Discovery of Benzo[f]indole-4,9-dione Derivatives as New Types of Anti-Inflammatory Agents

Certain benzo[f]indole-4,9-dione derivatives were synthesized and evaluated for their inhibitory effects on superoxide anion generation and neutrophil elastase (NE) release in formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils. Results indicated that (Z)-1-benzyl-4-(hydroxyimino)-1H-benzo[f]indol-9(4H)-one (10) showed a potent dual inhibitory effect on NE release and superoxide anion generation with IC50 value of 2.78 and 2.74 μM respectively. The action mechanisms of 10 in human neutrophils were further investigated. Our results showed that compound 10 did not alter fMLF-induced phosphorylation of Src (Src family Y416). Notably, phosphorylation of Akt (S473) and mobilization of [Ca2+]i caused by fMLF was inhibited by compound 10. Further structural optimization of 10 is ongoing.


Introduction
Human neutrophils play an important role in the defense system against invasion by microorganisms and in the pathogenesis of various diseases such as rheumatoid arthritis, ischemia-reperfusion injury, chronic obstructive pulmonary disease, and asthma [1][2][3][4][5]. In response to diverse stimuli, activated neutrophils secrete a series of cytotoxins, such as superoxide anion, a precursor of other reactive oxygen species (ROS), granule proteases, and bioactive lipids [2,6,7]. Of these, neutrophil elastase (NE) is stored in the azurophil granules of neutrophils and is released following neutrophil exposure to inflammatory stimuli. High concentrations of ROS and NE have been implicated in the pathogenesis of many acute and chronic pulmonary diseases including asthma, chronic obstructive pulmonary disease, cystic fibrosis, and acute respiratory distress syndrome [2,[8][9][10]. Therefore, inhibition of neutrophils activation and the following release of inflammatory mediators provide a promising strategy for the development of potential anti-inflammatory agents.

Biological Results and Discussion
Certain benzo[f]indole-4,9-dione derivatives were synthesized and evaluated for their inhibitory effects on superoxide anion generation and neutrophil elastase (NE) release in formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF)-activated human neutrophils and results are shown in Table 1. 1-Benzyl-1H-benzo [f]indole-4,9-dione (8) and its 2-methyl derivative 9 exhibited weak inhibitory effects on superoxide anion generation and were inactive on the inhibition of NE release. (Z)-1-Benzyl-4-(hydroxyimino)-1H-benzo [f]indol-9(4H)-one (10) showed a potent dual inhibitory effect on NE release and superoxide anion generation with IC50 value of 2.78 and 2.74 μM respectively. In contrast, its 2-methyl derivative 11 exhibited only marginal activity on superoxide anion generation and was inactive on the inhibition of NE release. These results indicated that the oxime moiety enhanced anti-inflammatory activities while methyl substituent at C-2 position was unfavorable especially in the inhibition of NE release. The same structure-activity relationships were observed in which 3-acetyl-1-benzyl-2-methyl-1H-benzo[f]indole-4,9dione (12) was inactive while its oxime derivative 13 exhibited a weak dual inhibitory effect on NE release and superoxide anion generation. Compound 14, the methyl derivative of 13, was inactive. Among these benzo (15) was the most potent dual inhibitor on NE release and superoxide anion generation with IC50 value of 0.51 and 2.05 μM respectively. (16) exhibited a strong inhibitory effect on superoxide anion generation with an IC50 value of 0.52 μM, it induced NE release of human neutrophils.
Reagents and conditions: i) benzylamine, EtOH, reflux; ii) MeCHO or Me 2 CO, Mn(Ac) 3 , iii) NH 2 OH-HCl, K 2 CO 3 , EtOH; iv) acetylacetone, CAN; v) NH 2 OR-HCl (R = H or Me), K 2 CO 3 , EtOH; vi) procainamide, TiCl 4 , CH 2 Cl 2. .  These benzo[f]indole-4,9-dione derivatives were evaluated in vitro against a panel of cell lines consisting of MCF7 (breast), NCI-H460 (lung), and SF-268 (CNS) as described previously [27]. Compounds which reduced the growth of any one of the cell lines to 50% or less at the concentration of 4 μg/mL were considered cytotoxic and subjected to further evaluation for their dose-response effects and IC50 measurement. Results from Table 2 indicated compounds 15 and 16 were cytotoxic and their IC50 against three cancer cells and a normal cell, Detroit 551, were shown in Table 3. The IC50 value of compounds 15 and 16 ranged between 3.16 and 27.41 μM and were much less cytotoxic than that of camptothecin (CPT).  Compound 15 (3, 10 and 30 μM) showed cytotoxicity effects in human neutrophils, as measured by lactate dehydrogenase (LDH) release. In contrast, compound 10, even at high concentration of 30 μM, showed no cytotoxicity effects in human neutrophils (data not shown). The action mechanisms of 10 in human neutrophils were further investigated. Human neutrophil activations, such as respiratory burst and degranulation, are regulated by Akt and calcium signal pathways [28,29]. Therefore, calcuim and Akt are considered as therapeutic target for developing anti-inflammatory agents. Compound 10 did not alter fMLF-induced phosphorylation of Src (Src family Y416) (Figure 2A). Notably, phosphorylation of Akt (S473) and mobilization of [Ca 2+ ]i caused by fMLF was inhibited by compound 10 (Figures 2B and 3).

Preparation of Human Neutrophils
Blood was taken from healthy human donors (20-30 years old) by venipuncture, using a protocol approved by the institutional review board at Chang Gung Memorial Hospital. Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes [30].

Superoxide Generation and Elastase Release
Superoxide generation and elastase release were carried out according to the procedures described previously [31]. Neutrophils (6 × 10 5 /mL) were equilibrated at 37 °C for 2 min and incubated with compounds for 5 min. Neutrophils were then activated by fMLF (100 nM) in the pretreatment of cytochalasin B (1 μg/mL for superoxide generation and 0.5 μg/mL for elastase release) for 10 min. Superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release was performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.

Western Analysis
Neutrophils were pretreated with compounds for 5 min before being stimulated with fMLF at 37 °C. The reaction was stopped by adding 5 × Laemmli's sample buffer [32,33]. Proteins derived from whole-cell lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using polyacrylamide gels and blotted onto nitrocellulose membranes. Immunoblotting was performed using the indicated antibodies and horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibodies (Cell Signaling Technology, Beverly, MA, USA). The immunoreactive bands were visualized by an enhanced chemiluminescence system (Amersham Biosciences, Bucks, UK) and detected by Ultraviolet Product (UVP) imaging system (Upland, CA, USA).