Synthesized Two New Water-soluble Fluorescents Calix[4]arene 4-sulfo-1,8-naphthalimide Derivatives Inhibit Proliferation of Human Colorectal Carcinoma Cells

Synthesis of 1,8-naphthalimide derivatives and biochemical studies using them as anti-cancer and cellular imaging agents have attracted signicant attention in recent years. This study aimed to synthesize two new uorescent lower rim-functionalized 4-sulfo-1,8-naphthalimide derivatives of calix[4]arenes [4 and 7] and examination of their cytotoxic properties for cancerous cells. For this purpose, p-tert-butylcalix[4]arene bearing dihydrazide [3] or diaminopropyl [6] units on its lower rim were reacted with 4-sulfo-1,8-naphthalic anhydrate. The structure of these synthesized compounds has been characterized using 1 H-NMR, 13 C-NMR, and FT-IR techniques. To investigate the anti-cancer properties, compounds 4, 7, and N,N-dimethylaminoethyl-1,8-naphthalimide [8] were tested on the human colorectal cancer cell line (DLD-1) as well as healthy colon epithelial cell line (CCD-18Co). The IC 50 values of 4 and 7 were determined as 12.95 µM and 16.13 µM, respectively, on DLD-1 cells. Furthermore, following the treatment of CCD-18Co cells with 4 and 7, the IC 50 values were determined as 508 µM and 269 µM, respectively. However, it was observed that compound 8 had no cytotoxic effect on both DLD-1 cells and CCD-18Co cells. Furthermore, the uorescence imaging of human colorectal cancer cells treated with the synthesized compounds [4, 7, and 8] was performed on living cells. 128.6, 128.1, 127.3, 125.5, 123.2, 122.5, 56.7, 45.8, 37.8. Anal. Calc.: C 16 H 15 KN 2 O 5 S. 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%.


Introduction
Cancer, one of the most important and dangerous diseases worldwide, possesses a high mortality rate due to lifestyle change and global population aging (9,47). World Health Organization reported that 9.6 million died in 2018 due to cancer, and this number would reach 15 million by 2030 (38, 24). Colon cancer ranks third, with a rate of 10% among cancer types worldwide (16). Colon cancer occurs with the formation of malignant and adenomatous cells in the colon (6). Chemotherapy has an important place in cancer treatment; however, its importance in treating and regressing different cancer types should not be ignored (12). The disadvantage of drugs used in cancer chemotherapy is that they are not selective. The main problem in cancer treatment is severe side effects for chemotherapotical medicines due to miss targeting (1,27). The high metabolite consumption rate of cancerous cells and increased signaling pathways make cancerous cells more resistant to many anticarcinogenic agents (32). Herein, developing new anti-cancer agents gain crucial importance to decrease the high mortality rate of cancer (26).
Calix [n]arenes, which are cyclic and exible compounds, are easily synthesized in large quantities by the base-catalyzed condensation reaction between formaldehyde and p-tert-butyl-phenol (23,46). Calixarenes have signi cant advantages over other macrocyclic compounds, such as synthetic versatility on both the lower and upper edges, a hydrophobic cavity, and being able to be decorated with a exible and wellde ned binding core. Calix[n]arene skeleton can have both hydrophilic and hydrophobic features simultaneously from the same molecule (36). They may interact with nucleic acids (25) and proteins (22), which modulate the activity of cancerous cells, metabolic pathways, and many enzymes (40,39 [4]arene inhibits the proliferation of several drug-resistant tumor cells (18). In another study, calix [4]arenes were functionalized with polyhydroxyamine, and their cytotoxic effects were investigated in different cancer cells. The results exhibited that the calix [4]arene is quite effective in human ovarian carcinoma cells (36). Recently, L-or D-proline derivatives of calix [4]arene have been used against human cervical cancer, and it was observed that L1 pentamer formation of HPV (Human papillomavirus) was inhibited by the L-proline derivative of calix [4]arene (22). In our previous study, we prepared L-proline derivatives of calix [4]arene and examined their cytotoxic properties on different human cancer cells, and clari ed the cell death mechanism (37).
It has been known that the substituted 1,8-naphthalimide structures exhibit therapeutic properties (15).  (44,49,50,5). These derivatives might change the DNA conformation and prevent DNA duplication or transcription, thus inhibiting cancer cell growth.

Materials and Instruments
The reagents used were provided by different commercial companies (Merck, Fluka, and Sigma-Aldrich) and used without further puri cation. 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. 1 H-NMR spectra were enrolled on a Varian 400 MHz spectrometer in CDCl 3 and DMSO-d 6 , and the vibration spectra of the compounds were recorded using a Bruker Vertex FT-IR spectrometer. The uorescence spectra were obtained on a Perkin Elmer LS 55 spectrometer.

Synthesis of compound [4]
Calix [4]arene derivative [3] (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 (N 2 ). 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 ltered through a lter with 0.45 µm pore size (Millipore®, USA), and the solvent was evaporated in a vacuum. Finally, 4 was formed as a yellowish Synthesis of compound [7] 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 N 2 . 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 ltered through a lter with 0.

Bio-Imaging Studies
To visualize the localization of compounds in DLD-1 cells, 1x10 5 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% CO 2 (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% CO 2 . 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).
To see the cytotoxic effect of the 4-sulfo-1,8-naphthalimide group before binding to calix [4]arene compound, compound 8 was synthesized. In the FT-IR spectrum of compound 8, the carbonyl group's band was observed at 1655 cm − 1 (Fig.S17). Besides, in the 1 H-NMR spectrum of 8, the signals of aromatic protons between 9.2 and 7.8 ppm and the protons of methyl groups at 2.24 ppm (Fig.S11), as well as the appearance of the carbonyl group at 164.2 and 163.5 ppm in the 13 C-NMR spectrum, con rm the compound (Fig.S14). 4 and 7 treatment of human CRC cell lines; DLD-1 signi cantly suppresses the cell growth in a dosedependent manner (Fig. 1) while the proliferation of healthy colon epithelial cells (CCD-18Co) was not affected with 4 and 7 treatment (Fig. 1). The IC 50 values of 4 and 7 were calculated as 12.95 µM and 16.13 µM, respectively, on DLD-1 cells. On the other hand, following the treatment of CCD-18Co cells with 4 and 7, the IC 50 values were determined as 508 µM and 269 µM, respectively (Fig. 1). A decrease in cell proliferation started 24h after treatment with the compounds. A reduction in the cell number proved that their action on the cancerous cells isn't cytostatic, it is cytotoxic.
Compounds 4 and 7 synthesized in this study showed cytotoxic effects against DLD-1 cells. In contrast, compound 8 did not show cytotoxic effects, which can be explained by the fact that compounds 4 and 7 contain hydrophilic (4-sulfo 1.8 naphthylimide) groups and hydrophobic (calix [4] (48). Bio-imaging and uorescence properties of the compounds were examined. From these images given Fig. 2, it is understood that compounds 4, 7, and 8 entered the cell and radiated into the uorescence emission.

Conclusion
In conclusion, we designed two new uorescence calixarene derivatives and examined their cytotoxic properties on DLD-1. Comparing the cytotoxic effects of compounds 4 and 7 on the DLD-1 cell, it was determined that compound 4 was more effective than compound 7. Besides, when 4 and 7 were examined in a healthy colon epithelial cell line, it was determined that these compounds did not show any signi cant cytotoxicity. No signi cant toxic effect was observed when N,N-dimethylaminoethyl-1,8naphthalimide derivative (8) was used in the DLD-1 cell line. The increase in cytotoxicity of 4-sulfo-1,8naphthalimide due to binding to the hydrophobic calix [4]arene unite indicates that the calix [4]arene unit plays a vital role in cell death. The bio-imaging study of compounds 4, 7, and 8 were performed, and a high uorescence imaging study of 4 and 7 were obtained. This study has developed two new selective and cost-effective uorescent anti-tumor agents and will pave the way for future studies. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
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Con ict of Interest The authors declare no competing interests.