3.1. Chemistry
General. “Melting points were measured with a Stuart melting point apparatus and are uncorrected. The IR spectra were recorded using a FTIR Bruker–vector 22 spectrophotometer as KBr pellets. The 1H and 13C NMR spectra were recorded in DMSO–d6 as a solvent on Varian Gemini NMR spectrometer at 400 MHz and 100 MHz, respectively, using TMS as an internal standard. Chemical shifts are reported as δ values in ppm. Mass spectra were recorded with a Shimadzu GCMS–QP–1000 EX mass spectrometer in EI (70 eV) model. Elemental analyses were performed on a Perkin-Elmer 240 microanalyzer at the Micro-analytical Center of Cairo University”.
Synthesis of tetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinolin-3-yl)-3-(4-fluorophenyl)prop-2-en-1-one (3–8)
A mixture of 3-acetyl-1-aryltetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinoline 1 (1 mmol) and 4-fluorobenzaldehyde 2 (1 mmol) was dissolved in 20 mL ethanol in the presence of potassium hydroxide (20%, 5 mL) at 0–5 oC. The reaction mixture was then stirred at room temperature for 10 h, then poured over cold water containing hydrochloric acid. The solid obtained was then filtered, washed with water, dried, and crystallized from proper solvent to afford chalcones 3–8.
![](https://myfiles.space/user_files/58677_ec8811c6b4185256/58677_custom_files/img1621344437.png)
( E )-1-(8,9-Dimethoxy-1-phenyl-1,5,6,10b-tetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinolin-3-yl)-3-(4-fluorophenyl)prop-2-en-1-one (3)
Yield: (81 %) as a pale-yellow solid (from acetonitrile); m.p 162–164 oC. IR (KBr, cm− 1): 1668 (CO); 1H NMR (400 MHz, DMSO-d6): δ, ppm: 2.64–2.84 (m, 2H, H6), 3.49 (s, 3H, OMe), 3.61–3.68 (m, 1H, H5), 3.72 (s, 3H, OMe), 4.20–4.26 (m, 1H, H5), 6.66 (s, 1H, H10b), 6.77 (s, 1H, H7), 6.97–7.88 (m, 12H, H10 + 2 vinyl-H + Ar-H);13C NMR (100 MHz, DMSO-d6): δ, ppm: 27.3, 41.9, 55.7, 56.0, 78.1, 109.3, 112.4, 115.4, 116.6, 121.6, 122.8, 127.4, 128.8, 129.8, 131.5, 140.6, 143.6, 147.5, 149.0, 149.8, 162.6, 165.1, 179.3; MS (EI): m/z = 457 (M+). Anal. Calcd. for C27H24FN3O3 (457.51): C, 70.88; H, 5.29; N, 9.18. Found: C, 70.71; H, 5.44; N, 9.32.
![](https://myfiles.space/user_files/58677_ec8811c6b4185256/58677_custom_files/img1621344616.png)
( E )-1-(8,9-Dimethoxy-1-( p -tolyl)-1,5,6,10b-tetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinolin-3-yl)-3-(4-fluorophenyl)prop-2-en-1-one (4)
Yield: (89 %) as a pale-yellow solid (from dioxane); m.p 156–158 oC. IR (KBr, cm− 1): 1665 (CO); 1H NMR (400 MHz, DMSO-d6): δ, ppm: 2.29 (s, 3H, CH3), 2.62–2.83 (m, 2H, H6), 3.41 (s, 3H, OMe), 3.61–3.72 (m, 1H, H5), 3.87 (s, 3H, OMe), 4.20–4.53 (m, 1H, H5), 6.64 (s, 1H, H10b), 6.77 (s, 1H, H7), 7.18–7.87 (m, 11H, H10 + 2 vinyl-H + Ar-H); 13C NMR (100 MHz, DMSO-d6): δ, ppm:20.8, 27.3, 41.9, 55.8, 56.0, 78.5, 109.5, 112.4, 116.6, 122.9, 126.9, 127.3, 128.9, 130.2, 130.7, 131.5, 140.4, 141.3, 147.4, 149.0, 149.6, 162.6, 165.0, 179.1; MS (EI): m/z = 471 (M+). Anal. Calcd. for C28H26FN3O3 (471.53): C, 71.32; H, 5.56; N, 8.91. Found: C, 71.51; H, 5.72; N, 8.79.
![](https://myfiles.space/user_files/58677_ec8811c6b4185256/58677_custom_files/img1621344729.png)
( E )-1-(8,9-Dimethoxy-1-(4-methoxyphenyl)-1,5,6,10b-tetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinolin-3-yl)-3-(4-fluorophenyl)prop-2-en-1-one (5)
Yield: (78 %) as a pale-yellow solid (from acetonitrile); m.p 138–140 oC. IR (KBr, cm− 1): 1665 (CO); 1H NMR (400 MHz, DMSO-d6): δ, ppm: 2.58–2.81 (m, 2H, H6), 3.39 (s, 3H, OMe), 3.49–3.56 (m, 1H, H5), 3.71 (s, 3H, OMe), 3.75 (s, 3H, OMe), 4.29–4.35 (m, 1H, H5), 6.57 (s, 1H, H10b), 6.73 (s, 1H, H7), 6.92 (s, 1H, H10), 6.97–7.85 (m, 10H, 2 vinyl-H + Ar-H); 13C NMR (100 MHz, DMSO-d6): δ, ppm: 27.3, 41.9, 55.7, 55.8, 55.9, 79.5, 109.8, 112.5, 115.1, 116.6, 118.4, 123.0, 126.7, 129.1, 131.4, 131.7, 137.0, 140.0, 147.3, 148.9, 149.5, 155.2, 162.5, 164.9, 178.8; MS (EI): m/z = 487 (M+). Anal. Calcd. for C28H26FN3O4 (487.53): C, 68.98; H, 5.38; N, 8.62. Found: C, 69.12; H, 5.51; N, 8.83.
![](https://myfiles.space/user_files/58677_ec8811c6b4185256/58677_custom_files/img1621344832.png)
( E )-1-(8,9-Dimethoxy-1-(4-nitrophenyl)-1,5,6,10b-tetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinolin-3-yl)-3-(4-fluorophenyl)prop-2-en-1-one (6)
Yield: (88 %) as a pale-yellow solid (from dioxane); m.p 194–196 oC. IR (KBr, cm− 1): 1668 (CO); 1H NMR (400 MHz, DMSO-d6): δ, ppm: 2.73–2.91 (m, 2H, H6), 3.50 (s, 3H, OMe), 3.74 (s, 3H, OMe), 3.86–3.95 (m, 1H, H5), 4.06–4.12 (m, 1H, H5), 6.61 (s, 1H, H10b), 6.87 (s, 1H, H7), 7.07 (s, 1H, H10), 7.28–8.26 (m, 10H, 2 vinyl-H + Ar-H); 13C NMR (100 MHz, DMSO-d6): δ, ppm: 27.4, 42.0, 56.0, 56.1, 76.8, 108.5, 112.8, 113.2, 116.7, 122.5, 126.4, 127.1, 128.6, 131.4, 131.9, 139.7, 142.5, 147.7, 148.6, 151.8, 162.9, 165.4, 180.0; MS (EI): m/z = 502 (M+). Anal. Calcd. for C27H23FN4O5 (502.50): C, 64.54; H, 4.61; N, 11.15. Found: C, 64.71; H, 4.84; N, 11.43.
![](https://myfiles.space/user_files/58677_ec8811c6b4185256/58677_custom_files/img1621344923.png)
( E )-1-(1-(4-chlorophenyl)-8,9-dimethoxy-1,5,6,10b-tetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinolin-3-yl)-3-(4-fluorophenyl)prop-2-en-1-one (7)
Yield: (85 %) as a pale-yellow solid (from dioxane); m.p 178–180 oC. IR (KBr, cm− 1): 1668 (CO); 1H NMR (400 MHz, DMSO-d6): δ, ppm: 2.66–2.85 (m, 2H, H6), 3.47 (s, 3H, OMe), 3.64–3.69 (m, 1H, H5), 3.72 (s, 3H, OMe), 4.13–4.19 (m, 1H, H5), 6.62 (s, 1H, H10b), 6.80 (s, 1H, H7), 6.93 (s, 1H, H10), 7.26–7.88 (m, 10H, 2 vinyl-H + Ar-H); 13C NMR (100 MHz, DMSO-d6): δ, ppm: 27.4, 41.9, 55.8, 56.0, 77.8, 109.1, 112.5, 116.5, 116.6, 122.7, 124.9, 127.4, 128.8, 129.6, 131.6, 141.1, 142.7, 147.6, 149.1, 150.2, 156.5, 162.7, 165.1, 179.5 ; MS (EI): m/z = 491 (M+). Anal. Calcd. for C27H23ClFN3O3 (491.95): C, 65.92; H, 4.71; N, 8.54. Found: C, 66.15; H, 4.56; N, 8.76.
![](https://myfiles.space/user_files/58677_ec8811c6b4185256/58677_custom_files/img1621345037.png)
( E )-1-(1-(4-Bromophenyl)-8,9-dimethoxy-1,5,6,10b-tetrahydro-[1, 2, 4]triazolo[3,4-a]isoquinolin-3-yl)-3-(4-fluorophenyl)prop-2-en-1-one (8)
Yield: (88 %) as a pale-yellow solid (from dioxane); m.p 174–176 oC. IR (KBr, cm− 1): 1666 (CO); 1H NMR (400 MHz, DMSO-d6): δ, ppm: 2.68–2.83 (m, 2H, H6), 3.49 (s, 3H, OMe), 3.73 (s, 3H, OMe), 3.89–3.91 (m, 1H, H5), 4.12–4.17 (m, 1H, H5), 6.62 (s, 1H, H10b), 6.80 (s, 1H, H7), 6.92 (s, 1H, H10), 7.26–7.89 (m, 10H, 2 vinyl-H + Ar-H); 13C NMR (100 MHz, DMSO-d6): δ, ppm: 27.4, 41.9, 55.8, 56.0, 66.8, 77.7, 109.1, 112.5, 112.6, 116.4, 116.7, 116.8, 127.4, 128.8, 131.6, 132.4, 141.1, 143.1, 147.6, 149.1, 150.2, 162.7, 165.2, 179.5; MS (EI): m/z = 536 (M+). Anal. Calcd. for C27H23BrFN3O3 (536.40): C, 60.46; H, 4.32; N, 7.83. Found: C, 60.61; H, 4.57; N, 7.97.
3.2. Biological assays
Cell culture
The human HCT-116, MCF7, PC3, A-549, and RPE-1 cell lines were purchased from American Tissue Culture Collection (Rockville, MD, USA). HCT-116, A-549, and MCF7 were maintained in DMEM media, while PC3 cancer cell line was in RPMI-1640 media (Lonza, Biowahittkar, Belgium). The human normal cell line RPE-1 was maintained in DMEM-F12 media. All the media were supplemented with 1 % antibiotic-antimycotic mixture (10,000 U/mL potassium penicillin, 10,000 µg/ml streptomycin sulfate, 25 µg/ mL amphotericin B, 1 % L-glutamine and 10 % fetal bovine serum (FBS) (Biowest, USA).
MTT cytotoxicity assay
Cell viability was assessed using MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (Bio Basic Canada Inc., Canada). Briefly, cells were seeded into a 96-well plate at the concentration of 104 cells/well and allowed to adhere for 24 h. Different concentrations of the chemical compounds tested at 100, 50, 25 and 12.5 µM were added to the cell monolayer in triplicate and incubated for 48 h. Subsequently, the medium was aspirated and fresh media with 40 µl MTT (2.5 µg/ ml) was added to each well and incubated for a further 4 h. Then, 200 µl of 10 % sodium dodecyl sulfate (SDS) was added to each well and incubated overnight at 37 ˚C to stop the reaction and dissolve the formed formazan crystals. The amount of formazan product was measured at a wavelength of 595 nm with a reference wavelength of 620 nm using a microplate reader (Bio-Rad Laboratories, model 3350, USA). Adrinamycin® (doxorubicin) was used as a positive control. Dimethyl sulfoxide (DMSO) was the vehicle used for dissolving all chalcone compounds whereby, its final concentration was less than 0.2 %. IC50 was evaluated using the Prism software program (Graph Pad software incorporated, version 3).
Molecular docking
The molecular docking studies were performed using the ‘‘Molecular Operating Environment (MOE) version 2009.10 release of Chemical Computing Group’s’’. The target compound was drawn using the MOE builder interface and subjected to energy minimization using the included MOPAC. The resulted model was subjected to Systematic Conformational Search where all items were set as default with RMS gradient of 0.01 kcal/mole and RMS distance of 0.1Ao. The X-ray crystallographic structure of the C-terminal binding protein (CtBP) 1 and histone demethylase (PLU-1/ JARID1B) complexed with their ligands (PDB ID: 4LCE and 5FUP, respectively) were obtained from the protein data bank. The proteins were prepared for molecular simulations where: Firstly, the hydrogen atoms were added to the target protein with their standard geometry. Then, the water chains and unwanted co-ligands were removed from the protein active site. The active site was determined by MOE alpha site finder and dummy atoms were made from the resulted alpha spheres. Finally, after the self-docking of the prepared protein with the co-crystallized ligand, it was then docked with the target compound to predict the ligand-protein interactions at the active site. The final results obtained in 2D form, which were then visualized through BIOVIA Discovery Studio V6.1.0.15350 in the 3D form.
Quantitative real-time PCR
Total RNA from the chalcone 8, and cisplatin-treated MCF7 cells, untreated cells was extracted using the RNA Purification Kit GeneJET (Thermo scientific, ON, Canada). One microgram of RNA was reverse transcribed into complementary DNA (cDNA) using High-Capacity cDNA Reverse Transcription Kit (Thermo scientific, ON, Canada). qPCR was performed using Brilliant SYBR Green qPCR master mix (Applied Biosystems, San Francisco, CA, USA) in a Step One Plus Real-Time PCR System (Applied Biosystems, San Francisco, CA, USA) in a 25 µL total reaction volume using 12.5 µL of SYBR green master mix, 1 µL of each primer (Bax, Bcl2, P53, MMP1, LC3B, ATG5, Caspase- 7, Caspase- 8, and Caspase- 9) with 10 pmol/µL (Qiagen, CA, USA), 2.5 µL of cDNA and 8 µL of RNase free water. The thermal profile of PCR started with an initial denaturation at 95oC for 10 min, followed by 40 cycles at 94oC for 15 sec, 55oC for 30 sec and 60oC for 30 sec. The relative gene expression was assessed using the 2−ΔΔCt method after normalization to expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Qiagen, CA, USA) (10 pmol/µL). Primer sequences used according to the published literature as follows: Bax 5'-CCACCCTGGTCTTGGATCCAGCCC-3' (forward) and 5'-GGGCTGGACATTGGACTTCCTCCGGGAGCG-3' (reverse); for Bcl2 5'-ATGTGTGTGGAGAGCGTCAACC-3' (forward) and 5'- TGAGCAGAGTCTTCAGAGACAGCC-3' (reverse); for p53 5'- CTGTCATCTTCTGTCCCTTC-3' (forward) and 5'- TGGAATCAACCCACAGCTGCA-3' (reverse); for MMP1 5'- AGCTAGCTCAGGATGACATTGATG-3' (forward) and 5'- GCCGATGGGCTGGACAG − 3' (reverse); for LC3B 5'- AGCAGCATC CAACCAAAATC − 3' (forward) and 5'- CTGTGTCCGTTCCCAACAG-3' (reverse); for ATG5 5'-TCTGCACTGTTCATCTAAGGATGCA-3' (forward) and 5'-TCCGATTGATGGCCCAAAACTGGT-3' (reverse); for Caspase-7 5'-GGACCGAGTGCCCACTTATC-3' (forward) and 5'-TCGCTTTGTCGAAGTTCTTGTT-3' (reverse); for Caspase-8 5'- GCCTCCCTCAAGTTCCT-3' (forward) and 5'- CCTGGAGTCTCTGGAATAACA-3' (reverse); for Caspase-9 5'- GGCTGTCTACGGCACAGATGGA-3' (forward) and 5'-CTGGCTCGGGGTTACTGCCAG-3' (reverse); for GAPDH 5'- ACGGATTTGGTCGTATTGGG-3' (forward) and 5'-GATTTTGGAGGGATCTCGC-3' (reverse).
Cell cycle assay:
DNA content was quantitatively assessed using propidium iodide (ab139418, Abcam, Cambridge, UK) and Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL). Firstly, cells were treated with chalcone 8 at IC50 and incubated for 48 h. Subsequently, cells were collected in a single cell suspension and fixed in 70% ethanol on ice. Then, the cells were centrifuged at 500 xg for 5 minutes, the pellet was washed by 1 ml 1x phosphate-buffered saline (PBS) and the supernatant was discarded. Afterward, the cells were centrifuged, the cell pellet was re-suspended gently in 200 µl 1X propidium iodide and incubated in dark at 37ºC for 20–30 minutes. Finally, the cells were analyzed using a flow cytometer. The cell cycle profile was analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA).
Apoptosis assay
Apoptosis was analyzed using the Annexin V-FITC kit (catalog number # 4830-01K, R&D Systems, Minneapolis, MN, USA). MCF7 cell line was treated with chalcone 8 at IC50 value 10.65 µM for 48 h. The cells were centrifuged at approximately 300 xg at room temperature for 10 min. Cells were washed in 500 µl cold 1X PBS buffer and then were collected by centrifugation as mentioned before. Then, the cells were gently resuspended in the annexin V incubation reagent comprising of 10 µL binding buffer (10X) + 10 µL propidium iodide + 1 µL annexin V-FITC + 79 µL deionized water. A total of 105-106 cells in 100 µL annexin V incubation reagent was incubated in dark at room temperature for 15 min. Finally, 400 µl of 1X binding buffer was added and samples were acquired on flow cytometry.
DNA fragmentation assay
The percentage of fragmented DNA was determined by using Diphenylamine (DPA) assay. The monolayer cell cultures were harvested directly into centrifuge tubes and centrifuged at 300 xg at 4°C for 10 min. Then, the cell pellet was resuspended in 0.8 mL of 10 mM PBS, pH 7.4, and 0.7 mL of ice-cold lysis buffer ((5 mM Tris-HCl, 20 mM EDTA, pH 8.0, 0.5% (v/v) Triton X-100. Afterward, the cell lysate was transferred to microfuge tubes and incubated on ice for 15 min. The lysate was centrifuged at 13,000 xg at 4°C for 15 min to separate fragmented DNA from high-molecular-weight DNA. The entire supernatant (about 1.5 ml containing fragmented DNA) was transferred to a 5 ml glass tube. The pellet containing intact DNA was resuspended in 1.5 ml TE buffer (1 mM EDTA, 10 mM Tris, pH 8.0), and again transferred to another 5 ml glass tube. 1.5 mL of 10% Trichloroacetic acid (TCA) was added to each tube and incubated for 10 min at room temperature. The tubes were centrifuged at 500 xg at 4°C for 15 min and the supernatant was discarded. The 10% TCA precipitates were resuspended in 0.7 ml of 5% TCA and boiled at 100°C for 15 min, then cooled to room temperature, and centrifuged at 300 xg at 4°C for 15 min. 0.5 mL of the supernatant was transferred without disturbing the precipitate to a new glass tube. 1 mL of DPA was added and incubated overnight at 30°C. The absorbance was measured at 600 nm. The percentage of DNA fragmentation was calculated using the following equation: % fragmented DNA = OD600 of the supernatant/ [OD600 of the supernatant + OD600 of the pellet] × 100.