Synthesis, characterization, DFT, antioxidant, antibacterial, pharmacokinetics and inhibition of SARS-CoV-2 main protease of some heterocyclic hydrazones.

Three hydrazone derivatives have been synthesized using condensation reaction of 4-hydrazinylbenzoic acid with three aromatic aldehydes namely: thiophene-2-carbaldehyde, thiophene-3-carbaldehyde and 2-furaldehyde in ethanol at 78 °C reflux. The synthesized molecules have been characterized using spectroscopic and physicochemical methods including UV-Vis, IR, 1H NMR, 13C NMR, 15N NMR and melting point determination. Optimized molecular structures, UV-Vis and IR spectra modeling, the reactivity, the stability and some quantum chemical parameters of the synthesized molecules were modeled utilizing density functional theory (DFT). The obtained theoretical results were found in good agreement with the experimental results. On the other hand, the antioxidant and antibacterial activities of the molecules under study were evaluated to better understand the associated mechanisms of action specifically. Also, predicted ADME-T and pharmacokinetic parameters indicated that these compounds showed good oral bioavailability. Finally, molecular docking has been used to predict the inhibitory activity of the studied hydrazone derivatives on the SARS-CoV-2 main protease (Mpro).

Introduction

Hydrazone derivatives are considered among the most important molecules in organic chemistry, which show an efficient activity in a wide range of applications including development of new drugs with anti-cancer [1], [2], [3], [4], antimicrobial [5], [6], [7], analgesic [8], antihypertensive [9], anticonvulsant [10], anti-inflammatory [11], antituberculosis [12,13], antitumoral [14,15], anti-HIV [16,17], antimalarial [18], antidepressant [19], vasodilatory [20], anti-Alzheimer [21,22], and anti-corrosion activities [23,24]. The presence of both N – N and C=N functional groups in the structure of hydrazones is responsible of their high therapeutic activity [25].

The most common process to prepare hydrazone compounds consists in heating the appropriate hydrazines with different aldehydes or ketones in various organic solvents such as ethanol and methanol. Gudasi et al. described the ineffectiveness of synthesizing hydrazone by condensing carbaldehyde with o-aminobenzoylhydrazide [26], [27], [28]. Another synthesis route of hydrazones consists in coupling aryldiazonium salt with active hydrogen compound [29].

Recently, DFT (Density Functional Theory) calculations have mostly been utilized to determine the active sites of organic compounds [30], [31], [32]. Also, the electronic and structural properties calculated by DFT can be used to correlate different activities of chemical species [33], [34], [35]. DFT calculations with B3LYP/6–311++G(d,p) as the basis set have also been used to determine the optimized geometry, dipole moment, polarizability, EHOMO-ELUMO energy, molecular electrostatic potential and excited state energy [36,37].

During the last two years, scientists have accelerated the researches to discover new drugs and vaccines to combat COVID-19 pandemic. In this context, the crystalline structure of SARS-CoV-2 main protease (Mpro) is considered as a target to discover therapeutic agents to COVID-19 [38]. Also, the molecular docking calculations are a useful pathway to predict the inhibitive activity of organic compounds towards Mpro, by studying the possible interactions and energies required to inhibit the activity of Mpro [38], [39], [40], [41].

This work reports the synthesis and spectral analysis of three hydrazone derivatives, namely: 4-{(2E)−2-[(thiophen-2-yl)methylidene]hydrazinyl}benzoic acid (HYDZ-1), 4-{(2E)−2-[(thiophen-3-yl)methylidene]hydrazinyl}benzoic acid (HYDZ-2) and 4-{(2E)−2-[(furan-2-yl)methylidene]hydrazinyl}benzoic acid (HYDZ-3). The synthesized molecules have been characterized by spectroscopic and physicochemical methods such as UV–Vis, IR, 1H NMR, 13C NMR, 15N NMR and melting point. Density functional theory was used to calculate the optimized molecular structures, UV–Vis and IR spectra, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), the reactivity, the stability, and some quantum chemical parameters of the investigated molecules. On the other hand, the biological activities of the synthesized hydrazones were evaluated. Also, the inhibition activity of SARS-CoV-2 main protease of the synthesized molecules has been evaluated in silico by molecular docking calculations. It is noted that is the first time that these molecules were tested.

Chemistry, materials and methods

Chemical reagents

All the compounds used in this work for the syntheses of the investigated hydrazones are commercially available and purchased from Sigma-Aldrich and Fluka. In addition, they were used without any further purification.

General procedure for the synthesis of hydrazone derivatives

4-{(2E)−2-[(thiophen-2-yl)methylidene]hydrazinyl}benzoic acid (HYDZ-1)

According to Fig. 1 , 1.0 mmol of 4-hydrazinylbenzoic acid 97% and 1.0 mmol of thiophene-2-carbaldehyde 98% are dissolved in ethanol. The mixture was refluxed at a temperature of 78 °C for 10 h. The resulting crude solution was cooled at room temperature and kept clear until a solid precipitate forms. The resulting product was purified by methanol crystallization. The yellow solid hydrazone is produced in yield 79%. MF: C12H10N2O2S, M.p. 240.02 °C, UV–Vis (Methanol), λmax (nm): 363. IR (ATR, ν (cm−1)): 3502 (O–H), 3311 (N–H), 3276 (C–HAr), 2930 (C–HAlph), 1661 (C=O), 1593 (NCAr), 1541 (C—H), 1273 (C—N), 1138 (N–N), 1090 (C—O), 852 (C-S), 685 (N–H). (400 MHz, DMSO, δ(ppm)): 7.01 (s, 1H, -C Ar5), 7.10 (s, 1H, C Ar3), 7.30 (d, 2H, C Ar8—C Ar9), 7.55 (s, 1H, C Ar4-S), 7.83 (d, 2H, C Ar10—C Ar11), 8.15 (s, 1H, C N), 10.75 (s, 1H, -NN), 12.23 (s, 1H, O) ; : (75 MHz, DMSO, δ(ppm)): 111.54 (d, 2C, HAr10— HAr11), 120.84 (s, 1C, Ar12), 127.13 (s, 1C, HAr5), 128.17 (s, 1C, -HN—N-), 128.30 (s, 1C, HAr4-S), 131.69 (d, 2C, Ar10— Ar11), 134.93 (s, 1C, HAr3), 140.71 (s, 1C, Ar1-S), 148.99 (s, 1C, Ar7—NH), 167.74 (s, 1C, OOH) ; , DMSO, δ(ppm) : 147.71 (s, 2 N, -H—  = C).

Fig. 1

Synthetic rout of the studied hydrazone derivatives.

4-{(2E)−2-[(thiophen-3yl)methylidene]hydrazinyl}benzoic acid (HYDZ-2)

HYDZ-2 is prepared by dissolving 1.0 mmol of 4-hydrazinylbenzoic acid 97% and 1.0 mmol of thiophene-3-carbaldehyde 98% in ethanol. The mixture was refluxed for 12 h at a temperature of 78 °C (Fig. 1). The obtained crude solution was cooled at room temperature and it remained clear until the formation of a solid precipitate. The resulting solid is purified by crystallization using ethanol. The yellow solid hydrazone is produced in yield 77%. MF: C12H10N2O2S, M.p. 252.1 °C, UV–Vis (Methanol), λmax (nm): 346. IR (ATR, ν(cm−1)): 3524 (O–H), 3313 (N–H), 3282 (C–HAr), 2954 (C–HAlph), 1654 (C = O), 1593 (N = C), 1541 (C—H), 1268 (C—N), 1150 (N–N), 1091 (C—O), 852 (C-S), 627 (N–H). (400 MHz, DMSO, δ(ppm)): 7.09 (s, 1H, -C Ar15), 7.53 (s, 1H, -C Ar11-S), 7.58 (d, 2H, C Ar2—C Ar3), 7.73 (s, 1H, C Ar4-S), 7.82 (d, 2H, C Ar4—C Ar5), 8.01 (s, 1H, C NH), 10.64 (s, 1H, -NN), 11.91 (s, 1H, OH) ; : (75 MHz, DMSO, δ(ppm)): 111.54 (d, 2C, HAr2— HAr3), 120.55 (s, 1C, Ar6), 125.12 (s, 1C, HAr11-S), 125.16 (s, 1C, HAr13), 127.61 (s, 1C, HAr14-S), 131.64 (d, 2C, Ar4— Ar5), 135.63 (s, 1C, HAr15), 138.82 (s, 1C, 16 N-), 149.38 (s, 1C, Ar1—NH), 167.77 (s, 1C, OOH) ; , DMSO, δ(ppm) : 147.15 (s, 2 N, -H— =C).

4-{(2E)−2-[(furan-2-yl)methylidene]hydrazinyl}benzoic acid (HYDZ-3)

The synthesis was performed according to the same procedure as before, in which we dissolved 1.0 mmol 4-hydrazinybenzoic acid 97% and 1.0 mmol 2-furaldehyde 99% in ethanol. The mixture was refluxed at a temperature of 78 °C for 4 h. The resulting crude precipitate was purified by crystallization in methanol. The obtained yellow solid hydrazone is produced in yield 77%. MF: C12H10N2O3, M.p. 187.9 °C; UV–Vis (Methanol), λmax (nm): 354. IR (ATR, ν(cm −1)) : 3564 (O–H), 3312 (N–H), 3277 (C–HAr), 2975 (C–HAlph), 1650 (C = O), 1590 (NCAr), 1527 (C—H), 1272 (C—N), 1162 (N–N), 1095 (C—O), 674 (N–H). (400 MHz, DMSO, δ(ppm)): 6.58 (s, 1H, -C Ar13), 6.74 (s, 1H, C Ar11), 7.06 (d, 2H, C Ar2—C Ar3), 7.75 (s, 1H, CH Ar12—O), 7.83 (d, 2H, C Ar4—C Ar5), 7.85 (s, 1H, CHN-), 10.75 (s, 1H, -NN), 12.23 (s, 1H, O) ; : (75 MHz, DMSO, δ(ppm)): 110.77 (s, 1C, HAr11), 111.60 (d, 2C, HAr2— HAr3), 112.42 (s, 1C, HAr13), 120.87 (s, 1C, Ar6), 129.98 (s, 1C, HAr12—O), 131.66 (d, 2C, Ar4— Ar5), 144.22 (s, 1C, Ar14 N-), 149.07 (s, 1C, Ar9—O), 150.76 (s, 1C, Ar1—NH), 167.73 (s, 1C, OOH) ; , DMSO, δ(ppm) : 148.63 (s, 2 N, -H— =C).

Characterization

The synthesized molecules have been characterized by spectroscopic and physicochemical methods such as UV–Vis, IR, 1H NMR, 13C NMR, 15N NMR and melting point. The open capillary technique in BÜCHI melting point B-540 was used to determine the melting point of the synthesized compounds. The UV–Vis spectra were evaluated in methanol solution using a Jasco V-650 spectrometer in range of 190–900 nm. Also, IR spectra of the synthesized molecules were realized in solid state at room temperature using the JASCO 4000 FTIR spectrometer in the region of 600–4000 cm–1. The NMR spectra were realized on a Bruker AVANCE III HD with sande BBO liquid at 400 MHz for 1H NMR, 13C NMR and 15N NMR in DMSO‑d 6 as a dissolvable.

DFT calculations

Recently, DFT calculations are considered the most generally utilized computational techniques because of their precision and less time utilization. In this context, the Gaussian 09 W program was utilized to perform all quantum chemical computations. Also, the DFT method at the B3LYP/6–311++G(d,p) level has been utilized to calculate the optimized molecular structures of HYDZ-1, HYDZ-2 and HYDZ-3 (Fig. 6) [42]. In addition, the calculated vibrational frequencies have been obtained at the optimized structures of the investigated hydrazones. On the other hand, the Time-dependent DFT (TD-DFT) with the B3LYP/6–311++G(d,p) method has been used to predict the electronic spectra of the studied hydrazones derivatives in methanol as solvent. The energy gap (ΔE GAP), electrophilicity index (ω), dipole momentum (µ), global softness (σ), global hardness (η) and absolute electronegativity (χ) are calculated using the obtained values of energies of HOMO and LUMO orbitals. All these parameters are calculated using the following equations [43], [44], [45]:

Fig. 6

Optimized molecular structures and frontier molecular orbitals density distributions of HYDZ-1, HYDZ-2 and HYDZ-3.

In vitro biological evaluation

The antioxidant activity of the synthesized hydrazones was evaluated in vitro using DPPH [46], ABTS [47], CUPRAC [48], FRAP [49] and phenantroline [50] assays. In these procedures, the methanol was used as a negative control, while BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene) and ascorbic acid were used as standards. All assays were performed in 96-well microplates using Perkin Elmer, Enspire microplate reader, in triplicate.

The in vitro antibacterial activity of these molecules was tested against four ATTC bacterial gram positive and Gram negative strains: Bacillus subtilis (ATCC-6633), Staphylococcus aureus (ATCC-6538P), Escherichia coli (ATCC-8739) and Pseudomonas aeruginosa (ATCC-9027). It was determined by the agar disk diffusion method [51,52]. First, the stock solution of the molecules tested was prepared by dissolving 1 mg of the product in 1 ml of DMSO, and then dilutions of 1, 2, 3 and 4 mg/ml were made. Filter paper discs of 5 mm of diameter were impregnated with the corresponding dilutions then are placed on the inoculated MH agar seeded previously by swabbing by an inoculum of ≈ 108 cells/ml of a young culture (18–24 h) of the strain tested. Petri dishes are then pre-incubated for 1/2 h at room temperature, allowing the complete diffusion of the product and then incubated at 37 °C for 24 h. The antibacterial activity was determined by measuring of inhibition zone diameters (mm); a seeding from the zone of inhibition is used to determine whether the activity is bacteriostatic or bactericidal. Gentamicin (10 µg) was used as a positive control and DMSO as a negative one.

Furthermore, it is well known in drug development that the absorption, distribution, metabolism, excretion and toxicity (ADME-T) properties of molecules are one of the main reasons for their failure in clinical trials. The ADME-T properties of the synthesized hydrazones and their pharmacokinetic parameters were evaluated and calculated using OSIRIS and Mol inspiration online property calculation toolkit (available at: http://www.molinspiration.com) [53].

Molecular docking

One of the best ways to understand the binding interaction of ligands and proteins in drug design industry is molecular docking research. Also, molecular docking is one of the best simulation methods for estimating complex of drug-target interactions by calculating energy minimization and binding energy [54,55]. The completion of the molecular docking check is to evaluate the binding affinity of the synthetic ligands to the SARS-CoV-2 main protease. The crystal structure of Mpro was provided from the PDB protein data base through code of 6LU7 (Fig. 2 ). Also, the optimized geometries of HYDZ-1, HYDZ-2 and HYDZ-3 obtained by DFT method at the B3LYP/6–311++G(d,p) level have been used in molecular docking calculations. Therefore, the molecular docking process has been executed by means of the iGEMDOCK program version 2.1. On the other hand, the obtained HYDZ-1/Mpro, HYDZ-2/Mpro and HYDZ-3/Mpro complexes were visualized with Accelry's Discovery Studio Visualizer version 16.1.0 [38].

Fig. 2

Crystal structure of SARS-CoV-2 main protease.

Results and discussion

Spectral analysis

Analysis of the UV–Vis spectra

Generally, the examination of the resulting experimental and calculated UV–Vis spectra (Fig. 3 ) of HYDZ-1, HYDZ-2 and HYDZ-3 recorded in methanol at room temperature indicate the presence of absorption bands in the ultraviolet region.

Fig. 3

Theoretical and experimental UV–Vis spectra of HYDZ-1, HYDZ-2 and HYDZ-3.

The experimental spectra of HYDZ-1, HYDZ-2 and HYDZ-3 illustrate bands at 202 nm, 204 nm and 204 nm, respectively, corresponded to the n→π* transitions produced by the carboxylic acid group (-COOH), where electrons are jump from n no bonding orbitals to π anti-bonding orbitals. Also, the observed shoulders at 225 nm, 228 nm and 237 nm, respectively, are corresponded to the n→σ* transitions created by oxole and thiole groups [56]. On the other hand, the appeared bands at 269 nm, 276 nm and 269 nm, respectively, are related to the π→π* transitions of the C=C group of the aromatic ring. The obtained shoulders at 300 nm, 302 nm and 303 nm, respectively, represent the n→π* transitions associated to the (N—NH) group [25]. Finally, the UV–Vis spectra of the three hydrazones reveal a broad band at 363 nm, 346 nm and 354 nm, respectively, which are related to the n→π* transitions produced by the imine group (C=N) [38].

Vibrational analysis

The experimental and calculated IR spectra of HYDZ-1, HYDZ-2 and HYDZ-3 are presented in Fig. 4 . Also, the obtained vibrational frequencies and their vibrational mode assignments groups are presented in Table 1 . On the other hand, a scaling factor of 0.9614 has been used to scale the calculated frequencies [57]. The examination of the results of Table 1 shows that the scaled theoretical frequencies are in good harmony with the experimental frequencies. The investigation of the experimental spectra of the synthesized hydrazones shows the existence of the following vibrational modes:

Fig. 4

Experimental and calculated IR spectra of HYDZ-1, HYDZ-2 and HYDZ-3.

Table 1

Experimental and theoretical frequencies of selected vibrations of HYDZ-1, HYDZ-2 and HYDZ-3.

Compounds	HYDZ-1			HYDZ-2				HYDZ-3
Assignement	Experimental frequencies (cm–1)	Calculated frequencies (cm–1)		Experimental frequencies (cm–1)	Calculated frequencies (cm–1)		Experimental frequencies (cm–1)	Calculated frequencies (cm–1)
		Unscaled	Scaled		Unscaled	Scaled		Unscaled	Scaled
(N-H)	3311	3497	3362	3313	3516	3380	3312	3498	3362
ν(C—H)Ar	3276	3248	3314	3282	3238	3113	3277	3244	3118
ν(C—H)Alph	2930	3029	2912	2954	3153	3031	2975	3049	2931
ν(C = O)	1661	1773	1704	1654	1774	1705	1650	1773	1704
ν(C = N)	1593	1646	1582	1593	1651	1587	1590	1643	1579
ν(C—N)	1273	1295	1245	1268	1291	1241	1272	1296	1245
(N—N)	1138	1193	1146	1150	1097	1054	1162	1218	1170
ν(C—O)	1090	1091	1048	1091	1090	1047	1095	1102	1059
ν(C-S)	852	858	824	852	823	791	–	–	–
ρout(C—H)	762	701	673	771	652	626	734	743	714
ρout(N—H)	685	642	617	627	442	424	674	500	480

ν: stretching, ρout: out of plane bending.

4-{(2E)−2-[(thiophen-2-yl)methylidene]hydrazinyl}benzoic acid

According to Fig. 4, the small peaks at 3502 and 3311cm–1 are attributed to the O–H and N—H stretching vibrations, respectively. A weak peak appeared at 3276 cm–1 can be referred to the stretching vibration of aromatic C—H groups. The stretching vibrations of aliphatic C—H groups are corresponded to the less intense peak appeared at 2930 cm–1. The presence of peaks within the range of 1600–2000 cm–1 is mostly due to the bending vibration of the C=N or aromatic C=C groups. The characteristic peak of HYDZ-1 is observed as a very intense peak at 1593 cm–1 which is assigned to the stretching vibration of the imine group (C=N). In addition, peaks with medium intensities obtained within the region 1200–1273 cm–1 can be explaining the vibration of C—N groups. The detected peaks in the region 1100–1138 cm–1 could be assigned to the presence of the N—N functional group. The intense thin peak located at 685 cm–1 may be due to N—H bending vibration [25,58].

4-{(2E)−2-[(thiophen-3yl)methylidene]hydrazinyl}benzoic acid

The week peaks located at 3524 and 3313 cm–1 could be attributed to the O–H and N—H stretching vibrations, respectively. A small peak appeared at 3282 cm–1 can be presents the vibration of aromatic C—H groups. The stretching vibrations of the aliphatic C—H groups are corresponded to the less intense peak appeared at 2954 cm–1. The presence of peaks within the range of 1600–2000 cm–1 is mostly due to the bending vibration of the C=N or aromatic C=C functional groups. The characteristic peak of HYDZ-2 is observed as a very intense peak at 1593 cm–1 which is assigned to the stretching vibration of the hydrazone group (C=N—N). In addition, peaks with medium intensities obtained within the region 1200–1268 cm–1 can be explaining the vibration of C—N group. The observed peaks in the region 1100–1150 cm–1 present the vibration of the N—N functional group. The N—H bending vibration is observed as an intense thin peak at 627 cm–1.

4-{(2E)−2-[(furan-2-yl)methylidene]hydrazinyl}benzoic acid

The small peaks situated at 3564 and 3312 cm–1 are attributed to the stretching vibrations of O—H and N—H groups, respectively. Also, the stretching vibrations of aromatic C—H groups are appeared as a weak peak at 3277 cm–1. On the other hand, the stretching vibrations of aliphatic C—H groups are observed as a less intense peak at 2975 cm–1. Generally, the bending vibrations of the C=N and aromatic C=C groups are obtained within the range of 1600–2000 cm–1. The very intense peak located at 1590 cm–1 represents the characteristic peak of HYDZ-3, which is referred to the stretching vibration of the imine group (C=N). Moreover, the stretching vibrations of the C—N group are appeared as medium peaks within the region of 1200–1272 cm–1. Generally, the characteristic peaks of the N—N group vibrations are situated between 1100 and 1162 cm–1. Finally, the intense thin peak sited at 674 cm–1 may be associated to the N—H bending vibration.

Fig. 5 shows the linear fit of the variation of scaling theoretical frequencies versus experimental results. Generally, straight lines have been obtained between theoretical and experimental frequencies with correlation coefficients of correlation coefficients of R2 = 0.9957, R2 = 0.9926 and R2 = 0.9932, respectively, of HYDZ-1, HYDZ-2 and HYDZ-3. We observe that the obtained values of R2 are approximately equal to 1, indicating that the scaling theoretical frequencies are in good agreement with the experimental frequencies.

Fig. 5

Correlation diagrams between the theoretical and experimental wavenumbers of HYDZ-1, HYDZ-2 and HYDZ-3.

NMR analysis

The examination of the NMR results of HYDZ-1, HYDZ-2 and HYDZ-3 (Table 2 and Fig. S3) recorded in DMSO‑d 6 at room temperature permits to determine the following characteristic signals:

Table 2

Experimental 1H NMR and 13C NMR chemical shifts (δ in ppm) of HYDZ-1, HYDZ-2 and HYDZ-3.

HYDZ-1		HYDZ-2		HYDZ-3
1H NMR	13C NMR	1H NMR	13C NMR	1H NMR	13C NMR
7.01	111.54	7.09	111.54	6.85	110.73
7.10	120.84	7.53	120.55	6.74	111.60
7.30	127.13	7.58	125.12	7.06	112.42
7.55	128.17	7.73	125.16	7.75	120.87
7.83	128.30	7.82	127.61	7.83	129.98
8.15	131.69	8.01	131.64	7.85	131.66
10.75	134.93	10.64	135.63	10.75	144.22
12.73	140.71	11.91	138.82	12.29	149.07

From 1H NMR results of HYDZ-1, HYDZ-2 and HYDZ-3, the observed signals between 6.58 and 7.83 ppm are attributed to the aromatic protons (C Ar-benzene and C Ar-heterocyclic). The proton of the hydrazone group (C N—N) appears as a single signal at 8.15, 8.01 and 7.85 ppm, respectively. In addition, single signals observed at 10.75, 10.64 and 10.75 ppm, respectively, are related to the proton of the NN- group. Also, the clarification of the obtained H of the carboxylic acid group (O) was observed as a single signal at 12.23, 11.91 and 12.29 ppm, respectively, for these three molecules [38].

13C NMR spectra of the three hydrazones derivatives show that the following characteristic signals are present: the carbon of the (Ar-NH-) group is observed as a single signal at 148.99, 149.38 and 150.76 ppm, respectively. The signal at 128.17, 138.82 and 144.22 ppm can be related to the carbon of the imine group (-N H-). Also, single signals observed at 167.74, 167.77 and 167.73 ppm, respectively, are related to the carbon atom of the carboxylic acid group (OOH). On the other hand, the aromatic ring carbon atoms are observed between 110.77 ppm and 140.71 ppm [25].

For the 15N NMR results, the obtained spectra of HYDZ-1, HYDZ-2 and HYDZ-3 (Fig. S3) show the presence of the following characteristic signals: single intense signals appeared at 147.71, 147.15 and 148.63, respectively, are attributed to the two nitrogen atoms of the hydrazone group (-H— =C).

DFT study

Table 3 combines the obtained values ​​of the chemical reactivity descriptors of the synthesized hydrazones determined by DFT calculations, such as total energy (ETot), energy gap (ΔEGAP), dipole momentum (µ), electrophilicity (ω) and chemical hardness (η) [59,60].

Table 3

Global chemical reactivity descriptors for HYDZ-1, HYDZ-2 and HYDZ-3 calculated by DFT method at B3LYP/6–311++G(d,p).

Quantum chemical descriptors	HYDZ-1	HYDZ-2	HYDZ-3
ETot (eV)	−30,521.49432	−30,521.364	−21,732.7029
EHOMO (eV)	−5.70487	−5.9002	−5.6689
ELUMO (eV)	−2.061263	−1.8198	−1.9548
ΔEGAP (eV)	3.6436	4.0804	3.7141
µ (Debye)	4.4728	5.5653	4.4565
η (eV)	1.8218	2.0402	1.8570
σ	0.5489	0.4901	0.5385
χ (eV)	3.8830	3.860	3.8118
ω	4.1382	3.6515	3.9122

Optimized molecular structures

According to the values of the total energy calculated at the optimal geometries of the studied compounds (Table 4 ), we observe that HYDZ-2 presents the minimum value of E Tot, which indicates that the conformation of HYDZ-2 presented in Fig. 6 is more stable than these of HYDZ-3 and HYDZ-1. Also, we can be seen from Table 3 that the value of the ΔE GAP of HYDZ-1 is low than these of HYDZ-2 and HYDZ-3, indicating that the HYDZ-1 has the good chemical reactivity and the lowest stability which indicate that the transfer of electrons between HUMO and LOMO is easiest for it and therefore it acts as a better bioactive molecule [61,62].

Table 4

Atomic NBO charges of HYDZ-1, HYDZ-2 and HYDZ-3 calculated by DFT method at B3LYP/6–311++G(d,p).

HYDZ-1		HYDZ-2		HYDZ-3
Symbol	NBO Charge	Symbol	NBO Charge	Symbol	NBO Charge
C1	−0.2196900	C1	−0.3600700	C1	0.2420600
C2	−0.2457100	S2	0.4482700	O2	−0.4609100
C3	−0.2439300	C3	−0.1716300	C3	−0.2562800
S4	0.4259500	C4	−0.3762300	C4	0.1305300
C5	−0.3827100	C5	−0.2621100	C5	−0.2944000
C6	0.0257600	C6	0.0447000	C6	0.0077900
H7	0.2199800	C7	−0.2430600	C7	−0.2448700
H8	0.2267800	C8	−0.1258000	C8	−0.1250500
C9	−0.2444200	C9	0.1904000	C9	0.1872100
C10	−0.1249300	C10	−0.2055000	C10	−0.2061700
C11	0.1877300	C11	−0.2491000	C11	−0.2489400
C12	−0.2062300	C12	−0.1409600	C12	−0.1402900
C13	−0.2489400	C13	0.7855200	C13	0.7850000
C14	−0.1400200	O14	−0.6124800	O14	−0.6127000
C15	0.7850000	O15	−0.6979600	O15	−0.6977500
O16	−0.6126400	N16	−0.4033300	N16	−0.3762900
O17	−0.6977300	N17	−0.2401700	N17	−0.2366500
N18	−0.3780000	H18	0.1945300	H18	0.2305500
N19	−0.2395200	H19	0.2300800	H19	0.2286600
H20	0.2303400	H20	0.2295300	H20	0.2028600
H21	0.2286700	H21	0.2257700	H21	0.2253900
H22	0.2028800	H22	0.2331300	H22	0.4836000
H23	0.2253700	H23	0.2283000	H23	0.3559200
H24	0.4836100	H24	0.2029600	H24	0.2308000
H25	0.3553200	H25	0.2248100	H25	0.2238900
H26	0.2289000	H26	0.4834000	H26	0.1978500
H27	0.1581900	H27	0.3670100	H27	0.1681800

Frontier molecular orbitals analysis

The stability and the chemical reactivity of bioactive compounds can be explained extensively via HOMO and LUMO orbitals and their energies [38]. Generally, a high value of E HOMO is related to the electron donor ability of the molecule [63], while a higher value of the E LUMO is related to the molecule ability to accept electrons [64]. Fig. 6 shows that the electron densities of the HOMO and LUMO orbitals are almost dispersed on the entire molecular structures of HYDZ-1, HYDZ-2 and HYDZ-3. On the other hand, the positive phases of each molecule are colored in red solid surfaces, while the negative phases are colored in green [33]. The HOMO densities are related to the strong electronegativity of oxygen and sulfur atoms. It was disturbed on the thiophene, furan ring and C=N function which have larger affinity to conserve their electronic pairs [25]. The high HOMO energy corresponds to a high capacity to lose an electron, whereas the smaller LUMO energy indicates the greatest affinity for electrons. We observe from Table 3, the order of E HOMO is HYDZ-3 (−5.6689) > HYDZ-1 (−5.70487) > HYDZ-2 (−5.9002), hence HYDZ-3 has better electron donating ability and liberates electrons more than HYDZ-1 and HYDZ-2 which expresses their capacity in antioxidant activity. Also, the lowest value of E LUMO is referred to the HYDZ-2 (−1.8198) which shows its elevated potential to receive electrons.

Although dipole moment importance as an indicator of the electronic distribution within a molecule is certain, studies have reported inconsistent findings on the association between dipole moment values and the inhibition efficiency of bioactive molecules [65]. In the current study, as expected, we did find this significant association between the dipole moment values and the order of the bioactivity: HYDZ-2 > HYDZ-1> HYDZ-3.

The chemical hardness indicates the resistance to deformation or polarization of the electron cloud of atoms, ions or molecules under a small disturbance of the chemical reaction. Also, hard molecules have a high energy gap and soft molecules have a small energy gap [66]. Generally, the low value of the hardness and the high value of the softness of a molecule can produce a high inhibitory efficiency [67]. The hardness and softness of our molecules follow the following order: HYDZ-1 < HYDZ-3 < HYDZ-2 and HYDZ-1 > HYDZ-3 > HYDZ-2, respectively. These results indicate that the HYDZ-1 with a lowest hardness and highest softness is the less stable and the most reactive species.

The values of electrophilicity index () of these compounds are between 3.6515 and 4.1382. Generally, the high value of proves better electrical properties, while a low value of indicates poor electrophiles. According to the Table 3, we find that our molecules have better electrophilicity which measures the energy lowering due to the electron flow between the donor and acceptor. Finally, we conclude that HYDZ-1 has a good inhibiting activity (antibacterial and inhibition of SARS-CoV-2 main protease) and HYDZ-3 have a good antioxydante activity, while HYDZ-2 shows a more stable conformer among these compounds.

Molecular electrostatic potential surfaces (MEP)

To determine the active sites that lead to electrophilic and nucleophilic attacks, we decided to use the molecular electrostatic potential surfaces (MEP) as a useful descriptor [58]. MEP describes the charge distribution of molecules in a three-dimensional manner, and correlates the total charge distribution with dipole moment, electronegativity, partial charge, and chemically reactive sites of molecules [67]. The zero potential area is represented by green color, and the evolution of potential follows the order of red < orange < yellow < green < blue [68]. Fig. 7 represents the calculated MEP surfaces ​​of HYDZ-1, HYDZ-2 and HYDZ-3 with potential ranges of [−5.944 10–2, 5.944 10–2], [−5.402 10–2, 5.402 10–2] and [−5.930 10–2, 5.930 10–2], respectively. So, surfaces under investigation appear in different colors. Generally, red, orange and yellow zones indicate the negative regions of MEP, corresponding to nucleophilic sites (carboxylic, thiophene, furane and hydrazone functional groups). On the other hand, green and blue zones present the positive regions responsible for the electrophilic attacks (hydrogen atoms and benzene ring).

Fig. 7

Molecular electrostatic potential maps of HYDZ-1, HYDZ-2 and HYDZ-3.

Atomic charges of HYDZ-1, HYDZ-2 and HYDZ-3 using NBO analysis

In general, the electron density of molecules can be affected by NBO charges. Also, to check bonds and interaction bonds within and between molecules, NBO analysis can be used to evaluate the delocalization of the electron density between occupied Lewis-type orbitals and empty non-Lewis NBOs, indicating the stability of the donor-acceptor interaction [69]. For the investigated hydrazones, the calculated natural atomic charges of all atoms are offered in Table 4. From the obtained results, it can be seen that oxygen and nitrogen atoms have the most negative charges, which is due to molecular relaxation [38]. In addition, the more positive charges are located on the hydrogen atoms. For the studied molecules, the negative charges are located on O16, O17, N18 and N19 atoms for HYDZ-1, O14, O15, N16 and N17 atoms for HYDZ-2 and O2, O14, O15, N16 and N17 atoms for HYDZ-3.

Biological activity

Evaluation of the antioxidant activity

The IC50 and A0.50 values were determined for all the compounds and presented in Table 6. In DPPH assay, all tested compounds showed a good antioxidative activity. Also, HYDZ-3 showed the lowest IC50 value among the synthesized hydrazones (38.33 ± 0.81 mM), and presents an antioxidant activity superior to that of the standard BHT (70.9 ± 6.6 mM), and comparable to that of the standard BHA (24.28 ± 1.28 mM). It is therefore possible to conclude that all the prepared hydrazones exhibit a high antioxidant activity (IC50 between 38.33 ± 0.81 and 143.07 ± 1.3 mM) [70].

Table 6

The antibacterial inhibition diameters in mm.

Strains	Diameter of the inhibition zones (mm)
	HYDZ-1	HYDZ-2	HYDZ-3	GM
P. aeruginosa	14±2.08	7 ± 0.57	<5	20
B. subtilis	<5	<5	<5	30
S. aureus	<5	<5	<5	25
E. coli	<5	<5	<5	22

*GM : gentamicine.

The results attained from ABTS assay attest that all tested compounds exhibit elevated antioxidant activities. In addition, HYDZ-3 was found to be the best antioxidant agent (IC50 1.27 ± 0.10 mM)). On the other hand, it can be seen from Table 5 that the investigated hydrazone derivatives have an antioxidant activity higher or comparable to that of the standards BHT and BHA.

Table 5

Determination of the antioxidant activity of the examined hydrazone derivatives by DPPH, ABTS, CUPRAC, FRAP and Phenantroline assays.

Compounds	DPPHIC50 mMa	ABTSIC50 mMa	CUPRACA0.50 mMa	FRAPA0.50 mMa	PhenanthrolineA0.50 mMa
HYDZ-1	87.91±3.34	8.29±0.32	49.23±1.2	41.12±0.40	11.49±1.45
HYDZ-2	143.07±1.30	7.46±0.39	31.59±0.30	77.40±0.30	14.06±0.87
HYDZ-3	38.33±0.81	1.27±0.10	30.95±1.05	56.38±0.49	15.89±1.12
BHAb	24.28±1.28	7.67±0.40	17.97±0.66	N.T	4.31±0.03
BHTb	70.9 ± 6.6	8.24±0.13	16.62±0.79	N.T	4.20±0.06
Ascorbic acidb	N.T	N.T	N.T	15.39±1.10	N.T

aValues expressed are means ± S.D. of three parallel measurements. (p<0.05). N.T: not tested.

bReferences.

According to the CUPRAC assay, we can see that all the prepared hydrazones exhibit a comparable antioxidant activity (A0.50 between 31.59 ± 0.30 and 49.23 ± 1.2 mM) with that of the standards BHA and BHT (17.97 ± 0.66 mM and 16.62 ± 0.79 mM, respectively).

As to the results of the FRAP assay, our hydrazones derivatives exhibit also a comparable antioxidant activity (A0.5° between 41.12 ± 0.40 and 77.40 ± 0.30 mM) to that of the standard ascorbic acid (15.39 ± 1.10 mM).

Regarding the phenantroline assay, the high antioxidant activity has been observed for HYDZ-1 with an A0.50 value of 11.49 ± 1.45 mM, which demonstrates that it has a good antioxidant activity comparable to that of standards BHA and BHT (4.31 ± 0.03 and 4.20 ± 0.06 µM, respectively).

In summary, from the results of the antioxidant evaluation by DPPH, ABTS, CUPRAC, FRAP and Phenanthroline assays, the following observations can be derived:

The studies carried out measuring the antioxidant activity did not correlate with each other. This result can be explained by the different mechanisms of action of the assays.

The prepared hydrazone derivatives exhibited a high antioxidant activity.

In DPPH and ABTS assay, compound HYDZ-3 showed a higher antioxidant activity than that of the standards BHA and BHT, due to presence of furan group within hydrazone compounds that exerted an abundant inhibitory effect against various free radicals.

The thiophene group contributed to the antiradical activity even if it is in position 2 or 3 on the hydrazone moiety (HYDZ-1 and HYDZ-2). It is to be noted that position 2 (HYDZ-1) is more reactive than position 3.

In FRAP and Phenanthroline assays, HYDZ-1 was regarded as the best antioxidant with comparable value of the standards BHA and BHT because they showed a low value of E Gap, which means that it is likely to react more, and the mechanism of this assays is consists in the electron transfer which explains the reactivity of this compound.

It is well known that the best anti-oxidant activity is observed with HYDZ-1 and HYDZ-3 due to the electronic resonance outcome of the hydrazone compounds [71].

The mechanism of DPPH and ABTS based on the transfer of H + (IC50) and CUPRAC, FRAP phenantroline based on the mechanism of transfer of electrons (A0.5) explain the different reactivities of these molecules.

Finally, except the ABTS assay, the other assays showed a smaller dynamic range in the data. This could also explain the non-correlation of the results.

Evaluation of the antibacterial activity

Among the examined strains (Table 6 ), only P. aeruginosa was susceptible to the HYDZ-1 (15 mm) and HYDZ-2 (7 mm). Although, P. aeruginosa is intrinsically resistant to many antibiotics [72], and is capable of rapidly acquire additional resistance during treatment is a frequent source of therapeutic failure [73], its double membrane structure specific to lipids makes it sensitive to lipophilic substances, which affect the membrane integrity and permeability and would lead to the lysis of the bacterial cell. On the other hand, according to Haenni et al. [73], the microbiological studies showed that the presence of an aromatic or a hetero aromatic moiety containing more lipophilic character significantly contributed to antibacterial activity. El-Etrawy et al. [74] attributed the inhibitory effect of the new synthesized hydrazones against pathogenic bacteria to its ability to activate enzymes and some cellular proteins essential for ATP synthesis and expression of ribosomal subunit proteins. Studying the structure-activity relationship suggests that different substitutions on aromatic rings affected drastically the antibacterial activity of the synthesized compounds [75]. In the same way, the presence of electronegative atoms and groups (-NO2, -Cl, S-H and N—H) on aromatic rings improves the antimicrobial activity of the compounds [76]. The presence of active functions on the aromatic ring, N=C groups of hydrazones and thiophene motif enhances the antibacterial activity of the studied compounds. This activity remains bacteriostatic and lower than gentamicin. This activity, with 4 mg (20 µl), is satisfying in comparison with other studies [75,77], where high concentrations were used (50 µL, 35 µL). These observations suggest that the molecular targets of HYDZ-1 and HYDZ-2 are very different from the other molecules as well as those of conventional antibiotics.

Evaluation of the ADME-T activity

Drug-likeness of the synthesized compounds compared with the known drugs was evaluated under Lipinski's rule. In general, an orally active drug fulfill the following criteria: Not more than 5 hydrogen bond donors (n-OH and n-NH), not more than 10 hydrogen bond acceptors (n-ONs), molecular weight (MW) less than 500 D, not more than one violation and octanol-water partition coefficient (milogP) should be not more than 5 [78]. As it can be seen from Table 7 , all our hydrazone derivatives did not show any violation of Lipinski's rules with showing a good solubility. Contrarily to the standards, and as an example, chloroquine drugs exhibited one violation, which poses risks of mutagenic and irritation. On the other hand, in the toxicity profile, the studied compounds were ranged as relatively safe species, except standards which showed a risk of toxicity. The tested compounds did not show any risk of toxicity. Consequently, they do not cause any mutagenic, tumorigenic, irritation, or reproductive effect. These results suggest that the synthesized hydrazones have good ADME-T parameters and can be conducive candidates as drugs.

Table 7

Calculated physicochemical and pharmacokinetic parameters (ADME-T) of the synthesized compounds, which important for a good oral bioavailability.

Compounds	miLogP<5	solubility	TPSA (oA) <500	MW	nON<10	nOHNH<5	MV	vio		Toxicity risks
										Mut	Tumo	Irr	R,E
HYDZ-1	4.57	−3.17	61.69	246.29	4	2	208.83		0	No	No	No	No
HYDZ-2	4.26	−3.06	61.69	246.29	4	2	208.83		0	No	No	No	No
HYDZ-3	3.93	−2.84	74.83	230.22	5	2	199.68		0	No	No	No	No
Chloroquine*	4.01	-4.06	28.16	319.88	3	1	313.12		1	Yes	No	Yes	No
Hydroxychl-oroquine*	3.08	-3.55	48.38	335.88	4	2	321.38		0	Yes	No	No	No

*: Drugs Reference, Topological polar surface area (TPSA); Number of rotatable bonds (n-rotb); Molecular weight (MW); Molecular volume (MV); Logarithm of partition coefficient between n-octanol and water (miLogP); Number of hydrogen bond donors (n-OHNH); Number of hydrogen bond acceptors (n-ON); Lipinski's violation (vio) (Mut) mutagenic (Tumo): tumorigenic (Irr) : irritant (R.E) reproductive effective.

Molecular docking analysis

The different interactions and their energies between an inhibitor and amino acids inside the active sites of enzymes have been determined by molecular docking simulations. In this context, the energy distribution of different interactions between the Mpro and the synthesized ligands are illustrated in Table 8 , which explains the different interactions between the active amino acids of the targeted receptor (Mpro), and HYDZ-1, HYDZ-2 and HYDZ-3 [79]. The obtained crystal structures of the best docked modes for Mpro-HYDZ-1, Mpro-HYDZ-2 and Mpro-HYDZ-3 complexes are represented in Fig. 8 . It is to be noted that the investigated ligands preferably bind in the external structure of Mpro [38]. Also, the 3D and 2D binding-interaction diagrams of HYDZ-1, HYDZ-2 and HYDZ-3 with SARS-CoV-2 main protease are shown in Fig. 9 . Generally, the presence of hydrogen bonds is crucial for the interaction between inhibitors and receptors. In this context, the interpretation of the 3D and 2D interaction diagrams indicates the presence of the following types of interactions:

Table 8

Energy distribution of the investigated hydrazones between van der Waals forces, hydrogen bonding and electrostatic interactions.

Compounds	Total energy	vdW		H-Bond		Elec
Mpro—HYDZ-1	−89.90	−70.17		−17.17		−2.56
Mpro-HYDZ-2	−85.66	−60.75	−22.03		−2.88
Mpro-HYDZ-3	−84.82	−71.07	−13.07		−0.69
Chloroquine*	-83.91	−74.41	−9.5		0
Hydroxychloroquine*	-82.27	−67.02	−15.25		0

Drugs Reference, H-bonds: hydrogen bonding, vdW: van der Waals forces and Elec: electrostatic interactions.

Fig. 8

Best docked poses visualization of HYDZ-1, HYDZ-2 and HYDZ-3 with SARS-CoV-2 main protease.

Fig. 9

3D and 2D Binding-interaction diagrams of HYDZ-1, HYDZ-2, HYDZ-3, Chloroquine and Hydroxychloroquine with SARS-CoV-2 main protease.

In Table 9 , HYDZ-1 ligand was found to interact with a single conventional hydrogen bond LEU141 (6.35 Å) with the oxygen of the carboxylic group, a Pi-sigma type with MET165 (5.24 Å) and the benzene group, a Pi-Sulfur with MET49 (5.44 Å), HIS41 (5.14 Å) and MET165 (5.32 Å) and thiophene group, a VDW bond with HIS172 (4.93 Å) and carboxylic group.

Table 9

Molecular docking results interactions and distance of the investigated inhibitors with SARS-CoV-2 main protease.

Compounds	Amino acid Interactions	Distance (Å)	Type of the interaction
HYDZ-1	- LEU141- HIS172- MET49, HIS141, MET 165- MET165	6.354.935.44, 5.14, 5.325.24	Hydrogen bondvdWPi-sulfurPi-sigma
HYDZ-2	- ARG 217, THY 304- GLN 256- VAL 303	5.44, 3.834.116.24	Hydrogen bondPi-sigmavdW
HYDZ-3	- CYS145, GLY143, SER144,SER 144, HIS163- CYS145- GLU166- MET165- MET 165	3.55, 3.41, 3.93, 3.36, 4.57.6.005.186.084.71	Hydrogen bondHydrogen bondPi-AlkylvdWPi-sigmaPi-cation
Chloroquine*	- ASN 142- HIS 164- LEU 27 CYS 145- CYS 145 CYS 145- GLY 143	4.129.655.00, 4.094.41, 6.794.95	Hydrogen bondPi-Pi T-shapedPi-AlkylPi-cationvdW
Hydroxychloroquine*	- GLY143, SER 144, CYS 145- CYS145 HIS163- HIS172 THR 26	2.95, 4.28, 3.076.69, 5.775.01, 4.28	Hydrogen bondPi-AlkylvdW

: Drugs Reference.

HYDZ-2 ligand was also observed to interact through two conventional hydrogen bonds ARG217 (5.44 Å) and THR304 (3.83 Å) with the oxygen of the carboxylic and amine groups of the hydrazone, respectively, a Pi-sigma type with GLN256 (4.11 Å) and a vdW with VAL303 (6.24 Å) with thiophene and C = N function, respectively.

As to HYDZ-3 ligand, it has interacted through five hydrogen bonds with CYS145 (3.55 Å), GLY143 (3.41 Å), SER144 (3.93 Å), SER144 (3.36 Å) and HIS163 (4.57 Å) which reacted on the carboxylic group, a Pi-Alkyl with CYS145 (6.00 Å), a Pi-Cation with MET165 (4.71 Å) reacted on the furan group, a VDW with GLU166 (5.18 Å) and Pi-Sigma with MET165 (6.08 Å) and benzene group.

For the standard Chloroquine, there is only one hydrogen bonding of ASN142 (4.12 Å) with amine group. Then, a Pi-Alkyl with LEU27 (5.00 Å) and CYS145 (4.09 Å) with the Chlorure atom, a Pi-Cation with CYS145 (4.41 Å), CYS145 (6.79 Å) react on the quinoleine group, a Pi-Pi T-shaped with HIS164 (9.65 Å), GLY143 (4.95 Å) of vdW interaction.

For Hydroxychloroquine, there are three hydrogen bonds of GLY143 (2.95 Å), SER144 (4.28 Å) and CYS145 (3.07 Å) with OH function, two bonds of Pi-Alkyl of CYS145 (6.69 Å), HIS163 (5.77 Å), two van der Waals bonding of HIS172 (5.01 Å), and THR (4.28 Å) interacted with Chlorure atom and amine groups of hydroxychloroquine, respectively.

Pi-Pi Cation, Pi-Sulfur and Pi-alkyl type interactions are included in the hydrophobic category of van der Waals forces, while the hydrophilic interactions included H-bonding forces [38,80]. From these results, we observe that HYDZ-1, HYDZ-2 and HYDZ-3 have a Pi-Sigma bond which does not appear in the standards. This means that the inhibitive effect has increased. We can conclude that the amino acids reacted more strongly at the nucliphilic sites (hydroxy, furan and thiophene groups).

Fig. 10 shows the interactions with the molecular surface around the studied ligands at the binding site of Mpro. Accordingly, the green area represents the electron acceptor region, while the pink area represents the electron donor one. The calculated inhibitor-receptor complex interactions showed very positive results [81]. Table 10 gathers comparative values of binding energies of the compounds according to the following order: HYDZ-1 > HYDZ-2 > HYDZ-3 > Chloroquine > Hydroxychloroquine drugs. As a result, the investigated ligands showed a good inhibitory ability against SARS-CoV-2 main protease than standards (Hydroxychloroquine and Chloroquine). Also, HYDZ-1 ligand showed a better docking score (−89.90 kcal/mol) than other compounds.

Fig. 10

Ligands interaction with their molecular surface maps inside the active site pocket.

Table 10

Comparison of the binding energy of HYDZ-1, HYDZ-2 and HYDZ-3 with these of some drugs.

Compounds	Binding energy in Kcal/mol
HYDZ-1	- 89.90
HYDZ-2	−85.66
HYDZ-3	- 84.82
Chloroquine*	- 83.91
Hydroxychloroquine*	- 82.27

: Drugs Reference.

Conclusion

In the present work, we have synthesized three new bioactive hydrazones. The obtained compounds were characterized by high melting points and their structures were determined by UV–Vis, IR, 1H MNR, 13C NMR and 15N NMR. Moreover, the quantum chemical study of the investigated hydrazones has been performed applying DFT method with B3LYP/6–311++G(d,p) basis set. IR and UV–Vis theoretical results were found in good agreement with the experimental results. Besides, the calculated values ​​of E HOMO indicated that the investigated HYDZ-3 is the more electron donor sharing electrons with the acceptor species. Also, the obtained values of ΔE GAP showed the high reactivity and non-stability of HYDZ-1. The calculated MEP illustrated that the electrophilic reaction possible sites are located at the O16, O17, N18 and N19 atoms for HYDZ-1, O14, O15, N16 and N17 atoms for HYDZ-2, and O14, O15, N16 and N17 atoms for HYDZ-3. According to the NBO charge results, oxygen and nitrogen atoms are the most negatively charged. The in vitro evaluation of the antioxidant activity of the examined hydrazones demonstrated that HYDZ-3 shows a higher activity with DPPH than standards, using ABTS assays, while in FRAP and Phenanthroline assays HYDZ-1 showed a good activity compared to standards. The antibacterial activity was obtained with all three hydrazone derivatives. HYDZ-1 showed a moderate pseudomonas bacteriostatic activity compared to gentamicin. Also, the predicted ADME-T and pharmacokinetic parameters indicated that these compounds have a good oral bioavailability and show a powerful biological activity. So, the biological study demonstrated that HYDZ-1 and HYDZ-3 can be regarded as promising new radical scavengers and oxidative stress inhibitors for the treatment of liver disorders and could be a lead for new, effective, and safe drugs. On the other hand, the calculated binding energies of Mpro-HYDZ-1, Mpro-HYDZ-2 and Mpro-HYDZ-3 complexes indicated the ability of HYDZ-1 to inhibit SARS-CoV-2 main protease. Finally, the docking results allowed to conclude that the investigated hydrazones derivatives may be developed as therapeutic agents against SARS-CoV-2.

CRediT authorship contribution statement

Lilia Adjissi: Writing – review & editing, Data curation, Formal analysis, Methodology, Resources, Investigation. Nadjib Chafai: Conceptualization, Methodology, Validation, Supervision, Project administration, Resources, Investigation, Writing – review & editing. Khalissa Benbouguerra: Supervision. Imene Kirouani: Writing – review & editing. Abdelkader Hellal: Supervision, Writing – review & editing. Houdheifa Layaida: Writing – review & editing. Meriem Elkolli: Supervision, Writing – review & editing. Chawki Bensouici: Supervision, Writing – review & editing. Salah Chafaa: Project administration, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.