Plausible mechanism of the T3P-DMSO mediated synthesis of title compounds.

<p>Plausible mechanism of the T3P-DMSO mediated synthesis of title compounds.</p

Physical characteristics of the synthesized 2-amino-chromene-3-carbonitriles.

a<p> Isolated yield.</p><p>MP- Melting point.</p><p>Physical characteristics of the synthesized 2-amino-chromene-3-carbonitriles.</p

Known small molecules that target Bcl-2.

<p>As can be seen, different bioactive scaffolds have been established, however both efficacy and avoiding off-target effects of this class of compounds still remains a challenge.</p

Compound 4g induces apoptosis of AML cells in a time-dependent manner.

<p><b>A</b>. Flow cytometry profile represents Annexin V-FITC staining on the X-axis and PI staining on the Y-axis. The upper left quadrants display the necrotic cells, upper right quadrants show the late apoptotic cells, lower left quadrants display the live cells and lower right quadrants show the early apoptotic cells. <b>B</b>. Western blot showed increased expression of cleaved caspase-9 protein in compound 4g treated AML cells compared to control cells.</p

Synthesis of a library of 2-amino-chromene-3-carbonitriles from alcohols.

<p>Synthesis of a library of 2-amino-chromene-3-carbonitriles from alcohols.</p

Cell cycle analysis of AML cell lines treated with compound 4g (5 µM, 72 hr).

<p>Panel (A), fluorescent activated cell sorter analyzed the percent cells in each phase of the cell cycle. Cell cycle analysis of three cell lines (MOLM14, MOLM14 and MV4-11) treated with diluents control and compound <b>4g</b> (5 µM) for 72 h. The figures are representative of three independent experiments. Data are presented in histograms as mean ±SD of three independent experiments. <b>*</b><i>P</i>≤0.005; <b>**</b><i>P</i>≤0.001.</p

Molecular docking analysis of the compound 4g in the navitoclax binding site of Bcl-2.

<p><b>A</b>. The 3-dimensional orientation of compound <b>4g</b> in the navitoclax binding site of Bcl-2. Amino acid side chains are shown as stick (elements are purple color except for carbon-pink), the inhibitor is shown as a ball and stick (elements are green color except for carbon-green). The hydrogen bonding is represented as a green dotted line. <b>B</b>. Navitoclax and compound 4g bound surface view of the Bcl-2, showing the interaction at the P2 and P4 hotspot of the protein. The electrostatic potential of the key amino acids is shown. The bound ball and stick version of the compound <b>4g</b> and navitoclax are represented.</p

Screening of active compounds affecting the proliferation of HL-60 AML cells from a library of 2-amino-chromene-nitriles derivatives.

<p>MTT assays were performed after incubation of HL-60 cells with indicated concentrations of chromene derivatives 4 (a-y) for 72 hr. For each concentration, percent inhibition values were calculated and data was normalized to diluent controls. The scale X-axis is non-linear and the data represent mean ±SD from three independent experiments done in quadruplicates. ** <i>P</i>≤0.001 (Student's t-test).</p

Anti-proliferative effect of 4g tested against AML cell lines in liquid culture.

<p>Panels (A–D): MTT assay determined cell viability of AML cells. 8,000 cells per well were seeded for MOLM13, MOLM14, MV4-11 and HL-60 cells in 96 well plates in quadruplicates. A series of dilutions (starting from 1.25 µm to 10 µm) of <b>4g</b> were added into the wells. Cell proliferation was measured after compound <b>4g</b> treatment relative to diluents controls. Results represent the mean ±SD of three independent experiments with quadruplicate wells per experiment point. ** <i>P</i>≤0.001; *** <i>P</i>≤0.0001 (Student's t-test).</p

Expression of Bcl-2 proteins in human AML cell lines and anti-proliferative effect of compound 4g (5, 10 µm against C57BL/6 mouse bone marrow cells in liquid culture.

<p><b>A</b>. MTT assay determined cell viability of C57BL/6 mouse total bone marrow cells. Results represent the mean ±SD of three independent experiments with quadruplicate wells per experiment point. <b>B</b>. MOLM13, MOLM14, MV4-11 and HL-60 AML cells were cultured either with compound <b>4g</b> (5 µM, 24 hr) or diluents control, and levels of Bcl-2 and Bcl-xL were examined by western blot. GAPDH was used as an internal loading control.</p