Novel c-Jun N-Terminal Kinase (JNK) Inhibitors with an 11 H-Indeno[1,2- b]quinoxalin-11-one Scaffold

Abstract

c-Jun N-terminal kinase (JNK) plays a central role in stress signaling pathways implicated in important pathological processes, including rheumatoid arthritis and ischemia-reperfusion injury. Therefore, inhibition of JNK is of interest for molecular targeted therapy to treat various diseases. We synthesized 13 derivatives of our reported JNK inhibitor 11H-indeno[1,2-b]quinoxalin-11-one oxime and evaluated their binding to the three JNK isoforms and their biological effects. Eight compounds exhibited submicromolar binding affinity for at least one JNK isoform. Most of these compounds also inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) activation and interleukin-6 (IL-6) production in human monocytic THP1-Blue cells and human MonoMac-6 cells, respectively. Selected compounds (4f and 4m) also inhibited LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. We conclude that indenoquinoxaline-based oximes can serve as specific small-molecule modulators for mechanistic studies of JNKs, as well as potential leads for the development of anti-inflammatory drugs.

Keywords: 11H-indeno[1,2-b]quinoxalin-11-one; c-Jun N-terminal kinase; interleukin-6; kinase inhibitor; nuclear factor-κB; oxime.

Figure 1

The reported scaffolds of JNK inhibitors and range of their inhibitory activity (IC₅₀ values) [25,32,33,34].

Scheme 1

Reagents and conditions: (a) AcOH, 100 °C, 30 min, 35–90%; (b) CDI, MeONa, dimethylformamide, 25 °C, 1 h, 80%; (c) NBS, CCl₄, 90 °C, 2 h, 80%; (d) R₂NH; THF; 25 °C, 25–30%; (e) NH₂OH∙HCl, C₅H₅N, EtOH, 100 °C, 4 h, 90–95%; (f) NH₂OH∙HCl, NaOH, 80 °C, 2 h, 90–95%. Abbreviations: MRF—morpholin-1-yl.

Figure 2

Molecular structure of compound 3b determined by X-ray diffraction analysis.

Figure 3

Effect of the compounds 4f and 4m on IL-6 production by human MonoMac-6 cells. MonoMac-6 cells were pretreated with the indicated compounds or DMSO (negative control) for 30 min followed by addition of 250 ng/mL LPS or buffer for 24 h. Production of IL-6 in the supernatants was evaluated by ELISA. The data are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of three independent experiments.

Figure 4

Effect of 4f and 4m on LPS-induced c-Jun (Ser63) phosphorylation. Human MonoMac-6 monocytic cells were pretreated with indicated concentrations of 4f or 4m for 30 min, followed by treatment with LPS (250 ng/mL) or control vehicle (1% DMSO) for another 30 min. The cells were lysed, and the lysates were analyzed by Western blotting. Total JNK (non-phosphorylated) was used as loading control for the lysates. A representative blot from two independent experiments is shown (Panel A). The blots were analyzed by densitometry, and the ratio of phospho-c-Jun/total c-Jun is shown in Panel B.

Figure 5

Bioavailability radar plots for compounds 4b, 4f, 4m, and SP600125. The plots depict LIPO (lipophilicity), SIZE (molecular weight), POLAR (polarity), INSOLU (insolubility), INSATU (unsaturation), and FLEX (rotatable bond flexibility) parameters.

Figure 5

Bioavailability radar plots for compounds 4b, 4f, 4m, and SP600125. The plots depict LIPO (lipophilicity), SIZE (molecular weight), POLAR (polarity), INSOLU (insolubility), INSATU (unsaturation), and FLEX (rotatable bond flexibility) parameters.