br Figure In ovo efficacy
Figure 3. In ovo efficacy of 8a compared to DOX after two intratumoral injections of each drug (20 nmol) twice a day for two consecutive days, white arrows indicate the tumors, scale bar: 0.5 cm. (A) left: tumor before injection of DOX; right: tumor three days post injection of DOX (50 % tumor regression). (B) left: tumor before injection of 8a; right: tumor three days post injection of 8a (tumor necrosis and 50 % regression). Image treatment and tumor measurements were fulfilled using FIJI software.
In order to confirm that the prodrug activity was due to its conversion into free DOX, a HPLC method for the measurement of DOX concentration in the tumors was elaborated. Tumors were extracted from the chorioallantoic membrane and sonicated to recover the free DOX. The same amount of DOX was found in both groups (30 % of the initial dose) which is coherent with the
tumor regression observed in ovo whereas prodrug 8a was not detected. These results show that, in presence of a well-developed tumor, the prodrug 8a is converted into DOX.
In this study, no strong tumor regression or total resection was observed. Indeed, in the HET-CAM assay, the dose escalation is difficult to study as it can lead to the death of the embryo resulting in a false positive result. Taking into account the ethical regulation and the small window between the drug injection and the end point before the embryo’s birth, observation of tumor evolution over a long period is not possible. Therefore, we were able to only monitor partial regression of the tested tumors.
A profluorescent probe was used to select a cancer cell line strongly producing ROS. Among the six cell lines tested, Hep G2 and MiaPaCa-2 were the most effective at oxidizing the benzeneboronate probe. Prodrugs of doxorubicin and controls were then synthesized and their cytotoxic activity was determined in vitro on our panel of cell lines. Prodrug 8a was selectively active on pancreatic cancer Pam3CSK4 MiaPaca-2 with a recovery of 67 % of the activity of the free doxorubicin. Further testing in ovo allowed us to observe the behavior of this prodrug on a MiaPaCa-2 tumor model. Similar tumor regression was observed using the same dose of both the prodrug 8a and the free drug counterpart on the available timescale of reading. Quantification of the doxorubicin released from 8a by oxidation in pancreatic tumor in ovo confirmed the latter result. The development of benzeneboronate-containing prodrugs must be pursued for the treatment of certain cancer types and we herein highlighted the promising potential of such prodrug in the treatment of pancreatic adenocarcinoma.
All chemical reagents were of analytical grade, obtained from Acros, Alfa Aesar, or Aldrich, and used without further purification. Solvents were obtained from SDS or VWR-Prolabo. Dichloromethane (DCM) was dried on molecular sieves and used immediately. Chromatography was performed using silica gel (35-70 μm, Merck). Concentration of solutions was performed under reduced pressure at temperature below 40°C using rotary evaporator. Analytical TLC was performed using Silica Gel 60 F254 pre-coated aluminum plates (Merck). Spots were visualized by treatment with ninhydrine revelator followed by heating and/or by absorbance of UV light at 254 nm or fluorescent light at 360 nm. NMR spectra were collected on Bruker DPX 250 (1H at 250 MHz and 13C at 62.5 MHz), AV 300 (1H at 300 MHz and 13C at 75 MHz) or AV 360 (1H at 360 MHz and 13C at 90 MHz) spectrometers and analyzed using MestReNova software. Chemical shifts are reported in ppm (δ) and coupling constants in Hz (J). NMR spectra were performed in CDCl3 or (CD3)2SO. High-resolution mass spectrometry (HRMS) analyses were performed by electrospray with positive or negative (ESI+ or ESI-). The purity of all compounds used for biological activity test was checked by reverse phase analytical HPLC and confirmed to be ≥ 95 %.
To a solution of bromo derivative 1 (41 mg, 0.13 mmol), coumarin 2 (30 mg, 0.13 mmol) in 5 mL of DMF was added Cs2CO3 (46 mg, 0.14 mmol). The reaction was kept under agitation at r.t. for 4 h before being quenched with a saturated solution of NaHCO3. The suspension was extracted with EtOAc (3 x 20mL), 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: 100-0 to 60-40 to give the expected product as a white solid (35 mg, yield: 60 %).