Synthesis of new calix[4]arene derivatives and evaluation of their cytotoxic activity

Since calixarenes are more easily synthesized and functionalized than other supramolecules, they are compounds of interest in organic chemistry. In this study, the dihydrazide (3a and 3b) and diamino propyl (6a and 6b) derivatives of p-tert-butylcalix[4]arene and calix[4]arene were synthesized. Then the L-proline methyl ester substituted chlorocyclopropenium was reacted with the calix[4]arene derivatives (3a, 3b, 6a, and 6b) at room temperature in CH2Cl2 to obtain calix[4]arene superbase derivatives (4a, 4b, 7a, and 7b) in 75%, 60%, 70%, and 55% yield, respectively. The synthesized compounds’ structure was elucidated using spectroscopic techniques (FTIR, 1H NMR, and 13C NMR). The cytotoxic properties of the calix[4]arene superbase derivatives were investigated against different human cancer cell, including A549, DLD-1, HEPG2, and PC-3 and human healthy epithelium cell line PNT1A. The cytotoxicity results showed that calix[4]arene superbase derivatives inhibited the proliferation of DLD-1, A549, HEPG2, and PC-3 cells in a dose-dependent manner. Compound 7a had the highest toxic effect on colorectal carcinoma (IC50: 4.7 µM), and the IC50 values were 18.5 µM and 74.4 µM against human prostate and lung cancer cells, respectively. Furthermore, compound 4b was found more effective on hepatocellular carcinoma cells (IC50: 210.2 µM). As a result, the synthesized calix[4]arene superbase derivatives can be developed to treat different human cancer cell. They can be considered as a preliminary result for molecular-level research.


Introduction
Organic bases have significant advantages over ionic bases due to milder reaction conditions and better solubility requirements in organic synthesis. In addition to this, they have various applications in functional material synthesis [1]. As the smallest ring system that complies with Huckel rules, the cyclopropenium ion (2π-electron) provides significant aromatic resonance stabilization to the conjugate acid of cyclopropenimine [2,3]. Strong organic bases often result from a planar cyclic π-electron system through an aromatization mechanism. Cyclic π-electron system present in cyclopropeneimines gives the fundamental property that enlists it to "superbase" family [4]. The prominent proton affinity of cyclopropene is due to the presence of imino group at the three-membered cyclic ring [5]. Lambert et al. reported the promising and simple method for preparing cyclopropenimines and recommended it for several organic synthesis applications [4][5][6][7].
Ding et al. prepared hydrophilic and hydrophobic groups surfaces on calix [4] arenes. They investigated these compounds' antitumor activities and found that compounds to which 2-dimethylaminoethyl groups were attached to the phenolic-O position showed significant cytotoxicity [24,25]. Because of the fascinating combination of calixarenes in chemical and structural terms, researchers began to apply calixarenes beyond the chemical field, especially in the pharmaceutical industry. In this context, the crystallization of drug molecules with calixarene derivatives helps to change the active pharmaceutical ingredients' physicochemical properties and control the drug structure [26]. p-Sulfonatocalix [4]arene increases its effectiveness by forming a complex with topoisomerase I inhibitor topotecan, which is used to treat many different types of cancer [27][28][29]. In connection to this application, series of polyhydroxyamine calix[n]arene derivatives were synthesized, and their application was reported in cytotoxicity study of different cancer cell lines. From their observations, it was deduced that calixarene derivatives were effectively induced cell death in human ovarian carcinoma cells [18]. Furthermore, the cytotoxic activities of calix [4]arene derivatives on various tumor cells (MU2, MU2F, HT1080, SP6.5, 1PC227, Jurkat, MEWO, Hl-60, Huh7, Hep-G2, MEWO, DLM.1) were compared with standard anticancer drugs. As a result, calix [4]arene derivatives were found to be potent anticancer agents, especially in lymphoblastic leukemia and melanoma cells [30]. Previous work reported that the Lproline functionalized calix [4]arenes used against human cervical cancer to prevent L1 pentamer formation of modified HPV (Human papillomavirus) [31]. In our recent study, calix [4]arene derivatives bearing L-proline on their upper and lower rim were prepared and investigated their antitumor activity for different human cancer cells [32] and observed that these compounds have a potent cytotoxic effect against human colon cancer cells (DLD-1).
From this point of view, to obtain the calix [4]arene superbase derivative, dimethyl(3-chlorocycloprop-1-ene-1,2-diyl)di-L-prolinate derivative of the calix [4]arenes were synthesized and used in cytotoxicity studies. In addition, the compounds were investigated for the proliferation of human colorectal carcinoma, human lung cancer cells, human hepatocarcinoma, and human prostate cancer cells.

Synthesis of calix[4]arene-super base derivatives
In recent years, significant advances have been made in drugbased cancer treatment. Individualized treatment methods using drugs suitable for the specific targets of a particular cancer are being developed, which are becoming more complex than general cytotoxic chemotherapy [33]. In addition to an increasing number of new generation antiproliferative cytotoxic drugs, anti-angiogenic agents, peptides, and therapeutic antibodies have been developed, and many of these have progressed to the clinic [34]. There are a limited number of antitumor activity studies of calixarenes derivatives functionalized with different groups. Dings et al. [24,25,35] reported that different calix [4]arene amide derivatives were highly influential in inhibiting various tumor cells.

Effects of calix[4]arene super base derivatives on viability and proliferation of human cancer cell and healthy cell
Forty-eight hours treatment of human cancer cell with TCP compounds significantly inhibited the cell viability (Fig. 2). All compounds inhibited cell viability in a dose-dependent manner (Fig. 2a). As seen in Fig. 2b, compound 7a was found to be the most potent inhibitor of the proliferation of DLD-1 cells and least inhibitory action on HEPG2 cells. The IC 50 values against the proliferation of DLD-1, A549, HEPG2, PC-3, and PNT1A were calculated as 4.7, 74.4, 240.7, 18.5, and 153.4 µM, respectively (Fig. 2c, d). Compound 7b was found to be the most and least potent inhibitor against DLD-1 and HEPG2 cells, respectively. The IC 50 values against the proliferation of DLD-1, A549, HEPG2, PC-3, and PNT1A were calculated as 16 Also, we compared the 8(a*,b*) compounds synthesized in our previous studies with compounds 4(a,b) and 7(a, b) synthesized in this study. As shown in Table 1, compounds 4a and 7(a,b) showed higher toxic effects on colon cancer cells (DLD-1) than compound 8(a*, b*). Furthermore, compounds 4a, 7(a,b) showed higher toxic effects on both lung cancer cells (A549) and prostate cancer cells (PC-3) than compound 8b*. Besides, compound 7a demonstrated significant cytotoxicity relative to compound 8(a*, b*) in prostate cancer cells (PC-3). However, compounds 8(a*,b*) showed higher toxicity in hepatocarcinoma cells (HEPG2) than compounds in this study. Finally, compounds 4(a,b) and 7(a,b) showed less toxicity in healthy cells (PNT1A) than compounds 8 (a*, b*). As a result, superbases derivatives of calix [4]arene showed much more cytotoxicity than L-proline derivatives [32].
In this study, L-proline methyl ester groups were reacted with tetrachlorocyclopropene and calix [4]arene aminopropyl and hydrazide derivatives to increase the proton binding capability of L-proline methyl ester groups. The better activity of 7a and 7b than 4a and 4b can be explained by the partial degradation of these compounds (4a and 4b) in the solution phase. Superbasetype compounds are relatively unstable and degradable in the solution phase [4,7]. These compounds are also degradable in solid form to a low degree. However, the HCl salts of these compounds can be stored for quite a long time. 1 H-NMR spectra proved that when both 7a-b and 4a-b were kept in the solution phase for 24 h, a significant proportion of cyclopropeneimines groups were separated from 7a-b (80%) 4a-b (65%) compounds were observed.

Materials and instruments
The standard analytical grade solvents and reagents used for the study were provided by various commercial companies and can be used without further purification unless otherwise stated. The 1 H and 13 C NMR spectra were obtained on a Varian 400 NMR instrument and were used CDCl 3 as the deuterated solvent. Infrared spectra (FTIR) were measured using a Bruker Vertex 70 spectrometer. Elemental analyses were calculated on a Gallenkamp. Cell viability and inhibitory potential of TCP compounds were investigated using Alamar Blue reactive (Thermo Fisher Scientific, USA).

The preparing of compounds
Schemes 1 and 2 represent the synthesis of various derivatives of calix [4]arenes. The synthesis of calixarene diaminopropyl and hydrazine derivatives were synthesized as precursor compounds (compound 3a, 3b, 6a, and 6b) using the reported procedures with slight modification [37,38]. Then, the target calix [4]arene super base derivatives (4a, 4b, 7a, and 7b) were synthesized according to the methods given below. All compounds were characterized by 1 H-NMR, 13 C-NMR, elemental analysis, and FT-IR.
The general procedure of the synthesis of calix [4] arene super base derivatives (4a, 4b, 7a, and 7b) To prepare the calix [4] arene superbase derivatives, 1.10 g (6.6 mmol) of L-proline methyl ester hydrochloride was dissolved in anhydrous CH 2 Cl 2 and an equivalent amount of NH 4 OH solution was added to the solution for remove of HCl salt. The mixture stirred at room temperature for 1 h, the organic phase was separated from the aqueous phase and dried over MgSO 4 . After that, tetrachlorocyclopropene (1.10 mmol) was slowly added to the solution under nitrogen atmosphere and stirred at room temperature for overnight and then the calix [4]arene derivative (3a, 3b, 6a, or 6b) (0.5 mmol in dichloromethane) was added into the reaction mixture. After complete the reaction, the most of solvent was evoporated under vacum and then the remaining solid was extracted with CHCl 3 /H 2 O several times, and the organic phase was dried over MgSO 4 in inert atmosphere. The calix [4]arene-super base derivative was formed as white solid.

Determination of cytotoxic potential of TCP compounds
The cell viability and cytotoxic potential of TCP compounds were carried out with Alamar Blue Assay [39]. TCP compounds were dissolved in growth media. 1 × 10 4 cells were seeded into a 96-well plate and treated with various concentrations of TCP compounds ranging from 0 to 250 µM and incubated at 37°C for 48 h. After the incubation, the media were removed, and the cells were washed with PBS and incubated with Alamar Blue (10%) for 3 h. The absorption was measured at 570 nm and 600 nm in an ELISA plate reader. Cell viability and IC 50 values were determined from the sigmoidal plot of cell viability vs. log concentration of the TCP compounds by GraphPad Prism 8.0.2 software.

Conclusion
In conclusion, to obtain the calix [4]arene superbase derivatives, dihydrazide (3a and 3b) and diamino propyl (6a and 6b) derivatives of calix [4]arene were successfully functionalized with L-proline methyl ester-substituted chlorocyclopropenium. The synthesized compounds (7a, 7b, and 4a) have a potent antiproliferative effect against human colorectal carcinoma cells. Besides, compound 7a significantly inhibited the proliferation of human prostate cancer cells. Compared with healthy cells, compound 7a was found 32.6-fold and 8.3-fold cytotoxic against DLD-1 and PC-3's viability. Herein, compound 7a is an advanced candidate for the cure of human colon and prostate cancer. As a result, superbase derivatives of calix [4]arene are much more cytotoxic than L-proline derivatives of calix [4]arene. Therefore, these compounds have a much higher potential to be a drug candidate that can be used in human cancer treatment due to their superbase properties. Following the advanced molecular studies, superbase derivatives of calix [4]arene might be used for preclinical studies.