Novel insights on [1,2]oxazolo[5,4-e]isoindoles on multidrug resistant acute myeloid leukemia cell line

Abstract

A series of [1,2]oxazolo[5,4-e]isoindole derivatives was evaluated against HL-60 cell line and its multidrug resistance (MDR) variant, HL-60R, resistant to doxorubicin and to other P-gp substrates by overexpressing the efflux pump. They displayed antiproliferative activities, with IC₅₀ values ranging from 0.02 to 5.5 µM. In particular, the newly synthesized compound 4k produced synergistic effects in terms of cell growth inhibition and cell death induction either in combination with a Vinca alkaloid, Vinblastine, and a Taxane, Paclitaxel in HL-60R cells. The study of the mechanism of action indicated that all compounds showed antimitotic activity through inhibition of tubulin polymerization. Thus, [1,2]oxazoles could represent a valuable tool to overcome MDR mechanism, confirming the potential use of this class of compounds.

Keywords: [1,2]oxazolo[5,4-e]isoindoles; antimitotic agents; multidrug resistance.

Scheme 1

Synthetic strategy of substituted [1,2]oxazolo[5,4‐e]isoindoles 3a–3l and 4a–4l. (a) NaH, DMF, 0°C to rt, 1 h, then benzyl halide at 0°C to rt, 1–24 h, 60–88%; (b) t‐BuOK, toluene, 0°C to rt, 3 h then HCOOEt, rt, 24 h, 64%−87%; or TBDMAM, toluene, reflux, 24 h; (c) NH₂OH·HCl, ethanol, reflux, 50 min, 57%−83%. DMF, dimethylformamide; TDBMAM, t‐butoxy‐bis‐(dimethylamino)‐methane.

Figure 1

Induction of cell death by 4k, Paclitaxel (PTX), and Vinblastine (VIN). Representative example of flow cytometry analysis in HL‐60R cells treated for 48 h with 4k, PTX, and VIN at different concentrations, either alone or in combination. Profiles of propidium iodide‐stained DNA are shown. Percentages of the events accumulated in the pre G₀‐G₁ position in each panel are indicated.

Figure 2

Western blot analysis of the levels of survivin, X‐linked inhibitor of apoptosis protein (XIAP), IAP‐1, and Bcl‐2 in HL‐60R cells. Quantitative estimations of the protein levels were determined by densitometry measurements after normalization with glyceraldehyde‐3‐phosphate dehydrogenase. The results expressed as mean ± standard error of three different experiments. Differences when treatments are compared to the control: *p < .01 (Tukey test).

Figure 3

Effects of compounds on intracellular accumulation of doxorubicin in HL‐60R cell line. Representative example of flow cytometry analysis of intracellular accumulation of doxorubicin (2 µM) after 1 h of incubation in HL‐60R cells pretreated with 4e 0.04 µM, 4k 5.50 µM or verapamil 10 µM for 24 h (Labbozzetta et al., ; Molinari et al., 1998).

Figure 4

Effects of compounds 4e and 4k (at their IC_50s) on verapamil‐stimulated P‐gp ATPase activity. The data are expressed as fold changes of P‐gp ATPase activity compared to basal one (ΔRLU_TC/ΔRLU_basal) and are presented as mean ± SE of three experiments, each in duplicate. Differences when treatments are compared to the basal activity, *p ˂ .05, **p < .01 (one‐way analysis of variance followed by Tukey's test).

Figure 5

Representative western blot showing the soluble (S) or polymerized (P) tubulin fraction in HL‐60 and HL‐60R cells. Cells were treated for 24 h with vinblastine (VIN), PTX, and 4k at the concentrations corresponding to the IC₅₀ at 72 h. Vinblastine and Paclitaxel were used as reference drugs due to their opposite mechanism of action on tubulin polymerization. The experiment repeated three times gave the same result.