S,8R)-guaiacylglycerol-8-O-4′-sinapyl ether 9′-O-β-D- glucopyranoside (1) and -syringylglycerol-8-O-4′-sinapyl ether 9′-O-β-D-gluco- pyranoside (2) were isolated from the leaves of Syringa velutina Kom.. Their structures were established by chemical properties and spectroscopic evidence.Two new neolignans, (7 Syringa (S.) velutina Kom. has been widely cultivated in the northern parts of China and Korea. Its leaves, flower buds, and bark have been used for many centuries in traditional Chinese medicine to treat infectious fevers, counteract inflamations, dampness and acute icteric hepatitis 23D -9.1° . The IR spectrum of 1 showed absorption bands at 3,321, 1,607, 1,500, 1,064, and 1,021 cm-1, ascribable to hydroxyl, aromatic and ether functions. The positive- and negative-ion HR-ESI-MS of 1 showed quasimolecular ion peaks at m/z 591.2062 [M+Na]+ and 567.2077 [M-H]-, which revealed the molecular formula of 1 to be C27H36O13. Acid hydrolysis of 1 with 1M HCl liberated D-glucose, which was identified by HPLC analysis using an optical rotation detector , one 1,3,4,5-tetrasubstituted aromatic ring [δ 6.73 ], which indicated a partial symmetric structure for 1, one trans-double bond , one anomeric proton [δ 4.20 ], three methoxy groups , along with other alkyl groups signals. The 13C-NMR spectrum showed 27 carbon signals, including one 1,2,4- trisubstituted aromatic ring , one 1,3,4,5-tetrasubstituted aromatic ring , two olefinic carbons , one glucose unit , three methyoxyl carbons , and four other alkyl carbon signals, the chemical shifts of which indicated the connection to an oxygen atom. The direct correlations between the proton and carbon were assigned by its HSQC spectrum  and δ 135.4 (C-4′), δ 4.20 (H-1″) and δ 68.8 (C-9′), connected the partial structures above together. Irritating δ 4.77 (H-7), δ 4.11 (H-8) showed NOE correlations in the difference Nuclear Overhauser Effect (dNOE) spectrum, which indicated that 1 possessed an erythro relative configuration. The relative structure was further confirmed by NOESY correlations between H-8/H-2, 7, 6, and H-9(δ 4.40)/H-2, 6. Enzymatic hydrolysis of 1 with cellulase gave the aglycone 1a. The erythro configuration was also confirmed by the J7, 8-value (3.8 Hz) of 1a [1 was determined to be  based on the negative cotton effects of 1 and 1a in its CD spectrum [1 as  guaiacylglycerol-8-O-4′-sinapyl ether 9′-O-β-D-glucopyranoside. Compound spectrum . Accordi2 was obtained as a white amorphous powder (MeOH) with a negative optical rotation [α]23D -9.4° . The IR spectrum of 2 showed absorption bands at 3,308, 1,647, 1,541, 1,489, 1,061, and 1,020 cm-1, ascribable to hydroxyl, aromatic and ether functions. The positive- and negative-ion HRESI-MS of 2 showed quasimolecuar ion peaks at m/z 621.2161 [M+Na]+ and 597.2182 [M-H]-, which revealed the molecular formula of 2 to be C28H38O14. Acid hydrolysis of 2 with 1 M HCl liberated D-glucose, which was identified by HPLC analysis using an optical rotation detector  was observed instead of the guaiacylglycerol unit in 1. Irridiating δ 4.81 (H-7), δ 4.15 (H-8) showed a NOE correlation in the dNOE spectrum, which indicated that 2 also possessed an erythro relative configuration. Thus, the structure was identified as  syringylglycerol- 8-O-4′-sinapyl ether 9′-O-β-D-glucopyranoside, which has the same stereostructure as 1 based on the J-value of H-7 and negative Cotton effect in its CD spectrum .Compound l = 5 cm); UV spectra, Shimadzu UV-1700 spectrometer; IR spectra, Bruker IFS-55 spectrometer; high-resolution ESI-MS, LC-MSD-Trap-SL mass spectrometer; CD detector, JASCO CD-2095-plus; 1H- and 13C-NMR spectra, Bruker AV-600 (600 MHz) spectrometer, with tetramethylsilane as an internal standard; and HPLC instrument, Shimadzu LC-9A and SPD-M6A PAD (UV-VIS)detector; HPLC column, COSMOSIL 5C18-PAQ (250×4.6 mm i.d.) and YMC-PACK ODS-a (250×10 mm i.d.) columns were used for analytical and preparative purposes, respectively. The following experimental conditions were used for chromatography: D101 macroporous resins ; ordinary-phase silica gel column chromatography, Silica gel ; TLC Silica gel GF254 ; and detection was achieved by spraying with 1 % Ce(SO4)2 -10 % aqueous H2SO4, followed by heating.The following instruments were used to obtain physical data: specific rotations, Perkin-Elmer 241MC . A voucher specimen (No. 20050517) was deposited in the Herbarium of Section of medicinal plants of Shenyang Pharmaceutical University.The leaves of 3-MeOH (100:0→50:1→30:1→10:1→0:100 v/v), and five fractions  were collected. Fraction 4 was further isolated by semi-prep. HPLC (MeOH:H2O 40:60) to give 1  and 2 , respectively. The compounds 1 and 2 were identified by [α]D, NMR, MS, CD dataThe air-dried material (5 kg) was extracted with 70% ethanol (20 L) at room temperature for two weeks. The extract was then filtered and concentrated under reduced pressure to afford a viscous mass , which was suspended in water and then subjected to D101 macroporous resin chromatography [ethanol-water (0:100→30:70→50:50→70:30→100:0 v/v)]. Five fractions were collected: the water soluble fraction , 30% ethanol-water soluble fraction , 50% ethanol-water soluble fraction , 70% ethanol-water soluble fraction  and 100% ethanol soluble fraction . A part of the 30% ethanol-water soluble fraction (300 g) was subjected to column chromatography on silica gel (2000 g). The column was eluted with CHCl-Guaiacylglycerol-8-O-4′-sinapyl ether 9′-O-β-D-glucopyranoside (1). White amorphous powder (MeOH); [α]23D -9.1° ; CD (MeOH) Δε -9.4 (236); IR (KBr) νmax cm–1 3,321, 1,607, 1,500, 1,064, 1,021; HRESI-MS m/z 591.2062 [M+Na]+ and 567.2077 [M-H]- ; 13C- (150 MHz) and 1H-NMR (600 MHz) spectral data see -Syringylglycerol-8-O-4′-sinapyl ether 9′-O-β-D-glucopyranoside (2). White amorphous powder (MeOH); [α]23D -9.4° ; CD (MeOH) Δε -9.8 (236); IR (KBr) νmax cm–1 3,308, 1,647, 1,541, 1,489, 1,061, 1,020; HRESI-MS m/z 621.2161 [M+Na]+ and 597.2182 [M-H]- ; 13C- (150 MHz), 1H-NMR (600 MHz) spectral data see 1 (4 mg) and 2 (2 mg) were refluxed with 1M HCl in 75% EtOH (2 mL) for 7 hrs, respectively. After cooling, the reaction mixture was neutralized with IRA-400 (OH- form). Then, the filtrate was extracted with EtOAc. The aqueous layer was subjected to HPLC analysis under the following conditions: HPLC column, YMC-Pack NH2 (NH12S05-2546WT), 250×4.6 mm i.d. ; detection, optical rotation [Jasco OR-2090 Chiral Detector ]; mobile phase, CH3CN-H2O ; flow rate 1.0 mL/min; column temperature: room temperature. Identification of D-glucose from 1 and 2 present in the aqueous layer was carried out by comparison of its retention time and optical rotation with those of authentic sample. tR: 11.4 min .Compound 1 (7.0 mg) and cellulase (36 mg) was incubated at 40°C for 4 d. The reaction mixture was extracted with EtOAc, and the EtOAc layer was evaporated under reduced pressure. The residue was purified by silica gel column chromatography [CHCl3-MeOH-H2O 30:3:1] to give  guaiacylglycerol-8-O-4′-sinapyl ether . White amorphous powder (MeOH), [α]23D +10.1° . CD(MeOH) Δε -4.6 (236). 1H-NMR  δ: 6.98 , 6.88 , 6.76 , 4.98 , 4.14 , 3.52 , 3.93 , 6.70 , 6.58 , 6.31 , 4.38 .An aqueous solution (8.0 ml) containing


Among them, compounds 15 and 16 demonstrated anticancer activity for the breast cancer cell line T47D, whereas compounds 6 and 10 which are thiosemicarbazide derivatives displayed anti-tumourigenic activity against the PC-3 cell line, consistent with the literature. However, no activity was observed for the HCT-116 cancer cell line with the tested thiosemicarbazide derivatives. Only compound 16 displayed activity against the HCT-116 cell line. Therefore, it was speculated that the diflunisal and thiosemicarbazide functionalities potentiate anticancer activity on prostate cancer and the thiosemicarbazide functionality decreases the anticancer activity of diflunisal on colon cancer cell lines. In order to gain insight into the anticancer activity and COX-2 inhibition, molecular docking studies were carried out for COX-1 and COX-2 enzymes utilizing the newly synthesized compounds 15, and 16. Both 15 and 16 showed high selectivity and affinity toward COX-2 isozyme over COX-1, which is in agreement with the experimental results.Cyclooxygenase enzymes play a vital role in inflammatory pathways in the human body. Apart from their relation with inflammation, the additional involvement of COX-2 enzyme with cancer activity was recently discovered. In some cancer types the level of COX-2 enzyme is increased indicating that this enzyme could be a suitable target for cancer therapy. Based on these findings, we have synthesized some new diflunisal thiosemicarbazides and 1,2,4-triazoles and tested them against androgen-independent prostate adenocarcinoma (PC-3), colon carcinoma (HCT-116), human breast cancer (T47D), breast carcinoma (MCF7) and human embryonic kidney (HEK-293) cell lines. Specifically, the diflunisal and thiosemicarbazide functionality are combined during the synthesis of original compounds anticipating a potency enhancement. Compounds  Cyclooxygenase (COX) enzymes play an important role in the synthesis of prostaglandins. So far, three isoenzymes of COX have been discovered; COX-1, COX-2 and COX-3. The constitutive COX-1 isoenzyme which can be found throughout the body, where it is responsible for the protection of the gastrointestinal track, whereas COX-2 is an inducible isoenzyme responsible for inflammation . The impDiflunisal , a salicylic acid derivative, is known as a NSAID. It is a selective inhibitor of COX-2 isoenzyme over COX-1 . DifluniSome of the known anti-cancer agents are not very effective, as many of them have inadequate ADMET properties and easily induce resistance. There is also a considerable diversity in resistance of various cells within the same tumor. Thus, development of safe and effective new agents with less side effects and reduced drug resistance for long-term treatment of cancer is highly required . Difluni1H-NMR and 13C-NMR. Their purity was proven by TLC, HPLC and elemental analysis. We have also investigated their anticancer activity on PC-3, HCT-116, T47D and MCF-7 cancer cell lines using cell proliferation assays. Among the tested compounds; compounds 6, 10, 15 and 16 showed anticancer activity against prostate adenocarcinoma (PC-3), colon carcinoma (HCT-116), and human breast cancer (T47D) cancer cell lines. None of the tested compounds were found active against the MCF-7 cell line. In order to correlate the anticancer activity and selectivity with COX-2 inhibition, molecular docking studies were performed utilizing both COX-1 and COX-2 as target enzymes with the newly synthesized compounds. These in silico studies, suggest that compounds 15 and 16 show high selectivity toward COX-2 isoenzyme over COX-1, agreeing with the experimental results.In this study, we hypothesized that the conversion of carboxylic acid group of diflunisal into thiosemicarbazides and 1,2,4-triazole-3-thiones would enhance the selectivity of the drug towards cancer cells. Accordingly, we have some synthesized new thiosemicarbazides and 1,2,4-triazole-3-thiones of diflunisal and elucidated their structures by FT-IR, 1) and diflunisal hydrazide (2) according to a reported procedure +. Beside the main fragment, OH radical and halogen losses were detected in both compounds.Compounds ly study . The str11–16, ring closures were carried out with 2 N NaOH for 4 h using the reported method +, [M-OH]+ and [M-RNCS]+ peaks , colon carcinoma (HCT-116), human breast cancer (T47D), breast carcinoma (MCF7) and a normal human embryonic kidney cell line (HEK293) were used to evaluate the anticancer activity of newly synthesized compounds.A number of studies have reported the association between COX-2 overexpression and cancer progression. Expression of the COX-2 isoenzyme is upregulated in many human cancers, including gastric, breast, lung, colon, esophageal, prostate and hepatocellular carcinomas . It has 50) values of all the synthesized compounds were determined for all the cell lines used in this study by a MTS assay. Compounds for which the IC50 value against HEK293 cell line was twice as high as the IC50 value for at least one cancer cell line  were considered active , however, no activity was observed for the HCT-116 cancer cell line with the thiosemicarbazide derivatives. Therefore, we assumed that diflunisal and thiosemicarbazide functionality enhance anti-prostate cancer activity but the thiosemicarbazide functionality decreases the activity of diflunisal on colon cancer cell lines hydrazine carbothioamide (6): White solid. Yield 83%; m.p. 199 °C; MW: 478.309 g/mol; Rf × 100 value: 21; Rt value: 4.82 min. FT-IR ν max. (cm−1): 3244 (N-H), 1626 (Amide C=O), 1274 (C=S), 1141 (Ar-F). 1H-NMR (DMSO-d6/TMS) δ ppm: 7.09 , 7.22 , 7.24–7.40 , 7.48–7.74 , 8.06 , 9.94 , 10.02 , 10.82 , 12.01 . 13C-NMR (DMSO-d6/TMS) δ ppm: 104.45 (C-9), 111.98 (C-11), 115.37 (C-5), 117.46 (C-3), 120.43 , 123.97 (C-7), 125.03 (C-1), 129.95 (C-13), 129.18 , 131.58 (C-12), 127.57 (C-2), 134.28 (C-6), 140.74 (C-15), 159.00 (C-4), 157.32–160.62 (C-8), 159.82–163.09 (C-10), 169.00 (C=O), 180.00 (C=S). HR-MS : 478.9937 (M++2), C20H14BrF2N3O2S 476.9952/476.9952 (M+), 383.0898/383.0902, 306.0295/306.0296, 264.0704/264.0700233.0403/233.0408. Anal. Cald. C20H14BrF2N3O2S Calculated/Found (%): C:50.22/50.22 H:2.95/3.27 N:8.79/8.77 S:6.70/6.60.′,4′-difluoro-4-hydroxybiphenyl-3-yl)carbonyl]hydrazine carbothioamideN-(4-Bromophenyl)-2-hydrazinyl}carbonothioyl)benzamide 8): White solid. Yield 91%; m.p. 233 °C; MW: 427.424 g/mol; Rf × 100 value: 16; Rt value: 4.90 min. FT-IR ν max. (cm−1): 3254 (N-H), 1676 (Benzoyl C=O) 1651 (Amide C=O), 1263 (C=S), 1139 (Ar-F). 1H-NMR (DMSO-d6/TMS) δ ppm: 7.13 , 7.19 , 7.35 , 7.52–7.69 , 7.98 , 8.12 , 11.96 , 12.03 , 13.71 . 13C-NMR (DMSO-d6/TMS) δ ppm: 104.48 (C-9), 112.08 (C-11), 115.74 (C-5), 117.32 (C-3), 123.82 (C-7), 125.71 (C-1), 128.50 (C-2), 129.69 (C-16), 130.15 , 130.39 (C-13), 131.48 (C-12), 131.73 , 134.20 (C-6), 140.74 (C-15), 159.75 (C-4), 157.32–160.62 (C-8), 159.96–163.09 (C-10), 168.45 (C=O), 171.50 (Benzoyl C=O), 180.00 (C=S). Anal. Cald. C21H15F2N3O3S Calculated/Found (%): C:59.01/58.38 H:3.54/3.98 N:9.83/9.61 S:7.50/7.27.2-[carbonyl]-N-(2-methoxyphenyl)hydrazine carbothioamide (9): White solid. Yield 82%; m.p. 195 °C; MW: 502.532 g/mol; Rf × 100 value: 68; Rt value: 4.48 min. FT-IR ν max. (cm−1): 3198 (N-H), 1645 (DMF C=O), 1633 (Amide C=O), 1240 (C=S), 1138 (Ar-F). 1H-NMR) (DMSO-d6/TMS) δ ppm: 2.73 , 2.88  methyl protons), 3.39 (water in solvent and O-CH3 protons), 6.95 , 7.08 , 7.16 , 7.21 , 7.36 , 7.57–7.69 , 7.95 , 8.07 , 9.36 , 9.84 , 10.85 , 11.44 , 11.99 . 13C-NMR (DMSO-d6/TMS) δ ppm: 55.63 (O-CH3), 104.38 (C-9), 111.44 (C-16), 112.00 (C-11), 115.83 (C-5), 117.32 (C-3), 119.79 (C-14), 123.94 (C-7), 125.17 (C-1), 126.12–126.72 , 127.51 (C-13), 131.55 (C-12), 129.42 (C-2), 134.01 (C-6), 159.50 (C-4), 157.31–160.58 (C-8), 159.78–162.06 (C-10), 166.20 (C=O), 180.00 (C=S). HR-MS : C21H17F2N3O3S 429.0953/429.0953, C21H18FN3O2S 395.1098/395.1098, C13H10F2N2O2 264.0704/264.0704, C13H7F2O2 233.0403/233.0408 Anal. Cald. C21H17F2N3O3S.DMF Calculated/Found (%): C:57.36/56.66 H:4.81/4.77 N:11.15/10.95 S:6.38/6.57.2-[carbonyl]-N-(4-ethylphenyl)hydrazine carbothioamide (10): White solid. Yield 89%; m.p. 185–186 °C; MW: 473.5454 g/mol; Rf × 100 value: 38; Rt value: 4.84 min. FT-IR ν max. (cm−1): 3234 (N-H), 1635 (Amide C=O), 1263 (C=S), 1139 (Ar-F). 1H-NMR) (DMSO-d6/TMS) δ ppm: 1.05 , 1.17 , 2.57 , 3.40 (water in solvent and Ethanol CH2), 4.39 , 7.06 , 7.15–7.25 , 7.32–7.40 , 7.55–7.64 , 8.06 , 9.82 , 10.85 , 12.04 . 13C-NMR) (DMSO-d6/TMS) δ ppm: 15.55–27.61 (CH2CH3) 104.38 (C-9), 111.93 (C-11), 115.59 (C-5), 117.38 (C-3), 123.96 (C-7), 125.04 (C-1), 127.44 , 128.03 , 129.19 (C-13), 131.56 (C-12), 134.14 (C-2), 136.55 (C-6), 139.17 (C-16), 158.50 (C-4), 157.32–160.60 (C-8), 159.81–163.07 (C-10), 168.00 (C=O), 180.00 (C=S). Anal. Cald. C22H19F2N3O2S. C2H5OH Calculated/Found (%): C:60.81/60.24 H:5.27/5.23 N:8.86/8.91 S:6.75/6.87.3–9 except for compound 8 and 10 (0.01 mol) were dissolved in sodium hydroxide solution  and heated under reflux for 4 to 6 h. The solution then was neutralized with concentrated hydrochloric acid. The crude product was precipitated, filtered and washed with distilled water. The compounds recrystallized twice from ethanol or DMF [Compounds l or DMF .5--4-propyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (11): White solid. Yield 69%; m.p. 177–179 °C; MW: 347.3823 g/mol; Rf × 100 value: 35; Rt value: 3.81 min. FT-IR ν max. (cm−1): 3190 (N-H), 1626 (triazole C=N), 1257 (C=S), 1141 (Ar-F). 1H-NMR (DMSO-d6/TMS) δ ppm: 0.66 , 1.54 , 3.84 , 7.12 , 7.17 , 7.32 , 7.47–7.61 , 13.76 . AR-OH exchanged with deuterium of solvent. Anal. Cald. C17H15F2N3OS Calculated/Found (%): C:58.78/58.28 H:4.35/4.50 N:12.10/11.99 S:9.23/9.56.5--4-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (12): White solid. Yield 98%; m.p. 244 °C; MW: 399.3889 g/mol; Rf × 100 value: 37; Rt value: 3.81 min. FT-IR ν max. (cm−1): 3250 (N-H), 1633 (triazole C=N), 1255 (C=S), 1139 (Ar-F). 1H-NMR (DMSO-d6/TMS) δ ppm: 6.84 , 7.12–7.18 , 7.19–7.31 , 7.32–7.53 , 10.32 , 14.14 . 13C-NMR) (DMSO-d6/TMS) δ ppm: 104.43 (C-9), 111.94 (C-11), 113.27 (C-5), 115.46 (C-17) 115.75 (C-16), 115.99 (C-3), 123.77 (C-7), 124.20 (C-14), 124.75 (C-1), 130.17 (C-13), 132.54 (C-2), 131.36 (C-12), 131.73 (C-18), 135.77 (C-6), 149.20 (C-4), 155.62 (C=N), 157.22–160.57 (C-8), 159.71–162.97 (C-10), 159.62 (C-15), 167.68 (C=S). Anal. Cald. C20H12F3N3OS Calculated/Found (%): C:60.15/59.85 H:3.03/3.27 N:10.52/10.46 S:8.03/8.16.4-(3-Chlorophenyl)-5--2,4-dihydro-3H-1,2,4-triazole-3-thione (13): White solid. Yield 95%; m.p. 243–244 °C; MW: 415.8435 g/mol; Rf × 100 value: 36; Rt value: 3.94 min. FT-IR ν max.(cm−1): 3188 (N-H), 1620 (triazole C=N); 1269 (C=S), 1139 (Ar-F). 1H-NMR (300 MHz) (DMSO-d6/TMS) δ ppm: 6.84 , 7.15 , 7.24–7.31 , 7.34 , 7.35 , 7.39–7.56 , 7.57 , 10.34 , 14.16 . 13C-NMR (DMSO-d6/TMS) δ ppm: 104.44 (C-9), 111.93 (C-11), 113.22 (C-5), 115.99 (C-3), 123.77 (C-7), 124.80 (C-1), 126.66–128.14–128.89 , 130.18 (C-13), 132.45 (C-2), 131.38 (C-12), 131.81 (C-18), 132.65 (C-15), 135.62 (C-6), 149.19 (C-4), 155.54 (C=N), 157.24–160.53 (C-8), 159.71–162.98 (C-10), 167.63 (C=S). Anal. Cald. C20H12ClF2N3OS Calculated/Found (%): C:57.77/57.54 H:2.91/3.25 N:10.10/10.13 S:7.71/8.03.4-(3-Bromophenyl)-5--2,4-dihydro-3H-1,2,4-triazole-3-thione (14): White solid. Yield 84%; m.p. 232–233 °C; MW: 460.2945 g/mol; Rf × 100 value: 36; Rt value: 4.18 min. FT-IR ν max.(cm−1): 3201 (N-H), 1620 (triazole C=N), 1240 (C=S), 1139 (Ar-F). 1H-NMR (300 MHz) (DMSO-d6/TMS) δ ppm: 6.85 , 7.14 , 7.28–7.59 , 10.42 , 14.12 . 13C-NMR (DMSO-d6/TMS) δ ppm: 104.43 (C-9), 111.93 (C-11), 113.19 (C-5), 116.05 (C-3), 123.81 (C-7), 124.69 (C-1), 127.09 , 130.44 (C-13), 130.96 , 131.36 (C-12), 131.68 (C-15), 132.46 (C-2), 135.91 (C-6), 149.21 (C-4), 155.69 (C=N), 157.22–160.52 (C-8), 159.69–162.95 (C-10), 167.59 (C=S). HR-MS : 460.9822 (M+2), C20H12BrF2N3OS 458.9846/458.9847 (M+), C20H12BrF2N3O 427.0126/427.0126, 347.0853/347.0856, 231.0490/231.0492. Anal. Cald. C20H12BrF2N3OS Calculated/Found (%): C:52.19/51.31 H:2.63/3.01 N:9.13/9.06 S:6.97/6.62.4-(4-Bromophenyl)-5--2,4-dihydro-3H-1,2,4-triazole-3-thione (15): White solid. Yield 69%; m.p. 263–264 °C; MW: 460.294 g/mol; Rf × 100 value: 60; Rt value: 4.38 min. FT-IR ν max. (cm−1): 3196 (N-H), 1622 (triazole C=N), 1242 (C=S), 1139 (Ar-F). 1H-NMR (DMSO-d6/TMS) δ ppm: 6.84 , 7.14 , 7.28 , 7.29–7.35 , 7.43–7.60 , 7.61 , 10.27 , 14.13 . 13C-NMR (DMSO-d6/TMS) δ ppm: 104.45 (C-9), 111.90 (C-11), 113.30 (C-5), 116.01 (C-3), 121.95 , 123.75 (C-7), 124.82 (C-1), 130.02 , 131.40 (C-12), 131.57 (C-13), 131.80 (C-16), 132.62 (C-2), 133.76 (C-6), 149.25 (C-4), 155.47 (C=N), 157.24–160.53 (C-8), 159.70–162.97 (C-10), 167.58 (C=S). Anal. Cald. C20H12BrF2N3OS Calculated/Found (%): C:52.19/52.27 H:2.63/2.91 N:9.13/8.80 S:6.97/6.77.5--4-(2-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (16): White solid. Yield 65%; m.p. 196 °C; MW: 457.493 g/mol; Rf × 100 value: 60; Rt value: 3.90 min. FT-IR ν max. (cm−1): 3198 (N-H), 1626 (triazole C=N), 1257 (C=S), 1141 (Ar-F). 1H-NMR (DMSO-d6/TMS) δ ppm: 1.05 (ethanol CH3), 3.43 (Ethanol CH2), 3.55 , 3.90 (ethanol OH), 6.85 , 6.80–7.04 , 7.13 , 7.25–7.39 , 10.31 , 14.02 . 13C-NMR (DMSO-d6/TMS) δ ppm: 55.41 (O-CH3), 104.42 (C-9), 111.93 (C-11), 112.28 (C-15), 116.02 (C-5), 120.14 (C-17) 122.68 , 123.80 (C-7), 124.39 (C-1), 130.34 (C-13), 130.80 (C-2), 131.23 (C-12), 132.14 (C-6), 133.50 (C-3), 149.89 (C-4), 154.21 (C-14), 155.75 (C=N), 157.19–160.48 (C-8), 159.64–162.90 (C-10), 168.19 (C=S). HR-MS : C21H15F2N3O2S 411.0847/411.0847 (M+), C21H14F2N3O2 378.1043/378.1048, 231.0490/231.0478. Anal. Cald. C21H15F2N3O2S. C2H5OH Calculated/Found (%): C:60.32/60.06 H:4.59/4.05 N:9.18/9.63 S:6.99/7.26.® CCL-247™), androgen-dependent prostate cancer cell line PC-3 (ATCC® CRL-1435™), breast cancer cell lines T47D (ATCC® HTB-133™), MCF7 (ATCC® HTB-22™) and finally, epithelial embryonic kidney cell line HEK293 (ATCC® CRL-1573™) were used. All media were completed with %10 fetal bovine serum (FBS) and %1 penicillin streptomycin amphotericin (PSA). All cells were cultured periodically at 37 °C, 5% (v/v) CO2 and 95% (v/v) air in T-75 flasks. Different media were used for the different cell lines based on ATCC standards: MCF7 and HEK293 cell lines were cultured in DMEM High Mmedium ; T47D and PC-3 cell lines were cultured in RPM 1640 medium ; HCT116 cell line was cultured in DMEM F12 medium . RPM 1640 medium was further completed with 0.2 unit/mL bovine insulin for the T47D cell line. Cells were passaged when they reached %80 confluency in order to support proliferation.In this project, the colorectal cancer cell line HCT116 (ATCCH-tetrazolium (MTS) is a yellow tetrazolium salt which is catabolized to formazan by dehydrogenase enzyme in the mitochondria of the living cells. Formazan is a purple compound and the detection in this assay is based on the measurement of formazan compounds by an ELISA plate reader. After incubating the cells for 72 h, 10 µL MTS reagent and 100 µL designated medium was given to the cells followed by 2 h of incubation at 37 °C. Thereafter, the absorbance at 490 nm was measured by an ELISA plate reader. . The activity doses of the compounds were determined according to 50% cell death rate when compared to DMSO, only after 72 h of incubation. Compounds that do not affect cell viability of healthy cell line (HEK293) but deathly to cancer cell lines were further tested with narrower test conditions for MTS assay to determine IC50 values. IC50 values are calculated by the GraphPad prism software.The effects of the synthesized compounds on the cell viability were tested. Stock solutions for each compound were prepared by measuring 20 mg of each compound and dissolving it in 1 mL DMSO to a final concentration of 20 mg/10 mL, followed by filtration through a 0.2-μm filter . Then stock solutions were diluted as 1/10, 1/100 and 1/1000 in the designated media and applied on the cell lines. In order to determine the effect of DMSO itself, the same dilutions were performed by using DMSO, only and applied on the cell lines. Cell densities used for each cell line in 96 well plate were as follows: for PC-3, HCT116 and HEK293 cell lines 5000 cells/well; for T47D cell lines 20,000 cells/well. Cell viability was measured by the MTS cytotoxicity assay  according to the manufacturer’s instructions. 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-250 values was followed by the investigation of effect of the synthesized compounds on apoptosis. Cells were seeded into six well plates at densities of 15,000 cells/well  and 60,000 cells/well (T47D cell lines). After the treatment cells were harvested and washed with ice cold PBS. 1 × Annexin-binding buffer was prepared while the cells were being washed. Working solution of PI (100 µg/mL) was prepared as well by diluting 5 µL of the 1 mg/mL PI stock solution in 45 µL 1 × Annexin-binding buffer. After the precipitation of the cells, they were resuspended in 500 µL 1 × Annexin-binding buffer. Resuspended cells were separated into four groups  each containing 100 µL of suspension. Five µL of the Annexin V conjugate was added to the proper groups. Samples were incubated for 15 min at room temperature. The incubation step was followed by adding 2 µL of the 100 µg/mL PI working solution to the proper groups then the fluorescence was observed by flow cytometry.Determination of IC® X-100 in PBS for 20 min at room temperature. Click-iT® reaction cocktail (0.5 mL) was added to the cells after washing with 3% BSA solution. Cells were incubated for 30 min at room temperature, then DNA synthesis was observed under a confocal microscope.The EdU staining protocol directly measures the level of active DNA synthesis of the cell cycle. This measurement enables one to see dividing cells under a fluorescence microscope. In order to perform EdU staining cells were seeded on the coverslips which were located in the wells of six well plates. As in the Annexin V procedure cells were seeded into six well plates at densities of 15,000 cells/well  and 60,000 cells/well (T47D cell lines). After drug treatment, cells that are on the coverslips were incubated with 10 µM EdU. The incubation step is followed by cell fixation and permeabilization. Fixation occurred with 3.7% formaldehyde in PBS for 15 min then cells were washed with 3% BSA in PBS. Permeabilization was performed by incubating cells in 0.5% Tritonhttp://www.rcsb.org/pdb/). All protein structures were cleaned of inhibitors, water molecules and non-interacting ions, while cofactors were retained. All hydrogens were added and proteins minimized using the “Clean Geometry” toolkit. For more complete optimization, proteins were submitted to the “Prepare Macromolecule” protocol. Missing hydrogen atoms were added based on the protonation state of the titratable residues at pH 7.4. Compounds were prepared and minimized in the same force field. After preparation, docking was performed in AutoDock 4.2 program (http://autodock.scripps.edu). All the compounds were set to be flexible, while the proteins were kept rigid. AutoDock adds a free-energy scoring function created from a linear regression analysis, the AMBER force field, and a large set of diverse protein-ligand complexes with known inhibition constants. The coordinates of alpha carbon of Tyr355 were used for grid-centering for COX enzymes, grid box was set to be 70 grid points (each grid point is 0.375 Å). For the docking studies the Lamarkian genetic algorithm was used as search algorithm. For visualization of non-bonded interactions, we also used BIOVIA Discovery Studio 4.5.The preparation of the protein, as well as the compounds were performed in BIOVIA Discovery Studio 4.5. The crystal structures of the enzymes COX-1 and COX-2 were downloaded from the PDB database  and 16  showed high inhibition on COX-2 enzyme. The highest selectivity towards COX-2 was shown by compound 13. Further experiments are still needed in order to improve our understanding of how these compounds elicit their anticancer activity.In this study, we have synthesized new diflunisal thiosemicarbazides and 1,2,4-triazole-3-thiones in order to determine their possible anticancer activity. The synthesis, structure characterization and antibacterial activity against ly study . Compoun


H-thiadiazolopyrimidine-6-carbonitrile derivatives. The in-vitro anticancer activities of these compounds were evaluated against four human tumor cell lines. Among all the synthesized derivatives, compound 4i, which has substituent 3-hydroxy-4-methoxyphenyl is found to have the highest GI50 value of 32.7 μM, 55.3 μM, 34.3 μM, 28.9 μM for MCF-7, K562, HeLa and PC-3 cancer cell lines respectively. A docking study of the newly synthesized compounds were performed, and the results showed good binding mode in the active site of thymidylate synthase enzyme. ADME properties of synthesized compounds were also studied and showed good drug like properties.Herein, we report an environmentally friendly, rapid, and convenient one-pot ultrasound-promoted synthesis of 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7 Cancer is a disease in which cells grow and proliferate in an uncontrolled manner. Cancer disease evokes a high level of mortality regardless of recent advances in the development of clinically authorized anticancer agents . On the H-thiadiazolopyrimidine-6-carbonitrile derivatives.Folate metabolism is considered as an important target for the development of new anticancer agents due to its role in the biosynthesis of nucleic acid precursors ,6. The iPyrimidine and its derivatives have been recognized as important heterocyclic compounds due to their variety of chemical and biological significance to medicinal chemistry ,13,14,15The conventional multistep methods for the preparation of complex molecules involve large synthetic operations, including extraction and purification processes for each individual step, that lead to synthetic inefficiency and the generation of large amounts of waste. Therefore, designing multicomponent reactions (MCRs) in one pot and the creation of several bonds in a single operation are the major challenges for modern organic chemistry.The synthesis of heterocycles using toxic and hazardous chemicals which cause pollution has given birth to “Green Chemistry,” coined by Paul Anastas in 1998 . UltrasoH-1,3,4-thiadiazolopyrimidine by two steps involving the addition of 2-aminothiadiazole derivatives to ethyl acetoacetate, tandem hydrolysis of the ester to the acid, and cyclization to give the ring-fused thiadiazolopyrimidines in PPA. Most of these are multistep protocols, which suffer from generation of by-products, low yields, and use of metal-containing reagents. Therefore, it is quite significant to develop the direct, efficient, and green alternative approaches to get the functionalized thiadiazolopyrimidine derivatives from viewpoint of green chemistry. Herein we reported a new, simple protocol for an environment friendly, rapid and convenient synthesis, and antitumor activity against MCF-7, K562, HeLa, PC-3 cancer cell lines, of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-thiadiazolopyrimidine-6-carbonitrile derivatives obtained in excellent yield through a one-pot three component condensation reaction of 5-(4-chlorophenyl)-1,3,4-thiadiazol-2 amine, aromatic aldehyde and malononitrile using sodium hydroxide as catalyst in ultrasound. Moreover, docking studies using thymidylate synthase (TS) enzyme are presented in this paper as well.The existing synthetic methodologies for thiadiazolopyrimidine nucleus in a modular fashion are not straightforward and the synthetic routes involve multiple steps. For example, 1,3,4-thiadiazolopyrimidine-7-sulfonamide derivatives were synthesized from 5-aminol-3,4-thiadiazole-2-sulfonamide via a two steps approach . SalimovH-thiadiazolopyrimidine-6-carbonitrile derivatives from three component reactions of an 5-(4-chlorophenyl)-1,3,4-thiadiazol-2 amine (1), aromatic aldehydes (3) and malononitrile (2) in presence of NaOH under reflux and ultrasonic irradiation as shown in 1), suitable aldehyde (3), malononitrile (2) were carried out using different solvents in the presence of NaOH as a catalyst at different mole percentage as shown in 2O was chosen as solvent and instead acetonitrile, methanol and dimethylformaide was chosen as solvent. The desired product was formed in low yield under reflux and ultrasonic irradiation as shown in 1H-NMR, 13C-NMR, mass spectroscopy and IR.Herein we report the one-pot synthesis of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-74a–j) were evaluated for their anticancer activity against MCF-7, K562, HeLa and PC-3 cancer cell lines. The GI50 values (concentration required to Growth inhibition of 50%) for the synthesized compounds were determined using SRB assays method. The anticancer evaluation results and GI50 values were listed in The target compounds , chloro  exhibited less activity compared to electron donating, polar groups. The anticancer activity for derivatives bearing an electron withdrawing group such as chloro group at ortho position (4b)  exhibited more activity than compound 4c (chloro group at meta position)  and compound 4a (chloro group at para position) . Replacement of the phenyl group in the parent compound by furan ring in 4j has shown decreased activity in comparison to the standard drug 5-FU . From SAR it can be considered that compounds containing electron donating, polar groups such as 4e, 4f, 4g, 4i have good anticancer activity in comparison to electron withdrawing groups such as 4a, 4b, 4c, 4d. It is also clear that the replacement of the phenyl ring with the furan ring decreases anticancer activity.Structural activity relationship (SAR) studies for these compounds demonstrated that electron withdrawing groups such as fluoro , 4c (−5.24), 4d (−5.14), 4f (−5.14), 4h (−5.38) and 4j (−4.98), all showed G-Score greater than 5-FU (−4.75).5-Fluorouracil derivatives and their structurally related compounds like pemetrexed, Capecitibine, Raltitrexed are well known to inhibit thymidylate synthase. We decided to carry out a molecular docking study on our newly synthesized 1,3,4-thiadiazolopyrimidine skeleton derivatives into the binding site of thymidylate synthase (PDB ID: 1JU6) using 5-FU as a reference for docking results. The docking results indicated that compounds were held in the active pocket by combination of various hydrogen and hydrophobic interactions with TS. The docking results revealed that the highest binding compound to TS was 4g, 4h and 4i had potential to inhibit thymidylate synthase. G-Score of all the synthesized derivatives is as shown in On the basis of the anticancer activity and docking results, it was found that compounds The prediction of the ADME parameters prior to the experimental studies is one of the most important aspects of drug discovery and development of the drug molecule. ADME studies have always played a critical role in helping to optimize the pharmacokinetic properties of new drugs, thereby increasing their success rate. The analysis of Lipinski’s rule of five was performed to indicate whether a chemical compound could be an orally active drug in humans. It was observed that the compounds exhibited a good % absorption (% ABS) ranging from 87.50% to 100% .4a–j had good absorption and were found to be nontoxic.The results of the prediction of ADME properties are depicted in 1H-NMR and 13C-NMR spectra of synthesized compounds were recorded on Bruker Avance II 400 NMR Spectrometer  at 400 MHz Frequency in deuterated DMSO and CDCl3 and using TMS as internal standard . Mass spectra of some compounds were scanned on FTMS + p ESI full mass (100.00–1500.00).All the reactions were performed in oven-dried glasswares. All reagents and solvents were used as obtained from the supplier or recrystallized/redistilled unless otherwise noted. The ultrasound sonicator  equipped with solid synthetic probe, 13 mm in tip diameter, operating at 20 kHz with a maximum power output of 500 W, was used for synthesis of final title compounds. The purity of the synthesized compounds was monitored by ascending thin layer chromatography (TLC) on silica gel-G  coated aluminum plates, visualized by iodine vapor and melting points were determined in open capillary tubes. Infrared (IR) spectra were recorded on a PS 4000 FTIR  using KBr pellets. Elemental analyses  were done with a FLASHEA 112 Shimadzu’ analyzer  and all analyses were consistent (within 0.4%) with theoretical values. The 1), malononitrile (0.01 mol) (2), suitable aldehyde (0.01 mol) (3) in ethanol (10–12 mL) and the catalyst NaOH (20% mmol) and the reaction mixture was refluxed . After completion of the reaction (monitored by TLC), the mixture was poured into ice cold water. The product obtained, was filtered and dried. The corresponding product was obtained in high purity after recrystallization of the crude product from ethanol. The authenticity of compounds was established by 1H-NMR, 13C-NMR, IR and HRMS.Method A: A 25 mL round bottom flask was charged with a mixture of an 5-(4-chlorophenyl)-1,3,4-thiadiazol-2 amine (0.01 mol) (1), malononitrile (0.01 mol) (2), suitable aldehyde (0.01 mol) (3) in ethanol (10–12 mL) and the catalyst NaOH (20% mmol) and the reaction mixture was kept inside an Ultrasonicator acoustic chamber at 80 °C at 20%. After completion of the reaction (monitored by TLC), the mixture was poured into ice cold water. The product obtained, was filtered and dried. The corresponding product was obtained in high purity after recrystallization of the crude product from ethanol. The authenticity of compounds was established by 1H-NMR, 13C-NMR, IR and HRMS.Method B: A 25 mL a beaker was charged with a mixture of an 5-(4-chlorophenyl)-1,3,4-thiadiazol-2 amine (0.01 mol) -7H-thiadiazolopyrimidine-6-carbonitrile4a. M.P: 237–240 °C. Rf value: 0.28. IR (KBr) υmax cm−1: 3400 (C-NH2), 3100 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic rings), 1H-NMR δ ppm: 10.00 , 8.00–7.02 , 3.35 , 13C-NMR δ: 172.11 (C), 158.31 (C), 143.21 (C), 139.50 (C), 136.51 (C), 131.00 (C), 130.52 (CH), 129.87 (CH), 128.53 (CH), 128.23 (C), 127.93 (CH), 118.27 (C), 60.22 (C), 54.53 (C), m/z 399 (100.0%), 401.01 (68.9%), 400.01 (22.7%) Molecular Formula: C18H11Cl2N5S. Elemental Analysis: Calculated:  54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.04, 2.74, 17.68, 17.51, 8.00.5-Amino-7-(2-chlorophenyl)-2-(4-chlorophenyl)7H-thiadiazolopyrimidine-6-carbonitrile4b. M.P: 240–242 °C. Rf value: 0.24. IR (KBr) υmax cm−1: 3400 (C-NH2), 3000 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic rings). 1H-NMR δ ppm: 9.98 , 8.00–7.09 , 3.35 , 13C-NMR δ ppm: 170.24 (C), 158.37 (C), 143.77 (C), 138.10 (C), 134.26 (C), 129.52 (CH), 129.00 (CH), 128.51 (CH), 128.03 (C), 127.99 (CH), 126.11 (CH), 117.59 (C), 60.00 (C), 55.02 (C), m/z 399 (100.0%), 401.01 (68.9%), 400.01 (22.7%) Molecular Formula: C18H11Cl2N5S. Elemental Analysis: Calculated:  54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.02, 2.76, 17.70, 17.52, 8.02.5-Amino-7-(3-chlorophenyl)-2-(4-chlorophenyl)7H-thiadiazolopyrimidine-6-carbonitrile4c. M.P: 235–238 °C. Rf value: 0.20. IR (KBr) υmax cm−1: 3400 (C-NH2), 3100 (Aromatic C-H stretching), 162.3 (C=N), 740.55 (C-Cl of aromatic rings), 1H-NMR δ ppm: 10.01 , 8.00–7.05 , 3.32 , 13C-NMR δ ppm: 172.21 (C), 158.20 (C), 155.00 (C), 142.50 (C), 137.69 (C), 136.17 (C), 134.09 (C), 130.03 (CH), 129.99 (CH), 129.02 (CH), 128.93 (CH), 128.37 (CH), 125.59 (CH), 123.95 (CH), 119.11 (CH), 117.57 (C), 59.97 (C), 52.00 (C), m/z 399 (100.0%), 401.01 (68.9%), 400.01 (22.7%), Molecular Formula: C18H11Cl2N5S. Elemental Analysis: Calculated:  54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.01, 2.77, 17.67, 17.49, 8.03.5-Amino-2-(4-chlorophenyl)-7-(4-fluorophenyl)-7H-thiadiazolopyrimidine-6-carbonitrile4d. M.P: 239–240 °C. Rf value: 0.37. IR (KBr) υmax cm−1: 3400 (C-NH2), 3000 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic ring), 1053 (C-F of aromatic rings), 1H-NMR δ ppm: 9.79 , 8.02–7.06 , 3.33 , 13C-NMR δ ppm: 172.22 (C), 159.07 (C), 158.31 (C), 143.77 (C), 136.62 (C), 136.99 (C), 130.67 (CH), 129.52 (CH), 128.99 (C), 128.77 (CH), 117.54 (C), 115.34 (CH), 60.07 (C), 52.97 (C), m/z 383.04 (100%), 385.04 (37%), 384.04 (22.7%), Molecular Formula: C18H11ClFN5S. Elemental Analysis: Calculated:  56.33, 2.89, 9.24, 4.95, 18.25, 8.35 Found: 56.37, 2.90, 9.20, 4.91, 18.23, 8.36.5-Amino-2-(4-chlorophenyl)-7-(4-methoxyphenyl)-7H-thiadiazolopyrimidine-6-carbonitrile4e. M.P: 210–212 °C. Rf value: 0.36. IR (KBr) υmax cm−1: 3400 (C-NH2); 3000 (Aromatic C-H stretching), 1623 (C=N), 1055 (C-OCH3), 740.55 (C-Cl of aromatic ring), 1H-NMR δ ppm: 9.99 , 8.02–6.79 , 3.36 , 3.56 , 13C-NMR δ ppm: 172.26 (C), 158.99 (C), 157.51 (C), 143.70 (C), 136.99 (C), 133.04 (C), 130.07 (CH), 129.55 (CH), 128.52 (CH), 128.00 (C), 117.37 (C), 114.26 (CH), 60.00 (C), 55.99 (CH3), 53.00 (C), m/z 395.06 (100%), 397.06 (37.0%), 396.06 (23.8%), Molecular Formula: C19H14ClN5OS. Elemental Analysis: Calculated:  57.65, 3.56, 8.96, 17.69, 4.04, 8.10 Found: 57.68, 3.59, 8.93, 17.67, 4.03, 8.11.5-Amino-2-(4-chlorophenyl)-7--7H-thiadiazolopyrimidine-6-carbonitrile4f. M.P: 220–222 °C. Rf value: 0.26. IR (KBr) υmax cm−1: 3400 (C-NH2), 3100 (Aromatic C-H stretching), 1623 (C=N), 1059 (C-OCH3), 740.55 (C-Cl of aromatic ring), 1H-NMR δ ppm: 10.00 , 8.02–6.79 , 3.36 , 3.56 , 13C-NMR δ ppm: 172.21 (C), 158.89 (C), 152.81 (C), 143.32 (C), 136.52 (C), 136.00 (C), 135.01 (C), 129.51 (CH), 128.52 (CH), 128.77 (C), 117.51 (C), 106.50 (CH), 61.00 (CH3), 60.52 (C), 56.00 (CH3), 53.00 (C), m/z 455.08 (100%), 457.08 (37.1%), 456.09 (23.7%), Molecular Formula: C21H18ClN5O3S. Elemental Analysis: Calculated:  55.32, 3.98, 7.78, 15.36, 10.53, 7.03 Found: 55.37, 3.99, 7.77, 15.33, 10.52, 7.00.5-Amino-2-(4-chlorophenyl)-7--7H-thiadiazolopyrimidine-6-carbonitrile4g. M.P: 225–228 °C. Rf value: 0.50. IR (KBr) υmax cm−1: 3400 (C-NH2), 3100 (Aromatic C-H stretching), 1623 (C=N), 1623 (C=N), 1052 (C-OCH3), 740.55 (C-Cl of aromatic ring), 1H-NMR δ ppm: 10.02 , 8.02–6.79 , 3.36 , 3.56 , 13C-NMR δ ppm: 172.07 (C), 158.00 (C), 149.07 (C), 146.72 (C), 143.54 (C), 136.58 (C), 134.51 (C), 129.59 (CH), 128.55 (CH), 128.70 (C), 122.01 (C), 117.56 (C), 114.99 (CH), 112.53 (CH), 59.11 (C), 56.09 (CH3), 53.00 (C), m/z 425.07 (100.0%), 427.07 (37.0%), 426.07 (24.09%), Molecular Formula: C20H16ClN5O2S. Elemental Analysis: Calculated:  56.40, 3.79, 8.32, 16.44, 7.51, 7.53 Found: 56.44, 3.82, 8.30, 16.00, 7.50, 7.52.5-Amino-2-(4-chlorophenyl)-7-phenyl-7H-thiadiazolopyrimidine-6-carbonitrile4h. M.P: 218–220 °C. Rf value: 0.43. IR (KBr) υmax cm−1: 3400 (C-NH2), 3000 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic ring), 1H-NMR δ ppm: 10.00 , 8.02–7.27 , 3.34 , 13C-NMR δ ppm: 172.12 (C), 158.37 (C), 143.77 (C), 141.00 (C), 136.50 (C), 129.55 (CH), 129.08 (CH), 128.61 (C), 128.52 (CH), 125.79 (CH), 125.60 (CH), 117.51 (C), 60.00 (C), 53.12 (C), m/z 365.05 (100.0%), 367.05 (37.0%), 366.05 (22.7%), Molecular Formula: C18H12ClN5S. Elemental Analysis: Calculated:  59.09, 3.31, 9.69, 19.14, 8.76 Found: 59.13, 3.33, 9.67, 19.10, 8.75.5-Amino-2-(4-chlorophenyl)-7-(3-hydroxy-4-methoxyphenyl)-7H-thiadiazolopyrimidine-6-carbonitrile4i. M.P: 240–245 °C. Rf value: 0.51. IR (KBr) υmax cm−1: 3400 (C-NH2), (C-OH) 3333, 3000 (Aromatic C-H stretching), 1623 (C=N), 1055 (C-OCH3), 740.55 (C-Cl of aromatic ring), 1H-NMR δ ppm: 10.00 , 8.00–6.57 , 5.35 , 3.56 , 3.34 , 13C-NMR δ ppm: 172.11 (C), 158.29 (C), 147.17 (C), 147.00 (C), 143.77 (C), 136.69 (C), 134.91 (C), 129.54 (CH), 128.69 (CH), 128.56 (C), 122.63 (CH), 117.58 (C), 115.07 (CH), 112.62 (CH), 60.00 (C), 56.02 (CH3), 53.07 (C), m/z 411.06 (100.0%), 413.05 (36.4%), 412.06 (22.2%), Molecular Formula: C19H14ClN5O2S. Elemental Analysis: Calculated:  55.41, 3.43, 8.61, 17.00, 7.77, 7.79 Found: 55.44, 3.44, 8.59, 17.00, 7.79, 7.80.5-Amino-2-(4-chlorophenyl)-7-(furan-2-yl)-7H-thiadiazolopyrimidine-6-carbonitrile4j. M.P: 200–210 °C. Rf value: 0.49. IR (KBr) υmax cm−1: (C-NH2), 3000 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic ring), 1H-NMR δ ppm: 9.08 , 7.77–7.55 , 7.52 d, 1H, furan ring), 6.40 , 6.08 , 3.71 , 13C-NMR δ ppm: 172.17 (C), 157.77 (C), 152.59 (C), 144.32 (C), 142.36 (CH), 136.71 (C), 129.75 (CH), 129.00 (CH), 128.70 (C), 117.29 (C), 111.00 (CH), 105.97 (CH), 59.60 (C), 54.37 (C), m/z 355.03 (100%), 357.03 (37.1%), 356.03 (20.5%), Molecular Formula: C16H10ClN5OS. Elemental Analysis: Calculated:  54.01, 2.83, 9.96, 19.68, 4.56, 9.01 Found: 54.05, 2.85, 9.95, 19.67, 4.56, 9.00.Experimental procedure for SRB assay .l-glutamine. For the present screening experiment, cells were inoculated into 96 well microtiter plates in 90 µL at 5000 cells per well. After cell inoculation, the microtiter plates were incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. Experimental drugs were solubilized in an appropriate solvent to prepare stock of 10−2 concentration. At the time of experiment, four 10-fold serial dilutions were made using complete medium. Aliquots of 10 µL of these different drug dilutions were added to the appropriate micro-titer wells already containing 90 µL of medium, resulting in the required final drug concentrations.The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM w/v) TCA  and incubated for 60 min at 4 °C. The supernatant was discarded; the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 min at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing five times with 1% acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on an Elisa plate reader at a wavelength of 540 nm with 690 nm reference wavelength.After compound addition, plates were incubated at standard conditions for 48 h and the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µL of cold 30% (50) was calculated from ((Ti − Tz)/(C − Tz)) × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz.Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells. Percent Growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells × 100. Using the six absorbance measurements (time zero (Tz), control growth (C), and test growth in the presence of drug in the four concentration levels (Ti)), the percentage growth was calculated at each of the drug concentration levels. The dose response parameters were calculated for each test article. Growth inhibition of 50% . All compounds were built using Maestro build panel and optimized to lower energy conformers using Ligprep v3.5.9  The coordinates for thymidylate synthase enzyme were taken from RCSB Protein Data Bank and prepared for docking using ‘protein preparation wizard’ in Maestro v10.3.  The bond orders and formal charges were added for heterogroups and hydrogens were added to all atoms in the structure. Side chains that are not close to the binding cavity and do not participate in salt bridges were neutralized and termini were capped by adding ACE and NMA residue. After preparation, the structure was refined to optimize the hydrogen bond network using OPLS_2005 force field. The minimization was terminated when the energy converged or the RMSD reached a maximum cutoff of 0.30 Å. The extra precision (XP) docking mode for all compounds was performed on generated grid of protein structure ,29,30,314a–j was performed for prediction of ADME properties. The absorption, distribution, metabolism and excretion (ADME) properties of all compounds were predicted using Qikprop v3.5 (Schrödinger LLC). In the present study, we have calculated the molecular volume (MV), molecular weight (MW), Predicted octanol-water partition coefficient (log Po/w), number of hydrogen bond acceptors (n-ON), number of hydrogen bonds donors (n-OHNH), Percentage human oral absorption (% ABS), Polar surface area (PSA). The above described properties help us in understanding the ADME properties of any drug/synthesized molecule. A molecule likely to be developed as an orally active drug candidate should show no more than one violation of the following four criteria: log Po/w (octanol-water partition coefficient) ≤ 5, molecular weight ≤ 500, number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5 [A computational study of synthesized compounds nors ≤ 5 .H-thiadiazolo pyrimidine-6-carbonitrile and gives an impression about a clue for the mode of action by performing molecular docking study. The present protocol is also extendable to a wide variety of substrates. The advantages of this protocol are the use of eco-friendly catalyst, short reaction time, easy work-up, ease of product isolation, and high yield. From the results of anticancer activity and docking study, it is observed that the 1,3,4-thiadiazolopyrimidine skeleton is essential for the anticancer activity. Compounds containing electron donating, polar groups such as 4e, 4f, 4g, 4i have good anticancer activity in comparison to electron withdrawing groups. Among all the synthesized derivatives, 4g and 4i were equipotent to standard drug 5-FU. Compound 4i, which has substituent 3-hydroxy-4-methoxyphenyl, is found to have the highest GI50 value of 32.7 μM, 55.3 μM, 34.3 μM, 28.9 μM for MCF-7, K562, HeLa and PC-3 cancer cell lines respectively. Compound 4g, which has 3,4-dimethoxyphenyl, is found to have GI50 values of 34.8 μM, 54.3 μM, 38.7 μM, 34.7 μM for MCF-7, K562, HeLa and PC-3 cancer cell lines respectively. Finally, the ADME study shows that the synthesized drugs showed good drug like properties and opens the gateway for further optimization of studied compounds.The present work reports green synthetic route leading to the formation of antitumor active 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7


For the UPE, the optimal extraction was developed with conditions including solvent of 90% ethanol, sample to solvent ratio of 1:20 g/mL, pressure of 200 MPa, and time of 2 min, which rendered the yields of compounds 4 and 5 were 15.0 and 78.0 mg/g, respectively. For the HSCCC separation, the strategy of changing flow rates between 1.0 and 2.0 mL/min allowed the acquisition for 2.7 mg of compound 1, 4.5 mg of compound 2, 6.8 mg of compound 3, 5.9 mg of compound 4, 11.2 mg of compound 5, and 2.2 mg of compound 6 in one separation run under the solvent system of ethyl acetate:n-butanol:water  from 200 mg of the UPE extract. The structures of these phenylethanoid and secoiridoid glycosides were elucidated by extensive spectroscopic methods.Three phenylethanoid glycosides, echinacoside ( Ligustrum lucidum Ait., an evergreen shrub from the family of Oleaceae, is mainly distributed in the southern regions of China, such as Jiangsu, Guangxi, Yunnan and so on. The dried mature fruits of L. lucidum, documented in the Chinese Pharmacopoeia as “Ligustri Lucidi Fructus”, have long been used in traditional Chinese medicine to invigorate muscles and bones, and tonify kidney and liver +: 809.1837; 1H-NMR  δ: 7.47 , 7.03 , 6.99 , 6.76 , 6.64 , 6.62, 6.51 , 6.21 , 5.03 , 4.71 , 4.37 , 4.16 , 3.90 , 2.70 , 0.95 ; 13C-NMR  δ: 166.5 (C-9′′′′), 149.2 (C-4′′′′), 146.2 (C-7′′′′), 145.8 (C-3′′′′), 145.4 (C-3), 144.0 (C-4), 129.4 (C-1), 125.8 (C-1′′′′), 122.0 (C-6′′′′), 120.0 (C-6), 116.8 (C-2), 116.2 (C-5′′′′), 115.9 (C-5), 115.3 (C-2′′′′), 113.6 (C-8′′′′), 103.8 (C-1′′′), 102.5 (C-1′), 101.6 (C-1′′), 79.2 (C-3′), 77.3 (C-5′′′), 77.0 (C-3′′′), 74.8 (C-2′), 73.9 (C-2′′′), 73.6 (C-5′), 72.1 (C-4′′), 71.0 (C-8), 70.8 (C-3′′), 70.7 (C-2′′), 70.4 (C-4′′′), 69.5(C-4′), 69.1 (C-5′′), 68.5 (C-6′), 61.5 (C-6′′′), 35.5 (C-7), 18.6 (C-6′′).Isonuezhenide (2): HRESI-MS m/z [M + Na]+: 709.1438; 1H-NMR  δ: 7.52, 7.30 , 6.99 , 6.18 , 5.97 , 4.81 , 4.30 , 4.11 , 3.81 , 2.94 , 2.74 , 2.51 , 1.75 ; 13C-NMR  δ: 170.4 (C-7), 167.2 (C-11), 153.4 (C-3), 148.5 (C-4′′′), 134.9 (C-1′′′), 129.6 , 129.5 (C-9), 123.5 (C-8), 121.1 , 107.9 (C-4), 103.0 (C-1′′), 99.3 (C-1′), 93.5 (C-1), 77.0 (C-5′), 76.6 (C-3′), 76.5 (C-5′′), 73.7 (C-3′′), 73.4 (C-2′), 70.3 (C-4′′), 70.08 , 70.03 (C-2′′), 61.39 (C-6′), 61.25 (C-6′′), 50.5 (OMe), 40.0 (C-6), 33.9 (C-β), 30.3 (C-5), 12.3 (C-10).Salidroside (3): HRESI-MS m/z [2M + Na]+: 623.2222; 1H-NMR  δ: 7.06 , 6.69 , 4.29 , 4.02 , 3.86 , 3.68 , 3.23-3.37 , 3.18, 2.83 ; 13C-NMR  δ: 155.4 (C-1), 129.6 , 129.4 (C-4), 114.7 , 103.0 (C-1′), 76.7 (C-4′), 76.6 (C-3′), 73.7 (C-2′), 70.7 (C-5′), 70.3 (C-8), 61.4 (C-6′), 35.0 (C-7).Nuezhenoside G13 (4): HRESI-MS m/z [2M + Na]+: 1095.4089; 1H-NMR  δ: 7.57 , 7.52 , 7.29 , 6.98 , 6.18 , 6.09 , 6.03 , 5.92 , 4.81 , 4.31 , 4.01 , 3.76 , 3.73 , 3.68 , 2.94 , 1.76 , 1.72 ; 13C-NMR  δ: 171.6 (C-7A), 170.3 (C-7B), 167.3 , 153.9 (C-3A), 153.8 (C-3B), 149.1 (C-4′′′), 136.6 (C-1′′′), 129.6 , 129.2 (C-9A), 129.1 (C-9B), 123.8 (C-8A), 123.6 (C-8B), 121.1 , 108.0 (C-4A), 107.9 (C-4B), 103.0 (C-1′′), 99.6 (C-1′A), 99.5 (C-1′B), 94.0 (C-1A), 93.8 (C-1B), 77.0 , 76.5 , 76.5 (C-3′′), 73.8 (C-5′′), 73.6 (C-2′′), 73.4 , 70.2 , 70.1(C-α), 70.0 (C-4′′), 63.6 (C-6′′), 61.34 (C-6′A), 61.27 (C-6′B), 50.6 , 39.9 (C-6A), 39.7 (C-6B), 35.2 (C-β), 30.4 , 12.4 (C-10A), 12.3 (C-10B).Specnuezhenide (5): HRESI-MS m/z [M + Na]+: 709.2308; 1H-NMR  δ: 7.52 , 7.05 , 6.63 , 6.09 , 5.92 , 4.81 , 4.30 , 3.68 , 2.83 , 2.74 , 2.49 , 1.72 ; 13C-NMR  δ: 171.6 (C-7), 167.3 (C-11), 155.5 (C-4′′′), 153.8 (C-3), 129.6 , 129.3 (C-4), 129.1 (C-1′′′), 123.6 (C-8), 114.8 , 108.0 (C-9), 103.0 (C-1′′), 99.5 (C-1′), 93.8 (C-1), 77.0 (C-3′′), 76.6 (C-5′′), 76.5 (C-5′), 73.8 (C-3′), 73.6 (C-2′), 73.4 (C-2′′), 70.8 (C-4′′), 70.2 (C-α), 70.1 (C-4′), 63.6 (C-6′′), 61.3 (C-6′), 50.6 (OMe), 39.9 (C-6), 35.0 (C-β), 30.4 (C-5), 12.3 (C-10).Acteoside (6): HRESI-MS m/z [M + Na]+: 647.1963; 1H-NMR  δ: 7.59 , 7.05 , 6.95 , 6.77 , 6.69 , 6.67 , 6.56 , 6.27 , 5.18 , 4.37 , 4.04 , 2.79 , 1.09 ; 13C-NMR  δ: 166.9 (C-9′′′), 148.4 (C-4′′′), 146.6 (C-7′′′), 145.4 (C-3′′′), 144.7 (C-3), 143.2 (C-4), 130.1 (C-1), 126.1 (C-1′′′), 121.8 (C-6′′′), 119.9 (C-6), 115.7 (C-2), 115.1 (C-5), 114.9 (C-5′′′), 113.8 (C-2′′′), 113.3 (C-8′′′), 102.8 (C-1′), 101.6 (C-1′′), 80.2 (C-3′), 74.8 (C-5′), 74.7 (C-2′), 72.4 (C-4′′), 71.0 (C-3′′), 70.9 (C-8), 70.7 (C-2′′), 69.2 (C-5′′), 69.0 (C-4′), 61.0 (C-6′), 35.2 (C-7), 17.0 (C-6′′).L. lucidum, was purchased from Zhonglu hospital of Shandong University of Traditional Chinese Medicine and authenticated by Prof. Jia Li . All solvents used, except for acetonitrile, which was of HPLC grade and used in HPLC analysis, were of analytical grade. Acetonitrile was purchased from Fisher Scientific , and other solvents were purchased from Fuyu Fine Chemical Co., Ltd . The water used was deionized by an osmosis Milli-Q system .Ligustri Lucidi Fructus, the dried mature fruits of The extraction was carried out by the HPP.L3-600 UPE with a High Hydrostatic Pressure Processor . The separations were performed on a TBE-300A high-speed counter-current chromatography equipment , fitted with a polytetrafluoroethylene (PTFE) multilayer coil 300 mL capacity, 1.6 mm in diameter, and a 20 mL manual sample loop. The revolution speed of the column coil was regulated to be 800 rpm. The HSCCC system was equipped with a Model 3057 portable recorder , a 8823A-UV detector at 254 nm , a TBP-5002 constant-flow pump , as well as a DC-0506 low constant temperature-circulating bath  to maintain the temperature at 25 °C.18Endiapped . The NMR data of the isolated compounds were collected using a Bruker AV-400 spectrometer  and HRESI-MS analysis was performed on a Bruker Impact II mass spectrometer .Waters e2695 equipment was used to perform HPLC analysis. The Waters 2695 system consisted of an Empower 3 ChemStation unit, a Waters 2695 solvent delivery unit, a Waters 2998 Photodiode Array Detection (DAD) detector, an autosampler, and a Waters 2695 column oven. The analytical column used was a PurospherSTAR RPThe extraction was carried out in a sterile polyethylene bag containing 1.0 g of Ligustri Lucidi Fructus powder and 20 mL of 90% ethanol. After being sealed up using a plastic-envelop machine, the packaged bag was subjected to a pressure vessel from the HPP.L 3-600 equipment, and then extracted for a certain period. After extraction, the extracts were removed from the bag and filtered under reduced pressure, and then the filtrate was concentrated to yield the initial UPE extract. After the UPE extraction factors were determined, 2.0 g Ligustri Lucidi Fructus powder was extracted under the determined UPE conditions, and 0.341 g of 90% ethanol crude extract was obtained, which was stored at 2–8 °C for the subsequent HSCCC separation.The procedure for heat extraction was as follows: The dried and powdered fruits (1.0 g) were extracted in a 50 mL flask by 90% ethanol (20 mL) at 60 °C for 1 h. The procedure for ultrasonic extraction was as follows: The dried and powdered fruits (1.0 g) were decocted in a 50 mL flask by 90% ethanol (20 mL) at room temperature for 1 h.n-butanol:water . Firstly, the solvent mixture was equilibrated in a separator funnel, resulting in two phases, with the upper one as the stationary phase and the lower one as the mobile phase. Next, the HSCCC separation began. The column was entirely filled with the upper phase at 20.0 mL/min, which was subsequently revolved at a speed of 800 rpm, while the lower phase was pumped into the column at 2.0 mL/min. When the lower phase was eluted out, the steady equilibrium state of the solvent system was reached; the sample solvent (each 5 mL for upper and lower phase) containing 200 mg of extract was injected into the apparatus through the sample loop, and the lower phase was pumped into the column with a changeable flow rate: 0–100 min, 2.0 mL/min; 100–170 min, 1.0 mL/min; 170–300 min, 2.0 mL/min. Separation curves were monitored by UV at 254 nm, and the effluents were collected together every 10 mL. Finally, when the HSCCC separation was over, ethanol was used as the mobile phase to completely elute the residual solvents to calculate the stationary phase retention.For the HSCCC experiment, a TBE-300A HSCCC apparatus was used, along with the selected solvent system of ethyl acetate:HPLC analysis of the crude sample by UPE and fractions obtained from the HSCCC separation were performed on a Waters e2695 apparatus. Acetonitrile (A)-water (B) was appointed as the mobile phase, and the gradient elution was: 0–2 min, 5–5% A; 2–40 min, 5–30% A; 40–45 min, 30–30% A; 45–53 min, 30–50% A, at a flow rate of 1.0 mL/min. The HPLC chromatograms were carried out by a Purospher STAR RP18 Endiapped  and detected at 282 nm. The structure elucidations of these compounds were achieved by analyzing the HRESI-MS and NMR spectroscopic data and comparing with data in the literature. 1), salidroside (3), and acteoside (6), and three secoiridoid glycosides, isonuezhenide (2), nuezhenoside G13 (4), and specnuezhenide (5), from Ligustri Lucidi Fructus, has been successfully achieved by UPE and HSCCC for the first time. The optimal UPE extraction was developed with conditions set at solvent of 90% ethanol, sample to solvent ratio of 1:20 g/mL, pressure of 200 MPa, and time of 2 min, which rendered the yields of 4 and 5 to be 15.0 and 78.0 mg/g, respectively. These experimental results indicated that UPE extraction needed shorter time and less solvent, however, produced higher yields when compared with conventional extraction methods. Meanwhile, the HSCCC separation strategy of changing flow rates between 1.0 and 2.0 mL/min allowed the acquisition of these six compounds in one separation run in about 300 min, which indicated that HSCCC could yield compounds of widely varied polarities with less time and solvent consumption compared with conventional column separation. The overall results showed that the combination of UPE and HSCCC is efficient for the extraction and isolation of phenylethanoid and secoiridoid glycosides from Ligustri Lucidi Fructus. To the best of our knowledge, this integration method of UPE and HSCCC was applied rapidly to obtain the active components from Ligustri Lucidi Fructus for the first time. Furthermore, this gradient flow rate method demonstrated good performance in isolating compounds with similar polarities and poor peak resolutions, which paved the pathway for one-run systematic separation of complex compounds.In this present work, a combined method for rapid extraction and efficient separation of three phenylethanoid glycosides, echinacoside (


O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 µM for HL-60 cells and 36 to >200 µM for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-dioxoloisoquino isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 µM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9- Berberis aristata, B. aquifolium, B. vulgaris, Hydrastis canadensis, Coptis chinensis and many others dioxoloisoquinoisoquinolin-7-ylium bromide) possesses the highest cytotoxic activity and ability to induce apoptosis of HL-60 leukemia cells. Therefore, this compound could become a promising candidate for anticancer therapy, and has been selected for the follow-up studies to reveal the molecular mechanisms of its antitumor action.In conclusion, we have synthesized a series of four novel 9-® , Alfa-Aesar® , Fluka®  and Mikrochem® . Solvents  were purified and/or dried using standard laboratory methods and stored over molecular sieves (4 Å). Column chromatography was performed using silicagel Nomasil—40–63 μm  and displayed eluent. Reaction progress was monitored by thin layer chromatography on Silufol or Alufol plates  with a UV indicator for λ = 254 nm.All reagents and solvents were purchased from Sigma-Aldrich®, Randor, PA, USA).Melting points (m. p.) of the prepared compounds were established on a Kofler block using digital thermometer TD 121 –[ppm] (parts per million) were referenced to residual signal of the solvent. Coupling constants (J) were given in [Hz] with multiplicity: s (singlet), d (doublet), dd (doublet of the doublet), t (triplet), q (quartet), q (quintet) and m (multiplet).Spectrometers INOVA 300 , VXR-300, Bruker , and VNMRS 600  were used for measurements of ®, Madison, WI, USA) was used to obtain ATR IR absorption bands (ν) in [cm−1]. High resolution mass spectra (HR-MS) were recorded with spectrometer LTQ Orbitrap XL . Microwave irradiation was realized in a CEM Discover microwave reactor .Spectrometer Nicolet NEXUS 470 FT-IR  was placed into a round-bottomed flask equipped with a cross head magnetic stirrer. The flask was inserted into an oil bath preheated to 200 °C and attached to a vacuum pump. The berberine was then intensively stirred at 195 °C for 40 min . After tThe yield of berberrubine was 1.25 g .1H NMR: (CD3OD) δ 9.28 , 8.70 , 7.54 , 7.49 , 6.91 , 6.85 , 6.04 , 4.60 , 3.89 , 3.12 .13C NMR: (CD3OD) δ 149.5 (C-13′), 149.4 (C-9), 148.0 (C-10), 145.9 (C-8), 134.2 (C-2), 132.2 (C-3), 129.2 (C-12′), 122.6 (C-8′), 121.4 (C-4′), 120.1 (C-13″), 118.3 (C-12), 107.7 (C-11), 107.1 (C-13), 104.4 (C-4), 101.8 (C-1), 56.9 (C-2′), 55.2 (C-10′), 54.1 (C-6), 27.5 (C-5).3CN and stirred at 40 °C for 15 min. After that, haloacetylhalides chloracetyl chloride  or bromacetyl bromide  were dropped into the stirred reaction mixture, followed by stirring for additional 2 h. After cooling the mixture to RT, the products were filtered off, washed with dry CH3CN and left to dry in a vacuum.Berberrubine  was dissolved in dry CH1 was 0.42 g .The yield of berberine derivative 1H NMR: (CD3OD) δ 10.03 , 8.72 , 8.54 , 8.19 , 7.27 , 7.16 , 6.34 , 5.12 , 4.19 , 3.78 , 3.14 .13C NMR: (CD3OD) δ 159.8 (C-9′), 152.6 (C-10), 147.9 (C-3), 147.1 (C-2), 146.3 (C-8), 140.1 (C-9), 135.5 (C-13′), 132.2 (C-12′), 131.9 (C-4′), 124.4 (C-11), 121.8 (C-12), 120.1 (C-8′), 119.1 (C-13″), 118.2 (C-13), 106.7 (C-4), 105.1 (C-1), 100.3 (C-2′), 61.1 (C-10′), 53.8 (C-6), 44.7 (C-9″) 21.77 (C-5).2 was 0.38 g .The yield of berberine derivative 1H NMR: (CD3OD) δ 9.83 , 8.71 , 8.64 , 7.87 , 7.23 , 7.14 , 6.72 , 5.13 , 4.22 , 3.92 , 3.03 .13C NMR: (CD3OD) δ 161.3 (C-9′), 149.2 (C-10), 148.1 (C-3), 145.7(C-2), 143.1 (C-8), 142.6 (C-9), 138.4 (C-13′), 134.8 (C-12′), 131.6 (C-4′), 129.1 (C-11), 123.3 (C-12), 122.7 (C-8′), 120.1 (C-13″), 117.8 (C-13), 109.2 (C-4), 107.4 (C-1), 100.9 (C-2′), 63.1 (C-10′), 52.5 (C-6), 45.9 (C-9″) 23.8 (C-5).3 was further purified using column chromatography (eluent CHCl3:MeOH—10:1).Berberrubine  was dissolved in dry DMF (10 mL) at 80 °C and stirred for 15 min. Then, 1,3-dibromopropane  was added dropwise and the reaction was stirred for 2 h. After cooling to RT, the diethyl ether (20 mL) was added. Separated precipitate was filtered off using Büchner funnel. Obtained berberine derivative 3 was 0.54 g .The yield of berberine derivative 1H NMR: (CD3OD) δ 9.79 , 8.94 , 8.20 , 8.00 , 7.79 , 7.09 , 6.17 , 4.94 , 4.42 , 4.06 , 3.94 , 3.21 , 2.33 .13C NMR: (CD3OD) δ 150.2 (C-10), 149.8 (C-3), 147.6 (C-2), 145.2 (C-8), 142.4 (C-9), 137.5 (C-13′), 133.0 (C-12′), 130.7 (C-4′), 126.7 (C-11), 123.5 (C-12), 121.4 (C-8′), 120.4 (C-13″), 120.2 (C-13), 108.4 (C-4), 105.4 (C-1), 102.0 (C-2′), 71.2 (C-9′), 57.1 (C-10′), 55.2 (C-6), 42.1 (C-9″′), 32.6 (C-9″), 26.3 (C-5).2Cl2 (20 mL) was slowly added and the suspension was allowed to exsiccate until dry using a rotary vacuum evaporator. The procedure was repeated 3 times to remove the excess of HCl and thionyl chloride. As the result, the target carbonyl chloride derived from ciprofloxacin 5 was obtained [A round bottomed flask  containing thionyl chloride  was cooled in ice bath and the apparatus was filled with argon. After cooling off the flask content, ciprofloxacin  was added. The colour of the suspension turned orange. The sample was stirred for 2 h. During stirring, the temperature was allowed to reach RT. Then dry CHobtained .5 was 0.085 g .The yield of chlorinated ciprofloxacin 1H NMR: (TFA-d) δ 9.63 , 9.47 , 8.13 , 3.93–4.49 , 3.13 , 2.43 , 1.47–1.85 .13C NMR: (TFA-d) δ 178.2 (C-4), 162.3 (C-3′), 155.4 (C-2), 149.9 (C-6), 149.5 (C-7), 148.1 (C-8′), 117.2 (C-3), 115.1 (C-4′), 112.8 (C-5), 107.6 (C-8), 52.1, 52.3 , 44.6, 44.9 , 32.6 (C-1′), 12.6, 12.8 .19F NMR: (TFA-d) δ −77.95 (F-6).5  were slowly added to dry dioxane (5 mL). The reaction mixture was then irradiated for 10 min using 300 W power at a temperature of 100 °C. After cooling, the mixture was exsiccated until dry and further purified using column chromatography on silicagel using MeOH:CHCl3 (1:10) as an eluent.During continuous stirring, berberrubine  and NaH  and after 15 min chlorinated ciprofloxacin 4 was 0.039 g .The yield of berberrubine-ciprofloxacin conjugate, also referred to as berberine derivative 1H NMR: (TFA-d) δ 9.43 , 9.31 , 8.98 , 8.47 , 8.27 , 7.98–8.12 , 6.63 , 5.28 , 4.25 , 3.25–4.18 , 2.98 , 1.53–1.72 .13C NMR: (TFA-d) δ 176.1 (C-4C), 167.5 (C-3′C), 159.7 (C-2C), 152.6 (C-10), 151.7 (C-6C), 150.3 (C-7C), 148.1 (C-3), 146.9 (C-2), 144.9 (C-8), 143.5, 144.1 , 140.0 (C-13′), 135.1 (C-12′), 131.7 (C-4′), 123.8 (C-11), 123.1 (C-12), 119.3, 120.7, 121.9 , 118.5 (C-13), 115.4 (C-8C), 106.3, 106.9, 108.2 , 100.1 (C-2′), 62.1 (C-10′), 53.8, 54.6, 55.1 , 48.7, 49.3 , 38.7 (C-1′C), 31.4 (C-5), 14.7, 14.9 .19F NMR: (TFA-d) δ −75.71 (F6).HRMS (ESI): calculated for C36H32ClFN4O6 (M + H+): 671.1994; found: 670.9106.v:v) were recorded using a UV-3600 UV-vis-NIR spectrophotometer  with a 1-cm square quartz cell.The UV/visible spectra of berberine, ciprofloxacin and newly synthesized berberine derivatives dissolved in the mixed solvent DMSO/water  reaching the irradiance value of 13 mW·cm−2 within the EPR cavity, determined using a UVX radiometer  or the visible-light source ) were used as the irradiation sources. The g-values were determined using a built-in nuclear magnetic resonance teslameter  and an integrated frequency counter. The EPR spectra were analyzed and simulated using Bruker software WinEPR and EasySpin toolbox working under Matlab software  [The formation of paramagnetic intermediates of the studied berberine derivatives upon their UVA irradiation was monitored in situ using the EPR spin trapping technique. The photoinduced production of singlet oxygen during the excitation of berberine derivatives was followed by the oxidation of a sterically-hindered amine 4-oxo-2,2,6,6-tetramethylpiperidine (TMPO). The solution of the berberine derivatives was mixed either with the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or TMPO solution, and immediately after a careful air or argon saturation using a slight gas stream, the prepared sample was transferred to a small quartz flat cell  optimized for the TE102 cavity  of the X-band EPR spectrometer . The photoexcitation of the samples took place directly in the EPR resonator at 295 K and the EPR spectra were recorded in situ during the continuous irradiation or after a defined exposure. A UV LED monochromatic radiator  .2 and 95% air at 37 °C. HeLa cells were maintained in a DMEM medium and HL-60 cells were maintained in RPMI 1640 medium, both supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, and 50 µg/mL streptomycin. Stock solutions of berberine and its derivatives were prepared in DMSO, except for a stock solution of ciprofloxacin, which was prepared in 0.1 M HCl. In all experiments, the final concentration of DMSO was kept ≤ 0.1% (v/v) and 0.1% DMSO alone was used as vehicle control. Cell viability was determined using a 3--2, 5-diphenyltetrazolium bromide (MTT) assay [4 cells/well) or HeLa cells (4 × 104 cells/well) were seeded in 96-well plates. Cells were treated with various concentrations of berberine, ciprofloxacin and berberine derivatives (0–100 µM) for 24 and 48 h. At the end of the treatment, MTT solution  was added to each well and the plates were incubated for another 4 h. The precipitated formazan was dissolved in DMSO and its concentration was measured at 595 nm using a microplate reader . Based on the curve fitting using nonlinear regression , the median inhibitory concentrations IC50 values (the concentration resulting in 50% inhibition of the cell proliferation that was recorded in control experiments) were determined separately for each experiment. The values were calculated from three independent experiments. Data are presented as a mean ± SD (n = 3).HL-60 (human promyelocytic leukemia) and HeLa (human cervix epithelioid carcinoma) cells were grown in a humidified atmosphere containing 5% COT) assay . HL-60 cThe morphology of cells treated with tested drugs was evaluated using light microscopy  .6) treated with various concentrations of tested derivatives were washed with ice-cold PBS containing 10 mM EDTA. Pellets of cells were resuspended in 250 µL of lysis buffer  supplemented with 0.4 mg/mL DNase-free RNase A, incubated at 65 °C for 15 min, cooled to RT for 10 min, and incubated at 37 °C for 90 min. After that, Proteinase K was added to a final concentration of 0.2 mg/mL and samples were incubated at 50 °C for an additional 1 h. The DNA was extracted with 200 µL of phenol:chloroform:isoamyl alcohol (25:24:1) for 1 min and centrifuged at 14,500× g for 5 min. The DNA was precipitated from the aqueous phase with 3 volumes of chilled EtOH containing 0.3 M sodium acetate. The DNA pellets were washed with 70% EtOH, dried, and dissolved in 100 µL of TE buffer. The DNA was separated on 1.5% agarose gel and visualized under UV illumination after being stained with GelRed Nucleic Acid Gel Stain  [Cells  .TM Assay System, Promega, Madison, WI, USA). Briefly, exponentially growing HL-60 cells were treated with tested derivatives (10–30 µM) for 48 h. Cells (5 × 106) were pelleted by centrifugation  and resuspended in 50 μL of lysis buffer . The lysed cell mixture was then incubated on ice for 15 min before centrifugation . 42 μL of reaction buffer supplemented with 10 mM DTT were mixed with 20 µL of cell lysates. The substrate DEVD-pNA (2 µL) was added and the samples were incubated for 4 h at 37 °C. The release of p-nitroaniline from specific caspase-3 substrate was measured at 405 nm using a microplate reader .Activation of caspase-3 was measured using a colorimetric caspase-3 assay kit (CaspACE6) treated with berberine derivatives (0–30 µM) for 24 and 48 h were washed with PBS and fixed in ice-cold 70% ethanol. Next, the cells were washed twice with PBS and resuspended in 0.1% Triton-X in PBS containing 50 µg/mL RNase A and incubated for 30 min at 37 °C. Afterwards, DNA was stained using PI (50 µg/mL) for 15 min at 4 °C. The cell cycle distribution was analyzed using a FACS Canto II flow cytometer . A minimum of 10,000 cells per sample was analyzed at a flow rate of 200 cells/s [Cells (0.5 × 10 cells/s .


E)-3-Ylideneoxindoles are prepared in methanol in reasonable to good yields, as adducts of photochemical 5-exo-trig of aryl radicals, in contrast to previously reported analogous radical cyclizations initiated by tris(trimethylsilyl)silane and azo-initiators that gave reduced oxindole adducts.( E)-(1-methyl-2-oxoindolin-3-ylidene)acetate (2a) have recently become privileged precursors in organic synthesis for organocatalyzed asymmetric Michael additions/cyclization ..exo-trigE)-3-ylidene- oxindoles, after 5-exo-trig cyclization is reported  matched the literature -4-oxobut-2-enoate (1d). 4-Methyl-morpholine  was added to a suspension of fumaric acid monoethyl ester  and 4--4-methylmorpholinium chloride  in THF (50 mL) and the suspension was stirred at room temperature for 20 min. A solution of 2-iodo–p-toluidine  in THF (5 mL) was added and the suspension was stirred overnight. The reaction mixture was diluted with water, extracted with diethyl ether washed with saturated NaHCO3, water, 2% hydrochloric acid, brine, dried (NaSO4), and evaporated. The residue was added to a mixture of sodium hydride  in THF (25 mL), which was cooled to 0 °C. The solution was stirred for 30 min at 0 °C and at room temperature for another 30 min. Methyl iodide  was added and the reaction was stirred for 2 h. The solvent was evaporated and the residue dissolved in ethyl acetate, washed with water, dried (MgSO4) and evaporated. The resulting crude was recrystallized from diethyl ether to give the title compound  as an off-white solid; mp 76–80 °C; vmax  2925, 1716 (C=O), 1663 (C=O), 1633, 1489, 1419, 1375, 1295, 1174, 1127, 1059, 1033; δH  1.24 , 2.35 , 3.23 , 4.15 , 6.63 , 6.87 , 7.10 , 7.20 , 7.74 ; δC  14.2 (CH3CH2), 20.7 (CH3), 36.7 (NCH3), 61.1 (OCH2), 99.1 (C), 128.7 (6-CH), 130.9 (5-CH), 131.6, 133.8 (CH), 140.8 (3-CH), 140.9, 142.1 (C), 164.2, 165.8 (C=O). HRMS (ESI) m/z (M+H)+, C14H17NO3I calcd. 374.0253, observed 374.0248.Ethyl (2E)-4-[benzyl(2-iodophenyl)amino]-4-oxobut-2-enoate (1e). Same procedure as for the synthesis of 1d was followed, except 2-iodoaniline  and benzyl bromide  were used, and the crude was purified by dry vacuum column chromatography with gradient elution of petroleum ether and ethyl acetate to give the title compound  as an off-white solid; Rf 0.52 (1:4 EtOAc/Pet); mp 64–66 °C; vmax  3031, 2981, 1720 (C=O), 1662 (C=O), 1637, 1577, 1468, 1388, 1292, 1160, 1082, 1023; δH  1.23 , 4.08 , 4.14 , 5.66 , 6.58 , 6.71 , 6.93 , 7.06 , 7.18–7.27 , 7.93 ; δC  14.2 (CH3), 52.3 (NCH2), 61.1 (OCH2), 100.3 (C), 127.9, 128.6, 129.4, 129.6, 130.5 (CH), 131.0 (6-CH), 132.2, 133.8 (CH), 136.3 (C), 140.5 (3-CH), 142.6 (C) 163.9, 165.6 (C=O). HRMS (ESI) m/z (M+H)+, C19H19NO3I calcd. 436.0410, observed 436.0414.(2E)-N-(2-iodophenyl)-N,N',N'-trimethylbut-2-enediamide (1f). 4-((2-Iodophenyl)(methyl)amino)-4-oxobut-2(E)-enoic acid amino}sulfonyl)benzoate (2c). 54 mg (26%) using MeCN and 49 mg (24%) using MeOH; red solid; Rf 0.61 (1:4 EtOAc/Pet); mp 134–137 °C; vmax  2954, 2925, 2853, 1735 (C=O), 1717 (C=O), 1682 (C=O), 1609, 1470, 1434, 1360 (SO2), 1298, 1265, 1167 (SO2), 1104, 1057; δH  3.20 , 3.43 , 3.90 , 6.73 , 6.92 , 7.27 , 7.30 , 7.63–7.65 , 7.77 , 8.32–8.34 ; δC  26.3, 33.2 (NCH3), 53.4 (OCH3), 108.3 (7-CH), 119.3 (C), 122.8, 123.5 (CH), 126.8 (4-CH), 129.7, 130.8, 132.3 (CH), 132.5 (C), 132.7, 133.8 (CH), 135.6, 136.9, 145.6 (C), 165.8, 166.7, 167.1 (C=O); HRMS (ESI) m/z (M + Na)+, C20H18N2O6SNa calcd. 437.0783, observed 437.0775.5-exo-trig reaction of aryl radicals proceeds to give 3-ylideneoxindoles. Higher yields are obtained when using methanol compared to acetonitrile as the reaction solvent. In the case of the 3-ylideneoxindole acetates a greater preponderance for the subsequent [2+2] photochemical cycloaddition occurs in acetonitrile, as well as some reduction of the cyclizing radical. Novel 3-ylideneoxindole acetamides are also accessed, but are found to degrade with prolonged UV-irradiation. In contrast, the same iodide radical precursors are reported to give reduced oxindole products when the 5-exo-trig is carried out using literature metal hydride and azo-initiator protocols [A photochemical “initiator and metal-free” rotocols ,21.


C-acylation of active methylene compounds and the resulting intermediates provided, through a cyclative cleavage reaction, the desired product.An efficient three-step solid-phase synthesis of diverse 3,5-disubstituted-2-aminofuranones has been developed. α-Hydroxy acids loaded on a nitrophenyl carbonate derivative of Wang resin are used as acylating agents for the  Multistep organic syntheses can be substantially facilitated by conducting reactions on substrates that are covalently attached to insoluble supports. The principles of “solid-phase synthesis” were introduced in 1963 by Merrifield ,3,4,5 suA crucial point in the design of compound libraries and, in particular, for cyclative cleavage strategies ,7, is thFour main factors contribute to the popularity of this technique: (i) the simplified reaction procedures − reactions can be accomplished in only three steps: addition of reagents, filtering and finally washing the resin; (ii) purification and isolation steps are eliminated as the only purification needed is the washing of the resin; (iii) the capability of using high concentrations of reagents, as well as (iv), automation of synthesis, which is a basic requirement for multiple parallel syntheses.S)-ketone cyanohydrins, their ureido derivatives as antitumor agents and their 5-hydroxy derivatives as antibiotics + calcd. for C8H12NO4 186.02663 found 186.02679.(S)-2-Amino-3-cyano-5-methyl-4-furanone (1d): White solid, mp: 240-241 °C, αD = -95.3 (c = 0.01 MeOH), Rf = 0.34 (DCM: MeOH = 95 :5), 1H-NMR (DMSO-d6): δ 1.32 , 4.82 , 9.18 ; 13C-NMR (CD3OD): δ 16.76 (CH3), 68.51 (C-5), 84.92 (C-3), 113.55 (CN), 179.91 (C-2), 196.80 (C-4); HRMS: m/z [M +NH4]+ calcd. for C6H10N3O2 156.07730 found 156.07689.(S)-2-Amino-3-ethoxycarbonyl-5-phenyl-4-furanone(1e) + calcd. for C9H14NO4 200.09228 found 200.09224.2-Amino-3-cyano-5,5-dimethyl-4-furanone (1h): Pale yellow solid, mp: 240-242 °C (dec.), Rf = 0.24 (DCM: MeOH = 95 :5), 1H-NMR (DMSO-d6): 1.33 , 9.16 ; 13C-NMR (DMSO-d6): 22.88 (CH3), 64.64 (C-5), 89.76 (C-3), 113.87 (CN), 176.03 (C-2), 195.05 (C-4); HRMS: m/z [M +NH4]+ calcd. for C7H12N3O2 170.09295 found 170.09265.


The structure of compound 1 was determined on the basis of 1D, 2D NMR and HR-ESI-MS data, as well as chemical method. Compounds 2–5, 8–12, 15, 17, and 19 were evaluated for anti-inflammatory activity. Only compounds 10, 15 and 17 showed slightly IL-1β expression inhibitory activities on LPS induced THP-1 cells, with inhibition rate of 15.8%, 37.3%, and 35.8%, respectively, at concentration of 50 μg/mL.A new triflavanoid, kandelin B-5 ( Smilax china L. (Liliaceae), a perennial climbing deciduous shrub, is widely distributed in Southern China, and Southeast Asian countries. The leaves of S. china are used as detoxication agent in folk China -4β--dihyro-2(3H)-pyranone (6) [e]-4α--dihyro-2(3H)-pyranone (7) , suggesting the existence of a phenylpropanoid moiety. The phenylpropanoid moiety was connected with A-ring of the upper flavan-3-ol unit since all the protons on A rings showed singlets in the 1H-NMR spectrum   . The 1D 1 were confirmed by hydrochloric acid-catalyzed degradation with cysteamine (1 afforded 4β-(2-aminoethylthio)-cinchonain Ib (1a), 4β-(2-aminoethylthio)-catechin (1b) −.4β-(2-aminoethylthio)-cinchonain Ib (1b): Amorphous powder,c = 0.06, MeOH); 1H-NMR : δ 6.91 , 6.75 , 6.70 , 5.91 , 5.31 , 4.00 , 3.88 , 3.60–2.40 .4β-(2-aminoethylthio)-catechin (1c): Amorphous powder; 1H-NMR : δ 6.91 , 6.81 , 6.77 , 6.04 , 5.89 , 4.57 , 4.00 , 2.93 , 2.90 .catechin . The quantity of IL-1β in culture supernatant was determined by using Enzyme Linked Immunosorbent Assay (ELISA) kit specific for IL-1β [The human acute monocytic leukemia (THP-1) cells  were cultured in Dulbecco’s Modefied Eagles Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) at 37 °C in a humidified atmosphere with 5% COor IL-1β . The inh2–5, 8–12, 15, 17, and 19, on the viability of THP-1 cells were evaluated using the Cell Counting Kit-8 (CCK-8) according to the manufacturer’s instructions. Briefly, THP-1 cells were seeded into 96-well plates at a density of 2.5 × 104 cells/well in the presence of sample solutions  or absence of compounds for 48 h. Thereafter, 10 μL CCK-8 solutions were added to each well for further incubation at 37 °C in 4 h. The optical density was measured at a wavelength of 450 nm using a microplate reader [The effect of compounds e reader . Cell vi1), was isolated from the rhizomes of Smilax china L., together with six known phenylpropanoid substituted flavan-3-ols (2–7), nine flavonoids (8–16), two stilbenoids , and two other compounds . The structure of compound 1 was determined on the basis of 1D, 2D NMR and HR-ESI-MS data, as well as chemical method. Compounds 2–5, 8–12, 15, 17, and 19 were evaluated for anti-inflammatory activities. Only neoastilbin (10), (-)-epicatechin (15), and scirpusin A (17) showed slight IL-1β expression inhibitory activities on LPS induced THP-1 cells.In this study, a new triflavanoid, kandelin B-5 (