An Eco-Friendly Ultrasound-Assisted Synthesis of Novel Fluorinated Pyridinium Salts-Based Hydrazones and Antimicrobial and Antitumor Screening

The present work reports an efficient synthesis of fluorinated pyridinium salts-based hydrazones under both conventional and eco-friendly ultrasound procedures. The synthetic approach first involves the preparation of halogenated pyridinium salts through the condensation of isonicotinic acid hydrazide (1) with p-fluorobenzaldehyde (2) followed by the nucleophilic alkylation of the resulting N-(4-fluorobenzylidene)isonicotinohydrazide (3) with a different alkyl iodide. The iodide counteranion of 5–10 was subjected to an anion exchange metathesis reaction in the presence of an excess of the appropriate metal salts to afford a new series of fluorinated pyridinium salts tethering a hydrazone linkage 11–40. Ultrasound irradiation led to higher yields in considerably less time than the conventional methods. The newly synthesized ILs were well-characterized with FT-IR, 1H NMR, 13C NMR, 11B, 19F, 31P and mass spectral analyses. The ILs were also screened for their antimicrobial and antitumor activities. Within the series, the salts tethering fluorinated counter anions 11–13, 21–23, 31–33 and 36–38 were found to be more potent against all bacterial and fungal strains at MIC 4–8 µg/mL. The in vitro antiproliferative activity was also investigated against four tumor cell lines (human ductal breast epithelial tumor T47D, human breast adenocarcinoma MCF-7, human epithelial carcinoma HeLa and human epithelial colorectal adenocarcinoma Caco-2) using the MTT assay, which revealed that promising antitumor activity was exhibited by compounds 5, 12 and 14.


Introduction
In recent years, the development of clean, safe and efficient eco-friendly protocols has become a major challenge in green chemistry. Ultrasound (US) has been extensively adopted as a promising green pathway [1] in several organic transformations. US was reported to drastically increase reaction rates, improve yields and provide high purity of products with an easy work-up [2][3][4]. An enhanced Int. J. Mol. Sci. 2016, 17, 766 2 of 20 selectivity and reduction of chemical hazards using this safe ultrasound method have been well documented [2][3][4].
Ionic liquids (ILs) have emerged as fascinating new green solvents as alternatives to volatile organic solvents due to their sought-after properties such as negligible vapor pressure, high thermal stability, high ionic conductivity and high ability to solvate both polar and non-polar compounds [2,5]. Their most attractive features are their very low volatility, nonflammability and stability, which make them suitable for applications in diverse fields, such as organic synthesis, catalysis and biocatalysis, analytical chemistry, nanotechnology, food science and as function fluids (e.g., lubricants, heat transfer fluids and corrosion inhibitors) [4,5]. Recently, some acidic task-specific ILs were used as solvents and catalysts for the hydrolysis/conversion of cellulose and lignocellulosic biomass [6].
ILs have also demonstrated promising applications for medicinal chemistry, including antimicrobial, antiseptic, anticancer and anti-inflammatory activities [7].
Because the synthesis of ionic liquids [5,8] is not easy, chemists have developed several green protocols for the clean and safe synthesis of IL liquids including microwave (MW), ultrasound (US) and solvent-free reactions.
Hydrazones are an important class of Schiff bases and are widely used as antimalarial [9], anticancer [10], antibacterial [11], antifungal [12], antitubercular [13], antimicrobial [14] and antiviral [15] agents. The azomethine linkage on the Schiff base structures are responsible for their bioactivity, enabling them to serve as models for biologically important scaffolds [16]. In addition, there are many commonly used drugs incorporating hydrazone groups in their structures.
In view of the emerging importance of ILs and hydrazones as antimicrobial and anticancer agents and our general interest in ultrasound-assisted organic synthesis, we focus on developing a straightforward, safe and ecofriendly method for the synthesis of fluorinated pyridinium ILs tethering a hydrazone linkage under ultrasound irradiation and conventional heating. To the best of our knowledge, the synthesis of ionic liquids carrying a hydrazone functionality has not been previously reported in the literature. However, the synthesized compounds were found to be salts rather than ILs and were screened for their antimicrobial and antitumor activities in order to evaluate the synergistic effect resulting from the clubbing of these salts with azomethine hydrazone functionality in a single molecular frame work.

Chemistry
In the present work, several attempts to find the optimum conditions for the synthesis of new classes of fluorinated pyridinium ionic liquid-based hydrazone have been investigated under both conventional and ultrasound methods. These attempts led to the finding that the alkylation of isonicotinic acid hydrazide with several alkyl iodides in different solvents such as acetonitrile, toluene and methanol afforded very poor yields (24%-28%) of compound 4, under either conventional heating or US. In addition, no reaction was observed under several attempts to condensate the resulting 4 with p-fluorobenzaldehyde (2) in the presence of a catalytic amount of HCl in boiling ethanol and/or under ultrasonic conditions (Scheme 1).
Conversely, the successful strategy for synthesizing the target 5-10 was based on the alkylation of N-(4-fluorobenzylidene)isonicotinohydrazide (3) with the appropriate alkyl halides under both conventional and US conditions. Thus, the condensation of acid hydrazide 1 with p-fluorobenzaldehyde (2) afforded the corresponding hydrazone 3 in excellent yield (90%) in refluxing ethanol for 1 h; a comparable yield has been obtained within 30 min under ultrasound irradiation. The resulting hydrazone 3 has been alkylated with different alkyl iodides, furnishing the target halogenated pyridinium salts 5-10 in 83%-91% yields, as shown in Scheme 1.
When the alkylation was carried out under ultrasound irradiation, great reductions in reaction time (12-14 h) were observed with higher yields (90%-94%) compared to those obtained under the classical method (72 h) ( Table 1).
The structures of the hydrazones 5-10 have been established based on their mass and spectroscopic data ( 1 H NMR, 13 C NMR, 19 F NMR). The NMR spectra of the synthesized compounds 5-10 measured in DMSO-d6 revealed the presence of a diastereomeric mixture (i.e., E/cis and E/trans) for each imino-amide moiety.  The N-hexyl derivative 9 was selected to discuss the NMR data used to confirm the success of the quaternization reaction. From its 1 H NMR spectrum, the appearance of the diagnostic CH3 and NCH2 as a triplet at δH 0.88 ppm and a doublet of doublets at δH 4.70 ppm, respectively, are clear evidence for the success of the alkylation reaction. The remaining methylene groups were also observed.
The spectrum also revealed the presence of two singlets at δH 8.16 and 8.50 ppm, with a ratio of 1:3, which have a total integration of one proton characteristic of the imine proton (HC=N). In addition, the NH group split into two singlets at δH 12.47 and 12.52 ppm that have a total integration of one proton with the same ratio ( Figure 1). Moreover, eight aromatic protons resonated in their Scheme 1. Synthesis of halogenated pyridinium salts tagged with hydrazone 5-10 under conventional method (CM) and ultrasound irradiation (US).
When the alkylation was carried out under ultrasound irradiation, great reductions in reaction time (12-14 h) were observed with higher yields (90%-94%) compared to those obtained under the classical method (72 h) ( Table 1). The structures of the hydrazones 5-10 have been established based on their mass and spectroscopic data ( 1 H NMR, 13 C NMR, 19 F NMR). The NMR spectra of the synthesized compounds 5-10 measured in DMSO-d 6 revealed the presence of a diastereomeric mixture (i.e., E/cis and E/trans) for each imino-amide moiety.
The N-hexyl derivative 9 was selected to discuss the NMR data used to confirm the success of the quaternization reaction. From its 1 H NMR spectrum, the appearance of the diagnostic CH 3 and NCH 2 as a triplet at δ H 0.88 ppm and a doublet of doublets at δ H 4.70 ppm, respectively, are clear evidence for the success of the alkylation reaction. The remaining methylene groups were also observed.
The spectrum also revealed the presence of two singlets at δ H 8.16 and 8.50 ppm, with a ratio of 1:3, which have a total integration of one proton characteristic of the imine proton (HC=N). In addition, the NH group split into two singlets at δ H 12.47 and 12.52 ppm that have a total integration of one proton with the same ratio ( Figure 1). Moreover, eight aromatic protons resonated in their appropriate chemical shifts with a similar isomeric pattern to that observed for the NH and H-C=N groups. To confirm the solvent effect for the isomerism of hydrazones, the 1 H NMR spectrum of compound 9 was recorded in a less polar solvent (CDCl 3 ). Consequently, one singlet signal was observed at δ H 12.25 ppm for the NH proton and at δ H 9.13 ppm for the HC=N proton, corresponding to the cis and trans conformers of the E isomer ( Figure 2). These results agree with those previously reported in our work, where the hydrazone functionality was proven to exhibit E/cis and E/trans geometrical isomerism in polar solvents such as DMSO-d 6 , while only the E/cis or E/trans isomer was recorded in a less polar solvent (CDCl 3 ) [17,18]. appropriate chemical shifts with a similar isomeric pattern to that observed for the NH and H-C=N groups. To confirm the solvent effect for the isomerism of hydrazones, the 1 H NMR spectrum of compound 9 was recorded in a less polar solvent (CDCl3). Consequently, one singlet signal was observed at δH 12.25 ppm for the NH proton and at δH 9.13 ppm for the HC=N proton, corresponding to the cis and trans conformers of the E isomer ( Figure 2). These results agree with those previously reported in our work, where the hydrazone functionality was proven to exhibit E/cis and E/trans geometrical isomerism in polar solvents such as DMSO-d6, while only the E/cis or E/trans isomer was recorded in a less polar solvent (CDCl3) [17,18].    appropriate chemical shifts with a similar isomeric pattern to that observed for the NH and H-C=N groups. To confirm the solvent effect for the isomerism of hydrazones, the 1 H NMR spectrum of compound 9 was recorded in a less polar solvent (CDCl3). Consequently, one singlet signal was observed at δH 12.25 ppm for the NH proton and at δH 9.13 ppm for the HC=N proton, corresponding to the cis and trans conformers of the E isomer ( Figure 2). These results agree with those previously reported in our work, where the hydrazone functionality was proven to exhibit E/cis and E/trans geometrical isomerism in polar solvents such as DMSO-d6, while only the E/cis or E/trans isomer was recorded in a less polar solvent (CDCl3) [17,18].   Further assignment of the diastereomer formation for compound 9 was supported by 13 C NMR and dept-135 experiments. In the 13 C NMR spectrum, each peak of compound 9 appeared as two sets of signals due to the presence of the diastereomeric mixture. In the aliphatic region, the methyl and NCH 2 carbons resonated as two sets of signals at 61.43 and 61.51, respectively. The C=N and C=O groups of the E/cis and E/trans diastereomers also resonated as double peaks at δ C 159.27-165.72 ppm.
The 19 F NMR spectrum ( Figure 3) also proved the formation of a diastereomeric mixture (E/cis and E/trans) through the appearance of two characteristic multiplets at δ F´1 09.90 to´109.82 and 109.44 to´109.36 ppm, attributed to the aromatic fluorine atom. Further assignment of the diastereomer formation for compound 9 was supported by 13 C NMR and dept-135 experiments. In the 13 C NMR spectrum, each peak of compound 9 appeared as two sets of signals due to the presence of the diastereomeric mixture. In the aliphatic region, the methyl and NCH2 carbons resonated as two sets of signals at 61.43 and 61.51, respectively. The C=N and C=O groups of the E/cis and E/trans diastereomers also resonated as double peaks at δC 159.27-165.72 ppm.
The 19 F NMR spectrum ( Figure 3) also proved the formation of a diastereomeric mixture (E/cis and E/trans) through the appearance of two characteristic multiplets at δF −109.90 to −109.82 and −109.44 to −109.36 ppm, attributed to the aromatic fluorine atom. The structure of compound 9 was also confirmed by the electron impact mass spectrum, which showed a molecular ion peak at 459.38 [M + ].
All the newly synthesized iodonated pyridinium salts 5-10 have been subjected to a metathesis reaction in which the iodide anion has been displaced by different anions. The anion exchange reactions were carried out by their refluxing with different metal salts such as NaBF4, KPF6, NaOCOCF3, NaSCN, or NaNO3, in acetonitrile as solvent for 16 h to give new specific-based hydrazones 11-40 in 80%-98% yields (Scheme 2). Scheme 2. Synthesis of specific-based hydrazones 11-40.  The structure of compound 9 was also confirmed by the electron impact mass spectrum, which showed a molecular ion peak at 459.38 [M + ].
All the newly synthesized iodonated pyridinium salts 5-10 have been subjected to a metathesis reaction in which the iodide anion has been displaced by different anions. The anion exchange reactions were carried out by their refluxing with different metal salts such as NaBF 4 , KPF 6 , NaOCOCF 3 , NaSCN, or NaNO 3 , in acetonitrile as solvent for 16 h to give new specific-based hydrazones 11-40 in 80%-98% yields (Scheme 2). Further assignment of the diastereomer formation for compound 9 was supported by 13 C NMR and dept-135 experiments. In the 13 C NMR spectrum, each peak of compound 9 appeared as two sets of signals due to the presence of the diastereomeric mixture. In the aliphatic region, the methyl and NCH2 carbons resonated as two sets of signals at 61.43 and 61.51, respectively. The C=N and C=O groups of the E/cis and E/trans diastereomers also resonated as double peaks at δC 159.27-165.72 ppm.
The 19 F NMR spectrum (Figure 3) also proved the formation of a diastereomeric mixture (E/cis and E/trans) through the appearance of two characteristic multiplets at δF −109.90 to −109.82 and −109.44 to −109.36 ppm, attributed to the aromatic fluorine atom. The structure of compound 9 was also confirmed by the electron impact mass spectrum, which showed a molecular ion peak at 459.38 [M + ].
All the newly synthesized iodonated pyridinium salts 5-10 have been subjected to a metathesis reaction in which the iodide anion has been displaced by different anions. The anion exchange reactions were carried out by their refluxing with different metal salts such as NaBF4, KPF6, NaOCOCF3, NaSCN, or NaNO3, in acetonitrile as solvent for 16 h to give new specific-based hydrazones 11-40 in 80%-98% yields (Scheme 2). Scheme 2. Synthesis of specific-based hydrazones 11-40. When these reactions were assisted by ultrasound irradiation, 6 h were required to afford comparable yields of the same ILs (Table 2). The analysis of the NMR spectra of compounds 11-40 revealed that their 1 H and 13 C NMR are practically the same as those recorded for their precursors 5-10, with the isomeric splitting pattern. Accordingly, the 31 P NMR, 19 F NMR and mass spectra analyses have supported the success of the metathesis reaction. In the 31 P NMR spectrum of compound 31, the appearance of a characteristic multiplet signal at δ P´1 57.37 to´131.02 ppm confirms the presence of the PF 6´a nion. In addition, its 19 F NMR spectrum displays two characteristic singlets at δ F´7 1.10 and´69.22 ppm, confirming the presence of a fluorine atom in its PF 6´f orm, while the aromatic fluorine atom was assigned as two multiplets at δ F (´109.90 to´109.82) ppm and (´109.44 to´109.36) ppm. In addition, the presence of a molecular ion peak at 473.39 [M + ] in its mass spectrum supports the structure of compound 31. The iodide anion exchange of 9 using NaBF 4 as a metal salt led to the formation of compound 32, with its structure supported by its 11 B NMR and 19 F NMR spectra. The appearance of a characteristic doublet at δ B´1 .28 ppm in the 11 B NMR spectrum confirmed the incorporation of the boron anion in its structure.
The 19 F NMR spectrum displays two doublets at δ F´1 48.30 and´145.25 ppm, attributed to the fluorine anion (BF 4´) , while the aromatic fluorine was recorded at δ F´1 09.90 to´109.82 ppm and 109.45 to´109.37 ppm as two multiplets. The structure of IL 32 has also been established based on its electron impact mass spectrum, which shows a molecular ion peak at 415. 22 [M + ]. The anion exchange with trifluoroacetate has also been investigated and gave IL 33, as was also confirmed by its 19 F NMR spectrum, which clearly shows a singlet at δ F´7 3.50 ppm due to the CF 3 COO´anion. The aromatic fluorine atom resonated at the expected area. The mass spectral data reveals the presence of the molecular ion peak at 441.18 [M + ] as evidence for the formation of compound 33.
Because 34 and 35 carrying NO 3´a nd/or SCN´anion head-groups display similar 1 H and 13 C NMR spectra compared to their precursor 9, their formation becomes more evident based on their mass spectra. The mass spectra of compounds 34 and 35 display molecular ion peaks at 390.37 [M + ] and 386.56 [M + ], respectively.

Antimicrobial Activity
Compounds 5-15, 21-25 and 31-40 were assessed in vitro for their efficacy as antimicrobial agents by the minimum inhibitory concentration (MIC) using the broth dilution method [19,20] against six standard bacterial strains (Streptococcus pneumonia, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeuroginosa, Escherichia coli and Klebsiella pneumonia) and two fungi (Aspergillus fumigatus and Candida albicans). The MIC results are summarized in Table 3.
The antibacterial activity screening for the halogenated pyridinium salts 5-10 against all of the bacterial strains demonstrated that all the compounds reveal promising antibacterial activities, with an MIC range of 8-16 µg/mL. In contrast, the opposite result was observed for the two fungal species, towards which the compounds showed no activity.
From the antibacterial activity results of compounds 11-40, it can be stated that those resulting from the metathetical anion exchange with fluorinated metal salts (PF 6´, BF 4´o r CF 3 COO´) are more effective against all bacterial strains at an MIC of 4-8 µg/mL.
The antifungal bioassay results summarized in Table 3 reveal that, among the tested salts 11-40, compounds 21-23, 31-33 and 36-38 show good to excellent potency against all of the tested fungal strains, with an MIC range of 8-16 µg/mL. In fact, the highest antifungal activity, with an MIC of 8 µg/mL, was exhibited by compounds 33 and 38, possessing a CF 3 COO´counter anion and a C 6 to C 7 alkyl chain in the cation head group.
The antimicrobial activity and structure activity relationship reveal that the promising activity displayed by the halogenated 5-10 against all of the bacterial strains is presumably due to the chain length. The incorporation of a fluorine atom was found to dramatically increase the antimicrobial activity, as exhibited by the fluorinated 11-13, 21-23, 31-33 and 36-38 carrying PF 6´, BF 4´a nd/or CF 3 COO´. In addition, the metathetic exchange with these fluorinated metal salts resulted in higher antifungal activity.
In the antimicrobial screening, it was observed that compounds with long alkyl side chains possessing a fluorine atom in their anion head-group (PF 6´, BF 4´a nd CF 3 COO´) exhibit excellent activity compared to the corresponding halogenated precursors against all of the bacterial strains, indicating the influence of the presence of the fluorine atom in the structure of the ionic liquids.

Antiproliferative Activity
An in vitro evaluation of the antiproliferative activities of the newly synthesized compounds was investigated against four human tumor cell lines by using the protocol described in ISO 10993-5 [21]. The results are presented as IC 50˘S D values (Table 4). Each experiment was repeated three times. IC 50 concentrations were obtained from the dose-response curves using Graph Pad Prism Software 5. Only the compounds shown in Table 4 demonstrated a measurable IC 50 against the tested cancer cell lines and thus can be used as model compounds for the construction of novel anticancer drugs. Interestingly, reducing the chain length of the compounds yielded more potent cytotoxic activities, suggesting a steric factor mediating either transport or molecular interaction with the cellular targets.

General
Melting points were recorded on a Stuart Scientific SMP1 apparatus (Stuart, Red Hill, UK) and are uncorrected. The IR spectra were recorded using an SHIMADZU FTIR-8400S spectrometer (SHIMADZU, Boston, MA, USA). The NMR spectra were measured with a Bruker spectrometer (400 and 600 MHz, Brucker, Fällanden, Switzerland) using Tetramethylsilane (TMS) (0.00 ppm) as an internal standard. The ESI and EI mass spectra were measured by Finnigan LCQ and Finnigan MAT 95XL spectrometers (Finnigan, Darmstadt, Germany), respectively. Ultrasound-assisted reactions were performed in a Kunshan KQ-250B ultrasound cleaner (50 KHz, 240 W, Kunshan Ultrasonic Instrument, Kunshan, China).

Ultrasound Method
A mixture of isonicotinic acid hydrazide (1) (1 mmol) in ethanol (25 mL) and 4-fluorobenzaldehyde (2) (1.2 mmol) with a few drops of hydrochloric acid was irradiated by ultrasound irradiation for 30 min at room temperature. The reaction proceeded as described above to furnish the same compound 3.

Conventional Method
A mixture of compound 3 (1 mmol) and the appropriate alkyl iodide with chain lengths ranging from C 2 to C 7 (1.5 mmol) in acetonitrile (30 mL) was refluxed for 72 h. After cooling, the solvent was removed under reduced pressure, and the solid formed was collected by filtration, washed with acetonitrile, and crystallized from dichloromethane to afford the desired pyridinium hydrazones 5-10.

Ultrasound Method
A mixture of compounds 5-10 (1 mmol) in acetonitrile (8 mL) and potassium hexafluorophosphate, sodium tetrafluoroborate, sodium nitrate, sodium thiocyanate and/or sodium trifluoroacetate (1.2 mmol) was irradiated by ultrasound irradiation for 5-6 h at room temperature. The reaction proceeded as described above to afford the same compounds 11-40.