THE PHARMACOLOGICAL IMPACT OF "NOVEL" DIACEREIN DERIVATIVES

Abstract

Abstract Background: Today, cancer is regarded as one of the leading causes of death worldwide. Therefore, new anticarcinogenic drugs that are safer and more effective should be discovered. Cyclooxygenase-2 or COX-2 overexpression is common in many tumors and is associated with increased proliferation and invasiveness of cancer cells resulting from angiogenesis. Diacerein, a mild anti-inflammatory drug, acts as a prodrug by converting to rhein in the body; however, it is primarily used for the treatment of osteoarthritis. Anthraquinone derivatives demonstrated anti-inflammatory and anti-tumor activity, but their chemical structure should be optimized to improve the therapeutic efficiency. In addition, eugenol is a bioactive phenolic compound that can enhance the anticarcinogenic properties of other medicinal plants with apoptotic effects. Methods: A new amide derivative of diacerein, N-(4-tert-butylphenyl)-4,5-bis(acetyloxy)-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (NT4), was designed and synthesized via an EDCI/DMAP-mediated carbodiimide coupling of diacerein with 4-tert-butylaniline in anhydrous dichloromethane at room temperature. Its biological activities were evaluated in comparison with laboratory-made rhein. Following antioxidant activity assessment via the DPPH assay, the in vitro cytotoxic potential of the compounds was evaluated against MCF-7, HeLa, Hep3B, and B16F1 tumor cell lines, both alone and in combination with an eugenol vehicle. Additionally, in vitro enzyme inhibition assays and in silico studies were conducted to determine the selectivity of NT4 and the reference drug toward COX-1 and COX-2 isoenzymes, as well as to evaluate the drug-like properties of the lead compound. Results: All data are reported as mean ± SD (n = 3). In the DPPH assay, NT4 displayed significant antioxidant activity (IC50 = 9.99 μg/mL; maximum inhibition 61.8 ± 2.9% at 100 μg/mL) compared to Trolox (IC50 = 4.31 μg/mL; 99.1 ± 4.8%). NT4 demonstrated notable antiproliferative activity against HeLa (IC50 = 25.75 μg/mL; 76.8 ± 3.9% inhibition at 50 μg/mL) and MCF-7 cell lines (IC50 = 58.71 μg/mL). Co-administration with eugenol markedly enhanced cytotoxicity against MCF-7 cells, reducing the IC50 of diacerein from 41.37 to 25.17 μg/mL (89.1 ± 4.5% inhibition at 50 μg/mL) and NT4 from 58.71 to 38.19 μg/mL (74.1 ± 3.6% inhibition at 50 μg/mL). Against B16F1 melanoma cells, eugenol combination improved diacerein cytotoxicity, decreasing its IC50 from 48.68 to 17.52 μg/mL (78.9 ± 3.9% inhibition at 50 μg/mL). Non-cancerous HEK293T assays showed safety optimization, with NT4 exhibiting a higher IC50 in eugenol (22.44 μg/mL) than alone (11.23 μg/mL). Biochemically, NT4 displayed superior COX-2 isoenzyme inhibition (IC50 = 1.79 ± 0.08 μg/mL; SI = 47.96 ± 2.15), representing over 5-fold greater potency and higher selectivity relative to diacerein (IC50 = 9.62 ± 0.45 μg/mL; SI = 26.77 ± 1.20). Off-target COX-1 inhibition was minimal for both agents (IC50 = 85.85 ± 4.12 μg/mL for NT4 vs. 257.54 ± 12.80 μg/mL for diacerein). Conclusion: Compound NT4 is a novel, selective COX-2 inhibitor featuring promising cytotoxic potency and synergistic enhancement when paired with eugenol. Future work will focus on developing optimized nano-formulation drug delivery vehicles and conducting preliminary in vivo pharmacokinetic and therapeutic trials. Keywords: Diacerein; Rhein; COX-2 inhibitors; Eugenol; Cytotoxicity; DPPH; NSAIDs.