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Three new branched peptides, namely, H-Gly-Dap(H-Gly)-Gly-NH2 (3G), H-His-Dap(H-His)-Gly-NH2 (2HG), and H-Gly-Dap(H-Gly)-His-NH2 (2GH), where Dap stands for the 2,3-diaminopropionic acid residue, were synthesized by solid phase procedures. Because of the junction at Dap these peptides have three available pending arms for metal chelation. The complex formation between these peptides and 1 equiv of Cu(2+) was investigated as a function of pH by potentiometry ultraviolet-visible absorption, circular dichroism, and X-band electron paramagnetic resonance spectroscopy in aqueous medium. Our results clearly demonstrate that cooperation between all three peptide arms essentially contributes to the stability of copper(II) complexes.

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The long-term use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in treatment of different chronic inflammatory disorders is strongly restricted by their serious gastrointestinal adverse effects. Therefore, there is still an urgent need to search for new, safe, and efficient anti-inflammatory agents. Previously, we have reported the Mannich base-type derivatives of pyrrolo[3,4-d]pyridazinone which strongly inhibit cyclooxygenase, have better affinity to COX-2 isoenzyme and exert promising anti-oxidant activity. These findings encouraged us to perform further optimization of that structure. Herein, we present the design, synthesis, molecular docking, spectroscopic, and biological studies of novel pyrrolo[3,4-d]pyridazinone derivatives bearing 4-aryl-1-(1-oxoethyl)piperazine pharmacophore 5a,b–6a,b. The new compounds were obtained via convenient, efficient, one-pot synthesis. According to in vitro evaluations, novel molecules exert no cytotoxicity and act as selective COX-2 inhibitors. These findings stay in good correlation with molecular modeling results, which additionally showed that investigated compounds take a position in the active site of COX-2 very similar to Meloxicam. Moreover, all derivatives reduce the increased level of reactive oxygen and nitrogen species and prevent DNA strand breaks caused by oxidative stress. Finally, performed spectroscopic and molecular docking studies demonstrated that new compound interactions with bovine serum albumin (BSA) are moderate, formation of complexes is in one-to-one ratio, and binding site II (subdomain IIIA) is favorable.

Despite the availability of the current drug arsenal for pain management, there is still a clinical need to identify new, more effective, and safer analgesics. Based on our earlier study, newly synthesized 1,3,4-oxadiazole derivatives of pyrrolo[3,4-d]pyridazinone, especially 10b and 13b, seem to be promising as potential analgesics. The current study was designed to investigate whether novel derivatives attenuate nociceptive response in animals subjected to thermal or chemical noxious stimulus, and to compare this effect to reference drugs. The antinociceptive effect of novel compounds was studied using the tail-flick and formalin test. Pretreatment with novel compounds at all studied doses increased the latency time in the tail-flick test and decreased the licking time during the early phase of the formalin test. New derivatives given at the medium and high doses also reduced the late phase of the formalin test. The achieved results indicate that new derivatives dose-dependently attenuate nociceptive response in both models of pain and exert a lack of gastrotoxicity. Both studied compounds act more efficiently than indomethacin, but not morphine. Compound 13b at the high dose exerts the greatest antinociceptive effect. It may be due to the reduction of nociceptor sensitization via prostaglandin E2 and myeloperoxidase levels decrease.

Neuroinflammation is considered to be one of the potential causes for the development of neurodegenerative diseases, including Alzheimer's disease. In this study, we evaluated the effect of four newly synthesized pyrrolo [3,4-d]pyridazinone derivatives on the neuron-like PC12 cells under simulated inflammation conditions by preincubation with lipopolysaccharide (LPS). Our novel derivatives are selective cyclooxygenase-2 (COX-2) inhibitors and have similar effects to nonsteroidal anti-inflammatory drugs (NSAIDs). We assessed viability (LDH assay), metabolic activity (MTT assay), DNA damage (number of double-strand breaks measured by fast halo assay), and the neuronal features of cells (average neurite length and neurite outgrowth measured spectrofluorimetrically). DCF-DA and Griess assays were also performed, which allowed determining the impact of the tested compounds on the level of oxygen free radicals and nitrites. LPS administration significantly negatively affected the results in all tests performed, and treatment with the tested derivatives in most cases significantly reduced this negative impact. Multiple-criteria decision analysis indicated that overall, the best results were observed for compounds 2a and 2b at a concentration of 10 µM. The new derivatives showed intense activity against free oxygen radicals and nitrites. Reduced reactive oxygen species level also correlated with a decrease in the number of DNA damage. The compounds improved neuronal features, such as neurite length and outgrowth, and they also increased cell viability and mitochondrial activity. Our results suggest that derivatives 2a and 2b may also act additionally on mechanisms other than 3a and 3b.

Potentiometric, spectroscopic (UV-Vis, CD and EPR), mass spectrometric and electrochemical data indicate that in monomeric Cu II -3H complexes the metal is bound with higher affinity compared to its structural domains indicating that the effect of 3D branching should be taken as an important factor for future studies on Cu II peptide constructs.

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