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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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ACS Chemical Neuroscience

Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors Young-Hwan Jung,† 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,† Myung Ha Yoon, *,‡,§ and Yong-Chul Kim*,†, ║ †

School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712,

Republic of Korea ‡

Department of Anesthesiology and Pain Medicine, §Center for Creative Biomedical Scientists at

Chonnam National University, Medical School, Gwangju 501-757, Republic of Korea ║

Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology

(GIST), Gwangju 500-712, Republic of Korea ┴ #

College of Pharmacy, Dankook University, Cheonan 330-714, 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 130-650, Republic of Korea ¶

New Drug Development Center (NDDC), Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF),

80 Cheombok-ro, Dong-gu, Daegu 41061, Republic of Korea

ACS Paragon Plus Environment

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ABSTRACT: 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 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 chemotherapyinduced 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, structure-activity relationship


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INTRODUCTION

P

2X receptors are ligand-gated 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

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 X-ray 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 cysteine-rich extracellular loop containing ATP binding sites, and intracellular N- and C-termini. 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 Among the P2X receptor subtypes, the P2X3 receptor (P2X3R) exists as homomeric P2X3 and heteromeric P2X2/3 receptors in small to medium diameter C- and Aδ-fiber primary afferent neurons, suggesting a high degree of specificity to the pain-sensing system.8,9 In fact, a number of studies, including knock-out animal studies, have shown that P2X3R is closely related to pain sensation. For example, the administration of ATP or a P2X3R-selective agonist, αβ-methylene ATP, to the hind paws of rats caused nociceptive behaviors.10–12 In neuropathic or inflammatory pain models, P2X3R knock-out or deficient animal studies with P2X3R-selective antisense or short interfering RNA (siRNA) resulted in greatly diminished pain-related behaviors.13–15 The crucial role of P2X3R in pain sensation was further investigated using P2X3 receptor antagonists, TNP-ATP (2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′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 non-nucleotide dual P2X3-P2X2/3 antagonist A-317491 (1),17,18 which showed pain-reducing effects in chronic and inflammatory pain animal models. Roche reported diaminopyrimidine-based analogues, RO-4 (2a),19,20 RO-51 (2b)21 and RO-85 (3),22 as the first allosteric antagonists with high potency and selectivity for P2X3-P2X2/3Rs. AstraZeneca reported structure-activity relationship (SAR) studies of pyrrolo-pyrimidinone derivatives (4)23 for improving the potency and physicochemical properties of P2X3 receptor antagonists. Afferent Pharmaceutical’s clinical drug candidate, AF-219,24,25 which is a potent and orally bioavailable selective P2X3-P2X2/3 receptor antagonist, has been studied in several proof-of-concept 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 non-competitive peptide antagonists, Spinorphin (5, IC50 = 8.3 pM)26 and 5-hydroxy pyridine derivatives (7a-c),27,28 which were developed from the modification of the strong anionic phosphate sulfonic acid groups and an unstable azo (-N=N-) linkage of a non-selective P2X antagonist, PPADS (6a), to a weak anionic carboxylic acid and a stable carbon-carbon bond, respectively. In this article, we report further structure-activity relationship (SAR) investigations aimed at the optimization of drug properties of the 5-hydroxy pyridine derivatives (7a-c). First, substituents at the 6position of the 5-hydroxy pyridine skeleton was modified to optimize antagonistic potency. Second, various substituents at the 4-position 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 The syntheses of the 3,4-dicarboxypyridine derivatives, 13a-l, are depicted in Scheme 1. To explore the SAR of the 6-position of the 3,4-dicarboxypyridine derivatives, we replaced the methyl group of the 6position with various alkyl and arylalkyl groups. The synthetic procedures of the previous report27 were employed, starting from various natural and unnatural amino acids. Briefly, p-methoxy-phenyl propionic acid, 8, was conjugated with esters of the amino acids to yield 10a-k, which were subsequently cyclized to oxazole analogs, 11a-k, by treatment with phosphorous pentoxide. The dimethyl pyridine-3,4dicarboxylate derivatives, 12a-k, were synthesized through Diels-Alder reactions of 11a-k in the presence of dimethyl maleate. Through a subsequent oxidation reaction of the sulfide group of 12k using Oxone®, 12l was obtained. Finally, the 3,4-dicarboxypyridine derivatives, 13a-l, were synthesized by the hydrolysis of 12a-l using 20% aqueous KOH (Scheme 1). For the synthesis of compounds 16-20, which have diverse functionalities at the 3,4-positions, DielsAlder reactions of oxazole-based diene (11b) were conducted with dienophiles, ethyl cis-betacyanoacrylate, maleic anhydride and methyl vinyl sulfone to produce compounds 14, 18 and 20, respectively. In the case of compound 14, only one regio-isomer was obtained, which should be a 4cyano-3-carboxylate substituted isomer, as it has been known29,30 that a higher electron-withdrawing group, such as the nitrile group of ethyl cis-beta-cyanoacrylate, is oriented at the 4-position of pyridine during the reaction. In addition, the nitrile group of 14 was further transformed to a tetrazole moiety (15), a bio-isostere of carboxylic acid, using sodium azide. The final compounds, 16 and 17, were obtained by the base-catalyzed 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 4position and the -CH3 or –H groups at the 3-position of the pyridine ring was accomplished following Scheme 3. Compounds 21a-21c were obtained from the same Diels-Alder reaction using methyl acrylate


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and methyl crotonate as dienophiles. Subsequently, 21a-21c were hydrolyzed using 20% aqueous KOH to yield 22a-22c, and 21c was further converted to the carboxamide analog 23 using 30% aqueous ammonia. The 2-phenoxybenzyl 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. Diels-Alder reactions of oxazole-based 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 5-OH group with a benzyl group, LiAlH4 reduction of the 4-methylester group to a 4-hydroxymethyl group (compound 31), MnO2 oxidation to 4-carboxaldehyde (compound 33) after a deprotection reaction of the 5-benzyl ether moiety with Pd/C under H2 gas, and a Wittig-Honor reaction with triethyl phosphonoacetate to yield trans region-isomer compound 34 (J value = 16.4 Hz in 1H NMR). The methyl propionate analog, 36, was prepared from the hydrogenation reaction of the α,β-unsaturated 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), 4-carboxylic acid (29), 4-propenoic acid (35) and 4-propionic 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 4-position of pyridine. Methyl crotononitrile and 25 were used as the starting materials for the Diels-Alder reaction to give 38. After the protection of the 5-OH 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 5-benzyl ether moiety using Pd/C under an H2 gas condition.


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■ RESULTS AND DISCUSSION Structure-Activity Relationships for In Vitro P2XR Antagonism. To explore the structure-activity relationship (SAR) of the synthesized compounds, the antagonistic activities were evaluated by twoelectrode 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 NF-44931 and AZD905632 were tested together as positive control antagonists for hP2X3, mP2X1 and hP2X7Rs, respectively. 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 4aldehyde group. Therefore, we have tried to optimize the combinations of moieties at the 3, 4 and 6positions of the 5-hydroxy pyridine skeleton, including modifications of the 4-aldehyde group. As shown in Table 1, the P2XR antagonistic effects of substituents at the 6-position of 3,4dicarboxypyridine derivatives were initially investigated with various alkyl, aromatic and electronegative atom-containing 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 4-5-fold compared with compounds 13a-c. 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 6-position. Thus, an approximately 100-fold selectivity (13d and 13e) for hP2X3R vs mP2X1R was achieved from non-selective 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 6position, the activity at the hP2X3R of 13f (IC50 = 466 nM) was similar to that of 13b (6-methyl 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 non-polar phenyl groups with different carbon chain at the 6-position, displayed weaker antagonistic effects against hP2X3R, with IC50 values in the 700 nM range. Derivatives with different electrostatic potential at the 6-position, 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 6-substituents for the antagonistic effect. Next, we investigated the positional preference and bio-isostere 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 18-23 (Table 2). Initially, we introduced nitrile (16) and methanesulfonyl (20) groups as hydrogen bond acceptors and tetrazole (17) as a bioisostere of the 4-carboxylic 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 4-position may be essential for binding at the receptors. Therefore, modifications of the carboxylic acid at the 3-position 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 monocarboxylic acid analog at the 4-position of the pyridine group, showed a nearly 2-fold decrease in antagonistic activity against hP2X3R compared with the corresponding 3,4-dicarboxylic acid analog, 13b (IC50 = 460 nM), the 3-methylpyridine-4-carboxylic acid derivative, 22b (IC50 = 384 nM), maintained hP2X3R antagonistic activity. The attempt to increase the potency against hP2X3R by the replacement of the 6-methyl group of 22b by a 6-n-propyl group (22c, IC50 = 303 nM) resulted in only a slight increase in


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the antagonistic activity. In the case of a 4-carboxamide derivative, 23 (IC50 = 709 nM), with less acidic but similar H-bond donor properties, a 2-fold decrease in antagonistic activity at hP2X3R was observed. These results suggest that possible ionic interactions with pharmacophores at the 4-position may be more important than the H-bonding interaction for the antagonistic activity at hP2X3R. Next, we introduced an m-phenoxyphenyl group at the 2-position of the pyridine moiety (Table 3), which was previously reported as an alternative pharmacophore to a p-methoxyphenethyl group with improved selectivity profiles.28 However, a 3,4-dicarboxy-2-m-phenoxyphenyl derivative, compound 27 (IC50 = 301 nM), enhanced neither the hP2X3R antagonistic effect nor the selectivity vs mP2X1R compared with the corresponding 2-p-methoxyphenethyl analog, 13d. Additionally, we introduced –CH3 (29) instead of 3-carboxylic acid of 27 to decrease polarity. Unlike the case of compound 22c and 13d, the 4-carboxy-3-methyl-2-m-phenoxyphenyl 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 4-position of 29 was performed, including carbon chain-elongated carboxylic acids such as 4-propenoic acid (35) or 4propionic acid (37), 4-nitrile (38), 4-methylamine (41) and 4-methanesulfonamide (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 4-position of pyridine ring may be composed of amino acids with the property of ionic interaction with a narrow binding pocket. The 5-hydroxy 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 0-43% 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.

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 drug-drug 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 permeabilityPAMPA45 (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 permeability-PAMPA (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.

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 placingstepping 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.

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 anti-nociceptive 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 in-vivo 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 brain-to-plasma ratios of 29 (0.24, Table 6) by i.v. administered compound 28 was 12-fold 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 chemotherapy-induced 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 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 pro-drug itself would be effective or not, and to verify any possible off-target activities of it. As the results, i.t. administered compound 28 did not show any antiallodynic effects (Figure 5), indicating that the in-vivo 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 in-vivo pharmacological effect (Figure 5). Regarding the PK-PD 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. The results of in-vivo animal experiments and the species differences of in-vitro 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 pro-moieties 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, ex-vivo 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.

■ 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 5hydroxypyridine skeleton with 6-n-propyl, 4-carboxylic acid and 3-methyl substituents resulted in the discovery of compound 29, which showed significantly reduced allodynia in SNL by intrathecal administration. Furthermore, the 4-methyl 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 hydroxypyridine-based P2X3R antagonists.

■ METHOD Two-Electrode Voltage-Clamp Assay on Recombinant mP2X1 and hP2X3 Receptors. Xenopus oocytes were isolated and incubated with 270 U/mg collagenase for 1.5 h at 23-24 °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 cRNA-injected oocytes were kept for up to 12 days at 4 °C. Two-electrode voltage-clamp technique (Axoclamp 2B amplifier) recorded ATP-activated membrane currents (Vh = 70


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mV) using the cRNA-injected oocytes. Voltage recording (1-2 MΩ tip resistance) and current-recording microelectrodes (5 MΩ 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 60-120 s and then washed out for 20 min. For data analysis, the ATP-evoked 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 ATP-evoked current in the presence of an antagonist. The data are expressed as the mean ± SEM (n ≥ 3) from different batches of oocytes.

Measurement of Ethidium Bromide Accumulation in hP2X7-Expressing 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 L-glutamine, and antibiotics (50 U/mL penicillin and 50 µg/mL streptomycin). BzATP-induced pore formation was determined using a fluorescence plate reader by measuring the cellular uptake of ethidium in hP2X7-expressing HEK293 cells. The hP2X7-expressing 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 µL. The cells were resuspended at 2.5 × 106 cells/mL in an assay buffer consisting of 10 mM HEPES, 5 mM N-methyl-D-glucamine, 5.6 mM KCl,10 mM Dglucose, 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 5-hydroxyl-pyridine derivatives and the reference compound (AZD9056), these compounds were added to cells at 10 µM 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 Bio-Tek 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.

Drug Distribution. Male SD rats (weighing 250–290 g) of 7-8 weeks of age were purchased from SAMTACO (Osan, Kyungki-Do, Korea) and were given free access to a commercial rat chow diet (SAMTACO, Osan, Kyungki-Do, Korea) and tap water. The animals were housed three per cage at a temperature of 23 ± 2 °C and a relative humidity of 50-60%, under a 12:12 hr light-dark 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 4-fold volume of distilled water. The homogenized brain samples were centrifuged for 10 min at 12,000 rpm, and 500 µl aliquots were stored at -20 °C until analysis. Quantification of samples was performed using an LC-MS/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 Q-trapⓇ mass spectrometer equipped with an electrospray ionization source (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.

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 - post-injured baseline threshold)/(cutoff threshold - post-injured baseline threshold)] X 100. The dose-response data were analyzed using one-way analysis of variance (ANOVA) with Scheffe’s post hoc test.

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 placing-stepping 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 placing-stepping 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 5-hydroxypyridine derivatives (Scheme S1), the general synthetic protocol for the compounds, 1H and MS (ESI) of synthetic intermediates and ﬁnal products, 13C 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 in-vitro PK and hERG channel studies.

■ AUTHOR INFORMATION Corresponding Authors


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*

Y.C.K. Phone: 82-62-715-2502, Fax: 82-62-715-2484, E-mail: [email protected]

*

M.H.Y. Phone: 82-62-220-6893, Fax: 82-62-232-6294, E-mail:[email protected]

Authors Contributions 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 patch-clamp 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 14075-3) 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’)-O-(4-benzoylbenzoyl)-ATP; CIPN, chemotherapy-induced 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, structure-activity relationship; SNL, spinal nerve ligation; TEVC, two-electrode voltage clamp; TM, transmembrane

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Table 1. Antagonistic effects of 3,4-dicarboxypyridine derivatives with modifications of R position at mP2X1, hP2X3 and hP2X7 receptors.

Compounds

mP2X1

R

a

hP2X3

a

hP2X7 f

e

1g

-

35 ± 4 nM

-

NF-449h

307 ± 93 pM

-

-

AZD9056i

-

-

0.23 ± 0.11 nM

d

561 ± 99 nM

f

460 ± 69 nM

c

550 ± 93 nM

c

93 ± 36 nM

d

inactive

c

95 ± 45 nM

d

inactive

c

466 ± 99 nM

c

32.3 ± 1.6%

13a

-H

236 ± 78 nM

13b

-CH3

446 ± 32 nM

13c

-CH2CH3

48.1 ± 8.8%

13d

-CH2CH2CH3

42.8 ± 7.4%

13e

-CH2CH2CH2CH3

41.9 ± 9.1%

13f

-CH(CH3)2

37.3 ± 8.2%

13g

-CH2CH(CH3)2

23.4 ± 0.8%

13h

-CH2C6H5

Inactive

13i

-CH2CH2C6H5

Inactive

13j

-COOH

36.7 ± 2.8%

13k

-CH2CH2SCH3

Inactive

d

Inactive

f

Inactive

d

inactive

d

inactive

b

inactive

c

751 ± 83 nM

d

Inactive

c

742 ± 73 nM

d

inactive

319 ± 81 nM

d

Inactive

287 ± 87 nM

d

inactive

c

c


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

13l

-CH2CH2-S(=O)2-CH3

c

Inactive

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721 ± 96 nM

d

Inactive

The 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 for mP2X1 and hP2X3 receptors (mean ± SEM, n ≥3). eThe ethidium bromide accumulation was measured in hP2X7-expressing 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 (A317491, IC50 value = 97 nM against the hP2X3 receptor) was reported.18 hNF-449 (IC50 value = 280 pM against the rP2X1 receptor) was reported.31 iAZD9056 (IC50 value = 13 nM against the hP2X1 receptor) was reported.38 a

Table 2. Antagonistic effects of 5-hydroxy pyridine derivatives with various modifications of pyridine at mP2X1, hP2X3 and hP2X7 receptors.

Compounds

R1

R2

R3

16

-CH3

-CN

-COOH

Inactive

17

-CH3

-COOH

Inactive

mP2X1a

hP2X7

hP2X3a

c

33.3 ± 3.1%

c

27.3 ± 1.8% c

18

25.3 ± 2.8%

19

Inactive

e

b

Inactive

b

inactive

b

inactive

b

inactive

b

inactive

27.8 ± 1.3%

c

16.3 ± 3.2%

c

20.4 ± 3.7%

c

681 ± 98 nM

d

Inactive

c

384 ± 99 nM

d

inactive

303 ± 66 nM

d

Inactive

709 ± 60 nM

d

inactive

20

-CH3

-S(=O)2-CH3

-H

inactive

22a

-CH3

-COOH

-H

Inactive

22b

-CH3

-COOH

-CH3

Inactive

22c

-CH2CH2CH3

-COOH

-CH3

22.7 ± 1.9%

23

-CH2CH2CH3

-CONH2

-CH3

Inactive

c

c

The 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 for mP2X1 and hP2X3 receptors (mean ± SEM, n ≥3). eThe ethidium bromide accumulation was a


26

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measured in hP2X7-expressing HEK293 cells, and the % inhibition of the accumulation by 10 µM of the compounds was evaluated (mean ± SEM, n ≥3).

Table 3. Antagonistic effects of 5-hydroxy pyridine derivatives with bulky m-phenoxyphenyl group at 2position of pyridine for mP2X1, hP2X3 and hP2X7 receptors.

Compounds

R

mP2X1a

hP2X3a c

301 ± 65 nM

d

Inactive

c

245 ± 29 nM

d

Inactive

b

inactive

b

inactive

b

inactive

b

inactive

b

inactive

27.5 ± 4.5%

27

hP2X7e

29

-COOH

23.3 ± 4.8%

35

-CH=CHCOOH

Inactive

37

-CH2CH2COOH

inactive

38

-CN

inactive

41

-CH2NH2

inactive

43

-CH2NH-S(=O)2-CH3

43.1 ± 12.9%

c

37.0 ± 1.0%

c

29.7 ± 3.1%

c

32.4 ± 0.8%

c

34.4 ± 1.2% c

42.4 ± 2.1%

The 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 for mP2X1 and hP2X3 receptors (mean ± SEM, n ≥3). eThe ethidium bromide accumulation was measured in hP2X7-expressing HEK293 cells, and the % inhibition of the accumulation by 10 µM of the compounds was evaluated (mean ± SEM, n ≥3). a


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Table 4. In-vitro PK and hERG channel studies Property

Compound 28

Compound 29

0

3

rat

33

0

mouse

35

2

99.5 ± 0.82

99.9 ± 0.06

32.5 ± 8.36

99.8 ± 0.13

-4.80

-7.04

3A4 isozyme

12 ± 2

10 ± 1

2C9 isozyme

28 ± 3

26 ± 2

1A2 isozyme

4±1

36 ± 3

Not determined

> 100

Metabolic stability Stability-microsomes human

Plasma protein binding Human

Units % remaining after 30 min

Plasma protein binding rate (%)

Rat Permeability-PAMPA

log Pe

CYP450 inhibition

% inhibition at 1µM

hERG

IC50 (µM)


28

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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

0.5 hr

2 hr

4 hr

8 hr

Plasma(µg/mL)

0.29 ± 0.04

0.04 ± 0.02

0.02 ± 0.01

0.004 ± 0

Brain (µg/g)

0.005 ± 0.002

0.001 ± 0

0.0001 ± 0

0.0000 ± 0

Brain to plasma ratio

0.02 ± 0.01

0.02 ± 0

0.004 ± 0

0.007 ± 0

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

0.5 hr

2 hr

4 hr

8 hr

Plasma (µg/mL)

0.62 ± 0.14

0.08 ± 0.02

0.06 ± 0.01

0.02 ± 0.01

Brain (µg/g)

1.11 ± 0.36

0.09 ± 0.01

0.05 ± 0.01

0.03 ± 0.01


1.79 ± 0.44

1.13 ± 0.25

0.94 ± 0.21

1.19 ± 0.41

Compound 29

0.5 hr

2 hr

4 hr

8 hr

Plasma(µg/mL)

0.11 ± 0.05

0.03 ± 0

0.02 ± 0.01

0.01 ± 0


29


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Page 30 of 39

Brain (µg/g)

0.022 ± 0.003

0.002 ± 0.001

0.001 ± 0

0.001 ± 0.001


0.24 ± 0.13

0.08 ± 0.02

0.05 ± 0.03

0.1 ± 0.12

Figure 1. The structure of P2X3 receptor antagonists.


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Figure 2. Antiallodynic effects of SNL rats by intrathecal administration of pregabalin, AF353 and compound 29. The withdrawal threshold or the percentage of maximal possible effect (% MPE at 1µg) represents the mean ± SEM of 5-6 rats for each experimental group. The antiallodynic efficacies of pregabalin (65 ± 9%), AF353 (32 ± 11%) and compound 29 (58 ± 19%) were observed in SNL rats. Compared with vehicle, * P < 0.05.


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Figure 3. Antiallodynic effects of SNL rats by intravenous administration of compound 28. The withdrawal threshold or the percentage of maximal possible effect (% MPE) represents the mean ± SEM of 5-6 rats for each experimental group. The antiallodynic efficacy of compound 28 (ED50 values of 2.62 mg/kg) was observed in SNL rats. Compared with vehicle, ** P < 0.01, *** P < 0.001.


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Figure 4. Antiallodynic effects of CIPN rats by intravenous administration of compound 28. The withdrawal threshold or the percentage of maximal possible effect (% MPE) represents the mean ± SEM of 5-6 rats for each experimental group. The antiallodynic efficacy of compound 28 (ED50 values of 2.93 mg/kg) was observed in CIPN rats. Compared with vehicle, *** P < 0.001.


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Figure 5. Antiallodynic effects of SNL rats by intrathecal (A,B) or oral (C,D) administration of compound 28. The withdrawal threshold or the percentage of maximal possible effect (% MPE) represents the mean ± SEM of 5-6 rats for each experimental group. Intrathecal or oral administration of compound 28 did not show any antiallodynic effects.


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Scheme 1. Synthesis of 3,4-dicarboxypyridine derivatives. Reagents and conditions: (a) EDC, TEA, DCM, RT, 2 h, 55-99%; (b) P2O5, CHCl3, reflux, 5 h, 58-99%; (b’) triphenylphosphine, iodine, TEA, DCM, RT, 12 h, 50-63%; (c) dimethyl maleate, neat, reflux, 5 h, 21-52%; (d) Oxone®, MeOH/THF = 3:1,


35


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RT, 4 h, 63-88%; (e)

Page 36 of 39

20% KOH (aq), RT, 6 h,

35-90% .


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Scheme 2. Synthesis of 5-hydroxypyridine derivatives. Reagents and conditions: (a) cis-betacyanoacrylate, neat, reflux, 5 h, 27-55%; (b) sodium azide, ammonium chloride, DMF, 90 °C, 12 h, 4862%; (c) 20% KOH (aq), RT, 6 h, 30-52%; (d) maleic anhydride, benzene, 60 °C, 2 h, 43-54%; (e) hydrazine hydrate (aq), sodium acetate anhydrous, acetic acid, reflux, 2 h, 35-46%; (f) methyl vinyl sulfone, neat, reflux, 5 h, 49-55%.

Scheme 3. Synthesis of 5-hydroxypyridine derivatives. Reagents and conditions: (a) methyl acrylate, neat, 5 h, 37-46%; (a’) methyl crotonate, neat, 5 h, 32-52%; (b) 20% KOH (aq), RT, 6 h, 40-57%; (c) 30% ammonia (aq), RT, 12 h, 32-45%.


37


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Scheme 4. Synthesis of 5-hydroxypyridine derivatives. Reagents and conditions: (a) P2O5, CHCl3, reflux, 5 h, 85-99%; (b) methyl crotonate, neat, reflux, 5 h, 22-52%; (c) benzyl bromide, K2CO3, acetone, reflux, 2 h, 73-96%; (d) LAH, ether, 0 °C, 1 h, 67-88%; (e) Pd/C, H2, RT, 0.5 h, 54-91%; (f) MnO2, DCM, RT, 2 h, 66-85%; (g) NaH, triethyl phosphonoacetate, THF, RT, 1 h, 63-78%; (h) 20% KOH (aq), RT, 6h, 3794%; (i) dimethyl maleate, neat, reflux, 5 h, 32-54%.


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127x72mm (300 x 300 DPI)


Discovery of Potent Antiallodynic Agents for ... - ACS Publications

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