Design, synthesis and biological evaluation of novel O-substituted tryptanthrin oxime derivatives as c-Jun N-terminal kinase inhibitors

Abstract

The c-Jun N-terminal kinase (JNK) family includes three proteins (JNK1-3) that regulate many physiological processes, including inflammatory responses, morphogenesis, cell proliferation, differentiation, survival, and cell death. Therefore, JNK represents an attractive target for therapeutic intervention. Herein, a panel of novel tryptanthrin oxime analogs were synthesized and evaluated for JNK1-3 binding (K_d) and inhibition of cellular inflammatory responses (IC₅₀). Several compounds exhibited submicromolar JNK binding affinity, with the most potent inhibitor being 6-(acetoxyimino)indolo[2,1-b]quinazolin-12(6H)-one (1j), which demonstrated high JNK1-3 binding affinity (K_d = 340, 490, and 180 nM for JNK1, JNK2, and JNK3, respectively) and inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) transcription activity in THP-1Blue cells and interleukin-6 (IL-6) production in MonoMac-6 monocytic cells (IC₅₀ = 0.8 and 1.7 μM, respectively). Compound 1j also inhibited LPS-induced production of several other proinflammatory cytokines, including IL-1α, IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF) in MonoMac-6 cells. Likewise, 1j inhibited LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. Molecular modeling suggested modes of binding interaction of selected compounds in the JNK3 catalytic site that were in agreement with the experimental JNK3 binding data. Our results demonstrate the potential for developing anti-inflammatory drugs based on these nitrogen-containing heterocyclic systems.

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

FIGURE 1

Chemical structures of several JNK inhibitors.

SCHEME 1

Synthetic routes to tryptanthrin derivatives.

FIGURE 2

Effect of 1j on LPS-induced c-Jun (Ser63) phosphorylation. Human MonoMac-6 monocytic cells were pretreated with the indicated concentrations of compounds or 0.5% DMSO for 30 min, followed by treatment with LPS (250 ng/ml) or control buffer for another 30 min. Controls with cells alone (no DMSO) or cells treated with LPS alone were also included. The cells were lysed, and the lysates were analyzed by Western blotting on 10% SDS-PAGE gels. A representative blot from three independent experiments is shown (A). The blot was analyzed by densitometry as described under Materials and Methods, and the ratio of phospho-c-Jun/total c-Jun is shown in (B). Values are expressed as mean ± SD of three independent experiments.

FIGURE 3

Effect of the compound 1j on proinflammatory cytokine production in MonoMac-6 cells. Human MonoMac-6 monocytic cells were pretreated with 10 μM of 1j or DMSO for 30 min, followed by addition of 250 ng/ml LPS or buffer for 24 h. Production of cytokines in the supernatants was evaluated using a Multiplex human cytokine ELISA kit. The relative level of cytokine production is shown as fold increase over background (i.e., 1% DMSO control). The data are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of two independent experiments.

FIGURE 4

Docking poses of compounds 1c (A), 1f (B), 1i (C), 1o (D), TRYP-Ox (E), and 1j (F) in JNK3 (PDB code 1PMV). H-bonds are shown in blue dashed lines. Residues within 3 Å of each pose are visible.

FIGURE 5

Correlation of docking scores for the best docking poses of the compounds in JNK3 and binding affinities (K_d) with JNK3. Binding affinities are represented as inverse (1/K_d) values. Dashed lines indicate area of the 95% confidence band.