Discovery of 2-Substituted 3-Arylquinoline Derivatives as Potential Anti-Inflammatory Agents Through Inhibition of LPS-Induced Inflammatory Responses in Macrophages

We describe herein the preparation of certain 2-substituted 3-arylquinoline derivatives and the evaluation of their anti-inflammatory effects in LPS-activated murine J774A.1 macrophage cells. Among these newly synthesized 2-substituted 3-arylquinoline derivatives, 2-(4-methoxy- benzoyl)-3-(3,4,5-trimethoxyphenyl)quinoline (18a) and 2-(4-fluorobenzoyl)-3-(3,4,5-trimethoxy- phenyl)quinoline (18b) are two of the most active compounds which can inhibit the production of NO at non-cytotoxic concentrations. Our results have also indicated that compounds 18a and 18b significantly decrease the secretion of pro-inflammatory cytokines (TNF-á and IL-6), inhibit the expression of iNOS, suppress the phosphorylation of MAPKs, and attenuate the activity of NF-êB by LPS-activated macrophages. Through molecular docking analysis, we found that 18b could fit into the middle of the TNF-á dimer and form hydrophobic interactions with Leu55, Leu57 chain A and B, Tyr59, Val123 chain B and D, Ile 155. These results suggest that both 18a and 18b are potential lead compounds in inhibiting LPS-induced inflammatory responses. Further structural optimization to discover novel anti-inflammatory agents is ongoing.


Introduction
Macrophages are the cornerstone of the innate immune system, which crucially deal with infection of microorganisms and repair of tissue damage by inflammatory responses. It has been considered that macrophages not only contribute to metabolic homeostasis in adipose tissue, liver and pancreas, but are also involved in the development of several diseases such as atherosclerosis, diabetes and cancers [1]. The inflammatory response contributes to antimicrobial defense, tissue repair and metabolism by activated macrophages. The pro-inflammatory mediators and cytokines are secreted when macrophages recognize components of pathogens and debris of dead cells [2]. Lipopolysaccharide (LPS), the Gram-negative bacteria membrane component, is recognized by Toll-like receptor 4 (TLR4) and triggers inflammatory responses via nuclear factor kappa light chain enhancer of activated B cells (NF-êB) and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (p38 MAPK)) signaling activation, resulting in the expression of inflammatory mediators in mammals such as nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on activated macrophages [3,4]. Uncontrolled inflammatory responses induced by LPS have been demonstrated to trigger pathogenesis of acute infection diseases such as sepsis and septic shock [5], and also contribute to the development of chronic inflammatory diseases such as type 2 diabetes and cardiovascular diseases [6].
Cyclooxygenase-2 (COX-2) is selectively induced by pro-inflammatory cytokines at the site of inflammation. The pro-inflammatory mediators, NO and cytokines such as TNF-α and IL-6, are generated by activated macrophages, which exert antimicrobial effects and connect to adaptive immunity system [7]. Noteworthy, uncontrolled pro-inflammatory mediator and cytokine production leads to tissue injury and inflammatory diseases [1]. Activated macrophages are major sources of NO production. NO reacts with superoxide O 2 − , and converts into cytotoxic molecule peroxynitrite anion (ONOO − ). The immoderate production of ONOO − causes tissue damage and dysfunction-+ [8].

Effect of 2-substituted 3-arylquinoline derivatives on NO production and cell survival in macrophages
NO plays an important role in mediating multiple aspects of inflammatory responses [26]. To examine the effects of 2-substituted 3-arylquinoline derivatives on NO production by LPS-activated macrophages, J774A.1 cells were pre-treated with series of 2-substituted 3-arylquinoline derivatives 16a-23 at concentration a 10 μM for 1 h, and were incubated with or without LPS (1 μg/mL) for 24 h. The cell culture supernatants were then harvested and quantified NO concentration using Griess's method [27], and the cells were used to determine the cell survival by MTT assays. As shown in Table 1 and Figure 2A, LPS stimulation dramatically increased NO production to 15.43 μM compared to the negative control of 1.17 μM. In addition, our results showed that all 2-substituted 3-arylquinoline derivatives significantly reduced LPS-induced NO production by J774A.1 cells (Table 1 and Figure 2A

Compounds 18a and 18b Significantly Suppresses the Production of TNF-α and IL-6 by LPS-Activated Macrophages
LPS stimulates the secretion of proinflammatory cytokines by macrophages such as TNF-á and IL-6 which are critical to the initiation of the inflammatory response; however, hypersecretion of proinflammatory cytokines ultimately result in inflammatory-related diseases or autoimmune diseases [27,28]. The above experiments indicated that compounds 18a and 18b were candidate anti-inflammatory compounds among the prepared 2-substituted 3-arylquinoline derivatives.
Thus, we investigated the effects of compounds 18a and 18b on LPS-induced TNF-α and IL-6 production by macrophages. J774A.1 cells were pre-treated with compounds 18a or 18b at different concentrations (0, 2.5, 5 and 10 µM) for 1 h, and were incubated with or without LPS (1 µg/mL) for 24 h. The cell culture supernatants were harvested and quantified TNF-α and IL-6 concentrations by ELISA. Our experimental results indicated that both compounds 18a and 18b significantly reduced the production of TNF-α and IL-6 by LPS-activated macrophages ( Figure 3). Thus, we investigated the effects of compounds 18a and 18b on LPS-induced TNF-α and IL-6 production by macrophages. J774A.1 cells were pre-treated with compounds 18a or 18b at different concentrations (0, 2.5, 5 and 10 μM) for 1 h, and were incubated with or without LPS (1 μg/mL) for 24 h. The cell culture supernatants were harvested and quantified TNF-α and IL-6 concentrations by ELISA. Our experimental results indicated that both compounds 18a and 18b significantly reduced the production of TNF-α and IL-6 by LPS-activated macrophages ( Figure 3).

Compounds 18a and 18b Significantly Attenuate the Activity of NF-êB by LPS-Activated Macrophages
NF-êB is a downstream transcription factor of TLR4, which regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory responses [29]. We further examined whether compounds 18a and 18b affected the activity of NF-êB. J-blue cell, a NF-êB reporter J774A.1 macrophage cell line that stably expresses the gene for secreted embryonic alkaline phosphatase (SEAP) inducible by NF-ê B, which was used to detect the activity of NF-êB [30]. J-blue cells were pre-treated with compounds 18a or 18b at different concentrations (0, 2.5, 5 and 10 µM) for 1 h, and were incubated with or without LPS (1 µg/mL) for 24 h. The cell culture supernatants were harvested, and the activity of NF-êB was examined by SEAP assay. Our experimental results showed that both compounds 18a and 18b significantly attenuated the activity of NF-êB by LPS-activated macrophages ( Figure 4). Data are presented as means ± SD of three independent experiments. "+"means treated with LPS; "−"means untreated. Statistical significance was represented as follows: * p < 0.05 and ** p < 0.01 vs LPS alone.

Compounds 18a and 18b
Significantly Attenuate the Activity of NF-êB by LPS-Activated Macrophages NF-êB is a downstream transcription factor of TLR4, which regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory responses [30]. We further examined whether compounds 18a and 18b affected the activity of NF-êB. J-blue cell, a NF-êB reporter J774A.1 macrophage cell line that stably expresses the gene for secreted embryonic alkaline phosphatase (SEAP) inducible by NF-ê B, which was used to detect the activity of NF-êB [31]. J-blue cells were pre-treated with compounds 18a or 18b at different concentrations (0, 2.5, 5 and 10 μM) for 1 h, and were incubated with or without LPS (1 μg/mL) for 24 h. The cell culture supernatants were harvested, and the activity of NF-êB was examined by SEAP assay. Our experimental results showed that both compounds 18a and 18b significantly attenuated the activity of NF-êB by LPS-activated macrophages ( Figure 4). Data are presented as means ± SD of three independent experiments. "+"means treated with LPS; "−"means untreated. Statistical significance was represented as follows: * p < 0.05 and ** p < 0.01 vs LPS alone.

Compounds 18a and 18b Inhibit the Expression of iNOS and Suppress the Phosphorylation of MAPKs
To further examine whether compounds 18a and 18b affect iNOS, COX2 and MAPKs expression, J774A.1 cells were pre-treated with compound 18a or 18b at different concentrations (0, 5 and 10 µM) for 1 h, and then incubated with or without LPS (1 µg/mL) for 2h (for MAPKs expression detection) or 24 h (for iNOS and COX2 expression detection). The protein level of iNOS, COX2, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-p38 and p38 was measured by western blot. As shown in Figure 5A, compounds 18a and 18b inhibit LPS-induced iNOS expression in J774A.1 cells, but not COX2. In addition, we have also found that compounds 18a and 18b suppress the phosphorylation of ERK, JNK and p38 by LPS-activated J774A.1 cells ( Figure 5B).

Compounds 18a and 18b Inhibit the Expression of iNOS and Suppress the Phosphorylation of MAPKs
To further examine whether compounds 18a and 18b affect iNOS, COX2 and MAPKs expression, J774A.1 cells were pre-treated with compound 18a or 18b at different concentrations (0, 5 and 10 μM) for 1 h, and then incubated with or without LPS (1 μg/mL) for 2h (for MAPKs expression detection) or 24 h (for iNOS and COX2 expression detection). The protein level of iNOS, COX2, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-p38 and p38 was measured by western blot. As shown in Figure 5A, compounds 18a and 18b inhibit LPS-induced iNOS expression in J774A.1 cells, but not COX2. In addition, we have also found that compounds 18a and 18b suppress the phosphorylation of ERK, JNK and p38 by LPS-activated J774A.1 cells ( Figure 5B). To understand the interaction between our hit compounds 18a-b and TNF-á (PDB code 2AZ5) [31,32], we performed a molecular docking study using the Achilles Blind Docking Server. The docking pose with the lowest binding energy of compounds 18a-b was shown in Figure 6. The results showed that both compounds 18a and 18b could fit into the middle of TNF-á dimer. According to the docking results, compound 18a formed hydrophobic interactions with Leu55, Tyr59, Ile 155 and pi-stacking interaction with Tyr59, while compound 18b also formed hydrophobic interaction with Leu55, Leu57 chain A and B, Tyr59, Val123 chain B and D, Ile 155. The lowest binding energy score between compound 18a, 18b and TNF-á was-7.00 and −8.30 kcal/mol individually.

Discussion
The innate immune response constitutes the first line of defense against invading pathogens and plays an important role in inflammatory diseases [34]. Sepsis is a severe illness in which the bloodstream is overwhelmed with bacteria, resulting in inflammation throughout the body and causing more than 215,000 deaths annually just in the United States [35]. LPS is well known to trigger an inflammatory response leading to release of inflammatory mediators and occasionally sepsis, or even septic shock [4]. In the present study, we synthesized a total of sixteen 2-substituted 3-arylquinoline derivatives and evaluated the anti-inflammatory effect in LPS-activated murine macrophage cell line J774A.1 cells. We firstly examined the effect of 2-substituted 3-arylquinoline

Discussion
The innate immune response constitutes the first line of defense against invading pathogens and plays an important role in inflammatory diseases [33]. Sepsis is a severe illness in which the bloodstream is overwhelmed with bacteria, resulting in inflammation throughout the body and causing more than 215,000 deaths annually just in the United States [34]. LPS is well known to trigger an inflammatory response leading to release of inflammatory mediators and occasionally sepsis, or even septic shock [4]. In the present study, we synthesized a total of sixteen 2-substituted 3-arylquinoline derivatives and evaluated the anti-inflammatory effect in LPS-activated murine macrophage cell line J774A.1 cells. We firstly examined the effect of 2-substituted 3-arylquinoline derivatives on the production of NO by LPS-activated macrophages. We found that compounds 18a and 18b significantly reduced the production of NO by LPS-activated macrophages at 10 ìM with no toxicity. In addition, compounds 18a and 18b also repressed the expression of iNOS by LPS-activated macrophages. NO has been regarded that is crucial in pathogenesis of autoimmune diseases such as rheumatoid arthritis and chronic inflammatory diseases such as atherosclerosis [35]. NO converts to ONOO − by O 2 − , and ONOO − exerts cytotoxicity effects. The overproduction of NO strongly contributes to tissue damage and augmentation of inflammation. Our results suggest that compounds 18a and 18b have potential in inhibiting the production of NO in LPS-induced sepsis. Abnormal macrophage activity causes overproduction of pro-inflammatory mediators and cytokines, and triggers several inflammatory diseases [1].
To reduce production of proinflammatory mediators and cytokines by activated macrophages is a critical role in prevention of immoderate inflammation. TNF-α and IL-6 are major pro-inflammatory cytokines that are produced by LPS-activated macrophages [3], and critically augment inflammatory cascades [36]. The uncontrolled TNF-α and IL-6 production lead to metabolic disorders such as type 2 diabetes and atherosclerosis [10,37]. Thus, to inhibit the overproduction of pro-inflammatory mediators and cytokines is a key point for prevention of inflammatory diseases. We further investigated the effects of compounds 18a and 18b on LPS-induced TNF-α and IL-6 productions. Our experimental results indicated that compounds 18a and 18b exert inhibitory effects on LPS-induced TNF-α and IL-6 productions. These results suggest that compounds 18a and 18b might have benefit on the prevention of overactive inflammatory responses induced by microbe's infection.
Activation of the transcription factor NF-êB and MAPKs is commonly thought to be critical to LPS-stimulated macrophage inflammatory mediator production including NO, TNF-α and IL-6 [4][5][6]. Therefore, we investigated whether compounds 18a and 18b decrease the production of NO, TNF-α and IL-6 through regulating NF-êB and MAPKs signaling pathways. Our experimental results demonstrated that compounds 18a and 18b attenuate the activity of NF-êB and suppress the phosphorylation of MAPKs in LPS-activated macrophages. These results suggest that compounds 18a and 18b inhibit the production of NO, TNF-α and IL-6 by LPS-activated macrophages through NF-êB and MAPKs signaling pathways.
In the molecular docking study, the scores of compound 18b are better than those of compound 18a. Moreover, compound 18b showed an excellent lowest binding energy with no hydrogen bond interaction. It could be explained that our hit compounds 18a and 18b, were able to form a stable complex with TNF-α by docking into the middle pocket of dimer. The previous study showed that the complex retained the same basic structural subunit fold compared to native dimer, but the introduction of small molecular may cause the angle between the subunits slightly widened [38]. In our study, we suggested that both compound 18a and 18b may change the angle of TNF-α dimer between the subunits and lead to inactivation to affect the downstream inflammatory reaction.

General Information
Melting points were determined on an IA9100 melting point apparatus (Dubuque, IA, USA) and are uncorrected. Nuclear magnetic resonance ( 1 H and 13 C) spectra were recorded on a Gemini 200 or Unity-400 spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts were expressed in parts per million (δ) with tetramethylsilane (TMS) as an internal standard. Analytical TLC was performed on Art. 5554 Kieselgel 60 GF254 produced by Merck (Darmstadt, Germany) and the spots of compounds were detected with UV light indicator irradiated at 254 and 366 nm. Art. 7734 Kieselgel 60 GF254 (70-400 mesh, Merck) was used for column chromatography. The elemental analyses were performed in the Instrument Center of National Science Council at National Cheng-Kung University and National Taiwan University using Heraeus CHN-O Rapid EA, and all values are within ± 0.4% of the theoretical compositions.

General Procedure for the Preparation of 3-aryl-2-methylquinoline-4-carboxylic Acids 10 and 11
A mixture of isatin 7 (40 mmol), phenylacetone 8 or 9 (48 mmol) and KOH (6.74 g, 120 mmol) in EtOH was heated at 80 • C for 48 h (TLC monitoring). After cooling, the solvent was removed in vacuo and the residue dissolved in H 2 O (50 mL), and the aqueous solution was washed twice with Et 2 O (30 mL). The ice-cold aqueous phase was acidified to pH 1 with 37% HCl, and the precipitate was collected by suction filtration, washed with H 2 O and recrystallized with EtOH to give 3-aryl-2-methylquinoline-4-carboxylic acids 10 and 11. The suspension of quinoline-4-carboxylic acid 10 or 11 (5.0 mmol) in 10 mL Dowtherm was heated to 280 • C for 4 h (TLC monitoring). After cooling, the reaction mixture was added n-hexane (50 mL) and the precipitate was collected by suction filtration and washed with n-hexane. The crude product was purified by flash chromatography on silica gel (hexane/CH 2 Cl 2 = 1/1) and recrystallized from EtOH to give compounds 12 and 13.

General Procedure for the Preparation of quinoline-2-carbaldehydes 14 and 15
A mixture 12 or 13 (3.0 mmol) and selenium dioxide (0.66 g, 6.0 mmol) in 1,4-dioxane (50 mL) was heated to 100 • C for 2 h (TLC monitoring). The mixture was diluted with saturated aqueous NaHCO 3 (50 mL) and extracted with CH 2 Cl 2 (100 mL × 3). The combined organic layers were washed with H 2 O followed by brine, dried with MgSO 4 and the solvent was removed in vacuo. The residue was recrystallized with EtOH to give quinoline-2-carbaldehydes 14 and 15. , and THF (30 mL) was stirred at 0 • C for 12 h (TLC monitoring). The reaction was quenched by addition of water (3 mL) and partitioned between H 2 O (50 mL) and CH 2 Cl 2 (50 mL). The organic layer was washed with brine, dried over MgSO 4 and the solvent was removed in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/CH 2 Cl 2 = 3/2) and recrystallized from EtOH to give compounds 16a-c and 17a-c.

General Procedure for Preparation of 2-benzoyl-3-phenylquinolines 18a-c and 19a-c
A mixture of 16a-c or 17a-c (1.0 mmol) and MnO 2 (10 mmol) in CH 2 Cl 2 (20 mL) was stirred at room temperature for 12 h (TLC monitoring). The reaction mixture was partitioned between H 2 O (50 mL) and CH 2 Cl 2 (50 mL). The organic layer was washed with brine, dried over MgSO 4 and the solvent was removed in vacuo. The residue was recrystallized from MeOH to give compounds 18a-c and 19a-c.

Molecular Docking Study
The crystal structure of TNF-á (PDB ID: 2AZ5) was acquired from the RCSB Protein Date Bank. The 3D conformation of target compound 18a and 18b was produced by ChemBio 3D Ultra 14.0. The molecular docking was performed by Achilles Blind Docking Server (http://bio-hpc.ucam.edu/ achilles/). The "blind docking" approach was used for the docking of the small molecule to the targets, which was done without a priori knowledge of the location of the binding site by the system [39]. Visual representation of molecules was created with 3Dmol by Nicholas Rego and David Koes [40].

Statistical Analysis
All data are presented as means ± SD. Each value is the mean of three independent experiments. Statistical analysis was assessed via one-way ANOVA followed by Tukey post-hoc test by GraphPad Prism 5 (San Diego, CA, USA), and the significant difference was set at *: p < 0.05; **: p < 0.01.

Conclusions
We have synthesized a total of sixteen 2-substituted 3-arylquinoline derivatives and evaluated their anti-inflammatory effect in LPS-activated murine macrophage cell line J774A.1 cells. Among these 2-substituted 3-arylquinoline derivatives, we found that compounds 18a and 18b have significant anti-inflammatory activities on LPS-activated macrophages through inhibition of the production of NO, TNF-α and IL-6 and attenuating the activity of NF-êB, repressing the expression of iNOS, and suppressing the phosphorylation of MAPKs. Compounds 18a and 18b might have potential starting points for the development of anti-inflammatory and immunosuppressive drugs in the treatment of sepsis and septic shock.