were incubated in a thermostat-controlled fluo-
rometer cuvette (37 °C) in a modified Ca2+-free saline solution
containing EGTA for 20 min in the dark at a concentration of 106
cells/mL in the presence of 20 µM ethidium bromide and stimulated
with 1 mM ATP. Cell suspension was incubated with synthesized
antagonists in the range 5-1000 nM for 10 min at 37 °C before
fluorimetric analysis in a LS50, Perkin-Elmer (Norwalk, CT)
spectrofluorimeter at 37 °C. Fluorescence changes were monitored
at the wavelength pair 360 and 580 nm. All experiments were
repeated five times.
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C. F. The cytokine IL-1 beta transiently enhances P2X7 receptor
expression and function in human astrocytes. Glia 2005, 49, 245-
258.
Acknowledgment. We thank King Pharmaceuticals Inc.,
Research and Development, for its generous financial support
of this work. O.C.-L. is grateful for the financial support from
the Ramon Areces Foundation.
Supporting Information Available: Synthesis and spectro-
scopic data for compounds 2 and 3; elemental analysis results of
compounds 4a-ap. This material is available free of charge via
the Internet at http://pubs.acs.org.
(23) Gargett, C. E.; Wiley, J. S. The isoquinoline derivative KN-62: a
potent antagonist of the P2Z receptor of human lymphocytes. Br. J.
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G. R. Isoquinolines as antagonists of the P2X7 nucleotide receptor:
high selectivity for the human versus rat receptor homologues. Mol.
Pharmacol. 1998, 54, 22-32.
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JM070443E