Research paperSynthesis and structure-activity relationships of quinolinone and quinoline-based P2X7 receptor antagonists and their anti-sphere formation activities in glioblastoma cells
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Introduction
Purinergic receptors are plasma membrane proteins that can be classified into three different families: P1, P2X and P2Y. Broadly, P1 (adenosine) and P2Y receptors are G protein-coupled receptors (GPCR), and the P2X receptors are ligand-gated ion channels that are activated by extracellular ATP molecules [[1], [2], [3], [4]].
In the case of P2X receptors (P2XR), seven different subtypes, P2X1‒P2X7, have been identified in the immune and nervous systems. Among the P2XR subtypes, P2X7R has drawn particular interest in terms of its distinctive molecular structure, such as a longer C-terminus with 100–200 amino acids and pathological functions related to inflammation. In the tertiary structure of the ion channel, P2X7R is composed of homotrimeric subunits, and the receptor is mainly expressed in hematopoietic cells, including mast cells, lymphocytes, erythrocytes, and macrophages [5]. The P2X7Rs also play important pathophysiological functions in the human monocyte cell line THP-1, epidermal langerhans cells, fibroblasts, and cells in the central nervous system (CNS) such as microglia and Schwann cells, which suggests that the receptor is involved in diseases such as chronic inflammation, neurodegeneration, and chronic pain [6].
P2X7R is activated not only by the endogenous ligand ATP but also by other agonists such as BzATP, ADP, UTP and AMP [3,7,8]. As a unique function of the receptor, the activation of P2X7R results in the formation of permeable pores and the induction of signaling cascades downstream of the receptor. Upon the activation of P2X7R, cations such as Ca2+, Na+ and K+ can permeate through the ion channel or pore. This process leads to the activation of related inflammatory signals, including phospholiphase A2, phospholiphase D, mitogen-activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [9]. More importantly, P2X7R activation ultimately stimulates the release of interleukin-1β (IL-1β) [10,11], which is a master cytokine for the mediation of inflammatory signals, along with cell proliferation, differentiation and apoptosis [12]. In addition, P2X7R triggers several other signaling cascades, which ultimately lead to macrophage fusion, superoxide production in microglia and lymphoid cells [13,14]. Thus, therapeutic interventions targeting P2X7R have been explored as a novel approach for the prevention or treatment of inflammatory disorders such as arthritis, chronic inflammatory pain, neuropathic pain, and neurodegenerative diseases [[15], [16], [17], [18]]. In addition, P2X7R is over-expressed in various cancer cells, in which studies have reported to show important functions, such as improved survival, invasiveness, and metastasis, in cancer microenviroments [19,20]. Recently, P2X7R was reported to be involved in the activation of microglia located near glioma cells [[21](a), [21](b), [21]]. The inhibition of P2X7R function showed a decrease in the number of glioma cells, which can induce glioblastoma. Glioblastoma is the most common type of brain tumor with high morbidity [[22](a), [22](b), [22]].
According to the above diverse pathophysiological functions of the P2X7 receptor, several P2X7R antagonists have been discovered. For example, KN-62 (1, Fig. 1), previously known as a type II Ca2+/calmodulin-dependent kinase inhibitor, was developed as an early-generation P2X7R antagonist [[23](a), [23](b), [23]]. The antagonists developed by the pharmaceutical industry for the purpose of new drug discovery are compounds 2–5 in Fig. 1. AZD9056 (2) is an orally bioavailable P2X7R antagonist with an IC50 value of 10–13 nM (IL-1β ELISA assay), targeting rheumatoid arthritis (RA), chronic obstructive pulmonary disease (COPD) and Crohn's disease. However, AZD9056 (2) failed in phase 2 clinical trials because it did not show appreciable efficacy compared with placebo [[24](a), [24](b), [24]]. Another potent and highly selective P2X7R antagonist, CE-224535 (3), also entered clinical trials for treating patients with RA that was inadequately controlled by methotrexate, but it failed to show significant efficacy [25]. A-438079 (4) and GSK314181A (5) were reported to have anti-nociceptive effects in neuropathic [26] and inflammatory pain [27,28] and are currently in the discovery stage.
We have reported diverse classes of P2X7R antagonists, including dichloropyridine (6) [29], and 2,5-dioxoimidazolidine derivatives (7) [30]. Recently, we reported potent imidazole-based P2X7R antagonists (8) [31] with significant anti-migration and invasion activities against metastatic breast cancers.
To discover new chemical entities as P2X7R antagonists, a quinolinone library [32] that was previously reported by our group was screened to identify a hit compound (11a, Table 1) as a weak P2X7R antagonist. The quinolinone scaffold has been studied extensively to develop agents for treating immune disorders [[33], [33](a), [33](b), [34], [35], [36]]. Moreover, we recently reported the optimization of quinolinone derivatives for their inhibitory effects on IL-2 secretion and their functional mechanism of immunomodulation [37]. In this report, we describe the development of new P2X7R antagonists by structure-activity relationships (SAR) and the optimization of the quinolinone-based hit compound, 11a. In addition, the functional effects of the representative compound, including the inhibition of IL-1β release, which is closely related to immune disorders, and anti-sphere formation activities against glioblastoma cells, in which P2X7 receptors are overexpressed and function as activation of microglia located near glioblastoma cells, were examined [21,22].
Section snippets
Chemistry
The general synthetic procedures for the quinolinone and quinoline analogs are described in Scheme 1, Scheme 2, and a full list of synthesized analogs is provided in Table 1, Table 2, Table 3, Table 4, Table 5.
The starting materials, 5-nitroquinolin-2(1H)-one (9a) [38] and methyl 5-nitro-2-oxo-1,2-dihydroquinoline-3-carboxylate (9b) [32], were prepared following the previously reported procedure [32,38]. 5-Nitro groups of 9a‒b were first reduced to the corresponding amines with SnCl2, and the
Conclusions
Screening a quinolinone-based compound library led to the identification of 11a as a new lead compound for P2X7R antagonism. To optimize P2X7R antagonistic potency, modifications at R1, R2 and R3 of the quinolinone structure were performed. SAR analysis of the various derivatives suggested that a carboxylate ethyl ester group at R1, adamantyl carboxamide at R2 and 4-methoxy substitution at R3 gave the best substituents for the antagonism of P2X7R activity. Because the quinolinones showed low
Chemistry
All the reagents and solvents were purchased from Sigma-Aldrich and TCI and used without further purification. 1H NMR spectra were determined with a JEOL JNM-ECX 400P spectrometer at 400 MHz, and 13C NMR spectra were recorded using a FT-NMR spectrometer at 125 MHz (Korea Basic Science Institute, Gwangju); spectra were taken in CDCl3, DMSO-d6 or MeOH-d4. Unless otherwise noted, chemical shifts are expressed as δ units downfield from internal tetramethylsilane or relative parts per million (ppm)
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1D1A4A01015910) and this research was supported by GIST-Caltech Research Collaboration grant funded by the GIST in 2017.
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2022, Pharmacology and TherapeuticsCitation Excerpt :AstraZeneca disclosed two patents covering the synthesis of a novel class of quinoline derivatives targeting the P2X7R that were also identified during high-throughput screening in 2003 (Ebden & Guile, 2011; Guile & Thompson, 2012; Park & Kim, 2017). The US patents for the compounds have since been extended until 2030 and cover one novel P2X7R antagonist, LDD-2598, which is reportedly being investigated in preclinical studies as a treatment for atherosclerosis, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis and cancer (Kwak et al., 2018). While interest in P2X7R antagonists has somewhat dissipated since trials evaluated the efficacy of AZD9056, the significance of the P2X7R in the pathogenesis of common degenerative and life-threatening conditions has resulted in the persistent investment of pharmaceutical companies and institutes in the development of novel classes of P2X7R antagonists.
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2021, European Journal of PharmacologyCitation Excerpt :Further, extra modifications of triterpenoids scaffold at 6th position carbon were also not well tolerated. A recent study by Kwak et al. also demonstrated the structure-activity relationship of quinolinone and quinoline-based compounds as P2X7 receptor antagonists (Kwak et al., 2018). Earlier screening of a library of quinolinone derivatives led to the identification of Ph-(CH2)2CO substituted compound as P2X7 receptor antagonists.
Quinolines, a perpetual, multipurpose scaffold in medicinal chemistry
2021, Bioorganic ChemistryCitation Excerpt :The SAR studies had shown the substitution of carboxylic acid ethyl ester group, carboxamide group and 4-methoxy group substituted at 2-position of quinoline scaffold necessary for P2X7R receptor inhibition. Further, compound 29 displayed the functional activity in reducing the sphere size of TS15-88 glioblastoma cells [36]. Cytokine is a key mediator of inflammation, cell proliferation, differentiation and cell apoptosis process.
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Present address: Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.
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Present address: COSMAX, #701, Pangyo inno valley E, 255, Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea.
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Present address: New Drug Development Center (NDDC), Daegu-Gyeongbuk, Medical Innovation Foundation (DGMIF), 80 Cheombok-ro, Dong-gu, Daegu 41061, Republic of Korea.