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(2018) 12:118 Al‑Blewi et al. Chemistry Central Journal https://doi.org/10.1186/s13065-018-0489-z RESEARCH ARTICLE Chemistry Central Journal Open Access Novel amphiphilic pyridinium ionic liquids‑supported Schiff bases: ultrasound assisted synthesis, molecular docking and anticancer evaluation Fawzia Faleh Al‑Blewi1, Nadjet Rezki1,2*, Salsabeel Abdullah Al‑Sodies1, Sanaa K. Bardaweel3, Dima A. Sabbah4, Mouslim Messali1 and Mohamed Reda Aouad1* Abstract 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 coun‑ teranions 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 synthe‑ sized 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 apop‑ totic cellular death pathway. Conclusions: Quantum-polarized ligand docking studies against phosphoinositide 3-kinase α displayed that com‑ pounds 2–6 bind to the kinase site and form H-bond with S774, K802, H917 and D933. Keywords: Cationic, Amphiphilic, Pyridinium, Hydrazones, Ultrasound, Anticancer, QPLD docking 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=CR1R2) are one of the versatile and *Correspondence: nadjetrezki@yahoo.fr; aouadmohamedreda@yahoo.fr 1 Department of Chemistry, Faculty of Science, Taibah University, Al‑Madinah Al‑Munawarah, Medina 30002, Saudi Arabia Full list of author information is available at the end of the article 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 ­(PF6−, ­BF4− and ­CF3COO−). © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Encouraged by these findings and in continuation of our efforts in designing highly active heterocyclic hydrazones [17–19], 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. Results and discussion 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 ­C8 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 1H NMR analysis showed one methyl and Page 2 of 18 methylene groups resonating as two multiplets between δH 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 δH 4.68– 4.78 ppm assigned to NCH2 protons. In addition, the imine proton (H–C=N) resonated as two set of singlets at δH 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 13C NMR data also confirmed the appearance of E/cis and E/trans diastereomers through the presence of two peaks at δH 58.60 and 62.74 ppm for NCH2. In the downfield region between δC 156.38–165.76 ppm, the carbonyl and the imine carbons of the hydrazone linkage resonated as two sets of signals. In their 19F NMR spectra, the aromatic fluorine atom appeared as two mutiplet signals between δH (− 107.98 to − 109.89 ppm) and (− 107.72 to − 109.37 ppm). Treatment of the halogenated pyridinium hydrazones 2–9 with fluorinated metal salts ­(KPF6, ­NaBF4 or ­NaOOCCF3) 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 1H NMR and 13C NMR spectra because they displayed virtually the same characteristic proton and carbon signals. Consequently, other spectroscopic techniques (19F, 31P, 11 B NMR and mass spectroscopy) have been adopted to confirm the presence of fluorinated counteranions ­(PF6−, ­BF4− and ­CF3COO−) in the structure of the resulted ILs 10–33. Thus, the presence of ­PF6− in ILs 10, 13, 16, 19, 22, 25, 28 and 31 has been established by their 31P and 19 F NMR analysis. Thus, the resonance of a diagnostic Scheme 1 Synthesis of pyridinium hydrazones 2–9 carrying iodide counter anion Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 3 of 18 Table 1 Times and yields of halogenated pyridinium hydrazones 2–9 under conventional and ultrasound Compound no R Conventional method CM Ultrasound method US Time (h) Yield (%) Time (h) Yield (%) 2 C8H17 72 84 10 92 3 C9H19 72 90 10 96 4 C10H21 72 88 12 92 5 C11H23 72 92 12 98 6 C12H25 72 88 12 92 7 C14H29 72 85 12 92 8 C16H33 72 89 12 94 9 C18H37 72 83 12 96 multiplet between δP − 152.70 and − 135.76 ppm in the 31 P NMR spectra confirmed the presence of ­PF6− in their structure. On the other hand, the 19F NMR analysis of the same compounds revealed the appearance of new doublet at δF − 70.39 and − 69.21 ppm attributed to the six fluorine atoms in P ­ F6− anions. The formation of ionic liquids 11, 14, 17, 20, 23, 26, 29 and 32 carrying ­BF4− in their structures were supported by the 11B and 19F NMR experiments. Thus, their 11B NMR spectra exhibited a multiplet between δB − 1.30 and − 1.29 ppm confirming the presence of boron atom in its ­BF4− form. Two doublets were recorded at δF − 149.12 and − 148.12 ppm in their 19F NMR spectra. Structural elucidation of the ionic liquids containing trifluoroacetate (CF3COO−) was investigated by the 19F NMR analysis which revealed the presence of characteristic singlet ranging from − 73.50 to − 75.30 ppm. The physical (state of product and melting points) and photochemical (fluorescence and λmax 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 (Caco2) 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. Docking results In order to explain the anticancer activity of the verified compounds 2–9 against the examined cancer cell lines, we recruited the crystal structure of PI3Kα (PDB ID: 2RD0) [21] to determine the binding interaction of these compounds in PI3Kα kinase domain. Noting that these cell lines express phosphatidylinositol 3-kinase (PI3Kα) particularly MCF-7 [22–26], T47D [22, 25–32], Caco-2 [33–35] and Hela [36–38]. The binding site of 2RD0 is composed of M772, K776, W780, I800, K802, L807, D810, Y836, I848, E849, V850, V851, S854, T856, Q859, M922, F930, I932 and D933 [39]. The hydrophobic and polar residues are located in the binding domain. It’s worth noting that the exposed hydrophilic and hydrophobic surface areas of the cocrystallized ligand agree with the surrounding residues. Scheme 2 Synthesis of pyridinium hydrazones 10–33 carrying fluorinated counteranions Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 4 of 18 Table 2 Times and yields of pyridinium hydrazones 10–33 carrying fluorinated counter anions under conventional and ultrasound Compound no R Y Conventional method CM Ultrasound method US Time (h) Yield (%) Time (h) Yield (%) 10 C8H17 PF6 16 83 6 90 11 C8H17 BF4 16 98 6 98 12 C8H17 COOCF3 16 80 6 88 13 C9H19 PF6 16 90 6 94 14 C9H19 BF4 16 85 6 90 15 C9H19 COOCF3 16 87 6 92 16 C10H21 PF6 16 98 6 98 17 C10H21 BF4 16 88 6 90 18 C10H21 COOCF3 16 86 6 92 19 C11H23 PF6 16 94 6 98 20 C11H23 BF4 16 93 6 94 21 C11H23 COOCF3 16 90 6 94 22 C12H25 PF6 16 87 6 90 23 C12H25 BF4 16 82 6 90 24 C12H25 COOCF3 16 88 6 92 25 C14H29 PF6 16 95 6 98 26 C14H29 BF4 16 93 6 96 27 C14H29 COOCF3 16 97 6 98 28 C16H33 PF6 16 89 6 92 29 C16H33 BF4 16 90 6 94 30 C16H33 COOCF3 16 88 6 92 31 C18H37 PF6 16 88 6 92 32 C18H37 BF4 16 87 6 90 33 C18H37 COOCF3 16 84 6 90 The polar residues furnish hydrogen-bonding, ion–dipole and dipole–dipole interactions. Furthermore, the polar acidic or basic residues mediate an ionic (electrostatic) bonding. The nonpolar motif such as the aromatic and/or hydrophobic residue affords π-stacking aromatic and hydrophobic (van der Waals) interaction, respectively. 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 [41–45] 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 ­(R2 = 0.828) between the QPLD docking scores against PI3Kα and ­IC50. 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 Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 5 of 18 Table 3 Physical and analytical data for the newly synthesized pyridinium hydrazones 2–33 Comp R no Y mp °C λmax (nm) Fluorescence 2 C8H17 I 104–105 222, 330, 430 + 3 C9H19 I 91–93 220, 332, 432 + 4 C10H21 I 110–112 220, 332, 430 + 5 C11H23 I 82–83 220, 332, 430 + 6 C12H25 I 72–73 220, 330, 430 + 7 C14H29 I 86–88 220, 332, 430 + 8 C16H33 I 78–80 220, 332, 430 + 9 C18H37 I 98–99 220, 332, 430 + 10 C8H17 PF6 Yellow crystals 64–65 220, 330, 430 + 11 C8H17 BF4 Yellow crystals 80–82 220, 332, 430 + 12 C8H17 COOCF3 Yellow crystals 74–76 220, 332, 430 + 13 C9H19 PF6 Yellow crystals 69–70 220, 330, 428 + 14 C9H19 BF4 Yellow crystals 88–90 222, 328, 426 + 15 C9H19 COOCF3 Yellow crystals 96–98 222, 332, 424 + 16 C10H21 PF6 Yellow syrup 220, 330, 428 + 17 C10H21 BF4 Colorless syrup 220, 330, 428 + 18 C10H21 COOCF3 Yellow syrup 222, 334, 432 + 19 C11H23 PF6 Yellow syrup 220, 330, 428 + 20 C11H23 BF4 Yellow syrup 220, 330, 426 + 21 C11H23 COOCF3 Colorless syrup 222, 332, 430 + 22 C12H25 PF6 Yellow syrup 222, 330, 430 + 23 C12H25 BF4 Yellow syrup 218, 332, 430 + 24 C12H25 COOCF3 Colorless syrup 220, 336, 428 + 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. Experimental Apparatus and analysis The Stuart Scientific SMP1 apparatus (Stuart, Red Hill, UK) was used in recording of the uncorrected melting points. The SHIMADZU FTIR-8400S spectrometer (SHIMADZU, Boston, MA, USA) was used on the IR measurement. The Bruker spectrometer (400 and 600 MHz, Brucker, Fällanden, Switzerland) was used in the NMR analysis using Tetramethylsilane (TMS) (0.00 ppm) as an internal standard. The Finnigan LCQ and Finnigan MAT 95XL spectrometers (Finnigan, Darmstadt, Germany) were used in the ESI and EI measurement, respectively. The Kunshan KQ-250B ultrasound cleaner (50 kHz, 240 W, Kunshan Ultrasonic Instrument, Kunshan, China) was used for carrying out all reactions. 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. Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 6 of 18 Ultrasound method (US) Table 3 (continued) Comp R no Y mp °C λmax (nm) Fluorescence 25 C14H29 PF6 Yellow syrup 220, 332, 428 + 26 C14H29 BF4 Yellow syrup 220, 336, 430 + 27 C14H29 COOCF3 Colorless syrup 220, 330, 428 + 28 C16H33 PF6 Yellow syrup 220, 338, 432 + 29 C16H33 BF4 Yellow syrup 218, 332, 428 + 30 C16H33 COOCF3 Colorless syrup 220, 334, 430 + 31 C18H37 PF6 Yellow syrup 220, 330, 428 + 32 C18H37 BF4 Yellow syrup 220, 330, 432 + 33 C18H37 COOCF3 Colorless syrup 220, 332, 430 + Table 4 IC50 values (µM) on 4 different cancer cell lines Code 4 5 6 7 8 9 16 17 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 MCF-7 153 ± 12 136 ± 7 134 ± 9 T47D 145 ± 10 134 ± 10 139 ± 7 Caco-2 156 ± 9 139 ± 9 139 ± 9 Hela 155 ± 11 142 ± 6 129 ± 11 120 ± 6 123 ± 7 128 ± 7 119 ± 8 20 ± 3 23 ± 4 18 ± 3 25 ± 3 61 ± 5 179 ± 15 176 ± 12 137 ± 8 132 ± 4 178 ± 10 129 ± 4 128 ± 10 131 ± 10 134 ± 10 59 ± 7 172 ± 13 170 ± 10 133 ± 11 139 ± 9 176 ± 19 129 ± 8 120 ± 9 139 ± 6 133 ± 9 67 ± 6 171 ± 19 168 ± 12 139 ± 6 134 ± 5 171 ± 15 125 ± 9 121 ± 14 145 ± 7 132 ± 5 68 ± 5 177 ± 10 177 ± 11 141 ± 10 138 ± 5 169 ± 17 124 ± 13 128 ± 11 132 ± 12 131 ± 9 123 ± 10 127 ± 15 127 ± 12 129 ± 11 39 ± 5 40 ± 6 32 ± 4 36 ± 4 67 ± 4 21 ± 3 45 ± 6 71 ± 3 39 ± 7 41 ± 5 61 ± 2 20 ± 4 46 ± 4 77 ± 8 34 ± 4 48 ± 7 67 ± 4 19 ± 1 41 ± 3 74 ± 5 38 ± 7 44 ± 3 Values are expressed as mean ± SD of three experiments 68 ± 6 26 ± 2 48 ± 6 79 ± 2 35 ± 7 49 ± 5 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. 4‑(2‑(4‑Fluorobenzylidene) hydrazinecarbonyl)‑1‑oc‑ tylpyridin‑1‑ium iodide (2) It was obtained as yellow crystals; mp: 104–105 °C. FT-IR (KBr), ­cm−1: ῡ = 1595 (C=N), 1670 (C=O), 2870, 2960 (Al–H), 3071 (Ar–H). 1 H NMR (400 MHz, DMSO-d6): δH = 0.83–0.87 (m, 3H, CH3), 1.25–1.32 (m, 10H, 5× CH2), 1.94–1.99 (m, 2H, ­N CH2CH2), 4.68 (t, 2H, J = 8 Hz, NCH2), 7.22 (t, 0.5H, J = 8 Hz, Ar–H), 7.34 (t, 1.5H, J = 8 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.88 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H–C=N), 8.39 (d, 0.5H, J = 4 Hz, Ar–H), 8.50 (s, 0.75H, H–C=N), 8.53 (d, 1.5H, J = 8 Hz, Ar–H), 9.25 (d, 0.5H, J = 8 Hz, Ar–H), 9.33 (d, 1.5H, J = 4 Hz, Ar–H), 12.47 (bs, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 13.89 (CH3), 21.99, 25.36, 25.41, 28.30, 28.40, 30.50, 30.63, 31.08 (6×CH2), 60.95, 61.02 (NCH2), 115.74, 115.95, 116.17, 126.14, 127.11, 129.36, 129.44, 129.73, 129.81, 130.21, 130.24, 145.08, 145.67, 147.33, 149.36, 149.63 (Ar–C), 158.76, 162.28, 164.75, 165.21 (C=N, C=O). 19 F NMR (377 MHz, DMSO-d6): δF = (− 109.72 to − 109.65), (− 109.20 to − 109.12) (2m, 1F, Ar–F). MS (ES) m/z = 483.32 ­[M+]. 4‑(2‑(4‑Fluorobenzylidene) hydrazinecarbonyl)‑1‑non‑ ylpyridin‑1‑ium iodide (3) It was obtained as yellow crystals; mp: 91–93 °C. FT-IR (KBr), c­m−1: ῡ= 1598 (C=N), 1682 (C=O), 2872, 2969 (Al–H), 3078 (Ar–H). 1 H NMR (400 MHz, DMSO-d6): δH = 0.83–0.87 (m, 3H, CH3), 1.25–1.32 (m, 12H, 6× CH2), 1.94–1.99 (m, 2H, ­NCH2CH2), 4.69 (dd, 2H, J = 4 Hz, 8 Hz, NCH2), 7.25 (dd, 0.5H, J = 8 Hz, 12 Hz, Ar–H), 7.37 (dd, 1.5H, J = 8 Hz, 12 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.89 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.15 (s, 0.25H, H–C=N), 8.40 (d, 0.5H, J = 8 Hz, Ar–H), 8.50 (s, 0.75H, H–C=N), 8.53 (d, 1.5H, J = 8 Hz, Ar–H), 9.25 (d, 0.5H, J = 8 Hz, Ar–H), 9.33 (d, 1.5H, J = 8 Hz, Ar–H), 12.46 (s, 0.75H, CONH), 12.51 (s, 0.25H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 13.92 (CH3), 22.03, 25.36, 25.41, 28.35, 28.52, 28.70, 30.51, 30.64, 31.18 (7×CH2), 60.93, 61.01 (NCH2), 115.74, 115.96, 116.18, 126.15, 127.11, 129.35, 129.43, 129.73, 129.82, 130.20, 130.23, 145.06, 145.69, 147.31, 149.33, 149.64 (Ar–C), 158.75, 162.28, 164.76, 165.23 (C=N, C=O). 19F NMR (377 MHz, DMSO-d6): δF = (− 109.94 to − 109.86), (− 109.42 to − 109.34) (2m, 1F, Ar–F). MS (ES) m/z = 497.10 ­[M+]. Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 7 of 18 140 Table 5 The QPLD docking scores (Kcal/mol) and H-bond interactions between the verified compounds 2–9 and PI3Kα 120 Compound no Chamges in Caspase-3 acvity (%) 160 100 Control 80 100 uM 60 4 5 40 6 20 0 2 3 7 8 SI16 SS7 SS8 SS14 Fig. 1 Caspase3 activity in MCF7 cells after 48 h. The results are the means of two independent experiments. P < 0.05 was considered significant 1‑Decyl‑4‑(2‑(4‑fluorobenzylidene) hydrazinecarbonyl) pyridin‑1‑ium iodide (4) It was obtained as yellow crystals; mp: 110–112 °C. FT-IR (KBr), c­ m−1: ῡ = 1615 (C=N), 1690 (C=O), 2873, 2966 (Al–H), 3074 (Ar–H). 1 H NMR (400 MHz, DMSO-d6): δH = 0.83–0.87 (m, 3H, CH3), 1.25–1.32 (m, 14H, 7× CH2), 1.94–1.99 (m, 2H, ­N CH2CH2), 4.68 (t, 2H, J = 8 Hz, NCH2), 7.23 (t, 0.5H, J = 8 Hz, Ar–H), 7.38 (dd, 1.5H, J = 8 Hz, 12 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.89 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H– C=N), 8.40 (d, 0.5H, J = 4 Hz, Ar–H), 8.50 (s, 0.75H, H–C=N), 8.54 (d, 1.5H, J = 8 Hz, Ar–H), 9.25 (d, 0.5H, J = 4 Hz, Ar–H), 9.34 (d, 1.5H, J = 8 Hz, Ar–H), 12.48 (bs, 1H, CONH). 13C NMR (100 MHz, DMSOd6): δC = 12.40, 12.42 (CH3), 20.55, 23.85, 23.89, 26.84, 27.11, 27.24, 27.28, 27.32, 28.99, 29.13, 29.72 (8×CH2), 59.42, 59.49 (NCH2), 114.24, 114.46, 114.68, 124.63, 125.59, 127.84, 127.92, 128.22, 128.31, 128.55, 128.68, 128.71, 143.54, 144.18, 145.78, 147.80, 148.12 (Ar–C), 9 Docking score (Kcal/mol) − 6.03 − 5.93 − 5.78 − 6.16 − 5.69 − 5.68 − 5.36 − 4.58 H-bond K802 K802 D933 H917, D933 S774, D933 NA K802 NA 157.25, 160.77, 163.24, 163.73 (C=N, C=O). 19F NMR (377 MHz, DMSO-d6): δF = (− 109.94 to − 109.85), (− 109.42 to − 109.34) (2m, 1F, Ar–F). MS (ES) m/z = 511.05 ­[M+]. 4‑(2‑(4‑Fluorobenzylidene)hydrazinecarbonyl)‑1‑unde‑ cylpyridin‑1‑ium iodide (5) It was obtained as yellow crystals; mp: 82–83 °C. FT-IR (KBr), c­m−1: ῡ = 1598 (C=N), 1677 (C=O), 2872, 2967 (Al–H), 3078 (Ar–H). 1 H NMR (400 MHz, DMSO-d6): δH = 0.83–0.87 (m, 3H, CH3), 1.24–1.32 (m, 16H, 8× CH2), 1.96–1.99 (m, 2H, ­NCH2CH2), 4.68 (t, 2H, J = 8 Hz, NCH2), 7.22 (t, 0.5H, J = 8 Hz, Ar–H), 7.34 (t, 1.5H, J = 8 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.89 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H–C=N), 8.39 (d, 0.5H, J = 4 Hz, Ar–H), 8.50 (s, 0.75H, H–C=N), 8.53 (d, 1.5H, J = 8 Hz, Ar–H), 9.25 (d, 0.5H, J = 8 Hz, Ar–H), 9.34 (d, 1.5H, J = 8 Hz, Ar–H), 12.45 (bs, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 12.39 (CH3), 20.53, 23.86, 26.83, 27.13, 27.23, 27.37, 27.40, 28.98, 29.12, 29.74 (9×CH2), 59.46, 59.53 (NCH2), 114.23, 114.44, 114.66, 124.63, 125.61, 127.85, 127.93, 128.22, 128.31, 128.53, Fig. 2 The catalytic kinase domain of (a) 2RD0 harbors the QPLD docked poses of some of the verified molecules 2–9 and (b) superposition of the QPLD docked pose 2 and the co-crystallized ligand represented in red and blue colors, respectively Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 8 of 18 Fig. 3 The ligand/protein complex of a 2, b 3, c 6, and d 9 − 109.88), (− 109.35 to − 109.37) (2m, 1F, Ar–F). MS (ES) m/z = 525.10 ­[M+]. Fig. 4 The correlation between the QPLD docking scores and between ­IC50 for the tested compounds 128.56, 128.71, 128.74, 143.58, 144.18, 145.82, 147.88, 148.15 (Ar–C), 157.23, 160.78, 163.26, 163.69 (C=N, C=O). 19F NMR (377 MHz, DMSO-d6): δF = (− 109.95 to 1‑Dodecyl‑4‑(2‑(4‑fluorobenzylidene) hydrazinecar‑ bonyl)pyridin‑1‑ium iodide (6) It was obtained as yellow crystals; mp: 72–73 °C. FT-IR (KBr), ­cm−1: ῡ = 1605 (C=N), 1688 (C=O), 2883, 2961 (Al–H), 3074 (Ar–H). 1 H NMR (400 MHz, DMSO-d6): δH = 0.83–0.87 (m, 3H, CH3), 1.24–1.32 (m, 18H, 9× CH2), 1.96–1.99 (m, 2H, ­NCH2CH2), 4.70 (dd, 2H, J = 4 Hz, 8 Hz, NCH2), 7.22 (t, 0.5H, J = 8 Hz, Ar–H), 7.34 (t, 1.5H, J = 8 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.88 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H–C=N), 8.39 (d, 0.5H, J = 4 Hz, Ar–H), 8.50 (s, 0.75H, H–C=N), 8.53 (d, 1.5H, J = 8 Hz, Ar–H), 9.25 (d, 0.5H, J = 4 Hz, Ar–H), 9.34 (d, 1.5H, J = 8 Hz, Ar–H), 12.46 (bs, 1H, CONH). 13 C NMR (100 MHz, DMSO-d6): δC = 11.54, 11.59 (CH3), Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 9 of 18 Fig. 5 PI3Kα inhibitor pharmacophore model with a 2, b 3, c 6, and d 9. Aro stands for aromatic ring; Acc for H-bond acceptor; and Hyd for hydrophobic group. Picture made by ­MOE52 127.29 (Ar–C), 156.38, 159.93, 162.40, 162.83 (C=N, C=O). 19F NMR (377 MHz, DMSO-d6): δF = (− 109.95 to − 109.88), (− 109.44 to − 109.36) (2m, 1F, Ar–F). MS (ES) m/z = 539.40 ­[M+]. Fig. 6 The superposition of KWT QPLD-docked pose and its native conformation in 3HHM. The native coordinates are represented in orange and the docked pose in green color. Picture visualized by PYMOL 19.68, 23.00, 25.98, 26.30, 26.38, 26.51, 26.60, 28.13, 28.27, 28.88 (10× CH2), 58.60, 58.67 (NCH2), 113.37, 113.59, 113.80, 123.78, 124.75, 127.00, 127.08, 127.36, 127.45, 127.86, 127.89, 142.72, 143.33, 144.97, 147.02, 4‑(2‑(4‑Fluorobenzylidene)hydrazinecarbonyl)‑1‑tetra‑ decylpyridin‑1‑ium iodide (7) It was obtained as yellow crystals; mp: 86–88 °C. FT-IR (KBr), ­cm−1: ῡ = 1590 (C=N), 1679 (C=O), 2878, 2964 (Al–H), 3078 (Ar–H). 1 H NMR (400 MHz, DMSO-d6): δH = 0.83–0.86 (m, 3H, CH3), 1.24–1.32 (m, 22H, 11× CH2), 1.94–1.98 (m, 2H, ­NCH2CH2), 4.68 (t, 2H, J = 8 Hz, NCH2), 7.22 (t, 0.5H, J = 8 Hz, Ar–H), 7.34 (t, 1.5H, J = 8 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.89 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H–C=N), 8.39 (d, 0.5H, J = 4 Hz, Ar–H), 8.50 (s, 0.75H, H–C=N), 8.53 (d, 1.5H, J = 8 Hz, Ar–H), 9.25 (d, 0.5H, J = 8 Hz, Ar–H), 9.33 (d, 1.5H, J = 4 Hz, Ar–H), 12.44 (s, 0.75H, CONH), 12.49 (s, 0.25H, CONH). 13C NMR (100 MHz, DMSOd6): δC = 13.89 (CH3), 22.03, 25.36, 27.80, 28.34, 28.65, 28.74, 28.86, 28.96, 28.99, 29.77, 30.48, 30.62, 31.24, 32.85 (12×CH2), 60.96, 61.03 (NCH2), 115.73, 115.94, Al‑Blewi et al. Chemistry Central Journal (2018) 12:118 Page 10 of 18 116.16, 126.13, 127.11, 129.34, 129.43, 129.72, 129.81, 130.21, 130.24, 145.08, 145.68, 147.31, 149.38, 149.65 (Ar–C), 158.73, 162.29, 164.29, 165.18 (C=N, C=O). 19 F NMR (377 MHz, DMSO-d6): δF = (− 109.96 to − 109.89), (− 109.44 to − 109.36) (2m, 1F, Ar–F). MS (ES) m/z = 567.20 ­[M+]. (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. 4‑(2‑(4‑Fluorobenzylidene)hydrazinecarbonyl)‑1‑hexade‑ cylpyridin‑1‑ium iodide (8) It was obtained as yellow crystals; mp: 78–80 °C. FT-IR (KBr), ­cm−1: ῡ = 1610 (C=N), 1677 (C=O), 2887, 2969 (Al–H), 3076 (Ar–H). 1H NMR (400 MHz, DMSO-d6): δH = 0.83–0.86 (m, 3H, CH3), 1.23– 1.30 (m, 26H, 13× CH2), 1.96–1.98 (m, 2H, ­NCH2CH2), 4.68 (t, 2H, J = 8 Hz, NCH2), 7.22 (t, 0.5H, J = 8 Hz, Ar–H), 7.34 (t, 1.5H, J = 8 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.89 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H–C=N), 8.39 (d, 0.5H, J = 4 Hz, Ar–H), 8.50 (s, 0.75H, H– C=N), 8.53 (d, 1.5H, J = 8 Hz, Ar–H), 9.25 (d, 0.5H, J = 8 Hz, Ar–H), 9.34 (d, 1.5H, J = 4 Hz, Ar–H), 12.45 (s, 0.75H, CONH), 12.49 (s, 0.25H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 13.88 (CH3), 22.03, 25.36, 28.34, 28.64, 28.74, 28.87, 28.96, 29.00, 30.49, 30.62, 31.24 (12×CH2), 60.96, 61.03 (NCH2), 115.73, 115.94, 116.16, 126.14, 127.11, 129.34, 129.43, 129.72, 129.81, 130.04, 130.24, 145.08, 145.69, 147.31, 149.37 (Ar–C), 158.72, 162.29, 164.76, 165.18 (C=N, C=O). 19F NMR (377 MHz, DMSO-d6): δF = (− 109.97 to − 109.89), (− 109.45 to − 109.37) (2m, 1F, Ar–F). MS (ES) m/z = 595.30 ­[M+]. A mixture of equimolar of IL 2–9 (1 mmol) and fluorinated metal salt ­ (KPF6, ­NaBF4 and/or ­ NaCF3COO) (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. 4‑(2‑(4‑Fluorobenzylidene)hydrazinecarbonyl)‑1‑octade‑ cylpyridin‑1‑ium iodide (9) It was obtained as yellow crystals; mp: 98–99 °C. FT-IR (KBr), ­cm−1: ῡ= 1612 (C=N), 1678 (C=O), 2887, 2955 (Al–H), 3086 (Ar–H). 1H NMR (400 MHz, ­CDCl3): δH = 0.79–0.82 (m, 3H, CH3), 1.16–1.20 (m, 30H, 15× CH2), 1.96–2.00 (m, 2H, ­NCH2CH2), 4.78 (dd, 2H, J = 4 Hz, 8 Hz, NCH2), 6.97 (t, 2H, J = 8 Hz, Ar–H), 7.71 (dd, 2H, J = 4 Hz, 8 Hz, Ar–H), 8.87 (d, 2H, J = 4 Hz, Ar–H), 9.08 (s, 1H, H–C=N), 9.12 (d, 2H, J = 8 Hz, Ar–H), 12.18 (bs, 1H, CONH). 13C NMR (100 MHz, ­CDCl3): δC = 14.08 (CH3), 22.66, 26.10, 28.96, 29.31, 29.33, 29.48, 29.57, 29.63, 29.68, 31.67, 31.90 (16× CH2), 62.74 (NCH2), 115.85, 116.07, 127.88, 129.47, 130.14, 130.22, 144.82, 147.91, 151.67 (Ar– C), 158.57, 163.22, 163.25, 165.76 (C=N, C=O). 19F NMR (377 MHz, ­CDCl3): δF = (− 107.98 to − 107.89), (− 107.72 to − 107.65) (2 m, 1F, Ar–F). MS (ES) m/z = 623.30 ­[M+]. 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 ­ (KPF6, ­NaBF4 and/or ­ NaCF3COO) Ultrasound method (US) 4‑(2‑(4‑Fluorobenzylidene) hydrazinecarbonyl)‑1‑oc‑ tylpyridin‑1‑ium hexafluorophosphate (10) It was obtained as yellow crystals; mp: 64–65 °C. 1H NMR (400 MHz, DMSO-d6): δH = 0.82–0.88 (m, 3H, CH3), 1.26–1.30 (m, 10H, 5×CH2), 1.94–2.00 (m, 2H, ­NCH2CH2), 4.68 (t, 2H, J = 8 Hz, NCH2), 7.26 (dd, 0.5H, J = 8 Hz, 12 Hz, Ar–H), 7.38 (dd, 1.5H, J = 8 Hz, 12 Hz, Ar–H), 7.62 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.89 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H–C=N), 8.40 (d, 0.5H, J = 4 Hz, Ar–H), 8.50 (s, 0.75H, H–C=N), 8.53 (d, 1.5H, J = 4 Hz, Ar–H), 9.25 (d, 0.5H, J = 4 Hz, Ar–H), 9.33 (d, 1.5H, J = 4 Hz, Ar–H), 12.50 (bs, 1H, CONH).13C NMR (100 MHz, DMSO-d6): δC = 13.09 (CH3), 22.00, 25.36, 25.41, 28.30, 28.40, 30.51, 30.64, 31.09 (6×CH2), 60.95, 61.02 (NCH2), 115.75, 115.96, 116.18, 126.14, 127.11, 129.35, 129.44, 129.73, 129.81, 130.05, 130.24, 130.24, 145.06, 145.67, 147.35, 149.35, 149.63 (Ar–C), 158.78, 162.28, 164.75, 165.22 (C=N, C=O). 31P NMR (162 MHz, DMSO-d6): δP = − 152.70 to − 135.29 (m, 1P, PF6). 19F NMR (377 MHz, DMSO-d6): δF = − 69.98 (d, 6F, PF6), (− 109.72 to − 109.65), (− 109.20 to − 109.12) (2m, 1F, Ar–F). MS (ES) m/z = 501.20 ­[M+]. 4‑(2‑(4‑Fluorobenzylidene) hydrazinecarbonyl)‑1‑oc‑ tylpyridin‑1‑ium tetrafluoroborate (11) It was obtained as yellow crystals; mp: 80–82 °C. 1H NMR (400 MHz, DMSO-d6): δH = 0.84–0.88 (m, 3H, CH3), 1.26–1.31 (m, 10H, 5×CH2), 1.95–2.00 (m, 2H, ­NCH2CH2), 4.70 (dd, 2H, J = 4 Hz, 8 Hz, NCH2), 7.26 (dd, 0.5H, J = 8 Hz, 12 Hz, Ar–H), 7.38 (dd, 1.5H, J = 8 Hz, 12 Hz, Ar–H), 7.63 (dd, 0.5H, J = 4 Hz, 8 Hz, Ar–H), 7.90 (dd, 1.5H, J = 4 Hz, 8 Hz, Ar–H), 8.16 (s, 0.25H, H–C=N), 8.41 (d, 0.5H, J = 8 Hz, Ar–H), 8.51 (s, 0.75H, H–C=N), 8.54 (d, 1.5H, J = 4 Hz, Ar–H), 9.27 (d, 0.5H, J = 8 Hz, Ar–H), 9.36 (d, 1.5H, J = 8 Hz, Ar–H), 12.49 (s, 0.75H, CONH), 12.53 (s, 0.25H, CONH).13C NMR (100 MHz, DMSO-d6): δC = 13.87 (CH3), 21.97, 25.32, 25.38, 28.27, 28.37, 28.40, 30.48, 30.61, 31.06 (6× CH2), 60.89, 60.96