Journal Information
Journal ID (publisher-id): chemical
Title: Journal of the Korean Chemical Society
Translated Title (ko): 대한화학회지
ISSN (print): 1017-2548
ISSN (electronic): 2234-8530
Publisher: Korean Chemical Society대한화학회
INTRODUCTION▼
Antioxidants protect the cells from oxidative injury, and it decreases or neutralizes the free radicals.1 Free radical are ions, atoms, or molecules having unpaired electrons in the external shell of electrons.2,3 They are produced in the human body regularly and become toxic when generated in surplus amount and cause damage to all macromolecules, including proteins, lipids, enzymes, and nucleic acids. It can lead to many diseases such as diabetes, cancer, autoimmune diseases, cardiovascular, aging, neurodegenerative disorders, through the aggressive reactivity of the free radicals.4–6 In addition, oxidative stress induce either direct or indirect reactive oxygen species (ROS) and damage the macromolecules and has been found in various pathophysiological conditions like inflammation, cancer, neurodegeneration, cardiovascular disorders and rheumatoid arthritis.7−16 There are two types of antioxidant mechanisms, either mediated by enzymatic or non-enzymatic. The enzymatic group includes several enzymes, such as catalase, glutathione, and superoxide dismutase (SOD).17 The non-enzymatic groups are antioxidants and directly act on oxidative agents and acquired from dietary sources like vitamin C, vitamin E, carotenoids, flavonoids, polyphenols, etc.18−21 So far, various natural compounds have been reported to limit radical reactions, to reduce or to prevent oxidative stress-related diseases and as a stabilizer for foods during dispensation and storage.12−16,23
Catechin, obtained from the plant Camellia sinensis, is a significant constituent in tea polyphenols, has multiple favorable health-beneficial properties, such as the antiviral, 24−27 antibacterial,28−32 antifungal,33,34 anticancer, Parkinson’s diseases, anti-Alzheimer’s diseases, and the control of obesity.35−38
(-)-Epigallocatechin-3-gallate (Fig. 1), a catechin derivative showed the most outstanding antioxidant and free radical scavenging activity.39 Therefore, substantial research has aimed at the identification of new antioxidants compounds to check radical-induced damage. We synthesized (−)-catechin derivatives (1a-l), evaluated antioxidant activities, and performed docking studies of the compounds with STAT1 to generate information of binding.
EXPERIMENTAL▼
Materials and Methods
Chemistry. A sequence of (–)-catechin derivatives (1a-l) (Fig. 2) were synthesized from 2a-l. The reductive debenzylation of different alkyl/aryl heterocyclic substitutes 2a-l was carried in the presence of a 10% Pd/C in ethyl acetate as solvent to obtain the desired (–)-catechin analogues 1a-l in 75–87% yield (Fig. 2). All the compounds were completely characterized by 1H and 13C NMR spectroscopy and mass spectrometry.35
Antioxidant activity by DPPH free radical scavenging method. Antioxidant activity of synthesized catechin compounds (1a-l) was assessed for free radical scavenging using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method40 with some modifications. The diluted working solutions of the catechin derivatives (1a-l) were prepared in methanol. One milliliter of DPPH (0.004% prepared in methanol) was mixed with 1 mL of test compound. The compounds were shaken vigorously and left to stand in the dark for 30 min. The absorbance of the resulting solution was measured at 517 nm in a Lambda 25 UV-visible spectrophotometer (Perkin-Elmer, Shelton, CT, USA). The radical scavenging activity was measured as a decrease in the absorbance of DPPH. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The radical scavenging activity was calculated using the following formula.41
Radical scavenging activity was expressed as EC50 value, which represents the test compound concentration at which 50% of the DPPH radicals scavenged. BHT were used as positive controls. All tests were performed in triplicate, and values are represented as mean ± S.D.
Docking studies. To studies the interactions and behavior of small molecules, molecular docking method was used to monitor the binding site of protein targets. With the aim of binding mode prediction and estimation of binding affinities, the compounds 1d, 1e and 1j were docked into STAT1 binding cavity, as described previously.42 Docking studies were performed using GOLD v5.2.2 program. Initially, the X-ray crystal structure of STAT1 (PDB code: 1YVL) was downloaded from the protein data bank (www.rcsb.org). All heteroatoms and water molecules were removed. Protein was cleaned using Discovery Studio 2017 (DS) software. Hydrogen atoms were added to the protein using the CHARMM force field. The binding site on the protein was defined as per the previous report.43 Ligands were minimized using the steepest descent algorithm and prepared in DS environment. Every histidine tautomer was set to ND1H protonation as observed in the crystal structure. To predict the binding affinity of the ligands to their target protein, Goldscore was used as the default scoring function while rescoring was done using the Chemscore scoring function. The best-docked poses were selected based on high Gold fitness score, low Chemscore dG, the formation of hydrogen bonds, and other molecular interactions between the ligand and the binding site residues of the protein.
RESULTS AND DISCUSSION▼
Twelve compounds of (−)-catechin derivatives (1a-l) (Fig. 2) were synthesized in 10 steps with an yields of 75–86% using the starting compound 2,4,6-trihydroxyacetophenone and 3,4-bis(benzyloxy) benzaldehyde and characterized according to our reported method.35 The in vitro antioxidant activities of the synthesized compounds 1a-l using DPPH scavenging activity with respect to the standard BHT (butylated hydroxyl toluene) using a spectrometric assay are reported in Table 1 and Fig. 3.40,41
Table1.
In vitro antioxidant activity by DPPH method
Based on the experimental results we found that all the compounds except 1g and 1l showed antioxidant activities better than BHT. Especially compounds 1d, 1e and 1j showed very good activity with EC50 <15 μg mL−1, whereas other compounds 1a, 1b, 1c, 1f, 1h, 1i and 1k exhibited good activity with an EC50 <20 μg mL−1 when compared with control sample BHT.
Three of all tested compounds, 1d, 1e and 1j showed good antioxidant activity. The cLog P values of all compounds were intended using ACD/ChemSketch software (listed in Table 1 and used for structure-activity relationship (SAR) studies. According to SAR analyses, compound 1d showed good antioxidant activity with an EC50 < 14.9 μg mL−1 and cLog P 6.27 as compared to the other derivatives. The enhancement in the activity may be attributed due to benzoyl ester group and also due to increase in hydrophilicity of the compound. The similar ester linked analogues 1c and 1l showed less activities than 1d due to aliphatic unit, even though 1l showed good hydrophilicity.
While, compound 1e containing a morpholine moiety also showed good activity (EC50 < 15 μg mL−1 and cLog P 3.67), but less activities than 1d due to the decrease in hydrophilicity. When the morpholine group was replaced with substituted piperazine moieties (1f-i), we observed decreased activities, which may be due to the presence of aryl group on nitrogen. On the other hand, 1j showed good activity than 1d (cLog P 7.10). The activity may be due to the presence of electron-withdrawing functions like trifluoromethyl- and chloro- groups on pyridine ring. An electron-donating group like amine on pyridine moiety in 1k has reduced the activities, which shows the importance of substituent on pyridine. The primary compound 1a (cLog P 3.67) that has no ether or ester link to a hydroxyl group also produced significant activity but lesser than 1d, 1e, and 1j.
Molecular docking studies of STAT1 with compounds 1d, 1e, and 1j and were undertaken to obtain information of binding interactions at the atomic level. Previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) directly interacts and inhibits signal transducer activator of transcription 1 (STAT1) activation.43,45−46 The EGCG binding site on STAT1 was determined by several site-directed mutagenesis and surface plasmon resonance (SPR) analyses.43 The binding site was formed between the SH2 domain and linker domain of STAT1 comprising of residues such as Lys566, His568, Leu570, Pro571, Tyr680, Leu639, Phe644, and Lys679. Compounds 1d, 1e and 1j were subjected to docking with X-ray crystal structure of STAT1 (PDB ID: 1YVL) using GOLD program.47 The range of Gold fitness score for the compounds was 30.45 to 55.29. The Goldscore of reference compound BHT was 30.45. Compounds 1d, 1e and 1j showed a Goldscore of 55.29, 48.47, and 51.10, respectively. Chemscore was used as the rescoring function. This program estimates the total free energy change that occurs upon ligand binding. The Chemscore for the reference inhibitor, 1d, 1e and 1j were -29.24, -16.79, and -20.59 kJ/mol, respectively. Compounds 1d, 1e and 1j showed better Goldscore and Chemscore values in comparison to the reference compound.
All compounds in their representative structures were superimposed (left) and enlarged (right) with solvent accessible surface representation. The coiled-coiled domain, DNA-binding domain, linker domain, and SH2 domain of the STAT1 protein were shown as solid ribbons colored in orange, blue, green, and purple color, respectively. The compounds 1d, 1e, 1j, and reference inhibitor are depicted by sticks in blue, orange, yellow, and grey, respectively.
Docking simulations revealed that all compounds bind to the same binding site cavity and the binding patterns of the compounds were similar to that of the reference inhibitor BHT (Fig. 4). BHT formed a hydrogen bond with residue His568 (Fig. 5A) and showed electrostatic interactions with Pro645 and hydrophobic interactions with Leu569, Leu570, Leu572, Leu639, and Val642. Compound 1d formed hydrogen bonds with important amino acids such as Lys566, His568, Leu570, and Phe644 (Fig. 5B), also showed electrostatic interactions with Pro571, Pro645, and Ile647. Besides hydrogen bond formation, 1d showed hydrophobic interactions with residues such as Leu569, Leu570, Leu639, Ala641, Val642, and Ile647. Compound 1e formed hydrogen bonds with residues such as Lys566, His568, Leu570, and Arg649 (Fig. 5C). 1e was involved in electrostatic interactions with Pro571, also showed hydrophobic interactions with residues such as Leu569, Leu570, Leu572, Leu639, Ala641, and Val642. Binding mode of compound 1j showed hydrogen bonding with residues including Lys566, His568, Leu570, and Phe644 (Fig. 5D) and formed electrostatic interactions with Pro571, Val642, and Pro645, also interacted with STAT1 by forming hydrophobic interactions with residues such as Leu569, Leu570, Leu572, Leu639, Ala641, and Val642. The binding of compounds with STAT1 was also facilitated by Van der Waals interactions with residues such as Ile565, Lys567, Leu569, Pro571, Leu572, Leu639, Ser640, Ala641, Val642, Thr643, Phe644, Pro645, Asp646, Asn650, Tyr680, Tyr681, Ser682, and Arg683. The details of molecular interactions formed by compounds with binding site residues are summarized in Table 2.
Figure4.
The binding modes of 1d, 1e, 1j, and reference inhibitor in the binding site at SH2 domain and linker domain of STAT1.
Figure5.
The binding conformation and hydrogen bonding interactions of 1d, 1e, 1j, and reference inhibitor in the binding site of STAT1. Color representations (A) BHT: grey (B) 1d: blue (C) 1e: orange and (D) 1j: yellow. Hydrogen bond interactions between proteins and compounds are shown as black dotted lines. Only polar hydrogen atoms are shown for clarity. The SH2 and linker domains of STAT1 are represented as lines in pink and green, respectively.
Table2.
Molecular interactions formed by compounds 1d, 1e, and 1j with binding site residues of STAT1
CONCLUSION▼
(−)-Catechin derivatives were synthesized and characterized. Most of the compounds showed significant activities, among which compounds 1d, 1e, and 1j exhibited excellent activity. Docking and binding mode analysis of the hit compounds revealed that they bind to STAT1 via various molecular interactions such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and Van der Waals interactions. The compounds 1d, 1e, and 1j displayed a high Gold score, lower Chemscore, and a large number of molecular interactions as compared to the reference compound. Moreover, few of the synthesized compounds showed a novel hydrogen bond interaction with Arg649, in addition to previously reported amino acids, such as Lys566, His568, Leu570, and Phe644. These results explain the difference between the activity values of the compounds and the reference compound. Therefore, compounds 1d, 1e, and 1j are potential antioxidant candidates.