Design, Synthesis, and Biological Evaluation of 3-Substituted-Indolin-2-One Derivatives as Potent Anti-Inflammatory Agents

This study aimed to synthesize and evaluate the anti-inflammatory activity of 3-substituted-indolin-2-one derivatives. Cell viability of 3-substituted-indolin-2-one derivatives was measured with the EZ-Cytox reagent; interleukin (IL)-6, tumor necrosis factor (TNF)-α, and inducible NOS mRNA levels were measured using Taqman qRT-PCR; pro-inflammatory cytokine IL-6 and TNF-α levels were determined using ELISA kits; the phosphorylation of Akt, JNK, ERK, p38, p65, and IκB protein levels were measured by immunoblotting. Among the nineteen 3-substituted-indolin-2-one derivatives synthesized, 3-(3-hydroxyphenyl)-indolin-2-one showed the highest anti-inflammatory activity, inhibiting the nitric oxide production related to inflammation, suppressing the production of TNF-α and IL-6 in a concentration-dependent manner and mRNA expression. Moreover, 3-(3-hydroxyphenyl)-indolin-2-one significantly inhibited lipopolysaccharide (LPS)-induced signal pathways such as the Akt, MAPK, and NF-κB signaling pathways. Our findings revealed that a 3-substituted-indolin-2-one derivative, 3-(3-hydroxyphenyl)-indolin-2-one, possesses excellent anti-inflammatory activity and can be considered for future research.


Introduction
Inflammation is a physiological and pathological response that activates immune and non-immune cells that protect host cells or organs from infectious species, including bacteria, viruses, and toxins, by eliminating pathogens and promoting tissue repair and recovery [1][2][3]. In well-controlled inflammation, the initiation phase is involved in host defense by rapid and robust immune activation. The resolution phase curtails inflammation and restores tissue homeostasis when the danger signal is eliminated [4]. Uncontrolled inflammation can cause chronic inflammatory diseases, such as arthritis, colitis, and asthma. These diseases are associated with severe tissue damage and an increased risk for the development of cardiovascular diseases, cancer, and osteoporosis [5][6][7][8].
The inflammatory response involves various cells, including neutrophils, macrophages, and lymphocytes [9]. In macrophages, inflammation is initiated by lipopolysaccharide (LPS) which is a lipid and a polysaccharide of Gram-negative bacteria. LPS can increase the expression of tumor necrosis factor α (TNF-α) and the release of inflammatory cytokines such as interleukin (IL)-6, IL-1, IL-1β, and nitric oxide (NO) [10,11]. As a result, LPS can induce an acute inflammatory response. Activated macrophages enhance the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), which act as inflammatory mediators and immune modulators. The structure of the aurones consists of benzofuran-3-one and benzylidene, which are attached to two positions on benzofuran-3-one. Previous studies synthesized azaaurones as aurone derivatives and evaluated their biological effects [32][33][34][35]. In addition, an azaaurone scaffold has been utilized as an antimalarial agent [36].
The structure of the aurones consists of benzofuran-3-one and benzylidene, which are attached to two positions on benzofuran-3-one. Previous studies synthesized azaaurones as aurone derivatives and evaluated their biological effects [32][33][34][35]. In addition, an azaaurone scaffold has been utilized as an antimalarial agent [36].
To identify structurally similar aurones with anti-inflammatory activity, we designed 3-substituted-indolin-2-one derivatives based on the aurone and azaaurone structures ( Figure 2). The nineteen designed 3-substituted-indolin-2-one derivatives were synthesized and assayed for their anti-inflammatory activities.  The compounds described in this study were prepared using straightforward chemical synthesis ( Figure 3). The nineteen derivatives were synthesized following a previously described method with minor modifications [37,38]. The reaction mixtures were purified by preparative high-performance liquid chromatography to afford major compounds. The desired derivatives were characterized by 1 H nuclear magnetic resonance (NMR), 13 C NMR, and liquid chromatography-mass spectrometry (Supplementary Figure S1). In the previous report, chemical shifts of H-2 and H-6 protons in the phenyl ring at the C-3 position of 3-substituted-indolin-2-one were observed to determine E and Z isomers. We followed the reported method to determine the configurations of nineteen synthesized analogs [37,38].
pounds. The desired derivatives were characterized by 1 H nuclear magnetic resonance (NMR), 13 C NMR, and liquid chromatography-mass spectrometry (Supplementary Figure  S1). In the previous report, chemical shifts of H-2′ and H-6′ protons in the phenyl ring at the C-3 position of 3-substituted-indolin-2-one were observed to determine E and Z isomers. We followed the reported method to determine the configurations of nineteen synthesized analogs [37,38].

Effect of 3-(3-Hydroxyphenyl)-Indolin-2-One on iNOS Expression in LPS-Induced RAW264.7 Cells
NO is an inorganic vitreous substance produced by nitric oxide synthase and is involved in various biological processes such as the immune response, cytotoxicity, and neurotransmission. NO is produced from l-arginine by nitric oxide synthases (NOS), neuronal NOS, endothelial NOS, and iNOS present in various tissues and cells.
Therefore, we attempted to confirm the inhibition of LPS-induced iNOS protein expression by 3-(3-hydroxyphenyl)-indolin-2-one. RAW264.7 cells were treated with various concentrations of 3-(3-hydroxyphenyl)-indolin-2-one for 2 h and then treated with LPS for 12 h. As shown in Figure 6a, iNOS protein expression was strongly suppressed by 3-(3-hydroxyphenyl)-indolin-2-one treatment in a concentration-dependent manner, sim-ilar to that observed for nitric oxide and iNOS mRNA levels. The bar chart displays the intensity of the immunoblot bands visualized using Image J software. In addition, this result correlated with the inhibition of nitric oxide (Figure 4b) and mRNA expression of iNOS (Figure 5b). ronal NOS, endothelial NOS, and iNOS present in various tissues and cells.
Therefore, we attempted to confirm the inhibition of LPS-induced iNOS protein expression by 3-(3-hydroxyphenyl)-indolin-2-one. RAW264.7 cells were treated with various concentrations of 3-(3-hydroxyphenyl)-indolin-2-one for 2 h and then treated with LPS for 12 h. As shown in Figure 6a, iNOS protein expression was strongly suppressed by 3-(3-hydroxyphenyl)-indolin-2-one treatment in a concentration-dependent manner, similar to that observed for nitric oxide and iNOS mRNA levels. The bar chart displays the intensity of the immunoblot bands visualized using Image J software. In addition, this result correlated with the inhibition of nitric oxide (Figure 4b) and mRNA expression of iNOS (Figure 5b).
To elucidate the molecular mechanisms underlying the anti-inflammatory signaling pathway induced by 3-(3-hydroxyphenyl)-indolin-2-one, we first investigated the phosphorylation of Akt and MAPK (ERK, JNK, p38) proteins, which are representative signaling proteins activated by LPS in RAW264.7 cells.
As shown in Figure 7a, phosphorylation of MAPK (JNK, ERK, p38) and Akt was induced after 30 min of LPS treatment and was strongly inhibited by 3-(3-hydroxyphenyl)indolin-2-one treatment at 80 µM. We also confirmed that 3-(3-hydroxyphenyl)-indolin-2-one inhibited LPS-induced phosphorylation of JNK, ERK, and p38 in a concentrationdependent manner (20-80 µM). In addition, LPS-induced phosphorylation of Akt protein was significantly inhibited in a concentration-dependent manner by 3-(3-hydroxyphenyl)indolin-2-one treatment (Figure 7a). Next, we analyzed the NF-κB pathway, a representative pathway that induces LPS-induced inflammation. When a cell is in a normal state, IκBα protein exists in a complex with p65 and p50 proteins in the cytoplasm; however, when stimulated by LPS, IκBα is phosphorylated and then degraded [7]. At the same time, p65 is phosphorylated and translocated into the nucleus to act as a transcription factor. As shown in Figure 7b, when cells were treated with LPS, IκBα phosphorylation, IκBα degradation, and p65 phosphorylation were observed. However, treatment with 3-(3-hydroxyphenyl)indolin-2-one strongly suppressed IκBα phosphorylation, IκBα degradation, and p65 phosphorylation in a concentration-dependent manner in RAW264.7 cells (Figure 7b). Taken together, these results indicate that 3-(3-hydroxyphenyl)-indolin-2-one inhibits the production of inflammatory factors such as nitric oxide, IL-6, and TNF-α by strongly inhibiting the phosphorylation of Akt, MAPKs (JNK, ERK, and p38), and the NF-κB pathway (Figure 7).
2-one inhibited LPS-induced phosphorylation of JNK, ERK, and p38 in a concentrationdependent manner (20-80 µM). In addition, LPS-induced phosphorylation of Akt protein was significantly inhibited in a concentration-dependent manner by 3-(3-hydroxyphenyl)indolin-2-one treatment (Figure 7a). Next, we analyzed the NF-κB pathway, a representative pathway that induces LPS-induced inflammation. When a cell is in a normal state, IκBα protein exists in a complex with p65 and p50 proteins in the cytoplasm; however, when stimulated by LPS, IκBα is phosphorylated and then degraded [7]. At the same time, p65 is phosphorylated and translocated into the nucleus to act as a transcription factor. As shown in Figure 7b, when cells were treated with LPS, IκBα phosphorylation, IκBα degradation, and p65 phosphorylation were observed. However, treatment with 3-(3-hydroxyphenyl)-indolin-2-one strongly suppressed IκBα phosphorylation, IκBα degradation, and p65 phosphorylation in a concentration-dependent manner in RAW264.7 cells ( Figure  7b). Taken together, these results indicate that 3-(3-hydroxyphenyl)-indolin-2-one inhibits the production of inflammatory factors such as nitric oxide, IL-6, and TNF-α by strongly inhibiting the phosphorylation of Akt, MAPKs (JNK, ERK, and p38), and the NF-κB pathway (Figure 7). (c) pAKT, pJNK, pERK, and pp38 protein levels were validated and total protein levels were obtained using the ImageJ software. (d) pIκB, IκB, and p65 protein levels were validated using the Image J software. β-actin was used as an internal control. Data are presented as the mean ± standard deviation (SD) of three independent experiments. White bar: non-treated group, black bar: LPS treated group, gray bar: sample treated group. # p < 0.0001 vs. the control group. *** p < 0.0001, ** p < 0.001 or * p < 0.05 vs. the LPS group.

Discussion
Inflammation is a defense mechanism against harmful stimuli in the body. When cells and tissues are stimulated externally, vasoactive substances are released locally, causing inflammation, fever, pain, and loss of function [39]. NSAIDs with analgesic, antipyretic, and anti-inflammatory actions are commonly used globally in acute and chronic inflammatory conditions [40]. NSAIDs are commonly used to treat various types of diseases related to inflammation, pain, and fever by reducing the synthesis of prostaglandins via blocking the COX enzyme [41]. However, their extensive use has associated side effects such as gastrointestinal disorders and edema [42]. Accordingly, many studies have (c) pAKT, pJNK, pERK, and pp38 protein levels were validated and total protein levels were obtained using the ImageJ software. (d) pIκB, IκB, and p65 protein levels were validated using the Image J software. β-actin was used as an internal control. Data are presented as the mean ± standard deviation (SD) of three independent experiments. White bar: non-treated group, black bar: LPS treated group, gray bar: sample treated group. # p < 0.0001 vs. the control group. *** p < 0.0001, ** p < 0.001 or * p < 0.05 vs. the LPS group.

Discussion
Inflammation is a defense mechanism against harmful stimuli in the body. When cells and tissues are stimulated externally, vasoactive substances are released locally, causing inflammation, fever, pain, and loss of function [39]. NSAIDs with analgesic, antipyretic, and anti-inflammatory actions are commonly used globally in acute and chronic inflammatory conditions [40]. NSAIDs are commonly used to treat various types of diseases related to inflammation, pain, and fever by reducing the synthesis of prostaglandins via blocking the COX enzyme [41]. However, their extensive use has associated side effects such as gastrointestinal disorders and edema [42]. Accordingly, many studies have been conducted in recent years to discover new active materials with low toxicity and excellent antiinflammatory action using natural product derivatives [43,44].
The nineteen 3-subsituted-indolin-2-one derivatives were examined at various concentrations (10-80 µM) based on their ability to preserve cell viability and induce the inhibitory activity of nitric oxide using RAW264.7 cells. Among the nineteen 3-subsituted-indolin-2one derivatives, 3-(3-hydroxyphenyl)-indolin-2-one showed the lowest cytotoxicity and was confirmed to exhibit excellent NO inhibitory activity. Activated macrophages produce inflammatory mediators such as NO, TNF-α, and IL-6, and increase the inflammatory response at the early stage of infection [45]. TNF-α is a representative cytokine plays an important role in the inflammatory response, and induces the production of other cytokines to sustain the inflammatory response [46,47]. Fever symptoms appear when IL-6 is excessively produced in an inflammatory response to LPS. The production of TNF-α and IL-6 in LPS-stimulated RAW264.7 cells was decreased in a concentration-dependent manner compared with the LPS-treated group using 3-(3-hydroxyphenyl)-indolin-2-one treatment at 20, 40, and 80 µM. This result is consistent with previous studies, and the inflammatory response in the early stages of infection can be effectively controlled by regulating the production of TNF-α and IL-6. These results imply that the regulation of MAPKs and NF-kB suppresses the inflammatory response [48,49]. In macrophages, iNOS, overexpressed by LPS, produces nitric oxide, intensifying the inflammatory response. Analysis of iNOS expression in LPS-stimulated RAW264.7 cells showed that iNOS expression was decreased in a concentration-dependent manner by 3-(3-hydroxyphenyl)-indolin-2-one treatment at 40 or 80 µM. Therefore, 3-(3-hydroxyphenyl)-indolin-2-one is expected to ultimately reduce NO production by inhibiting iNOS expression in RAW264.7 cells. The MAPK pathway is activated by external stimuli, and various intracellular responses occur through the phosphorylation of specific substrates in the cell. ERK, JNK, and p38 belong to the MAPK subfamily. p38 and SAPK are protein kinases belonging to the MAP kinase family [50][51][52]. SAPK is also called JNK because it can phosphorylate the N-terminal region of c-Jun. p38 and JNK kinases are activated by inflammatory cytokines, such as TNF-α and IL-1β, various stress stimuli, ultraviolet rays, and chromosome-damaging substances, and induce various biological responses, such as cell differentiation, apoptosis, and inflammatory responses. ERK, another MAP kinase, is involved in the signaling pathways that promote cell growth and differentiation [50][51][52]. Analysis of MAPK phosphorylation and the NF-κB pathway by LPS shows that the increased phosphorylation of MAPKs, NF-κB subunit p65, and IκBα, along with degradation of IκBα are significantly prevented by treatment with 3-(3-hydroxyphenyl)-indolin-2-one in a concentration-dependent manner in RAW264.7 cells. During the inflammatory response, NF-κB regulates the expression of iNOS and COX-2. p65 protein acts as a transcriptional regulator and increases the expression of iNOS and COX-2 to regulate the inflammatory response [53,54]. Therefore, we hypothesize that the inhibition of phosphorylation and degradation of IκBα and phosphorylation of p65 induced by 3-(3-hydroxyphenyl)-indolin-2-one decrease the expression of iNOS.

Conclusions
Considering the above results, 3-(3-hydroxyphenyl)-indolin-2-one was confirmed as a non-toxic compound in the RAW264.7 cell line. Moreover, it was verified that 3-(3hydroxyphenyl)-indolin-2-one effectively inhibits pro-inflammatory mediator (NO, TNF-α, IL-6) production by LPS stimulation. In addition, the intracellular signal transduction of anti-inflammatory activity of 3-(3-hydroxyphenyl)-indolin-2-one was attenuated by regulating the Akt, MAPK, and NF-κB signaling pathways in RAW264.7 cells. These results show that 3-(3-hydroxyphenyl)-indolin-2-one could be developed as a drug for the prevention of diseases based on inflammation (e.g., arthritis, inflammatory bowel disease, inflammatory skin disease, gastritis, etc.). However, these experiments were performed by cell lines in vitro; further research applied to animal experiments will be conducted to verify the results.

Cell Culture
RAW264.7 cells (Korean Cell Line Bank, Seoul, Republic of Korea) were cultured in Dulbecco's modified Eagle's medium (Corning, NY, USA) supplemented with 10% fetal bovine saline and 1% penicillin and streptomycin. Cell growth conditions were determined at 37 • C and humidified at 5% CO 2 .

Cell Viability
RAW264.7 macrophage cells were seeded into 96-well plates. The nineteen 3-substitutedindolin-2-one derivatives were diluted with culture medium to 20, 40, and 80 µM concentrations and then treated with cells for 2 h. Subsequently, the cells were treated with LPS (500 ng/mL) for 20 h. Cell viability was determined using the EZ-Cytox reagent. Absorbance was measured at 450 nm using a microplate reader.

Determination of Nitric Oxide (NO)
NO production from cell culture supernatants treated with nineteen 3-substitutedindolin-2-one derivatives was analyzed using the Griess method. Briefly, cells were stimulated with 20, 40, and 80 µM of the nineteen 3-substituted-indolin-2-one derivatives for 2 h and then treated with LPS (500 ng/mL) for 20 h. Nitric oxide in the cell supernatants was reacted with an equal volume of Griess reagent.

Preparation of Cell Lysate and Immunoblotting
RAW264.7 cells were plated into a 6-well plate (2 × 10 6 cells/well) and then treated with 3-(3-hydroxyphenyl)-indolin-2-one for 2 h, followed by treatment with LPS for 30 min (for NF-κB p65, IκB-α and MAPKs proteins) or 18 h (for iNOS protein). Then, washed cells were lysed using radioimmunoprecipitation assay buffer containing a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), dithiothreitol (Wako, Tokyo, Japan), and complete™ Mini Protease Inhibitor Cocktail (Roche Diagnostics Corp., IN, USA). The soluble proteins from cell lysates were collected after centrifugation at 13,000 rpm for 20 min at 4 • C. The SDS-PAGE separated the proteins, and proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked using skim milk at 4 • C overnight. The PVDF membranes were incubated with specific primary antibodies, diluted with TBS with Tween-20 (0.5%). The membranes were washed three times with TBS-T buffer, followed by treatment with a secondary antibody linked to horseradish peroxidase (HRP). Protein was detected using Super Signal ® West Femto Substrate (Thermo Fisher, CA, USA) and developed using a Fusion Solo Chemiluminescence System (Vilber Lourmat, Paris, France) ECL detection system.

Statistical Analysis
The results are expressed as mean ± standard deviation of triplicate experiments. Results were analyzed statistically using the Mann-Whitney U-test in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA), with p < 0.05 considered statistically significant.