Synthesis and cytotoxic activity of ethyl ferulate derivatives as potent anti-inflammatory agents

Abstract While a range of pharmacological agents are currently used to alleviate inflammation, the clinical administration of many of these anti-inflammatory drugs is associated with high rates of adverse side effects that make them poorly suited to long-term use. Therefore, there is a critical need for the development of novel anti-inflammatory agents. Natural compounds and derivatives like ethyl ferulate have risen to prominence as a foundation for many drug discovery efforts owing to their structural diversity and wide-ranging biological activities. In the present study, 24 ethyl ferulate derivatives were synthesized. Their anti-inflammatory activity was evaluated in vitro using RAW264.7 cells and CCK-8, ELISA, and Western blotting assays. These analyses revealed that most of the synthesized compounds exhibited moderate to high anti-inflammatory activities. In particular, c10 and c23 exerted more pronounced activity than ethyl ferulate or dexamethasone with respect to the suppression of tumour necrosis factor-α production by RAW264.7 cells through the targeting of the NF-κB and MAPK signalling pathways, suggesting that these compounds warrant further investigation. Graphical Abstract


Introduction
Inflammation is a key physiological defense response engaged in response to adverse stimuli, resulting in symptoms including redness, swelling, heat, pain, and dysfunction.Inflammatory activity has been linked to conditions including cardiovascular disease, cancer, and diabetes.Natural products play a critical role in drug discovery, particularly in the treatment of inflammation, as these products are inexpensive, harbour diverse compounds, and are associated with few adverse reactions (Bagherniya et al. 2021;Li et al. 2013;Yang et al. 2017).Moreover, synthesised and structural modification natural product compounds was beneficial to improve the bioactivity and properties (Duan et al. 2021;Mahal et al. 2019).
Ethyl ferulate (ethyl-3-hydroxy-4-methoxycinnamate) is a phenylpropanoid derivative of ferulic acid that is found at high levels in maize, rice, and other grains (Cunha et al. 2019).It has emerged as a focus of clinical interest owing to its beneficial pharmacological properties including antioxidant, anti-inflammatory, renoprotective, and neuroprotective activities (Pang et al. 2021).As it harbours an ester group, ethyl ferulate is highly lipophilic and thus more readily able to cross lipid-rich cellular membranes.These lipophilic properties make ethyl ferulate of particular value when seeking to transport specific compounds across the blood-brain barrier (Scapagnini et al. 2004).Under primary cell culture conditions, treatment with ethyl ferulate has been shown to reduce the upregulation of inducible nitric oxide synthase (iNOS) in response to amyloid-beta exposure, thereby suppressing intracellular nitration and associated nitrosative stress (Sultana et al. 2005).Consistently, multiple studies have demonstrated that ethyl ferulate can decrease the expression of iNOS, COX2, and PGE2.When used to treat macrophages that have been stimulated with lipopolysaccharide (LPS), it can further inhibit the production of pro-inflammatory IL-1b, IL-6, and TNF-a.
Consistent with such anti-inflammatory activity, ethyl ferulate suppresses p-IrBa expression together with NF-rB p65 nuclear translocation, while additionally promoting Nrf2/HO-1 pathway activation and reducing reactive oxygen species (ROS) production within cells.When applied in vivo, ethyl ferulate can further reduce MPO activity, constrain leukocyte infiltration, and reduce both the expression and secretion of TNF-a and IL-6 (Islam et al. 2009;Nazare et al. 2014;Shi et al. 2021), protecting against murine acute lung injury in response to LPS treatment (Wu et al. 2021).
Thiadiazole is a bioisostere derived from oxadiazole and pyrimidine with significant potential therapeutic utility.As thiadiazole contains a sulfur atom, it exhibits superior liposolubility, making it well-equipped to cross the cell membrane owing to its mesoionic nature (Li et al. 2013;Mahal et al. 2017).Owing to these properties, thiadiazole derivatives possess a range of antitumor, antiviral, antibacterial, antifungal, antiparasitic, antidiabetic, anticonvulsant, and anti-inflammatory activities (Li et al. 2013;Hu et al. 2014;Dawood and Farghaly 2017).Moreover, specific thiadiazole ring-containing compounds (Figure 1B) have been shown to effectively inhibit COX2 enzymes with IC 50 values of 0.42-0.1 lM (Said, Mohd. & Naira 2017;Yasser et al. 2020).As such, the preparation of a thiadiazole-ethyl ferulate hybrid and modified versions thereof has the potential to be of interest as a means of producing novel compounds with potentially robust anti-inflammatory activity.The amide functional group is an important structural component of many anti-inflammatory drugs in current clinical use including indomethacin and piroxicam (Figure 1C).As such, a series of novel ethyl ferulate derivatives (C1-C24) were synthesized in this study via the amide bond-based conjugation of ethyl ferulate and thiadiazole.

Chemistry
Synthesis strategies used to generate the target compounds analyzed in the present study are outlined in Figure 2. Overall, synthesis reactions were conducted through three main steps.First, ethyl ferulate was treated with bromoacetic acid for 4 h at 60 C via the Williamson reaction, yielding intermediate B. Intermediates A1-A24 were generated using commercial aniline, phosphorus oxychloride, thiosemicarbazide, and replaced benzoic acid.The finalized product manufacturing phase was conducted concomitantly, with water-soluble EDCI and HOBt being used for activation of carboxylic acid, together with treatment using a relevant amine in conjunction with DMAP. 1 H-NMR, 13 C-NMR, and HRMS were used for the structural characterization of all prepared compounds.All synthesized target compounds were successfully validated.In hydrogen spectra, the singlet with a hydrogen number of two near the 5 ppm shift corresponds to the methylene hydrogen introduced into ethyl ferulate, and the singlet hydrogen near the 13 ppm shift corresponds to the hydrogen attached to the nitrogen on the amide group.

Structure-activity relationships (SARs)
Toxicological experiments (Supplementary Information S2) showed that the toxicity of the compounds increased when the benzothiadiazole benzene ring was unsubstituted or when Cl, CH 3 , and OCH 3 substituents were introduced alone on the benzene ring, however, when the benzene ring is polysubstituted, the toxicity of the compound will be reduced.LPS-induced TNF-a production by RAW264.7 cells treated with these 24 novel compounds was additionally assessed (Supplementary material Figure S4), revealing that c9-11, c15, and c23 significantly inhibited the release of inflammatory TNF-a.This suggests that structural activity increases with the introduction of a Br group into the benzothiadiazole benzene ring, with the following order of activity: 4-Br > 3-Br > 2-Br.The activity of this series of derivatives also rose with benzene ring 2-CH 3 or 2-CF 3 substitution.

Analysis of the effects of synthesized compounds on RAW264.7 macrophage survival
To assess the ability of these 24 synthesized compounds to alter macrophage survival, we treated RAW264.7 cells with different synthesized compounds at a dose of 40 lg/ mL and then evaluated cytotoxicity via MTT assay.This analysis revealed c1-4, c9, c11-15, c18, and c22 to induce significant cytotoxicity at the 40 lg/mL dose (Baker et al) (Supplementary material Figure S1).As such, we further examined the anti-inflammatory activity of the 24 synthesized compounds at the 20 lg/mL dose level.
2.4.Analysis of the effects of synthesized compounds upon LPS-driven TNF-a discharge from RAW264.7 TNF-a consists of a critical inflammatory cytokine that is secreted by macrophages and other immune cell types in response to bacterial infection or other stimuli such as LPS, thereupon regulating local and systemic responses associated with autoimmunity and chronic inflammation (Zhang et al. 2008;Hung et al. 2017;Shi et al. 2020).An ELISA approach was next used to examine the effects of the 24 different synthesized compounds on LPS-induced TNF-a release from macrophages (Supplementary material Figure S2).LPS treatment (20 lg/mL) was associated with the robust production of this cytokine, while its release was significantly inhibited by treatment with c9-11, c15, and c23 as compared to ethyl4-hydroxy-3-methoxycinnamate.Of these, compounds c10 and c23 exhibited the most robust inhibitory efficacy comparable to that of dexamethasone, and these two compounds were thus selected for further analysis.

Assessment of the effects of compounds c10 and c23 on IL-6/TNF-a expression by LPS-treated RAW264.7 cells
Given such observed effects of c10 and c23 on LPS-induced TNF-a secretion, their impact on TNF-a and IL-6 expression was next examined via Western blotting.While LPS promoted the upregulation of both of these inflammatory cytokines, both c10 and c23 markedly suppressed their expression (Supplementary Material Figure S3).
2.6.Analysis of the impact of compounds c10 and c23 on MAPK signaling pathway activity in LPS-treated RAW264.7 cells MAPK serine-threonine protein kinases can be activated in response to multiple extracellular stimuli, including hormones, stress-related factors, neurotransmitters, cytokines, and cell adhesion activity (Cuadrado and Nebreda 2010;Yue and L opez 2020).The ERK, JNK, and p38 MAPK subfamilies can be activated in response to LPS stimulation, in turn promoting the activation of myriad transcription factors and thereby modulating IL-1, IL-6, IL-8, and TNF-a expression (Lai et al. 2017;Wu et al. 2020).In order to probe the anti-inflammatory effects of c10 and c23 treatment in further detail, the phosphorylation of ERK, p38, and JNK was assessed.Significant decreases in phosphorylated ERK/JNK/p38 levels were observed in LPS-treated RAW264.7 macrophages that had been treated with c10 or c23 (Supplementary Material Figure S4), confirming that the anti-inflammatory effects of these novel compounds are at least partially mediated via the MAPK pathway.
2.7.The effects of c10 and c23 on LPS-driven NF-jB signalling activity within RAW264.7 macrophages Several studies have shown that LPS-induced NF-jB activation is central to multiple pathogenic conditions (Mitchell and Carmody 2018).NF-jB serves as the central regulator of many aspects of inflammatory signalling (Baker et al), and Western blotting was thus used to evaluate the impact of c10/c23 on LPS-driven NF-jB signalling in RAW264.7 cells.As both c10 and c23 were able to significantly inhibit LPS-induced NF-jB p65 activation (Supplementary material Figure S5), this suggests that both of these compounds can suppress NF-jB signalling.

General procedures
Chemicals and spectral-grade solvents used in the present study were purchased from commercial sources and subject to no additional purification.Standard protocols were used to dry and utilize solvents.TLC was used as a means of monitoring all chemical reactions, with TLC plate visualization being achieved via ultraviolet light exposure (254 and 365 nm).Flash column chromatography using silica gel (200-300 mesh) was employed to purify compounds.An RY-1 MP apparatus was used to measure compound melting point values, while an AV-300 (Bruker, Switzerland) instrument was used to assess uncorrected 1 H-NMR and 13 C-NMR spectra, with tetramethylsilane serving as a reference standard.Chemical shifts were registered as parts per million relative to tetramethylsilane.A Q Exactive instrument (Thermo Scientific, USA) was used for mass spectra measurements.

Preparation of compounds A1-A24
A mixture of different forms of substituted benzoic acid (1.0 mmol), thiosemicarbazide (91.1 mg, 1.0 mmol), and POCl 3 (0.6 mL) was warmed (150 min at 80 C), cooled to room temperature, and then 2 mL of water was then added to this solution.Solution was then refluxed for 240 min, cooled, and adjusted to a pH of 8 via adding NaOH (40%) in a dropwise manner with constant stiring.Resultant precipitate/residue was then filtered/passed through column chromatography for generating purified A1-A24 with yields from 74-88%.

Synthesis of compound B
A solution containing ethyl ferulate (2.22 g, 0.01 mol), bromoacetic acid (1.39 g, 0.01 mol), and NaOH (0.8 g, 0.02 mol) in 40 mL of acetone was heated for 3-4 h at 60 C. Upon reaction termination, the solvent was cooled to room temperature and HCl was added for neutralization.The resultant precipitate/residue was then filtered/ re-crystallized using aqueous ethanol (5:1), producing a white solid consisting of purified compound B with a yield of 93%.

Synthesis of compounds C1-C24
A mixture of compound B (140 mg, 0.5 mmol) and A1-A24 (1.0 mmol) was prepared in CH 2 Cl 2 (15 mL), with HOBt (35.1 mg, 0.26 mmol), EDCI (105.4 mg, 0.55 mmol), DMAP (67.1 mg, 0.55 mmol), and triethylamine (0.55 mmol) being introduced into these solutions.The mixtures were then stirred for 12 h at room temperature under nitrogen, followed by the addition of water and CH 2 Cl 2 extraction.Solvent evaporation was performed to produce precipitates that were additionally purified via flash chromatography, enabling the isolation of the final pure product.Purified compound yields in this study ranged from 77.3% to 85% (Supplementary material: Compounds C1-C24).

Cytotoxicity assay
The cytotoxic effects of the synthesized compounds were assessed via CCK-8 assay.Briefly, RAW264.7 cells were added to 96-well plates (1 Â 10 4 cells/well in 100 mL/well) and treated for 24 h with the 24 synthesized compounds (40 lg/mL) for one day.Then, 10 mL of CCK-8 reagent was added per well, followed by a 4 h incubation (37 C).Absorbance (570 nm) was then measured via microplate reader (Thermo Scientific, MA, USA).

Statistical analysis
Data are means ± standard error (SE), and were compared via one-way ANOVAs or Student's t-tests with GraphPad Prism 8.0.p < 0.05 was the threshold of significance.

Conclusions
In conclusion, in this study, 24 novel derivatives of ethyl 4 0 -hydroxy-3 0 -methoxycinnamate-derived compounds were synthesized, profiled through 1 H-NMR, 13 C-NMR, and HRMS assays, and assessed for anti-inflammatory and cytotoxic activity.Of these compounds, c10 and c23 were found to exhibit more pronounced activity than parental ethyl 4 0 -hydroxy-3 0 -methoxycinnamate in multiple assays, suppressing LPS-driven TNFa expression by RAW 264.7 macrophage cultures via regulating the NF-jB/MAPK signaling pathways.These data thus suggest that c10 and/or c23 may represent novel anti-inflammatory drug candidates with the potential for clinical development.
Natural Science Foundation of Inner Mongolia of China;

Disclosure statement
No potential conflict of interest was reported by the authors.

Figure 1 .
Figure 1.The design of new compounds with increased anti-inflammatory activity.(A) The Structure of Ethyl ferulate; (B) Structures of anti-inflammatory derivatives containing thiadiazole; (C) Structures of anti-inflammatory drugs containing amide functional group; Designing Ethyl ferulate derivatives to achieve potent anti-inflammatory activity.