Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors

Article abstract of DOI:10.1021/acschemneuro.6b00401

Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED₅₀ values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

Full text of DOI:10.1021/acschemneuro.6b00401

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Article
Discovery of Potent Antiallodynic Agents for
Neuropathic Pain Targeting P2X3 Receptors
Young-Hwan Jung, Yong-Chul Kim, Yeo Ok Kim, Hai Lin, Joong-heui Cho, Jin-Hee Park, So-Deok Lee, Jinsu
Bae, Koon Mook Kang, Yoon Gyoon Kim, Ae Nim Pae, Hyojin Ko, Chul-Seung Park, and Myung Ha Yoon
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00401 • Publication Date (Web): 21 Mar 2017
Downloaded from http://pubs.acs.org on March 26, 2017
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Discovery of Potent Antiallodynic Agents for Neuropathic Pain
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Targeting P2X3 Receptors
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YoungꢀHwan Jung,† Yeo Ok Kim,‡ Hai Lin,‡ JoongꢀHeui Cho,¶ JinꢀHee Park,¶ SoꢀDeok Lee,† Jinsu Bae,║
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Koon Mook Kang,† YoonꢀGyoon Kim,⊥ Ae Nim Pae,# Hyojin Ko,† ChulꢀSeung Park,† Myung Ha Yoon,
*,‡,§ and YongꢀChul Kim*,†, ║
†School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500ꢀ712,
Republic of Korea
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‡Department of Anesthesiology and Pain Medicine, Center for Creative Biomedical Scientists at
Chonnam National University, Medical School, Gwangju 501ꢀ757, Republic of Korea
║Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology
(GIST), Gwangju 500ꢀ712, Republic of Korea
┴College of Pharmacy, Dankook University, Cheonan 330ꢀ714, Republic of Korea
#Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of
Science and Technology, PO Box 131, Cheongryang, Seoul 130ꢀ650, Republic of Korea
¶New Drug Development Center (NDDC), DaeguꢀGyeongbuk Medical Innovation Foundation (DGMIF),
80 Cheombokꢀro, Dongꢀgu, Daegu 41061, Republic of Korea
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ABSTRACT: Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the
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management of neuropathic pain because P2X3 receptors are predominantly localized on small to
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medium diameter Cꢀ and Aδ fiber primary afferent neurons, which are related to the painꢀsensing system.
ꢀ
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In this study, 5ꢀhydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro
biological activities by twoꢀelectrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3
receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin
(calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation
of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by
intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the
corresponding methyl ester analog, 28, which could be converted to compound 29 in vivo, was
investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic
effects, with ED50 values of 2.62 mg/kg and 2.93 mg/kg in spinal nerve ligation and chemotherapyꢀ
induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the
P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.
KEYWORDS: Neuropathic pain, antiallodynic effect, P2X3 receptor, adenosine 5'ꢀtriphosphate,
antagonist, structureꢀactivity relationship
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INTRODUCTION
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2X receptors are ligandꢀgated cation channels, which are activated in response to the binding of
extracellular adenosine 5'ꢀtriphosphate (ATP).1,2 It has been well established that the opening of the
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transmembrane pore of the P2X receptors allows the influx of cations such as Ca2+, Na+ and K+ into the
cytoplasm, which results in membrane depolarization and cell excitation.3–5 In mammals, seven P2X
receptor subtypes have been identified with a wide expression in vascular smooth muscles, platelets,
peripheral and central nervous systems, and immune cells, which mediate various physiological processes
including synaptic transmission, presynaptic modulation, smooth muscle contraction, cell proliferation
and death, intestinal motility, platelet aggregation, taste, nociception, and inflammation.6,7 A recent study
of the Xꢀray crystal structure of the zebra fish P2X4 receptor clarified the common topology of P2X
receptors, which includes two transmembrane (TM1 and TM2) domains, a large glycosylated and
cysteineꢀrich extracellular loop containing ATP binding sites, and intracellular Nꢀ and Cꢀtermini. In
addition, the gating mechanism upon the binding of ATP to the zP2X4 receptor was determined by the
conformational changes between the apo and the open state of the crystal structures.4,6
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Among the P2X receptor subtypes, the P2X3 receptor (P2X3R) exists as homomeric P2X3 and
heteromeric P2X2/3 receptors in small to medium diameter Cꢀ and Aδ fiber primary afferent neurons,
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suggesting a high degree of specificity to the painꢀsensing system.8,9 In fact, a number of studies,
including knockꢀout animal studies, have shown that P2X3R is closely related to pain sensation. For
example, the administration of ATP or a P2X3Rꢀselective agonist, αβꢀmethylene ATP, to the hind paws
of rats caused nociceptive behaviors.10–12 In neuropathic or inflammatory pain models, P2X3R knockꢀout
or deficient animal studies with P2X3Rꢀselective antisense or short interfering RNA (siRNA) resulted in
greatly diminished painꢀrelated behaviors.13–15 The crucial role of P2X3R in pain sensation was further
investigated using P2X3 receptor antagonists, TNPꢀATP (2′,3′ꢀOꢀ(2,4,6ꢀtrinitrophenyl)adenosineꢀ5′ꢀ
triphosphate) and PPADS, which showed reduced pain responses in chronic neuropathic pain animal
models.16 Thus, these data suggest that antagonism of P2X3R could be one of the potential therapeutic
strategies for the management of chronic pain.
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In the last decade, potent and selective P2X3 receptor antagonists were developed by pharmaceutical
industries and academic institutes, including our group (Figure 1). Abbott Laboratories disclosed the first
selective nonꢀnucleotide dual P2X3ꢀP2X2/3 antagonist Aꢀ317491 (1),17,18 which showed painꢀreducing
effects in chronic and inflammatory pain animal models. Roche reported diaminopyrimidineꢀbased
analogues, ROꢀ4 (2a),19,20 ROꢀ51 (2b)21 and ROꢀ85 (3),22 as the first allosteric antagonists with high
potency and selectivity for P2X3ꢀP2X2/3Rs. AstraZeneca reported structureꢀactivity relationship (SAR)
studies of pyrroloꢀpyrimidinone derivatives (4)23 for improving the potency and physicochemical
properties of P2X3 receptor antagonists. Afferent Pharmaceutical’s clinical drug candidate, AFꢀ219,24,25
which is a potent and orally bioavailable selective P2X3ꢀP2X2/3 receptor antagonist, has been studied in
several proofꢀofꢀconcept Phase 2 clinical trials, including interstitial cystitis/bladder pain syndrome and
chronic cough, which showed a meaningful pharmacological effect. Our group reported potent selective
and nonꢀcompetitive peptide antagonists, Spinorphin (5, IC50 = 8.3 pM)26 and 5ꢀhydroxy pyridine
derivatives (7aꢀc),27,28 which were developed from the modification of the strong anionic phosphate
sulfonic acid groups and an unstable azo (ꢀN=Nꢀ) linkage of a nonꢀselective P2X antagonist, PPADS (6a),
to a weak anionic carboxylic acid and a stable carbonꢀcarbon bond, respectively.
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In this article, we report further structureꢀactivity relationship (SAR) investigations aimed at the
optimization of drug properties of the 5ꢀhydroxy pyridine derivatives (7aꢀc). First, substituents at the 6ꢀ
position of the 5ꢀhydroxy pyridine skeleton was modified to optimize antagonistic potency. Second,
various substituents at the 4ꢀposition of the pyridine ring were explored to replace the aldehyde moiety of
7c, which may be problematic for metabolic stability. Third, we introduced –CH3 or ꢀH at the 3 position
of pyridine to decrease polarity while maintaining the antagonistic activity. Thus, novel hP2X3 receptor
antagonists with improved drug properties were developed by appropriate combinations of the moiety at
each position of the pyridine skeleton.
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■ CHEMISTRY
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The syntheses of the 3,4ꢀdicarboxypyridine derivatives, 13aꢀl, are depicted in Scheme 1. To explore the
SAR of the 6ꢀposition of the 3,4ꢀdicarboxypyridine derivatives, we replaced the methyl group of the 6ꢀ
position with various alkyl and arylalkyl groups. The synthetic procedures of the previous report27 were
employed, starting from various natural and unnatural amino acids. Briefly, pꢀmethoxyꢀphenyl propionic
acid, 8, was conjugated with esters of the amino acids to yield 10aꢀk, which were subsequently cyclized
to oxazole analogs, 11aꢀk, by treatment with phosphorous pentoxide. The dimethyl pyridineꢀ3,4ꢀ
dicarboxylate derivatives, 12aꢀk, were synthesized through DielsꢀAlder reactions of 11aꢀk in the presence
of dimethyl maleate. Through a subsequent oxidation reaction of the sulfide group of 12k using Oxone®,
12l was obtained. Finally, the 3,4ꢀdicarboxypyridine derivatives, 13aꢀl, were synthesized by the
hydrolysis of 12aꢀl using 20% aqueous KOH (Scheme 1).
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For the synthesis of compounds 16ꢀ20, which have diverse functionalities at the 3,4ꢀpositions, Dielsꢀ
Alder reactions of oxazoleꢀbased diene (11b) were conducted with dienophiles, ethyl cisꢀbetaꢀ
cyanoacrylate, maleic anhydride and methyl vinyl sulfone to produce compounds 14, 18 and 20,
respectively. In the case of compound 14, only one regioꢀisomer was obtained, which should be a 4ꢀ
cyanoꢀ3ꢀcarboxylate substituted isomer, as it has been known29,30 that a higher electronꢀwithdrawing
group, such as the nitrile group of ethyl cisꢀbetaꢀcyanoacrylate, is oriented at the 4ꢀposition of pyridine
during the reaction. In addition, the nitrile group of 14 was further transformed to a tetrazole moiety (15),
a bioꢀisostere of carboxylic acid, using sodium azide. The final compounds, 16 and 17, were obtained by
the baseꢀcatalyzed hydrolysis of 14 and 15, respectively. A pyridazine derivative, 19, was synthesized by
the reaction of maleic anhydride moiety of compound 18 with hydrazine hydrate (Scheme 2).
The synthesis of derivatives in combination with carboxylic acid and carboxamide groups at the 4ꢀ
position and the ꢀCH3 or –H groups at the 3ꢀposition of the pyridine ring was accomplished following
Scheme 3. Compounds 21aꢀ21c were obtained from the same DielsꢀAlder reaction using methyl acrylate
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and methyl crotonate as dienophiles. Subsequently, 21aꢀ21c were hydrolyzed using 20% aqueous KOH to
yield 22aꢀ22c, and 21c was further converted to the carboxamide analog 23 using 30% aqueous ammonia.
The 2ꢀphenoxybenzyl derivatives 27, 29, 35 and 37 were synthesized as shown in Scheme 4 from a
common intermediate, 25, which was prepared via compound 24 using a similar synthetic procedure as in
Scheme 1. DielsꢀAlder reactions of oxazoleꢀbased diene (25) were conducted with dienophiles, methyl
maleate and methyl crotonate, to produce compounds 26 and 28, respectively. Starting from the
intermediate 28, the following sequential reactions were performed: protection of the 5ꢀOH group with a
benzyl group, LiAlH4 reduction of the 4ꢀmethylester group to a 4ꢀhydroxymethyl group (compound 31),
MnO2 oxidation to 4ꢀcarboxaldehyde (compound 33) after a deprotection reaction of the 5ꢀbenzyl ether
moiety with Pd/C under H2 gas, and a WittigꢀHonor reaction with triethyl phosphonoacetate to yield trans
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regionꢀisomer compound 34 (J value = 16.4 Hz in H NMR). The methyl propionate analog, 36, was
prepared from the hydrogenation reaction of the α,βꢀunsaturated double bond of 34 with Pd/C under H2
gas. Finally, hydrolysis reactions of the ester groups of 26, 28, 34 and 36 using 20% KOH yielded 3,4
dicarboxylic acid (27), 4ꢀcarboxylic acid (29), 4ꢀpropenoic acid (35) and 4ꢀpropionic acid derivatives (37),
respectively.
Scheme S1 of the Supporting Information shows the synthesis for the introduction of nitrile, primary
amine and methanesulfonamide groups at the 4ꢀposition of pyridine. Methyl crotononitrile and 25 were
used as the starting materials for the DielsꢀAlder reaction to give 38. After the protection of the 5ꢀOH
group of pyridine with a benzyl group, the nitrile group was reduced to a primary amine moiety using
LiAlH4 to yield 40, of which the benzyl group was subsequently deprotected with Pd/C and H2 gas to
yield 41. Compound 43 was synthesized from a sulfonylation reaction of the primary amine group of 40
with methanesulfonyl chloride and a subsequent deprotection of the 5ꢀbenzyl ether moiety using Pd/C
under an H2 gas condition.
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■ RESULTS AND DISCUSSION
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StructureꢀActivity Relationships for In Vitro P2XR Antagonism. To explore the structureꢀactivity
relationship (SAR) of the synthesized compounds, the antagonistic activities were evaluated by twoꢀ
electrode voltage clamp (TEVC) assays in Xenopus oocytes expressing recombinant mP2X1 or hP2X3,
respectively. The hP2X7R antagonistic activity was evaluated using an assay of ethidium ion
accumulation in human HEK293 cells stably expressing recombinant human P2X7R. Compound 1,17,18
NFꢀ44931 and AZD905632 were tested together as positive control antagonists for hP2X3, mP2X1 and
hP2X7Rs, respectively.
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Although we previously reported a potent antagonist, 7c, with an IC50 value of 60 nM against hP2X3R
for its inhibitory pain signaling activity,27 the in vitro metabolic stability of 7c was determined to be
extremely low (% remaining after 30 min incubation with S9 microsomal fraction), likely due to the 4ꢀ
aldehyde group. Therefore, we have tried to optimize the combinations of moieties at the 3, 4 and 6ꢀ
positions of the 5ꢀhydroxy pyridine skeleton, including modifications of the 4ꢀaldehyde group.
As shown in Table 1, the P2XR antagonistic effects of substituents at the 6ꢀposition of 3,4ꢀ
dicarboxypyridine derivatives were initially investigated with various alkyl, aromatic and electronegative
atomꢀcontaining groups. In the SAR analysis of derivatives with straight carbon chain lengths, the
hP2X3R antagonistic activity increased along with the increase of carbon chains from 0 to 4. In particular,
compounds 13d and 13e, with carbon chain lengths of 3 and 4, showed dramatically improved IC50 values
(93 and 95 nM, respectively) by 4ꢀ5ꢀfold compared with compounds 13aꢀc. Interestingly, the order of
mP2X1R antagonistic activity of this series of derivatives was reverse in comparison with hP2X3R,
showing that the potency at mP2X1R significantly decreased in serial order by increasing the carbon
chain lengths and bulkiness at the 6ꢀposition. Thus, an approximately 100ꢀfold selectivity (13d and 13e)
for hP2X3R vs mP2X1R was achieved from nonꢀselective compounds 13a and 13b by optimizing the
carbon chain lengths from 0 to 4.
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In the case of substitutions of bulky carbon chains such as isopropyl (13f) or isobutyl (13g) at the 6ꢀ
position, the activity at the hP2X3R of 13f (IC50 = 466 nM) was similar to that of 13b (6ꢀmethyl analog),
while compound 13g showed a significantly decreased antagonism (32% inhibition, 1 µM), suggesting
that there might be a narrow binding pocket with a certain distance. Thus, compounds 13h and 13i,
having nonꢀpolar phenyl groups with different carbon chain at the 6ꢀposition, displayed weaker
antagonistic effects against hP2X3R, with IC50 values in the 700 nM range. Derivatives with different
electrostatic potential at the 6ꢀposition, such as carboxylic acid (13j), sulfide (13k) and sulfone (13l), did
not provide a beneficial influence for hP2X3R antagonistic activity compared with compound 13d,
suggesting that the corresponding binding pocket in the receptor may be mainly accessed based on the
steric management of the 6ꢀsubstituents for the antagonistic effect.
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Next, we investigated the positional preference and bioꢀisostere substitution of the carboxylic acid
moiety at the 3,4 positions. Additionally, further modifications at these positions were pursued, with the
groups for various electronic effects, such as compounds 18ꢀ23 (Table 2). Initially, we introduced nitrile
(16) and methanesulfonyl (20) groups as hydrogen bond acceptors and tetrazole (17) as a bioisostere of
the 4ꢀcarboxylic acid group, resulting in markedly decreased antagonistic activity against P2X1R and
P2X3R. The derivatives with fused heterocyclic groups such as maleic anhydride (18) and pyridazine (19),
which have a potential to interact with a lysine residue in the ATP binding site of the P2X3R, also
showed a significant loss of the antagonistic activities. These results suggest that some appropriate degree
of acidic functionality at the 4ꢀposition may be essential for binding at the receptors. Therefore,
modifications of the carboxylic acid at the 3ꢀposition with ꢀH or –CH3 were made, aimed at reducing the
polar negative property of the dicarboxylic acid groups of compound 13. While compound 22a, a monoꢀ
carboxylic acid analog at the 4ꢀposition of the pyridine group, showed a nearly 2ꢀfold decrease in
antagonistic activity against hP2X3R compared with the corresponding 3,4ꢀdicarboxylic acid analog, 13b
(IC50 = 460 nM), the 3ꢀmethylpyridineꢀ4ꢀcarboxylic acid derivative, 22b (IC50 = 384 nM), maintained
hP2X3R antagonistic activity. The attempt to increase the potency against hP2X3R by the replacement of
the 6ꢀmethyl group of 22b by a 6ꢀnꢀpropyl group (22c, IC50 = 303 nM) resulted in only a slight increase in
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the antagonistic activity. In the case of a 4ꢀcarboxamide derivative, 23 (IC50 = 709 nM), with less acidic
but similar Hꢀbond donor properties, a 2ꢀfold decrease in antagonistic activity at hP2X3R was observed.
These results suggest that possible ionic interactions with pharmacophores at the 4ꢀposition may be more
important than the Hꢀbonding interaction for the antagonistic activity at hP2X3R.
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Next, we introduced an mꢀphenoxyphenyl group at the 2ꢀposition of the pyridine moiety (Table 3),
which was previously reported as an alternative pharmacophore to a pꢀmethoxyphenethyl group with
improved selectivity profiles.28 However, a 3,4ꢀdicarboxyꢀ2ꢀmꢀphenoxyphenyl derivative, compound 27
(IC50 = 301 nM), enhanced neither the hP2X3R antagonistic effect nor the selectivity vs mP2X1R
compared with the corresponding 2ꢀpꢀmethoxyphenethyl analog, 13d. Additionally, we introduced –CH3
(29) instead of 3ꢀcarboxylic acid of 27 to decrease polarity. Unlike the case of compound 22c and 13d,
the 4ꢀcarboxyꢀ3ꢀmethylꢀ2ꢀmꢀphenoxyphenyl derivative (29, IC50 = 245 nM) maintained the hP2X3R
antagonistic potency of compound 27. Furthermore, compound 29 also showed antagonistic effects at
heterogeneous rP2X2/3Rs, which are more physiologically relevant receptors (Figure S1 of the
Supporting Information). More exploration of the effects of other substituents at the 4ꢀposition of 29 was
performed, including carbon chainꢀelongated carboxylic acids such as 4ꢀpropenoic acid (35) or 4ꢀ
propionic acid (37), 4ꢀnitrile (38), 4ꢀmethylamine (41) and 4ꢀmethanesulfonamide (43). Unfortunately, all
of the compounds showed less than 50% inhibition in 1 µM against hP2X3R. Therefore, it is speculated
that the binding partners for the pharmacophore at the 4ꢀposition of pyridine ring may be composed of
amino acids with the property of ionic interaction with a narrow binding pocket. The 5ꢀhydroxy pyridine
derivatives were evaluated for subtype selectivity against mP2X1R and hP2X7R. As a result, all
compounds at a concentration of 10 µM in Table 2 and 3 exhibited only 0ꢀ43% inhibition toward
mP2X1R and to inactivity toward hP2X7R.
Among the novel hP2X3R antagonists, compound 29 was selected for further evaluation of functional
activities in in vivo NeP (Neuropathic pain) animal models. A more potent antagonist, 13d, was excluded
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because of its weaker in vivo efficacy in the initial animal tests, presumably due to its highly polar
physicochemical property.
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In Vitro PK and hERG Channel Studies of Compound 29. In the evaluation of in vitro inhibitory
activities against CYP enzymes (3A4, 2C9 and 1A2) and the hERG channel, compound 29 showed weak
and no inhibition, respectively, indicating that the compound had reasonable profiles for both drugꢀdrug
interactions and cardiac toxicity (table 4). However, compound 29 displayed strong plasma protein
binding (> 99%), low metabolic stability (< 5% remaining after 30 min) and low cell permeabilityꢀ
PAMPA45 (log Pe = ꢀ7.04). Therefore, the corresponding methyl ester analog, 28, which could be partially
converted to compound 29 in vivo, was found to result in medium cell permeabilityꢀPAMPA (log Pe = ꢀ
4.8) with improved metabolic stability (33% remaining after 30 min) and plasma protein binding (33%) in
rat. In addition to these results, the lack of antiallodynic effects of compound 29, even by intraperitoneal
(i.p.) administration in NeP animal models (see the next section), led to the decision to evaluate
compound 28 for in vivo experiments.
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In Vivo Behavioral Responses of Compound 28 and 29. The analysis of behavioral responses of
compounds 28 and 29 after intrathecal or intravenous administration showed that righting and placingꢀ
stepping reflexes were normal and that pinna and corneal reflexes were present at the highest dosage used
in this study. Additionally, no overt abnormal behavioral changes were observed.
In Vivo Pharmacology of Compound 29 in Neuropathic Pain Animal Models. The antiallodynic
efficacy of compound 29 was evaluated using the von Frey method after intrathecal administration in
spinal nerve ligation (SNL). The results for compound 29 showed an increase in paw withdrawal
thresholds as the indication of antiallodynic effects in the SNL rats (Figure 2). The antiallodynic efficacy
of compound 29 (%MPE at 1 µg (0.0026 µmol) = 58±19) turned out to be more potent than AF353 (P2X3
receptor antagonist, %MPE at 1 µg (0.0023 µmol) = 32±11) and parallel antiallodynic efficacy compared
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with pregabalin (calcium channel modulator, %MPE at 1 µg (0.0063 µmol) = 65±9) in SNL rats.
However, no pharmacological activity was observed by intraperitoneal administration of compound 29 in
the NeP animal models.
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In Vivo Pharmacology of Compound 28 in Neuropathic Pain Animal Models. Although intrathecal
administration of compound 29 resulted in potent antiallodynic effects in the NeP rats, intraperitoneal
injection of compound 29 did not provide antiallodynic effects (Figure S2 of the Supporting Information).
Recently, AbbVie, Inc. reported that P2X3R antagonists should penetrate blood brain barrier (BBB) to be
efficacious for antiꢀnociceptive responses in neuropathic pain but not necessarily in inflammatory pain
animal models.37 In addition, a review paper described the possible importance of CNS penetration of
P2X receptor antagonists as a factor of physicochemical properties for pain management.44 For that
reason, we have performed additional experiment to determine the brain distribution of intravenous (i.v.)
administered compound 29 and found that the concentration of compound 29 at 0.5 hr in the brain tissue
was only 0.005 µg/g brain tissue (0.29 µg/mL plasma). Thus, the BBB penetration of compound 29 might
be too low to generate inꢀvivo antiallodynic effects in NeP animal models even by i.p. administration. To
improve the physicochemical properties of 29 for BBB penetration, a prodrug strategy was explored using
the methyl ester derivative of 29, compound 28, which could be partially converted to 29 in the CNS to
show antiallodynic efficacy.39 In fact, the concentration of 29 (0.022 µg/g tissue, Table 6) in the brain
tissue at 0.5 hr after i.v. administration of compound 28 was 4 times higher than that of 29 (0.005 µg/g
tissue, Table 5) at 0.5 hr after i.v. administration of compound 29, and the brainꢀtoꢀplasma ratios of 29
(0.24, Table 6) by i.v. administered compound 28 was 12ꢀfold higher than those of 29 (0.02, Table 5) by
i.v. administered compound 29. Therefore, we investigated the in vivo pharmacological effects of i.v.
administered compound 28 in NeP models, including SNL and chemotherapyꢀinduced peripheral
neuropathy (CIPN) rats, and observed potent antiallodynic effects, with ED50 values of 2.62 mg/kg
(Figure 3) and 2.93 mg/kg (Figure 4) in the SNL and CIPN rats, respectively. The in vivo efficacy of 28
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was higher than reported values of current clinical drugs for NeP (pregabalin (ED50 = 4.21 mg/kg) and
tramadol (ED50 = 4.67 mg/kg)).40,41
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However, since the brain concentration of compound 29 is lower than the corresponding effective dose
shown in Figures 3 and 4, we performed additional experiments to investigate whether compound 28, the
proꢀdrug itself would be effective or not, and to verify any possible offꢀtarget activities of it. As the
results, i.t. administered compound 28 did not show any antiallodynic effects (Figure 5), indicating that
the inꢀvivo efficacy in Figure 3 and 4 may be more likely related with the metabolite, compound 29. Also,
oral administration of compound 28 could not provide the inꢀvivo pharmacological effect (Figure 5).
Regarding the PKꢀPD correlation, we observed that the peak amount time of compound 28 in brain was
0.5 hr (Table 6) and the peak antiallodynic effects of compound 28 reached at 1 hr and maintained at 3 hr
(Figure 3 and 4). One of the considerable factors for explaining the time courses disconnection between
PK and PD data could be the sampling site used in our PK study. Since the concentrations of compounds
in whole brain were measured in our study, a report regarding the general distribution of compounds in
the spinal cord, which could be the relevant target site of compound 29, was referred.42 The report showed
that CSF flow descends via the cisterna magna into the spinal subsrachnoid space, reaching the low
cervical–high thoracic region at 10–20 min, thoracolumbar area at 30–40 min, lumbosacral cul de sac at
60–90 min, and eventually into basal cisterns at 2–2.5 h when using radioisotope labeled CSF flow in
human. These data indicate that the distribution time across spinal cord is considerable due to slow CSF
circulation, thus we suppose that the lag time between PK and PD may be explained in this point of view.
Therefore, these results implied that overall improvement of physicochemical properties for BBB
penetration, metabolic stability and plasma protein binding properties of compound 28 enabled its potent
antiallodynic efficacy in NeP animal models by intravenous injection.
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The results of inꢀvivo animal experiments and the species differences of inꢀvitro PK properties of the
novel P2X3R antagonists described in this study suggest the needs and direction of further optimization
for clinical use. Based on the report by Salameh et al.,39 regarding the development of prodrugs targeting
the central nervous system, clinical candidates may be developed by the optimization of ester groups by
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the introduction of clinically used proꢀmoieties as following criteria: 1) possibility of the BBB penetration
2) ability of the conversion to compound 29 in CNS 3) similar bioavailability and metabolic fates of
prodrug in both of rat and human systems. Also, exꢀvivo studies of pain signals using human DRG
system43 were recently introduced to predict the efficacy of compounds in human and investigate the gap
of species, which should be the way of direction to finalize the decision of clinical candidate compounds
as pain killers.
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■ CONCLUSIONS
In this study, we reported the development of novel P2X3R antagonists through extensive SAR and
optimization studies of previously reported hydroxypyridine derivatives, in vitro and in vivo PK analysis
and in vivo pharmacological studies using NeP animal models. Thus, the optimization of 5ꢀ
hydroxypyridine skeleton with 6ꢀnꢀpropyl, 4ꢀcarboxylic acid and 3ꢀmethyl substituents resulted in the
discovery of compound 29, which showed significantly reduced allodynia in SNL by intrathecal
administration. Furthermore, the 4ꢀmethyl ester prodrug of 29, compound 28, showed a potent
antiallodynic effect by intravenous administration. This study could be applied to contribute to the field
with highly unmet needs, new drug discovery for NeP patients and to further drug development programs
currently in progress using the novel hydroxypyridineꢀbased P2X3R antagonists.
■ METHOD
TwoꢀElectrode VoltageꢀClamp Assay on Recombinant mP2X1 and hP2X3 Receptors. Xenopus
oocytes were isolated and incubated with 270 U/mg collagenase for 1.5 h at 23ꢀ24 °C. The defolliculated
oocytes were kept at 18 °C in Barth’s solution. After 1 day, each defolliculated oocyte was cytosolically
microinjected with mP2X1 and hP2X3 receptor cRNA (40 nL, 1 µg/mL) and incubated for 24 h at 18 °C
in Barth’s solution. The cRNAꢀinjected oocytes were kept for up to 12 days at 4 °C. Twoꢀelectrode
voltageꢀclamp technique (Axoclamp 2B amplifier) recorded ATPꢀactivated membrane currents (Vh = 70
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mV) using the cRNAꢀinjected oocytes. Voltage recording (1ꢀ2 Mꢀ tip resistance) and currentꢀrecording
microelectrodes (5 Mꢀ tip resistance) contained 3.0 M KCl. Oocytes were placed in an
electrophysiological chamber continuously superfused with Ringer’s solution (5 mL/min, at 18 °C)
containing 110 mM NaCl, 2.5 mM KCl, 5 mM HEPES and 1.8 mM BaCl2, adjusted to pH 7.5. To induce
an evoked current, 2 µM ATP was superfused for 60ꢀ120 s and then washed out for 20 min. For data
analysis, the ATPꢀevoked current was normalized at pH 7.5. The compounds were applied to the fixed
oocyte in an electrophysiological chamber for 20 min prior to the ATP exposure. The IC50 values (the
concentration of compounds that inhibit the ATP response by 50%) were determined by Hill plots
constructed using the formula log (I/Imax ꢀ I), where I is the ATPꢀevoked current in the presence of an
antagonist. The data are expressed as the mean ± SEM (n ≥ 3) from different batches of oocytes.
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Measurement of Ethidium Bromide Accumulation in hP2X7ꢀExpressing HEK293 Cells. HEK293
cells stably transfected with the human P2X7R were maintained in a humidified atmosphere of 5% CO2 at
37 °C in DMEM supplemented with 10% (v/v) FBS, 2 mM ꢀglutamine, and antibiotics (50 U/mL
L
penicillin and 50 ꢁg/mL streptomycin). BzATPꢀinduced pore formation was determined using a
fluorescence plate reader by measuring the cellular uptake of ethidium in hP2X7ꢀexpressing HEK293
cells. The hP2X7ꢀexpressing HEK293 cells were rinsed once in the appropriate assay buffer, which was
also removed before performing the subsequent assay additions. All studies were performed at room
temperature, and the final assay volume was 100 ꢁL. The cells were resuspended at 2.5 × 106 cells/mL in
an assay buffer consisting of 10 mM HEPES, 5 mM NꢀmethylꢀDꢀglucamine, 5.6 mM KCl,10 mM ꢀ
D
glucose, and 0.5 mM CaCl2 (pH 7.4) and supplemented with either 280 mM sucrose or 140 mM NaCl. To
determine the antagonistic activities of the 5ꢀhydroxylꢀpyridine derivatives and the reference compound
(AZD9056), these compounds were added to cells at 10 ꢁM with BzATP but without preincubation. After
incubation for 2 h in a humidified 5% CO2 atmosphere, ethidium dye uptake was observed by measuring
the fluorescence with a BioꢀTek Instrument FL600 fluorescence plate reader (excitation wavelength of
530 nm and emission wavelength of 590 nm). The results are expressed as percentages relative to the
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maximum accumulation of ethidium bromide when stimulated with BzATP only.
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Drug Distribution. Male SD rats (weighing 250–290 g) of 7ꢀ8 weeks of age were purchased from
SAMTACO (Osan, KyungkiꢀDo, Korea) and were given free access to a commercial rat chow diet
(SAMTACO, Osan, KyungkiꢀDo, Korea) and tap water. The animals were housed three per cage at a
temperature of 23 ± 2 °C and a relative humidity of 50ꢀ60%, under a 12:12 hr lightꢀdark cycle. The
experiments were started after acclimation under these conditions for at least 1 week. The rats were fasted
for at least 12 hr prior to the start of the experiments and had free access to tap water. A 5 mg/kg dose of
compound was administered by tail vein. At 0.5, 2, 4 and 8 hr after i.v. injection, each animal was
sacrificed by cervical dislocation. The blood samples were centrifuged at 12,000 rpm for 5 min and
immediately stored at ꢀ70 °C until analysis. Following collection of blood samples, the brains were
collected. Brain tissue were accurately weighed and homogenized (IKA T10 basic homogenizer; IKA,
Staufen, Germany) with a 4ꢀfold volume of distilled water. The homogenized brain samples were
centrifuged for 10 min at 12,000 rpm, and 500 ꢁl aliquots were stored at ꢀ20 °C until analysis.
Quantification of samples was performed using an LCꢀMS/MS system. The HPLC system was comprised
of an Agilent 1290 Series rapid resolution LC System, including a binary pump (Model G4220A), an
autosampler (Model G4226A) and a column oven (Model G1316C). The mass spectrometry analysis was
carried out with a Sciex 4000 QꢀtrapⓇ mass spectrometer equipped with an electrospray ionization source
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(Applied Biosystems Sciex, Toronto, Ontario, Canada). Data acquisition and quantification were
conducted using the Analyst software version 1.6 from AB SCIEX. The chromatographic separation was
achieved using a Gemini NX C18 (2.0 x 100 mm, 3 µm, Phenomenex, USA). The mobile phases
consisted of ACN (0.1% formic acid) and water (0.1% formic acid) (50:50, v/v). The flow rate was set at
0.25 mL/min, and the injection volume was set at 5 µL. The mass spectrometry was optimized and
performed using multiple reaction monitoring (MRM) scan in positive ion mode. The m/z transitions
were set as 392.1 → 360.2 for compound 28 and 378.2 → 360.2 for compound 29. Plasma and brain
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homogenate sample preparations involved protein precipitation with acetonitrile. In 1.5 mL polyethylene
microtubes, an aliquot 50 µL of plasma or tissue homogenate was spiked with 5 µL of IS (cimetidine, 10
µg/ml). Subsequently, 150 µL of acetonitrile (extraction solvent) was added to the tubes. After vortexing
for 30 sec, the mixture was centrifuged at 12,000 rpm for 5 min. Ten µL of the supernatant was directly
injected onto HPLC column.
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In Vivo Efficacy. The Institutional Animal Care and Use Committee, Medical Science of Chonnam
National University approved the study protocol. The antiallodynic effects (mechanical allodynia) were
assessed using rats following a previously reported protocol. Briefly, the antiallodynic effects of rats
(SNL and CIPN)33,34 were evaluated at 14 days after neuropathic pain development. The rats were placed
on a plastic mesh floor of the individual plastic cages to evaluate withdrawal thresholds. The rats were
acclimated to the environment for 30 min and tested after intrathecal35 or intraperitoneal or intravenous or
oral administration. The control groups (SNL and CIPN) received the same volume of the vehicle (saline
or DMSO for intrathecal administration, DMSO:PEG:PBS=1:6:3, v/v% for intraperitoneal or intravenous
administration or oral) as the experimental groups. Evaluations of mechanical withdrawal thresholds were
measured as responses to the application of calibrated von Frey filaments (Stoelting, Wood Dale, IL,
USA) to the hind paw through openings in the mesh floor underneath in the cage using the up and down
method.36 A series of eight von Frey filaments (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15 g) were applied
perpendicularly to the plantar underside of the ipsilateral hind paw for 5 s while the hair was bent. And
also, the withdrawal threshold of contralateral hind paw was measured in each experimental animal group.
The contralateral uninjured hind paws (the cutoff value of 15g) of each experimental animal group were
unaffected. Brisk withdrawal or paw flinching was regarded as a positive response. The absence of a
response in the rats at a pressure of 15 g von Frey filaments was used as the cutoff value. Only rats with
definite allodynia were tested (i.e., a mechanical withdrawal threshold: < 4 g after SNL or < 5 g after
CIPN). The rats were euthanized using sevoflurane overdose at the end of the experiment. The
withdrawal threshold data of the von Frey filament testing were converted to %maximal possible effect
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(%MPE) according to the formula %MPE = [(postdrug threshold ꢀ postꢀinjured baseline threshold)/(cutoff
threshold ꢀ postꢀinjured baseline threshold)] X 100. The doseꢀresponse data were analyzed using oneꢀway
analysis of variance (ANOVA) with Scheffe’s post hoc test.
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General Behavior. Additional rats received the highest doses of drugs used to evaluate behavioral
changes at 5, 10, 20, 30, 40, 50, and 60 min after intrathecal or intravenous tail vein administration. Motor
functions were examined using the righting and placingꢀstepping reflexes. The righting reflex was
evaluated by placing the rat horizontally with its back on the table, which normally elicits an immediate
coordinated twisting of the body to an upright position. The placingꢀstepping reflex was induced by
drawing the dorsum of either hind paw across the edge of the table. Rats normally attempt to put their
paws forward into a position for walking. Pinna and corneal reflexes were also assessed and judged as
present or absent. Other abnormal behaviors, such as serpentine movement or tremors, were also
evaluated.
■ ASSOCIATED CONTENT
Supporting information
Synthetic scheme for 5ꢀhydroxypyridine derivatives (Scheme S1), the general synthetic protocol for the
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compounds, H and MS (ESI) of synthetic intermediates and final products, C NMR, HRMS (ESI) and
HPLC analysis for target compounds, the ion current response of compound 29 at hP2X3 and rP2X2/3
receptors, antiallodynic effects of SNL rats by i.p. administration of 29 and experimental procedures for
inꢀvitro PK and hERG channel studies.
■ AUTHOR INFORMATION
Corresponding Authors
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*Y.C.K. Phone: 82ꢀ62ꢀ715ꢀ2502, Fax: 82ꢀ62ꢀ715ꢀ2484, Eꢀmail: yongchul@gist.ac.kr
*M.H.Y. Phone: 82ꢀ62ꢀ220ꢀ6893, Fax: 82ꢀ62ꢀ232ꢀ6294, Eꢀmail:mhyoon@chonnam.ac.kr
Authors Contributions
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Y.H.J. synthesized and analyzed the compounds with the help of H.K.. Y.H.J. wrote the manuscript.
Y.H.J., J.B., and K.M.K. performed the TEVC experiments and analyzed the data with the help of C.S.P..
J.H.P. and S.D.L. performed and analyzed the measurement of ethidium bromide accumulation. J.H.P.
performed the CYP inhibition assay. Y.O.K. and H.L. did the animal behavioral tests, and interpreted the
results. Y.G.K. carried out the drug distribution study, and analyzed the results. Y.H.J. analyzed the in
vitro PK data. A.N.P. was responsible for the hERG K+ channel conventional patchꢀclamp assay. J.H.C.
was involved in the research planning. Y.C.K. and M.H.Y. coordinated the project as supervisors and
were responsible for the study of design, analysis and interpretation of data, and manuscript reviewing.
All authors have given approval to the final manuscript.
Funding
This research was supported by the GIST Research Institute(GRI) in 2017 and by a grant of the Korea
Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI),
funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI14C1234) and by a
grant (CRI 14075ꢀ3) Chonnam National University Hospital Biomedical Research Institute.
Notes
The authors declare no competing financial interest.
■ ABBREVIATIONS
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ACN, acetonitrile; ANOVA, analysis of variance; ATP, adenosine 5'ꢀtriphosphate; BBB, blood brain
barrier; BzATP, 2’(3’)ꢀOꢀ(4ꢀbenzoylbenzoyl)ꢀATP; CIPN, chemotherapyꢀinduced peripheral neuropathy;
CYP, cytochrome P450; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; i.p., intraperitoneal
administration; i.v., intravenous administration; MPE, maximal possible effect; NeP, neuropathic pain;
PBS, phosphate buffered saline; PEG, polyethylene glycol; P2XR, P2X receptor; SAR, structureꢀactivity
relationship; SNL, spinal nerve ligation; TEVC, twoꢀelectrode voltage clamp; TM, transmembrane
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■ REFERENCES
(1) Khakh, B. S., and North, R. A. (2006) P2X receptors as cellꢀsurface ATP sensors in health and
disease. Nature 442, 527–532.
(2) Evans, R. J., Derkach, V., and Surprenant, A. (1992) ATP mediates fast synaptic transmission in
mammalian neurons. Nature 357, 503–505.
(3) Surprenant, A., and North, R. A. (2009) Signaling at Purinergic P2X Receptors. Annual Review of
Physiology 71, 333–359.
(4) Hattori, M., and Gouaux, E. (2012) Molecular mechanism of ATP binding and ion channel activation
in P2X receptors. Nature 485, 207–212.
(5) North, R. A. (2002) Molecular physiology of P2X receptors. Physiological reviews 82, 1013–67.
(6) KaczmarekꢀHájek, K., Lörinczi, E., Hausmann, R., and Nicke, A. (2012) Molecular and functional
properties of P2X receptorsꢀꢀrecent progress and persisting challenges. Purinergic signalling 8, 375–417.
(7) Dal Ben, D., Buccioni, M., Lambertucci, C., Marucci, G., Thomas, A., and Volpini, R. (2015)
Purinergic P2X receptors: Structural models and analysis of ligandꢀtarget interaction. European Journal
of Medicinal Chemistry 89, 561–580.
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(8) Dunn, P. M., Zhong, Y., and Burnstock, G. (2001) P2X receptors in peripheral neurons. Progress in
Neurobiology 65, 107–134.
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(9) Bradbury, E. J., Burnstock, G., and McMahon, S. B. (1998) The expression of P2X3 purinoreceptors
in sensory neurons: effects of axotomy and glialꢀderived neurotrophic factor. Molecular and cellular
neurosciences 12, 256–268.
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(10) Chen, M., and Gu, J. G. (2005) A P2X receptorꢀmediated nociceptive afferent pathway to lamina I of
the spinal cord. Molecular pain 1, 4.
(11) Tsuda, M., Koizumi, S., Kita, A, Shigemoto, Y., Ueno, S., and Inoue, K. (2000) Mechanical
allodynia caused by intraplantar injection of P2X receptor agonist in rats: involvement of heteromeric
P2X2/3 receptor signaling in capsaicinꢀinsensitive primary afferent neurons. The Journal of
neuroscienceꢁ: the official journal of the Society for Neuroscience 20, RC90.
(12) Kennedy, C. (2005) P2X Receptors: Targets for Novel Analgesics? The Neuroscientist 11, 345–356.
(13) Honore, P., Kage, K., Mikusa, J., Watt, A. T., Johnston, J. F., Wyatt, J. R., Faltynek, C. R., Jarvis, M.
F., and Lynch, K. (2002) Analgesic profile of intrathecal P2X3 antisense oligonucleotide treatment in
chronic inflammatory and neuropathic pain states in rats. Pain 99, 11–19.
(14) Dorn, G., Patel, S., Wotherspoon, G., HemmingsꢀMieszczak, M., Barclay, J., Natt, F. J. C., Martin,
P., Bevan, S., Fox, A., Ganju, P., Wishart, W., and Hall, J. (2004) siRNA relieves chronic neuropathic
pain. Nucleic acids research 32, e49.
(15) Barclay, J., Patel, S., Dorn, G., Wotherspoon, G., Moffatt, S., Eunson, L., Abdel’al, S., Natt, F., Hall,
J., Winter, J., Bevan, S., Wishart, W., Fox, A., and Ganju, P. (2002) Functional downregulation of P2X3
receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory
pain. The Journal of neuroscienceꢁ: the official journal of the Society for Neuroscience 22, 8139–8147.
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(16) Honore, P., Mikusa, J., Bianchi, B., McDonald, H., Cartmell, J., Faltynek, C., and Jarvis, M. F.
(2002) TNPꢀATP, a potent P2X3 receptor antagonist, blocks acetic acidꢀinduced abdominal constriction
in mice: Comparison with reference analgesics. Pain 96, 99–105.
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(17) Jarvis, M. F., Burgard, E. C., McGaraughty, S., Honore, P., Lynch, K., Brennan, T. J., Subieta, A.,
Van Biesen, T., Cartmell, J., Bianchi, B., Niforatos, W., Kage, K., Yu, H., Mikusa, J., Wismer, C. T.,
Zhu, C. Z., Chu, K., Lee, C. H., Stewart, A. O., Polakowski, J., Cox, B. F., Kowaluk, E., Williams, M.,
Sullivan, J., and Faltynek, C. (2002) Aꢀ317491, a novel potent and selective nonꢀnucleotide antagonist of
P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proceedings
of the National Academy of Sciences of the United States of America 99, 17179–17184.
(18) McGaraughty, S., Wismer, C. T., Zhu, C. Z., Mikusa, J., Honore, P., Chu, K. L., Lee, C. H.,
Faltynek, C. R., and Jarvis, M. F. (2003) Effects of Aꢀ317491, a novel and selective P2X3/P2X2/3 receptor
antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and
intraplantar administration. British Journal of Pharmacology 140, 1381–1388.
(19) Gever, J. R., Soto, R., Henningsen, R. A., Martin, R. S., Hackos, D. H., Panicker, S., Rubas, W.,
Oglesby, I. B., Dillon, M. P., Milla, M. E., Burnstock, G., and Ford, A. P. D. W. (2010) AFꢀ353, a novel,
potent and orally bioavailable P2X3/P2X2/3 receptor antagonist. British Journal of Pharmacology 160,
1387–1398.
(20) Carter, D. S., Alam, M., Cai, H., Dillon, M. P., Ford, A. P. D. W., Gever, J. R., Jahangir, A., Lin, C.,
Moore, A. G., Wagner, P. J., and Zhai, Y. (2009) Identification and SAR of novel diaminopyrimidines.
Part 1: The discovery of ROꢀ4, a dual P2X3/P2X2/3 antagonist for the treatment of pain. Bioorganic and
Medicinal Chemistry Letters 19, 1628–1631.
(21) Jahangir, A., Alam, M., Carter, D. S., Dillon, M. P., Bois, D. J. Du, Ford, A. P. D. W., Gever, J. R.,
Lin, C., Wagner, P. J., Zhai, Y., and Zira, J. (2009) Identification and SAR of novel diaminopyrimidines.
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Part 2: The discovery of ROꢀ51, a potent and selective, dual P2X3/P2X2/3 antagonist for the treatment of
5
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pain. Bioorganic and Medicinal Chemistry Letters 19, 1632–1635.
7
8
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(22) BrothertonꢀPleiss, 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., and Zhai, Y. (2010) Discovery and optimization of
ROꢀ85, a novel drugꢀlike, potent, and selective P2X3 receptor antagonist. Bioorganic and Medicinal
Chemistry Letters 20, 1031–1036.
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(23) Cantin, L. D., Bayrakdarian, M., Buon, C., Grazzini, E., Hu, Y. J., Labrecque, J., Leung, C., Luo, X.,
Martino, G., Paré, M., Payza, K., Popovic, N., Projean, D., Santhakumar, V., Walpole, C., Yu, X. H., and
Tomaszewski, M. J. (2012) Discovery of P2X3 selective antagonists for the treatment of chronic pain.
Bioorganic & medicinal chemistry letters 22, 2565–71.
(24) Ford, A. P. (2012) In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent
sensitization. Purinergic Signalling 8, 3–26.
(25) Abdulqawi, R., Dockry, R., Holt, K., Layton, G., McCarthy, B. G., Ford, A. P., and Smith, J. A.
(2015) P2X3 receptor antagonist (AFꢀ219) in refractory chronic cough: a randomised, doubleꢀblind,
placeboꢀcontrolled phase 2 study. Lancet 385, 1198–205.
(26) Jung, K. Y., Moon, H. D., Lee, G. E., Lim, H. H., Park, C. S., and Kim, Y. C. (2007) Structure ꢀ
activity relationship studies of spinorphin as a potent and selective human P2X3 receptor antagonist. J.
Med. Chem. 50, 4543–4547.
(27) Cho, J. H., Jung, K. Y., Jung, Y., Kim, M. H., Ko, H., Park, C. S., and Kim, Y. C. (2013) Design and
synthesis of potent and selective P2X3 receptor antagonists derived from PPADS as potential pain
modulators. European Journal of Medicinal Chemistry 70, 811–830.
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(28) Jung, K. Y., Cho, J. H., Lee, J. S., Kim, H. J., and Kim, Y. C. (2013) Synthesis and structure–activity
relationships of carboxylic acid derivatives of pyridoxal as P2X receptor antagonists. Bioorganic &
Medicinal Chemistry 21, 2643–2650.
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(29) Bondock, S. (2005) Oneꢀpot synthesis of pyridine derivatives via dielsꢀalder reactions of 2,4ꢀ
dimethylꢀ5ꢀmethoxyoxazole. Heteroatom Chemistry 16, 49–55.
(30) Sandford, G., Wilson, I., and Timperley, C. M. (2004) DielsꢀAlder reactions of trifluoromethyl
alkenes with 5ꢀethoxyoxazoles: synthesis of trifluoromethylated pyridine derivatives. Journal of Fluorine
Chemistry 125, 1425–1430.
(31) Braun, K., Rettinger, J., Ganso, M., Kassack, M., Hildebrandt, C., Ullmann, H., Nickel P.,
Schmalzing G., and Lambrecht G. (2001) NF449ꢂ: a subnanomolar potency antagonist at recombinant rat
P2X1 receptors. NaunynꢀSchmiedeberg's Archives of Pharmacology 364, 285–290.
(32) Alcaraz, L., Caffrey, M., Furber, M., Luker, T., Mortimore, M., Pimm, A., Thorne, P., and Willis, P.
(2000) Adamanatane derivatives. (Astrazeneca CO., UAS). PCT Patent PCT/SE/2000/002505.
(33) Lin, H., Heo, B. H., and Yoon, M. H. (2015) A new rat model of cisplatinꢀinduced neuropathic pain.
Korean Journal of Pain 28, 236–243.
(34) Kim, S. H., and Chung, J. M. (1992) An experimental model for peripheral neuropathy produced by
segmental spinal nerve ligation in the rat. Pain 50, 355–363.
(35) Yaksh, T. L., and Rudy, T. A. (1976) Chronic catheterization of the spinal subarachnoid space.
Physiology and Behavior 17, 1031–1036.
(36) Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., and Yaksh, T. L. (1994) Quantitative
assessment of tactile allodynia in the rat paw. J. Neurosci. methods 53, 55–63.
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(37) Wang, X., Wakefield, B. D., Liu, B., Brown, B., Li, T., Kym, P., Scott, V., Fan, Y., Vortherm, T.,
Niforatos, W., Mills, C., Joshi, S., Schrimpf, M. R., Lee, C. H., and Brioni, J. (2015) Identification of
pyridazinone analogs as selective P2X3 antagonists for pain management. (AbbVie Inc., USA) 250th
American Chemical Society, Medi 14.
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(38) Park, J. H., Lee, G. E., Lee, S. D., Hien, T. T., Kim, S., Yang, J. W., Cho, J. H., Ko, H., Lim, S. C.,
Kim, Y. G., Kang, K. W., and Kim, Y. C. (2015) Discovery of Novel 2,5ꢀDioxoimidazolidineꢀBased
P2X7 Receptor Antagonists as Constrained Analogues of KN62. Journal of Medicinal Chemistry 58,
2114ꢀ2134.
(39) Salameh, F., Karaman, D., Mecca, G., Scrano, L., Bufo, S. A., and Karaman, R. (2015) Prodrugs
targeting the central nervous system (CNS). World Journal of Pharmacy and Pharmaceutical Sciences 4,
208–237.
(40) Christoph, T., Vry, J. De, Schiene, K., Tallarida, R. J., and Tzschentke, T. M. (2011) Synergistic
antihypersensitive effects of pregabalin and tapentadol in a rat model of neuropathic pain. European
Journal of Pharmacology 666, 72–79.
(41) Wrzosek, A., Obara, I., Wordliczek, J., and Przewlocka, B. (2009) Efficacy of tramadol in
combination with doxepin or venlafaxine in inhibition of nociceptive process in the rat model of
neuropathic pain: an isobolographic analysis. Journal of physiology and pharmacology 60, 71–78.
(42) Shen, D. D., Artru, A. A., and Adkison, K. K. (2004) Principles and applicability of CSF sampling
for the assessment of CNS drug delivery and pharmacodynamics. Advanced Drug Delivery Reviews 56,
1825–1857.
(43) Davidson, S., Copits, B. A., Zhang, J., Page, G., Ghetti, A., and Gereau, R.W. (2014) Human sensory
neurons: Membrane properties and sensitization by inflammatory mediators. Pain 155, 1861–1870.
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(44) Gum, R. J., Wakefield, B., and Jarvis, M. F. (2012) P2X receptor antagonists for pain managementꢂ:
5
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examination of binding and physicochemical properties. Purinergic Signalling 8 (Suppl 1), S41–S56.
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(45) Kerns, E. H., Di, L. I., Petusky, S., Farris, M., Ley, R. O. B., and Jupp, P. (2004) Combined
Application of Parallel Artificial Membrane Permeability Assay and Cacoꢀ2 Permeability Assays in Drug
Discovery. Journal of Pharmaceutical Sciences 93, 1440–1453.
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Table 1. Antagonistic effects of 3,4ꢀdicarboxypyridine derivatives with modifications of R position at
mP2X1, hP2X3 and hP2X7 receptors.
Compounds
1g
R
mP2X1a
ꢀ
hP2X3a
hP2X7e
ꢀ
35 ± 4 nMf
ꢀ
NFꢀ449h
AZD9056i
13a
307 ± 93 pM
ꢀ
ꢀ
ꢀ
0.23 ± 0.11 nM
Inactive
Inactive
inactive
inactive
inactive
inactive
inactive
Inactive
inactive
Inactive
inactive
236 ± 78 nMd
446 ± 32 nMf
48.1 ± 8.8%c
42.8 ± 7.4%c
41.9 ± 9.1%c
37.3 ± 8.2%c
23.4 ± 0.8%c
Inactivec
561 ± 99 nMd
460 ± 69 nMf
550 ± 93 nMd
93 ± 36 nMd
95 ± 45 nMd
466 ± 99 nMd
32.3 ± 1.6%b
751 ± 83 nMd
742 ± 73 nMd
319 ± 81 nMd
287 ± 87 nMd
ꢀH
ꢀCH3
13b
ꢀCH2CH3
13c
ꢀCH2CH2CH3
ꢀCH2CH2CH2CH3
ꢀCH(CH3)2
ꢀCH2CH(CH3)2
ꢀCH2C6H5
13d
13e
13f
13g
13h
Inactivec
ꢀCH2CH2C6H5
ꢀCOOH
13i
13j
36.7 ± 2.8%c
Inactivec
ꢀCH2CH2SCH3
13k
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13l
Inactivec
721 ± 96 nMd
ꢀCH2CH2ꢀS(=O)2ꢀCH3
Inactive
aThe ion current responses evoked by 2 µM ATP at the recombinant P2X receptors expressed in Xenopus
oocytes, and the % inhibition effects of compounds at 1 µMb, 10 µMc or the IC50 valuesd were measured
5
6
7
8
e
for mP2X1 and hP2X3 receptors (mean ± SEM, n ≥3). The ethidium bromide accumulation was
measured in hP2X7ꢀexpressing HEK293 cells, and the % inhibition of the accumulation by 10 µM of the
compounds was evaluated (mean ± SEM, n ≥3). f We reported the IC50 values in a previous study.27 g1 (Aꢀ
317491, IC50 value = 97 nM against the hP2X3 receptor) was reported.18 hNFꢀ449 (IC50 value = 280 pM
against the rP2X1 receptor) was reported.31 iAZD9056 (IC50 value = 13 nM against the hP2X1 receptor)
was reported.38
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34
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36
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Table 2. Antagonistic effects of 5ꢀhydroxy pyridine derivatives with various modifications of pyridine at
mP2X1, hP2X3 and hP2X7 receptors.
hP2X7e
Inactive
Compounds
R1
R2
R3
mP2X1a
Inactivec
hP2X3a
33.3 ± 3.1%b
ꢀCH3
ꢀCN
ꢀCOOH
16
Inactivec
25.3 ± 2.8%c
Inactivec
27.3 ± 1.8%b
27.8 ± 1.3%b
16.3 ± 3.2%b
20.4 ± 3.7%b
681 ± 98 nMd
384 ± 99 nMd
303 ± 66 nMd
709 ± 60 nMd
ꢀCH3
ꢀCOOH
inactive
inactive
inactive
inactive
Inactive
inactive
Inactive
inactive
17
18
19
inactivec
ꢀCH3
ꢀCH3
ꢀS(=O)2ꢀCH3
ꢀCOOH
ꢀH
20
Inactivec
ꢀH
22a
22b
22c
23
Inactivec
ꢀCH3
ꢀCOOH
ꢀCH3
ꢀCH3
ꢀCH3
22.7 ± 1.9%c
Inactivec
ꢀCH2CH2CH3
ꢀCH2CH2CH3
ꢀCOOH
ꢀCONH2
aThe ion current responses evoked by 2 µM ATP at the recombinant P2X receptors expressed in Xenopus
oocytes, and the % inhibition effects of compounds at 1 µMb, 10 µMc or the IC50 valuesd were measured
e
for mP2X1 and hP2X3 receptors (mean ± SEM, n ≥3). The ethidium bromide accumulation was
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measured in hP2X7ꢀexpressing HEK293 cells, and the % inhibition of the accumulation by 10 µM of the
compounds was evaluated (mean ± SEM, n ≥3).
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8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
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31
32
33
34
35
36
37
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Table 3. Antagonistic effects of 5ꢀhydroxy pyridine derivatives with bulky mꢀphenoxyphenyl group at 2ꢀ
position of pyridine for mP2X1, hP2X3 and hP2X7 receptors.
Compounds
R
mP2X1a
27.5 ± 4.5%c
23.3 ± 4.8%c
Inactivec
hP2X3a
hP2X7e
Inactive
Inactive
inactive
inactive
inactive
inactive
inactive
301 ± 65 nMd
245 ± 29 nMd
37.0 ± 1.0%b
29.7 ± 3.1%b
32.4 ± 0.8%b
34.4 ± 1.2%b
42.4 ± 2.1%b
27
29
35
37
38
41
43
ꢀCOOH
ꢀCH=CHCOOH
ꢀCH2CH2COOH
ꢀCN
inactivec
inactivec
inactivec
ꢀCH2NH2
43.1 ± 12.9%c
ꢀCH2NHꢀS(=O)2ꢀCH3
aThe ion current responses evoked by 2 µM ATP at the recombinant P2X receptors expressed in Xenopus
oocytes, and the % inhibition effects of compounds at 1 µMb, 10 µMc or the IC50 valuesd were measured
e
for mP2X1 and hP2X3 receptors (mean ± SEM, n ≥3). The ethidium bromide accumulation was
measured in hP2X7ꢀexpressing HEK293 cells, and the % inhibition of the accumulation by 10 µM of the
compounds was evaluated (mean ± SEM, n ≥3).
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Table 4. Inꢀvitro PK and hERG channel studies
Property
Metabolic stability
Stabilityꢀmicrosomes
human
Units
Compound 28
Compound 29
% remaining
after 30 min
0
3
0
2
rat
33
35
mouse
Plasma protein binding
rate (%)
Plasma protein binding
Human
99.5 ± 0.82
32.5 ± 8.36
ꢁ4.80
99.9 ± 0.06
99.8 ± 0.13
ꢁ7.04
Rat
log Pe
PermeabilityꢀPAMPA
CYP450 inhibition
3A4 isozyme
2C9 isozyme
1A2 isozyme
hERG
% inhibition at 1µM
12 ± 2
28 ± 3
10 ± 1
26 ± 2
36 ± 3
> 100
4 ± 1
Not determined
IC50 (µM)
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Table 5. Concentration (mean ± SD) and ratio (brain to plasma, mean ± SD) of compound 29 after
intravenous administration of compound 29 (5 mg/kg) in rats.
Compound 29
Plasma(µg/mL)
Brain (µg/g)
0.5 hr
2 hr
4 hr
8 hr
0.29 ± 0.04
0.005 ± 0.002
0.02 ± 0.01
0.04 ± 0.02
0.001 ± 0
0.02 ± 0
0.02 ± 0.01
0.0001 ± 0
0.004 ± 0
0.004 ± 0
0.0000 ± 0
0.007 ± 0
Brain to plasma
ratio
Table 6. Concentration (mean ± SD) and ratio (brain to plasma, mean ± SD) of compound 28 (prodrug)
and 29 (active) after intravenous administration of compound 28 (5 mg/kg) in rats.
Compound 28
Plasma (µg/mL)
Brain (µg/g)
0.5 hr
2 hr
4 hr
8 hr
0.62 ± 0.14
1.11 ± 0.36
1.79 ± 0.44
0.5 hr
0.08 ± 0.02
0.09 ± 0.01
1.13 ± 0.25
2 hr
0.06 ± 0.01
0.05 ± 0.01
0.94 ± 0.21
4 hr
0.02 ± 0.01
0.03 ± 0.01
1.19 ± 0.41
8 hr
Brain to plasma
ratio
Compound 29
Plasma(µg/mL)
0.11 ± 0.05
0.03 ± 0
0.02 ± 0.01
0.01 ± 0
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