All of the synthetic steps were previously reported by our research group (Mateev et al. 2024). The reagents were obtained from Merk without further purification. The resynthesised compounds were confirmed by IR spectroscopy.

The antioxidant capacities of the resynthesised hydrazide-hydrazones were initially tested with the DPPH in vitro assay. DPPH (2,2- diphenyl-1-picrylhydrazyl) is a stable free radical which gives an intensive violet colour when dissolved in a solvent. Therefore, the radical-scavenging capacities of the organic compounds can be measured with UV/VIS. The absorption maximum is at 515 nm. In the current work the standard protocol reported by Brand-Williams et al. was followed (Brand-Williams et al. 1995). Briefly, the title compounds were dissolved in methanol to form three different concentrations 31–250 µM, followed by the addition of 1 mL methanol solution of DPPH (1 mmol/L). Each reaction mixture was incubated for 30 min in a dark room. Three measurements for each sample were carried out. Trolox was used as a standard. The percentile inhibition of the tested samples was calculated by the following formula (1):

DPPH _{scavenging activity} = Abs_control – Abs_sample / Abs_control × 100% (1)

Where Abs_control is the absorbance of DPPH radical in methanol and Abs_sample is the absorbance of DPPH radical solution mixed with sample.

The ABTS radical scavenging capacities of the hydrazide-hydrazones were measured according to a modified method of Arnao et al. (Arnao et al. 1996). The absorbance was measured at λ = 734 nm. The stable radical cation (ABTS^+•) was generated by mixing 7 mmol/L of ABTS and 2.4 mmol/L of potassium persulphate. The solution was allowed to react for 14 hr in the dark at room temperature. 1 ml of the ABTS working solution was reacted with the hydrazide-hydrazones for 15 minutes. Thereafter, the absorbance was measured. Trolox was used as a standart. The inhibition percentage was calculated applying the same formula as the DPPH assay.

The molecular docking calculations were performed in the active site of NADPH oxidase with the docking module of Schrödinger - Glide (Schrödinger). The crystal structure of NADPH was downloaded from the PDB (PDB:2CDU) together with a co-crystallized ligand which was used to generate the grid space through the Receptor Grid Generation module. The hydrazide-hydrazone based antioxidants were drawn with 2Dsketch and prepared for the docking simulations with the module of Meastro - LigPrep. The crystal structure of NADPH was also prepared with the Protein Preparation module in Maestro. The extra precision module of Glide was used for the calculation of the energies and the complexes were visualised with the XP Visualized Maestro.

DFT computation was performed via the Gaussian 09 program (Frisch et al. 2009). Compounds 5b and 5k were optimised through B3LYP/6311G⁺⁺(d,p) basis setup at the ground state. Thereafter, energy computation was performed with the same setup. The highest occupied molecular orbital (HOMO) energy and the lowest unoccupied molecular orbital (LUMO) energy values were fetched from the computation in atomic units (a.u.). These values were converted into electron volts (eV). Thereafter, the related parameters were computed with these values. Molecular electrostatic potential (MEP) and frontier molecular orbital (FMO) analysis were then performed after visualisation was undertaken via GaussView 5.0 (Dennington et al. 2008; Muhammed and Aki-Yalcin 2024).

Several recent works noted the antioxidant potential of the hydrazide-hydrazone moiety (Amine Khodjaz and Boulebd 2020). Introducing electron-donating moieties was also considered as a potential approach for increasing the overall antioxidant effects. The antioxidant effects of the pyrrole ring were also discussed (Kundu and Pramanik 2020). Moreover, the presence of a tryptophan ring in molecules with good antioxidant properties directed us in including the former ring in the final structures. Considering the discussed research works, we resynthesised a series of pyrrole-based hydrazide-hydrazones with a subsequent test for their radical-scavenging activities. The synthesis of the title compounds was previously reported by our research group (Mateev et al. 2024) (Scheme 1) (Fig. 2). The final reaction step was optimised by using microwave irradiation which led to fast reaction times and good yields. Essentially, microwave irradiation leads to high temperature which is related to the direct heating effect (Shi and Hwang 2003). Importantly, all hydrazide-hydrazones were prepared for 30 seconds with excellent yields when the MW radiation was employed in the process. The infrared spectra of the synthesised compounds were compared with the previously reported data.

Scheme 1. 

Synthesis of the pyrrole-based hydrazide-hydrazones.

Figure 2. 

Aldehydes used for the condensation reactions.

The antioxidant effects of the synthesized hydrazide-hydrazones were assessed through DPPH and ABTS testing.

The radical scavenging properties during the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay is mainly associated with the hydrogen donating capacity of the tested compounds which could be detected spectrophotometrically with a decrease of the absorbance at 517 nm. For the current testing three concentrations were used (ranging from 31 to 250 μM). The DPPH radical scavenging effects of compounds 5a-m are displayed in Fig. 3. Trolox was used as a standard.

Figure 3. 

DPPH radical capacity of the hydrazide-hydrazones at concentrations 31, 125 and 250 µM. Standard deviation (SD) (n = 3).

The most active compound after evaluations with the DPPH test was the hydrazide-hydrazone 5b, which contains a hydroxyphenyl group. In the highest applied concentration (250 µM), the former structure showed 61.27% radical-scavenging property. Using the same concentration, Trolox displayed 92.94% effect which is considerably higher. Interestingly, none of the other compounds demonstrated any radical scavenging activities after using the DPPH test. The main hypothesis is that the presence of a hydroxyl group causes the good antioxidant effect of 5b. Recently reported paper confirms that the introduction of a hydroxy moiety enhances the DPPH performance of the compounds (Yamauchi et al. 2024).

During the ABTS test, the tested antioxidants could neutralise the ABTS cation and the change could be detected by the discoloration of the initial colour. Decreased absorbance at 734 nm indicates that the tested structures are with good antioxidant capacities. The ABTS radical-scavenging test of the hydrazide-hydrazones is provided in Fig. 4.

Figure 4. 

ABTS radical capacity of hydrazide-hydrazones at concentrations 31, 125 and 250 µM. Standard deviation (SD) (n = 3).

Overall, all of the applied hydrazide-hydrazone showed moderate to excellent ABTS radical scavenging properties. The most active compound was the hydrazide-hydrazone condensed with salicylaldehyde - 5b, which showed better results compared with the applied standard Trolox. It was noted that 5k also demonstrated moderate antioxidant activity and it could be further evaluated. At concentrations of 250 µM, 125 µM, and 31 µM, compound 5b displayed inhibition of 90.49, 60.44, and 35.77%, respectively. Numerous studies have shown that the introduction of a hydroxyl functional group leads to significantly enhanced antioxidant effects (Chen et al. 2020). Moreover, both tests showed different results and it was concluded that the ABTS test is more suitable for the tested hyrazide-hydrazones. The differences could be discussed with the ability of the ABTS test to examine both hydrophobic and hydrophilic compounds (Thaipong et al. 2006; Floegel et al. 2011). An article reported by Nayak et al. discussed that the tryptophan fragment increases the antioxidant effects (Nayak et al. 2016). Interestingly, the implementation of a tryptophan ring in the current study led to good results only after the utilisation of the ABTS test, however the DPPH assay showed little to none antioxidant effects of the applied molecules.

NADPH oxidases (NO) are enzymes that are mainly involved in the transfer of electrons to molecular oxygen. That process leads to the generation of reactive oxygen species (Tarafdar and Pula 2018). Moreover, it is noted that NO generates ROS during the metabolism of arachidonic acid. Therefore, the inhibition of NO lowers the oxidative stress and the maintenance of the redox homeostasis (Costa et al. 2018). A confirmation of the latter statement is noted in several papers that have reported the in silico simulations of ligands with free-radical scavenging activity in the active site of NO (Irfan et al. 2021). Thus, the in silico simulations in the current paper were conducted in the active pocket of NO (PDB code: 2CDU). Only the best antioxidant according to the DPPH and ABTS assays was tested - 5b. The calculated interactions are given in both 2D and 3D panels (Fig. 5).

Figure 5. 

Visualised intermolecular interactions between 5b and the active site of NO. A. 2D interaction; B. 3D interaction.

Two hydrogen bonds between 5b and Phe245 were observed. Moreover, the distances were optimal - 1.95 and 2.15 Å which shows the stability of the formed bonds. Lys187 was introduced into p-cation bonding with the benzene ring of the tryptophan ring, therefore it could be hypothesized that the tryptophan moiety enhances the antioxidant properties of 5b. In addition, the active residues Tyr188, Phe245, Tyr296, Ile297, Pro298, Leu346 and Ala349 participated in hydrophobic interactions with 5b. Overall, the formation of two stable hydrogen bonds with the hydroxyl group in the salicylaldehyde moiety demonstrates the importance of –OH function group for the formation of low energy complexes with NO.

The DFT calculation gave the HOMO and LUMO energies of compounds 5b and 5k. Based on these values, related electrical parameters of the compounds were computed. The computed parameters were harnessed to figure out the reactivity and stability of the compounds. The HOMO energy is correlated to the electron donating capacity of an optimized structure. The LUMO energy is the reflection of the ability of the optimised compound for gaining electrons. The energy gap between the LUMO and HOMO energies (ΔE) is crucial in understanding the relative stability and reactivity of compounds (Kınaytürk et al. 2023). The energy gap value of compound 5b was found to be higher than the value of 5k implying a higher stability and a lower reactivity for it (Table 1). The ionization potential shows the energy required to remove electron from a compound’s ground state. A compound with a higher ionization potential needs a higher energy to remove electrons from its ground state. So, this compound will be resistant for donating electrons. In this computation, compound 5k gave a higher ionization energy suggesting a higher energy requirement for electron donation. Compound 5k had a lower HOMO energy relative to compound 5b (Table 1). Hence, the electron donating capacity of compound 5k was found to be lower. The HOMO energy and the ionization potential results suggested that compound 5k would show a lower electron giving capacity. A higher electron affinity implies a higher electron absorbing capacity and a lower electron giving capacity. In this study, compound 5b had a lower electron affinity (Table 1). This in turn shows a higher electron giving capacity for it. This was reflected in its higher HOMO energy value (Table 1).

The relative hardness and softness of compounds are used to compare the stability and reactivity of the compounds. The hardness of a compound reflects its resistance for electron distribution implying a higher stability (Muhammed et al. 2023). In this study, compound 5b gave a higher hardness value than that of 5k (Table 1). Hence, compound 5b is anticipated to be more stable. This result was in line with the result of evaluation via energy gap. On the other hand, a higher softness reflects a higher propensity for reactivity. In this study, compound 5k gave a higher softness value (Table 1). Hence, compound 5k is expected to have a higher propensity for reactivity. In short, the DFT study revealed that 5b would be more stable and 5k would be more reactive.

The HOMO and LUMO orbitals of compound 5b were distributed differently. The HOMO orbitals were mainly concentrated around the indole heterocyclic ring. On the other hand, the LUMO orbitals were mainly concentrated around the 2-hydroxybenziledenehydrazinyl group of the compound (Fig. 6). Some degree of similarity between the orbital distributions of compounds 5b and 5k was observed. In this respect, the HOMO orbitals of compound 5k were mainly concentrated around the indole ring. On the other hand, the LUMO orbitals were mainly concentrated around the 5-ntirofuran heterocyclic ring (Fig. 7).

Figure 7. 

HOMO-LUMO orbitals of compounds 5b and 5k with corresponding energy values at B3LYP/6311G++(d,p) basis set.