br JOURNAL PRE PROOF br boron based
boron-based prodrugs is central since none of these molecules has entered clinical trial so far. In this work, we decided to develop potent arylboronate-drug conjugates and further select the most receptive target among several cancer cell lines in order to identify the type of cancer that responds well towards these precursors. First, we selected doxorubicin (DOX), a good candidate for the design of prodrug. Indeed, DOX is a very effective antitumor drug with limited therapeutic efficacy due to stochastic repartition in the patient organs resulting in important cardiotoxicity. Arylboronate-based self-immolative spacers were then used in order to branch the free amine function of DOX (Figure 1). Several arylboronate moieties were used in order to study their influence on the kinetic of DOX release in presence of H2O2. Three different scaffolds were envisioned: (i) the unsubstituted benzeneboronate; (ii) a fluorinated benzene homolog since electron withdrawing Mitoquinone were shown to increase the kinetic of oxidation; (iii) a furan ring, described as effective self-immolative spacer, to add a strong structural modification in comparison with the two aforementioned benzene moieties.
Dororubicin (DOX) OMe O OH O H O
B Arylboronate promoiety
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
The arylboronate profluorescent probe 3 was prepared in one step by reacting coumarin 2 with 4-bromomethylphenylboronic acid pinacol ester 1 in the presence of Cs2CO3 at room temperature for 4h (Scheme 2).
B HO O O a
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. 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).
O O B
O O Br B
HO OH O