Unraveling the molecular structure, spectroscopic properties, and antioxidant activities of new 2,4-dinitrophenylhydrazone derivatives through a comprehensive investigation

1.1 Experimental

1.1.1

1.1.1 General

The novel 2,4-DNPH compounds were synthesized in a series of reactions. During the experimental procedure, a solution containing equimolar amounts of 2,4-dinitrophenylhydrazine (1 ml) and terephthalaldehyde (1 ml) was refluxed in ethanol for 2.5 h in presence of acetic acid. The stepwise progress of the reaction was observed by TLC. Upon completion of the reaction, the resulting product was purified via recrystallization. These experimental steps are illustrated in Scheme 1.

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Scheme 1

Reaction of 2,4-DNPH and terephthalaldehyde.

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Scheme 1: Schiff base formation from 2,4-dinitrophenylhydrazine and terephtaldehyde.

In the second step, different substituted aromatic aldehydes were refluxed with hydrazine hydrate in the presence of ethanol for 1 hr (Scheme 2).

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Scheme 2

Reaction of hydrazine hydrate and substituted aldehydes.

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In the last step, different aromatic aldehydes reacted with 4((2-(2,4-dinitrophynl)hydrazono)methyl)benzaldehydes in the presence of ethanol for 3 to 5 hrs at a refluxed temperature, resulting in the formation of different compounds (Scheme 3). The compounds obtained from the synthesis were subjected to purification and recrystallization with the use of ethanol. Spectroscopic methods such as ¹H NMR and EI-Spectrometry were utilized to identify the structures of the samples, which were subsequently validated through DFT calculations. Four different compounds were successfully synthesized through the incorporation of different substituents, as detailed in Table 1.

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Scheme 3

Formation of new derivatives of 2,4-DNPH.

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Table 1 Substituents in the synthesized derivatives.

Derivatives	Substituents	Reaction time (hrs)	Derivatives	Substituents	Reaction time (hrs)
2,3-diEt-DNPH		5.0	3-NO2-DNPH		4.0
4-NO2-DNPH		3.0	2-NO2-DNPH		3.5

*Et = Ethoxy group.

1.2 Material and measurements

All the solvents, reagents, and chemicals (2,4-dinitrophenyl hydrazine CAS = 119–26-6, 97 % pure, 2,3-diethoxybenzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, and 2-nitrobenz- aldehyde 97–99 % pure) used in this work, were of analytical grade and brought from Sigma Aldrich, BDH, and Merck. An examination of the structures of the synthesized derivatives was conducted by utilizing Nuclear Magnetic Resonance (NMR) and Electron Impact Mass Spectra (EIMS) techniques. These analyses were performed in Germany using the Advance Bruker AM 500 MHz and Finnigan MAT-311A instruments, respectively. In the determination of the mass spectrum, an energy value of approximately 70 eV was utilized. The melting points of the synthesized derivatives were found to be in the range of 239–241 °C, with an average value. Furthermore, all the synthesized compounds were examined using the Density Functional Theory (DFT) method.

1.3 Spectral interpretation

1.4 Antioxidant activities

The antioxidant activity of the obtained derivatives was determined using the stable DPPH free radical as reported. Different concentrations (50, 100, 200, and 400 µM) of the synthesized compounds were combined with an ethanolic solution of 85 µM DPPH radical. The mixture was incubated at room temperature for 30 min, and the decrease in absorbance was measured at 518 nm using a UV spectrophotometer. Ascorbic acid was used as a positive control at the same concentrations. The experiment was repeated three times, and the percentage inhibition of the compounds and ascorbic acid was calculated using the relation (1);

The DPPH inhibition effect was represented by “X*”, while the control and sample absorbance readings were labeled as “AC” and “AS”, respectively. The ferrous ion chelating activity and ferric ion reducing power of the synthesized derivatives were determined using a standard method previously reported (Puntel et al., 2005). The compounds were mixed with 0.2 ml of 3.6 mM ferrous sulfate, 0.3 ml of 100 mM Tris-HCl (pH = 7.4), and 0.1 ml of 9 mM O-Phenanthroline at various concentrations (50, 100, 200, and 400 µM) and diluted up to 3.0 ml with ultra-pure distilled water. The mixture was then incubated at room temperature for 10 min. The absorbance decrease and increase of the synthesized derivatives for ferrous ion chelating activity and ferric ion reducing power were measured at 510 nm using a UV spectrophotometer. EDTA and ascorbic acid were used as reference standards for ferrous and ferric ions, respectively. The chelating capacity of Fe²⁺ and reducing power comparable with ascorbic acid were calculated using relation (1) with “X*” representing the chelating effect and reducing power, respectively.

The overall antioxidant capacities of the four compounds were assessed through a phosphomolybdenum assay (Saha et al., 1970). The reagent solution with various concentrations of compounds (50, 100, 200, and 400 µM) in ethanol and ultra-pure distilled water, along with 0.7 ml of 0.6 M sulphuric acid, 1.0 mM ammonium molybdate, and 1.0 ml of 28 mM potassium phosphate was incubated at 95 °C for 90 min. The increase in absorbance of the mixture was measured at 695 nm using a UV spectrophotometer to evaluate the total antioxidant capacities of the compounds. The reducing power was calculated using the same relation (1) as mentioned earlier. The scavenging activity for hydroxyl radicals was measured using the Fenton reaction mechanism (Li et al., 2011).

The reaction mixture for all compounds contained 0.1 ml of 7.5 mM O-phenanthroline, 0.5 ml of 0.2 M phosphate buffer (pH 6.6), 0.1 ml of 7.5 mM ferrous sulfate, and 0.1 ml of 0.1% H2O2 and was diluted up to 3 ml with distilled water. The reaction mixture was incubated at room temperature for 30 min, and the absorbance was measured at 510 nm using a UV spectrophotometer. The reaction mixture with Schiff bases complexes was used as the control and a blank was prepared without Schiff bases complexes and H₂O₂. The DPPH radical scavenging activity of the synthesized compounds and ascorbic acid was calculated using the following relation:

Where “A_s” and “A_c” are the absorbance reading of sample and control. While “A_b” is the absorbance reading of the blank.

1.5 Computational methods

The DFT method was employed for the computational studies using the G09 of Gaussian. The geometry optimizations were carried out at B3LYP (Lee et al., 1988) with a 6–311++G(d,p) basis set (E. T. Aljohani et al., 2021b, 2021a; F. S. Aljohani et al., 2021; Rassolov et al., 1998). Grimme’s D3 method was included for the account of dispersion forces(Grimme et al., 2010).The fundamental vibrational analysis was used to confirm true minima without imaginary frequency. A potential energy surface scan was carried out for all derivatives to confirm the lowest energy structure with a transition structure. The Gauge-Invariant Atomic Orbital (GIAO) method was utilized to carry out theoretical NMR calculations in DMSO solvent at B3LYP/311 + G(2d,p).The TD-DFT method was employed to determine the HOMO and LUMO energies with 6-31G and 6–311++G(d,p) basis sets, using the B3LYP functional. The energy gap between HOMO and LUMO was then calculated as,

2.1 Geometry optimization

To understand the chemical characteristics of the synthesized compounds, all the structures were optimized using the g09 package of Gaussian (Frisch, 2009). The optimized structures are shown in Fig. 1. The structure optimization was verified through frequency calculation, with no imaginary frequency. The optimized structures reveal that the bond lengths of sp² carbons (C = C) fall within the range of 1.39 to 1.41 Å, while the bond length of sp³ carbons (C–C) is 1.51 Å. Additionally, the C–H bond lengths for both aromatic and non-aromatic carbons were computed to be 1.08 Å and 1.09 Å, respectively. The presence of nitro groups at the 2nd and 4th positions leads to the elongation of the C2-C3 and C3-C4 bond lengths to 1.43 Å.

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Fig. 1

Optimized structures of 2,4-DNPH derivatives at B3LYP/6–311++G(d,p).The * sign indicates the position of the NO₂ group.

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The synthesized compounds exhibit a nitrogen atom positioned at varying locations, resulting in differing carbon–nitrogen (C-N) bond lengths. The C-N bond length between carbon and nitrogen of NO₂ was found to be 1.45 Å, consistent with reported values (Ji et al., 2010).The calculated length of the C = N bond is 1.28 Å, which is less than the length of a C-N single bond, which is 1.35 Å. Additionally, the nitrogen-nitrogen single bonds were calculated in a range of 1.36 Å, as presented in Table 2. To evaluate the discrepancy between the computed and experimental values, the root mean square deviation (RMSD) was determined for the computed bond lengths, and the results are shown in Table 2. A good correlation between the experimental and calculated data, as evidenced by an RMSD value of 0.019 at the 6–311++G(d,p) basis set. The calculated data was found well matched with the existing experimental data.

The bond angles between sp² carbons were calculated to range from 119.0° to 121.9°. The inclusion of substituents resulted in a minor modification of the bond angle. However, the experimental value of the bond angle between sp² hybridized carbons is 120°. Furthermore, the dihedral angles for C3-N12-N14-C15 and C26-N27-N28-C34 were computed to be 179.9°.

2.2 Potential energy surface scan (PES)

The evaluation of conformation is essential because different conformations of a compound are correlated with different properties such as physical, chemical, and medicinal properties. Conformational stability is also useful in determining the reaction mechanism and rate (Barton and Cookson, 1956). Herein, to identify the most stable conformation and transition structure, the PES scan was carried out around the dihedral angles D(C26-N27-N28-C34) by rotating the torsional angle every 10° for all compounds. The shapes of the potential energy curves as a function of torsional angles are illustrated in Fig. 2.

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Fig. 2

Potential energy surface (PES) scan of selected torsional angle D(C26-N27-N28-C34). Plots (a-d) correspond to 2,3-diEt-DNPH, 3NO₂-DNPH, 4NO₂-DNPH, and 2NO₂-DNPH, respectively.

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The PES curve of 2,3-diEt-DNPH shows the two stable conformers at dihedral angles ± 180.0° with the energy of −1746.17 Hartree (Ha). However, the transition structure appears asan energy barrier of −1746.14 Ha at an angle of 0°. The energy barrier to this rotation is determined to be 0.03 Ha, which is the energy difference between the transition state and the stable conformers. Similarly, for 3NO₂-DNPH, 4NO₂-DNPH, and 2NO₂-DNPH, the two stable conformers were found with the same energy of −1643.00 Ha and with dihedral angles of −179.42° and 180.0°, respectively. The transition structure for these three derivatives was found at an energy barrier of −1642.98 Ha at dihedral angles of 0°, as shown in Fig. 2. It has been calculated that the energy difference between the stable conformers and transition structure is 0.02 Ha. Figure S1 in the supporting information file displays all of the stable conformers that have corresponding transition structures.

The similarity of the potential energy surfaces (PES) of nitro compounds is in line with the similarities in their chemical structures. The only distinguishing factor among them is the position of NO₂. The presence of the -OC₂H₅ group in 2,3-diEt-DNPH results in its distinct electronic properties compared to other derivatives. Despite this difference, the PES was conducted at the same dihedral angle for all compounds, indicating that the structures were stable. These stable structures were then utilized for subsequent electronic calculations and spectroscopic analysis.

2.3 NMR studies

The ¹H NMR was calculated at B3LYP/6–311 + G (2d,p) GIAO method. The ¹H NMR chemical shifts (δ) values were compared with experimental ¹H NMR as given in Table 3. The shielding and deshielding of a proton are crucial in ¹H NMR. The electronegativity of an atom is directly linked to the deshielding of neighboring protons which results in downfield chemical shifts.

Herein, the most downfield chemical shift value noted for H-13, both experimentally and theoretically. This was attributed to the presence of electronegative atoms and hydrogen bonding, resulting in values of 11.57 and 10.88 ppm, respectively. Further investigation revealed that H-13 was a deshielded proton, likely satirically hindered by the neighboring NO₂ group, with a possible hydrogen bonding between the oxygen atom of the NO₂ group as shown in Figure S2. The ¹H NMR peak for H-33 was found experimentally as a singlet peak at 8.57 ppm, while the theoretically calculated value ranged from 7.36 to 7.44 ppm downfield from TMS. Furthermore, the experimental ¹H NMR spectrum for the H-C = N (H-35) proton was confirmed by a singlet peak appearing within 8.28–8.83 ppm range, while the computed peak was found within 7.05–8.06 ppm.

The chemical shift values for the aromatic protons appeared as multiplets within the range of 7.10–8.57 ppm, experimentally. The computed values for the same protons fell within the range of 7.04–8.54 ppm. The experimental chemical shift (δ) value for the CH₂ proton was 4.10 ppm, while the computed value fell between 3.7 and 4.22 ppm. Similarly, the triplet peak observed in the experimental spectrum for the CH₃ proton appeared at 1.34 and 1.45 ppm, while the calculated chemical shift range for CH₃ was 1.08–1.60 ppm. These findings provide insights into the shielding and deshielding of protons, contributing to a well understanding the nature of synthesized compounds.

The shielding values for ¹³C NMR were determined for the ring carbons and appeared in the range of 118.50–158.0 ppm due to the different chemical positions of ¹³C in the molecule. The chemical shift for the C-N carbon was found to be downfield in the range of 151.50 to 169 ppm. Additionally, the ¹³C chemical shift for –HC = N- was explored and found to be at 169.53 ppm downfield. A detailed analysis of the ¹³C chemical shift values with root mean square deviation (RMSD) is given in Table S1.

2.4 Vibrational analysis

The B3LYP/6–311++G (d,p) method was utilized to calculate the infrared (IR) spectroscopic analysis of the synthesized compounds.The wave numbers of observed and calculated spectra with tentative assignments are provided in Table S2. Frequencies below 4000 cm⁻¹ in the calculated spectra were adjusted by a scaling factor of 0.958 (Scott and Radom, 1996).The existing experimental data was used as a reference to assign the vibrational spectra (Bakir, 2018). Figure S18 shows the calculated spectra of synthesized compounds. The observed vibration modes were identified as C–H (aromatic and non-aromatic), C = C, C = N, N-N, N–H, and NO₂ by comparison with known experimental values.

2.5 Frontier molecular orbitals (FMO) and electronic properties

To obtain a better understanding of the electronic transitions of the synthesized compounds, TD-DFT calculations were conducted utilizing the B3LYP functional with 6-31G and 6–311++G (d,p) basis sets. The calculated spectra were assigned to the compounds shown in Figure S19. The absorption band for HOMO-LUMO transition was observed at a wavelength of 480.25, 454.90, 417.16, and 419.31 nm for 2,3-diEt-DNPH, 3NO₂-DNPH, 4NO₂-DNPH, and 2NO₂-DNPH, respectively. A detailed analysis of the HOMO-LUMO transition with absorption spectra and oscillator strength is presented in Figure S19. These results provide valuable information about the electronic properties of the synthesized compounds and can aid in understanding their potential applications.

TD-DFT calculations were performed to obtain various reactivity parameters, which are important for understanding the chemical nature of the synthesized compounds (Parr et al., 1999; Parr and Yang, 1983). The HOMO and LUMO energies are important descriptors for determining the electronic density in a compound, and their energies correspond to the ionization potential and electron affinity, respectively (Rahmani et al., 2018). The HOMO and LUMO energy gap is a significant factor that influences the electronic properties of a molecule. Molecules exhibiting a larger HOMO-LUMO energy gap tend to be less reactive compared to those molecules with a smaller energy gap. The computed HOMO and LUMO energies for the synthesized compounds are presented in Figures S3 and S4.

Reactivity parameters were determined through FMO analysis and are presented in Table 4. These parameters include the HOMO and LUMO gap (ΔE), ionization potential (IP), electron affinity (EA), electronegativity (χ), chemical hardness (η), and chemical softness (σ) (Blanchard and Brüning, 2015; Parr and Pearson, 1983; Pearson, 1986). The ΔEgaps for the synthesized compounds in the gas phase were calculated as 2.518, 3.083, 3.156, and 3.111 eV at B3LYP/6-31G.Whereas, in the aqueous phase, the gaps were 2.582, 2.887, 2.940, and 2.938 eV, respectively (see Figure S3). However, the HOMO-LUMO gaps calculated using 6–311++G(d,p) basis sets were 2.908, 3.099, 3.418, and 3.397 eV, as shown in Figure S4. A molecule has a larger HOMO-LUMO gap, it is typically less reactive than one with a smaller gap so the transfer of an electron from the HOMO to the LUMO becomes energetically unfavorable. The smaller energy gap observed for 2,3-diEt-DNPH indicates a higher reactivity compared to the other synthesized derivatives.

The ionization potential (IP) refers to the minimum energy needed to remove an electron from a molecule and is associated with the E_HOMO, as per equation (4).

The present study indicates that low ionization potential and electron affinity (EA) values suggest a higher reactivity of 2,3-diEt-DNPH relative to the other derivatives.. The electron affinity the amount of energy released by the addition of an electron to an atom or a molecule and it is calculated from equation (5).

Moreover, knowledge of the ionization potential and electron affinity values enables one to determine electronegativity (x), which reflects the ability of an atom or group of atoms to attract electrons toward itself and it is calculated according equation (6)

The calculated electronegativity for 2,3-diEt-DNPH is lower than that of the other derivatives. The FMO analysis clearly indicates the high reactivity of 2,3-diEt-DNPH compared to the other synthesized compounds.

In addition, the softness (Σ) and hardness (H) values of a molecule can provide insight into its polarizability, with high softness and low hardness indicating increased polarizability. In this study, the calculated data for all the derivatives showed nearly the same level of polarizability as given in Table 4. The hardness and softness is calculated according equation (7) and (8).

The reactivity information obtained from the TD-DFT calculations provides a significant implication for the potential applications of the synthesized compounds. By changing the nature and position of substituent brings the changes in the chemical reactivity of compounds (Avcı et al., 2023). The relation between SAR and TD-DFT analysis lies in the understanding of how changes in the molecular structure (as revealed in the SAR analysis) influence the electronic properties (as obtained from TD-DFT calculations) and subsequently impact the observed activities of the compounds. SAR analysis indicates that the change in nature/position of substituent exhibited change in the antioxidant activity as discussed in the section 2.7.

2.6 Molecular electrostatic potential (MEP)

By analyzing the distribution of charged particles, the MEP calculation provides a useful method for identifying the electrophilic and nucleophilic sites of a molecule (Luque et al., 2000). The same DFT method employed for FMO calculations was used to perform MEP calculations. The colorized figures presented in Figure S5 offer a clear description of the nucleophilic and electrophilic sites located on the molecular surface. The blue region of the molecular surface indicates the absence of electrons, making it a good site for nucleophilic attack. In contrast, the red region shows the availability of electrons, indicating a favorable site for electrophilic attack.

The red region in Figure S5 is occupied by oxygen atoms of the nitro group, confirming the availability of electrons and a more favorable site for an electrophilic attack. The blue color of the molecule shows the unavailability of electrons, indicating the suitable region for a nucleophilic attack.

2.7 Antioxidant activity

In vitro, free radical scavenging studies were conducted on all the synthesized compounds, and varying degrees of scavenging activity was observed, with IC₅₀ values ranging from 160.66 ± 13.75 µM to 192.40 ± 14.14 µM (see Table 5). The standard Vitamin C exhibited an IC₅₀ value of 181.43 ± 18.59 µM. 2,3-diEt-DNPH showed activity values that were close to the standard Vitamin C, with an IC₅₀ value of 160.66 ± 13.75 µM. However, the other synthesized compounds, 3NO₂-DNPH, 4NO₂-DNPH, and 2NO₂-DNPH, were comparatively less active than the standard Vitamin C, with IC₅₀ values of 183.92 ± 13.71 µM, 184.61 ± 13.97 µM, and 192.40 ± 14.14 µM, respectively.

Table 6 displays the analyzed values for the ferrous ion chelation activity of all compounds. According to the results, the 3NO₂-DNPH, 4NO₂-DNPH, and 2NO₂-DNPH exhibited activity values of 187.70 ± 7.77 µM, 189.68 ± 8.20 µM, and 181.41 ± 9.09 µM, respectively. However, 2,3-diEt-DNPH showed a value of 199.95 ± 6.42 µM as shown in Table 6.

Table 7 presents the results of the ferric ion-reducing activity of the synthesized compounds. Among all compounds, 3NO₂-DNPH exhibited higher ferric ion-reducing activity, with an IC₅₀ value of 164.06 ± 13.46 µM. The other three compounds showed almost similar values compared to the standard vitamin C, with IC₅₀ values ranging from 170.10 ± 17.10 µM to 171.26 ± 15.79 µM.

The synthesized compounds were subjected to molybdate ion reduction potential analysis. Among the synthesized compounds, 3NO₂-DNPH showed comparable potential to the standard vitamin C, with an IC₅₀ value of 178.46 ± 13.13 µM. However, the other compounds exhibited less reduction potential compared to the standard values as given in Table 8.

The hydroxyl radical scavenging activity for all the compounds are presented in Table 9. Among the synthesized compounds, 3NO₂-DNPH, 4NO₂-DNPH, and 2NO₂-DNPH showed higher activity than the standard vitamin C, with IC₅₀ values of 170.18 ± 12.27 µM, 196.27 ± 13.19 µM, and 186.82 ± 13.89 µM, respectively. However, 2,3-diEt-DNPH showed activity close to the standard Vitamin C, with an IC₅₀ value of 197.84 ± 18.02 µM. 2,3-diEt-DNPH was found to be less active than the standard Vitamin C as shown in Table 9.