Novel amphiphilic pyridinium ionic liquids-supported Schiff bases: ultrasound assisted synthesis, molecular docking and anticancer evaluation

Background Pyridinium Schiff bases and ionic liquids have attracted increasing interest in medicinal chemistry. Results A library of 32 cationic fluorinated pyridinium hydrazone-based amphiphiles tethering fluorinated counteranions was synthesized by alkylation of 4-fluoropyridine hydrazone with various long alkyl iodide exploiting lead quaternization and metathesis strategies. All compounds were assessed for their anticancer inhibition activity towards different cancer cell lines and the results revealed that increasing the length of the hydrophobic chain of the synthesized analogues appears to significantly enhance their anticancer activities. Substantial increase in caspase-3 activity was demonstrated upon treatment with the most potent compounds, namely 8, 28, 29 and 32 suggesting an apoptotic cellular death pathway. Conclusions Quantum-polarized ligand docking studies against phosphoinositide 3-kinase α displayed that compounds 2–6 bind to the kinase site and form H-bond with S774, K802, H917 and D933. Electronic supplementary material The online version of this article (10.1186/s13065-018-0489-z) contains supplementary material, which is available to authorized users.


Introduction
Schiff bases have been widely investigated due to a broad spectrum of relevant properties in biological and pharmaceutical areas [1]. In addition, a number of molecules having azomethine Schiff base skeleton are the clinically approved drugs [2]. Meanwhile, carbohydrazide hydrazone and their derivatives an interesting class of Schiff bases, represented reliable and highly efficient pharmacophores in drug discovery and played a vital role in medical chemistry due to their potency to exhibit significant antimicrobial [3], anticancer [4,5], anti-HIV [6], and anticandidal [7] activities. Azomethine hydrazone linkages (RCONHN=CR 1 R 2 ) are one of the versatile and attractive functional groups in organic synthesis [8,9]. Their ability to react with electrophilic and nucleophilic reagents make them valuable candidates for the construction of diverse heterocyclic scaffolds [10]. Some pyridine hydrazones have been reported to possess fascinating chemotherapeutic properties [11,12]. On the other hand, biological and toxicity of pyridinium salts have been well documented due to their increasing applications. More specifically, pyridinium salts carrying long alkyl chains were found to be outstanding bioactive agents as antimicrobial [13], anticancer [14] and biodegradable [15] agents. Recently, we have reported a green ultrasound synthesis of novel fluorinated pyridinium hydrazones using a series of alkyl halides ranging from C2 to C7 [16]. The biological screening results revealed that the activity increased with increasing the length of the alkyl side chains, especially for hydrazones tethering fluorinated counteranions (PF 6 − , BF 4 − and CF 3 COO − ).
Encouraged by these findings and in continuation of our efforts in designing highly active heterocyclic hydrazones [17][18][19], we aim to introduce a lipophilic long alkyl chain to a hydrazone skeleton to develop a new class of bioactive molecules. In the present work, a series of novel cationic fluorinated pyridinium hydrazone-based amphiphiles tethering different fluorinated counteranions were designed, synthesized and screened for their anticancer activities against four different cell lines. Additionally, their activities were further characterized via investigating the Caspase-3 signaling pathway, a hallmark of apoptosis that is commonly studied to understand the mechanism of cellular death. Molecular quantum-polarized ligand docking (QPLD) studies were carried out employing MAESTRO [20] software against the kinase domain of phosphoinositide 3-kinase α (PI3Kα) [21] to identify their structural-basis of binding and ligand/receptor complex formation.

Synthesis
The methodology for affecting the sequence of reactions utilized ultrasound irradiations which have been widely used by our team as an alternative source of energy. Starting from fluorinated pyridine hydrazone 1, the quaternization of pyridine ring through its conventional alkylation with various long alkyl iodide with chain ranging from C 8 to C 18 , in boiling acetonitrile as well as under ultrasound irradiation and gave the desired cationic fluorinated pyridinium hydrazones 2-9 tethering lipophilic side chain and iodide counteranion in good yields (Scheme 1). Short reactions time were required (10-12 h) when the ultrasound irradiations were used as an alternative energy source ( Table 1).
The structure of newly designed pyridinium cationic surfactants 2-9 have been elucidated based on their spectral data (IR, NMR, Mass). Their IR spectra revealed the appearance of new characteristic bands at 2870-2969 cm −1 attributed to the aliphatic C-H stretching which confirmed the presence of alkyl side chain in this structure. The 1 H NMR analysis showed one methyl and methylene groups resonating as two multiplets between δ H 0.74-0.87 ppm and 1.16-1.32 ppm, respectively. The spectra also showed the presence of characteristic triplet and/or doublet of doublet ranging between δ H 4.68-4.78 ppm assigned to NCH 2 protons.
In addition, the imine proton (H-C=N) resonated as two set of singlets at δ H 8.15-8.50 ppm with a 1:3 ratio. The presence of such pairing of signals confirmed that these compounds exist as E/cis and E/trans diastereomers.
The 13 C NMR data also confirmed the appearance of E/cis and E/trans diastereomers through the presence of two peaks at δ H 58.60 and 62.74 ppm for NCH 2 . In the downfield region between δ C 156.38-165.76 ppm, the carbonyl and the imine carbons of the hydrazone linkage resonated as two sets of signals.
Treatment of the halogenated pyridinium hydrazones 2-9 with fluorinated metal salts (KPF 6 , NaBF 4 or NaOOCCF 3 ) afforded the targeted cationic amphiphilic fluorinated pyridinium hydrazones 10-33 carrying variant fluorinated counteranions (Scheme 2). The reaction involved the anion exchange and was carried out in short time (6 h) under ultrasound irradiation and gave comparative yields with those obtained using classical heating (16 h) ( Table 2).
Structural differentiation between the metathetical products 10-33 and their halogenated precursors 2-9 was very difficult on the basis of their 1 H NMR and 13 C NMR spectra because they displayed virtually the same characteristic proton and carbon signals.
Consequently, other spectroscopic techniques ( 19 F, 31  The physical (state of product and melting points) and photochemical (fluorescence and λ max in UV) data of the synthesized pyridinium hydrazones 2-33 were investigated and recorded in Table 3.

Biological results
Attempting to characterize any potential biological activity associated with the newly synthesized compounds, an in vitro assessment of their antiproliferative activity was conducted on four different human cancerous cell lines; the human breast adenocarcinoma (MCF-7), human breast carcinoma (T47D), human colon epithelial (Caco-2) and human uterine cervical carcinoma (Hela) cell lines. Only compounds shown in Table 4 demonstrated a reasonably high antiproliferative activity against the model cancer cell lines used.
Remarkably, increasing the length of the hydrophobic chain appears to significantly potentiate the antiproliferative activities associated with the examined analogues, probably owing to their better penetration into the cellular compartment.
To determine the apoptotic effects of cytotoxic compounds and to evaluate modulators of the cell death cascade, activation of the caspase-3 pathway, a hallmark of apoptosis, can be employed in cellular assays. According to the demonstrated results ( Fig. 1) and in response to 48 h treatment with the most potent compounds, significant increase in caspase-3 activity is yielded suggesting that the antiproliferative activities of the examined compounds are most likely mediated by an apoptotic cellular death pathway.
Further exploration of possible pathways by which these compounds exert their antiproliferative activities should shed light onto prospective molecular targets with which the compounds may interrelate.
In order to identify the structural-basis of PI3Kα/ ligand interaction of the verified compounds in the catalytic kinase domain of PI3Kα, we employed QPLD docking [40,41] against the kinase cleft of 2RD0. Our QPLD docking data show that some of the synthesized molecules 2-9 bind to the kinase domain of PI3Kα (Fig. 2, part a). Indeed, compounds having side chain alkyl group more than twelve carbon atoms 7-9 extend beyond the binding cleft boundary.
Moreover, a part of the docked pose of 2 superposes that of the co-crystalized ligand (Fig. 2, part b).
Some of key binding residues are shown and H atoms are hidden for clarity purpose. Picture is captured by PYMOL. The backbones of 2-9 tend to form H-bond with S774, K802, H917, and D933 (Table 5) (Fig. 3). Additionally, 2-9 showed comparable QPLD binding affinity thus referring that the flexibility of the side-chain carbon atoms might ameliorate the steric effect. Other computational [41][42][43][44][45] and experimental studies [21] reported the significance of these residues in PI3Kα/ligand formation.
Noticing that the whole synthesized compounds, 2-18 and 22-23, share the core nucleus but differs in the sidechain carbon atoms number as well as the counterpart anion, for example 2 matches 10, 11, and 12. It's worth noting that the effect of salt enhances compound solubility and assists for better biological investigation.
Contrarily, in silico modeling neglects the effect of the counterpart anion thus we carried out the docking studies for 2-9 as representative models for the whole dataset. Figure 4 shows that there is a positive correlation factor (R 2 = 0.828) between the QPLD docking scores against PI3Kα and IC 50 .
In order to get further details about the functionalities of 2-9, we screened them against a reported PI3Kα inhibitor pharmacophore model [42]. The verified compounds 2-9 sparingly match the fingerprint of active PI3Kα inhibitors; three out of five functionalities for 2-9 ( Fig. 5a, b) whereas two out of five functionalities for 6-9 ( Fig. 5c, d). This finding explains their moderate to weak PI3Kα inhibitory activity and recommends optimizing the core skeleton of this library aiming to improve the biological activity. Strikingly, the biological activity of 8-9 would suggest that the hydrophobicity of the attached alkyl group as well as the lipid membrane solubility parameter might affect their attachment to the cell line membrane.
In order to evaluate the performance of QPLD program, we compared the QPLD-docked pose of KWT in the mutant H1047R PI3Kα (PDB ID: 3HHM) [46] to its native conformation in the crystal structure. Figure 6 shows the superposition of the QPLD-generated KWT pose and the native conformation in 3HHM. The RMSD for heavy atoms of KWT between QPLD-generated docked pose and the native pose was 0.409 Å. This demonstrates that QPLD dock is able to reproduce the native conformation in the crystal structure and can reliably predict the ligand binding conformation.

Apparatus and analysis
The Stuart Scientific SMP1 apparatus (Stuart, Red Hill, UK) was used in recording of the uncorrected melting points.
The Finnigan LCQ and Finnigan MAT 95XL spectrometers (Finnigan, Darmstadt, Germany) were used in the ESI and EI measurement, respectively.

General alkylation procedure for the synthesis of cationic amphiphilic fluorinated pyridinium hydrazones 2-9 Conventional method (CM)
To a mixture of pyridine hydrazone 1 (1 mmol) in acetonitrile (30 ml) was added an appropriate long alkyl iodides with chain ranging from C 8 to C 18 (1.5 mmol) under stirring. The mixture was refluxed for 72 h, then the solvent was reduced under pressure. The obtained solid was collected by filtration and washed with acetonitrile to give the target ILs 2-9.

Ultrasound method (US)
To a mixture of pyridine hydrazone 1 (1 mmol) in acetonitrile (30 ml) was added an appropriate long alkyl iodides with chain ranging from C 8 to C 18 (1.5 mmol) under stirring. The mixture was irradiated by ultrasound irradiation for 10-12 h. The reaction was processed as described above to give the same target ILs 2-9.  13 19 13 19 (7) 13 C=N, C=O). 19 13 C=N, C=O). 19 1H, H-C=N C=N, C=O). 19  General metathesis procedure for the synthesis of pyridinium hydrazones 10-33

Conventional method (CM)
A mixture of equimolar of IL 2-9 (1 mmol) and fluorinated metal salt (KPF 6 , NaBF 4 and/or NaCF 3 COO) (1 mmol) in dichloromethane (15 ml) was heated under reflux for 12 h. After cooling, the solid formed was collected by extraction and/or by filtration. The solid was washed by dichloromethane to afford the task-specific ILs 10-33.

Ultrasound method (US)
A mixture of equimolar of IL 2-9 (1 mmol) and fluorinated metal salt (KPF 6 , NaBF 4 and/or NaCF 3 COO) (1 mmol) in dichloromethane (15 ml) was irradiated by ultrasound irradiation for 6 h. The reaction was processed as described above to give the same task-specific ILs 10-33.