Synthesis and structure-activity relationship studies of tyrosine-based antagonists at the human P2X7 receptor

Article abstract of DOI:10.1016/j.bmcl.2007.11.077

Analogues of the P2X₇ receptor antagonist KN-62, modified at the piperazine and arylsulfonyl groups, were synthesized and assayed at the human P2X₇ receptor for inhibition of BzATP-induced effects, that is, uptake of a fluorescent dy

Full text of DOI:10.1016/j.bmcl.2007.11.077

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 571–575
Synthesis and structure–activity relationship studies
of tyrosine-based antagonists at the human P2X7 receptor
Ga Eun Lee,a Bhalchandra V. Joshi,b Wangzhong Chen,b Lak Shin Jeong,c
Hyung Ryong Moon,d Kenneth A. Jacobsonb and Yong-Chul Kima,*
aDepartment of Life Science, Gwangju Institute of Science and Technology (GIST), 1 Oryong-dong,
Buk-gu, Gwangju 500-712, Republic of Korea
bLaboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD, USA
cLaboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
dCollege of Pharmacy, Pusan National University, Pusan 609-735, Republic of Korea
Received 24 August 2007; revised 18 October 2007; accepted 20 November 2007
Available online 28 November 2007
Abstract—Analogues of the P2X7 receptor antagonist KN-62, modified at the piperazine and arylsulfonyl groups, were synthesized
and assayed at the human P2X7 receptor for inhibition of BzATP-induced effects, that is, uptake of a fluorescent dye (ethidium bro-
mide) in stably transfected HEK293 cells and IL-1b release in differentiated THP-1 cells. Substitution of the arylsulfonyl moiety with
a nitro group increased antagonistic potency relative to methyl substitution, such that compound 21 was slightly more potent than
KN-62. Substitution with D-tyrosine in 36 and sterically bulky tyrosyl 3,5-dimethyl groups in 9 enhanced antagonistic potency.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The P2 purinergic receptor family is divided into P2X
and P2Y subfamilies. The P2X receptors are ligand-
gated cation channels activated by extracellular ATP
(adenosine-5-triphosphate). P2X receptors are further
classified into seven receptor subtypes, P2X1–P2X7.1,2
In particular, the P2X7 receptor, formerly designated as
the P2Z receptor,3,4 is highly expressed in cells of the im-
mune system such as macrophages,5 lymphocytes,6 mast
cells,7 and microglia.8 The P2X7 receptor, which displays
35–40% structural homology with other subtypes of P2X
receptors, consists of two hydrophobic membrane-span-
ning domains (M1,M2), a large extracellular loop, a
short intracellular N-terminus, and a long intracellular
C-terminus.9 Interestingly, the C terminus of the P2X7
receptor is 239 amino acids longer (AA 352–595) than
that of other P2X receptor subtypes. The long intracellu-
lar C-terminal domain of the P2X7 receptor modulates
several functions, including signal transduction events
and cytolytic pore formation.10–12 The activation of
P2X7 receptors results in two distinct responses, depend-
ing on the exposure time to agonist. A brief stimulation
of the receptor by extracellular ATP opens a membrane
channel permeable to small cations (Na+, Ca2+, K+).
However, repeated or sustained activation by ATP in-
duces the formation of a pore permeable to large molec-
ular weight molecules/ions up to 900 Da in mass, such as
ethidium bromide (314 Da) and YO-Pro-1 (376 Da),
leading to cellular death or cell lysis.13–15
The P2X7 receptor is implicated in the regulation of the
expression and secretion of cytokines and inflammatory
mediators including interleukin (IL)-1,16 IL-2,17 IL-18,18
and tumor necrosis factor (TNF)-a.19 Notably, the P2X7
receptor plays an important role in the processing and
release of the proinflammatory cytokine IL-1b in the im-
mune system by a complex sequence of events: Initially,
the activated P2X7 receptor causes the depletion of K+
leading to the stimulation of IL-1b converting enzyme
(caspase-1), which converts LPS-activated pro-IL-1b to
mature IL-1b.16 Therefore, development of antagonists
for inhibition of the P2X7 receptor could be a therapeu-
tic strategy to treat inflammatory diseases (Fig. 1).
Keywords: P2X7 receptor; Tyrosine-based antagonists; Ethidium bro-
mide uptake; IL-1b release.
Among potent and specific non-competitive antagonists
at the human (h) P2X7 receptor developed so far20 is a
*
Corresponding author. Tel.: +82 62 970 2502; fax: +82 62 970
2484; e-mail: yongchul@gist.ac.kr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.077

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G. E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 571–575
OH
N
N
N
O
OH
S
O S O
O
O
N
S
O
O
O
O
a
H
N
b
N
H
N
O
H
N
Cbz
R2O
N S
O
O
N
O
O
OH
O
O
N
R1 N
N
O
O
4
7
O
O
N
R1 = C6H5, R2 = CH3,
KN-62, 1
R1 = Boc, R2 = H, MRS2306, 2
R1 = p-F-C6H4, R2 = CH3, 3
8
9
Scheme 1. Synthesis of a representative P2X7 receptor antagonist 9
consisting of a 3,5-dimethyltyrosyl derivative. Reagents and condi-
tions: (a) benzoyl piperazine, Et3N, Bop-Cl, rt, 95%; (b) p-meth-
ylbenzenesulfonyl chloride, DMAP, CH2Cl2, rt, 77%.
N
H
N
N
Cl
N
Cl
N
N
N
5
6
Figure 1. P2X7 receptor antagonists.21–27
R5
OH
OH
O
S
O
N
tyrosyl derivative, the isoquinoline KN-62 (1-(N,O-
bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl)-4-phe-
nylpiperazine) 1, first known as an inhibitor of
Ca2+/calmodulin-dependent protein kinase II (CaMK
II).21 Compound 2 (MRS2306), modified from KN-
62, with the general structure R1-Tyr(OR2)-piperazi-
nyl-R3, exhibited an IC50 value of 40 nM and was more
potent than KN-62 in an ATP-induced K+ efflux assay
in hP2X7-expressing HEK293 cells.22 Another series of
conformationally constrained and N-arylpiperazine-
modified analogues was reported.23,24 An N-p-fluor-
ophenylpiperazine analogue, 3, displayed higher antag-
onistic potency than KN-62 in an ethidium bromide
uptake assay and significantly inhibited ATP-stimulated
secretion of IL-1b from human macrophages. Other
P2X7 receptor antagonists, such as the thiazolidine-
2,4-dione derivative 4, the 4,5-diarylimidazoline deriva-
tive 5, and the 1-benzyl-5-phenyltetrazole derivative 6,
have been reported by several industrial pharmaceutical
laboratories.25–27
O
R1
N
a
R1
N
b
*
O
R1
N
Cbz
*
O
*
O
HO
10,
11,
12, R1=H, (R)
O
N
O
1
N
R2
O
R4
R =CH3, (S)
O
R4
1
R =H, (S)
R3
N
R2
13-15
R3
R1=CH3, (S)
13,
16-36
R5= -CH3, -NO2,
-CN, -F, -Br, -I, -
OCF3, -CH2Br
R2=Ph-CO, R3=H, R4=H
R1=H, (S)
14a, R2=Ph-CO, R3=H, R4=H
14b, R2=Ph-CO, R3=CH3, R4=H
R2=Ph-CO, R3=H, R4=CH3
R = p-(CH3)2N-Ph-CO, R =CH3, R =H
14c,
14d,
2
3
4
14e, R2=p-CF3-Ph-CO, R3=H, R4=H
14f, R2=p-F-Ph-CO, R3=H, R4=H
14g, R2=p-Br-Ph-CO, R3=H, R4=H
2
3
4
14h,
R =Boc, R =H, R =H
R1=H, (R)
15, R2=Boc, R3=H, R4=H
Scheme 2. Synthesis of various P2X7 receptor antagonists consisting
of tyrosyl derivatives. Reagents and conditions: (a) substituted
arylpiperazine or Boc-piperazine, HOBt, EDCÆHCl, DMF, rt, 93–
98%; (b) substituted benzenesulfonyl chloride, DMAP, CH2Cl2, rt, 70–
90%.
In the present study, we report the structure–activity
relationship (SAR) of a novel series of Cbz-substituted
tyrosyl derivatives having substituted piperazine and
arylsulfonyl groups. The compounds were evaluated in
an assay of the uptake of a fluorescent dye ethidium bro-
mide induced by 20- and 30-O-(4-benzoyl-benzoyl)-ATP
(BzATP), which activates the hP2X7 receptor stably
expressed in HEK293 cells, and in an ELISA for
IL-1b release from differentiated THP-1 cells.
of probing the proximity of adjacent binding sites on
the receptor.22 The nitro group of compound 21 was
reduced with NaBH4-copper (II) bisacetylacetonate to
an amine, which was subsequently converted to the
isothiocyanate in compound 38 by a reaction with
thiophosgene. The isothiocyanate group in 38 was
reacted with 1,4-diaminobutane to produce the primary
amine congener 39. Finally, to make the dimer 40 a
R5–R5 linker was created by a coupling reaction
between 38 and 39.
Two variable positions of the skeleton, that is, the
arylpiperazine and arylsulfonate moieties, were system-
atically modified through facile acylation and sulfonyla-
tion reactions as shown in Schemes 1–3. The starting
materials, the compounds 7, 10, 11, and 12, were first
reacted with a variably substituted arylpiperazine or
Boc-piperazine to produce an amide in the presence of
coupling reagents such as EDCÆHCl or Bop-Cl. Then,
the phenol was derivatized with various sulfonamide
groups by the reaction of a para-substituted arylsulfonyl
chloride in the presence of DMAP as catalyst. Com-
pound 21 was used as the starting material in a synthetic
route that led to the dimerized compound 40 (Scheme
3). This symmetric dimer was designed for the purpose
The SAR studies of tyrosine-based derivatives 9, 16–36,
and 40, which have various substitutions at the toluene-
sulfonyl, substituted piperazine, and benzyloxycarbonyl
(Cbz) groups, were assessed in the ethidium+ accumula-
tion assay. Experiments were performed using HEK293
cells stably transfected with cDNA encoding the hP2X7
receptor. As shown in Table 1, compound 9 with two
methyl groups at the phenyl ring of tyrosine displayed
a 3-fold increase of the antagonistic activity compared

G. E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 571–575
573
b
a
A
r-NH2
c
Ar-NO2
Ar-NCS
Ar-NHCSNH-(CH2)4NH2
37
21
38
39
S
HN
H
N
HN
S
HN
O S O
d
O
38
+ 39
O S O
O
O
NH
O
O
N
N
H
N
O
O
O
N
N
O
O
40
Scheme 3. Dimerization of P2X7 receptor antagonists through formation of a thiourea linkage. Reagents and conditions: (a) NaBH4, Cu(acac)2,
methanol, rt, 80–85%; (b) CSCl2, chloroform, water, sodium bicarbonate, 70–85%; (c) 1,4-diaminobutane, CH2Cl2, rt, 40–45%; (d) dry DMF, at
80 ꢁC, 52%.
with compound 16. In addition, when the methyl group
was adjacent to the benzoyl piperazine group (e.g., 17
and 18), the antagonist effects were also increased by 2-
fold compared with compound 16 regardless of the ste-
reochemistry of the chiral carbon of the piperazine
group. Thus, the steric effects near the sulfonyl ester of
the phenyl group of tyrosine and the benzoyl group of
piperazine ring were tolerated in the antagonism of the
receptor. However, unlike compound 17 or 18, their reg-
ioisomer, 19, contained a methyl group adjacent to the a-
Table 1. Activities of synthesized tyrosine-based derivatives on ethidium accumulation in hP2X7-expressing HEK293 cells28
O
S O
O
R5
O
R1
N
O
O
*
N
R4
R3
N
R2
16 - 36
R3
R1
R2
R4
R5
IC50
SE (nM)
a
Compound
R/S
KN-62 (positive control)
175 35
219 69
599 22
9b
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
40d
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(R)
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph-CO
Ph-CO
Ph-CO
Ph-CO
Ph-CO
Ph-CO
Ph-CO
H
H
CH3
CH3
CH3
CH3
CH3
CH3
NO2
CN
H
(S)CH3
H
H
260 77
256 96
(R)CH3
H
H
(R)CH3
H
504 136
1360 580
117 44
H
H
(S)CH3
H
p-(CH3)2N-Ph-CO
p-(CH3)2N-Ph-CO
H
539 90
(S)CH3
(S)CH3
H
H
I
55 5%c
54 3%c
2010 340
724 171
319 65
p-(CH3)2N-Ph-CO
p-(CH3)2N-Ph-CO
H
CH3
CH3
CH3
CH3
CH3
F
H
p-CF3-Ph-CO
p-F-Ph-CO
p-Br-Ph-CO
Boc
H
H
H
H
H
H
481 41
5880 3280
517 18
H
H
Boc
Boc
H
H
Br
H
H
I
CN
3540 907
780 107
725 118
2460 470
1540 102
861 123
28 1%c
Boc
Boc
H
H
H
H
OCF3
CH2Br
CH3
CH3
Boc
Boc
H
H
H
H
Boc
H
H
a IC50 = 50% inhibitory concentrations were obtained from concentration–response curves. Data values are expressed as means SD. All experi-
ments were repeated at least 3–11 times.
b This compound has two methyl groups at the phenyl ring of the tyrosyl moiety (see Scheme 1).
c The compounds 23, 24, and 40 represented the % inhibition at 10 lM concentration level.
d This compound is a dimer linked through formation of a thiourea linkage (see Scheme 3). 16, MRS2427; 21, MRS2447; 27, MRS2544.

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G. E. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 571–575
Table 2. Antagonistic effects of tyrosyl derivatives against BzATP-
stimulated IL-1b release in LPS/IFNc-differentiated human THP-1
cells29
100
90
80
70
60
50
40
30
20
10
0
KN 62
28
9
Compound
% inhibitiona
Compound
% inhibition
65
68 10
78
KN-62
9
16
83
87
9
8
26
27
28
29
30
31
32
33
34
35
36
40
6
70 14
69 12
6
17
18
56 13
68 12
65
45
3
4
19
20
72
60
46
52
4
8
9
5
47 12
48
59 11
21
22
7
100
101
102
103
104
23
24
61
68
6
7
72 15
56
37 20
Compound [ nM ]
5
25
65 12
Figure 2. Concentration-dependent inhibition of BzATP-stimulated
IL-1b release in LPS/IFNc-differentiated human THP-1 cells by
compounds 9, 28, and KN-62. Data points represent means SD of
values obtained (n = 3).
a The values represent the % inhibition at a 1 lM concentration
expressed as means SD against 1 mM Bz-ATP (n = 3).
carbonyl of the tyrosyl moiety, which reduced the antag-
onistic effect. In a comparison between compounds 20
and 16, the presence of a methyl group at the tyrosyl a-
nitrogen decreased the antagonistic activity by 2-fold at
the hP2X7 receptor, which is consistent with previously
reported results.23 The 4-nitro group at the R5 position
(compound 21) increased the antagonistic effect by 5-fold
compared with the corresponding 4-methyl group of 16;
compound 21 was slightly more potent than KN-62.
tion of 19 and the methyl group on the a-nitrogen of 20
seem to affect the inhibitory effect on the release of IL-1b
as a comparison between them and compound 16 shows.
Compounds 9 and 28, which showed inhibitory effects
similar to KN-62 against the release of IL-1b by BzATP,
were further evaluated with full concentration–response
curves to compare IC50 values (Fig. 2). The IC50 values
of compounds 9 (481 nM) and 27 (881 nM) were 2- and
4-fold higher than that of KN-62 (188 nM).
The rank order of potency according to IC50 values of
para-substituents at the benzoyl group was F (27) > Br
(28) > CF3 (26) > (CH3)2N (25). Comparing compounds
17 versus 24 and 16 versus 25 show that a 4-dimethyl-
amino group significantly reduced the antagonistic activ-
ity. Thus, basic or bulky groups seem to be inappropriate
recognition motifs for the antagonism. Compounds that
were Boc-substituted at the R2 position showed different
antagonistic effects depending on phenyl sulfonyl ring
para-substitution (i.e., R5-substituent) with the following
order of potency: Br (30) > OCF3 (33) > CN (32) >>
CH3 (35) > CH2Br (34) > I (31) > F (29).
In conclusion, a novel series of tyrosine-based P2X7
receptor antagonists with modifications at the pipera-
zine and arylsulfonyl groups were synthesized and eval-
uated in ethidium accumulation assays and ELISAs of
the release of IL-1b. Substitution of the arylsulfonyl
moiety with a nitro group, in comparison to a methyl
group, increased antagonistic potency in compound
21, which was slightly more potent than KN-62. Several
derivatives, including the dimethylphenylsulfonate ana-
log 9 and the methylpiperazine analogs 17 and 18, were
nearly equipotent to the parent compound KN-62 and
their functional inhibitory activity against the release
of the inflammatory cytokine, IL-1b, was confirmed.
Substitution with D-tyrosine in 36 and sterically bulky
tyrosyl 3,5-dimethyl groups in 9 favored P2X7 receptor
antagonistic potency.
In the study of the effect of chirality of the tyrosine by
comparing R- and S-isomers 35 and 36, the R-(D)-iso-
mer was the more potent P2X7 receptor antagonist.
The substitution with a D-tyrosyl residue might add sta-
bility to the compound in biological systems. The di-
meric compound 40 showed only marginal antagonism
at a 10 lM concentration.
Acknowledgments
The functional antagonism of the tyrosine-based deriva-
tives (9, 16–36, and 40) was evaluated with an ELISA for
the detection of BzATP-activated IL-1b release in LPS/
IFNc-differentiated human THP-1 cells (Table 2). Most
of the compounds with low submicromolar IC50 values
in the ethidium accumulation assay (e.g., 9, 17, 18, 27,
28, etc.) except for 21, resulted in the parallel correlation
with the inhibitory activity against the release of IL-1b
by BzATP. However, compounds 24 and 31, which
had very low antagonistic activity in the ethidium accu-
mulation assay, showed a relatively high inhibitory effect
on the release of IL-1b. The methyl group at the R4 posi-
This work was supported by the Korea Research Foun-
dation Grant (KRF-2004-042-E00169) and by the Intra-
mural Program of NIDDK, NIH for Dr. K.A.
Jacobson.
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