M.H. Ahagh, et al.
Bioorganic Chemistry 93 (2019) 103329
Found (%): C 67.42, H 7.32, N 4.81, Fe 20.51.
200 µL of Dimethylsulfoxide was added to disperse the dissoluble
crystals and the absorbance was recorded at 570 nm by a plate reader
[35].
2
.1.2. General procedure for synthesis of 3-amino-1-aryl-1H-benzo[f]
chromene-2-carbonitrile derivatives (4a-f)
To a mixture of 2-naphthol (1 mmol), appropriate arylaldehyde
2.2.3. Morphological assessment of the apoptotic cells
(
1 mmol) and malononitrile (1.2 mmol), 10 mg of 1, 4-bis(4-ferroce-
To study the effect of 4c on apoptosis, the cells were stained using
fluorescent acridine orange/ethidium bromide (AO/EtBr). For mor-
nylbutyl) piperazine was added as catalyst. The reaction mixture was
stirred at 100 °C in an oil bath under solvent free condition. After
completion of the reaction (monitored by TLC), the reaction mixture
was cooled and the obtained precipitate was washed with di-
chloromethane. Finally, the material was recrystallized from ethanol.
5
phological analysis, HT-29 cells (5 × 10 cells/well) were pipetted into
6 wells, and maintained with the respective IC50 concentration of 4c for
24 and 48 h. The HT-29 Cells were washed with 200 µL of cold phos-
phate-buffered saline (PBS). Cells (10 µL) were put on a glass slide and
mixed with the AO/Et Br stock solution (1:1, v/v) at a ultimate con-
centration of 100 µg/m. Samples were examined by fluorescence mi-
croscopy (Olympus BX 41, Hamburg, Germany) [36].
2
.1.3. Selected spectral data
2
.1.3.1. 3-Amino-1-phenyl-1H-benzo[f]chromene-2-carbonitrile
1
(
4a). White solid. m. p. 278–280 °C HNMR (400 MHz, DMSO‑d
6
): δ
5
.29 (1H, s, CH), 6.99 (2H, s, NH
1H, d, J = 8.91 Hz, Ar-H), 7.38–7.45 (2H, m, Ar-H), 7.82–7.84 (1H, m,
Ar-H), 7.89–7.94 (2H, m, Ar-H) ppm.
2
), 7.12–7.27 (5H, m, Ar-H), 7.33–7.35
2.2.4. Cell cycle study
(
Flow cytometry method was applied to study the apoptosis and cell
cycle dispersal of HT-29 cells incubated with 4c at IC50 concentration.
5
The HT-29 cells (5 × 10 cells/well) were pipetted into 24 wells for
2
.1.3.2. 3‐Amino‐1‐(4-methoxyphenyl)‐1H-benzo[f]
24 h earlier to incubation with 60 µM of 4c for 24 and 48 h. Later HT-29
cells were gathered and stained with 70% cold ethanol and maintained
at −20 °C for 30 min. Following the incubation, cells were collected,
suspended with PBS, and dyed with 1 mg/mL propidium iodide (PI) and
10 mg/mL RNase and maintained in dark room temperature for 15 min.
Finally, cell cycle distribution was recorded by flow cytometry. The
fraction of HT-29 cells in the sub-G1, G0/G1, S and G2/M phases were
evaluated by Multicycle Cell Cycle software (Partec PAS, Munster,
Germany) [37,38].
1
chromene‐2‐carbonitrile (4d). White solid. m. p. 191–193 °C H NMR
(
400 MHz, DMSO‑d
6 3
): δ 3.65 (3H, s, OCH ), 5.24 (1H, s, CH), 6.79–6.81
(
2H, d, J = 8.49 Hz, Ar-H), 6.94 (2H, s, NH
2
), 7.09–6.11 (2H, d,
J = 8.56 Hz, Ar-H), 7.31–7.32 (2H, d, J = 8.93 Hz, Ar-H), 7.38–7.45
(
2H, m, Ar-H), 7.83–7.85 (1H, d, J = 7.93 Hz, Ar-H), 7.89–7.92 (2H, m,
Ar-H) ppm.
2
.1.3.3. 3‐Amino‐1‐(thiophen‐2‐yl)‐1H-benzo[f]chromene‐2‐carbonitrile
1
(
4f). Yellow solid. m.p. 257–259 °C HNMR (400 MHz, DMSO‑d
6
): δ
5
.71 (1H, s, CH), 6.86–6.88 (1H, m, Ar-H), 7.01 (1H, d, J = 2.90 Hz, Ar-
2.2.5. Annexin-V-FITC/PI double staining assays
H), 7.14 (2H, s, NH
2
), 7.24–7.26 (1H, m, Ar-H), 7.30 (1H, d,
To further verify cell death, Annexin V/PI staining was utilized to
detect the localization of phosphatidyl serine (PC) on the outside of the
cell membrane [39]. Annexin-V/PI staining allows the quantitative
determination of apoptotic cells on a single-cell basis using flow cyto-
J = 8.93 Hz, Ar-H), 7.42–7.45 (1H, m, Ar-H), 7.48–7.52 (1H, m, Ar-
H), 7.90–7.93 (2H, m, Ar-H), 8.03 (1H, d, J = 8.38 Hz, Ar-H) ppm.
5
2.2. Biological activities
metry. HT-29 cells (5 × 10 cells/well) were seeded in 24 well cell
culture plates for 24 h, and then incubated with the respective IC50
concentration of 4c for 24 and 48 h. Following collection, the cells were
suspended with PBS, re-suspended in Annexin binding buffer (10 mM
The cell culture medium (RPMI 1640), fetal bovine serum (FBS) and
penicillin-streptomycin were obtained from Gibco (life technologies,
Paisley, Scotland). The culture plates were purchased from Nunc
HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ), and 10 µL of
2
(
Kamstrupvej, Denmark). Annexin-V FITC apopotosis kit was obtained
Annexin-V FITC and 10 µL PI (according to the manufacture’s re-
commendations) were added. The HT-29 cells were kept in the covert
area for 30 min, and studied by flow cytometry (Partec PAS) [4,40].
from Roche (Mannheim, Germany). Dimethylsulfoxide (DMSO), Tris-
HCl, ethidium bromide (EtBr), propidium iodide (PI), MTT assay kit,
acridine orange (AO), and Calf thymus DNA (ctDNA) were obtained
from Sigma-Aldrich (St. Louis, MO, USA). The human HT-29 cell line
was purchased from Pasteur Institute of Iran (Tehran, Iran).
2.2.6. Evaluation of Bax and Bcl-2 gene expression upon 4c treatment
Total RNA was prepared from treated and untreated cells using
Tripure isolation reagent (Roche, Cat No. 11667165001) based on the
protocol and stored at −80 °C. The cDNA was prepared using TAKARA
complementary DNA synthesis kit (TAKARA, Cat No. 6130). SYBR
green PCR master mix (TAKARA, Cat No. RR820W) and primer set for
each gene (Table 1) were used for qPCR. GAPDH (housekeeping gene)
expression level was utilized for normalization. Finally, the mean of
2
.2.1. Cell culture
The human HT-29 cancer cells were cultured in a cell culture
medium and added 10% fetal bovine serum, 100 IU/ml penicillin and
1
00 µg/ml streptomycin. The HT-29 cells were incubated at 37 °C under
a 5% CO atmosphere [34].
2
duplicated C
Bax, Bcl-2 and caspase-3, -8 and -9 genes were calculated by the com-
parative C method.
t
values was determined and the relative expression level of
2
.2.2. Cytotoxicity of chromene derivatives
MTT (water soluble tetrazolium salt assay) is an assay to specify
t
viability of cells with the changing of the water-soluble tetrazolium salt
to an unsolvable purple formazan by respiring cells. HT-29 cells
2.2.7. DNA binding experiments
4
(
4 × 10 cells/well) were pipetted into 96-well cell culture plates for
2.2.7.1. Preparation of stock solution. A stock solution of 4c was
prepared by dissolution a proper quantity of 4c in Tris-HCl buffer
containing 0.3% DMSO. The ctDNA stock solution was made by
dispersing a proper quantity of ctDNA in Tris-HCl buffer (10 mM, pH
7.4) and stored at 4 °C. A solution of ctDNA presented a proportion of
2
4 h before to adding 4a-f and were incubated at 37 °C and 5% CO
After treatments with 4c, at different doses for various time intervals,
0 µL of the tetrazolium salt (5 mg/ml in PBS) stock solution were
added to each plate and maintained for 3 h. During maintenance,
2
.
2
3