Novel Benzenesulfonamide Derivatives of 5'-Aminospirotriazolotriazine Exhibit Anti-Inflammatory Activity by Suppressing Pro-Inflammatory Mediators: In Vitro and In Vivo Evaluation Using a Rat Model of Carrageenan-Induced Paw Edema

Abstract

Background/Objectives: Inflammation is a crucial and complex mechanism that protects the body against infections. In our study, we propose to provide scientific evidence for the anti-inflammatory properties of 1,3,5-triazine derivatives. Methods: Initially, we ensured the safety of the three synthesized derivatives by administering graded doses of up to 2000 mg/kg intraperitoneally in Wistar rats. Thus, the three derivatives were considered generally safe. We also evaluated their ability to reduce carrageenan-induced rat paw edema. Results: Compounds 1, 2, and 3 demonstrated stronger anti-inflammatory activity than indomethacin (10 mg/kg), achieving maximum inhibition at the fourth hour with percentages of 96.31%, 72.08%, and 99.69%, respectively, at a dose of 200 mg/kg, compared to 57.66% for the standard drug. To explore the mechanism, levels of pro-inflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6, CRP) and oxidative stress markers were measured in paw tissue. All three compounds significantly reduced these markers more effectively than indomethacin and enhanced antioxidant levels (SOD and GSH) beyond those achieved by the standard treatment. Additionally, the compounds reduced COX-1 and COX-2 levels to values comparable to those in the normal (non-inflamed) control group. Conclusions: Compounds 1, 2, and 3 at doses of 200 mg/kg significantly (p < 0.05) inhibited the heat-induced hemolysis of red blood cell (RBC) membranes by 94.6%, 93.9%, and 95.2%, respectively, compared to 94.5% produced by indomethacin. Consequently, we concluded that 1,3,5-triazine derivatives are a safe antioxidant agent with significant anti-inflammatory activity.

Keywords: 1,3,5-triazine derivatives; MTT assay; anti-inflammatory; carrageenan; cox isoenzymes; indomethacin.

Figure 1

Purine and triazolotriazine isomers’ structures.

Scheme 1

Synthesis of new 5-amino-7-(substituted)-N-(4-sulfamoylphenyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a][1,3,5]triazine-2-carboxamide derivatives (1–3).

Scheme 2

Proposal reaction mechanism for synthesized derivatives of [1,2,4] triazolo [1,5-a][1,3,5] triazine-2’-carboxamide.

Figure 2

In vitro anti-inflammatory activity of synthesized compounds 1, 2, and 3 by using (A) bovine serum albumin denaturation, (B) protein denaturation, and (C) erythrocyte hemolysis methods. All values are expressed as mean ± SD (n = 3), using one-way ANOVA followed by Tukey’s posttest. C1, C2, C3: compound 1–3; RBC: red blood cell. * p ≤ 0.05 (significant), ** p ≤ 0.01 (very significant), *** p ≤ 0.001 (highly significant) vs. indomethacin.

Figure 3

Effect of compounds 1, 2, and 3 on (A) cyclooxygenase-1 inhibition and (B) cyclooxygenase-2 inhibition by in vitro study. All values are expressed as mean ± SD (n = 3), using one-way ANOVA followed by Tukey’s posttest. C1, C2, C3: compound 1–3. * p ≤ 0.05 (significant), ** p ≤ 0.01 (very significant), *** p ≤ 0.001 (highly significant) vs. Indomethacin.

Figure 4

Representative micrographs of in vitro cell cytotoxicity assessment of different concentrations of compounds 1–3 on WI-38 human lung fibroblast cells. (A) Control image showing untreated WI-38 cells with typical fibroblast morphology (elongated and spindle-shaped). (B–D) Morphological changes in WI-38 cells treated with increasing concentrations of compound 1–3 (31.25 to 1000 µg/mL). (E) Dose–response curve for compound 1, showing cell viability (blue line) and toxicity (red line). IC₅₀ = 362.78 ± 3.88 µg/mL. (F) Dose–response curve for compound 2. IC₅₀ = 327.88 ± 1.92 µg/mL. (G) Dose–response curve for compound 3. IC₅₀ = 148.13 ± 1.48 µg/mL. Note: Cell viability and toxicity were determined using a standard MTT assay. A dose-dependent decrease in viability and increase in toxicity were observed for all compounds, with compound 3 showing the highest cytotoxicity based on the lowest IC₅₀ value.

Figure 5

(A) Representative photo showing edema inhibition activity of compounds 1, 2, and 3 after 1, 2, 3, and 4 h from the carrageenan injection, which caused swelling and redness of the injected paw (B,C). Indo: indomethacin; C1, C2, C3: compound 1–3.

Figure 6

Paw tissue oxidative damage biomarkers of rats in groups (A) MDA, (B) SOD, and (C) GSH. Each value represents the mean ± SD for 6 rats per group. Results are considered significantly different when p < 0.05. ^a significant difference compared to the control. ^b significant difference compared to the carrageenan group. ^c significant difference compared to the Indo group. Carr: carrageenan; Indo: indomethacin; MDA: malondialdehyde; SOD: superoxide dismutase; GSH: reduced glutathione.

Figure 7

Compounds 1, 2, and 3 reduced significantly (A) COX-1 and (B) COX-2 levels in the carrageenan-injected paw. Each value is the mean ± SD for 6 rats per group. Above each column, different letters mean statistical significance at (p < 0.05). ^a significant difference compared to the control. ^b significant difference compared to the carrageenan group. ^c significant difference compared to the Indo group. Carr: carrageenan; Indo: indomethacin; COX: cyclooxygenase.

Figure 8

Photomicrograph of paw tissue of rats from experimental groups stained with HE: (A) The normal control group shows the normal appearance of the epidermis (white arrowheads) and dermis without any lesion (star) and normal dermal vessels (arrows). (B) Carrageenan-injected paw tissue shows a thin epidermis (white arrow), massive edema (stars), and marked vasodilation with severe inflammatory cellular infiltration (arrow). (C) The carrageenan-injected paw of rats treated with indomethacin shows a thin epidermis (white arrow), marked edema (stars), and dilated and congested dermal vessels (arrow). (D) The carrageenan-injected paw of rats treated with compound 1 shows a normal epidermis (white arrow) and mild edema (stars). (E) The carrageenan-injected paw of rats treated with compound 2 shows more or less epidermal thickness (white arrow), moderate edema (stars), and more or less normal dermal vessels (arrow). (F) The carrageenan-injected paw of rats treated with compound 3 shows normal epidermal thickness (white arrow), normal dermis (stars), and normal dermal vessels (arrow).

Figure 9

Photomicrograph of paw tissue of rats from experimental groups stained with HE: (A) The normal control group shows the normal appearance of the epidermis and dermis without any lesion, with normal dermal vessels (arrows). (B) The carrageenan-injected paw tissue shows massive edema (stars) and marked inflammatory cellular infiltration (arrowheads). (C) The carrageenan-injected paw of rats treated with indomethacin shows marked edema (stars) and inflammatory cellular infiltration (arrowheads). (D) The carrageenan-injected paw of rats treated with compound 1 shows a mild inflammatory dermal edema (stars). (E) The carrageenan-injected paw of rats treated with compound 2 shows moderate edema (stars), mild dermal vessels, and inflammatory cellular infiltration (arrows). (F) The carrageenan-injected paw of rats treated with compound 3 shows normal epidermal thickness and normal dermis without any lesion (star).

Figure 10

Photomicrograph of paw tissue of rats from experimental groups stained with Sirius Red stain: (A) The normal control group shows the normal appearance of dermal collagen (stars). (B) The carrageenan-injected paw tissue shows few dispersed collagenous fibers in the edematous dermis (stars). (C) The carrageenan-injected paw of rats treated with indomethacin shows marked dispersed collagenous fibers (stars). (D) The carrageenan-injected paw of rats treated with compound 1 shows normal collagen quantity and distribution (stars). (E) The carrageenan-injected paw of rats treated with compound 2 shows normal dermal collagen (stars). (F) The carrageenan-injected paw of rats treated with compound 3 shows normal dermal collagenous fibers without any lesion (star).

Figure 11

Histomorphometry graph showing semiquantitative measurements of lesion scores recorded in PAW tissue sections among the experimental groups. (A) Inflammation, (B) edema, (C) vascular alterations, (D) total lesion scores. Data are expressed as means ± standard deviations. Significant differences vs. the control group are marked by different asterisks, and significant differences vs. Carr. The treated group is marked by different # through the Kruskal–Wallis test with Dunn’s Multiple Comparison post hoc test: (*^,# p ≤ 0.05, **^,## p ≤ 0.01, ***^,### p ≤ 0.001), ns refers to non-significant. Carr: carrageenan; Indo: indomethacin.