The transcription factor Nuclear factor erythroid-2-related factor 2 (NRF2) and its principal repressive regulator, Kelch-like ECH-associated protein 1 (KEAP1), are perilous in the regulation of inflammation, as well as maintenance of homeostasis. Thus, NRF2 activation is involved in cytoprotection against many inflammatory disorders. N′-Nicotinoylquinoxaline-2-carbohdyrazide (NQC) was structurally designed by the combination of important pharmacophoric features of bioactive compounds reported in the literature. NQC was synthesised and characterised using spectroscopic techniques. The compound was tested for its anti-inflammatory effect using Lipopolysaccharide from Escherichia coli (LPSEc) induced inflammation in mouse macrophages (RAW 264.7 cells). The effect of NQC on inflammatory cytokines was measured using enzyme-linked immune sorbent assay (ELISA). The Nrf2 activity of the compound NQC was determined using ‘Keap1:Nrf2 Inhibitor Screening Assay Kit’. To obtain the insights on NQC’s activity on Nrf2, molecular docking studies were performed using Schrödinger suite. The metabolic stability of NQC was determined using mouse, rat and human microsomes. NQC was found to be non-toxic at the dose of 50 µM on RAW 264.7 cells. NQC showed potent anti-inflammatory effect in an in vitro model of LPSEc stimulated murine macrophages (RAW 264.7 cells) with an IC50 value 26.13 ± 1.17 µM. NQC dose-dependently down-regulated the pro-inflammatory cytokines [interleukin (IL)-1β (13.27 ± 2.37 μM), IL-6 (10.13 ± 0.58 μM) and tumor necrosis factor (TNF)-α] (14.41 ± 1.83 μM); and inflammatory mediator, prostaglandin E2 (PGE2) with IC50 values, 15.23 ± 0.91 µM. Molecular docking studies confirmed the favourable binding of NQC at Kelch domain of Keap-1. It disrupts the Nrf2 interaction with kelch domain of keap 1 and its IC50 value was 4.21 ± 0.89 µM. The metabolic stability studies of NQC in human, rat and mouse liver microsomes revealed that it is quite stable with half-life values; 63.30 ± 1.73, 52.23 ± 0.81, 24.55 ± 1.13 min; microsomal intrinsic clearance values; 1.14 ± 0.31, 1.39 ± 0.87 and 2.96 ± 0.34 µL/min/g liver; respectively. It is observed that rat has comparable metabolic profile with human, thus, rat could be used as an in vivo model for prediction of pharmacokinetics and metabolism profiles of NQC in human. NQC is a new class of NRF2 activator with potent in vitro anti-inflammatory activity and good metabolic stability.

中文翻译：

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构建新型喹喔啉作为新型 Nrf2 激活剂

转录因子核因子红细胞 2 相关因子 2 (NRF2) 及其主要抑制调节因子，Kelch 样 ECH 相关蛋白 1 (KEAP1)，在调节炎症和维持体内平衡方面是危险的。因此，NRF2 激活参与了针对许多炎症性疾病的细胞保护。N'-Nicotinoylquinoxaline-2-carbohdyrazide (NQC) 是通过结合文献中报道的生物活性化合物的重要药效学特征而设计的。使用光谱技术合成和表征 NQC。使用来自大肠杆菌的脂多糖 (LPSEc) 在小鼠巨噬细胞 (RAW 264.7 细胞) 中诱导炎症测试该化合物的抗炎作用。使用酶联免疫吸附试验 (ELISA) 测量 NQC 对炎性细胞因子的影响。使用“Keap1:Nrf2 Inhibitor Screening Assay Kit”测定化合物 NQC 的 Nrf2 活性。为了深入了解 NQC 对 Nrf2 的活性，使用薛定谔套件进行了分子对接研究。使用小鼠、大鼠和人微粒体测定 NQC 的代谢稳定性。发现 NQC 在 RAW 264.7 细胞上以 50 µM 的剂量是无毒的。NQC 在 LPSEc 刺激的小鼠巨噬细胞（RAW 264.7 细胞）的体外模型中显示出有效的抗炎作用，IC50 值为 26.13 ± 1.17 µM。NQC 剂量依赖性地下调促炎细胞因子 [白细胞介素 (IL)-1β (13.27 ± 2.37 μM)、IL-6 (10.13 ± 0.58 μM) 和肿瘤坏死因子 (TNF)-α] (14.41 ± 1.83 μM) ); 和炎症介质，前列腺素 E2 (PGE2) 的 IC50 值为 15.23 ± 0.91 µM。分子对接研究证实了 NQC 在 Keap-1 的 Kelch 结构域的有利结合。它破坏了 Nrf2 与 keap 1 的 kelch 域的相互作用，其 IC50 值为 4.21 ± 0.89 µM。NQC 在人、大鼠和小鼠肝微粒体中的代谢稳定性研究表明，它具有相当稳定的半衰期值；63.30 ± 1.73、52.23 ± 0.81、24.55 ± 1.13 分钟；微粒体内在清除值；1.14 ± 0.31、1.39 ± 0.87 和 2.96 ± 0.34 µL/min/g 肝脏；分别。观察到大鼠具有与人相当的代谢特征，因此，大鼠可用作预测人体内NQC的药代动力学和代谢特征的体内模型。NQC是一类新型的NRF2激活剂，具有有效的体外抗炎活性和良好的代谢稳定性。