# Spectroscopic Methods in Evaluation of Antioxidant Potential, Enzyme Inhibition, Cytotoxicity, and Antimicrobial Activity of the Synthesized N3-Substituted Amidrazones

## TL;DR

This paper reports the synthesis and biological evaluation of amidrazones, showing their antioxidant, enzyme inhibition, cytotoxicity, and antimicrobial properties.

## Contribution

The study introduces new amidrazones with substituted moieties and demonstrates their diverse biological activities through experimental and multivariate analysis.

## Key findings

- Compound 2d exhibited potent antioxidant activity with DPPH = 90.43% and FRAP = 4.73 mM FeSO4.
- Compound 2d showed high inhibition of α-amylase (86.8%) and moderate inhibition of lipase and pepsin.
- Unsupervised multivariate analysis confirmed that Ar1 and Ar2 substituents significantly influence amidrazone biological activity.

## Abstract

Seven amidrazones containing a characteristic NH2–N=C(Ar1)–NHAr2 moiety, where Ar1, Ar2 are phenyl, 4-methylphenyl, 4-nitrophenyl, 2-pyridyl, and 4-pyridyl substituents, denoted as 2a–2g, were synthesized by the reactions between thioamides and hydrazine. Their molecular structures were confirmed by 1H, 13C, 1H-13C HMQC, 1H-13C HMBC, and 1H-15N HMBC NMR spectroscopy, with complete assignment of the detected signals, as well as by high-resolution mass spectra. The biological activity of all compounds was studied, exhibiting antioxidant properties determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods, inhibitory potential against digestive tract enzymes (α-amylase, lipase, pepsin), cytotoxicity (hemolysis), and antimicrobial activities (against Gram-positive and Gram-negative bacteria, and a fungus). The antioxidant activity of the studied amidrazones varied from 83.34% to 93.27% and 1.01–5.79 mM FeSO4 for the DPPH and FRAP methods, respectively. Moreover, these derivatives revealed inhibition potential against α-amylase (28.6–86.8%), lipase (28.0–60.0%), and pepsin (34.1–76.6%), which increased when increasing their concentrations from 0.2 to 1 mg/mL. Among them, compound 2d (possessing 2-pyridyl and 4-nitrophenyl substituents) stood out in particular, as a potent antioxidant (DPPH = 90.43%, FRAP = 4.73 Mm FeSO4) with the highest activity against Gram-positive bacteria: S. aureus (MIC = 64 μg/mL), G. rubripertincta (MIC = 64 μg/mL), and fungus: C. albicans (MIC = 32 μg/mL); high α-amylase (86.8%) inhibition at the highest concentration (1 mg/mL); and lipase (38.0%) and pepsin (43.8%) inhibition at the lowest concentration (0.2 mg/mL). The obtained results were analyzed by unsupervised multivariate techniques to confirm significant differences in the biological activity of amidrazones depending on the Ar1 and Ar2 substituents.