Spectroscopic studies on the antioxidant and anti-tyrosinase activities of anthraquinone derivatives

2.2.1 Synthesis of compound 1

To mixture of 1,4-dihydroxyanthracene-9,10-dione (0.01 mmol of 2.40 g), benzamide (0.01 mmol of 1.21 g), and benzaldehyde (0.01 mmol of 1.02 mL) were added in ethanol (10 mL) at reflux 1 h at 60 °C. The obtained solid material was washed with water. The final product was identified and conformed by using thin-layer chromatography. The solid substance is separated using column chromatography with ethyl acetate and hexane (4:3 ratio).

Yellow solid; yield 87 %; mp: 165–169 ⁰C; IR (KBr, cm⁻¹): 3359 (NH), 1765 (CO), 3353 (–OH). ¹H NMR (DMSO‑d₆), δ(ppm): 8.29–7.88 (4H, dd, Ar-CO ring), 8.03 (–NH, 1H, s), 8.03–7.63 (Ar-ring, 5H, m), 7.37–7.26 (Ph-ring, 5H, m), 7.09 (=CH, 5H, m), 6.16 (NH-CH, 5H, m), 5.35 (2-OH, 5H, m); ¹³C NMR (DMSO‑d₆), δ(ppm): 187.1 (1C, CO), 186.9 (1C, C = O), 166.1 (1C, CO), 155.0, 153.44, 124.3, 118.7, 116.6, 112.2 (6C, Hq-ring), 141.3, 129.2, 128.2, 126.2 (6C, Ph-ring), 134.2, 132.1, 128.8, 127.5 (6C, Ar-CO ring), 133.6, 132.1, 126.8 (6C, Ar-ring), 51.6 (1C, NH-C). EI-Ms, m/z (Relative intensity %): 449.13 (M⁺, 30.7 %). Anal. Cacld. C₂₈H₁₉NO₅: C, 74.83; H, 4.25; N, 3.11, Found: C, 74.85; H, 4.26; N, 3.13 %.

2.3.1 Synthesis of compound 2

To mixture of 1,4-dihydroxyanthracene-9,10-dione (0.01 mmol of 2.40 g), benzamide (0.01 mmol of 1.21 g), and cinnamaldehyde (0.01 mmol of 1.25 mL) were added in ethanol (10 mL) at reflux 1 h at 60 °C. The obtained solid material was washed with water. The final product was identified and conformed by using thin-layer chromatography. The solid substance is separated using column chromatography with ethyl acetate and hexane (4:3 ratio).

Yellow solid; yield 84 %; mp: 172–174 ⁰C; IR (KBr, cm⁻¹): 3495 (NH), 1860 (CO), 3540 (–OH). ¹H NMR (DMSO‑d₆), δ(ppm): 8.29–7.88 (Ar-CO ring, 4H, dd), 8.03 (–NH, 4H, dd), 8.03–7.63 (Ar-ring, 4H, dd), 7.40–7.24 (Ph-ring, 4H, dd), 7.09 (=CH, 4H, dd), 6.66 (–CH, 1H, s), 6.37 (Ph-CH, 1H, s), 5.56 (NH-CH, 1H, s), 5.35 (2-OH, 1H, s); ¹³C NMR (DMSO‑d₆), δ(ppm): 187.1 (1C, CO), 186.9 (1C, C = O), 167.8 (1C, CO), 154.3, 152.1, 127.6, 117.4, 115.9, 112.7 (6C, Hq-ring), 136.4, 128.6, 128.5, 127.9 (6C, Ph-ring), 134.2, 132.1, 128.8, 127.5 (6C, Ar-CO ring), 133.6, 132.1, 126.8 (6C, Ar-ring), 129.5 (1C, Ph-C), 123.3 (1C, =C-), 52.4 (1C, NH-C). EI-Ms, m/z (Relative intensity %): 475.14 (M⁺, 32.9 %). Anal. C₃₀H₂₁NO₅: C, 75.78; H, 4.43; N, 2.95, found: C, 75.75; H, 4.45; N, 2.93 %.

2.4.1 DPPH free radical scavenger assay

The vivid purple type and absorption at 517 nm of compounds 1 and 2 against DPPH assays. As a result, anti-radical activity and the amount of leftover DPPH are inversely associated. Several quantities of the test compounds (varying from 25 to 100 µg/mL in methanol) were added to a 0.004 % solution in 4 mL (w/v) methanolic solution of DPPH. The absorbance at 517 nm was measured against a blank after a 30-minute incubation period at room temperature (Kumar et al., 2017).

To calculate the percentage of free radical scavenging (%), the following equation was used: $$\mathit{Scavenging}\%DPPHScavengingeffect\left(\%\right)=\frac{A_{\mathit{control}}-A_{\mathit{sample}}}{A_{\mathit{control}}}\times 100$$

Where,

A_sample represents the measured material's absorbance and A_control represents the DPPH solution's absorbance, which contains all of the reagents other than the test substance. There were three runs of each test.

2.5.1 Method for hydroxylating benzoic acid

The Fenton reaction, which produces –OH at the C₃ or C₄ position of the benzene ring, converts benzoic acid into hydroxyl. FeSO₄ (10 mmol), 7H₂O (10 mmol), EDTA (10 mmol), and the representative solution were combined in 0.2 mL in a test tube with a screw top. The sample solution, phosphate buffer (pH 7.4, 0.1 mol), and 0.2 mL of sodium benzoate (10 mmol) were then mixed together to get a final volume of 1.8 mL. Finally, 0.2 mL of a 10 mmol H₂O₂ solution was added, and the combination was pre-incubated for 2 h at 37 °C using BHT as the standard for excitation following this incubation, fluorescence was assessed at 407 nm release (305 nm). $$\mathit{OHradicalscavengingactivity}=\left(1-\frac{{F.I.}_s-{F.I.}_o}{{F.I.}_c-{F.I.}_o}\right)\times 100$$

Where,

Fluorescence intensity at 305 and 407 nm without treatment, or F.I._O. F.I._C. stands for fluorescence intensity for the treatment control at 305 and 407 nm. Fluorescence intensity of the treated sample at 305 and 407 nm (Wang et al., 2022).

2.5.2 Activation of hydrogen peroxide scavengers (H₂O₂^. Scavenging activity)

The calculation of the H₂O₂ scavenging capacity is explained by Lateef (Lateef et al., 2017). In brief, an H₂O₂ solution with a 40 mM concentration was produced using phosphate buffer (pH 7.4). The H₂O₂ solution was subsequently exposed to test substances at concentrations between 25 and 100 µg/mL. At 230 nm, the absorbance values of the reaction mixture were calculated. To calculate the percentages of H₂O₂ scavenging, use the equation below. Percent scavenging A_control A_sample = 100 A_control where A_sample denotes the absorbance of the test compounds and A_control denotes the absorbance of the control reaction, which uses all reagents other than the test compound. Each test was done three times. $${\mathit{IC}}_{50}Inhibition=\frac{A_{\mathit{control}}-A_{\mathit{sample}}}{A_{\mathit{sample}}}\times 100$$

Where,

A_sample is the test substances' absorbance, and A_control is the absorbance of the control reaction (which contains all reagents but the test compound).

2.5.3 Activity for scavenging nitric oxide (NO^. Scavenging activity)

With a few minor adjustments, the measurement of nitric oxide scavenging activity adhered to the standards set by Ghosh (Ghosh and Tiwari, 2018). NO^.were created by the reaction of sodium nitroprusside. The test chemicals (1 and 2) were introduced in various concentrations (range from 25 to 100 µg/mL) and incubated for 150 min at 258 °C with C₅FeN₆Na₂O (10 mM, 1 mL) and 1.5 mL of phosphate buffer saline (0.2 M, pH 7.4). The reaction mixture was added to the Griess reagent after it had been incubating for 1 mL (1 % sulphanilamide, 2 % H₃PO₄, and 0.1 % naphthylethylenediaminedihydrochloride). The chromosphere absorption was measured at 546 nm. The nitric oxide scavenging activity was calculated using the following equation. $$\%NOScavenging=\frac{A_{\mathit{control}}-A_{\mathit{sample}}}{A_{\mathit{control}}}\times 100$$

Where, A_control represents the absorbance of the control reaction and A_sample represents the absorbance of the test chemicals (which contains all reagents but the test compound.

2.5.4 Ferric reducing antioxidant power (FRAP) assay

Using the previously described technique, the FRAP test was carried out by Benzie and Strain. Freshly prepared FRAP reagent was created by mixing 2.5 mL of TPTZ (10 mM), 40 mM HCl, 20 mL of FeCl₃, and 25 mL of acetate buffer (300 mM concentration and pH 3.6). The presence of Fe²⁺-TPTZ in the light blue reagent indicates that Fe³⁺-TPTZ is present, which changes to dark blue when it interacts with antioxidants. The increase in absorbance seen at different concentrations of the compounds (1 and 2) in FRAP reagent (100, 50, and 25 µg/ml) at a wavelength of 593 nm was the cause of these modifications (Benzie and Devaki, 2018).

2.5.5 Anti-tyrosinase activity

All of the compounds were put through an inhibition of tyrosinase test with L-DOPA as the substrate for purpose of evaluating the tyrosinase inhibition, in accordance with the method described by Bradford with a few minor modifications (Selvaraj et al., 2020). As a result, the reference ingredient was chosen to be kojic acid, a substance that is frequently used to whiten the skin due to its significant inhibitory activity against tyrosinase.

2.5.6 Molecular docking analysis

The most potent anthraquinone series compounds (1 and 2) and the proteins 2Y9X were subjected to molecular docking investigations using Auto Dock Vina 1.1.2 to examine the mechanism of binding and interactions (Chidambaram et al., 2021). The collected data were then assessed in relation to the molecular docking models of the reference medications, which comprised Kojic acid. The Compounds 1, 2, and Kojic acid all have three-dimensional models made using the Chem Draw Ultra 12.0 application.

3.8.1 Ligand preparation

Chem 3D pro and Chem draw 12.0 were used to draw the ligands (Compound 1, 2, and Kojic acid). For use in docking investigations, the ligand molecules were then transformed into a Protein Data Bank (PDB) file.

3.8.2 Receptor preparation

The protein data bank was used to download this structure, which represents a protein that binds mosquito odorant. Water molecules and ligands are taken out of the utilising Discovery Studio 2019 as a receptor. The SWISS PDB Viewer was used to lower the energy of the receptor. After that, the receptor underwent molecular docking.

3.8.3 Identification of binding pocket

The Discovery Studio 2019 application and a co-crystallized ligand were used to examine the target protein's binding pocket. Asp 368, Asp 438, Asp 368, Ser 407, Asn 323, and Asn 332 residues were found in the binding pocket.

3.8.4 Docking

In order to investigate the interactions between compounds 1, 2, and kojic acid, the mosquito odorant binding protein, molecular docking experiments were carried out using Schrodinger Maestro 9.2. The amino acid residues that made up the binding pocket were taken into account when choosing the docking grid boxes. With an exhaustiveness score of 8, the interactions were visually inspected in the PyMOL and Discovery Studio programmes. The software utilized to analyze the docking behaviour of compounds 1, 2, and kojic acid with 2Y9X protein is 9.2 for the Schrodinger Maestro. A 2D illustration of a molecule coupled with a receptor for compounds 1 and 2 comparatively, the binding affinity of compound 2 for the 2Y9X protein is substantially lower (-1.7 kcal/mol) than that of kojic acid (-4.7 kcal/mol). Kojic acid was employed as a benchmark because it is a widely available insecticide for controlling mosquitoes.

The stability of protein–ligand interaction is greatly influenced by the hydrogen bond, and the ideal distance amongst the H-acceptor and H-donor atoms is lower than 3.5. In compounds 1 and 2 having strong hydrogen connections to the corresponding 2Y9X proteins, hydrogen bond lengths of less than 3.5 were discovered. Compound 2 created hydrogen-bond interactions with receptor 2Y9X. The hydrogen-interacting residues Asp 368 and Asp 438 have a bond length of 2.14 and 2.73, respectively. VAL 370, GLY 375, VAL 377, VAL 404, GLU 405, ARG 406, SER 407, ALA 413, TYR 415, LYS 435, PRO 436, ASP 438, and PRO 439 are among the amino acid residues. According to Fig. 8a, the receptor's docking score (-1.7 kcal/mol) and a 3D representation of the inhibitor molecule were implicated in hydrophobic interactions. Compound 2 is more active than compound 1 and kojic acid. As a result, the complex of Asp 368 and Ser 407, which has a bond length of 1.88, 219, is an interacting residue that interacts with hydrogen. LYS 435, PRO 436, ASP 438, PRO 439, THR 440, GLU 441, and LYS 443 The amino acid residues are THR 321, ASN 323, ASN 332, THR 333, PRO 334, ASP 368, VAL 370, GLY 375, VAL 377, VAL 404, ARG 406, SER 407, ALA 413, and TYR 415. Inhibitor molecules docked into receptors were depicted in 3D in Fig. 8b, and their hydrophobic contacts were indicated by their docking score (-2.7 kcal/mol).

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Fig. 8

(a) Molecular docking study of Compound 1, (b) Compound 2, and (c) Kojic acid with 2Y9X protein (2d and 3d interaction structures).

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As a result, the Asn 323, Asn 332, of kojic acid, which has a bond length of 1.70, 1.78, is an interfering residue that interacts with hydrogen. THR 321, ASN 323, GLY 331, ASN 332, THR 333, PRO 334, VAL 404, GLU 405, SER 407, SER 412, ALA 413, TYR 415, ASP 438, and PRO 436 are among the amino acid residues. Inhibitor molecules docked into receptors were depicted in 3D in Fig. 8c, and their hydrophobic contacts were indicated by their docking score (-4.7 kcal/mol). Table 1 shows that docking socre, interacting the residues between the compounds with2Y9X proteins (Akbar et al., 2022).

3.8.5 Structure–activity relationship (SAR) analysis

The correlation between the biological activities of physiologically significant substances and their chemical composition in the test system is referred to as the structural-activity relationship (SAR). We discovered a number of critical factors while analysing the relationships between structure and activity.

Fig. 9 illustrate the SAR of compound 1 and 2. The compound 2 was highly active against all other activities compared with standard and compound 1.

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Fig. 9

Structural activity relationship.

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Compound 2, which has anthraquinone connected with benzamide and benzaldehyde, it is highly active against all the activity, but lowly active against previously reported anthraquinone derivative (Selvaraj et al., 2020) was highly active (IC₅₀ = 6.26 µg/mL) against anti-tyrosinase activity.

Compound 2, which has anthraquinone connected with benzamide and benzaldehyde, it is highly active against all the activity, but lowly active against previously reported anthraquinone derivative (Zarren et al., 2021) was highly active (IC₅₀ = 21.88 µg/mL) against antioxidant activity (DPPH).

Furthermore study is required since lead compound 2 constitute the novel class of very effective antioxidant, and anti-tyrosinase.