Fig 1.
LASSBio-1524 and the design concept of the novel N-acylhydrazone derivatives (2a-c).
Fig 2.
Synthesis of N-acylhydrazone derivatives 2a-c.
Reagents and conditions: a) MeOH, H2SO4 cat. 70°C, 6 h, 90%; b) NH2NH2 H2O 80%, EtOH, 80°C, 4 h, 84–88%; c) ArCHO, EtOH, HCl 10%, 3 h, 75–94%; d) MeOH, r.t., 2 h, 86%.
Table 1.
Yields and physical properties of N-acylhydrazone derivatives 2a-c.
Fig 3.
NOESY experiment of N-acylhydrazone derivative LASSBio-1760 (2c).
Table 2.
Aqueous solubility and other drug-like properties of N-acylhydrazone derivatives (2a-c).
Fig 4.
Effect of N-acylhydrazone derivatives in leukocyte migration induced by carrageenan in subcutaneous air pouch (SAP).
The animals were pretreated orally 60 minutes before carrageenan injection in the SAP, with vehicle (Polysorbate 80) or compounds at doses of 0.3, 3, 10 or 30 mg/kg, IKK-β inhibitor SC-514 (10 mg/kg) or intraperitoneally pretreated with dexamethasone (2.5 mg/kg). Results were expressed as mean ± standard deviation of the number of total leukocytes (x 103/μL). Statistical significance (p <0.05) was calculated by analysis of variance (ANOVA) followed by Bonferroni post-test. # p<0.05 when compared to the vehicle-treated animals in the group of PBS in SAP; *p<0.05 compared to the vehicle-treated animals in the group of carrageenan in the SAP.
Table 3.
ED50 values of the N-acylhydrazone derivatives LASSBio-1760 (2c), LASSBio-1763 (2b) and LASSBio-1764 (2a) and the LASSBio-1524 (1) in the subcutaneous air pouch (SAP) model in mice, after oral administration.
Fig 5.
Effect of N-acylhydrazone derivatives in nitric oxide production induced by carrageenan at SAP.
The animals were pretreated orally 60 minutes before carrageenan injection in the SAP, with vehicle (Polysorbate 80) or N-acylhydrazone derivatives at doses of 0.3, 3, 10 or 30 mg/kg, IKK-β inhibitor SC-514 (10 mg /kg) or intraperitoneally pretreated with dexamethasone (2.5 mg /kg). Results were expressed as mean ± standard deviation of the number of total leukocytes (x 10³/μL). Statistical significance (p <0.05) was calculated by analysis of variance (ANOVA) followed by Bonferroni post-test. # p<0.05 when compared to the vehicle-treated animals in the group of PBS in SAP; *p<0.05 compared to the vehicle-treated animals in the group of carrageenan in the SAP.
Fig 6.
Effect of N-acylhydrazone derivatives in TNF-α production induced by carrageenan at SAP.
The animals were pretreated orally 60 minutes before carrageenan injection in the SAP, with vehicle (Polysorbate 80) or N-acylhydrazone derivatives at doses of 0.3, 3, 10 or 30 mg/kg, IKK-β inhibitor SC-514 (10 mg /kg) or intraperitoneally pretreated with dexamethasone (2.5 mg /kg). Results were expressed as mean ± standard deviation of the number of total leukocytes (x 103/μL). Statistical significance (p <0.05) was calculated by analysis of variance (ANOVA) followed by Bonferroni post-test. # p<0.05 when compared to the vehicle-treated animals in the group of PBS in SAP; *p<0.05 compared to the vehicle-treated animals in the group of carrageenan in the SAP.
Fig 7.
Oxidative metabolism of leukocytes collected from the SAP 24 hours after carrageenan injection, stimulated with PMA.
Intracellular ROS levels were quantified and reading was performed by flow cytometry counting 10,000 events. DCF expression is presented in values of the geometric mean fluorescence intensity in FL1.
Fig 8.
Cell lysates were prepared and Western blots were probed for phospho-NFkB p65 antibody.
Western blot and densitometric analysis of NF-κB activation in LPS stimulated RAW 264.7 cells. Cells were stimulated with LPS (1 μg/mL) for 2 hours. β-actin protein levels were used as loading control. Representative Western blot of β-actin and phospho-NF-κB p65 (Ser536) expression. Equal amounts of nuclear proteins were loaded onto each lane. Graphical densitometry representation of ratio between β-actin and phospho-NF-κB p65 levels after incubation with 1, 10 or 30 μM for 2 hours. Negative control without LPS treatment. Each value is expressed as mean± SD of triplicate experiments. Statistical significance (*p <0.05) was calculated by analysis of variance (ANOVA) followed by Bonferroni post-test. Each Fig is representative of 3 independent experiments.