Chemistry
All the chemicals, reagents and solvents used for experimental work were analytical grade. The formation of product was monitored by performing TLC (using Silica gel F254 Aluminium sheets from Merck Company). Column chromatography was carried out by using Silica gel 100-200 mesh. Open capillary method was performed to determine the melting point of the synthesised quercetin derivatives and was expressed in units of ºC. IR spectral study was carried out using Shimadzu FTIR 8400-S spectrophotometer by KBr Press method and IR data obtained was expressed in cm-1. 1H NMR and 13C NMR studies were carried out on Bruker 400 MHz FT-NMR spectrophotometer using trimethylsilane as internal standard and DMSO (D6) and CDCl3 as solvent systems. Mass spectra were recorded on LC-MS ACQUITY UPLC mass spectrometer using AP and ESI method and TOF detector.
Synthesis of quercetin derivatives
Synthesis of 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl benzoate (QB1)
Quercetin dihydrate (2 mmol), benzoic acid (6 mmol) and zinc chloride (2 mmol) were taken in a RBF. 10 ml of phosphorous oxychloride was gradually added to the mixture and was stirred for 15 hrs at 75 °C. The reaction completion was supervised by TLC. Upon formation of the product, stopped stirring and the product was allowed to cool. Ice cold water was added and then product was extracted using ethyl acetate.10 The separated ethyl acetate layer was collected and evaporated. The obtained brownish yellow product was subjected to preparative TLC, using chloroform: methanol (70:30) as the mobile phase. The pure product was separated, collected and dried. Yield: 80 mg (10%); m.p. 220-223 ˚C; FT-IR (cm-1): 3360.11 (alcohol O–H stretch), 3070.78 (Ar – H), 1695.49 (C=O stretch), 1506.46 (C=C stretch), 1174.59 (C–O ester);1H NMR (400 MHz [D6]DMSO): δ 6.18 (s, 1H, ArH(6)), 6.45 (s, 1H, ArH(8)), 7.13 (d, J = 6.4 Hz, 1H, ArH(14)), 7.47 (s, 1H, ArH(11)), 7.59 (t, J = 2.4 Hz, 2H, ArH(19)), 7.74 (d, J = 5.6 Hz, 1H, ArH(15)), 7.93 (t, J = 8.4 Hz, 1H, ArH(20)), 8.13 (d, J = 3.2 Hz, 2H ArH(18)), 9.32 (s, 3H, OH(7,12,13)), 12.46 (s, 1H, OH(5)); 13C NMR (400 MHz [D6]DMSO): δ 93.97 (C8), 98.85 (C6), 104.89 (C4), 113.24 (C11), 116.15 (C14), 122.72 (C15), 124.32 (C10), 128.97 (C19), 129.39 (C18), 129.7 (C2), 130.1 (C20), 130.33 (C17), 131.4 (C12), 133.2 (C13), 155.81 (C1), 157.95 (C9), 161.12 (C5), 164.21 (C16), 164.53 (C7), 167.86 (C3); LC-MS, m/z: calculated for C22H14O8 is 406.07; found 407.01 [M+H] +.
Synthesis of 2-(3,4-dihydroxyphenyl)-7-hydroxy-4-oxo-4H- chromene-3,5-diyl bis(2-chlorobenzoate) (QB2)
Quercetin dihydrate (2 mmol), 2-chlorobenzoic acid (5 mmol) and zinc chloride (2 mmol) were taken in a round bottom flask (RBF). Then, 10 ml of phosphorous oxychloride was gradually added to the reaction mixture and stirred for 15 hrs at 75 °C. The reaction completion was supervised by thin layer chromatography (TLC). Upon formation of the product, stopped stirring and the product was allowed to cool. Ice cold water was added to the mixture and product was extracted using ethyl acetate. The separated ethyl acetate layer was collected and evaporated.10 The obtained yellow product was subjected to preparative TLC, using chloroform: methanol (60:40) as the mobile phase. The pure product was separated, collected and dried. Yield: 100 mg (8.7%); m.p. 230-232 °C; FT-IR (cm-1): 3344.68 (alcohol O–H stretch), 2924.18 (Ar – H), 1726.35 (C=O stretch), 1654.99 (C=C stretch), 1172.76 (C–O ester), 725.78 (C–Cl ); 1H NMR (400 MHz [D6]DMSO): δ 6.037 (s, 1H, ArH(8)), 6.179 (s, 1H, ArH(6)), 6.45 (s, 1H, ArH(11)), 6.72 (d, J = 4.4 Hz, 1H, ArH(14)), 7.20 (d, J = 3.6 Hz, 1H, ArH(15)), 7.69 (t, J = 8 Hz, 4H, ArH(20, 21)), 8.08 (d, J = 10.4 Hz, 2H ArH(19)), 8.23 (d, J = 2.4 Hz, 2H ArH(22)), 9.375 (s, 3H, OH(7,12,13)); 13C NMR (400 MHz [D6]DMSO): δ 94.43 (C8), 98.95 (C6), 102.12 (C4), 114.97 (C14), 115.08 (C11), 119.87 (C10), 121.27 (C15), 128.02 (C21), 129.41 (C17), 129.76 (C20), 130.87 (C19), 131.65 (C22), 133.59 (C18), 141.73 (C2), 144.58 (C12), 144.67 (C13), 147.58 (C5), 153.33 (C1), 153.42 (C9), 160.16 (C7), 166.21 (C16), 174.03 (C3); LC-MS, m/z:exact mass calculated for C29H16Cl2O9 is 578.02; found 578.85.
Synthesis of 2-(3,4-dihydroxyphenyl)-7-hydroxy-4-oxo-4H-chromene-3,5-diyl bis(4-chlorobenzoate) (QB3)
The quercetin dihydrate (3 mmol), 4-chlorobenzoic acid (8 mmol) and zinc chloride (3 mmol) were transferred in a RBF. Then 10 ml of phosphorous oxychloride was gradually added and the reaction mixture was stirred for 15 hrs at 75 °C. The reaction completion was monitored by TLC and upon formation of the product the reaction was stopped and mixture was allowed to cool. Ice cold water was added and then extracted using ethyl acetate. The separated ethyl acetate layer was collected and evaporated. The obtained pale yellow product was subjected to column chromatography using dichloromethane: ethyl acetate (50:50)) as the mobile phase. The pure product was separated, collected, and dried. Yield: 154 mg (9%); m.p. 240-242˚C; FT-IR (cm-1): 3363.97 (alcohol O–H stretch), 2924.18 (Ar – H), 1743.71 (C=O stretch), 1687.77 (C=C stretch), 1139.97 (C–O ester), 725.82 (C–Cl ); 1H NMR (400 MHz CDCl3): δ 6.07 (s, 1H, ArH(8)), 6.25 (s, 1H, ArH(6)), 6.54 (s, 1H, ArH(11)), 6.85 (d, J = 5.6 Hz, 1H, ArH(14)), 7.25 (d, J = 4.8 Hz, 1H, ArH(15)), 7.51 (d, J = 2.4 Hz, 2H, ArH(24)), 7.74 (d, J = 2 Hz, 2H, ArH(19)), 8.18 (d, J = 4.8 Hz, 2H, ArH(23)), 8.35 (d, J = 4.8 Hz, 2H, ArH(18)), 9.672 (s, 3H, OH(7,12,13)); 13C NMR (400 MHz CDCl3): δ 94.2 (C8), 99.15 (C6), 103.95 (C4), 116.22 (C11), 116.23 (C14), 119.65 (C10), 121.99 (C15), 125.72 (C17), 126.41 (C22), 129.12 (C24), 129.24 (C19), 130.685 (C23), 130.72 (C18), 133.54 (C25), 133.76 (C20), 140.54 (C2), 144.81 (C12), 145.79 (C13), 148.47 (C5),153.34 (C1), 153.46 (C9), 161.1 (C7), 166.21 (C16), 166.32 (C23), 176.13 (C3); LC-MS, m/z: exact mass calculated for C29H16Cl2O9 is 578.02; found 578.92.
Synthesis of 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 4-methyl benzoate (QB4)
Quercetin dihydrate (3 mmol), 4-methylbenzoic acid (8 mmol), zinc chloride (3 mmol) and 10 ml of phosphorous oxychloride was gradually added in the RBF. The reaction mixture was stirred for 15 hrs at 75 °C. The reaction completion was supervised by TLC. Upon formation of the product, stopped stirring and the product was allowed to cool. Ice cold water was added and to extract the product ethyl acetate was used. The separated ethyl acetate layer was collected and evaporated. The obtained orange-yellow product was subjected to column chromatography using dichloromethane: ethyl acetate (50:50)) as mobile phase. The pure product was separated, collected and dried.10 Yield: 127 mg (10.2%); m.p. 210-214 °C; FT-IR (cm-1): 3295.21 (alcohol O–H stretch), 2924.18 (Ar – H), 2860.53 (aliphatic C–H stretch), 1743.71 (C=O stretch), 1680.05 (C=C stretch), 1143.53 (C–O ester); 1H NMR (400 MHz CDCl3): δ 2.61 (s, 3H, ArH(21)) 6.34 (s, 1H, ArH(6)), 6.62 (s, 1H, ArH(8)), 7.694 (s, 1H, ArH(11)), 7.82 (d, J = 2 Hz, 1H, ArH(14)), 8.19 (d, J = 1.2 Hz, 1H, ArH(15)), 8.40 (d, J = 3.6 Hz, 2H, ArH(19)), 8.73 (d, J = 2.4 Hz, 2H, ArH(18)), 10.16 (s, 3H, OH(7,12,13)), 12.18 (s, 1H, OH(5)); 13C NMR (400 MHz CDCl3): δ 22.32 (C21), 91.48 (C8), 97.16 (C6), 103.76 (C4), 114.24 (C11), 117.39 (C14), 120.51 (C15), 122.25 (C10), 124.62 (C17), 129.79 (C19), 131.31 (C18), 133.42 (C2), 142.97 (C20), 143.84 (C12), 143.85 (C13), 149.17 (C1), 152.86 (C9), 160.93 (C5), 164.22 (C16), 165.49 (C7), 174.34 (C3); LC-MS, m/z: exact mass calculated for C23H16O8is 420.08; found 420.06.
Physicochemical, Pharmacokinetic and Drug-likeness Properties Screening and Prediction
The molecular descriptors, physicochemical properties and pharmacokinetic properties were evaluated using Swiss ADME web server (http://www.swissadme.ch/ accessed on 22 July 2021)11 and Pre-ADME (https://preadmet.bmdrc.kr/) were used to predict physicochemical, lipophilicity, water solubility, pharmacokinetic and drug-likeness properties including absorption, distribution, metabolism and excretion (ADME) and toxicity. The screening criteria of Drug-likeness compounds state that the synthesized compounds should be aqueous-soluble, should have high gastrointestinal absorption, orally active drug should satisfy Lipinski’s rule of five and low hERG inhibition risk (low toxicity).11
Biological Activity
In vitro antioxidant activity
The methods for in vitro antioxidant activity are based on the inhibition of free radicals. The inhibition of free radicals is measured when the samples are added to a system where free radicals are generated. The inhibition is relative to the antioxidant capacity of each sample. In vitro antioxidant methods provide an indication of antioxidant capacity of the compounds. In the present study, the synthesized quercetin derivatives were tested for in vitro antioxidant activity using DPPH and H2O2 assay.
Scavenging of H2O2 assay
A solution of H2O2 was prepared in phosphate buffer saline pH 7.4. Various concentrations (100-12.5 μg/ml) of samples and standards (ascorbic acid and quercetin) in methanol were prepared and added to H2O2 solution in PBS. After 10 min, the absorbance was measured at 230 nm.12,13
Scavenging of DPPH assay
The 100 μM of DPPH solution was prepared using methanol. The different concentrations of standard (ascorbic acid and quercetin) and quercetin derivatives in DMSO were prepared (100-12.5 μg/ml) and then added to DPPH solution. After 30 min, the absorbance for each was measured at 490 nm.12,13
In vitro cytotoxicity by MTT assay method
In vitro cytotoxicity was carried out in the Animal Tissue Culture Laboratory, Department of Pharmaceutical Biotechnology at JSS College of Pharmacy, Ootacamund, Tamilnadu, India. The in vitro cytotoxicity study involved the determination of mitochondrial synthesis by MTT assay and was performed on MCF-7 breast cancer cell line.
Preparation of test solutions
All the quercetin derivatives (QB1-4) were dissolved in distilled dimethyl sulphoxide (DMSO) separately and Dulbecco's Modified Eagle Medium (DMEM) was used to make volume with 2% inactivated fetal bovine serum (FBS) to obtain a stock solution of 1000 µg/ml concentration and sterilized by filtration and stored at ─20 °C till use. Serial dilutions were made to obtain lower dilutions.
Cell lines and culture medium
MCF-7 cell line was procured from National Centre for Cell Sciences (NCCS), Pune, India. For culturing cell lines, DMEM supplemented with 10% inactivated FBS, Penicillin (100 IU/ml), Amphotericin B (5 mg/ml) and Streptomycin (100 mg/ml) were used in 5% CO2 at 37 °C in a humidified atmosphere. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% Ethylene diamine tetraacetic acid, 0.05% glucose in phosphate buffer solution). The cultures were grown in 25 ml flat bottles and experiment was carried out in 96 well microtitre plates, considered as stock culture.
The monolayer cell culture was trypsinized and by using DMEM containing 10% FBS the cell count was adjusted to 1.0 x 105 cells/ml. The 0.1 ml of diluted cell line (approximately 10,000 cells) was added to a 96 well microtitre plate. The partial monolayer was formed after 24 hrs and the supernatant was flicked off. The monolayer was washed with DMEM medium. To the partial monolayer, 100 ml of different concentrations of synthesized compounds were added in microtitre plates. The plates were kept for incubation at 37 oC for 72 hrs in 5% CO2 atmosphere. The microscopic examination was carried out and in an every 24 hrs interval observations were made. The drug solutions in the wells were discarded after 72 hrs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS (50 ml) was added to each well and gently shaken. The plates were again incubated for 3 hrs at 37 oC in 5% CO2 atmosphere. The supernatant was removed and 100 ml of propanol was added to the plates and gently shaken to solubilize the formed formazan. The plates were subjected for absorbance using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC50) values was generated from the dose-response curve of selected cell line.14,15
Molecular Docking
The X-ray crystallographic structure of human iNOS protein with resolution of 2.25 Å was collected from RCSB Protein Data Bank (PDB ID: 4NOS) and selected as the target protein for docking of the four designed compounds and quercetin. All the ligands were sketched using ChemDraw Professional 15.0 software. Ligand and protein were prepared and docking was done through BIOVIA Discovery Studio 2019 software. Molecular docking was performed using CDOCKER algorithm which is a simulated annealing docking method that uses CHARMm force field based molecular dynamics to generate random conformations. Different conformations for ligand adopting rigid body rotations were generated and
–CDOCKER energy and interaction energies (kcal/mol) for the molecules were obtained.16