S-allyl Cysteine methyl, ethyl and propyl ester-based non-steroidal anti-inflammatory drugs and their structures were elucidated by spectroscopic analysis. The chemopreventive potential of all compounds was evaluated against SW480 human colon adenocarcinoma cells and the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 10b-c, 11b and 12b displayed the best anticancer activity with IC50 values between 0.131-0.183 mM and selectivity indices higher than 1 after 48 h of treatment. Selectivity indices were comparable to those reported for the reference drug, 5-fluorouracil (SI > 1). The SAR analysis showed that compounds with two carbon atom alkylic chains displayed the best activity . Modeling studies including drug-likeness, bioactivity score and ADME/tox studies using online tools like molinspiration and Osiris suggested that these designed hybrids have a good pharmacological profile and can be considered as promising scaffolds for further studies in the search for new therapeutic alternatives to treat colorectal cancer.We synthesized twelve hybrids,  Colorectal cancer (CRC) is one of the leading causes of morbidity and mortality worldwide . AccordiS-allyl cysteine (SAC) is constituent of garlic, which has exhibited antioxidant properties both in-vitro  are a chemically heterogeneous group of compounds sharing certain therapeutic actions related to nociception, inflammation and pyretic processes. Among NSAIDs are included aspirin, diclofenac, naproxen and ibuprofen. The principal therapeutic effect of NSAIDs is the inhibition of cyclooxygenase (COX) family proteins which catalyze the rate-limiting steps in prostaglandin synthesis. Prostaglandins are important mediators of signal transduction pathways, and they are involved in cellular adhesion, growth and differentiation. In recent years, the possible role of NSAIDs in the prevention of malignancy has aroused interest -15. Seve1) exhibited good radical scavenging activity, being superior to the standards ferulic acid, eugenol and isoeugenol (28). SAC derivative (2) exhibited butyrylcholinesterase (BuChE) inhibitory activity (3), is an NO- and H2S-releasing agent. NOSH–aspirin inhibited cell growth, proliferation, induced apoptosis and caused G0/G1 cell cycle arrest in HT-29 colon cancer cells  inhibited the proliferation of liver cancer SMMC-7721 cells but not in normal liver LO2 cells, displaying selective inhibition and inducing cancer cell apoptosis  were used as reference. Chemical shifts (δ) are expressed in ppm with the solvent peak as reference and TMS as an internal standard; coupling constants (J) are given in Hertz (Hz). Carbon atom types  were determined using the DEPT pulse sequence. High-resolution mass spectra were recorded using electrospray ionization mass spectrometry (ESI-MS). A QTOF Premier instrument with an orthogonal Z-spray-electrospray interface  was used operating in the W-mode. Silica gel 60  was used for column chromatography, and precoated silica gel plates (Merck 60 F254 0.2 mm) were used for thin-layer chromatography (TLC). Monitoring of the reaction progress and product purification was carried out by TLC. Procedure for the synthesis of S-Allyl cysteine (7)4OH (20 mL). The resulting mixture was stirred at room temperature for 20h. Then, the reaction mixture was concentrated to precipitate the product as a white solid. The solid was filtered, washed with ethanol (3-10 mL) and dried under reduced pressure, affording 818 mg (80%) of compound 2. This compound was used in the following step without further purification. S-Cysteine hydrochloride  was added to allyl bromide  in 2M NH1H NMR : δ 2.48 (NH2), 2.72 , 2.87 , 3.06 , 3.56 , 4.29 (-NH2), 4.98-5.13 , 5.59-5.75 ; 13C NMR : δ 31.33 (S-CH2CHN), 33.93 (S-CH2CH=CH2), 53.45 (CH-N), 118.48 (S-CH2CH=CH2), 133.77 (S-CH2CH=CH2), 171.33 (-C=O).Procedure for the synthesis of S-Allyl cysteine methyl ester (8a)S-allyl cysteine  was added, and the mixture was stirred for 10 min. The resulting solution was stored at -10 °C for 2 h, kept at room temperature for a further 24 h and then poured into ether (100 mL) and refrigerated for 2 h. The product  separated as colorless needles and was removed by filtration.Thionyl chloride  was added over 5 min. to dry methanol (15 mL) cooled to -10 °C and the resulting solution stored for a further 5 min. Then 25 + 4.778 ; 1H NMR : δ 2.51 (NH2), 2.84 , 2.93 , 3.09 , 3.70 , 4.14 , 5.04-5.15 , 5.59-5.77 ; 13C NMR : δ 30.09 (S-CH2CHN), 34.20 (S-CH2CH=CH2), 52.09 (OCH3), 54.52 (CH-N), 118.86 (S-CH2CH=CH2), 133.37 (S-CH2CH=CH2), 168.96 (-C=O).m.p. 114-116 °C; [α] General procedure for the synthesis of S-Allyl cysteine esters (8b and 8c) S-allyl cysteine  was added, and the resulting mixture was stored at -10 °C for 2 h and kept at room temperature for a further 24 h. Then, the excess alcohol was removed by distillation. The residue was purified by column chromatography over silica gel eluting with dichloromethane-methanol (95:5 ratio) to obtain S-allyl cysteine ethyl ester and S-allyl cysteine propyl ester in 60% (352 mg) and 71% (447 mg) yields, respectively. Monitoring of the reaction progress and product purification was carried out by TLC.Thionyl chloride (3 eq) was added over 5 min. to dry ethyl or propyl alcohol (15 mL) cooled to -10 °C, and the resulting solution was stored for a further 5 min. Then, Ethyl S-prop-2-en-1-ylcysteinate (3b)25 + 1.40 ; 1H NMR : δ 0.96 , 2.63 (NH2), 3.22-3.25 , 3.18-3.22 , 3.29 , 4.29 , 5.15 , 5.24 , 5.74-5.85 ; 13C NMR : δ 14.05 (CH3), 30.48 (S-CH2CHN), 35.10 (S-CH2CH=CH2), 52.69 (CH-N), 62.94 (OCH2), 118.51 (S-CH2CH=CH2), 133.37 (S-CH2CH=CH2), 167.95 (-C=O). m.p. 121-123 °C; [α]Propyl S-prop-2-en-1-ylcysteinate (3c)25 + 3.750 ; 1H NMR : δ 0.91 , 1.53-1.72 , 1.98 (NH2), 2.83 , 2.65 , 3.11 , 3.58 , 4.05 , 5.07-5.16 , 5.64-5.82 ; 13C NMR : δ 10.41 (CH3), 21.97 (CH2), 35.14 (S-CH2CHN), 35.82 (S-CH2CH=CH2), 54.11 (CH-N), 66.82 (OCH2), 117.61 (S-CH2CH=CH2), 134.01 (S-CH2CH=CH2), 174.15 (-C=O).m.p. 115-117 °C; [α]General procedure for condensation using HBTU S-allyl cysteine ester (8a-8c) (1.2 mmol) was added, and the resulting mixture was allowed to stir for 15 h. The solvent was removed under reduced pressure, and the residue was purified by chromatography on silica gel afforded compounds 9-12 in yields ranging 25-75%. A solution of carboxylic acid  (1 mmol) and triethylamine (4 mmol) in THF (10 mL) was stirred for 15 min. Then, HBTU (1.5 mmol) was added, and the resulting mixture was stirred for 10 min. Then, Methyl S-allyl-N-(2-hydroxybenzoyl)-L-cysteinate (9a)1H NMR  δ 9.82 , 7.87 , 7.32 , 6.95 , 6.79 , 5.74 , 5.17 – 5.05 , 4.76 , 3.68 , 3.19 , 2.99 , 2.90 . 13C NMR  δ 171.60 (CH-C=O)-O), 167.72 (-NH-C=O), 161.21 (Ar), 134.58 (2C-Ar), 133.84 (S-CH2CH=CH2), 129.77 (Ar), 118.34 (S-CH2CH=CH2), 118.06 (Ar), 116.96 (Ar), 52.71 (OCH3), 52.51(CH-N), 34.49 (S-CH2CH=CH2), 31.78 (S-CH2CHN). ESI–MS: m/z 296.0956 [M + H]+, Calc. For C14H18NO4S: 296.0951.Yield 35%, yellow solid; m.p. 112-114 °C; Ethyl S-allyl-N-(2-hydroxybenzoyl)-L-cysteinate (9b)1H NMR  δ 7.91 , 7.36 , 7.02 – 6.94 , 6.85 , 5.82 – 5.65 , 5.17 – 5.04 , 4.69 , 4.14 , 3.18 , 2.99 , 2.91 , 1.20 . 13C NMR  δ 170.95 (CH-C=O)-O), 167.78 (-NH-C=O), 162.63 (Ar), 134.56 (2C-Ar), 134.01 (S-CH2CH=CH2), 129.71 (Ar), 118.57 (S-CH2CH=CH2), 118.06 (Ar), 116.66 (Ar), 115.84 (Ar), 61.46 (OCH2CH3), 52.63 (CH-N), 34.44 (S-CH2CH=CH2), 31.63 (S-CH2CHN), 14.51 (CH3). ESI–MS: m/z 310.1117 [M + H]+, Calc. For C15H20NO4S: 310.1107.Yield 30%, yellow oil; Propyl S-allyl-N-(2-hydroxybenzoyl)-L-cysteinate (9c)1H NMR  δ 9.14 , 7.77 , 7.25 , 6.83 , 6.75 , 5.62 , 5.02 – 4.90 , 4.55 , 3.90 , 3.03 , 2.85 , 2.77 , 1.44 , 0.72 . 13C NMR  δ 170.92 (CH-C=O)-O), 167.94 (-NH-C=O), 162.72 (Ar), 134.57 (2C-Ar), 134.22 (S-CH2CH=CH2), 129.56 (Ar), 118.05 (S-CH2CH=CH2), 118.0 (Ar), 117.77 (Ar), 116.40 (Ar), 66.86 (OCH2CH3), 52.68 (CH-N), 34.41 (S-CH2CH=CH2), 31.52 (S-CH2CHN), 21.94 (CH2), 10.68 (CH3). ESI–MS: m/z 324.1264 [M + H]+, Calc. For C16H22NO4S: 324.1264.Yield 75%, yellow oil; Methyl S-allyl-N-amino)phenyl)acetyl)-L-cysteinate (10a)1H NMR  δ 8.95 , 8.00 , 7.52 , 7.23 , 7.17 , 7.04 , 6.89 – 6.81 , 6.28 , 5.79 – 5.61 , 5.11 – 5.00 , 4.59 – 4.46 , 3.67 , 3.64 , 3.13 , 2.83 , 2.73 . 13C NMR  δ 172.05 (-NH-C=O), 171.40 (CH-C=O)-O), 143.37 (Ar), 137.59 (Ar), 134.55 (S-CH2CH=CH2), 130.96 (Ar), 130.10 (2C-Ar), 129.66 (2C-Ar), 127.77 (Ar), 125.72 (Ar), 121.14 (Ar), 118.02 (S-CH2CH=CH2), 116.34 (Ar), 52.65 (OCH3), 52.52 (CH-N), 37.98 (Ar-CH2-C=O), 34.56 (S-CH2CH=CH2), 31.73 (S-CH2CHN). ESI–MS: m/z 453.0831 [M + H]+, Calc. For C21H23Cl2N2O3S: 453.0801.Yield 30%, red solid; m.p. 90-91 °C; Ethyl S-allyl-N-amino)phenyl)acetyl)-L-cysteinate (10b)1H NMR  δ 8.94 , 8.02 , 7.52 , 7.29 – 7.20 , 7.16 , 7.04 , 6.96 , 6.90 – 6.79 , 6.28 , 5.12 – 5.00 , 4.47 , 4.09 , 3.67 , 3.14 , 2.83 , 2.73 , 1.13 . 13C NMR  δ 172.08 (-NH-C=O), 170.86 (CH-C=O)-O), 143.37 (Ar), 137.60 (Ar), 134.53 (S-CH2CH=CH2), 130.95 (Ar), 130.09 (2C-Ar), 129.66 (2C-Ar), 127.75 (Ar), 125.71 (Ar), 121.11 (Ar), 118.02 (S-CH2CH=CH2), 116.33 (Ar), 61.34 (OCH2), 55.92 (CH-N), 36.67 (Ar-CH2-C=O), 34.55 (S-CH2CH=CH2), 31.69 (S-CH2CHN), 14.38 (CH3). ESI–MS: m/z 467.0956 [M + H]+, Calc. For C22H25Cl2N2O3S: 467.0957.Yield 32%, yellow solid; m.p. 88-90 °C; Propyl S-allyl-N- amino)phenyl)acetyl)-L-cysteinate (10c)1H NMR  δ 8.90 , 8.02 , 7.52 , 7.23 , 7.17 , 7.06 – 7.02 , 6.85 , 6.31 – 6.26 , 5.11 – 5.03 , 4.49 , 4.09 , 3.67 , 3.17 – 3.13 , 2.83 , 2.74 , 1.58 – 1.49 , 0.84 . 13C NMR  δ 172.07 (-NH-C=O), 170.93 (CH-C=O)-O), 143.40 (Ar), 137.64 (Ar), 134.55 (S-CH2CH=CH2), 130.95 (Ar), 130.06 (2C-Ar), 129.67 (2C-Ar), 127.76 (Ar), 125.49 (Ar), 121.15 (Ar), 118.00 (S-CH2CH=CH2), 116.40 (Ar), 66.74 (OCH2), 52.69 (CH-N), 34.56 (Ar-CH2-C=O), 31.72 (S-CH2CH=CH2), 29.99 (S-CH2CHN), 21.88 (CH2), 10.65 (CH3). ESI–MS: m/z 481.1120 [M + H]+, Calc. For C23H27Cl2N2O3S: 481.1114Yield 60%, red oil; Methyl S-allyl-N-propanoyl)-L-cysteinate (11a)1H NMR  δ 8.56 , 7.83 – 7.68 , 7.45 , 7.27 , 7.20 – 7.09 , 5.83 – 5.51 , 5.16 – 4.89 , 4.53 – 4.37 , 3.86 , 3.64 (3.55) , 3.15 (3.01) , 2.84 , 2.71 , 1.41 . 13C NMR  δ 174.08 (174.04) (-NH-C=O), 171.70 (171.59) (CH-C=O)-O), 157.45 (Ar), 137.36 (Ar), 134.64 (134.50) (Ar), 133.60 (S-CH2CH=CH2), 129.56 (Ar), 128.80 (Ar), 127.07 (126.99) (2C-Ar), 125.83 (Ar), 119.02 (Ar), 117.95 (117.81) (S-CH2CH=CH2), 106.10 (Ar), 55.60 (OCH3), 52.45 (52.55) (CH-N), 52.29 (OCH3), 44.95 (CH-CH3), 34.44 (S-CH2CH=CH2), 31.74 (31.65) (S-CH2CHN), 19.14 (18.94) (CH-CH3). ESI–MS: m/z 388.1580 [M + H]+, Calc. For C21H26NO4S: 388.1577Yield 60%, yellow oil; Ethyl S-allyl-N-propanoyl)-L-cysteinate (11b)1H NMR  δ 8.55 , 7.82 – 7.69 , 7.45 , 7.28 , 7.14 , 5.82 – 5.50 , 5.18 – 4.89 , 4.15 – 4.04 , 3.99 , 3.85 , 3.15 (3.02) , 2.83 , 2.66 , 1.41 , 1.16 (1.04) . 13C NMR  δ 174.10 (173.99) (-NH-C=O), 171.14 (171.04) (CH-C=O)-O), 157.44 (Ar), 137.36 (137.34) (Ar), 134.62 (134.49) (Ar), 133.61 (S-CH2CH=CH2), 129.55 (Ar), 128.80 (Ar), 127.02 (126.97) (2C-Ar), 125.84 (125.82) (Ar), 119.01(Ar), 117.94 (117.82) (S-CH2CH=CH2), 106.10 (Ar), 61.22 (61.11) (CH3CH2O), 55.59 (OCH3), 52.58 (52.36) (CH-N), 44.94 (CH-CH3), 34.44 (S-CH2CH=CH2), 31.70 (31.61) (S-CH2CHN), 19.03 (18.94) (CH-CH3), 14.44 (14.32) (OCH2CH3). ESI–MS: m/z 402.1737 [M + H]+, Calc. For C22H28NO4S: 402.1733.Yield 25%, yellow oil; Propyl S-allyl-N-propanoyl)-L-cysteinate (11c)1H NMR  δ 8.55 , 7.81 – 7.69 , 7.45 , 7.27 , 7.13 , 5.82 – 5.53 , 5.17 – 4.89 , 4.50 – 4.35 , 4.08 – 3.93 , 3.85 , 3.16 (3.03) , 2.84 , 2.72 , 1.63-1.48 , 1.41 , 0.85 (0.72) . 13C NMR  δ 174.08 (173.95) (-NH-C=O), 171.21 (171.10) (CH-C=O)-O), 157.42 (Ar), 137.32 (Ar), 134.62 (134.48) (Ar), 133.61(S-CH2CH=CH2), 129.53 (Ar), 128.79 (Ar), 126.96 (125.81) (2C-Ar), 118.99 (Ar), 117.79 (117.90) (S-CH2CH=CH2), 106.08 (Ar), 66.61 (66.51) (CH3CH2O), 55.58 (OCH3), 52.55 (52.32) (CH-N), 44.95 (CH-CH3), 34.41(S-CH2CH=CH2), 31.80 (31.59) (S-CH2CHN), 21.91 (CH3CH2CH2O), 19.02 (18.91) (CH-CH3), 10.67 (10.54) (CH3CH2CH2O). ESI–MS: m/z 416.1891 [M + H]+, Calc. For C23H30NO4S: 416.1890.Yield 62%, yellow oil; Methyl S-allyl-N-(2-(4-isobutylphenyl)propanoyl)-L-cysteinate (12a)1H NMR  δ 8.49 , 7.22 , 7.07 , 5.82 – 5.53 , 5.17 – 4.91 , 4.49 – 4.37 , 3.74 – 3.65 , 3.63 (3.57) , 3.14 (3.01) , 2.83 , 2.74 , 2.39 2), 1.86 – 1.70 2), 1.30 , 0.85 2), 0.84 2). 13C NMR  δ 174.12 (174.07) (-NH-C=O), 171.68 (171.57) (CH-C=O)-O), 139.67 (139.70) (Ar), 139.48 (139.45) (Ar), 134.63 (134.51) (S-CH2CH=CH2), 129.16 (2C-Ar), 127.52 (2C-Ar), 117.93 (117.83) (S-CH2CH=CH2), 52.51 (CH-N), 52.41 (52.26) (OCH3), 44.70 (CH-CH3), 44.61 (Ar-CH2-CH(CH3)2), 34.49 (34.44) (S-CH2CH=CH2), 31.74 (31.58) (S-CH2CHN), 30.11(Ar-CH-(CH3)2), 22.63 (CH-(CH3)2), 19.19 (18.98) (CH-CH3). ESI–MS: m/z 364.1952 [M + H]+, Calc. For C20H30NO3S: 364.1940.Yield 31%, yellow solid; m.p. 89-91 °C; Ethyl S-allyl-N-(2-(4-isobutylphenyl)propanoyl)-L-cysteinate (12b)1H NMR  δ 8.48 , 7.22 , 7.07 , 5.84 – 5.55 , 5.19 – 4.91 , 4.47 – 4.30 , 4.01 , 3.75 – 3.62 , 3.14 (3.03) , 2.82 , 2.70 , 2.39 2), 1.89 – 1.71 2), 1.30 , 1.16 (1.08) , 0.84 2). 13C NMR  δ 174.13 (174.0) (-NH-C=O), 171.13 (171.03) (CH-C=O)-O), 139.71 (139.67) (Ar), 139.47 (139.45) (Ar), 134.62 (134.52) (S-CH2CH=CH2), 129.16 (2C-Ar), 127.53 (127.50) (2C-Ar), 117.93 (117.84) (S-CH2CH=CH2), 61.19 (61.10) (OCH2CH3), 52.62 (52.34) (CH-N), 44.69 (Ar-CH2-CH(CH3)2), 44.62 (CH-CH3), 34.49 (34.44) (S-CH2CH=CH2), 31.54 (31.68) (S-CH2CHN), 30.12 (CH-(CH3)2), 22.62 (CH-(CH3)2), 19.08 (18.97) (CH-CH3), 14.44 (14.34) (CH2CH3). ESI–MS: m/z 378.2099 [M + H]+, Calc. For C21H32NO3S: 378.2097Yield 42%, yellow solid; m.p. 65-67 °C; Propyl S-allyl-N-(2-(4-isobutylphenyl)propanoyl)-L-cysteinate (12c)1H NMR  δ 8.47 , 7.22 , 7.06 , 5.79 – 5.54 , 5.17 – 4.88 , 4.39 , 4.00 , 3.74 – 3.62 , 3.14 (3.03) , 2.82 , 2.71 , 2.39 2), 1.87-1.71 2),1.62-1.40 ,1.30 , 0.89 – 0.74 2). 13C NMR  δ 174.13 (173.97) (-NH-C=O), 171.21 (171.11) (CH-C=O)-O), 139.70 (139.66) (Ar), 139.47 (139.45) (Ar), 134.62 (134.51) (S-CH2CH=CH2), 129.15 (2C-Ar), 127.53 (127.49) (2C-Ar), 117.90 (117.82) (S-CH2CH=CH2), 66.60 (66.53) (OCH2CH3), 52.61 (52.32) (CH-N), 44.70 (Ar-CH2-CH(CH3)2), 44.64 (CH-CH3), 34.47 (34.43) (S-CH2CH=CH2), 31.70 (31.55) (S-CH2CHN), 30.12 (CH-(CH3)2), 22.62, (CH-(CH3)2), 21.92 (21.85) (OCH2CH2CH3), 19.11 (18.98) (CH-CH3), 10.68 (10.63) (CH2CH3). ESI–MS: m/z 392.2259 [M + H]+, Calc. For C22H34NO3S: 392.2254.Yield 40%, yellow solid; m.p. 58-60 °C; In-vitro biological assaysCell lines and culture medium2 flasks containing Dulbecco’s Modified Eagle Medium, supplemented with 10% heat-inactivated (56 °C) horse serum, 1% penicillin/streptomycin and 1% non-essential amino acids . For all experiments, horse serum was reduced to 3%, and the medium was supplemented with 5 mg/mL transferrin, 5 mg/mL selenium and 10 mg/mL insulin   and non-malignant (CHO-K1) cell lines obtained from The European Collection of Authenticated Cell Cultures . Cells were cultured in 25-cmad, USA) .Growth inhibition (SRB)2. All cultures were allowed to grow for 24 h. Afterward, they were treated with 1% of DMSO (vehicle control) or increasing concentrations  of the compounds. After treatment, cells were fixed with trichloroacetic acid  for one hour at 4 °C. Cell proteins were determined by staining with SRB , then they were washed with 1% acetic acid to remove unbound SRB and left for air-drying. Protein-bound SRB was solubilized in 10 mM Tris-base, and the absorbance was measured at 492 nm in a microplate reader (Mindray MR-96A) , by the ratio of IC50 values in non-malignant CHO-K1 cells to IC50 of SW480 cells. The growth inhibition of the ethanol extract, the Chromone and 5-fluorouracil  was evaluated through sulforhodamine B (SRB) assay, a colorimetric test that is based on staining of total cellular protein of adherent cells. Cells were seeded to a ﬁnal density of 20.000 cells/well in 96-well tissue culture plates and incubated at 37 °C in a humidified atmosphere at 5% CO MR-96A) , 34. AllStatistical analysis p ≤ 0.05 were considered significant. Data were analyzed with GraphPad Prism version 7.04 for Windows .All experiments were performed at least three times. Data are reported as mean ± SE (standard error). Statistical differences between the control group (non-treated) and treated cells were evaluated by one-way ANOVA followed by the Dunnett’s test. Values with Computational MethodsIn-silico Pharmacokinetic and ADME-tox studiesIn-silico drug-likeness prediction along with further ADMET  tools present an array of opportunities that help accelerate the discovery of new anti-cancer candidates. To find out the drug-like properties, the title hybrids 9-12 were screened for their pharmacokinetic properties using opensource cheminformatics toolkits such us Molinspiration software (o/w descriptor), Pre-ADMET 2.0 program to predicted various pharmacokinetic parameters and pharmaceutical relevant properties such as apparent predicted intestinal permeability (App. Caco-2), binding to human serum albumin (LogKHSA), MDCK cell permeation coefficients and intestinal or oral absorption (%HIA). These important parameters define absorption, permeability, motion and action of the drug molecule. The interpretation of two predicted ADMET properties using the Pre-ADMET program was as shown below: Value of Caco-2 permeability is classified into three classes: (1) If permeability < 4, low permeability; (2) if permeability < 70, moderate permeability; and (3) if permeability > 70, higher permeability. Value of MDCK cell permeability can be classified into three classes: 1) If permeability < 25, low permeability; 2) if 25 < permeability < 500, moderate permeability; and 3) if permeability > 500, higher permeability. Likewise, the Molinspiration web server was also used to predict the potential interaction of novel compounds with the most common human receptors G protein-coupled receptor (GPCR), ion channel, kinase, nuclear receptor, protease, and enzymes. Next to it, the OSIRIS Property Explorer (free open source) was used to evaluate the overall drug-score and the most common toxicity human parameters like mutagenic, tumorigenic, irritant effect, and possible injuries can affect the reproductive system.software   were determined by using the DEPT or APT pulse sequence. ESI-MS spectra showed characteristic [M+H]+ peaks corresponding to their molecular weights. The assignments of all the signals to individual H or C-atoms have been performed based on typical δ-values and J-constants. The 1H-NMR spectra of hybrids 9-12 dissolved in DMSO showed signals of S-CH2CHN (2.90 and 3.00 ppm) which appear as dd, S-CH2CH=CH2 , OCH2 or OCH3 , -CH-N , S-CH2CH=CH2 (5.10-5.22 ppm), S-CH2CH=CH2 , -CH-NH-C=O  and Ar-H . Additionally, compounds 10 and 12 showed the signal corresponding to the methylene group (Ar-CH2- 3.67 ppm for 10 and 2.39 ppm for 12). Finally, both hybrids 11a-11c and 12a-12c exhibited a doublet to 1.41 and 1.30 ppm, respectively, due to the -CHCH3 group. 13C-NMR spectra of the hybrids showed the signals corresponding to S-allyl cysteine esters and aromatic ring: (S-CH2CHN), (S-CH2CH=CH2), (CH-N), (OCH2 or OCH3), (S-CH2CH=CH2), (S-CH2CH=CH2), (Ar-C=C), (-NH-C=O) and ((CH-C=O)-O). Also, compounds 10 and 12 exhibited the signal corresponding to the methylene group, and hybrids 11 showed the signal duo the isopropylidene group. It is important to note that in the synthesis, we used racemic mixtures of ibuprofen and naproxen. Therefore, duplicate signals are observed in both the proton and carbon.The structures of all compounds have been established by a combined study of ESI-MS, Biological activityCytotoxic effect of S-allyl Cysteine methyl ester-based non-steroidal anti-inflammatory drugs on SW480 and CHO-K1 cell lines9a-c , 10a-c (based on diclofenac), 11a-c (based on naproxen) and 12a-c (based on ibuprofen), these were evaluated against SW480 human colon carcinoma cells and the non-malignant CHO-K1 cell line, through the sulforhodamine B assay. Cytotoxicity was reported as 50% inhibitory concentration . 5-FU was used as the reference drug. All results regarding the cytotoxic effect are summarized in To determine the cytotoxic effect of the synthesized hybrids, 10b-c together with 11b and 12b displayed the best cytotoxic activity with IC50 values ranging from 0.131 ± 0.012 to 0.183 ± 0.005 mM on SW480 cells, after 48 h of treatment. These compounds demonstrate the objective of molecular hybridization, improving the biological activity since compounds 10b-c and 11b were significantly more active than parental compounds , and their selectivity indices were comparable to the reference drug (SI > 1). These results are in accordance with those previously reported by Castrillon and colleagues, which also reported cytotoxic and selective activity of different S-allyl cysteine hybrids using the same model , which also found better cytotoxic activity and selectivity with some styrylcoumarin hybrids when tested using an in-vitro model of colorectal cancer , this value was also low in non-malignant cells (0.112 ± 0.008 mM), and thus the selectivity indices were lower than 1 both in 24 and 48 h after treatment. On the other hand, compound 9b decreased activity after 48 hours of treatment, evidenced by increased IC50 value, with loss in selectivity. The other hybrids evaluated  displayed activity neither 24 h nor 48 h after treatment.Among the tested compounds, it was observed that hybrids me model . Moreovel cancer . Further9a-c), which did not show any activity (IC50 = >0.20 mM), we proved the synergistic action of the parent subunits when they were linked to form a single structure in the hybrid, as in the case of compounds 10b-c, 11b, 12bvs. diclofenac, naproxen and ibuprofen, respectively. In addition, it was possible to denote that compounds with a two-carbon atom alkylic chain displayed the best activity . In the structure-activity relationship (SAR) study, excluding the hybrids based on salicylic acid  , the teso/w) were found for all the compounds (3.364 - 5.780), which fits well within the optimal range of 95% of approved drugs (–2.0 to 6.0). As expected, an increased degree of lipophilicity  was observed to be susceptible to those compounds with long alkyl chains. A close view of the LogPo/w numbers showed that high degrees of lipophilicity within each homologous series appears to correlate strongly with  in-vitro biological data. This behavior can be explained by considering that high lipophilicity frequently leads to compounds readily taken up into cancer cells in culture, indicating that these compounds can thereafter easily interact with intracellular targets, conducing to more cytotoxic compounds. An estimated 100% for human intestinal absorption (HIA%) suggests that all hybrids could be absorbed efficiently throughout the intestinal segments upon oral administration. Additionally, high degrees of lipophilicity , suggesting that these smaller compounds could penetrate more efficiently through neoplastic cells. Moreover, the in-silico passive transmembrane permeation was calculated for all hybrids using Caco-2 cell monolayers or MDCK cells as a model. Currently, both models are recommended as a simplified in-vitro model of intestinal absorption in drug development (Polar Surface Area (PSA) is the most important physicochemical parameter used to correlate passive molecular transport through membranes and drug-membrane interactions. Predicted PSA numbers  for the elopment -45. It wHSA), which is the most significant parameter for distribution and transport for anti-cancer formulations in the systemic circulation and play an important role in the early stage of drug discovery. For therapeutic uses, LogKHSA values in the range of -1.5 to 1.5 are recommended for potential drugs , ion channel, kinase, nuclear receptor, proteases and enzymes. As a general rule, the most promising drug candidates display larger numbers of bioactivity scores, higher than 0.00, which increases the probability that evaluated compounds would be biologically active. In our study, the novel hybrids based on S-allyl cysteine coupled with different molecules FDA-approved for use as anti-inflammatory drugs  were evaluated via virtual high-throughput screening (vHTS) with the help of Molinspiration chemoinformatic software to demonstrate that the synthesized compounds could be active and thus, could be used to run further biological assays focused on colorectal cancer drug discovery. The pharmacological response for a bioactive compound requires its binding to a specific target, which could provide a potential health benefit. In that context, bioactivity score prediction is currently the most important 9-12 were moderately active according to the bioactivity score (from -5.0 to 0.0). Interestingly, hybrids displayed excellent protease inhibition in the range of 0.04 to 0.29, including the most promising compounds 10a-c, which exhibited bioactivity scores higher than 0.10. In this scenario, since proteolytic enzymes are strongly associated with colorectal cancer development and progression, it is necessary to highlight the importance of designing novel candidates that could inhibit these proteases during cancer chemoprevention , drug-likeness and drug score. The toxicity risk predictions locate substructures within a molecule that could enhance the risks of adverse drug reactions on a cell, organ or organism. Notably, toxicity risk assessment revealed that combining S-allyl cysteine and NSAIDs fragments in a unique structural unity can lead to synthesized substances with low or null toxicity regarding mutagenicity, tumorigenicity, irritant effect and injury of reproductive system (Green suggests low toxicity risk by Osiris online software).Prediction of drug relevant properties and the overall toxicity of novel hybrids 9c, 9d, 10c, 11c, 12a and 12c showed positive drug-likeness values ranging from 0.18 to 3.07 and thus, they could be safe drug candidates, or their structures could be used as models for scaffold-based drug discovery in colorectal cancer chemotherapy. On the other side, drug-likeness measures based on the OSIRIS web tool were explored based on a drug-likeness model score revealing the potential therapeutic for all hybrids compared to marketed drugs. Osiris database considered that around 15000 nondrug-like chemicals and 3300 FDA-approved drugs have had positive values of the drug-likeness parameter. In contrast, almost all the nondrug like chemicals had negative values . A posit9c and 12a were significantly higher (>65%) than the remaining hybrids, which showed values lower than 56%. Finally, from the Osiris property explorer website, we estimated the aqueous solubility value  for all the designed compounds. The aqueous solubility is one of the most important biopharmaceutic properties associated with the absorption and distribution of a potential drug. It is, therefore, no surprise that the aqueous solubility plays a key role in the bioavailability of bioactive compounds. In fact, it is well known that more than 95% of the approved drugs can take an ideal range of -6.5 to 0.5 estimated for the LogS parameter. In our case, the LogS values of studied compounds are within the reference range (ranging from -2.42 and -6.30), suggesting their potential as drug-like.In addition, to guarantee safety and optimal drug-likeness indices for the new compounds, data from the Osiris property explorer website indicated that the potential drug score values of compounds 9-12 would suggest that these compounds have a great chance to advance through the earlier preclinical testing as well as may serve as attractive candidates for future drug development treatments focus on the initiation, progression, and migration in colorectal cancer. Taken together, the optimal results of safety and drug-likeness together with the acceptable drug score and LogS values estimated for hybrids 10b-c, 11b and 12b displayed significant cytotoxic activity against human colon adenocarcinoma cells being significantly more active than parental compounds and exhibiting selectivity comparable to a conventional chemotherapeutic (5-FU), highlighting the potential of the strategy based on molecular hybridization. The SAR analysis showed that the presence of two-carbon atom alkylic chains in the compounds increased the activity, as in hybrids 10b, 11b and 12b.Our results show that hybrids S-allyl cysteine with NSAIDs fragments leads to the formation of compounds with optimal druggability. Besides, the positive bioactivity scores higher than 0.10 against proteases suggest that there could be a possible therapeutic target for the novel hybrids. Moreover, the Osiris Drug Score (>65%) and toxicity risk calculations could indicate that the most active hybrids here evaluated could be used in further studies in the search for different therapeutics alternative against colorectal cancer. Calculated pharmacokinetics parameters suggest that merging The authors declare no conflict of interest.


H)–one (1), and they are evaluated for their inhibitory activity against β-tubulin polymerization. The target molecules 2–5 d were screened for their cytotoxic activity against breast cancer MCF-7 cell line. The results of cytotoxicity screening revealed that compounds 4e and 5d showed good cytotoxic profile against MCF-7 cells. Compounds 4e produced significant reduction in cellular tubulin with excellent β-tubulin polymerization inhibition activity. In addition, compound 4e exhibited cytotoxic activity against MCF-7 cells by cell cycle arrest at pre-G1 and G2/M phases, as shown by DNA flow cytometry assay. Aiming to enhance the limited aqueous solubility and, hence, poor oral bioavailability of the prepared lead acrylamide molecule, 4e-charged PEGylated bilosomes were successfully fabricated via thin film hydration techniques as an attempt to improve these pitfalls. 23 full factorial designs were manipulated to examine the influence of formulation variables: types of bile salt including either sodium deoxy cholate (SDC) or sodium tauro cholate (STC), amount of bile salt (15 mg or 30 mg) and amount of DSPE–mPEG-2000 amount (25 mg or 50 mg) on the characteristics of the nanosystem. The F7 formula of entrapment efficiency (E.E% = 100 ± 5.6%), particle size (PS = 280.3 ± 15.4 nm) and zeta potential (ZP = −22.5 ± 3.4 mv) was picked as an optimum formula with a desirability value of 0.868. Moreover, prominent enhancement was observed at the compound’s cytotoxic activity (IC50 = 0.75 ± 0.03 µM) instead of (IC50 = 2.11 ± 0.19 µM) for the unformulated 4e after being included in the nano-PEGylated bilosomal system.Cancer is a multifaceted disease. With the development of multi drug resistance, the need for the arousal of novel targets in order to avoid these drawbacks increased. A new series of acrylamide derivatives was synthesized from starting material 4-(furan-2-ylmethylene)-2-oxazol-5(4 Microtubules are one of the most important cellular protein scaffolds ,2. MicroBreast cancer characteristically displays uncontrolled or abnormal cell proliferation due to excessive microtubule synthesis ,12. KnowI, combretastatin A-4 (CA-4) II and chalcone III  cone III  exhibitef 0.6 μM . In addiund CA-4 .Unfortunately, most of chemotherapeutic drugs that suffer from a lack of persistent clinical and therapeutic outcomes. Moreover, they are associated with extensive adverse effects and diminished bioavailability . In ordeThe incorporation of bile salt in the vesicular structure aims to bypass the stability issue and other drawbacks associated with the other conventional vesicular systems, particularly for liposomes and niosomes . BilosomI–V. The incorporation of furan moiety as the second aryl ring into the diamide scaffold was performed to augment the anticancer activity of the produced compounds since oxygen in furan ring has the potential for hydrogen bonding with the target -3,4,5-trimethoxybenzamide (3). The 1H-NMR spectrum of compound 3 revealed the presence of doublet and multiplet signals at δ 1.12 and 3.94–4.04 ppm related to two methyl (2CH3) and methylidene (CH) of isopropyl group, respectively, in addition to two exchangeable protons (2NH) at δ7.78 and 9.67 ppm. Furthermore, compounds 4a–e were obtained from the reaction of oxazolone 1 with appropriate primary aromatic amines in absolute ethanol. The structures of compounds 4a–e were confirmed using IR and NMR spectra. 1H-NMR spectrum of compound 4c as a representative example showed the presence of characteristic two exchangeable proton (2NH) at δ 9.94 ppm. The 13C-NMR spectrum of compound 4c showed the presence of extra signals related to aromatic carbons. Finally, oxazolone 1 upon treatment with various substituted benzyl amines in glacial acetic acid in the presence of anhydrous sodium acetate produced the corresponding diamide derivatives 5a–d. The structures of compounds 5a–d were confirmed on the basis of IR, 1H-NMR and 13C-NMR spectral data. 1H-NMR spectra of compounds 5a–d revealed the presence of doublet signal assigned to methylene CH2 at δ 4.33–4.37 ppm. Further structural evidence of compounds 5a–d stemmed from the 13C-NMR spectra, which showed the presence of peaks at δ 37.74–42.55 ppm related to CH2 carbon in addition to other signals due to aromatic carbons.The designed molecules reported . The str2–5d were evaluated for their cytotoxic activities against breast cancer MCF-7 cell line by using an MTT colorimetric assay. The obtained results expressed as IC50 values were summarized in 2 revealed weak activity (IC50 > 50 μM) that is increased in the N-isopropylamide derivative 3 (IC50 = 4.73 μM). Furthermore, N-aryl amide derivatives 4a–e exhibited IC50 = 2.11–26.83 μM. Compound 4e (IC50 = 2.11 μM) showed superior activity at the submicromolar level than other derivatives 4a–d. Regarding N-benzyl amide derivatives 5a–d, 3,4-dimethoxybenzyl amide derivative 5c (IC50 = 2.61 μM) displayed higher cytotoxic activity against MCF-7 compared to other N-benzyl amide derivatives. Additionally, the therapeutic safety of N-(3-hydroxy-4-methoxy) aryl amide derivative 4e was determined by screening its cytotoxic activity against normal breast cell line (MCF-10A) using an MTT colorimetric assay. The results found that compound 4e revealed mild cytotoxic effects (IC50 = 29.27 μM). These results indicate the selectivity of N-(3-hydroxy-4-methoxy) aryl amide derivative 4e for tumor cells and its relative safety for normal breast cells.Compounds 4e at its IC50 concentration (2.11 μM) and Col at a concentration of 5 μM for 48 h and then submitted to immunofluorescence analysis under apotome fluorescence microscope. As shown in 4e-treated MCF-7 cells showed abnormal tubulin expression patterns, and the cell–cell microtubules mesh is reduced and fragmented with malformed distribution compared with the untreated control.In order to explore the cytotoxic activity of the prepared molecules for inhibition of tubulin polymerization, the localization tubulins were visualized in MCF-7 cells after treatment with compound 4e and colchicine (Col) using 5 μM concentration. β-tubulin inhibition analysis showed that compound 4e had good β-tubulin inhibition activity with 89% inhibition percentage that was nearly equipotent to Col, which had β-tubulin inhibition activity ≈ 93%  was used as the reference drug in this study. As revealed in 4e resulted in interference of the normal cell distribution in the cell cycle profile of MCF-7 cells. Compound 4e increased the percentage of cells in G2/M phase from 8.39% to 42.50% compared with the untreated control sample. This effect was accompanied by an increase in the percentage of cells in pre-G1 phase from 1.73% to 24.62%. Moreover, the capability of compound 4e in the induction of apoptosis in MCF-7 cells was superior over that of PTX. These results suggested that compound 4e induced cancer cell death via G2/M phase arrest with apoptosis inducing activity marked by the presence pre-G1 peak in the cell cycle profile of MCF-7 cells, and it is in line with the previously discussed data.In order to study the mechanism of the cytotoxic activity of compound 4e, a biparametric cytofluorimetric analysis was performed for the selected candidate at IC50 (2.11 μM) using AnnexinV/Propidium iodide (PI). The obtained data in 4e increased the percentage of cells at early and late stages compared to the untreated control. The percentage of early apoptotic cells increased by 6.23-fold compared to the untreated control. In addition, compound 4e increased the percentage of late apoptotic cells by 21.10-fold more than the untreated control. The data in this study indicate that compound 4e is an effective inducer of apoptosis in MCF-7 cells.Apoptosis is a process of cell death with characteristic morphological and biochemical features distinguishable from those associated with necrosis or accidental death . In orde4e,caspase 3/7 stimulation assay were carried out for compound 4e at its IC50 (2.11 μM) by using green flow cytometry analysis. The results showed that compound 4e highly stimulated caspase 3/7 by 6.95-fold more than the control. This experiment confirms that compound 4e stimulates caspase-3/7 activation and, hence, is involved in apoptosis induction in the MCF-7 cells (To confirm the apoptosis induced by compound -7 cells .4e into the active site of colchicine binding site in the tubulin protein (PDB code: 1SA0) in order to assess the possible binding interactions with the amino acid residue of the target protein. 4e interacts through hydrogen bonding with the amino acids Thr 340 and Phe 296 by its C=O and amide NH groups, respectively. It also interacts through hydrophobic interaction by its furan and 3-hydroxy-4-methoxyphenyl functions with the amino acid Pro 298 and Arg 339, respectively. Furthermore, the hydrophobicity of N-(3-hydroxy-4-methoxy) aryl amide molecule 4e resulted in the docking score of −13.31 kcal/mol. These findings revealed remarkably high binding affinity of N-(3-hydroxy-4-methoxy) aryl amide molecule 4e into the colchicine binding site of tubulin protein.Molecular docking simulation was performed for compound http://www.swissadme.ch/, accessed on 7 July 2021). These studies are correlated to the chemical structure of 4e, where certain parameters investigated included aqueous solubility level (LogS-SILICOS-IT), absorption Level, atom-based Log P (LogP 98), 2D polar surface area (2D TPSA), blood–brain barrier level (BBB LEV) and the probability of cytochrome P450 2D6 (CYP PROB) and cytochrome P450 2D6 (CYP2D6). The obtained results were depicted as an ADMET plot in The exploration of drug-likeness of compound and in silico pharmacokinetic properties was conducted via free web-based tool Swiss ADME  was distinguished as a significant parameter that might impede the compound’s full potential cytotoxic activity and oral bioavailability. This was affirmed by the predicted oral bioavailability (PSA = 145.2). In general molecules with PSA > 140 are poor in bioavailability; thus, 4e possesses limited oral bioavailability. The CYP2D6 value predicts the inhibitory and non-inhibitory aspect of 4e on Cytochrome P450 2D6 enzyme. Compound 4e is predicted to be an inhibitor for CYP2D6; thus, drug–drug interactions and the side effects such as liver dysfunction are expected upon administration.BBB and human intestinal absorption (HIA) plots were examined for 4e. 4e. Bile salt stabilized nanovesicular systems are established via the incorporation of bile salts into the lipid bilayers of the ordinary customized nano-vesicular systems  (DSPE–mPEG-2000) was a generous gift from Lipoid GmbH . Cholesterol was obtained from ITX Biomedicals . Sodium hydroxide, potassium dihydrogen orthophosphate and absolute ethanol were purchased from El-Nasr Chemical Co., Cairo, Egypt. The Spectra/PoreVR dialysis membrane  was purchased from Spectrum Laboratories Inc., Los Angeles, CA, USA. All chemicals and solvents were of HPLC grade and were used as received.Melting points were determined on open glass capillaries using Gallenkamp apparatus and are uncorrected. Infrared spectra were measured on a Shimadzu FT-IR 8400s infrared spectrophotometer , 2-acetic acid (0.01 mol), freshly fused sodium acetate (0.01 mol) and acetic anhydride (0.03 mol), was placed in a 500 mL conical flask, and the reaction mixture was heated for about 3 h. Moreover, ethanol (50 mL) was added slowly to the reaction mixture, and the mixture was left overnight in a refrigerator. The crystalline product that was obtained was filtered off, washed with ice-cold ethanol (30 mL) and dried. Recrystallization from 1,4-dioxane was conducted in order to obtain compound νmax cm−1): 3071, 3052 (arom.CH), 2897 , 1803 (C=O lactone), 1606, 1593 (C=C). 1H-NMR : 3.80 , 3.92 , 6.77–6.93 , 7.21 , 7.37 , 7.66 , 8.00 – 8.15 . 13C-NMR : 56.20 (2OCH3), 60.35 (OCH3), 105.18 , 114.25 (C3 furan), 117.11 (C4 furan), 120.05 (C1 trimethoxyphenyl), 120.52 (olefinic CH), 130.03 (olefinic CH), 142.20 (C4 trimethoxyphenyl), 147.93 (C5 furan), 149.87 (C2 furan), 153.28 , 161.94 (C2 oxazolone), 166.50 (C=O oxazolone). Analytically calculated for C17H15NO6 (329.30). Calculated: C, 62.00; H, 4.59; N, 4.25. Found: C, 61.95; H, 4.68; N, 4.33.Yellow powder ; m.p 183–185 °C; IR , Et3N (0.005 mol) in absolute ethanol (30 mL) was heated under reflux for 1 h. After completion of the reaction, the reaction mixture was cooled and poured into ice cold water and left to stand at room temperature for an additional 1 h. The solid product was collected, washed with water and dried to produce compound 2, which was purified by crystallization from absolute ethanol.A mixture of oxazolone νmax cm−1): 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 1.23 , 3.89 , 3.91 , 4.19 , 6.63 , 6.87 , 7.25 , 7.34 , 7.86 , 9.89 . 13C-NMR : 14.14 (CH3), 56.05 (2OCH3), 60.10 (OCH3), 60.86 (CH2), 105.27 , 112.58 (C3 ethyl acrylate), 115.91 (C3 furan), 120.81 (C4 furan), 123.72 (C2 ethyl acrylate), 128.51 (C1 trimethoxyphenyl), 140.47 (C4 trimethoxyphenyl), 145.59 (C5 furan), 149.17 (C2 furan), 152.71 , 164.47 (C1 ethyl acrylate), 164.96 (C=O). Analytically calculated for C19H21NO7 (375.37). Calculated: C, 60.79; H, 5.64; N, 3.73. Found: C, 61.05; H, 5.72; N, 3.55.White powder ; m.p 126–128 °C; IR -one (1) (0.005 mol) and isopropylamine (0.005 mol) in ethanol (30 mL) was refluxed for 1 h. The crystals that separated upon cooling the reaction mixture were collected and recrystallized from absolute ethanol to produce compound 3.A mixture of 4-(furan-2-ylmethylene)-2-oxazol-5(4νmax cm−1): 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 1.12 , 3.74 , 3.86 , 3.94–4.06 , 6.57 , 6.66 , 7.03 , 7.38 , 7.75 , 7.87 , 9.67 . 13C-NMR : 22.23 (2CH3), 40.97 (methylidene CH), 56.07 (2OCH3), 60.10 (OCH3), 105.49 , 112.19 (olefinic CH), 113.36 (C3 furan), 116.27 (C4 furan), 128.06 (C1 trimethoxybenzamide), 128.96 (olefinic CH), 140.33(C4 trimethoxybenzamide), 144.22 (C5 furan), 149.85 (C2 furan), 152.58 , 163.58 (C=O), 164.75 (C=O trimethoxybenzamide). Analytically calculated for C20H24N2O6 (388.41). Calculated: C, 61.84; H, 6.23; N, 7.21. Found: C, 61.62; H, 6.35; N, 7.26.White powder ; m.p 168–170 °C; IR -one (1) (0.005 mol) and the appropriate aromatic amines (0.005 mol) in absolute ethanol (25 mL) was refluxed for 3–4 h. The crystals that separated upon cooling the reaction mixture were collected and recrystallized from absolute ethanol to produce products 4a–e.A mixture of 4-(furan-2-ylmethylene)-2-oxazol-5(4νmax cm−1): 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 2.51 , 3.78 , 3.89 , 6.85 , 7.06 , 7.20 , 7.31 , 7.36–7.39 , 7.59 , 7.66 , 8.07–8.12 , 8.30 , 10.18 . 13C-NMR : 21.05 (CH3), 56.04 (2OCH3), 60.28 (OCH3), 105.62 , 114.08 (olefinic CH),114.24 (C3 furan),114.45 (C4 furan), 117.10 (C6 tolyl), 119.40 (C5 tolyl), 120.04 (C1 trimethoxybenzamide), 122.27 (C4 tolyl), 130.03 (C3 tolyl), 133.42 (C2 tolyl), 140.94 (olefinic CH), 147.09 (C1 tolyl), 147.91 (C4 trimethoxybenzamide), 149.87 (C5 furan), 152.81 , 157.30 (C5 furan), 166.50 (C=O trimethoxybenzamide), 167.70 (C=O amide). Analytically calculated for C24H24N2O6 (436.46). Calculated: C, 66.04; H, 5.54; N, 6.42. Found: C, 66.13; H, 5.61; N, 6.36.White crystals ; m.p 222–224 °C; IR : 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 2.28 , 3.74 , 3.87 , 6.61 , 6.77 , 6.89 , 7.10 , 7.19 , 7.42 , 7.49–7.59 , 7.77–7.85 , 10.06 , 10.15 . 13C-NMR : 21.19 (CH3), 56.06 (2OCH3), 60.12 (OCH3), 105.57 , 112.35 (olefinic CH), 113.98 (C3 furan), 116.69 (C4 furan), 117.35 (C6 tolyl), 120.68 (C5 tolyl), 124.10 (C1 trimethoxybenzamide), 128.32 (C4 tolyl), 128.82 (olefinic CH), 137.60 (C2 tolyl), 139.14 (C1 tolyl), 140.40 (C4 trimethoxybenzamide), 144.54 (C5 furan), 149.77 (C2 furan), 152.61 , 163.57 (C=O trimethoxybenzamide), 165.12 (C=O amide). Analytically calculated for C24H24N2O6 (436.46). Calculated: C, 66.04; H, 5.54; N, 6.42. Found: C, 66.10; H, 5.47; N, 6.39.White crystals ; m.p 217–219 °C; IR : 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 3.73 , 3.74 , 3.87 , 6.61 , 6.75 , 6.82–6.95 , 7.11 , 7.41 , 7.62 , 7.80 , 9.94 . 13C-NMR : 55.16 (OCH3), 56.07 (2OCH3), 60.12 (OCH3), 105.57 , 112.33 (olefinic CH), 113.61 , 113.61 (C3 furan), 116.58 (C4 furan), 121.78 , 128.22 (C1 trimethoxybenzamide), 128.84 (C1 methoxyphenyl), 132.26 (olefinic CH), 140.40 (C4 trimethoxybenzamide), 144.52 (C5 furan), 149.79 (C2 furan), 152.61 , 155.40 (C4 methoxyphenyl), 163.15 (C=O trimethoxybenzamide), 165.09 (C=O amide). Analytically calculated for C24H24N2O7 (452.46). Calculated: C, 63.71; H, 5.35; N, 6.19. Found: C, 63.88; H, 5.27; N, 6.24.Pale yellow crystals ; m.p 249–251 °C; IR : 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 3.74 , 3.86 , 6.60 , 6.71 , 6.74 , 7.10 , 7.41 , 7.47 , 7.73–7.88 , 9.83 , 9.97 . 13C-NMR : 56.09 (2OCH3), 60.14 (OCH3), 105.59 , 112.33 (olefinic CH), 113.61 (C3 furan), 114.89 , 116.56 (C4 furan), 122.07 , 128.34 (C1 trimethoxybenzamide), 128.89 (C1 hydroxyphenyl), 130.70 (olefinic CH), 140.41 (C4 trimethoxybenzamide), 144.48 (C5 furan), 149.84 (C2 furan), 152.62 , 153.62 (C4 hydroxyphenyl), 163.00 (C=O trimethoxybenzamide), 165.13 (C=O amide). Analytically calculated for C23H22N2O7 (438.43). Calculated: C, 63.01; H, 5.06; N, 6.39. Found: C, 62.91; H, 5.12; N, 6.47.White crystals ; m.p 214–216 °C; IR : 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 33.73 , 3.74 , 3.87 , 6.60 , 6.75 , 6.84 , 7.04–7.10 , 7.28 , 7.41 , 7.79 , 9.81 , 9.93 . 13C-NMR : 55.87 (OCH3), 56.08 (2OCH3), 60.13 (OCH3), 105.56 , 108.63 (C2 hydroxymethoxyphenyl), 111.14 (olefinic CH), 112.18 (C3 furan), 112.32 (C4 furan), 113.87 (C5 hydroxymethoxyphenyl), 116.49 (C6 hydroxymethoxyphenyl), 128.31 (C1 trimethoxybenzamide), 128.85 (C1 hydroxymethoxyphenyl), 132.69 (olefinic CH), 140.41 (C4 trimethoxybenzamide), 144.00 (C5 furan), 144.49 (C4 hydroxymethoxyphenyl), 146.20 (C3 hydroxymethoxyphenyl), 149.80 (C2 furan), 152.62 , 163.03 (C=O trimethoxybenzamide), 165.08 (C=O amide). Analytically calculated for C24H24N2O8 (468.46). Calculated: C, 61.53; H, 5.16; N, 5.98. Found: C, 61.61; H, 5.21; N, 5.91.Pale yellow crystals ; m.p 226–228 °C; IR , substituted benzyl amines (0.005 mol) and anhydrous sodium acetate (0.006) in glacial acetic acid (30 mL) was heated under reflux for 1–2 h. The reaction mixture was cooled, and the resulting solid was filtered off, dried and crystallized from acetic acid/water to provide compounds 5a–d.A mixture of oxazolone νmax cm−1): 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 2.29 , 3.74 , 3.87 , 4.35 , 6.59 , 6.72 , 7.04 , 7.10 , 7.13 , 7.16–7.25 , 7.41 , 7.78 , 8.61 , 9.80 . 13C-NMR : 21.05 (CH3), 42.55 (methylene CH2), 56.09 (2OCH3), 60.13 (OCH3), 105.61 , 112.31 (olefinic CH), 113.96 (C3 furan), 117.66 (C4 furan), 124.23 (C6 methylbenzyl), 127.18 (C4 methylbenzyl), 127.50 (C5 methylbenzyl), 127.81 (C1 trimethoxybenzamide), 128.04 (C2 methylbenzyl), 129.00 (olefinic CH), 137.12 (C1 methylbenzyl), 139.65 (C3 methylbenzyl), 140.38 (C4 trimethoxybenzamide), 144.57 (C5 furan), 149.79 (C2 furan), 152.58 , 164.39 (C=O amide), 165.22 (C=O trimethoxybenzamide). Analytically calculated for C25H26N2O6 (450.48): C, 66.65; H, 5.82; N, 6.22. Found: C, 66.92; H, 5.91; N, 6.29.White powder ; m.p 175–177 °C; IR : 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 3.74 , 3.79 , 3.86 , 4.34 , 6.58 , 6.72 , 6.90 , 6.96 , 7.18–7.28 , 7.40 , 7.77 , 8.43 , 9.82 . 13C-NMR : 37.74 (methylene CH2), 55.27 (OCH3), 56.11 (2OCH3), 60.14 (OCH3), 105.60 , 110.21 (olefinic CH), 112.32 (C3 methoxybenzyl), 114.07 (C3 furan), 117.74 (C4 furan), 120.07 (C5 methoxybenzyl), 126.93 (C1 methoxybenzyl), 127.14 (C4 methoxybenzyl), 127.39 (C6 methoxybenzyl), 127.69 (C1 trimethoxybenzamide), 129.01 (olefinic CH), 140.39 (C4 trimethoxybenzamide), 144.62 (C5 furan), 149.78 (C2 furan), 152.59 , 156.25 (C2 methoxybenzyl), 164.46 (C=O amide), 165.36 (C=O trimethoxybenzamide). Analytically calculated for C25H26N2O7 (466.48): C, 64.37; H, 5.62; N, 6.01. Found: C, 64.29; H, 5.55; N, 6.09.White powder ; m.p 166–168 °C; IR : 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 3.74 , 3.76 , 3.87 , 4.37 , 6.59 , 6.73 , 6.78 , 6.88 , 6.92 , 7.20 , 7.23 , 7.41 , 7.78 , 8.64 , 9.83 . 13C-NMR : 42.46 (methylene CH2), 54.95 (OCH3), 56.07 (2OCH3), 60.12 (OCH3), 105.59 , 112.18 (olefinic CH), 112.29 (C2 methoxybenzyl), 112.43 (C4 methoxybenzyl), 113.97 (C3 furan), 117.53 (C4 furan), 119.18 (C6 methoxybenzyl), 127.53 (C1 trimethoxybenzamide), 128.94 (C5 methoxybenzyl), 129.10 (olefinic CH), 140.38 (C1 methoxybenzyl), 141.41 (C4 trimethoxybenzamide), 144.57 (C5 furan), 149.77 (C1 furan), 152.56 , 159.30 (C3 methoxybenzyl), 164.38 (C=O), 165.25 (C=O trimethoxybenzamide). Analytically calucated for C25H26N2O7 (466.48): C, 64.37; H, 5.62; N, 6.01. Found: C, 64.47; H, 5.79; N, 6.93.White powder ; m.p 164–166 °C; IR : 3470 (NH), 3071, 3052 (arom.CH), 2897 , 1756, 1692 (C=O), 1606, 1593 (C=C). 1H-NMR : 3.72 , 3.74 , 3.77 , 3.87 , 4.33 , 6.59 , 6.72 , 6.82 , 6.87 , 6.97 , 7.17 , 7.41 , 7.78 , 8.58 , 9.84 . 13C-NMR : 42.19 (methylene CH2), 55.35 (OCH3), 55.58 (OCH3), 56.06 (2OCH3), 60.11 (OCH3), 105.57 , 111.13 (C2 dimethoxybenzyl), 111.62 (olefinic CH), 112.27 (C5 dimethoxybenzyl), 113.87 (C3 furan), 117.27 (C4 furan), 119.01 (C6 dimethoxybenzyl), 127.71 (C1 trimethoxybenzamide), 128.88 (C1 dimethoxybenzyl), 132.24 (olefinic CH), 140.37 (C4 trimethoxybenzamide), 144.51 (C5 furan), 147.52 (C4 dimethoxybenzyl), 148.62 (C2 furan), 149.77 (C3 dimethoxybenzyl), 152.56 , 164.30 (C=O amide), 165.20 (C=O trimethoxybenzamide). Analytically calculated for C26H28N2O8 (496.51): C, 62.89; H, 5.68; N, 5.64. Found: C, 63.03; H, 5.62; N, 5.56.White powder ; m.p 124-126 °C; IR  in volume of 100 µL was added to each well to dissolve the purple formazan that had formed. The color intensity of the formazan product, which represents the growth condition of the cells, is quantified by using an ELISA plate reader  at 570 nm absorbance. The experimental conditions were carried out with at least three replicates, and the experiments were repeated at least three times.Cytotoxic activity of the newly prepared acrylamide derivatives 4e and Col were evaluated against tubulin polymerization inhibitory activity according to manufacturer’s instructions [Compound ructions .5/well) were harvested and washed twice in PBS. After that, the cells were incubated at 37 °C and 5% CO2. The medium was replaced with DMSO 1% v/v containing the tested compound 4e (2.11 μM) and PTX (0.1 μM), then incubated for 48 h, washed twice in PBS, fixed with 70% ethanol, rinsed again with PBS and then stained with DNA fluorochrome PI for 15 min at 37 °C. The samples were analyzed by flow cytometry on a FACS Calibur flow cytometer .MCF-7 cells (2 × 105) after incubation for 12 h were used. Cells were treated with compound 4e (2.11 μM) and PTX (0.1 μM) for 48 h, then the cells were harvested and stained with Annexin V-FITC/ PI dye for 15 min in the dark at 37 °C. The samples were analyzed by using FACS Calibur flow cytometer .Apoptotic cells death was investigated by using fluorescent Annexin V-FITC/ PI detection kit by flow cytometry assay. Briefly, MCF-7 cells (2 × 10caspase 3/7 in MCF-7 cell line was detected in the presence of compound 4e (2.11 μM) and PTX (0.1 μM) using caspase 3/7 green flow cytometry assay kit as per the manufacturer’s instructions. Briefly, PTX and compound 4e treated MCF-7 cells (2.5 × 105/mL) were washed with ice cold PBS, and cell lysates were prepared and combined with reaction buffer and incubated with specific colorimetric substrates (Caspase 3/7 Detection Reagent) at 37 °C for 6 h. The samples were analyzed at 488 nm in a BD FACS Calibur flow cytometer. All experiments were performed in triplicates.The enzymatic activities of Molecular docking study was performed using MOE software program (MOE 2009.10). The tubulin crystal structure (PDB entry: 1SA0) was obtained from a protein data bank .Minute modifications were stuck to thin film hydration technique, which was manipulated for the development of 4e-loaded PEGylated bilosomes ,23. More® HPLC system , and HPLC conditions were in mobile phase: water (50:50); flow rate: 1 mL/min [Drug stock solution of 1 mg/mL in methanol was prepared, and a calibration curve was constructed utilizing six dilutions that were prepared in concentrations of 100, 200, 400, 600, 800 and 1000 µg/mL. All solutions were filtered using 0.22 µm syringe filter and then 10 µL was subjected to HPLC analysis using Waters-2690 Alliance1 mL/min . A disti4e charged within the formulated PEGylated bilosomal dispersion precisely, 1ml of 4e-loaded PEGylated bilosomal dispersion (resembling 2 mg of the drug) was diluted with 5 mL distilled water and manually agitated for 2 min. Cooling centrifugation technique for one hour was used to decouple the unembedded 4e from 4e-loaded PEGylated bilosome at 15,000 rpm and 4 °C  [4e within the vesicles was allocated via HPLC at λmax 254 nm (EE%) and was calculated as follows.In order to investigate the percentage of  Canada) . The sed4e-loaded PEGylated bilosome droplet size, zeta potential and PDI were investigated by utilizing Malvern sizer . The amount of 10 mL distilled water was utilized in order to dilute 0.1 mL of 4e-loaded PEGylated bilosomal dispersion within a glass tube that was manipulated, and then it was convulsed manually for 5 min. The manipulated technique was the dynamic laser scattering technique used to determine the distribution size at 25 °C by using 45 mm focus lens and beam lengths of 2.4 mm. The test was performed in triplicate [The tailored iplicate .3 factorial experiment was developed to assess the impact of multiple factors in the fabrication of PEGylated bilosome via Design Expert® software version 7 . The tailoring of 8 runs was yielded from the constructed design. Three factors were considered, bile salt type (A), bile salt amount (B) and DSPE–mPEG-2000 amount (C), to be independent variables, whereas EE% (Y1), PS (Y2), ZP (Y3) and Q8h (Y4) were picked as dependent variables. Moreover, based on the maximum EE%, ZP and minimum globule size, the optimum 4e-loaded PEGylated bilosomal formula was primed. Statistical analysis of the data was performed by utilizing Design Expert® 7 software. In addition, the statistical analysis conducted via ANOVA was implemented to highlight the prime impacts of the variables under exploration; the significance of each variable was analyzed, and the best formula with the superior desirability value was picked for further assessments [A 2essments .4e-loaded PEGylated bilosomal formula was manipulated via lyophilization technique , where mannitol (5% w/v) as lyoprotectant was utilized to disrupt the lysis of the vesicles. Accordingly, the PEGylated bilosomal suspension freezed overnight at −80 °C and was dried for a period of 24 h under vacuum [The solidification of the optimum r vacuum . The fre4e, plain optimum formula and 4e-loaded PEGylated bilosomal formula was examined adopting differential scanning calorimeter . The calibration of the equipment was conducted using purified indium (99.9%). About 10 °C was elevated each minute, surrounded by nitrogen in a temperature range of 20–400 °C [The thermal behavior of pure 0–400 °C .The configuration of the optimal PEGylated bilosomal formula was visualized by TEM . The stained vesicles’ dispersion was attached to a carbon grid with copper coat and kept to dry in order to obtain a thin film. The sheet of copper was enrolled into the TEM .4e-loaded PEGylated bilosomal formula, and then 1mL equivalent to 1 mg 4e from the diluted dispersion was transmitted to a 10 cm in length and 2.5 cm in diameter glass cylinder. A presoaked cellulose membrane was then allocated at its bottom where the dispersion prevailed over. The glass cylinder was mounted on the shaft of the dissolution tester  and hanged in 900 mL dissolution media  at 37 ± 0.5°C and speed of 50 rpm [4e release was conducted in triplicates.Concisely, 1mL of sorensen phosphate buffer (pH 7.4) was gathered with 1 mL of the optimum f 50 rpm . The wit4e and 5d showed good cytotoxic profile against MCF-7 cells. Compounds 4e produced significant reduction in cellular tubulin with excellent β-tubulin polymerization inhibition activity. In addition, compound 4e exhibited cytotoxic activity against MCF-7 cells by cell cycle arrest at pre-G1 and G2/M phases, as shown by the DNA flow cytometry assay. Additionally, compound 4e upregulated the expression of active caspase 3/7 percentages, as revealed by green flow cytometry analysis. PEGylated bilosomes were tailored as a nano platform for oral delivery of 4e as a remarkable anticancer. Eight formulae were tailored in accordance to 23 full factorial design. F7 was elected as the optimum formula due to the fundamentality of E.E%, PS and ZP. F7 exhibited the highest E.E%, smaller PS and absolute value of ZP. In vitro drug release studies affirmed the significant superior drug solubility of F7 over the 4e suspension. F7 was outstanding with respect to both higher solubility and enhanced cytotoxicity. Accordingly, 4e is developed as a lead molecule with potential anti-cancer activity, and PEGylated bilosomes can be considered as prosperous nanocarriers for compound 4e, hence improving its biopharmaceutical characteristics and cytotoxic activity after being loaded on a vesicular nanocarrier; thus, they worthy of future investigations via in vivo preclinical investigations.In conclusion, a series of novel acrylamide derivatives was designed and synthesized to be evaluated for their inhibitory activity against β-tubulin polymerization. The results of cytotoxicity screening over MCF-7 revealed that compounds


S-aralkylated bi-heterocyclic hybrids, 7a-l, was synthesized in a convergent manner. The structures of newly synthesized compounds were characterized by 1H-NMR, 13C-NMR, IR, and EI-MS spectral studies. The structure-activity relationship of these compounds was envisaged by analyzing their inhibitory effects against tyrosinase, whereby all these molecules exhibited potent inhibitory potentials relative to the standard used. The Kinetics mechanism was ascertained by Lineweaver-Burk plots, which revealed that 7g inhibited tyrosinase non-competitively by forming an enzyme-inhibitor complex. The inhibition constants Ki calculated from Dixon plots for this compound was 0.0057µM. These bi-heterocyclic molecules also disclosed good binding energy values  when assessed computationally. So, these molecules can be considered promising medicinal scaffolds for the treatment of skin disorders.Considering the diversified pharmacological importance of thiazole and triazole heterocyclic moieties, a unique series of  Elemental analyses were performed on a Foss Heraeus CHN-O-Rapid instrument and were within ± 0.4% of the theoretical values. IR peaks were recorded on a Jasco-320-A spectrometer by using the KBr pellet method. EI-MS spectra were measured on a JEOL JMS-600H instrument with a data processing system. 1H-NMR spectra  were recorded at 600 MHz  in DMSO-d6 using the Bruker Avance III 600 As- cend spectrometer using BBO probe. The abbreviations used in the interpretation of 1H NMR spectra are as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br.t, broad triplet; q, quartet; m, multiplet; dist. distorted.Chemicals were purchased from Sigma Aldrich and Alfa Aesar (Germany), and solvents of analytical grades were supplied by local suppliers. By using the open capillary tube method, melting points were taken on Griffin and George apparatus and were uncorrected. Using thin layer chromatography acetohydrazide (2)1) in methanol (60 mL) and hydrazine monohydrate  was taken in a 500 mL round bottom flask. The reaction mixture was refluxed for 2-3 h. After complete conversion, the acid hydrazide was attained by distilling methanol off from the reaction mixture. The precipitates were filtered, washed with cold n-hexane, and air-dried to get purified 2.Ethyl 2-acetate : 3358 (NH2 str.), 3351 (N-H str.), 3032 (C-H str.), 2950 (-CH2- str.), 1566 (C=C str.), 1587 (C=N str.), 1162 (C-N-C str.), 648 (C-S str.); 1H-NMR : 9.02 , 6.85 , 6.23 , 4.19 , 3.19 ; 13C-NMR : 168.97 (C-1′), 168.51 (C-2), 146.43 (C-4), 102.76 (C-5), 37.32 (C-2′). Anal. Calc. for C5H8N4OS2 (172.21): C, 34.87; H, 4.68; N, 32.53. Found: C, 34.98; H, 4.84; N, 32.69; EI-MS: m/z 172 [M]+, 130 (C4H6N2OS)+, 113 (C4H5N2S)+.White crystalline solid; Yield: 90%; m.p. 170-171 Synthesis of 2-[2-acetyl]-N-ethyl-1-hydrazinecarbothioamide (4)2; 0.13 mol.) was dissolved in methanol (50 mL) in a 500 mL round flask by heating, and after that, ethyl isothiocyanate  was added. Reaction mixture was kept on refluxing for 1 h. After completing the reaction, precipitates of the uncyclized compound, 4, were obtained by filtration and then dried for further use.The hydrazide : 3372 (N-H str.), 3052 (C-H str.), 2922 (-CH2- str.), 1585 (C=C str.), 1533 (C=N str.), 1158 (C-N-C bond str.), 622 (C-S str.); 1H-NMR : 9.87 , 9.21 , 7.87 , 6.88 , 6.31 , 3.50-3.48 , 1.03 ; 13C-NMR : 180.25 , 169.05 (C-2), 168.93 , 145.60 (C-4), 103.18 (C-5), 38.83 (C-1′′′), 37.36 (C-2′), 14.96 (C-2′′′). Anal. Calc. for C8H13N5OS2 (259.06): C, 37.05; H, 5.05; N, 27.00. Found: C, 37.18; H, 5.12; N, 27.13; EI-MS: m/z 259 [M+], 215 (C6H7N4OS2)+, 141 (C5H5N2OS)+, 113 (C4H5N2S)+.White amorphous solid; Yield: 86%; m.p. 176-177 Synthesis of 5-[methyl]-4-ethyl-4H-1,2,4-triazole-3-thiol (5)4; 0.13 mol.) was dissolved in 10% NaOH (100 mL) and slightly heated the solution until the compound 4 was dissolved and this solution was filtered. The precipitates of desired cyclized product, 5, were obtained by neutralizing the filtrate of the aforementioned solution with conc. HCl in a cold state.The intermediate compound : ʋ 3341 (N-H str.), 3062 (C-H str.), 2912 (-CH2- str.), 1580 (C=C str.), 1533 (C=N str.),1158 (C-N-C bond str.), 628 (C-S str.); 1H-NMR: δ 13.51 , 6.99 , 6.40 , 3.93 , 1.04 ; 13C-NMR: δ 169.22 (C-2), 166.69 (C-3′), 150.46 (C-5′), 145.55 (C-4), 103.46 (C-5), 38.92 (C-1′′), 28.26 (C-6), 13.36 (C-2′′). Anal. Calc. for C8H11N5S2 (241.34): C, 39.81; H, 4,59; N, 29.02. Found: C, 39.76; H, 4.65; N, 29.15; EI-MS: m/z 241 [M]+, 139 (C5H5N3S)+, 113 (C4H5N2S)+.Light brown amorphous solid; Yield: 94%; m.p. 206-207 General Synthesis of 4-({4-ethyl-5-[((un)functionalized-benzyl)sulfanyl]-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amines (7a-l)5; 0.2 g) was dissolved in DMF (3 mL) in a 100 mL round bottom flask at room temperature. After that, added one pinch of LiH and stirred it for 15-20 min. Then different un-functionalized benzyl halides (6a-l) were added in equimolar amounts (one in each reaction separately) and stirred for 6-8 hrs. A single spot on TLC indicated the completion of the reaction; the reaction mixture was quenched with ice cold water (50 mL). The desired derivatives, 7a-l, were obtained through filtration or solvent extraction according to the nature of the product.The thiol (4-{methyl}-1,3-thiazol-2-amine (7a)15H17N5S2; Mol. Mass.: 331 gmol-1. IR : 3388 (N-H str.), 3062 (C-H str. of aromatic ring), 2935 (-CH2- str.), 1592 (C=C str. of aromatic ring), 1522 (C=N str.), 1134 (C-N-C bond str.), 601 (C-S str.); 1H-NMR : 7.36-7.25 , 6.94 , 6.23 , 4.34 , 3.93 , 3.73 , 0.94 ; 13C-NMR : 168.56 (C-2), 153.09 (C-3′), 148.20 (C-5′), 146.17 (C-4), 137.31 (C-1′′′), 129.33 (C-2′′′ & C-6′′′), 128.65 (C-3′′′ & C-5′′′), 127.36 (C-4′′′), 102.35 (C-5), 38.45 (C-1′′), 35.14 (C-7′′′), 27.59 (C-6), 14.60 (C-2′′). Anal. Calc. for C15H17N5S2 (331.09): C, 54.35; H, 5.17; N, 21.13. Found: C, 54.65; H, 5.33; N, 21.24; EI-MS: m/z 331 [M]+, 240 (C8H10N5S2)+, 139 (C5H5N3S)+, 113 (C4H5N2S)+, 240 (C8H10N5S2)+, 91 (C7H7)+, 77 (C6H5)+.Dark brown gummy liquid; Mol. Formula: C4-({4-Ethyl-5-[(2-methylbenzyl)sulfanyl]-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7b) 16H19N5S2; Mol. Mass.: 345 gmol-1. IR : 3321 (N-H str.), 3022 (C-H str. of aromatic ring), 2966 (-CH2- str.), 1542 (C=C str. of aromatic ring), 1528 (C=N str.), 1122 (C-N-C bond str.), 622 (C-S str.); 1H-NMR : 7.23-7.17 , 6.94 , 6.23 , 4.34 , 3.93 , 3.72 , 2.24 , 0.94 ; 13C-NMR : 168.56 (C-2), 153.08 (C-3′), 148.13 (C-5′), 146.15 (C-4), 136.45 (C-1′′′), 134.74 (C-2′′′), 130.12 (C-3′′′), 127.78 (C-4′′′), 127.06 (C-6′′′), 125.86 (C-5′′′), 102.37 (C-5), 38.44 (C-1′′), 35.85 (C-7′′′), 27.57 (C-6), 18.77 (CH3-2′′′), 14.62 (C-2′′). Anal. Calc. for C16H19N5S2 (345.11): C, 55.62; H, 5.54; N, 20.27. Found: C, 55.68; H, 5.82; N, 20.38; EI-MS: m/z 345 [M]+, 240 (C8H10N5S2)+, 139 (C5H5N3S)+, 113 (C4H5N2S)+, 105 (C8H9)+, 91 (C7H7)+.Dark brown gummy liquid; Mol. Formula: C4-({4-Ethyl-5-[(3-methylbenzyl)sulfanyl]-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7c) 16H19N5S2; Mol. Mass.: 345 gmol-1. IR : 3338 (N-H str.), 3011 (C-H str. of aromatic ring), 2948 (-CH2- str.), 1572 (C=C str. of aromatic ring), 1541 (C=N str.), 1172 (C-N-C bond str.), 619 (C-S str.); 1H-NMR : 7.13-7.06 , 6.94 , 6.22 , 4.30 , 3.93 , 3.77 , 2.24 , 0.94 ; 13C-NMR : 168.56 (C-2), 153.04 (C-3′), 148.28 (C-5′), 146.18 (C-4), 137.57 (C-1′′′), 137.09 (C-3′′′), 129.42 (C-5′′′), 128.52 (C-4′′′), 128.21 (C-2′′′), 125.93 (C-6′′′), 102.32 (C-5), 39.09 (C-1′′), 37.26 (C-7′′′), 27.53 (C-6), 20.80 (CH3-3′′′), 14.61 (C-2′′). Anal. Calc. for C16H19N5S2 (345.11): C, 55.62; H, 5.54; N, 20.27. Found: C, 55.68; H, 5.82; N, 20.38; EI-MS: m/z 345 [M]+, 240 (C8H10N5S2)+, 139 (C5H5N3S)+, 113 (C4H5N2S)+, 105 (C8H9)+, 91 (C7H7)+.Dark brown gummy liquid; Mol. Formula: C4-({4-Ethyl-5-[(4-methylbenzyl)sulfanyl]-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7d)16H19N5S2; Mol. Mass.: 345 gmol-1. IR : 3348 (N-H str.), 3051 (C-H str. of aromatic ring), 2931 (-CH2- str.), 1565 (C=C str. of aromatic ring), 1504 (C=N str.), 1139 (C-N-C bond str.), 605 (C-S str.); 1H-NMR : 7.16 , 7.09 , 6.94 , 6.21 , 4.29 , 3.93 , 3.74 , 2.25 , 0.94 ; 13C-NMR : 168.55 (C-2), 153.01 (C-3′), 148.25 (C-5′), 146.19 (C-4), 136.62 (C-1′′′), 134.16 (C-4′′′), 129.01 (C-3′′′ & C-5′′′), 128.92 (C-2′′′ & C-6′′′), 102.31 (C-5), 38.43 (C-1′′), 37.09 (C-7′′′), 27.56 (C-6), 20.67 (CH3-4′′′), 14.61 (C-2′′). Anal. Calc. for C16H19N5S2 (345.11): C, 55.62; H, 5.54; N, 20.27. Found: C, 55.68; H, 5.82; N, 20.38; EI-MS: m/z 345 [M]+, 240 (C8H10N5S2)+, 139 (C5H5N3S)+, 113 (C4H5N2S)+, 105 (C8H9)+, 91 (C7H7)+.Dark brown gummy liquid; Mol. Formula: C4-({5-[(2-Chlorobenzyl)sulfanyl]-4-ethyl-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7e)15H16ClN5S2; Mol. Mass.: 365 gmol-1. IR : 3381 (N-H str.), 3012 (C-H str. of aromatic ring), 2908 (-CH2- str.), 1561 (C=C str. of aromatic ring), 1547 (C=N str.), 1148 (C-N-C bond str.), 628 (C-S str.); 1H-NMR : 7.42-7.30 , 6.98 , 6.25 , 4.41 , 3.94 , 3.75 , 0.94 ; 13C-NMR : 168.51 (C-2), 153.21 (C-3′), 147.65 (C-5′), 146.13 (C-4), 131.48 (C-1′′′), 129.94 (C-5′′′), 129.37 (C-3′′′), 127.55 (C-6′′′), 127.29 (C-4′′′), 127.17 (C-2′′′), 102.37 (C-5), 38.42 (C-1′′), 35.67 (C-7′′′), 27.45 (C-6), 14.54 (C-2′′). Anal. Calc. for C15H16ClN5S2 (365.05): C, 49.24; H, 4.41; N, 19.14. Found: C, 49.37; H, 4.52; N, 19.20; EI-MS: m/z 367 [M+2]+, 365 [M]+, 240 (C8H10N5S2)+, 139 (C5H5N3S)+, 125 [C7H6Cl]+, 113 (C4H5N2S)+, 111 [C6H4Cl]+.Dark brown gummy liquid; Mol. Formula: C4-({5-[(4-Chlorobenzyl)sulfanyl]-4-ethyl-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7f) 15H16ClN5S2; Mol. Mass.: 365 gmol-1. IR : 3355 (N-H str.), 3049 (C-H str. of aromatic ring), 2945 (-CH2- str.), 1592 (C=C str. of aromatic ring), 1584 (C=N str.), 1188 (C-N-C bond str.), 637 (C-S str.); 1H-NMR : 7.49 , 7.32 , 6.68 , 6.29 , 4.34 , 3.97 , 3.79 , 0.91 ; 13C-NMR : 168.82 (C-2), 152.81 (C-3′), 148.14 (C-5′), 146.17 (C-4), 136.60 (C-4′′′), 132.39 (C-1′′′), 131.01 (C-3′′′ & C-5′′′), 128.62 (C-2′′′ & C-6′′′), 102.81 (C-5), 38.51 (C-1′′), 36.28 (C-7′′′), 25.40 (C-6), 14.63 (C-2′′). Anal. Calc. for C15H16ClN5S2 (365.05): C, 49.24; H, 4.41; N, 19.14. Found: C, 49.37; H, 4.52; N, 19.20; EI-MS: m/z 367 [M+2]+, 365 [M]+, 240 (C8H10N5S2)+, 139 (C5H5N3S)+, 125 [C7H6Cl]+, 113 (C4H5N2S)+, 111 [C6H4Cl]+.Dark brown gummy liquid; Mol. Formula: C4-sulfanyl]-4-ethyl-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7g)oC; Mol. Formula: C15H15Cl2N5S2; Mol. Mass.: 399 gmol-1. IR : 3362 (N-H str.), 3021 (C-H str. of aromatic ring), 2904 (-CH2- str.), 1543 (C=C str. of aromatic ring), 1527 (C=N str.), 1168 (C-N-C bond str.), 622 (C-S str.); 1H-NMR : 7.63 , 7.38 , 7.33 , 6.95 , 6.23 , 4.39 , 3.94 , 3.79 , 0.96 ; 13C-NMR : 168.50 (C-2), 153.26 (C-3′), 147.37 (C-5′), 145.95 (C-4), 134.00 (C-4′′′), 133.88 (C-1′′′), 133.04 (C-2′′′), 132.44 (C-6′′′), 128.82 (C-3′′′), 127.32 (C-5′′′), 102.31 (C-5), 38.47 (C-1′′), 34.65 (C-7′′′), 27.45 (C-6), 14.56 (C-2′′). Anal. Calc. for C15H15Cl2N5S2 (400.35): C, 45.00; H, 3.78; N, 17.49. Found: C, 45.13; H, 3.82; N, 17.61; EI-MS: m/z 403 [M+4]+, 401 [M+2]+, 399 [M]+, 240 (C8H10N5S2)+, 159 [C7H5Cl2]+, 145 [C6H3Cl2]+, 139 (C5H5N3S)+, 113 (C4H5N2S)+.Light brown amorphous solid; Yield: 86%; m.p.:116-117 4-sulfanyl]-4-ethyl-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7h) 15H15Cl2N5S2; Mol. Mass.: 400 gmol-1. IR : 3366 (N-H str.), 3047 (C-H str. of aromatic ring), 2943 (-CH2- str.), 1608 (C=C str. of aromatic ring), 1550 (C=N str.), 1165 (C-N-C bond str.), 609 (C-S str.); 1H-NMR : 7.63 , 7.55 , 7.24 , 6.93 , 6.21 , 4.35 , 3.95 , 3.81 , 0.97 ; 13C-NMR : 168.58 (C-2), 153.26 (C-3′), 147.81 (C-5′), 146.15 (C-4), 133.38 (C-1′′′), 131.53 (C-4′′′), 131.07 (C-3′′′), 130.85 (C-5′′′), 130.45 (C-2′′′), 129.25 (C-6′′′), 102.31 (C-5), 38.56 (C-1′′), 35.74 (C-7′′′), 27.51 (C-6), 14.59 (C-2′′). Anal. Calc. for C15H15Cl2N5S2 (400.35): C, 45.00; H, 3.78; N, 17.49. Found: C, 45.13; H, 3.82; N, 17.61; EI-MS: m/z 403 [M+4]+, 401 [M+2]+, 399 [M]+, 240 (C8H10N5S2)+, 159 [C7H5Cl2]+, 145 [C6H3Cl2]+, 139 (C5H5N3S)+, 113 (C4H5N2S)+.Dark brown gummy liquid; Mol. Formula: C4-({5-[(2-Bromobenzyl)sulfanyl]-4-ethyl-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7i) oC; Mol. Formula: C15H16BrN5S2; Mol. Mass.: 410 gmol-1. IR : 3392 (N-H str.), 3074 (C-H str. of aromatic ring), 2940 (-CH2- str.), 1532 (C=C str. of aromatic ring), 1541 (C=N str.), 1194 (C-N-C bond str.), 625 (C-S str.); 1H-NMR : 7.62 , 7.35 , 7.29 , 7.21 , 6.92 , 6.25 , 4.41 , 3.93 , 3.76 , 0.96 ; 13C-NMR : 168.47 (C-2), 153.19 (C-3′), 147.60 (C-5′), 146.03 (C-4), 136.15 (C-1′′′), 132.65 (C-3′′′), 131.23 (C-4′′′), 129.64 (C-6′′′), 127.77 (C-5′′′), 123.73 (C-2′′′), 102.33 (C-5), 38.43 (C-1′′), 37.98 (C-7′′′), 27.46 (C-6), 14.53 (C-2′′). Anal. Calc. for C15H16BrN5S2 (410.36): C, 43.90; H, 3.93; N, 17.07. Found: C, 43.98; H, 3.99; N, 17.22; EI-MS: m/z 411 [M+2]+, 409 [M]+, 240 (C8H10N5S2)+, 169 [C7H6Br]+, 155 [C6H4Br]+, 139 (C5H5N3S)+, 113 (C4H5N2S)+.Light brown amorphous solid; Yield: 88%; m.p.: 108-109 4-({5-[(3-Bromobenzyl)sulfanyl]-4-ethyl-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7j) oC; Mol. Formula: C15H16BrN5S2; Mol. Mass.: 4106 gmol-1. IR : 3371 (N-H str.), 3022 (C-H str. of aromatic ring), 2935 (-CH2- str.), 1588 (C=C str. of aromatic ring), 1562 (C=N str.), 1192 (C-N-C bond str.), 613 (C-S str.); 1H-NMR : 7.56 , 7.45 , 7.32 , 7.24 , 6.93 , 6.22 , 4.35 , 3.93 , 3.78 , 0.95 ; 13C-NMR : 168.53 (C-2), 153.11 (C-3′), 147.88 (C-5′), 146.11 (C-4), 140.34 (C-1′′′), 131.57 (C-4′′′), 130.43 (C-2′′′), 130.10 (C-5′′′), 127.94 (C-6′′′), 121.34 (C-3′′′), 102.27 (C-5), 38.44 (C-1′′), 36.08 (C-7′′′), 27.44 (C-6), 14.50 (C-2′′). Anal. Calc. for C15H16BrN5S2 (410.36): C, 43.90; H, 3.93; N, 17.07. Found: C, 43.98; H, 3.99; N, 17.22; EI-MS: m/z 411 [M+2]+, 409 [M]+, 240 (C8H10N5S2)+, 169 [C7H6Br]+, 155 [C6H4Br]+, 139 (C5H5N3S)+, 113 (C4H5N2S)+.Bright orange amorphous solid; Yield: 84%; m.p.: 158-159 -({5-[(4-Bromobenzyl)sulfanyl]-4-ethyl-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7k)oC; Mol. Formula: C15H16BrN5S2; Mol. Mass.: 410 gmol-1. IR : 3344 (N-H str.), 3072 (C-H str. of aromatic ring), 2927 (-CH2- str.), 1568 (C=C str. of aromatic ring), 1520 (C=N str.), 1135 (C-N-C bond str.), 635 (C-S str.); 1H-NMR : 7.48 , 7.27 , 6.93 , 6.21 , 4.32 , 3.93 , 3.76 , 0.95 ; 13C-NMR : 168.49 (C-2), 153.07 (C-3′), 147.87 (C-5′), 146.07 (C-4), 136.97 (C-1′′′), 131.18 (C-3′′′ & C-5′′′), 130.98 (C-2′′′ & C-6′′′), 120.44 (C-4′′′), 102.24 (C-5), 38.42 (C-1′′), 36.23 (C-7′′′), 27.46 (C-6), 14.53 (C-2′′). Anal. Calc. for C15H16BrN5S2 (410.36): C, 43.90; H, 3.93; N, 17.07. Found: C, 43.98; H, 3.99; N, 17.22; EI-MS: m/z 411 [M+2]+, 409 [M]+, 240 (C8H10N5S2)+, 169 [C7H6Br]+, 155 [C6H4Br]+, 139 (C5H5N3S)+, 113 (C4H5N2S)+.Bright yellow amorphous solid; Yield: 90%; m.p.: 150-151 4-({4-Ethyl-5-[(4-fluorobenzyl)sulfanyl]-4H-1,2,4-triazol-3-yl}methyl)-1,3-thiazol-2-amine (7l) 15H16FN5S2; Mol. Mass.: 349 gmol-1. IR : 3368 (N-H str.), 3062 (C-H str. of aromatic ring), 2947 (-CH2- str.), 1573 (C=C str. of aromatic ring), 1534 (C=N str.), 1122 (C-N-C bond str.), 619 (C-S str.); 1H-NMR : 7.35-7.32 , 7.12 , 6.93 , 6.23 , 4.33 , 3.93 , 3.75 , 0.94 ; 13C-NMR: 168.48 (C-2), 162.13 & 162.01 , 153.04 (C-3′), 147.99 (C-5′), 146.08 (C-4), 133.65 & 133.64 (C-1′′′), 130.82 & 130.80 (C-2′′′ & C-6′′′), 115.17 & 115.03 (C-3′′′ & C-5′′′), 102.28 (C-5), 38.38 (C-1′′), 36.29 (C-7′′′), 27.47 (C-6), 14.52 (C-2′′). Anal. Calc. for C15H16FN5S2 (349.08): C, 51.56; H, 4.61; N, 20.04. Found: C, 51.70; H, 4.83; N, 20.21; EI-MS: m/z 349 [M]+, 240 (C8H10N5S2)+, 139 (C5H5N3S)+, 113 (C4H5N2S)+, 109 [C7H6F]+, 95 [C6H4F]+.Dark brown gummy liquid; Mol. Formula: CTyrosinase assay50). Each concentration was analyzed in three independent experiments. IC50 values were calculated by nonlinear regression using GraphPad Prism 5.0.The inhibition of mushroom tyrosinase was determined by modifying the dopachrome method using L-DOPA as a substrate . In detaThe inhibition% of tyrosinase was calculated as follows:Inhibition (%) = [(B - S)/B] × 100Here, the B and S are the absorbance’s for the blank and samples. Protocol for kinetics7g, was subjected to kinetic analysis. A series of experiments were performed to determine the inhibition kinetics of 7g, following the already reported methods  versus the inverse of substrate concentration 1/[L-DOPA] mM-1. The EI dissociation constant Ki was determined by the secondary plot of 1/V versus inhibitor concentrations.The most potent compound,  methods . The conFree radical scavenging assay3OH. After that, the assay reaction was then incubated for 30 minutes at room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The assay measurements were carried out using a microplate reader  at 517 nm. The reaction rates were compared, and the percent inhibition caused by tested inhibitors was calculated. All experiments were repeated thrice.Radical scavenging activity was determined by modifying the already reported method by 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay . The assComputational methodology Retrieval of tyrosinase in maestro The target protein structure was retrieved from Protein Data Bank (PDB) (www.rcsb.org) having PDBIDs 2Y9X. The protein structure was prepared using the “Protein Preparation Wizard” workflow in Schrödinger Suite. The bond orders were assigned, and hydrogen atoms were added to the protein molecule. The water molecules were removed from the protein structure. The structure was then minimized to reach the converged root mean square deviation (RMSD) of 0.30 Å with the OPLS_2005 force field. The prepared structure was employed for the further grid and docking analysis. Grid generation and molecular docking 7a-l) against receptor molecules. The synthesized molecules were sketched by a 2D sketcher in the Maestro interface and utilized in docking procedure. The default docking setup parameters were employed for the ligand docking experiment , and the varying groups are listed in (1) with hydrazine in methanol to get 2-acetohydrazide (2). The precipitates of 2 were collected, by filtration, and it was further refluxed with ethyl isothiocyante (3) in methanol to obtain an intermediary compound, 2-[2-acetyl]-N-ethyl-1-hydrazinecarbothioamide (4) which was cyclized to get a solid nucleophile, 5-[methyl]-4-ethyl-4H-1,2,4-triazole-3-thiol (5). This bi-heterocyclic nucleophile (5) was dissolved in DMF, and one pinch of LiH was added. The solution was stirred for 15-20 minutes to activate the mercapto position of this molecule. Then, in the last step, it was treated with equimolar amounts of various aralkyl halides (6a-l), acting as electrophiles, to acquire the targeted hybrid molecules (7a-l).The synthesis of designed isted in . The con7g was synthesized as a light brown amorphous solid with a melting point of 116-117 oC, and its molecular formula, C15H15Cl2N5S2, was corroborated by the molecular ion peak at m/z 399 along with CHN analysis data. The count of the number of protons in its 1H-NMR spectrum and the number of carbon resonances in its 13C-NMR spectrum was also coherent with this assignment. The functional groups were ascertained by its IR spectrum, whereby the characteristic peaks appeared at 3362 (N-H stretching), 3021 (C-H stretching of aromatic ring), 2904 (-CH2- stretching), 1543 (C=C stretching of aromatic ring), 1527 (C=N stretching), 1168 (C-N-C bond stretching), 622 (C-S stretching), indicating the presence of heterocyclic rings. In 1H-NMR spectrum, two peculiar signals of an ethyl group, attached to a nitrogen atom of triazole ring, appeared at δ 3.79  and 0.96 , while an AMX spin system of 2,4-dichlorobenzyl group was signified by three typical signals in aromatic region at δ 7.63 , 7.38  and 7.33 . The benzylic methylene, attached with sulfur atom, appeared at δ 4.39 . 2-Amino-1,3-thiazol-4-yl heterocycle was categorized by two signals at δ 6.95  and 6.23  while the signal δ 3.94  was assignable to a methylene group connecting the two heterocycles in the molecule. 1H-NMR spectrum of this molecule has been shown in The structure analysis of one of the compounds is elaborated hereby in detail for the benefit of the reader. Compound 13C-NMR spectrum sulfanyl was also indicated by two quaternary signals at δ 153.26 (C-3’) and 147.37 (C-5’). The methylene connecting the two heterocycles was apparent at δ 27.45 (C-6). The 2,4-dichlorobenzyl moiety was also obvious with three quaternary signals at δ 134.00 (C-4’’’), 133.88 (C-1’’’) and 133.04 (C-2’’’) along with three methine signals at δ 132.44 (C-6’’’), 128.82 (C-3’’’) and 127.32 (C-5’’’), in addition to a distinct signal at δ 34.65 (C-7’’’) for a benzylic methylene, attached to sulfur atom. The ethyl group connected with the triazole ring was also designated by one methylene signal at δ 38.47 (C-1’’) and one methyl signal at δ 14.66 (C-2’’). The C-H connectivities in the carbon skeleton were thoroughly corroborated by its HMBC spectrum, and the important correlations are illustrated on this spectrum -1,3-thiazol-2-amine. Likewise, the structures of all other compounds were characterized by their spectral data.All these assignments are also verified by its spectrum , which espectrum . These sBiologyTyrosinase inhibition and structure-activity relationship7a-l) were investigated against tyrosinase enzyme to explore their inhibitory potentials, and the results obtained so are tabulated (50 (µM) values, relative to the standard (Kojic acid), having IC50 value of 16.8320 ± 1.1600 µM. Although the displayed activity is characteristic of the whole molecule, a limited structure-activity relationship (SAR) was anticipated by perceiving the effect of varying (un) substituted-benzyl entities on the inhibitory potential. This approximation was made because it was the only part which was varying in all molecules. The general structural parts of the studied compounds are demarcated in , having unsubstituted-benzyl part. However, among the methylated regio-isomers, the compound 7c with meta-methyl group was found to be superb inhibitor (IC50 = 0.0341 ± 0.0012 µM), as compared to ortho-isomer , as well as para-isomer . It means, when a small-sized electron-donating group was present at 3-position in benzylic part  or at para-position , the molecules render some analogous interactions to the enzyme. The tivities . It mean7g (IC50 = 0.0018 ± 0.0005 µM), having ortho and para-positioned chloro groups behaved as a superb inhibitor than 7h (IC50 = 0.0142 ± 0.0013 µM) in which the two chloro groups were present at adjacent positions i.e. meta and para  was a suitable option. Furthermore, this compound was also recognized as the second most active molecule (IC50 = 0.0021 ± 0.0013 µM) in the synthetic series. The shifting of bromo group from ortho position  to para position  resulted in a decrease in the activity. Likewise, the presence of a highly electronegative fluoro group in 7h (IC50 = 0.0115 ± 0.0056 µM) was also not a superior choice   . The kinFree radical scavenging7a-g, has been determined using Ascorbic acid (Vitamin C) as a reference to compare the antioxidant activity of the synthesized compounds. The results obtained from DPPH assay have been presented  and hydrogen/hydrophobic interaction pattern. Results showed that all the ligands, 7a-l, exhibited good docking energy values and showed their interaction within the active region of the target protein. GlideScore is based on ChemScore (fitness function), but includes a steric-clash term, adds buried polar terms devised by Schrödinger to penalize electrostatic mismatches. The GScore is calculated from the Equation 1.Molecular docking is a significant approach to study the interactive behavior of newly synthesized ligands within the active region of target proteins , 62. TheGScore = vdW + Coul + Lipo + Hbond + Metal + BuryP + RotB + Site Equation 1.vdW = Van der Waals energy, Coul = Coulomb energy, Lipo = Lipophilic, Hbond = Hydrogen-bonding, Metal = Metal-binding, BuryP = Penalty for buried polar groups, RotB = Penalty for freezing rotatable bonds and Site = Polar interactions in the active site. in-vitro and in-silico docking energy results, 7c was ranked as the best ligands, which showed good inhibitory potential against targeted enzymes as compared to all other derivatives. Although the basic nucleus of all the synthesized compounds was the same, most compounds possess good efficient energy values and have no big energy fluctuations difference. The docking binding energy values are depicted here  was selected to check its binding and conformational position within the active region of the target protein (The binding pocket analysis showed that ligands,  protein  and 14.  protein , 57. TheIn conclusion, a new series of bi-heterocyclic hybrids were synthesized as propitious tyrosinase inhibitors. Significantly, the compounds bearing 3-bromobenzyl, 2-chlorobenzy, 4-chlorobenzyl, or 2,4-dichlorobenzyl groups possessed very superb activities. Moreover, molecular docking results also revealed good binding interactions and docking energy values. Therefore, it was concluded generally that these bi-heterocyclic molecules might be deliberated as commendable medicinal scaffolds for treating tyrosinase related ailments, particularly skin disorders.


Laminaria japonica, commonly known as edible brown seaweed, and their structures were identified based on detailed chemical methods and spectroscopic analyses. Spectroscopic analyses characterized 5 sterols as 29-Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol, saringosterol , 24-methylenecholesterol, fucosterol , and 24-Hydroperoxy-24-vinyl-cholesterol. The bioactivities of these sterols were tested using lipid peroxidation (LPO) and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Fucosterol exhibited the highest COX-1 and -2 enzyme inhibitory activities at 59 and 47%, respectively. Saringosterol, 24-methylenecholesterol and fucosterol showed higher LPO inhibitory activity at >50% than the other compounds. In addition, the results of molecular docking revealed that the 5 sterols were located in different pocket of COX-1 and -2 and fucosterol with tetracyclic skeletons and olefin methine achieved the highest binding energy  through hydrophobic interactions and hydrogen bond. Our results confirm the presence of 5 sterols in L. japonica and its significant anti-inflammatory and antioxidant activity.In this study, 5 sterols were isolated and purified from  Laminaria japonica, a commercial edible brown seaweed, is widely cultivated and consumed as a health food resource in China, Japan, and Korea. Over the past decades, L. japonica is one of the most important marine medicinal foodstuffs and a rich source of various functional compounds, which have been widely investigated by both in vitro and in vivo experiments  phenylpropionic acid was purchased from Molecular Probes  and 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine (SLPC) was purchased from Avanti Polar Lipids .Waters 2010 HPLC (high pressure liquid chromatography) system  equipped with XTerra Prep MS C-18 column   was used for the HPLC purification and all solvents used for HPLC purification were of ACS reagent grade . The NMR (Nuclear magnetic resonance) spectra were obtained using Agilent DirectDrive2 500 MHz  in deuterated chloroform (CDClL. japonica (GS-02-004-2013) used in this study were cultivated at Yantai  and harvested in July 2018. Fresh raw L. japonica was dried immediately after harvest under sun light and then shipped to the lab. The raw L. japonica in the lab was first washed with running tap water and deionized water, and then dried in an oven at 50°C. The dry sample was further pulverized with a blender to obtain the dry L. japonica power through a 50-mesh screen.L. japonica power (5 kg) was extracted with Hexane  and evaporated under vacuum to afford hexanes extract HE (22 g). An aliquot of HE (13 g) was pretreated with activated charcoal and then fractionated using silica gel VLC by eluting with hexanes-ethyl acetate , followed by methanol (100%), to yield fractions A (855 mg), B (4 g), C (1.44 g), D (374 mg), E (205 mg), and F (3 g), respectively. An aliquot of fractions A (68 mg) was fractionated by Pre-TLC with hexanes-acetone  to yield the main fraction A-1(31 mg). An aliquot of fractions B (76 mg) was fractionated by Pre-TLC with hexanes-acetone  to yield the main fraction B-1(50 mg), which was characterized as fatty acid and hence were kept aside. Fraction D (374 mg) fractionated by MPLC with hexanes-acetone  and Pre-TLC with chloroform: methanol  yielded three main fractions D-1, D-2 and D-3, which were the same compounds that from fraction C. And fraction E and F were found to be salt and put away.The dry Fraction C (1.44 g) was further purified with silica gel MPLC by eluting with hexanes-acetone , to yield fractions G (301 mg), H (599 mg), I (137 mg), J (160 mg), and K (166 mg), respectively. Fraction G had been put away for it was too complicated.H (215 mg) was fractionated by silica gel VLC and eluted under gradient conditions using chloroform: methanol (100:1 and 4.1 v/v) to yield fractions LJ-1 (161 mg) and LJ-2 (51 mg). An aliquot of fraction LJ-1 (160 mg), purified by HPLC by eluting with acetonitrile: methanol 50:50 v/v, 4 mL/min under isocratic conditions yielded fraction LJ-1(a) (62 mg), compound 3  (40 mg) eluting with acetonitrile:methanol 95:5 v/v, 3 mL/min under isocratic conditions yielded compound 1 -dien-3β-ol, 2  was further separated by recrystallization from hexane: acetone to afford compound 5  + in its positive-ion HRESITOFMS consistent with the molecular formula C29H48O3, This confirmed six indices of hydrogen deficiency in compound 1  spectrum exhibited two olefin methine proton signal at δH 5.33  and δH 4.53 , one oxygenated methine proton signal at δH 3.49 , and five methyl proton signals at δH 0.99 , 0.97 , 0.95 , 0.89 , and 0.65   spectrum exhibited 29 carbon signals including 2 olefin quaternary carbon signals at δC 155.3 (C-24) and 140.7 (C-5), two olefin methine carbon signals at δC121.7 (C-6) and δC114.8 (C-28), one oxygenated methine carbon signal at δC 71.8 (C-3), one oxygenated methylene carbon signal at δC 73.5 (C-29) and 5 methyl carbon signals at δC 21.9 (C-26), 21.8 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18) -dien-3β-ol  . The 13C8 (C-18) . Accordien-3β-ol  22]..1 was is2 was isolated as a white powder and exhibited a molecular ion peak at m/z 427.3562 [M+H-2H]+ in its positive-ion HRESITOFMS consistent with the molecular formula C29H48O2, This confirmed six indices of hydrogen deficiency in compound 2  spectrum exhibited two olefin methine proton signals at δH 5.34  and 5.75 , one olefin methylene signal at δH 5.11–5.27 , one oxygenated methine proton signal at δH 3.49 , and five methyl proton signals at δH 0.98 , 0.90 , 0.89 , 0.85 , and 0.66   spectrum exhibited 29 carbon signals including one olefin quaternary carbon signal at δC 140.7 (C-5), one olefin methylene carbon signals at δC121.7 (C-29), two olefinic methines carbon signals at δC137.1 (C-5) and δC116.3 (C-28), two oxygenated methine carbon signal at δC 71.7 (C-3) and δC 89.1 (C-24), and five methyl carbon signals at δC 21.0 (C-26), 19.3 (C-27), 18.7 (C-19), 17.5 (C-21), and 11.8 (C-18)   . The 13C8 (C-18) . Therefo24-diol)  22]..2 was is3 was isolated as a white powder and exhibited a molecular ion peak at m/z 381.3507 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the molecular formula C28H46O, This confirmed six indices of hydrogen deficiency in compound 3  spectrum exhibited one olefin methine proton signal at δH 5.33 , one olefinic methylene proton signals at δH 4.69  and 4.64 , one oxygenated methine proton signal at δH 3.49 , and five methyl proton signals at δH 0.98 , 0.97 , 0.96 , 0.94 , and 0.66   spectrum exhibited 28 carbon signals including 2 olefin quaternary carbon signals at δC 156.9 (C-24) and 140.7 (C-5), one olefin methine carbon signals at δC121.7 (C-6), one olefin methylene carbon signals at δC105.9 (C-28), one oxygenated methine carbon signal at δC 71.8 (C-3), and 5 methyl carbon signals at δC 21.9 (C-26), 21.8 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18)  . The 13C8 (C-18) . Accordilesterol  22, 23], 233 was4 was also isolated as a white powder and exhibited a molecular ion peak at m/z 395.3669 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the molecular formula C29H48O, This confirmed six indices of hydrogen deficiency in compound 4  spectrum exhibited two olefin methine proton signals at δH 5.33  and 5.16 , one oxygenated methine proton signal at δH 3.49 , one allyl methyl proton signal at δH 1.55 , and five methyl proton signals at δH 0.98 , 0.97 , 0.96 , 0.94 , and 0.66   spectrum exhibited 29 carbon signals including 2 olefin quaternary carbon signals at δC 146.9 (C-24) and 140.7 (C-5), two olefin methine carbon signals at δC121.7 (C-6) and 115.5 (C-28), one oxygenated methine carbon signal at δC 71.7 (C-3), and six methyl carbon signals at δC 13.2 (C-29), 22.2 (C-26), 22.1 (C-27), 19.4 (C-19), 18.7 (C-21), and 11.8 (C-18)   . The 13C8 (C-18) . Therefoe-3β-ol)  22, 26], 264 was5 was also isolated as a white powder and exhibited a molecular ion peak at m/z 427.3562 [M-H2O+H]+ in its positive-ion HRESITOFMS consistent with the molecular formula C29H48O3, This confirmed six indices of hydrogen deficiency in compound 5  spectrum exhibited two olefin methine proton signals at δH 5.34  and 5.75 , one olefin methylene signal at δH 5.09–5.19 , one oxygenated methine proton signal at δH 3.49 , and five methyl proton signals at δH 0.99 , 0.91 , 0.89 , 0.86 , and 0.64   spectrum exhibited 29 carbon signals including one olefin quaternary carbon signal at δC 140.7 (C-5), one olefin methylene carbon signals at δC121.6 (C-29), two olefinic methines carbon signals at δC 112.8 (C-5) and δC 142.5 (C-28), two oxygenated methine carbon signal at δC 71.7 (C-3) and δC 77.7 (C-24), and five methyl carbon signals at δC 21.0 (C-26), 19.3 (C-27), 18.7 (C-19), 17.5 (C-21), and 11.8 (C-18)  . The 13C8 (C-18) . Therefolesterol  22]..5 was alL. japonica was revealed by their COX-1 and -2 enzyme inhibitions. In this study, compounds (1–5) at 25 μg/mL inhibited COX-1 enzyme by 32, 19, 50, 59, and 26% and COX-2 enzyme by 23, 14, 33, 47, and 9%, respectively  model system, the results of which are shown in ectively . CompareMolecular docking was used to predict the interactions between the 5 sterols and the COX-1 and -2 enzyme at the molecular level. Root-Mean-Squared-Deviation (RMSD) is a similarity metric between molecular conformations, the lower the RMSD value is, the more similar the molecular conformations of ligands (before and after the docking) is. As shown in The possible mechanisms beneath the inhibition of compounds 1–5 against COX-1 and -2 was presented through molecular docking Figs , Table 1Furthermore, the binding sites and interactions between compounds 1–5 and COX-2 are represented in Figs 2 (PGG2) in their cyclooxygenase active site [COX-1 and -2 are bifunctional enzymes that convert arachidonic acid (AA) to prostaglandin Give site . Our resive site . Thus, tL. japonica afforded 5 sterols, and their structures were identified using spectroscopic and chemical evidence. Spectroscopic methods characterized these compounds as 29-Hydroperoxy-stigmasta-5,24(28)-dien-3β-ol (compound 1), saringosterol  (compound 2), 24-methylenecholesterol (compound 3), fucosterol  (compound 4), and 24-Hydroperoxy-24-vinyl-cholesterol (compound 5). These pure isolates were tested for antioxidant and anti-inflammatory activities using LPO and COX-1 and -2 enzyme inhibitory assays. Compared to the other compounds, fucosterol (compound 4) exhibited the highest COX-1 and -2 enzyme inhibitory activities at 59 and 47%, respectively. The COX enzyme inhibitory activity of 24-methylenecholesterol (compound 3) and fucosterol (compound 4) was comparable to the activity of the NSAIDs aspirin and ibuprofen. For the LPO assays, saringosterol (compound 2), 24-methylenecholesterol (compound 3) and fucosterol (compound 4) showed higher LPO inhibitory activity at >50% than the other compounds. In addition, the results of molecular docking predicted that 5 sterols were located in different pocket of COX-1 and -2 and confirmed that fucosterol with tetracyclic skeletons and olefin methine achieved the best COX-1 and -2 enzyme inhibitory activities through hydrophobic interactions and hydrogen bond. Our results confirm the presence of 5 sterols in L. japonica and its significant anti-inflammatory and antioxidant activity. Therefore, it appears that L. japonica have the potential to be developed as a dietary supplement, but molecular tests are required to verify the activity of 5 sterols in further study.Purification of hexane extracts from S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(DOCX)Click here for additional data file.S9 Fig(DOCX)Click here for additional data file.S10 Fig(DOCX)Click here for additional data file.S11 Fig(DOCX)Click here for additional data file.S12 Fig(DOCX)Click here for additional data file.S13 Fig(DOCX)Click here for additional data file.S14 Fig(DOCX)Click here for additional data file.S15 Fig(DOCX)Click here for additional data file.S16 Fig(DOCX)Click here for additional data file.S17 Fig(DOCX)Click here for additional data file.S18 Fig(DOCX)Click here for additional data file.S1 Graphical abstract(TIF)Click here for additional data file.


Daphniphyllum alkaloid, daphnioldhanol A (1), together with three known ones, were isolated from the stem part of Daphniphyllum angustifolium Hutch. Their structures were elucidated by spectroscopic methods and comparing with the literature data. Compound 2 is a new natural product, but known by synthesis as a racemate. Compound 1 exhibited week cytotoxic activity against Hela cell line with IC50 of 31.9 μM.One new The online version contains supplementary material available at 10.1007/s13659-021-00309-w. Daphniphyllum alkaloids are a structurally diversified group of complex polycyclic natural products isolated from the Daphniphyllum genus + , with 10 degrees of unsaturation. The IR absorptions implied the presence of hydroxyl (3441 cm−1), and an imine moiety (1631 cm−1). The 13C NMR and DEPT data of 1 displayed 32 carbon signals  at higher field. According to the molecular formula and relative NMR data, one CH group  was ascribed to those bearing an N-atom, while one quaternary C-atom (δC 84.4) were attributed to those bearing an O-atom. Additionally, three sp2 quaternary carbons were attributable to one lactone carbonyl (δC 180.2), one ester carbonyl (δC 172.2), and one iminium group (δC 168.4), while taking into account the three degrees of unsaturation. The remaining seven degrees of unsaturation were accounted for the presence of the heptacyclic system of 1.Daphnioldhanol A  was obta1H and 13C NMR spectra of 1 were closely related to those of the known compound Daphnioldhanine I + peak at m/z 747. By comparison of its 1H and 13C NMR data with those of (±)-nitrone 17 in the literature, high similarity between them indicated that 2 shared the same structure as the latter. However, the compound 2 is a new chiral natural product with OR at − 31.75°, but known by synthesis is racemate.The known compounds were identified as (−)-nitrone 17 (2) , daphnil2)  + ; [α]D20 =  + 9.88° ; UV (MeOH) λmax (log ε) 265 (3.43) nm, 242 (3.56) nm, 215 (3.93) nm; IR: νmax (KBr) cm–1: 3440, 2928, 2869, 1772, 1743, 1713, 1631, 1383, 1226, 1057, 1028 cm–1.Daphnioldhanol A (2): Colorless oil; C23H35NO3; ESI-MS (positive): m/z 747 [2 M + H] +; 1H NMR  δH: 3.72 , 1.58 , 1.02 , 0.94 , 0.85 ; 13C NMR  δC: 157.2 (C-1), 48.8 (C-2), 27.0 (C-3), 39.0 (C-4), 51.7 (C-5), 52.5 (C-6), 84.1 (C-7), 50.9 (C-8), 52.5 (C-9), 52.9 (C-10), 33.4 (C-11), 22.7 (C-12), 26.2 (C-13), 31.6 (C-14), 25.7 (C-15), 37.0 (C-16), 38.9 (C-17), 31.5 (C-18), 21.0 (C-19), 20.6 (C-20), 23.3 (C-21), 174.1 (C-22), 51.9 (C-23).(−)-Nitrone 17  assay [Cytotoxic activity of compound T) assay . DoxorubDaphniphyllum alkaloid, daphnioldhanol A (1), together with three known ones, were isolated from the stem part of D. angustifolium Hutch. Compound 1 exhibited week cytotoxic activity against Hela cell line.In conclusion, one new Supplementary file1 (DOCX 2262 kb)Below is the link to the electronic supplementary material.