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  • br Compound a br To

    2020-08-12


    Compound 7a
    To a solution of 4-nitrophenyl chloroformate (264 mg, 1.26 mmol) in 3 mL of DCM at 0°C was added dropwise Et3N (177 µL, 1.26 mmol). The mixture was stirred during 20 min under argon then a solution of 6a (100 mg, 0.42 mmol) in 4 mL of DCM was added dropwise to the first solution and the mixture was stirred during 3 h at r.t. The reaction was quenched with brine, extracted with DCM (3 x 20 mL), the organic layers were combined, dried with MgSO4 and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography over silica gel with cyclohexane-EtOAc: 90-10 to 60-40 to give the expected product (135 mg, yield: 80 %) .
    JOURNAL PRE-PROOF
    Compound 6c
    To a solution of boronic Methoctramine 5 (3.5 mmol, 500 mg) in anhydrous THF (15 mL) was added and pinacol (3.5 mmol, 416 mg). The resulting solution was evaporated under reduced pressure at 40 °C. This procedure was repeated until TLC analysis indicated a complete conversion. The crude product was purified by column chromatography over silica gel with hexane-EtOAc: 90-10 to 50-50 to give the expected product (618 mg, yield: 79 %).
    Compound 7b
    To a solution of 4-nitrophenyl chloroformate (175 mg, 0.87 mmol) in 5 mL of DCM at 0°C was added pyridine (68 µL, 0.87 mmol), the solution was stirred during 5 min at 0°C then a solution of 6b[39] (73 mg, 0.29 mmol) in 1 mL of DCM was added dropwise to the mixture and the reaction was stirred at r.t. overnight. Methoctramine The reaction was then quenched with a saturated solution of NaHCO3, the aqueous layer was extracted with EtOAc (3 x 20 mL), the organic layers were combined, washed with brine, dried with MgSO4, and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography over silica gel with Cyclohexane-EtOAc: 95-5 to 60-40 to give the expected product (25 mg, yield: 20 %).
    Compound 7c
    To a solution of 4-nitrophenyl chloroformate (504 mg, 2.5 mmol) in 15 mL of DCM was added DMAP (54 mg, 0.44 mmol) and the reaction was stirred et 0°C during 15 min. In another round bottom flask, a solution 6c (500 mg, 2.2 mmol) and Et3N (612 µL, 4.4 mmol) in 15 mL of DCM was stirred at 0°C. The second mixture was added dropwise on the first mixture to give a yellow solution. The solution was stirred during 6 hours at r.t. before being quenched with water. The aqueous layer was extracted with DCM (3 x 20 mL), the organic layers were combined, washed with brine, dried with MgSO4, and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography over silica gel with DCM-MeOH: 100-0 to 90-10 to give the expected product (250 mg, yield: 30 %)
    Compound 8b
    JOURNAL PRE-PROOF
    stirred a r.t. during 6 hours. The reaction was quenched with brine, the aqueous layer was extracted with extracted with DCM (3 x 20 mL), the organic layers were combined, dried with MgSO4 and concentrated under reduced pressure. The crude product was purified over silica gel with DCM-MeOH: 90-10 to give the expected product (30 mg, yield: 60 %).
    Compound 8c
    Compound 9
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    Compound 11
    Compound 12
    H2O2 activation of the profluorescent probe
    kinetic was studied over 12 h by reading its fluorescence intensity at λexcitation / λemission: 390 / 500 nm every 5 minutes for the first hour and every 30 minutes during the eleven remaining hours
    (Infinite M200 Pro, Tecan trading AG, Switzerland). The final fluorescent intensity was calculated by subtracting the minor fluorescence of 3 in absence of H2O2 to the fluorescence obtained in presence of H2O2. The curves relating the release kinetic of coumarin were obtained using GraphPad Prism software (GraphPad Software Inc, San Diego, CA, USA).
    JOURNAL PRE-PROOF
    Cell line and culture
    Several cancer cell lines have been used for the in vitro evaluation of the designed prodrugs in comparison to the parent drug: doxorubicin (DOX). Six cell lines were selected for the cytotoxicity assay as follows: U87 (glioblastoma), A549 (lung carcinoma), MCF-7 (breast cancer), MCF-7 MDR (multi drug resistant breast cancer), MiaPaCa-2 (pancreatic cancer) and Hep G2 (liver cancer). All cells were grown in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10 % of fetal calf serum (FCS) and 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen) in a 5 % CO2 and 95 % hygrometry environment at 37 °C.
    In vitro Screening
    All seven cell line were investigated for their ability to produce ROS using profluorescent probe 3. Six well plates were used for this study, each well was seeded with 5.105 cell per well and treated with 10 µM of 3 (sixplicate). After a 6h incubation period at 37 °C in presence 5 % CO2 and 95 % hygrometry, each well was sonicated three times during 5s (Sonicator FB505, Fisher Scientific, USA) and 100 µL of the supernatant was transferred in a 96 well black plate. The