Nialamide (purity >99%), acetonitrile, methanol, formic acid [high-performance liquid chromatography (HPLC) grade], dimethyl sulfoxide (DMSO)-d₆, LPS and Griess reagent were purchased from MilliporeSigma. All other chemicals used were of analytical grade. Compound analyses were conducted using an Agilent HPLC 1200 system (Agilent Technologies, Inc.) equipped with a photodiode array detector (1200 Infinity series; Agilent Technologies, Inc.) and a series of YMC-Pack ODS A-302 columns (4.6 mm i.d.x150 mm, particle size 5 µm; YMC Co., Ltd.) were used to analysis the compounds. HPLC was performed using an ODS column (YMC-Pack A-302; YMC Co., Ltd.) using an elution gradient of 5-100% MeCN in 0.1% HCOOH (detection: 280 nm; flow rate: 1.0 ml/min; oven temperature: 40˚C). ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were measured using an Avance NEO-600 instrument (Bruker Corporation) operated at 600 and 150 MHz, respectively. Chemical shifts are reported as δ (parts per million) values relative to those of the solvent DMSO-d₆ (δ_H 2.50; δ_C 39.5) on a tetramethylsilane scale. High-resolution electrospray ionization (ESI) mass spectra were obtained using a Vanquish UPLS System (Thermo Fisher Scientific, Inc.) and an Infinite F200 microplate reader (Tecan Austria GmbH) was used to measure absorbances.

Nialamide irradiation was performed at the Advanced Radiation Technology Institute (ARTI; Jeongeup, Republic of Korea) using a cobalt-60 irradiator (point source AELC; IR-79; MDS Nordion International Co. Ltd.) with a source strength of ~320 kCi and a dose of 10 kGy/h. Pure nialamide (1 g) was initially dissolved in MeOH (1:l), placed in chapped glass bottles and then directly irradiated with a dose of 50 kGy. After irradiation, the sample solution was promptly concentrated using a rotary vacuum evaporator to eliminate the methanol and was subsequently lyophilized.

The irradiated reactant (965.6 mg) irradiated at a dose of 50 kGy was promptly subjected to open column chromatography on a YMC gel ODS AQ HG (2.5 cm i.d.x40 cm) column (YMC Co., Ltd.) with aqueous MeOH, to obtain fractions N1-N6. Subfraction N4 (225.4 mg), which was flowed with 50% MeOH, was isolated using preparative HPLC (YMC-prep column, 20 mm i.d.x250 mm; flow rate: 9 ml/min; solvent systems 78:22=H₂O:MeOH; YMC Co., Ltd.) to produce compounds 3 (2.7 mg, t_R 10.6 min), 5 (6.7 mg, t_R 10.0 min) and 7 (56.7 mg, t_R 15.5 min). Fraction N5 (67.0 mg) was separated using preparative-HPLC (YMC-prep column, 20 mm i.d.x250 mm; solvent, flow rate: 9 ml/min; solvent systems 75:25=H₂O:MeOH; YMC Co., Ltd.) to yield compounds 2 (2.0 mg, t_R 21.6 min), 4 (2.8 mg, t_R 21.6 min) and 6 (1.4 mg, t_R 21.6 min). Fractions N1-N3 were subjected to recrystallization and identified as HBPA (compound 5; 500 mg).

Canine DH82 macrophages were purchased from the American Type Culture Collection and murine RAW 264.7 macrophages were purchased from the Korean Cell Line Bank. The DH82 and RAW 264.7 macrophages cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and were incubated at 37˚C in an incubator with a humidified atmosphere of 5% CO₂.

Cell viability was measured using the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Macrophage cells were seeded in 24-well plates at a density of 1x10⁵ cells/well, cultured for 24 h and treated with various concentrations of nialamide and isolated compounds 2-7 for an additional 24 h. Thereafter, the MTT reagent was added to each well. The formazan crystals thus produced were dissolved in DMSO and the absorbance of the well contents was measured at 570 nm using a scanning a microplate reader. The cell viability was determined from the proportions of viable and dead cells (18).

RAW 264.7 and DH82 macrophage cells were cultured in 24-well plates at a density of 1x10⁵ cells/well for 24 h. Following this initial incubation, the cells were pre-treated with nialamide and derived compounds 2-7 for 1 h and subsequently treated with LPS (RAW 264.7 cells: 0.1 µg/ml; DH82 cells: 2 µg/ml) for an additional 24 h. NO production in the macrophage culture medium was assessed using the Griess reagent method with a standard curve for quantification (19). In addition, the macrophage supernatant was used to assess for levels of PGE₂ and cytokines using the respective enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences), according to the manufacturer's instructions.

RAW 264.7 and DH82 cells were cultured in six-well plates at a density of 2x10⁵ cells/well for 24 h. Following incubation at 37˚C, the cells were pre-treated with compounds 1 and 5 for 1 h and were thereafter treated with LPS (RAW 264.7 cell: 0.1 µg/ml; DH82 cell: 2 µg/ml) for an additional 24 h. Total proteins were extracted using RIPA buffer (Rockland Immunochemicals, Inc.) and the concentrations of the isolated proteins were determined using a bicinchoninic acid protein assay kit. Proteins (40 µg) were separated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and subsequently transferred to polyvinylidene difluoride membranes (Merck KGaA). The membranes were then blocked with 5% non-fat milk in Tris-buffered saline containing 10% Tween-20 (TBST) for 1 h at room temperature and subsequently probed overnight at 4˚C with the following primary antibodies at dilution of 1:1,000: Anti-COX-2 (cat. no. 4842), anti-iNOS (cat. no. 2977), anti-p65 (cat. no. 8242), anti-p-p65 (cat. no. 3033), anti-IκB (cat. no. 4812), anti-p-IκB (cat. no. 2859) and anti-GAPDH (cat. no. 2118; all obtained from Cell Signaling Technology, Inc.). For canine macrophages, the following primary antibodies were used at a dilution of 1:1,000: anti-COX-2 (cat. no. PA1-9032), anti-iNOS (cat. no. PA1-036) and anti-GAPDH (cat. no. PA1-987; Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation with the primary antibodies, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at room temperature. Protein bands were detected using a chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Inc.) and exposure to an X-ray film. The blots were scanned and densitometric analysis was performed using ImageJ software (version 1.51k; National Institutes of Health).

The production of intracellular ROS was measured using ROS detection reagents. In brief, RAW 264.7 macrophage cells were seeded in 96-well plates at a density of 1x10⁴ cells/well and treated with 0.1 µg/ml LPS in combination with varying concentrations of compound 5 (50 to 200 µM) at 37˚C for 2 h. On day 1, the cells were washed with phosphate-buffered saline (PBS) and incubated with 7'-dichlorofluorescein diacetate (DCFH-DA) at 37˚C for 45 min. DCF fluorescence was measured at 0, 10 and 30 min using a microplate reader at excitation and emission wavelengths of 492 and 520 nm, respectively (20).

The scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical of the derived compounds was assessed using a modified Blois method (21). Briefly, a solution of DPPH (200 µM) was prepared in ethanol (EtOH), 60 µl of which was mixed with 120 µl of each of the assessed compounds at concentrations from 50 to 200 µM in 70% EtOH. After incubation at 30˚C in the dark for 15 min, the reduction in absorbance at 517 nm was recorded using an ELISA reader. For the determination of 3-ethylbenzothiazoline-6-sulphonic acid (ABTS⁺) radical scavenging activity, an ABTS⁺) solution was prepared by mixing 5 ml of a 7 mM ABTS⁺ solution in ethanol with 5 ml of a 2.4 mM potassium persulfate solution and incubating the mixture in the dark at 25˚C for 24 h prior to use. The ABTS⁺ solution (100 µl) was thereafter added to the wells of 96-well plates containing different concentrations of compound 5 and mixed for 30 sec, followed by incubation for 30 min at 25˚C (22). The DPPH and ABTS⁺ radical-scavenging activities were calculated using the following formula:

radical scavenging activity (%)=[1-(A₂/A₁)]x100

where A₁ is the absorbance of reaction mixtures without samples and A₂ is the absorbance of reaction mixtures containing the assessed compounds.

Each experiment was performed at least three times and the results were expressed as the mean ± standard deviation. For multiple comparisons, one-way analysis of variance (ANOVA) was used followed by Tukey's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

Nialamide irradiated at a 50 kGy dose was established to be characterized by the most significantly enhanced inhibitory effects with respect to the production of NO and PGE₂ in LPS-stimulated RAW 264.7 cells, with IC₅₀ values of 98.7±0.9 and 88.2±0.9 µg/ml, respectively. Comparatively, an IC₅₀ value >200 µg/ml was obtained for the parent compound, nialamide. DH82 canine macrophages treated with irradiated nialamide displayed notable inhibitions in both NO and PGE₂ production (Table I). The molecular changes in irradiated nialamide were assessed using reverse-phase HPLC. Newly generated peaks in the HPLC chromatograms corresponding to the modified products of nialamide were accordingly detected (Fig. S1). Successive chromatographic separation of this sample resulted in the purification of a new cyclized product, designated compound 2, along with five known degradation products (compounds 3-7).

Compound 2 was obtained as a colorless oil. The pseudomolecular ion in the positive high-resolution electrospray ion mass spectrum (HRESIMS) at m/z 191.1176 (M+H)⁺ had the molecular formula C₁₂H₁₅N₂O. The ¹H NMR spectrum of compound 2 (Fig. S2) showed characteristic resonances of a benzene ring system at δ_H 7.34 (2H; t; J=1.8 Hz; H-3; 5), 7.26 (2H; dd; J=8.4; 1.8 Hz; H-2; 6) and 7.24 (1H; dd; J=8.4; 1.8 Hz; H-4) and a nitrogenated methylene proton at δ_H 4.44 (2H; s; H-7), indicating the presence of a benzylamide moiety in the molecule of compound 2. Additionally, the low-field region of the ¹H NMR spectrum displayed signals for doubly nitrogenated methylene protons at δ_H 4.03 (2H; d; J=6.0 Hz; H-13) and two cyclized methylene protons at δ_H 2.92 (2H; br d; J=6.0 Hz; H-11) and 2.25 (2H; t; J=6.0 Hz; H-10), which is consistent with a tetrahydropyrimidione skeleton (23).

In addition, ¹³C NMR combined with heteronuclear single quantum coherence (HSQC) analysis of compound 2 (Figs. S3 and S4) revealed 11 carbon signals, attributable to one carbonyl carbon signal at δ_C 167.5 (C-9), five benzylamide signals at δ_C 137.9 (C-1), 128.6 (C-3; 5), 127.6 (C-2; 6), 127.1 (C-4) and 46.3 (C-7) and three methylene carbons at δ_C 63.0 (C-13), 42.0 (C-11) and 32.9 (C-10). Collectively, the aforementioned informative results provide evidence to indicate that compound 2 is a tetrahydropyrimidione-substituted benzylamide. A detailed interpretation of the combination of ¹H-¹H correlated spectroscopy, heteronuclear multiple bond correlation (HMBC) and nuclear Overhauser effect spectroscopy data for compound 2 further indicated that it features a characteristic cyclized product (Fig. S5, Fig. S6 and Fig. S7). The proposed linkage between the benzylamide and tetrahydropyrimidione moieties is suggested to occur at the C-7 position, as supported by the HMBC correlations (Fig. 1). On the basis of the aforementioned analyses, the planar structure of compound 2 was assigned to nialamionsin, a novel benzylamide analog with a tetrahydropyrimidione unit (Fig. 1).

Nialaminosin (compound 2): Colorless oil: UV λ_max (MeOH): 207 (3.14), 220 (sh) and 249 (2.26) nm; ¹H NMR (DMSO-d₆; 600 MHz): δ 7.34 (2H; t; J=1.8 Hz; H-3; 5), 7.26 (2H; dd; J=8.4; 1.8 Hz; H-2; 6), 7.24 (1H; dd; J=8.4; 1.8 Hz; H-4), 4.44 (2H; s; H-7), 4.03 (2H; d; J=6.0 Hz; H-13), 2.92 (2H; br d; J=6.0 Hz; H-11), 2.25 (2H; t; J=6.0 Hz; H-10); ¹³C NMR (DMSO-d₆; 150 MHz): δ 167.5 (C-9), 137.9 (C-1), 128.6 (C-3; 5), 127.6 (C-2; 6), 127.1 (C-4), 63.0 (C-13), 46.3 (C-7), 42.0 (C-11), 32.9 (C-10); ESIMS m/z 191 (M+H)⁺; HRESIMS m/z 191.1176 (M+H)⁺ (calculated for C₁₁H₁₅N₂O, 191.1178; Fig. S8).

The five known isolated compounds were characterized as 3-amino-N-benzylpropanamide (compound 3; ABPA), 3-methoxy-N-benzylpropanamide (compound 4; MBPA), 3-hydroxy-N-benzylpropanamide (compound 5; HBPA), N-benzylpropanamide (compound 6; BPA) and isonicotinamide (compound 7; INA), based on comparisons of the respective spectroscopic data (NMR and MS) with reports from the literature (24-28).

ABPA (compound 3): Colorless oil: ¹H NMR (DMSO-d₆; 600 MHz): δ 7.33 (2H; t; J=1.2 Hz; H-3; 5), 7.27 (2H; dd; J=7.8; 1.2 Hz; H-2; 6), 7.23 (1H; dd; J=7.8; 1.2 Hz; H-4), 4.45 (2H; s; H-7), 2.93 (2H; br d; J=6.0 Hz; H-11), 2.26 (2H; t; J=6.0 Hz; H-10), ¹³C NMR (DMSO-d₆; 150 MHz): δ 167.7 (C-9), 137.0 (C-1), 127.2 (C-3; 5), 126.4 (C-2; 6), 126.0 (C-4), 46.2 (C-7), 42.2 (C-11), 32.3 (C-10); ESIMS m/z 179 (M+H)⁺ (24,25).

3-Methoxy-N-benzylpropanamide (compound 4): Colorless oil: ¹H NMR (DMSO-d₆, 600 MHz): δ 7.33 (2H; t; J=1.8 Hz; H-3; 5), 7.24 (2H; dd; J=7.8; 1.8 Hz; H-2; 6), 7.22 (1H; dd; J=7.8; 1.8 Hz; H-4), 4.26 (2H; d; J=6.0 Hz; H-7), 3.55 (2H; t; J=6.0 Hz; H-11), 3.22 (3H; s; 11-OCH₃), 2.38 (2H; t; J=6.0 Hz; H-10); ¹³C NMR (DMSO-d₆; 150 MHz): δ 170.2 (C-9), 139.6 (C-1), 128.4 (C-3; 5), 127.3 (C-2; 6), 125.8 (C-4), 65.5 (C-11), 58.0 (11-OCH₃), 42.1 (C-7), 35.1 (C-10), ESIMS m/z 194 (M+H)⁺ (26).

3-Hydroxy-N-benzylpropanamide (compound 5): Colorless oil: ¹H NMR (DMSO-d₆; 600 MHz): δ 7.32 (2H; t; J=1.8 Hz; H-3; 5), 7.26 (2H; dd; J=8.4; 1.8 Hz; H-2; 6), 7.22 (1H; dd; J=8.4; 1.8 Hz; H-4), 4.27 (2H; d; J=6.0 Hz; H-7), 3.65 (2H; t; J=6.0 Hz; H-11), 2.31 (2H; t; J=6.0 Hz; H-10); ¹³C NMR (DMSO-d₆; 150 MHz): δ 170.9 (C-9), 139.9 (C-1), 128.5 (C-3; 5), 127.5 (C-2; 6), 126.9 (C-4), 57.9 (C-11), 42.2 (C-7), 39.0 (C-10); ESIMS m/z 180 (M+H)⁺ (25).

N-Benzylpropanamide (compound 6): Colorless oil: ¹H NMR (DMSO-d₆; 600 MHz): δ 7.32 (2H; d; J=7.8 Hz; H-2; 6), 7.26 (2H; t; J=1.8 Hz; H-3; 5), 7.23 (1H; dd; J=8.4; 1.8 Hz; H-4), 4.27 (2H; d; J=6.0 Hz; H-7), 2.15 (2H; q; J=7.8 Hz; H-10), 1.03 (3H; t; J=7.8 Hz; H-11); ¹³C NMR (DMSO-d₆; 150 MHz): δ 173.3 (C-9), 140.2 (C-1), 128.7 (C-2; 6), 127.6 (C-3; 5), 127.1 (C-4), 42.4 (C-7), 18.9 (C-10), 10.4 (C-11); ESIMS m/z 164 (M+H)⁺ (27).

Isonicotinamide (compound 7): White amorphous powder; ¹H NMR (DMSO-d₆; 600 MHz): δ 8.73 (2H; d; J=8.4 Hz; H-3; 5), 7.72 (2H; d; J=8.4 Hz; H-2; 6); ESIMS m/z 123 (M+H)⁺ (28).

Inflammatory responses promote the excessive production of pro-inflammatory mediators, such as NO and PGE₂ (11), the abnormal overproduction of which is implicated in the development of a diverse range of disorders, including circulatory shock, cancer and atherosclerosis (29,30). Consequently, regulating the production of pro-inflammatory agents is a desirable pharmacological property when seeking to develop new drugs. To investigate the anti-inflammatory activities of isolated products 2-7, the present study treated both RAW 264.7 and DH82 macrophages with different concentrations of these compounds. Using the MTT assay, the cytotoxicity of these compounds was initially assessed at concentrations ranging from 10-200 µM and it was accordingly established that all assessed compounds showed negligible cytotoxicity toward these two cell types at concentrations of ≥200 µM (Table I).

In the current study, compounds were evaluated for their inhibitory activity on the production of NO and PGE₂ in LPS-stimulated macrophages. Among these compounds, compared with the parent nialamide (1) the novel cyclized product nialaminosin (2) showed enhanced inhibitory effects against the production of NO and PGE₂ in LPS-stimulated RAW 264.7 cells with respective IC₅₀ values of 86.2±1.1 and 80.7±1.0 µM (Table I). Similarly, enhanced anti-inflammatory effects were detected for benzylpropanamide derivatives 3-6, with IC₅₀ values ranging from 63.5-194.4 µM for NO production and from 55.4-176.4 µM for PGE₂ production (Table I). Among these derivatives, it was found that hydroxylation at the C-11 position of HBPA conferred the most potent inhibitory activity against the activation of pro-inflammatory mediators (Table I). Comparatively, the simple pyridine amide isonicotinamide (7) was characterized by relatively lower inhibitory activity against the production of both NO and PGE₂ in RAW 264.7 macrophages (Table I). Consistently, in canine DH82 macrophages, the hydroxyl-substituted benzylpropanamide derivative was established have the most potent inhibitory activity against the production of NO and PGE₂, with IC₅₀ values of 57.5±0.6 and 49.4±0.4 µM, respectively (Table I). On the basis of these observations, the potent compound 5 was accordingly selected for further evaluation of its inhibitory effects on the release of NO and PGE₂ using western blot analysis.

Within cells, iNOS and COX-2 are induced in response to a range of stimuli, including pro-inflammatory mediators, growth factors, oncogenes, carcinogens and tumor promoters, thereby indicating that these two proteins play roles in both inflammation and the control of cell growth (31,32). Accordingly, for the development of new anti-inflammatory drugs, it would be desirable to identify compounds that can inhibit both the activity and expression of iNOS and COX-2 (33,34). The present study found that, compared with nialamide (1), compound 5 (identified in this study as the most potent inhibitor of NO and PGE₂ production) promoted a significant dose-dependent suppression of the expression of iNOS and COX-2 in both LPS-stimulated RAW 264. (Fig. 2A-C) and canine DH82 (Fig. 2D-F) cells, thereby resulting in an inhibition of the pro-inflammatory mediators NO and PGE₂ (Fig. S9).

Pro-inflammatory cytokines serve as key signaling molecules in the immune system and thereby play important roles in inflammatory responses (35). However, an excessive release of pro-inflammatory cytokines such as TNF-α and IL-6 can lead to damage to the epithelial barrier, initiate epithelial cell apoptosis and induce inflammation (36,37). Several studies have reported that the anti-inflammatory activities of benzylamide derivatives are associated with the control of the levels of diverse pro-inflammatory cytokines (35,38). In macrophages, exposure to LPS can induce the upregulation of IL-10, a well-established anti-inflammatory cytokine, by promoting the activation of the p38, JNK and ERK1/2 MAPK signaling pathways, which typically lead to the phosphorylation and activation of the Sp1 transcription factor and, in turn, expression of the IL-10 gene. The analysis in the present study of the inhibitory effects of compound 5 on the production of the cytokines TNF-α, IL-6 and IL-10 in LPS-induced macrophages revealed that, compared with the original nialamide, product 5 was characterized by a potent dose-dependent suppression of all three of these cytokines in LPS-stimulated RAW 264.7 cells, with similar effects being detected in LPS-stimulated DH82 macrophages (Fig. 3D-F).

ROS are key regulators of inflammatory responses, particularly the activation of the transcription factor NF-κB and its downstream signals (39). However, given their high reactivity, when generated in excess, ROS can have detrimental effects, particularly those associated with peroxidation and oxidative damage to normal DNA and proteins (40). Several studies have nevertheless reported that modified drugs can contribute to reductions in ROS production and enhance antioxidant activity (41,42). To investigate the inhibitory effects of major product 5 on ROS generation, LPS-activated RAW 264.7 macrophages were stained with DCFH-DA and fluorescence microscopy observations performed. Treatment with compound 5 was found to effectively suppress ROS production in a dose-dependent manner (Fig. 4A and B). Previous study have demonstrated that LPS-stimulated ROS generation is associated with NF-κB activation (43) and, consistently, in the present study, significant increases in the phosphorylation levels of NF-κB and IκB in the treated RAW 264.7 cells were detected (Fig. S10). By contrast, treatment with compound 5 was found to promote a dose-dependent inhibition of the LPS-stimulated phosphorylation of NF-κB (Fig. 4C and D). Moreover, compared with cells treated with the parent compound nialamide, an enhancement of DPPH radical and ABTS⁺ scavenging activities were detected in those cells treated with compound 5 (Fig. 4E and F). Collectively, these findings thus provide evidence to indicate that the γ irradiation-mediated modification of nialamide contributes to enhancing the anti-inflammatory properties of this drug.