AS-10

Fourteen novel Schiff base compounds (AS‐1∼AS‐14) containing 5‐amino‐1H‐1,2,4‐triazole‐3‐carboxylic acid and substituted benzaldehyde were successfully synthesized, and their structures were verified by melting point, elemental analysis (EA) and spectroscopic techniques (Fourier Transform Infra‐Red (FT‐IR) and Nuclear Magnetic Resonance (NMR)). In vitro hyphal measurements were used to investigate the antifungal activities of the synthesised compounds against Wheat gibberellic, Maize rough dwarf and Glomerella cingulate. The preliminary studies indicated that all compounds had good inhibitory effect on Wheat gibberellic and Maize rough dwarf, among which the compounds of AS‐1 (7.44 mg/L, 7.27 mg/L), AS‐4 (6.80 mg/L, 9.57 mg/L) and AS‐14 (5.33 mg/L, 6.53 mg/L) showed better antifungal activity than that of the standard drug fluconazole (7.66 mg/L, 6.72 mg/L); while inhibitory effect against Glomerella cingulate was poor, only AS‐14 (5.67 mg/L) was superior to that of fluconazole (6.27 mg/L). The research of structure‐activity relationship exhibited that the introduction of halogen elements on the benzene ring and electron withdrawing groups at the 2,4,5 positions on the benzene ring was beneficial to the improvement of the activity against Wheat gibberellic, while the large steric hindrance was not conducive to the improvement of the activity. Additionally, except for AS‐1, AS‐3 and AS‐10, the other compounds had one or several ratio systems to achieve synergistic effect after recombination with pyrimethamine, among which AS‐7 had significant synergistic effect and was expected to be a combinated agent with application prospects. Finally, the molecular docking results of isocitrate lyase with Wheat gibberellic displayed that the presence of hydrogen bonds enabled stable binding of compounds to receptor proteins, and the residues of ARG A: 252, ASN A: 432, CYS A: 215, SER A: 436 and SER A: 434 were the key residues for their binding. Comparing the docking binding energy and biological activity results, it was revealed that the lower the docking binding energy was, the stronger the inhibitory ability of the Wheat gibberellic, when the same position on the benzene ring was substituted.

The novel selenium‐aspirin compound AS‐10 was recently reported by us with a cancer cell killing potency three orders of magnitude greater than aspirin in pancreatic cancer cell lines with caspase‐mediated apoptosis and a reasonable selectivity against malignant cells. Although we also observed its cytocidal activity against PC‐3 and DU145 androgen receptor (AR)‐negative and P53‐null/mutant aggressive human prostate cancer (PCa) cell lines in NCI‐60 screen, the potential involvement and targeting of AR and P53 pathways that are intact in early‐stage prostate carcinogenesis has not been examined, nor its primary molecular signaling after exposure.

Human LNCaP PCa cells with functional AR and intact P53 were used to examine their cell cycle and cell fate responses to AS‐10 exposure and upstream molecular signaling events including histone acetylation as a known aspirin effect. The AR‐positive 22Rv1 human PCa cells were used to validate key findings.

In addition to confirming AS‐10's superior cytocidal potency than aspirin against all four PCa cell lines, we report a rapid (within 5 min) promotion of histone acetylation several hours ahead of the suppression of AR and prostate‐specific antigen (PSA, coded by KLK3 gene) in LNCaP and 22Rv1 cells. AS‐10 decreased AR and KLK3 mRNA levels without impacting pre‐existing AR protein degradation or nuclear translocation in LNCaP cells. Sustained exposure to AS‐10 arrested cells predominantly in G1, and induced caspase‐mediated apoptosis without necrosis. The death induced by AS‐10 in LNCaP cells was attenuated by nontranscriptional activation of P53 protein or Jun N‐terminal Kinase cellular stress signaling and was mitigated modestly by glutathione‐boosting antioxidant N‐acetylcysteine. AS‐10 synergized with histone deacetylase inhibitor SAHA to suppress AR/PSA abundance and kill LNCaP cells. RNA‐seq confirmed AR suppression at the transcriptional level and suggested multiple oncogene, cyclin, and CDK/CKI transcriptional actions to contribute to the cellular consequences.

AS‐10 promotes histone acetylation as its probable primary mechanism of action to induce PCa cell‐cycle arrest and apoptosis, regardless of AR and P53 status. Nevertheless, the inhibition of AR signaling through mechanisms distinct from canonical AR antagonists may hold promise for combinatorial use with androgen deprivation therapy regimens or AR‐axis targeting drugs.