Materials and Instruments
The reagents used were provided by different commercial companies (Merck, Fluka, and Sigma-Aldrich) and used without further purification. The synthesized substances were followed by thin-layer chromatography (TLC) (Kieselgel 60 F254 (Merck)). The melting points of all solid products were measured on a Gallenkamp. 1H-NMR spectra were enrolled on a Varian 400 MHz spectrometer in CDCl3 and DMSO-d6, and the vibration spectra of the compounds were recorded using a Bruker Vertex FT-IR spectrometer. The fluorescence spectra were obtained on a Perkin Elmer LS 55 spectrometer.
The calixarene derivatives [2, 3, 5, and 6] were synthesized according to previously published methods (34, 14, 13). 4, 7, and 8 were also prepared as described below.
Synthesis of compound 
Calixarene derivative  (0.3 g, 0.39 mmol) was dissolved in 10 mL of DMF, and 0.747 g (2.36 mmol) of 4-sulfo-1,8-naphthalic anhydride potassium salt was added into this solution and stirred for 1 h at RT under inert atmosphere (N2). Then 1.5 mL of 25% ammonia solution was added into the solution and heated at 80° C for 24 h. The reaction was followed by TLC (hexane: acetone 3:1), and after the reaction was completed, 40 mL of acetone and 4 mL of deionized water were added, and then the reaction mixture was stirred at RT for 1h. The precipitate formed was filtered through a filter with 0.45 µm pore size (Millipore®, USA), and the solvent was evaporated in a vacuum. Finally, 4 was formed as a yellowish solid. Yield: 80 %. Mp > 3000C; FT-IR: 3496 cm− 1(O-H), 3196 cm− 1, 3093 cm− 1, 2955 cm− 1(C-H, N-H), 1673 cm− 1(C = O), 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 1.1 ( s, 18H, But ), 1.16 ( s, 18H, But ), 3.35–3.52 (m, 4H, Ar-CH2-Ar ), 3.9–4.32 (m, 4H, Ar-CH2-Ar ), 4.39–4.94 (m, 4H, -CH2- ), 7.09–7.16 (br, 8H, ArH ), 7.8 (t, 2H, J = 7.24 Hz ArHNap), 8.2 (t, 2H, J = 7.43 Hz, ArHNap), 8.38–8.43 (m, 4H, ArHNap), 9.2 (d, 2H, J = 1.27 Hz ArHNap), 11.7 (s,2H, Amide); 13C-NMR (400 MHz DMSO-d6) δ (ppm): 164.8, 164.3, 150.3, 134.6, 130.2, 129.8, 128.3, 127.1, 125.4, 123.6, 122.9, 31.8, 31.3. Anal. Calc.: C72H70K2N4O16S2. 4H2O. C, 59.16; H, 5.38; K, 5.35; N, 3.83; S, 4.39%. Found: C, 59.10; H, 5.31; K, 5.30; N, 3.75; S, 4.31%.
Synthesis of compound 
Compound 6 (0.3 g, 0.39 mmol) was dissolved in 10 mL of DMF, and 0.747 gram (2.36 mmol) of 4-sulfo-1,8-naphthalic anhydride potassium salt was added into this solution and stirred at RT for 1 h at RT under N2. Then 1.5 mL of 25% ammonia solution was added to the solution and heated at 80° C for 24 h. The reaction was monitored by TLC (hexane: acetone 1:1), and at the end of the reaction, the mixture was added dropwise into a mixture of 100 mL of acetone and 3 mL of deionized water. The mixture was stirred for 1 h at RT. The precipitate was filtered through a filter with 0.45 µm pore size (Millipore®, USA), and the resulting clear yellowish solution was evaporated in a vacuum. The target compound 7 was formed as a yellow solid. Yield: 85%. Mp > 3000C; FT-IR: 3330 cm-1, 3045 cm-1 2955 cm-1 (O-H, N-H, C-H) 1670 cm-1 (C = O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 1.1 ( s, 18H, But), 1.16 (s, 18H, But ), 2.36–2.48 (m, 4H, -C-CH2-C-), 3.39 (d, 4H, J = 10.34 Hz ArCH2Ar ), 4.01–4.04 (m,4H -CH2-), 4.10–4.26 (m, 4H, Ar-CH2-Ar ), 4.39 ( t, 4H, J = 7.43 Hz -CH2-), 7.03–7.12 (br, 8H, ArH ), 7.76–7.86 (m, 2H, ArHNap), 8.15–8.22 ( m, 2H, ArHNap), 8.36–8.62 (m, 4H, ArHNap), 9.17–9.2 ( m, 2H, ArHNap); 13C-NMR (400 MHz DMSO-d6) δ (ppm): 164.2, 150.7, 150.5, 134.6, 133.7, 130.5, 128.7, 126.1,125.3, 123.4,122.5, 75.4, 34.5, 31.4. Anal. Calc.: C74H76K2N2O14S2.4H2O. C, 62.08; H, 5.91; K, 5.46; N, 1.96; S, 4.48%. Found: C, 62.01; H,5.80; K, 5.40; N, 2.00; S, 4.40%.
Synthesis of Compound 
1.0 g (3.16 mmol) of 4-sulfo-1,8-naphthalic anhydride potassium salt was dissolved in 20 mL of water and stirred at RT for 1h. Then N,N-dimethylaminoethylamine (15.8 mmol) was added dropwise to the solution under N2, and the mixture was refluxed for 3h under N2. Then the solvent was removed in a vacuum. The residue was washed with ethanol. The product was dried in a vacuum oven overnight, and the yellowish solid 8 was obtained in 75 % yield. Mp > 3000C; FT-IR: 1655 cm− 1 (C = O) 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.24 ( s, 6H, –N(CH3)2 ), 2.55 ( t, 2H, J = 6.74 Hz -CH2-) 4.15 ( t, 2H, J = 5.8 Hz -CH2-N), 7.82–7.90 (m, 1H, ArH), 8.22 (d, 1H, J = 7.65 Hz ArH ), 8.44–8.49 ( m, 2H, ArH), 9.2 ( d, 1H, J = 8.83 Hz ArH); 13C-NMR (400 MHz, DMSO-d6) δ (ppm): 164.2, 163.5, 150.2, 134.6, 131.2, 130.7, 128.6, 128.1, 127.3, 125.5, 123.2, 122.5, 56.7, 45.8, 37.8. Anal. Calc.: C16H15KN2O5S. C, 49.73; H, 3.91; K, 10.12; N, 7.25; S, 8.30%. Found: C,49.77; H, 3.82; K, 10.10; S, 8.32%.
Analysis of Fluorescence [4, 7, and 8]
The stock solutions (1x10− 2 M) were prepared from the compound [4, 7, or 8], and the stock solution was diluted to 1x10− 5 M. The spectrophotometric measurements were in a mixture of DMSO/EtOH (1:100 v/v). When compounds 4 and 7 were excited at 350 nm, the main bands appeared at 383 nm for 4 and 384 nm for 7. Similarly, compound 8 was excited at 340 nm, the emission band was seen at 384 nm.
Cell Culture Studies
Human colorectal cancer cell line (DLD-1) and healthy colon epithelial cell line (CCD-18Co), purchased from the American Type Culture Collection, were cultured in RPMI-1640 and DMEM mediums, respectively, and supplemented with 10% FBS (ATCC, USA), 1% pen-strep. The cells were incubated at 37°C with 5% CO2. All standard cell culture laboratory chemicals and reagents were purchased from corning life Sciences (Massachusetts, USA).
Cell Proliferation Assays
The cell number was calculated by TC20™ Automated Cell Counter (Bio-Rad, USA), and 1x104 of the DLD-1 and CCD-18Co cells were seeded in a 96-well plate incubated for 24 h at 37°C with 5% CO2. The cells were washed with 10 mM PBS to remove the unattached cell, then treated with the newly synthesized compounds were also diluted with the growth mediums with eight different concentrations of up to 200 µM. and incubated for 48 h at 37°C with 5% CO2. The cells were then washed with 10 mM PBS to remove dead cells and incubated with 10% Alamar blue reagent, a resazurin-based cell viability dye, for 3 h. Conversion of resazurin to resorufin was spectrophotometrically monitored at 570 and 600 nm (28). The results were analyzed by GraphPad Prism 8.0 software, and the IC50 values were determined from sigmoidal plots of cell inhibition vs. log compound concentration.
To visualize the localization of compounds in DLD-1 cells, 1x105 of the cells were seeded into glass bottom-high quality imaging cell culture micro-Dishes and incubated for 24 h at 37°C with 5% CO2 (20). The cells were then washed with 10 µM PBS and treated with 5 µM of the compounds and incubated for 30 min at 37°C with 5% CO2. Following the incubation, the cells were washed with 10 µM PBS to remove the excess amount of compound and monitored with ZOE Fluorescent Cell Imager (Bio-Rad, USA).