Antioxidant, Antimicrobial, and Molecular Docking Studies of Novel 1, 4-naphthoquinone Derivatives

The synthesis of novel 1,4-naphthoquinone derivatives has attracted prominent interest in the eld of medicinal chemistry since these compounds exhibit potent pharmacological activity as antibacterial, antioxidant, antifungal, and anticancer. Herein, a series of novel 1,4-naphthoquinone derivatives 4-7, 8a-c, and 9a-d containing heterocyclic moieties were synthesized in good yields and characterized by spectral and elemental analyses. All the new synthesized compounds were subjected to in-vitro antimicrobial testing against gram-positive, gram-negative, and fungal strains by calculating the average of the zone of inhibition. The antimicrobial results showed that compounds 8b, 9b, and 9c displayed the highest ecacy against both bacterial and fungal strains. Further studies have been conducted to estimate the antioxidant activity of the compounds using DPPH scavenging assay. The obtained results revealed that compounds 9d, 9a, 9b, 8c, and 6 exhibited the highest radical scavenging activity. Docking studies of the most active antimicrobial compounds within GLN- 6-P, recorded good scores with several binding interactions with the active site. strong antimicrobial and antioxidant activities by simple chemical procedures, it becomes critical to explore new combinations of bioactive heterocycles. As an extension to our studies [37–39] aiming to synthesize novel heterocyclic ring systems having potent pharmacological activity, we describe herein the design, characterization, antimicrobial, and antioxidant activities of new Schiff bases, chalcones and pyrimidines derivatives bearing 1,4-naphthoquinone moiety. Molecular docking studies were realized to predict accurately the type of interactions and the binding anity between the docked synthesized ligands and the active site of the target protein to form a stable complex. To an equimolar mixture of compound 3 (3.48g, 0.01 mol) and p-substituted acetophenone (0.01mol) in ethanol (30 ml) was added an aqueous solution of NaOH (40%). The reaction mixture was kept under stirring overnight at room temperature. Then, the resulting precipitate was ltered, washed with diethyl ether and recrystallized from ethanol. TLC results showed that the reaction was completed using (benzene ethanol 2:1).

Compound (1) (2.15g, 0.01 mol) was subjected to a diazotation reaction, it was dissolved in a round bottom ask with 16 mL of 85% phosphoric acid and heated under stirring. Subsequently, the obtained solution was cooled to 0˚C in an ice bath and then was concentrated in 8 mL nitric acid. A solution of sodium nitrite (3.74 mmol) in 4 mL of water was added to the mixture under vigorous stirring at a temperature below 5˚C for 10 minutes. Afterward, salicylaldehyde (0.374 mmol) in 1 mL water was added dropwise to the mixture under magnetic stirring.
The resulting red solid was ltered, washed several times with water, and then dissolved in 30 mL 10% NaOH. The solution was ltered; the crude product was precipitated during the neutralization with 10% HCl, then ltered and washed with water several times. 1 H NMR (400 MHz, DMSO-d 6  General procedure for the synthesis of Schiff bases (4)(5)(6) Compound 3 (3.48g, 0.01mmol) was reacted with an aromatic amine and dissolved separately in a minimum amount of ethanol, then mixed together and followed by addition of three drop of glacial acetic acid. Then, the solution was re uxed for 6-8 h then cooled to room temperature and poured in to ice-cold water. The resulting product was recrystallized from ethanol.

Antimicrobial activity
In present study, the synthesized compounds 4-7, 8a-c, and 9a-d were screened in-vitro against gram-positive bacteria (Streptococcus pyogenes, Staphylococcus aureus, Bacillus subtilis, Rodococcus ruber, and Enterococcus faecalis) and gram-negative bacteria (B. subtilis, S. aureus, and gram-negative bacteria: E. coli, and P. aeruginosa). Further studies have been conducted to investigate the antifungal activity of the new compounds against P. chrysogenum, Aspergillus niger, and Candida albicans. All experiments were repeated and carried out in triplicate in the case of a signi cant difference in the results. The average inhibition diameters were measured in mm/mg sample ( Table 2 and Table 3) following Hammer et al. method [43].
In-vitro antioxidant activity DPPH antioxidant assay DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of the target compounds (4-7, 8a-c, and 9a-d) was calculated in terms of radical scavenging or hydrogen donating capacity using the stable radical DPPH. This assay is based on the measurement of the reducing ability of antioxidants toward DPPH. Solution of DPPH of 1mM in methanol was prepared and added to all the synthesized compounds (4-7, 8a-c, and 9a-d) at different concentrations (25,50,75, and 100 µg/ml). The sample solutions were incubated in the dark for 30 min in room temperature. Subsequently, the absorbance of the samples was measured at 517 nm against the blank solution and the percentage (%) of inhibition was calculated. Ascorbic acid was used as a standard antioxidant. All the spectrophotometric analyses were carried out in triplicate using a UV-Visible Spectrophotometer (Shimadzu 1700).
The percent of inhibition (I %) of free radical production from DPPH was calculated by the following equation Where, A = Absorbance of the control sample (containing all reagents except the test compound) Absorbance of the test sample after 30 min   The scavenging activity expressed as IC 50 value (Table 4), is de ned as the concentration (µg/ml) of the synthesized compound necessary for scavenging DPPH radicals by 50%. IC 50 values were calculated by linear regression analysis using at least four different concentrations in triplicate [44].
The structure identi cation of all the synthesized compounds was deduced from the spectroscopic data (Mass spectrometry, 13 C-NMR, 1 H-NMR, and FTIR).
The FTIR spectra of compound 3 revealed the presence of a stretching band at about 1550 cm ¹ corresponding to the N = N group and a stretching abroad band around 3550 − 3200 (O-H) cm − 1 due to the intramolecular hydrogen bonding of the hydroxyl group. On the other hand, the ¹H-NMR spectrum of compound 3 displayed singlets at 5.43 ppm and 7.02-8.02 ppm assignable to the O-H proton and the proton of salicylaldehyde and aromatic ring, respectively. Moreover, the ¹H-NMR spectrum of compound 3 exhibited a singlet at 10.41ppm corresponding to the proton of the aldehyde group.
The condensation of 5-((3-acetyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)diazenyl)-2-hydroxybenzaldehyde (3)  Due to the medicinal features of chalcone derivatives, many efforts have been made in this study to further design potent bioactive chalcones derivatives bearing 1,4-naphthoquinone moiety. For this purpose, compound 7 was reacted with p-substituted benzaldehyde to afford three-chalcone derivatives 8a-c (Scheme 2). All these reactions proceeded smoothly and provided the corresponding compounds 8a-c with good yields varying between 60 and 64%. These Cipro oxacin and uconazole were used as a reference drugs and the mean inhibition diameter values (n = 3) were presented in Table 2 and Table 3 in mm/mg sample.
Mainly, all the tested compounds exhibited a comparatively promising activity against gram-positive and gram-negative bacteria.
From Table 2 and Table 3, we can deduce that compounds 9b and 9c revealed the highest e cacy against both bacterial and fungal species with inhibition zone diameters exceeding that of the reference cipro oxacin and uconazole, respectively. Compound 9c exhibited the highest antibacterial activity against Rodococcus ruber, Bacillus subtilis bacteria, Enterococcus faecalis, Streptococcus Pyogenes with an inhibition zone diameter (d = 28 mm/mg sample) exceeding that of the reference (d = 25 mm/mg sample). Furthermore, compounds 6, 8a, 8b, 8c, 9a and 9d presented good antibacterial activity towards the tested bacterial and fungal strains.
Structure-activity relationship (SAR) studies were realized to better gure out the effect of substituents present at the para position of the phenyl moiety in compounds 8a-c and 9a-d on the gram-positive, gram-negative bacteria and fungal strains ( Table 2 and Table 3). It was suggested that the presence of electron-withdrawing chloro (8b, 9b) nitro group (9c) at the para position of the phenyl ring enhances meaningfully their antibacterial and antifungal activity.
On the other hand, the presence of electrodonating amino (9a, 9d) and hydroxyl group at the 4-position of the phenyl moiety decreased slightly their antibacterial and antifungal activity towards all the tested strains.
It is worth noting that compounds 4, 5, 6, and 7 displayed the least antibacterial and antifungal activity against the tested strains since they showed almost similar inhibition zone diameters.
Further SAR studies were conducted to understand better the effect of the aromatic substituents attached to the diazenyl group of compounds 4-6 on the bacterial and fungal species. It was found that the presence of pyridine 4, pyrimidine 5, 4-amino antipyrine (6) has moderate effect on their antibacterial and antifungal activity. In fact, the presence of the antipyrine moiety enhanced slightly the antibacterial activity of compound 6 compared to compounds 4 and 5 against Staphylococcus Aureus, Bacillus subtilis, Rodococcus ruber, Enterococcus faecalis, all the tested gram-negative bacteria, and P. Chrysogenum. In the other hand, the of pyridine ring attached to the diazenyl group ameliorated slightly the antibacterial and antifungal activity of compound 4 against Streptococcus Pyogenes and Aspergillus niger and Candida albicans compared to compounds 5 and 6.  Antioxidant activity Naphthoquinones possess redox and electrophile properties and can play the role of pro-or anti-oxidants through their direct molecular action or by their interactions with various enzymes, which can adjust the oxidative conditions of the cells [48]. Previous studies suggested that phenolic compounds have the capacity to act as hydrogen donors [49] and the presence of the phenol moiety in the structure of the target molecules was related but not constantly proportional to the scavenging capacity [50]. Free radical scavenging is among the best-known mechanisms for testing preliminary free radical-scavenging activity in which antioxidants inhibit lipid oxidation [51].
In this study, the in-vitro antioxidant activity of the synthesized compounds 4-7, 8a-c, and 9a-d was assessed using a UV-Visible spectrophotometry by DPPH radicals, and the obtained results are depicted in Table 4. In fact, DPPH are stable free radicals and their radical character is neutralized in the presence of molecules able of donating hydrogen atoms. In other words, DPPH method is commonly used to measure the ability of antioxidants to sweep free radicals. Previous studies [52] using DPPH method have highlighted that compounds exhibiting potent antioxidant properties must have strong hydrogen donor groups.
Consistent with this nding, we noted from Table 4 and Fig. 1 that the synthesized derivatives 9d, 9a, 9b, 8c, and 6 displayed the highest radical scavenging activity compared to the other compounds. Their behavior was ascribed to the presence of electron donating groups attached to the phenyl ring, such as amino (9a), chloro (9b), and hydroxyl (9d, 8c) groups, compared to the standard ascorbic acid. Besides, the good antioxidant activity of compound 6 might be related to the presence of the carbonyl group in the 4-aminoantipyrine attached to the diazenyl group, which is a potential donor due to the large dipole moment (5.48 D) and display strong basic characters [53]. Compound 9c showed moderate antioxidant activity compared to the previous compounds, this result was assigned to the presence of the withdrawing nitro group at the para position of the benzene ring. In contrast, compound 7 had the lowest antioxidant activity compared to ascorbic acid among all the tested compound, this nding was attributed to the presence of withdrawing acetyl group attached at the 2-position of the chromene ring (IC 50 = 23.30 ± 1.09). Similarly, we found that of pyridine ring, pyrimidine, and 4-aminoantipyrine attached to the diazenyl group of the Schiff bases 4, 5 and 6, respectively caused the inhibition to decrease compared to the ascorbic acid.
The obtained data from the DPPH radical scavenging method showed that compounds 9d and 9a exhibited the strongest inhibition mainly at C = 100 µg/mL as presented in Table 4 and their radical scavenging activities were very close from that of ascorbic acid (87.19 ± 0.54).

Molecular docking study
In the present work, docking studies were carried out to obtain accurate predictions on the optimized conformations for both the new synthesized derivatives and protein targets to form a stable complex and to have a better insight into the structure-activity relationships (SARs) of the novel compounds.
Glucosamine-6-phosphate synthase enzyme (GlcN-6-P) has been proposed as a promising target for many antimicrobial agents since this enzyme is important for ammonia transferring from L-glutamine to fructose-6-phosphate and then transform by isomerization the produced fructosamine to glucosamine-6-phosphate, which is essential for cell wall formation [54][55][56]. To explore the binding modes of the novel candidates, all these candidates 4-9ad were docked inside the active site of GlcN-6-P using a molecular operating environment (MOE, version 2008.10). 3D crystal structure of this enzyme was obtained from a protein data bank (PDB: 1XFF). The data obtained from docking the newly synthesized derivatives, inside GlcN-6-P, including hydrogen bonding, energy scores, and the essential functional groups, are listed in Table 5. The co-crystallized ligand glutamate was redocked within GlcN-6-P with RMSD = 1.2557, In addition, glutamate displayed hydrogen bondings with Gly99, Trp74, Cys1, His86, Arg73, Thr76 and, Asp123 with binding score energy = -15.11 kcal/mol. The compound 9c recorded four hydrogen-bonding interactions within the active site as follows: a) Trp74 with carbonyl group, b) Cys1 with the carbonyl group, c) Pro177 with hydroxyl group via a water molecule, and d) Ser176 with hydroxyl group via a water molecule (Fig. 1).
Furthermore, compound 9b showed a high binding score (-16.01 Kcal/mol) and was detected to form four hydrogen bonds with His71, Cys1, and Trp74 throughbinding with C = O and OH moieties (Fig. 2).

Conclusion
A convenient, e cient, and economical protocol has been described herein for the synthesis of new derivatives bearing 1,4-naphthoquinone moiety. All the designed compounds 4-7, 8a-c, and 9a-d were assessed for their in-vitro antimicrobial activity against gram-positive, gram-negative bacteria, and fungal strains. Further studies have been conducted to evaluate the antioxidant activity of the synthesized compounds using the DPPH method. Based on the overall results, it was found that compounds 8b, 9b, and 9c displayed the highest e cacy against both bacterial and fungal strains compared to the other compounds. The structure-activity relationship (SAR) studies suggested that the presence of electron-withdrawing groups (chloro and nitro) at the para position of the phenyl ring increases the antibacterial and antifungal activity of the compounds compared to those possessing electrodonating groups (amino and hydroxyl) at the same position. The antioxidant studies revealed that compounds 9d, 9a, 9b, 8c, and 6 displayed the highest radical scavenging activity. This nding was attributed to the presence of electron-donating substituents related to the phenyl ring, such as amino 9a, chloro 9b, and hydroxyl (9d, 8c) groups of these compounds. The conducted molecular docking studies ascertained that the most active antimicrobial compounds displayed good energy binding scores within the GLN-6-P active site, suggesting that they can act by the inhibition of DNA replication.