Synthesis, characterization, and anticancer activity of new quinazoline derivatives against MCF-7 cells

Abstract

Two new synthesized and characterized quinazoline Schiff bases 1 and 2 were investigated for anticancer activity against MCF-7 human breast cancer cell line. Compounds 1 and 2 demonstrated a remarkable antiproliferative effect, with an IC50 value of 6.246×10(-6) mol/L and 5.910×10(-6) mol/L, respectively, after 72 hours of treatment. Most apoptosis morphological features in treated MCF-7 cells were observed by AO/PI staining. The results of cell cycle analysis indicate that compounds did not induce S and M phase arrest in cell after 24 hours of treatment. Furthermore, MCF-7 cells treated with 1 and 2 subjected to apoptosis death, as exhibited by perturbation of mitochondrial membrane potential and cytochrome c release as well as increase in ROS formation. We also found activation of caspases-3/7, -8, and -9 in compounds 1 and 2. Moreover, inhibition of NF-κB translocation in MCF-7 cells treated by compound 1 significantly exhibited the association of extrinsic apoptosis pathway. Acute toxicity results demonstrated the nontoxic nature of the compounds in mice. Our results showed significant activity towards MCF-7 cells via either intrinsic or extrinsic mitochondrial pathway and are potential candidate for further in vivo and clinical breast cancer studies.

Figure 1

Quinazoline-4-one.

Figure 2

The reaction pathway for the synthesis of compounds (1) and (2).

Figure 3

Compound (1).

Figure 4

Compound (2).

Figure 5

¹H NMR spectrum of compound (1) in (400 MHz, DMSO-d6).

Figure 6

¹³C NMR spectrum of compound (1) in (100 MHz, DMSO-d6).

Figure 7

¹H NMR spectrum of a compound (2) in (400 MHz, DMSO-d6).

Figure 8

¹³C NMR spectrum of compound (2) in (100 MHz, DMSO-d6).

Figure 9

Crystal structure of compound (1).

Figure 10

The LDH release assay revealed the significant cytotoxicity of the quinazolinone-based compound on MCF-7 cells at concentrations 4 × 10⁻⁶, 8 × 10⁻⁶, and 16 × 10⁻⁶mol/L.

Figure 11

Fluorescent micrographs of AO/PI-double-stained MCF-7 cells. (a) Untreated MCF-7 cells exhibit normal structures. (b1) and (c1) Early apoptosis features, namely, blebbing and chromatin condensation as well as late apoptotic cells, were detected after 24 h of treatment with (1) and (2). (b2) and (c2) Late apoptosis and secondary necrosis were obsereved after 48 h of treatment with (1) and (2), respectively (magnification: 200x). VI: viable cells; CC: chromatin condensation; BL: blebbing of the cell membrane; LA: late apoptosis; SN: secondary necrosis.

Figure 12

Cell cycle analysis. (a) Effect of (1) and (2) on cell cycle arrest. After incubation with DMSO or different concentrations of (1) and (2) for 24 h, MCF-7 cells were stained with BrdU and phosphohistone H3 and subjected to the Cellomics ArrayScan HCS reader for cell cycle analysis. (b) Representative bar charts indicating that treatment of both compounds markedly decreased BrdU and phosphohistone H3 fluorescence intensities in treated MCF-7 cells. Data were expressed as the mean ± SD of fluorescence intensity readings for three independent experiments.

Figure 13

Effect of MMD compound on the generation of ROS. The level of ROS significantly elevated at 8 × 10⁻⁶ and 16 × 10⁻⁶mol/L concentrations.

Figure 14

Effects of the Quinazoline Schiff bases on nuclear morphology, membrane permeability, mitochondrial membrane potential (MMP), and cytochrome c release. (a) Representative images of MCF-7 cells treated with medium alone and at 4 × 10⁻⁶, 8 × 10⁻⁶, and 16 × 10⁻⁶mol/L concentrations of compounds and stained with Hoechst 33342 for nuclear, cytochrome c, membrane permeability, and MMP dyes. Both compound induced a noteworthy elevation in membrane permeability and cytochrome c release and a marked reduction in mitochondrial membrane potential (magnification: 200x). (b) Representative bar charts indicating dose-dependent increased cell permeability reduced MMP and increased cytochrome c release in treated MCF-7 cells.

Figure 15

Relative luminescence time-dependent expression of caspases-3/7, -8, and -9 in MCF-7 cells treated with (1) and (2) at concentrations of 8 × 10⁻⁶mol/L and 7.6 × 10⁻⁶mol/L, respectively, after 24 hours incubation.

Figure 16

NF-κB translocation. (a) Photographs of the intracellular targets of stained MCF-7 cells that were exposed to (1) and (2) at concentrations of 8 × 10⁻⁶mol/L and 7.6 × 10⁻⁶mol/L, respectively, for 3 hours and then stimulated for 30 minutes with 1 ng/mL TNF-α (NF-κB activation). (b) Representative bar chart showing translocation of TNF-α-induced NF-κB nuclear in MCF-7 cells for different concenataions.

Figure 17

Histological sections in acute toxicity test (H&E staining, 20x). Histological sections of liver (first row) and kidney (second row). Untreated mices (control group) received 5 mL/kg vehicle (5% Tween 20) ((a) and (d)). Animals treated with 250 mg/kg are ((b) and (e)) and ((c) and (f)) for compounds 1 and 2, respectively. There are no significant differences in structures of liver and kidney between treated and control group.