Novel Dipyridinium Lipophile-Based Ionic Liquids Tethering Hydrazone Linkage: Design, Synthesis and Antitumorigenic Study

Novel dicationic pyridinium ionic liquids tethering amphiphilic long alkyl side chains and fluorinated counter anions have been successfully synthesized by means of the quaternization of the dipyridinium hydrazone through its alkylation with different alkyl halides. The resulting halogenated di-ionic liquids underwent a metathesis reaction in order to incorporate some fluorinated counter anions in their structures. The structures of all the resulting di-ionic liquids were characterized by several spectroscopic experiments. The antitumorigenic activities of the investigated compounds were further studied against three different human lung cancer cell lines. Compared to the standard chemotherapeutic agent, cisplatin, the synthesized di-ionic liquids exerted equal, even more active, moderate, or weak anticancer activities against the various lung cancer cell lines under investigation. The observed anticancer activity appears to be enhanced by increasing the length of the aliphatic side chains. Moreover, dicationic pyridinium bearing a nine carbon chain as counter cation and hexafluoro phosphate and/or tetrafluoro bororate as counter anion were selected for further evaluation and demonstrated effective and significant antimetastatic effects and suppressed the colonization ability of the lung cancer cells, suggesting a therapeutic potential for the synthesized compounds in lung cancer treatment.


Introduction
Lung cancer (LC) is one of the most common cancers in both genders and appears to be a leading cause of cancer mortality globally [1,2]. Lung cancer is categorized depending on histopathological characteristics: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which is the most commonly diagnosed type of lung cancer, accounting for 85% of all lung cancer cases [3]. The incidence of LC fluctuates by age, gender, and race in different human populations.
Effective treatments for lung cancer vary depending on the histologic type of cancer, the clinical stage, and the patient's functional status [4]. Radiotherapy, targeted therapy, chemotherapy, and immunotherapy are common effective treatments, with clear limitations for each type of therapy and high tumor relapse rates among patients [5]. While surgery is the first line treatment option in the early stages of lung cancers, it is not effective when metastasis to other organs occurs [4]. Lung cancer patients have a five-year survival rate of less than 20% throughout all stages [1,2]. As a consequence, it is crucial to examine new, safe, and effective therapeutic options against lung cancer.
Over the last decade, evidence has emerged to support the efficacy of ionic liquids (ILs) as druggable molecules in a molten state [6]. The chemistry of ILs has become a hot topic since it is concerned with the design and development of newer scaffolds, as well as the ability to understand their medical properties and feature areas that guide their design and synthesis process [7,8].
Recently, significant effort has been expended on the design and synthesis of these fascinating molecules, which have revolutionized virtually all aspects of daily life [6]. ILs can now be sketched and constructed from bulky organic cations paired with either organic or inorganic anions to accomplish a structural framework with a particular melting point, density and lower toxicity [9,10] along with increasing profits in their medical application due to their safety and stability properties: nonflammable and nonvolatile, negligible vapor pressure, high ionic conductivity and thermal stability, easy recyclability and miscibility with aqueous and organic solvents [11][12][13][14][15].
As part of our ongoing progress in the field of potent ionic liquid frameworks bearing pyridine-hydrazone [16][17][18][19], these features have significantly improved our structural and functional expertise. Thus, we attempt in the present work to provide the design, synthesis and characterization of an array of dicationic pyridinium hydrazone incorporating long alkyl side chains.
Further to that, we assessed the cytotoxic properties of the targeted ILs against three different human lung cancer cell lines and established their antitumorigenic activity profiles, not only as cancer cell growth inhibitors but also as antimigratory and anticolonization substances. Upon careful inspection of the existing literature, this study is the first report that has investigated the ability of ILs to modulate cancer cells' metastasis and their ability to colonize.

The Rational Study
Ionic liquids (ILs) are ionic materials with distinct properties that make them a promising contender in a range of fields, particularly pharmaceutical sciences. ILs are classified into three generations based on their properties and characteristics [20]. The third generation of ILs have been reviewed and documented as fascinating active pharmaceutical ingredients (APIs) and have been widely used to develop biologically active candidatebased IL building blocks. Recently, the majority of research has focused mainly on the physical and chemical characteristics, and the toxicity and biological behavior of ILs have gained prominence as two of the most controversial issues in this area. Thereby, novel ILs have been developed using biologically active ions; however, the primary motivation for this research has been to investigate the use of well-known low-toxicity ions to establish ILs with the necessary details [21][22][23][24][25].
Melting temperature and solubility are significant considerations in the pharmaceutical industry because they are regularly monitored and have the potential to impact drug processing and bioavailability [26][27][28]. This is a useful approach for varying the composition of a drug using ionizable functional groups in order to overcome the undesirable aspects in the parent molecule. A drug's quality, safety, and performance are all impacted only by salt structure. The ion pair chosen could have a major impact on the pharmacokinetics of a medication candidate. That is one of the reasons regulatory agencies have launched the categorizing of novel salts of a licensed medicine as a new chemical entity [28].
The generation and structural analysis of the demanded medications were used to generate such (ILs) as APIs and became an effective approach for addressing the challenges of the pharmaceutical industry.
Despite the fact that several treatment options are available for lung cancer, high tumor relapse rates and resistance to chemotherapy are prevalent. Lung cancer patients have a five-year survival rate of less than 20%. As a consequence, it is imperative to develop new, safe, and effective lung cancer therapeutic approaches.
We proposed to construct a library of dipyridinium ionic liquids tethering numerous long alkyl side chains and some fluorinated anions and test them for antitumorigenic activity to find the obvious ideal candidate of the new advanced third generation of IL-based active units with promising antiproliferative activity ( Figure 1).

Synthesis and Characterization
The synthetic pathway adopted in this study is depicted in Scheme 1. In general, ionic liquids can be synthesized by quaternizing nitrogen-containing molecules followed by a metathetical reaction. The dipyridinium hydrazone 1 was selected as the starting material for the synthesis of the designed DiILs. It was noticeable that the dipyridinium hydrazone 1 was prepared through the condensation of 3-pyridinecarboxaldehyde and isonicotinic acid hydrazide in refluxing ethanol in the presence of a few drops of hydrochloric acid, according to reported literature [29] (Scheme 1). Scheme 1. Synthetic route for the synthesis of dicationic pyridinium lipophile-based ionic liquids incorporating hydrazone anion 9-36.
In refluxing acetonitrile (CH 3 CN), the two nitrogen pyridine atoms of the starting dipyridine hydrazone 1 were bis-alkylated with two equivalents of some selected alkyl iodides with eight to eighteen carbons in their aliphatic (Scheme 1), yielding the targeted dipyridinium ILs 9-15 bearing a hydrophobic side chain as counter cation and iodide as counter anion as listed in Table 1. The success of such a quaternization reaction was evidenced by the proton and carbon nuclear magnetic resonance ( 1 H and 13 C NMR) experiments. Thus, the appearance of diagnostic triplet and multiplet around δ H 0.84 ppm and 4.58-4.69 ppm attributed to the lateral CH 3 and NCH 2 , respectively, confirmed the incorporation of the alkyl chain in their structure. The spectroscopic data also revealed extra methylene protons in the aliphatic area belonging to the alkyl protons. The carbons of the same groups (CH 3 and NCH 2 ) were observed in their 13 C NMR spectra at δ C 14.43-14.44 ppm and δ C 61.51-61.61 ppm, respectively. Additional aliphatic carbons were also resonated at their expected chemical shifts (See experimental section).
The synthesized dicationic halogenated pyridinium ionic liquids 9-15 were then subjected to the metathetical process in order to displace the iodide anion and incorporate the new fluorinated counter anions (PF 6 − , BF 4 − , CF 3 COO − ), as detailed in Table 1. In this respect, the thermal treatment of DiILs 9-15 with some fluorinated metal salts, in acetonitrile for 16 h resulted in the elaboration of the desired dicationic pyridinium hydrazones tethering the targeted fluorinated counter anions 16-36 (Scheme 1) in good yields (88-96%). The structures of these task-specific DiILs 16-36 were deduced based on their spectroscopic results. Due to the similarity of the proton and carbon signals on the 1 H and 13 C NMR of 16-36 compared to their halogenated analogues 9-15, no difference was detected.  11 B NMR data showed a multiplet arround δ B −1.39 and −1.23 ppm, revealing that the boron atom existed in the form of tetrafluoroborate counter-anion (BF 4 − ). In addition, their 19 F NMR spectra exhibited two doublets at δ F −148.21 and −148.14 ppm, respectively, which also supported the encompassing of tetrafluoroborate anion as counter anion in such structural ILs.

Effect of Compounds on the Viability of Lung Cancer Cells
To evaluate the effect of the examined series on the viability of different lung cancer cells, MTT assay was conducted after exposing A549, H1299, and H661 cell lines to increasing concentrations of each compound for 48 h. The results showed potent cytotoxic activity, as treated cells had lower cell viability than untreated control cells. Table 2

Effect of Treatment with the Compounds on Migration of Lung Cancer
The wound healing assay was performed in A549 cells to evaluate the effect of the synthesized series on lung cancer cell migration, as shown in Table 3. The wound of the control sample was completely closed after 24 h. Compounds 22 or 23 were found to be the most effective against all cell lines tested and were thus investigated further. Cell migration was inhibited after 24 h of treatment with compounds 22 or 23 compared to untreated control cells. At 10 µM concentration, treatment of cells with each investigated compound resulted in approximately 67% inhibition of wound closure ( Figure 2).

Effect of Treatment with the Compounds on Colony-Formation Ability of Lung Cancer Cells
To investigate the effect of treatment with either potent compound 22 or 23 at two different concentrations (5 and 10 µM) on the colony-forming capability of lung cancer cells, A549 cells were treated with either concentration for 24 h. Afterwards, cells were grown on soft agar. As shown in Table 4, treatment with both compounds appears to inhibit cell colony formation by reducing the number and size of colonies compared to the control of untreated cells. Representative images taken at day 12 are shown in Figure 3.

Discussion
In consideration of any potential antitumor activity that could be related to the synthesized series, the growth inhibition of lung cancer cell lines (A549, H1299, H661) induced by treatment with each compound for 48 h was evaluated. The results demonstrated that the DiIL 14 harboring a 16 carbon alkyl chain substitution exerted the most potent antiproliferative effect against all examined cell lines when compared to analogues with shorter or longer alkyl chain substitutions. The derivatives 23 and 27 with alkyl side chains of 10 carbons and 12 carbons, respectively, were more effective as antitumor agents relative to their analogues harboring alkyl side chains of different lengths. The IC 50 values (micromolar) are presented in Table 2. Relative to the standard chemotherapeutic agent cisplatin, which demonstrated IC 50 values of 23, 18, and 32 µM against A549, H1299, and H661 cell lines, respectively, the examined compounds appear to have equal, even more active, moderate, and weak anticancer activities against the different lung cancer cells lines under investigation. Several reports in the literature have often shown that the cytotoxic effects of ILs to cancer cells are linked to the length of the substituted alkyl side chain [30]. Most commonly, DiILs with C-1 to C-18 alkyl side chains attached to various anions exhibit improved toxic effects with the extending alkyl side chain length.
Interestingly, the synthesized series demonstrated less cytotoxic effects against the normal human dermal cell line fibroblasts, revealing a unique mechanism of the observed antitumor activity. The ability of lung cancer cells to migrate between edges and close an artificial wound was assessed within 24 h of treatment to further investigate the antitumorigenic activity of the investigated DiILs. The results indicate that treatment with sub-IC 50 concentrations of DiILs effectively suppressed the capability of the examined cancer cells to move and seal a wound, suggesting that treatment may potentially inhibit lung cancer metastasis, a hallmark of lung cancer that increases the rate of cancer replacement and chemotherapy failure. Future studies should shed light on the mechanism underlying the potential inhibition of the multistep process of invasion and metastasis.
Impactful colonization of distant organs by circulating tumor cells (CTC) is an essential process that takes place before metastatic growth. Nonetheless, factors triggering tumor dissemination and distant colonization are still unclear. According to the observed results, two of the examined DiILs have shown potent and significant inhibition of A549 cancer cell-colonization ability, measured by the number of colonies formed and the size of one formed colony, in a dose-dependent manner, suggesting a potential therapeutic utility for the targeted DiILs.

Chemistry
The resulting NMR spectra were obtained with a Bruker spectrometer (and 400 Brucker, Fällanden, Switzerland) using Tetramethylsilane (TMS) as an internal standard (0.00 ppm). A LCMS/MS impact II was used to perform high resolution mass spectroscopy (HRMS).

Statistical Analysis
Data was analyzed by Graphpad Prism software (Graphpad Software Inc. Jolla, CA, USA) for the determination of IC 50 value. All data were expressed as the average of triplicate experiments.

Wound Healing Assay
A549 cells were seeded in inserts (Ibidi GmbH, Gräfelfing, Germany) at concentration of 30,000 per insert in 70 µL medium and incubated for 24 h. Afterward, inserts were removed and cells incubated with 10 µg/mL of mitomycin C for 2 h to stop cell proliferation [8]. The medium was removed, and cells were washed three times then cells were treated with 10 µM concentration of the examined compound. Images were captured at 24 h using BOECO microscope coupled with 5.0 Mega Cmos camera at 4× magnification. Digital images were taken using ISCapture software and wound width was measured using MIPAR software (MIPAR, Worthington, OH, USA).

Colony Formation in Soft Agar Assay
To carry out the soft agar assay, a bottom agar film of 1% (w/v) was prepared by mixing autoclaved 1% (w/v) agar solution with 2× DMEM medium in a 1:1 ratio and allowed to solidify at room temperature [33]. A mixture of 1 × 10 4 of A549 cells treated with either compound NV7 (5 µM and 10 µM) or NV8 (5 µM and 10 µM) and 0.6% (w/v) agar solution was prepared in 1:1 ratio and poured on top of the bottom layer and left to solidify. Plate was incubated for 2 weeks. Photos were captured after 12 days using EVOS XL Core imaging system (Invitrogen, Waltham, MA, USA).

Conclusions
Ionic liquids are becoming increasingly important as anticancer candidates. By quaternizing the dipyridinium hydrazone with different long alkyl halides, some novel dipyridinium iodide-based ionic liquids harboring different alkyl side chains were successfully synthesized and subjected to a metathetical anion exchange to make the targeted task DiILs tether the fluorinated counter anion. Their structures were elucidated based on several NMR experiments. The synthesized compounds were evaluated for their antitumorigenic activities, and the results indicated that DILs tethering an alkyl chain of 10 to 16 carbons had significant anticancer activities against the examined lung cancer cell lines. Moreover, some compounds were further investigated for their ability to suppress metastasis and the colony-formation ability of the cancer cells and the results revealed considerable inhibition for both cancer promoting events, suggesting therapeutic potential for the reported compounds in lung cancer treatment.