Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 1: Initial structure-activity relationship studies of a hit from a high throughput screening

Article abstract of DOI:10.1016/j.bmcl.2017.04.060

The P2X3 receptor is primarily expressed in the peripheral sensory nerves, and therefore, antagonists of this receptor may be useful for the treatment of chronic pain. Pyrrolinone derivatives have been identified as a novel class of P2X3 receptor antagonists. A lead structure with moderate activity was discovered through a high-throughput screening assay. A structure-activity study led to the discovery of several P2X3 receptor antagonists. Compound 34 showed potent and specific antagonistic activity and analgesic efficacy.

Full text of DOI:10.1016/j.bmcl.2017.04.060

Accepted Manuscript
Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 1:
Initial structure-activity relationship studies of a hit from a high throughput
screening
Hiroyuki Tobinaga, Takayuki Kameyama, Miho Oohara, Naotake Kobayashi,
Masahide Ohdan, Natsuki Ishizuka, Masaharu Kume, Maki Tomari, Yoshikazu
Tanaka, Fumiyo Takahashi, Haruki Kinoshita, Shinji Shimada, Shunji
Shinohara, Hiroyuki Kai
PII:
S0960-894X(17)30434-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.060
BMCL 24913
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 February 2017
12 April 2017
18 April 2017
Please cite this article as: Tobinaga, H., Kameyama, T., Oohara, M., Kobayashi, N., Ohdan, M., Ishizuka, N., Kume,
M., Tomari, M., Tanaka, Y., Takahashi, F., Kinoshita, H., Shimada, S., Shinohara, S., Kai, H., Pyrrolinone
derivatives as a new class of P2X3 receptor antagonists. Part 1: Initial structure-activity relationship studies of a hit
from a high throughput screening, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.04.060
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Pyrrolinone derivatives as a new class of P2X3 receptor antagonists.
Part 1: Initial structure-activity relationship studies of a hit from a high throughput
screening.
Hiroyuki Tobinagaa,*, Takayuki Kameyamab, Miho Ooharaa, Naotake Kobayashia, Masahide Ohdana, Natsuki
Ishizukaa, Masaharu Kumea, Maki Tomaric, Yoshikazu Tanakad, Fumiyo Takahashib, Haruki Kinoshitab, Shinji
Shimadac, Shunji Shinoharab, and Hiroyuki Kaia,*
a Medicinal Chemistry Research Laboratory, Shionogi & Co., Ltd, 1-1 Futaba-cho, 3-chome, Toyonaka, Osaka 561-0825, Japan
b Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd, 1-1 Futaba-cho, 3-chome, Toyonaka, Osaka 561-0825, Japan
c Shionogi Techno Advance Research Co., Ltd., 1-1 Futaba-cho, 3-chome, Toyonaka, Osaka 561-0825, Japan
d Global Innovation Office, Shionogi & Co., Ltd, 1-1 Futaba-cho, 3-chome, Toyonaka, Osaka 561-0825, Japan
*Corresponding authors. Tel.: +81-6-6331-6155; fax: +81-6-6332-6385; E-mail address: hiroyuki.tobinaga@shionogi.co.jp (H. Tobinaga)
Tel.: +81-6-6331-6203; fax: +81-6-6332-6385; E-mail address: hiroyuki.kai@shionogi.co.jp (H. Kai)
Key words: Purinergic receptors, ion channels, P2X3 receptor antagonists, pyrrolinone derivatives, pain
Abstract
The P2X3 receptor is primarily expressed in the peripheral sensory nerves, and therefore, antagonists
of this receptor may be useful for the treatment of chronic pain. Pyrrolinone derivatives have been
identified as a novel class of P2X3 receptor antagonists. A lead structure with moderate activity was
discovered through a high-throughput screening assay. A structure-activity study led to the discovery of
several P2X3 receptor antagonists. Compound 34 showed potent and specific antagonistic activity and
analgesic efficacy.
Adenosine triphosphate (ATP), produced in the mitochondria in cells, is used as a source of energy for
various physiological activities, as a phosphorylation substrate for proteins, and as an extracellular
neurotransmitter. ATP is released into the extracellular fluid by a variety of stimuli including
inflammatory or cell damage. Extracellular ATP acts as a neurotransmitter for P2 receptors at the nerve
terminal. P2 receptors are classified into two groups, P2X includes the ligand-gated ion channels1 and
P2Y includes G protein-coupled receptors2. P2X3, a subtype of P2X receptors, was first cloned in
19953,4. P2X3 receptors are primarily expressed in the peripheral sensory nerve. P2X3 receptor
knockout mice were reported for suppression of pain behaviors5. In 2002, A-317491, which is a specific
P2X3 receptor antagonist, showed an analgesic effect by subcutaneous administration to rats in both an
inflammation pain model and neuropathic pain model6. Thus, in an effort to develop a new analgesic
1

drug, P2X3 receptor antagonists with various chemotypes were reported (Figure 1)7-9. Among them,
AF-219 improved symptoms of pain and urinary urgency in patients with moderate to severe symptoms
of interstitial cystitis/bladder pain syndrome (IC/BPS) in a Phase 2 clinical trial. Furthermore, AF-219 is
in a Phase 2 clinical trial that examines its ability to improve refractory chronic cough10.
Figure 1. Structures of P2X3 receptor antagonists and hit compound 1
Building on the information gained from these previous studies, we aimed to develop a new P2X3
receptor antagonist as an analgesic drug free from the side effects observed for existing analgesics that
act on the central nervous system. To discover novel chemotype P2X3 receptor antagonists, we
performed high throughput screening of our chemical library using a FLIPR 384 Ca2+-influx assay
system. Hit compound 1, which has a pyrrolinone skeleton, was identified. The IC50 value of compound
1 showed the decrease by the increase in concentration of ATP. These results showed that compound 1
was orthosteric antagonist. In attempts to improve the antagonistic activity of compound 1, a series of
pyrrolinone derivatives were synthesized with various substituents to identify the structure exhibiting the
strongest activity. Herein, we describe the synthesis and structure-activity relationships of pyrrolinone
derivatives as P2X3 receptor antagonists.
Pyrrolinone skeletons were easily synthesized in a one-pot by condensation of an α-ketoester, aniline
and aldehyde (Scheme 1)11. The other pyrrolinone derivatives were synthesized from compound 2 as a
2

starting material according to Scheme 2. Compound 19 was prepared by methylation of compound 2
using iodomethane in the presence of potassium carbonate. Dehydroxylated compound 20 was
synthesized by acetylation of the enolic alcohol 2 and hydrogenation using Pd/C. Compound 2 was
reacted with hydroxylamine to give compound 16 and 21 in moderate yields. To change the linker L at
position-4 of the pyrrolinone ring, the Beckmann rearrangement was applied to the pyrrolinone
derivatives (Scheme 3). The enolic alcohol 2 was converted to the benzyl ether by alkylation with benzyl
bromide to give 35. Oxime 36 was obtained by using the same conditions as described for compound 16,
and then amide compounds 37 and 38 were synthesized by Beckmann rearrangement using cyanuric
12
chloride-ZnCl2 . Both amide compounds were debenzylated by hydrogenation to give 17 and 18.
Scheme 1. Reagents and conditions for the synthesis of the pyrrolinone skeltons: (a) AcOH, dioxane,
reflux.
O
O
O
OMe
OH
O
O
O
a
N
N
b, c
N
MeO
MeO
MeO
COOEt
COOEt
COOEt
19
2
20
d
O
NOH
NHOH
O
OH
O
N
N
MeO
+
MeO
16
21
COOEt
COOEt
Scheme 2. Reagents and conditions for the conversion of compound 2.: (a) MeI, K2CO3, DMF, rt 3h,
83%; (b) Ac2O, pyidine, rt 3h, 93%; (c) H2, Pd/C, THF, rt, 3 days, 11%; (d) 50% NH2OHaq, THF, reflux
7h, 16 (20%), 21 (25%).
3

Scheme 3. Reagents and conditions for the synthesis of amide derivatives. : (a) BnBr, K2CO3, DMF, rt
overnight 84%; (b) NH2OH·HCl, pyridine, 80°C 6h, 64%; (c) cyanuric chloride, ZnCl2, CH3CN, reflux
2.5h, 32 (57%), 33 (31%); (d) H2, Pd/C, dioxane, rt 2h, 83%; (e) H2, Pd/C, dioxane, rt 2h, 83%.
Table 1 shows the effects of substitution of the pyrrolinone ring at each position and the
ethoxycarbonyl moiety of the pyrrolinone derivatives on antagonistic activity13.
Among compounds with a substituent (R1) at position-5 of the pyrrolinone ring (1 – 9), the phenyl (2),
4-fluorophenyl (5), and cyclohexyl (7) derivatives were the most active, followed by the 3-fluorophenyl
derivative (4). These compounds showed higher activity than compound 1 (R1: 2-chlorophenyl). The
2-fluorophenyl (3), 4-chlorophenyl (6), and isopropyl (8) derivatives showed reduced antagonistic
activities. Non-substituted compound (9) was inactive. These findings suggested that the presence of a
bulky group and electron-donating substituent at position-5 on the pyrrolinone ring may contribute to the
high activity.
By modification of substituents (R2) at position-4 on the pyrrolinone ring (10 – 13), 4-methoxyphenyl
(2) showed higher antagonistic activity than phenyl (10), and tert-butyl (13). The smaller substituents
methyl (11) and n-butyl (12) led to over a 10-fold reduction in activity. Therefore, a bulky substituent
was necessary at position-4 on the pyrrolinone ring for high activity.
To explore the linker (L) at position-4 on the pyrrolinone ring, the carbonyl moiety was removed (14)
or converted to a methylene from the carbonyl (15). Neither 14 nor 15 had activity. Additionally, oxime
(16) maintained some activity, but amides (17, 18) showed significant decreases in activity.
The enolic hydroxyl group (R3) at position-3 of the pyrrolinone ring was converted to the methyl ether
(19), non-substituted (20) or hydroxyamino (21) compound, all of which showed large activity decreases.
From these results, it appears that the carbonyl moiety at position-4 and enolic hydroxyl group at
position-3 were essential for the antagonistic activity.
We also explored the substituent effects (R4) at the para position on the benzene ring (22 – 28). The
para-substituted trifluoromethyl compound (27) showed moderate activity, but the compounds with the
other functional groups at this position showed significant decreases of activity (22 – 26). The ortho or
4

meta substituted compounds were also synthesized, and all showed significant decreased activities (data
not shown).
Table 1. Structure activity relationship study of pyrrolinone derivatives
R1
R2
L
R3
R4
Compound
IC50 (µM)
0.09
0.02
0.13
0.03
0.02
0.13
0.01
0.14
>10
2-Cl-Ph
Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
Ph
CO
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
H
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
1
CO
2
2-F-Ph
3-F-Ph
4-F-Ph
4-Cl-Ph
c-Hexyl
i-Pr
H
CO
3
CO
4
CO
5
CO
6
CO
7
CO
8
CO
9
Ph
CO
0.37
4.79
1.02
0.26
>10
10
11
12
13
14
15
16
17
18
19
20
21
Ph
Me
CO
Ph
Bu
CO
Ph
t-Bu
CO
Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
none
CH2
C(=NOH)
NHCO
CONH
CO
Ph
>10
Ph
0.56
1.37
3.93
9.80
>10
Ph
Ph
Ph
Ph
CO
Ph
CO
NHOH
4.24
5

Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
CO
CO
CO
CO
CO
CO
OH
OH
OH
OH
OH
OH
COOH
H
>10
>10
3.28
5.92
>10
0.36
22
23
24
25
26
27
Ph
Ph
Ac
Ph
CONMe2
CH2OH
CF3
Ph
4-F-Ph
Ph
4-MeO-Ph
CO
OH
0.10
28
The ester group of R4 contributed largely to the antagonistic activity, although the excretion rate in
vivo would likely be large because of the metabolic instability of the ester group. To improve the
metabolic stability while maintaining high activity, we attempted to incorporate 1,2,4-oxadiazole rings
as a bioisostere for the ester groups14. The resulting oxadiazole compound (28) was highly active.
Pyrrolinone derivatives with an ester group or 1,3,4-oxadiazole ring at R4 showed the highest activity,
so other substitutions at R4 were explored (Table 2). We introduced a 5-membered hetero aromatic ring
at R4 while R1, R2 and R3 were maintained to ensure high activity (29 – 34). As a consequence, the IC50
values were less than 0.05 µM except for the 1,3,4-oxadiazole (31) and oxazole (32). We evaluated the
rat pharmacokinetics for a series of these compounds. Ester (5) was rapidly excreted in vivo and was not
orally absorbed. As expected, the change of R4 from an ester to a 5-membered hetero aromatic ring
greatly improved the total clearance. However, these compounds showed low oral bioavailability. This
result suggested the possibility that the isoxazole ring was a bioisostere of the ester group.
Table 2. Pharmacokinetics of selected compounds in rats
R4
t1/2 (hr)
CLt (ml/min/kg)*
BA (%)
Compound
IC50 (µM)
6

COOEt
0.020
0.019
0.021
0.072
0.135
0.009
0.025
0.1
0.8
1.0
1.7
NT
1.1
1.3
37
NC
7.1
5
5
29
30
31
32
33
34
3.4
5.5
NT
6.2
7.7
13.5
2.9
NT
0.5
10.2
Compound 5: Position-4 of the pyrrolinone ring has a 4-fluorophenyl group instead of a cyclohexyl.
CLt: total clearance; BA: oral bioavailability; NC: not calculated; NT: not tested.
a. All compounds were administered at 0.5 mg/kg iv and 1.0 mg/kg po.
These compounds were administered as a mixture of three to five compounds.
To evaluate the activity of the other purinergic receptors, isoxazole derivative (34) was selected from
the above compounds for further study. Compound 34 was a specific and potent antagonist for the P2X3
receptor (Table 3). Additionally, compound 34 did not inhibit any of the 41 receptors and 17 enzymes
tested at 10 µM (data not shown). To confirm the analgesic effect of our P2X3 antagonist, the sodium
salt of compound 34 was prepared,15 and evaluated for efficacy in an acetic acid induced writhing test16
in mice by intravenous administration. The sodium salt of 34 showed good analgesic efficacy (ED50; 2.6
mg/kg) in a dose-dependent manner (Figure 2).
Table 3. The selectivity of compound 34 for P2X receptors.
7

P2Xn
IC50 (µM)
P2X1
>10
P2X2
>10
P2X3
0.025
P2X2/3
4.32
P2X4
>30
P2X7
>10
25
20
15
10
5
*
*
**
10
0
Vehicle
1
3
Compound 34 (i.v. dose : mg/kg)
Mean+S.E.M, * p<0.05, ** p<0.01
Figure 2. Analgesic effects of compound 34 by an acetic acid induced writhing test in mice
In conclusion, a high throughput screen was performed of our chemical library in an effort to identify a
new P2X3 antagonist. Hit compound 1 was discovered, which had a novel pyrrolinone skeleton different
from previously reported P2X3 antagonists. In a series of hit to lead structure activity relationship
studies, we identified compound 34, which showed potent and selective activity for the P2X3 receptor.
Compound 34 showed analgesic efficacy in an acetic acid induced writhing test in mice, suggesting that
a P2X3 antagonist may be useful as a new analgesic drug. It is expected that further improvement of the
pharmacokinetics of compound 34 may lead to its development as a new analgesic drug. Further
optimization of these pyrrolinone derivatives and evaluation of their optical isomers are ongoing.
References and Notes
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2. Abbracchio, M. P.; Burnstock, G.; Boeynaems, J. A.; Barnard. E. A.; Boyer, J. L.; Kennedy. C.;
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B.; Cain, G.; Berson, A.; Kassotakis, L.; Hedley, L.; Lachnit, W. G.; Burnstock, G.; McMahon, S. B.;
Ford, A. P. D. W. Nature, 2000, 407, 1011
6. Jarvis, M. F.; Burgard, E.C.; McGaraughty, S.; Honore, P.; Lynch, K.; Brennan, T. J.; Subieta, A.;
Biesen, T. V.; Cartmell, J.; Bianchi, B.; Niforatos, W.; Kage, K.; Yu, H.; Mikusa, J.; Wismer, C. T.;
Zhu, C. Z.; Chu, K.; Lee, C.; Stewart, A. O.; Polakowski, J.; Cox, B. F.; Kowaluk, E.; Williams, M.;
Sullivan, J.; Faltynek, C. PNAS, 2002, 99, 17179
7. Brotherton-Pleiss, C. E.; Dillon, M. P.; Ford, A. P. D. W.;Gever, J. R.; Carter, D. S.; Gleason, S. K.;
Lin, C. J.; Moore, A. G.; Thompson, A. W.; Villa, M.; Zhai, Y. Bioorg. Med. Chem. Lett. 2010, 20,
1031
8. Burgey, C. S. 241st ACS Natl Meet (March 27-30, Anaheim) 2011 Abst MEDI 15
9. Szántó, G.; Makó, A.; Bata, I.; Farkas, B.; Kolok, S.; Vastag, M.; Cselenyák, A. Bioorg. Med. Chem.
Lett. 2016, 26, 3896
10. Abdulgawi, R.; Dockry, R.; Holt, K.; Layton, G.; McCarthy, B. G.; Ford, A. P.; Smith, J. A. Lancet,
2015, 385, 1198
11. Andreichikov, Yu. S.; Gein, V.; Anikina, I. N. Zh. Org. Khim. 1986, 22, 1749-1756
12. Furuya, Y.; Ishihara, K.; Yamamoto, H. J. Am. Chem. Soc. 2005, 127, 11240-11241
13. Evaluation of P2X3 receptor inhibitory activity: Stably expressing cell line (C6BU-1 cell transfected
with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were
seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium
(8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at
37 °C under 5 % carbon dioxide atmosphere. The medium was replaced with 4 µM Fluo-3-AM
solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM
D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37°C
under 5% dioxide carbon atmosphere for one hour. The Fluo-3-AM solution was replaced with
9

washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6
mM D-glucose, 2.5mM probenecid, pH7.5). Inhibitory activity of the test compound was measured
by High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). The test
compoundsolution was prepared with dilution buffer (washing buffer with 0.1% Pluronic F-127) and
dispensed to each well. Five minutes after starting the measurement in FDSS 3000, 50 nM ATP
solution prepared by dilution buffer was dispensed through the built-in automatic dispenser, and the
measurement of fluorescence intensity was continued for 3 min. For each well, the fluorescence ratio
was calculated as the ratio of the maximum fluorescence intensity to the fluorescence intensity at the
starting of the measurement. The 50% inhibitory concentration (IC50) of the test compound was
calculated using Microsoft Excel (Microsoft Corporation) and XLfit (ID Business Solutions Ltd.).
The known P2X3 antagonist, A-317491 showed the IC50 value of 92 nM in house.
14. Meanwell, N. A. J. Med. Chem., 2011, 54, 2529–2591
15. Experimental procedure for preparation of sodium salt of compound 34: The synthesis method and
data of compound 34 are as follows. To a solution of methyl 4-(4-methoxyphenyl)-2,4-dioxobutanone
(236 mg, 1 mmol), 4-(isoxazol-3-yl)aniline (160 mg, 1 mmol) in AcOH (1 ml) was added
cyclohexanecarbaldehyde (0.12 ml, 1 mmol) and stirred at 95 °C for 20 minutes, then yellow
precipitate was observed. The pricipitate was filtered and washed with Et2O, EtOAc and dried in
vacuo to give compound 34 (190 mg, yield 41%) as an yellow solid; 1H NMR (300 MHz, DMSO-d6)
δ: 0.71-1.04 (5H, m), 1.38-1.51 (2H, m), 1.53-1.61 (2H, m), 1.69-1.81 (2H, m), 3.90 (3H, s), 5.48 (1H,
d, J = 2.5 Hz), 7.13 (2H, d, J = 8.9 Hz), 7.23 (1H, d, J = 1.7 Hz), 7.84 (2H, d, J = 8.7 Hz), 7.91 (2H, d,
J = 8.7 Hz), 8.03 (3H, d, J = 8.6 Hz), 9.06 (1H, t, J = 1.6 Hz).; MS-ESI (m/z) = 459 [M+H]+.
To a suspension of compound 34 (183 mg, 0.4 mmol) in MeOH (10 ml), H2O (10 ml) was added 0.1
M NaOH aqueous solution and stirred at room temprature for 1 hr. To a reaction mixture was added
THF (5 ml) then turned to clear solution. The reaction mixtures were evaporated and lyophilized,
1
which gave sodium salt of 34 (205 mg, yield 93%) as an yellow powder; H-NMR (300 MHz,
DMSO- d6) δ: 0.78-1.05 (5H, m), 1.38-1.76 (5H, m), 1.82-1.97 (1H, m), 3.82 (3H, s), 5.25 (1H, s),
6.88 (2H, d, J = 8.5 Hz), 7.20 (1H, d, J = 1.6 Hz), 7.76 (2H, d, J = 8.5 Hz), 7.86 (2H, d, J = 8.8 Hz),
7.97 (2H, d, J = 8.5 Hz), 9.03 (1H, d, J = 1.6 Hz).; Anal. Found: C, 62.34; H, 5.63; N, 5.40; Na, 4.80,
Calcd. for C27H25N2O5Na·2H2O: C, 62.78; H, 5.66; N, 5.42; Na, 4.45
16. Koster, R.; Anderson, M.; De Beer, E. J. Federation Proc., 1959, 18, 412
10

11