Synthesis and structure-activity relationships of quinolinone and quinoline-based P2X7 receptor antagonists and their anti-sphere formation activities in glioblastoma cells

Highlights

• Quinoline derivatives were developed as P2X7 receptor antagonists.

• We performed the introduction of various substituents at the R₃ position of the quinoline structure.

• Compounds 16c, 17b−c and 17h−k showed potent antagonistic effects (EtBr IC₅₀ = 3–18 nM).

• Also, compounds 16c, 17b−c and 17h−k exhibited highly potent functional activities (IL-1β IC₅₀ = 7–33 nM).

• 16c has an acceptable in vitro anti-cancer activities against the glioblastoma cells, in which P2X7Rs are overexpressed.

Abstract

Screening a compound library of quinolinone derivatives identified compound 11a as a new P2X7 receptor antagonist. To optimize its activity, we assessed structure-activity relationships (SAR) at three different positions, R₁, R₂ and R₃, of the quinolinone scaffold. SAR analysis suggested that a carboxylic acid ethyl ester group at the R₁ position, an adamantyl carboxamide group at R₂ and a 4-methoxy substitution at the R₃ position are the best substituents for the antagonism of P2X7R activity. However, because most of the quinolinone derivatives showed low inhibitory effects in an IL-1β ELISA assay, the core structure was further modified to a quinoline skeleton with chloride or substituted phenyl groups. The optimized antagonists with the quinoline scaffold included 2-chloro-5-adamantyl-quinoline derivative (16c) and 2-(4-hydroxymethylphenyl)-5-adamantyl-quinoline derivative (17k), with IC₅₀ values of 4 and 3 nM, respectively. In contrast to the quinolinone derivatives, the antagonistic effects of the quinoline compounds (16c and 17k) were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β, from LPS/IFN-γ/BzATP-stimulated THP-1 cells (IC₅₀ of 7 and 12 nM, respectively). In addition, potent P2X7R antagonists significantly inhibited the sphere size of TS15-88 glioblastoma cells.

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].