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• Rational design and synthesis of novel derivatives of pyrrolo[3,4- d pyridazinone have been described. • All new derivatives inhibit both cyclooxygenase enzymes. • Comprehensive in vitro and in silico studies confirmed that the most potent is 7b. • Novel Mannich base derivatives prevent DNA strand breaks caused by an increase in intracellular ROS. • All new derivatives bind to the active site of the enzyme in a way very similar to meloxicam. Novel Mannich base analogues of pyrrolo[3,4- d pyridazinone 7a , b - 13a , b are designed and synthesized as potential anti-inflammatory agents. The title compounds are obtained via convenient one-pot synthesis with good yields. Their structures and properties are described by spectroscopic techniques and elemental analyses. The aim of this study is to evaluate the inhibitory activity of the new derivatives against both cyclooxygenase isoforms COX1 and COX2 as well as their cytotoxicity. The results clearly indicate that the tested compounds 7a , b - 13a , b are not toxic, all show better affinity towards isoform COX-2, and some of them act as selective COX-2 inhibitors. Moreover, every examined derivative of pyrrolo[3,4- d pyridazinone demonstrates better inhibitory activity towards COX-2 and a superior COX-2/COX-1 selectivity ratio compared to the reference drug meloxicam. Molecular docking studies confirm that compounds 7a,b-13a,b preferably bind COX-2 and all of them bind to the active site of cyclooxygenase in a way very similar to meloxicam. Subsequently, taking into account that inflammation is strongly correlated with oxidative stress and both of these processes can potentiate each other, synthesized Mannich bases are evaluated for potential antioxidant activity. Most of the investigated derivatives reduce induced oxidative and nitrosative stress. Moreover, compounds 7a , b , 8a , 10a,b , 11b , 12a , b - 13a , b protect chromatin from oxidative stress and decrease the number of DNA strand breaks caused by intracellular growth of free radicals. Finally, a study of the binding mechanism between compounds 7a , b - 13a , b and bovine serum albumin (BSA) was carried out. According to spectroscopic and molecular docking studies, all examined derivatives interact with BSA, which suggests their potential long half-life in vivo. [ABSTRACT FROM AUTHOR]

• The investigational drug IND has been patented with USPTO. • Plasma protein binding has a major role in drug therapy specifically the pharmacokinetics and pharmacodynamics. • The interaction between IND and BSA has been investigated by using spectroscopic methods. • Molecular docking studies suggested that IND binds mainly to the subdomain IIA of BSA. • The experimental results will be helpful in preclinical drug development of IND. The indole derivative 2-(5-methoxy-2-methyl-1H-indol-3-yl)-N'-[(E)-(3-nitrophenyl) methylidene]acetohydrazide (IND) was synthesized for its therapeutic potential to inhibit cyclooxygenase (COX)-II. Binding if IND to bovine serum albumin (BSA) was investigated was because most drugs bind to serum albumin in -vivo. Fluorescence, UV–vis spectrophotometry and molecular modeling methodologies were employed for studying the interaction mechanism. The intrinsic fluorescence of BSA was quenched by BSA and the quenching mechanism involved was static quenching. The binding constants between IND and BSA at the three studied temperatures (298, 301 and 306 K) were 1.09 × 105, 4.36 × 104 and 1.23 × 104 L mol−1 respectively. The most likely site for binding IND to BSA was Site I (subdomain IIA). The analysis of thermodynamic parameter revealed the involvement of hydrogen bonding and van der Waals forces in the IND-BSA interaction. Synchronous fluorescence spectroscopic (SFS) and UV–vis spectrophotometric studies suggested conformational change in BSA molecule post interaction to IND. Molecular docking and the experimental results corroborated one another. The study can prove as an insight for future IND drug development. [ABSTRACT FROM AUTHOR]