Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1β mRNA Expression

LPS induces inflammatory cytokines, including IL-1β, IL-6, and TNF-α, and causes an inflammatory response. The development of small molecules that have suppressive effect on those inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases. We synthesized 12 novel compounds with 4-amino-N-(4-(benzo[d]oxazol-2-ylamino)phenyl)butanamide moiety and evaluated their biological activities. Among them, 4 compounds (compound 5d, 5c, 5f, 5m and synthetic intermediate 4d) showed potent inhibition activities on IL-1β and IL-6 mRNA expression in vitro. Further, in vivo activity was evaluated with two compounds (5f and 4d) and mRNA levels of IL-1β, IL-6, and TNF-α were significantly decreased without hepatotoxicity. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for suppression of representative inflammatory cytokines.


Introduction
Inflammation is an essential immune response of the host to the exposure of tissues and organs to harmful stimuli such as microbial pathogens, irritants, or toxic cellular components that cause the ultimate restoration of tissue structure and function [1]. Innate and adaptive immune response are two main integral components of the host's defense system which are critical for pathogen-specific defense and immunological memory [2]. In the innate immune system, many classes of pathogens are recognized by their common molecular patterns. There are various types of pathogen-associated molecular patterns (PAMP), including lipopolysaccharides (LPS), aldehyde-derivatized proteins, mannans, teichoic acids, denatured DNA, and bacterial DNA. Pathogen-recognition receptors (PRR) are used for recognition of PAMP by a group of germline coded and evolutionary conserved proteins [3].
Lipopolysaccharide (LPS) is the major component of Gram-negative bacteria cell walls [4]. As it stimulates the release of inflammatory cytokines in various cell types, bacterial LPS has been extensively used to establish an inflammatory model [5]. LPS is initially extracted from bacterial membranes by LPS binding protein (LPB) in serum. LPS is transferred to CD14 by LBP, cleaved into monomeric molecules, and presented to the TLR4-MD-2 complex. Binding and aggregation of the TLR4-MD-2 complex with LPS leads to the activation of multiple signaling components such as NK-kB and IRF3. Subsequently, various pro-inflammatory cytokines are produced and inflammatory response occurs [6]. In our previous studies, we synthesized various benzoxazole derivatives and investigated their biological activities, particularly their anti-inflammatory effect [25][26][27][28][29]. We developed the compounds with 5-LOX inhibition effect. With an improvement effect of methacholine-induced airway hypersensitivity, it was confirmed that it has potential as an asthma treatment [27]. In addition, IL-6-mediated STAT3 phosphorylation inhibitors were developed. The compounds also showed the inhibitory effect on the secretion of inflammatory cytokines from effector Th1, Th2, and Th17 cells. From the results, we confirmed that the compounds have the potential to be candidates for rheumatoid arthritis (RA) treatment [28]. Further, we developed the compounds that have an inhibitory effect on IL-1β, IL-6, IL-13, TNF-α, perilipin (PLIN) 2, and PLIN 3 expression from bone marrowderived mast cells (BMMC) activated by LPS [29]. The N-phenylbenzo[d]oxazol-2-amine derivatives synthesized in previous studies have amino, methyl, or chloro substituents at the 5th position (R 2 ). In addition, relatively small substituents were present on the phenyl ring connected to the benzoxazole by an amino linker, such as 4-ethyl, 3-bromo or 2-methoxy, and 4-nitro groups.
Meanwhile, the compound synthesized in this study may have methyl substituent (R 1 ) at the 4th position (R 1 ), methyl, fluoro, t-butyl, methoxy or chloro substituent at the 5th position (R 2 ), or methyl substituent on the 6th position (R 3 ). Further, methoxy substituent may be present at the 2-position of the phenyl group (R 4 ) linked to the benzoxazole by amino linker. In particular, the 4-amino-butanamide substituent is characterized by a longer alkyl amino chain and is in the 4th position of the phenyl group (R 5 ).
This study synthesized benzoxazole derivatives with 4-amino-butanamide moiety and investigated their LPS-induced inflammatory response inhibitory effects. We investigated if and how benzoxazole derivatives can have an inhibitory effect on LPS-induced inflammation and whether it has the potential to be useful in the treatment of inflammation diseases by inflammation cytokines. We employed two types of cells and a mouse model of LPSinduced inflammation to examine whether the benzoxazole derivatives can have excellent suppressive effects in LPS-induced acute inflammation and how they can be modulated.

In Vitro Inhibition of mRNA Expression Levels of IL-6 and IL-1β
We screened 13 novel synthetic compounds (compounds 5a-m) and three of their intermediates (compounds 4d, 4e, and 4l) by measuring mRNA expression levels of IL-6 and IL-1β. Human keratinocyte HaCaT cell was used for the evaluation of suppressive effect of mRNA expression. Compounds 5d, 5c, 5f, 5m, and synthetic intermediate 4d showed the potent activities (Table 1). Compounds were administered at a concentration of 10 μM. IL-6 mRNA expression was 5.3 for compound 4d, 4.6 for compound 5c, 7.5 for compound 5d, 7.2 for compound 5f, and 9.0 for compound 5m. IL-6 mRNA expression was significantly lower than when inflammation was induced by administration of LPS alone. Table 1. Inhibitory effects of 4-amino-N-(4-(benzo[d]oxazol-2-ylamino)phenyl)butanamide derivatives on mRNA expression levels of IL-6 and IL-1β.
To assess the inhibitory effects of two compounds (4d and 5f) on the LPS-induced signaling of inflammation in human liver hepatocytes AML-12 cells, we tested their inhibition effects on the hepatocytes using a concentration range, where no cytotoxicity was observed. We performed a Western blotting assay to confirm changes in STAT3 or NF-κB activity and found that the phosphorylation of STAT3 or IκB-α was prevented in AML-12 cells treated with two compounds, and statistical significance was confirmed in the group treated with 10 µM compounds. As shown in Figure 2, the expression levels of pY-STAT3, p-IκBα, and p-NF-κB p65 in AML-12 cells were significantly downregulated by compounds 5f and 4f. Many studies have shown that LPS causes the inflammatory response by activating the STAT3 or NF-κB pathway and we expected that compounds of 4d and 5f modulate inflammation by inhibiting STAT3/NF-κB activity.

In Vivo Inhibition of mRNA Expression Levels of IL-6 and IL-1β in Mice
Two compounds (4d and 5f) were selected for the in vivo assay. Compounds were diluted with olive oil and injected into mice 1 h before LPS (1 mg/kg, 6 h) was administered to the mice. LPS was resuspended in saline solution and injected i.p. at a dose of 1 mg/kg of body weight. At the end of the trial, mice were sacrificed, and blood and liver were collected. For the evaluation of anti-inflammatory activity in mice, mRNA expression of IL-1β, IL-6, and TNF-α were measured. The results are shown in Figure 3.

Liver Morphology and Macrophage Infiltration
Liver histology of LPS treatment showed inflammatory activity compared to the control (Figure 3a). As a result of LPS administration, macrophage infiltration was observed in liver tissue. When compounds 4f and 5d were administered, it was observed that the inflammatory response was improved compared to when LPS alone was administered.
In order to characterize the phenotype of macrophages that accumulated in the LPSinduced inflammation, an immunohistochemical study was performed using antibodies against F4/80, a marker of mature macrophages (Figure 4b). When LPS alone was administered to induce inflammatory response, hepatic macrophage accumulation increased. As a result, there was a marked elevation of the number of F4/80-positive cells. When compounds 4f and 5d were administered, a significant decrease of F4/80 positive cells was observed.  In accordance with altered histological features, plasma alanine transferase (ALT) and aspartate transaminase (AST) levels were increased after LPS administration ( Figure 4c,d), and when compounds 4f and 5d were administered, ALT and AST levels decreased. Table 2. IC 50 (uM) values of the compounds 4d, 5c, 5d, 5f, and 5m on suppression of IL-6 mRNA expression.
Dose-response study was conducted with the 5 compounds at different concentrations to obtain IC50 (uM) ( Table 2). IC50 (uM) of compound 4d was 6.04 × 10 −5 , compound 5c was 1.64, compound 5d was 3.27 × 10 −2 , compound 5f was 4.44 × 10 −4 , and compound 5m was 1.99 × 10 −4 . Cell viability was measured by MTT assay, and the compounds did not show cytotoxicity (data now shown). Table 2. IC50 (uM) values of the compounds 4d, 5c, 5d, 5f, and 5m on suppression of IL-6 mRNA expression. To assess the inhibitory effects of two compounds (4d and 5f) on the LPS-induced signaling of inflammation in human liver hepatocytes AML-12 cells, we tested their inhibition effects on the hepatocytes using a concentration range, where no cytotoxicity was observed. We performed a Western blotting assay to confirm changes in STAT3 or NF-κB activity and found that the phosphorylation of STAT3 or IκB-α was prevented in AML-12 cells treated with two compounds, and statistical significance was confirmed in the group treated with 10 μM compounds. As shown in Figure 2, the expression levels of pY-STAT3, p-IκBα, and p-NF-κB p65 in AML-12 cells were significantly downregulated by compounds 5f and 4f. Many studies have shown that LPS causes the inflammatory response

Discussion
Here in this study, we synthesized novel compounds with benzoxazole moieties and investigated their suppressive effect on the mRNA expression of inflammatory cytokines and in vivo anti-inflammatory effect on LPS administration. A total of 13 compounds were synthesized by linking tert-butyloxycarbonyl (boc)-γ-butyric acid to the benzoxazole moieties with various substituents, followed by the removal of boc protection group by hydrolysis under acidic condition. Biological activity evaluation was carried out on 13 final compounds and three intermediates before boc protecting group hydrolysis.
To induce inflammatory response, lipopolysaccharide (LPS) was used for in vitro and in vivo tests. LPS is the bacterial cell wall of Gram-negative bacteria and is generally recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines. LPS is also known as pro-skin inflammation agent and many studies used LPS as the stimuli to construct in vitro cell skin inflammation model [30,31].
Bacterial LPS has been widely used to study inflammation due to the abundance of inflammatory effects that it generates through TLR signaling. TLRs are transmembrane receptors with an extracellular domain which interacts with a pathogen ligand and an intracellular domain involved in signaling. Especially, TLRs play an important role in the recognition of pathogens in keratinocytes. In this study, we used LPS to induce inflammation in human keratinocyte HaCat cells and investigated the inhibitory effects of the compounds on inflammatory cytokine expression [32,33].
We examined mRNA expression levels of IL-6 and IL-1β of the experimental group and compared with those of the control groups. The inhibitory effects of the compounds on IL-6 and IL-1β showed in similar pattern. Compounds that were most effective in inhibiting IL-6 mRNA expression (compounds 5c, 5d, 5f, 5m, and 4d) were also the most effective in inhibiting IL-1β mRNA expression. Except for compound 5m, the compounds with potent activity were compounds in which the 2-position of the benzene ring (R 4 ) was not substituted with a methoxy group. Compounds 5f and 4d, which showed high activity in both IL-6 and IL-1β, were substituted with a tert-butyl group and a methyl group at the 5th position of benzoxazole (R 2 ), respectively. Compound 4d, an intermediate without removing the boc protecting group, showed high anti-inflammatory activity compared to other intermediates (compounds 4e and 4l). Among the compounds, when the substituent at the R 2 position was a tert-butyl group, the activity was good regardless of the presence or absence of a methoxy group at the R 4 position (compounds 5f and 5m). In the case of a methyl group at the R 2 position, the activity was generally good regardless of the presence or absence of a methoxy group at the R 4 position and a boc group at the R 5 position. When there was a methyl group at the R 3 position, the activity was not very good.
From the in vitro tests, two compounds were selected and were evaluated in vivo tests to test inhibition activity on the expression of IL-6, IL-1β, and TNF-α by injecting LPS into mice. Following to the LPS administration, severe macrophage infiltration was observed in liver tissue while administration of compounds 5f and 4d alleviated the inflammatory response. We also checked mRNA levels of IL-6, IL-1β, and TNF-α levels in the liver tissue. Compounds 5f and 4d showed significant suppressive activity on IL-6, IL-1β, and TNF-α expression levels. In addition, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were examined to investigate the hepatotoxicity of the compounds, which was confirmed to be lower than the control group injected LPS only. We confirmed that the potent anti-inflammatory activity of the two compounds through in vivo study, and it was found that the difference in activity between the two compounds was not significant.
Through LPS-induced inflammatory responses, proinflammatory cytokines IL-6, IL-1β, and TNF-α are activated. It is important to effectively control the cytokines since the cytokines play an important role in the inflammatory response.
In this study, we developed novel benzoxazole derivatives and confirmed their antiinflammatory activity by modulating pro-inflammatory cytokine expressions. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for suppression of representative inflammatory cytokines such as IL-1β, IL-6 and TNF-α. Therefore, it is possible to develop small molecule modulators for inflammatory diseases from these compounds through further study in the future.

Materials and Methods
Melting points were measured on an electro thermal digital melting point (Büchi, Germany) without calibration. 1 H-NMR spectra were recorded on Varian NMR AS and Varian Unity Inova 400 MHz NMR spectrometers. Chemical shifts were reported in parts per million (δ) units relative to the solvent peak. The 1 H NMR data were reported as peak multiplicities: s for singlet; d for doublet; t for triplet; q for quartet; and m for multiplet. Coupling constants were recorded in hertz (Hz). MS spectra were measured using Jeol JMS 700 high resolution mass spectrometer from the Korea Basic Science Institute (Daegu). Reagents were of commercial grade and were purchased from Sigma-Aldrich Co. (St. Louis, MI, USA), Merck (Darmstadt, Geramny), Duksan Pure Chemical Co (Ansan, Korea).

Synthesis of Substituted-N-(4-nitrophenyl)benzo[D]oxazol-2-amine Derivatives (2a-g)
First, 5 mL of dried acetonitrile was added to potassium hydroxide (KO 2 , 5 eq.) under N 2 atmosphere. 1-(Substituted-2-hydroxyphenyl)-3-(4-nitrophenyl)thioureas (1a-g) (100 mg, 1 eq.) solution in acetonitrile was delivered dropwise to the KO 2 -acetonitrile mixture. The reaction mixture was vigorously stirred at room temperature for 16 h under N 2 atmosphere. After the reaction, cold water was added to the reaction mixture and extracted with dichloromethane, followed by washing with brine solution. After drying with anhydrous MgSO 4 and filtration, the organic solvent was removed under reduced pressure to prepare the compounds 2a-g.

Synthesis of N 1 -(Substitutedbenzo[D]oxazole-2-yl)benzene-1,4-diamine Derivatives (3a-g)
First, 10 mL of ethanol was added to substituted-N-(4-nitrophenyl)benzo[d]oxazol-2-amine (2a-g) (100 mg, 1 eq.) and Tin(II) chloride (12 eq.). The reaction mixture was ultrasonicated for 3 h at room temperature. Potassium hydroxide solution was added to the reaction solution, and the mixture was extracted with ethyl acetate, followed by washing with brine solution. After drying with anhydrous MgSO 4 and filtration, the organic solvent was removed under reduced pressure to prepare the compounds 3a-f. First, 10 mL of dimethylformamide was added to N 1 -(substitutedbenzo[d]oxazole-2-yl)benzene-1,4-diamine derivatives (3a-g) (100 mg, 1 eq.), Boc-GABA-OH (1 eq.) and PyBOP (1.2 eq.) and stirred at 0 • C. Diisopropylethylamine (2 eq.) to the reaction mixture and the reaction mixture was stirred for 16 h at room temperature. After the reaction, 10% HCl solution was added to the reaction mixture and extracted with ethyl acetate, followed by washing with sodium bicarbonate solution and brine solution. After drying with anhydrous MgSO 4 and filtration, the organic solvent was removed under reduced pressure to prepare the compounds 4a-g.

Quantitative Real-Time PCR Analysis
Total RNA was isolated from tissue with the use of the TRIzol reagent (Invitrogen) and was processed for reverse transcription (RT) and real-time PCR analysis with an ABI PRISM 7700 system (PE Biosystems). Data were normalized by the amount of GAPDH mRNA. Primers are listed in Table 3.

Statistical Analysis
All quantitative data were analyzed with GraphPad Prism software and represented as mean ± SD. The statistical significance of the data between control and treatment groups was determined using one-way analysis of variance (ANOVA) followed by the Tukey posthoc test was used to determine differences between experimental groups. A p-value < 0.05 was considered statistically significant.

Conclusions
We synthesized 13 novel compounds with 4-amino-N-(4-(benzo[d]oxazol-2-ylamino) phenyl)butanamide moiety and evaluated their biological activities. Five compounds (compound 5d, 5c, 5f, 5m, and synthetic intermediate 4d) showed potent inhibition activities on IL-1β and IL-6 mRNA expression in vitro. Further, two compounds (5f and 4d) significantly decreased mRNA levels of IL-1β, IL-6, and TNF-α without hepatotoxicity in vivo. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for the suppression of representative inflammatory cytokines.

Institutional Review Board Statement:
The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Ewha Womans University Animal Experimentation Ethics Committee (protocol code EWHA IACUC 19-001).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.