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  • br OH br Dororubicin DOX OMe O OH O H

    2020-08-12


    OH
    Dororubicin (DOX) OMe O OH O H O
    OH
    HN
    O
    O O
    B Arylboronate promoiety
    O
    Figure 1. General structure of the arylboronate prodrugs of doxorubicin.
    Six cancer cell lines originating from different organs (breast cancer MCF-7 and the resistant counterpart MCF-7 MDR, hepatocellular carcinoma Hep G2, lung adenocarcinoma A549, glioblastoma U87, pancreatic cancer cell line MiaPaCa-2) were compared. An arylboronate profluorescent probe was used to determine the most effective cell line for the oxidation of the carbon-boron bond. Prodrugs of doxorubicin were then prepared and their cytotoxicity was assessed on the different cell lines. The most promising compound was further tested using in ovo tumor model via the HET-CAM (Hen’s Egg Test-Chorioallantoïc Membrane) assay. This test has been widely used both for screening anticancer drugs[34-36] and for human tumor growth.[37,38]
    Results and Discussion
    Synthesis
    The arylboronate profluorescent probe 3 was prepared in one step by reacting coumarin 2 with 4-bromomethylphenylboronic DHR 123 pinacol ester 1 in the presence of Cs2CO3 at room temperature for 4h (Scheme 2).
    JOURNAL PRE-PROOF
    O
    B HO O O a
    O OO
    Br
    Scheme 2. Synthesis of the arylboronate profluorescent probe 3. Reagents and conditions: (a) Cs2CO3, DMF, r.t., 4h.
    Prodrugs 8a-c were synthesized in two steps, starting from aryl alcohols 6a-c(Scheme 3). Firstly, boronate 6b was prepared through palladium catalyzed bromine-boron exchange starting from compound 4.[39] Ester 6c was obtained in 79 % yield by reacting commercially available boronic acid 5 with pinacol in THF. Alcohol activation of 6a-c was carried out in presence of 4-nitrophenyl chloroformate to provide activated compounds 7a-c. DOX was then linked to the arylboronate promoiety through its amine group using an addition-elimination reaction to afford prodrug 8a-c. A similar reaction was used to prepare the control molecule 9, a benzeneboronate precursor of non-toxic phenylalanine. Another control molecule, the carboxylic acid 12 which is a bioisostere derivative of 8a, unable to liberate DOX was prepared:
    (i) activated carbonate 11 was obtained from commercially available 4-(hydroxymethyl)benzoic acid 10 in 74 % yield; (ii) DOX was then branched according to an addition-elimination reaction (Scheme 3).
    JOURNAL PRE-PROOF
    O O B
    OH
    O O Br B
    a
    OH OH
    HO OH O O B b
    B O
    O
    OH
    COOH c
    OH
    B
    O O
    B
    OH O
    HO OH
    O
    O NH
    c
    d O
    H
    O
    O
    O O
    HO
    OH
    O
    O
    OMe
    7b (fluorophenyl)
    8b (fluorophenyl)
    e
    O
    HO
    HN
    O
    O
    B
    O
    O
    COOH
    COOH
    OH O
    HO
    O
    OH O
    d N
    O H H
    O
    O
    OO HO OH
    O O
    OMe
    H2O2 activation of the profluorescent probe
    The in vitro activation of arylboronate profluorescent probe 3 was carried out using increasing equivalent of H2O2 (1, 5, 10 and 50 equivalents). We recorded the temporal evolution of the free coumarin 2 fluorescence after oxidation and further self-immolation from precursor 3 (Figure
    JOURNAL PRE-PROOF
    2A). We observed that high concentrations of H2O2 led to the fastest kinetic of coumarin release. The use of 5, 10 and 50 equivalents led to the highest fluorescence release with half time values of 5.5, 5 and 2.5 hours, respectively. As expected, these results showed a bimolecular kinetic of oxidation for our benzeneboronate profluorescent probe 3. The use of equimolar quantity of H2O2 leads to slow release of coumarin in line with the known sluggish kinetic of reaction between benzeneboronate and H2O2.[40] Moreover, the release is slowed down since the coumarin is linked to the self-immolative spacer via an ether link.[41]
    Cell line screening
    The capacity of each tested cell line to produce ROS was measured using the benzeneboronate profluorescent probe 3. The slow kinetic profile of disassembly is essential to generate enough contrast between the different cell lines toward their ability to produce different level of ROS. After six hours of incubation with each cell line, the release of coumarin from 3 was quantified by spectrofluorimetry (Figure 2B).
    B
    Fluorescentintensity 8000
    Fluorescentintensity 1500
    Hep MiaPaCa - MCF -
    MCF -
    Time H
    PRE
    Cell line