Ionic Liquid-Assisted Fabrication of Bioactive Heterogeneous Magnetic Nanocatalyst with Antioxidant and Antibacterial Activities for the Synthesis of Polyhydroquinoline Derivatives

Antibacterial materials have obtained much attention in recent years due to the presence of hazardous agents causing oxidative stress and observation of pathogens. However, materials with antioxidant and antibacterial activities can cause toxicity due to their low biocompatibility and safety profile, urging scientists to follow new ways in the synthesis of such materials. Ionic liquids have been employed as a green and environmentally solvent for the fabrication of electrically conductive polymers. In the present study, an antibacterial poly(p-phenylenediamine)@Fe3O4 (PpPDA@Fe3O4) nanocomposite was fabricated using [HPy][HSO4] ionic liquid. The chemical preparation of PpPDA@Fe3O4 nanocomposite was initiated through the oxidative polymerization of p-phenylenediamine by ammonium persulfate in the presence of [HPy][HSO4]. The PpPDA@Fe3O4 nanocomposite exhibited antibacterial properties against Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria. The PpPDA@Fe3O4 nanocomposite was employed as a heterogeneous nanocatalysis for one-pot synthesis of polyhydroquinoline derivatives using aromatic aldehyde, dimedone, benzyl acetoacetate, and ammonium acetate. Polyhydroquinoline derivatives were synthesized in significant yields (90–97%) without a difficult work-up procedure in short reaction times. Additionally, PpPDA@Fe3O4 nanocatalyst was recycled for at least five consecutive catalytic runs with a minor decrease in the catalytic activity. In this case, 11 derivatives of polyhydroquinoline showed in vitro antioxidant activity between 70–98%.


Introduction
The ionic liquids (ILs) are considered substances with a melting point below 100 • C composed of a cation and an anion [1]. The ions present in ILs are tunable and it is possible to develop solvents and catalysts from ILs. The early reports of using ILs in enzymatic reactions were performed in 2000 [2,3]. In addition to chemical synthesis and catalysis, ILs have been employed in electrochemistry, biotechnology, and pharmaceutics [4]. The ILs have been utilized as stabilizers for DNA storage [5]. ILs have been employed in chemical investigations owing to their nonvolatility, high thermal and electrochemical stability [6]. They are environmentally friendly salts that have been employed in the synthesis of organic compounds, catalytic reactions as well as the synthesis of intrinsically conductive polymers (ICPs) [7,8].

FTIR:
The FTIR spectra of the prepared PpPDA and PpPDA@Fe3O4 nanocomposite in the presence of the [HPy][HSO4] ionic liquid are shown in Figure 1a. The PpPDA and PpPDA@Fe3O4 nanocomposite showed very similar spectra with tiny differences. In the FTIR spectrum of PpPDA, the absorption peak at 3200-3450 cm −1 is related to the stretching vibration of the NH2 and N-H groups. Two characteristic absorption peaks at 1570 cm −1 and 1503 cm −1 are associated with the stretching mode of quinoid imine and benzenoid amine units, respectively [32]. The absorption peaks with different intensities in the areas of 1310 cm −1 , 1108-1118 cm −1 , and 830 cm −1 are related to the stretching vibrations of SO4, S=O, and S-OH in the [HPy][HSO4] ionic liquid, respectively [11]. The incorporation of iron oxide nanoparticles in the PpPDA matrix led to the appearance of an obvious absorption peak at around 505 cm −1 that related to the Fe-O-Fe stretching modes in Fe3O4 [33].
Elemental analysis: Elemental analysis (CHNSO) was employed for characterization of the prepared PpPDA and PpPDA@Fe3O4 nanocomposite in the presence of the [HPy][HSO4] ionic liquid (Table 1). According to the data in Table 1, the existence of sulfur and oxygen elements in the PpPDA approved the presence of [HPy][HSO4] ionic liquid in the structure of the PpPDA. The increase of oxygen element in the PpPDA@Fe3O4 nanocomposite compared with PpPDA shows the presence of Fe3O4 nanoparticles in the nanocomposite [11]. Scheme 1. Schematic illustration of the antibacterial and antioxidant nanocatalyst for synthesis of bioactive polyhydroquinoline derivatives.

FTIR:
The FTIR spectra of the prepared PpPDA and PpPDA@Fe 3 O 4 nanocomposite in the presence of the [HPy] [HSO 4 ] ionic liquid are shown in Figure 1a. The PpPDA and PpPDA@Fe 3 O 4 nanocomposite showed very similar spectra with tiny differences. In the FTIR spectrum of PpPDA, the absorption peak at 3200-3450 cm −1 is related to the stretching vibration of the NH 2 and N-H groups. Two characteristic absorption peaks at 1570 cm −1 and 1503 cm −1 are associated with the stretching mode of quinoid imine and benzenoid amine units, respectively [32]. The absorption peaks with different intensities in the areas of 1310 cm −1 , 1108-1118 cm −1 , and 830 cm −1 are related to the stretching vibrations of SO 4 , S=O, and S-OH in the [HPy] [HSO 4 ] ionic liquid, respectively [11]. The incorporation of iron oxide nanoparticles in the PpPDA matrix led to the appearance of an obvious absorption peak at around 505 cm −1 that related to the Fe-O-Fe stretching modes in Fe 3 O 4 [33].
Elemental analysis: Elemental analysis (CHNSO) was employed for characterization of the prepared PpPDA and PpPDA@Fe 3 O 4 nanocomposite in the presence of the [HPy] [HSO 4 ] ionic liquid (Table 1). According to the data in Table 1, the existence of sulfur and oxygen elements in the PpPDA approved the presence of [HPy] [HSO 4 ] ionic liquid in the structure of the PpPDA. The increase of oxygen element in the PpPDA@Fe 3 O 4 nanocomposite compared with PpPDA shows the presence of Fe 3 O 4 nanoparticles in the nanocomposite [11].    Figure 1c. According to the literature, the XRD pattern of Fe 3 O 4 nanoparticles indicated a crystalline nature [35]. A semicrystalline nature was observed in the XRD pattern of PpPDA owing to intermolecular interactions between the PpPDA chains and the [HPy][HSO 4 ] ionic liquid [36]. The XRD pattern of PpPDA@Fe 3 O 4 nanocomposite showed more crystallinity compared with PpPDA due to the presence of crystalline nanoparticles of Fe 3 O 4 [37]. in the FESEM micrographs of PpPDA, and PpPDA@Fe3O4 nanocomposite, respectively. VSM: Vibrating sample magnetometer (VSM) was employed for the evaluation of the magnetic property of PpPDA@Fe3O4 nanocomposite as shown in Figure 2d. In the VSM of the PpPDA@Fe3O4 nanocomposite, the amount of magnetic coercivity (Hc) and magnetic remanence (Mr) is equal to zero. This indicates the PpPDA@Fe3O4 nanocomposite has a superparamagnetic property with a magnetization saturation value of 30.01 emu/g [38]. .

Samples
Solvent

Evaluation of the Catalytic Activity of PpPDA@Fe3O4 Nanocomposite
In the current study, we offered a new and efficient technique for the synthesis of polyhydroquinolines using PpPDA@Fe3O4 nanocomposite. We investigated the fourcomponent Hantzsch condensation by aromatic aldehyde, dimedone, benzyl acetoacetate, and ammonium acetate.
The effect of various solvents on the reaction rate and yield of the products was investigated to optimize the reaction conditions.
To optimize the reaction conditions, firstly the various solvents' effect on the rate of reaction and products yield was investigated. The reaction of benzaldehyde (1, 1 mmol), dimedon, (2, 1 mmol), ammonium acetate (3, 1 mmol), and benzyl acetoacetate (4, 1 mmol) as a model reaction was catalyzed by 0.03 g of PpPDA@Fe3O4 in different solvents, e.g., water, ethanol (EtOH), chloroform (CHCl3), tetrahydrofuran (THF) and hexane at reflux conditions (Table 3). In aprotic solvents, e.g., CHCl3, THF, and hexane, the reaction rate was very slow and product yield was low whereas reaction rates, as well as product yields in protic solvents, were improved. In water and solvent-free conditions, the expected  Figure 2d. In the VSM of the PpPDA@Fe 3 O 4 nanocomposite, the amount of magnetic coercivity (Hc) and magnetic remanence (Mr) is equal to zero. This indicates the PpPDA@Fe 3 O 4 nanocomposite has a superparamagnetic property with a magnetization saturation value of 30.01 emu/g [38].

Evaluation of the Catalytic Activity of PpPDA@Fe 3 O 4 Nanocomposite
In the current study, we offered a new and efficient technique for the synthesis of polyhydroquinolines using PpPDA@Fe 3 O 4 nanocomposite. We investigated the fourcomponent Hantzsch condensation by aromatic aldehyde, dimedone, benzyl acetoacetate, and ammonium acetate.
The effect of various solvents on the reaction rate and yield of the products was investigated to optimize the reaction conditions.
To optimize the reaction conditions, firstly the various solvents' effect on the rate of reaction and products yield was investigated. The reaction of benzaldehyde (1, 1 mmol), dimedon, (2, 1 mmol), ammonium acetate (3, 1 mmol), and benzyl acetoacetate (4, 1 mmol) as a model reaction was catalyzed by 0.03 g of PpPDA@Fe 3 O 4 in different solvents, e.g., water, ethanol (EtOH), chloroform (CHCl 3 ), tetrahydrofuran (THF) and hexane at reflux conditions (Table 3). In aprotic solvents, e.g., CHCl 3 , THF, and hexane, the reaction rate was very slow and product yield was low whereas reaction rates, as well as product yields in protic solvents, were improved. In water and solvent-free conditions, the expected product was achieved only in low yield after 4 h. Furthermore, the above condensation reaction by the PpPDA@Fe 3 O 4 catalyst was also carried out in ethanol at reflux conditions. temperatures. The yield of the products was increased when the reaction temperature was raised from room temperature to reflux conditions. Moreover, when 0.02, 0.03, 0.04, and 0.06 of PpPDA@Fe3O4 nanocatalyst were used, the yield of the products was 60%, 85%, 94%, and 96%, respectively. Therefore, 0.04 g of PpPDA@Fe3O4 was an optimal amount for producing products with high yields. For better comparison, the reaction was also investigated in the absence of the catalyst. Results (Table 3) showed that the rate of reaction was very slow and product yield was low. To optimize the temperature of the reaction, the mixture was heated at various temperatures. The yield of the products was increased when the reaction temperature was raised from room temperature to reflux conditions. Moreover, when 0.02, 0.03, 0.04, and 0.06 of PpPDA@Fe 3 O 4 nanocatalyst were used, the yield of the products was 60%, 85%, 94%, and 96%, respectively. Therefore, 0.04 g of PpPDA@Fe 3 O 4 was an optimal amount for producing products with high yields. For better comparison, the reaction was also investigated in the absence of the catalyst. Results (Table 3) showed that the rate of reaction was very slow and product yield was low.
For better comparison, the synthesis of polyhydroquinoline derivatives was studied in the absence and presence of various catalysts, e.g., PpPDA, PpPDA@  4 ] nanocatalyst was selected as an effective catalyst to perform the reactions. Table 5 shows that aromatic aldehydes containing electron-donating and electronwithdrawing groups reacted with dimedone, benzyl acetoacetate, and ammonium acetate in the presence of PpPDA@Fe 3 O 4 magnetic nanocatalyst in optimal conditions and in a short time to produced polyhydroquinolines with excellent yields. Likewise, thiophenecarbaldehyde and furfural (heteroaromatic aldehydes) produced the desired product after 35 min with 92% and 90% yields respectively (Table 5, entries 12 and 14). PpPDA@Fe 3 O 4 magnetic nanocatalyst was also suitable for the synthesis of polyhydroquinolines from aliphatic aldehyde such as α-methyl cinnamaldehyde ( Table 5, entry 13). Dialdehydes such as para phenylene dialdehyde and 2,2 -(hexane-1,6-diylbis(oxy))dibenzaldehyde reacted under optimal conditions using PpPDA@Fe 3 O 4 magnetic nanocatalyst and produced products with high yields (Table 5, entries 23 and 24).   Table 5 shows that aromatic aldehydes containing electron-donating and electronwithdrawing groups reacted with dimedone, benzyl acetoacetate, and ammonium acetate in the presence of PpPDA@Fe3O4 magnetic nanocatalyst in optimal conditions and in a short time to produced polyhydroquinolines with excellent yields. Likewise, thiophenecarbaldehyde and furfural (heteroaromatic aldehydes) produced the desired product after 35 min with 92% and 90% yields respectively (Table 5, entries 12 and 14). PpPDA@Fe3O4 magnetic nanocatalyst was also suitable for the synthesis of polyhydroquinolines from aliphatic aldehyde such as α-methyl cinnamaldehyde ( Table 5, entry 13). Dialdehydes such as para phenylene dialdehyde and 2,2′-(hexane-1,6-diylbis(oxy))dibenzaldehyde reacted under optimal conditions using PpPDA@Fe3O4 magnetic nanocatalyst and produced products with high yields ( Table 5, entries 23 and 24).  Table 5 shows that aromatic aldehydes containing electron-donating and electronwithdrawing groups reacted with dimedone, benzyl acetoacetate, and ammonium acetate in the presence of PpPDA@Fe3O4 magnetic nanocatalyst in optimal conditions and in a short time to produced polyhydroquinolines with excellent yields. Likewise, thiophenecarbaldehyde and furfural (heteroaromatic aldehydes) produced the desired product after 35 min with 92% and 90% yields respectively (Table 5, entries 12 and 14). PpPDA@Fe3O4 magnetic nanocatalyst was also suitable for the synthesis of polyhydroquinolines from aliphatic aldehyde such as α-methyl cinnamaldehyde ( Table 5, entry 13). Dialdehydes such as para phenylene dialdehyde and 2,2′-(hexane-1,6-diylbis(oxy))dibenzaldehyde reacted under optimal conditions using PpPDA@Fe3O4 magnetic nanocatalyst and produced products with high yields (Table 5, entries 23 and 24).

] to form intermediate (II). In the next step, Michael's addition reaction of intermediate (II) to intermediate (I) causes the formation of intermediate (III). The intermediate (III) is then converted to the intermediate (IV) by tautomerization and the product (VI) is obtained after cyclization reaction.
Recovery and reusability: Recyclability is an important property of heterogeneous catalytic systems in terms of environmental protection and industrial application. To evaluate the reusability of PpPDA@Fe 3 O 4 , it was magnetically isolated from the reaction mixture, washed several times with distilled water and ethanol, dried at room temperature, utilized again in the next reaction. As is observed in Figure 4, the yield of the products was not reduced considerably after five successive catalytic runs and the catalyst has retained its efficacy and stability in the synthesis of polyhydroquinolines derivatives.

Recovery and reusability:
Recyclability is an important property of heterogeneous catalytic systems in terms of environmental protection and industrial application. To evaluate the reusability of PpPDA@Fe3O4, it was magnetically isolated from the reaction mixture, washed several times with distilled water and ethanol, dried at room temperature, utilized again in the next reaction. As is observed in Figure 4, the yield of the products was not reduced considerably after five successive catalytic runs and the catalyst has retained its efficacy and stability in the synthesis of polyhydroquinolines derivatives.   , and synthesized polyhydroquinoline derivatives was studied using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)radical scavenging model ( Figure 5). Results showed that all used materials to prepare magnetic nanocatalysts had antioxidant activities between 72% and 90%. In   4 ], and synthesized polyhydroquinoline derivatives was studied using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)radical scavenging model ( Figure 5). Results showed that all used materials to prepare magnetic nanocatalysts had antioxidant activities between 72% and 90%. In addition, the antioxidant activity of 24 synthesized derivatives was investigated. Only 11 derivatives showed antioxidant activity between 75% and 98%. These results suggest that these derivatives may play a role in the synthesis of immune-boosting drugs.  , and synthesized polyhydroquinoline derivatives was studied using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)radical scavenging model ( Figure 5). Results showed that all used materials to prepare magnetic nanocatalysts had antioxidant activities between 72% and 90%. In addition, the antioxidant activity of 24 synthesized derivatives was investigated. Only 11 derivatives showed antioxidant activity between 75% and 98%. These results suggest that these derivatives may play a role in the synthesis of immune-boosting drugs.  , and seven of polyhydroquinoline derivatives (5c, 5i, 5j, 5r, 5s, 5o, and 5v) were investigated against Escherichia coli and Bacillus subtilis, and results shown in Table 6 and Figure 6. The results showed that the bare PpPDA and Fe 3 O 4 NPs had good growth inhibitory effects against tested microorganisms, and among them, Fe 3 O 4 NPs exhibited the highest antibacterial activity against both microorganisms while PpPDA@IL was effective against Bacillus subtilis. The nanocatalyst showed lower antimicrobial activity than that of bare minerals against tested bacteria. Moreover, among polyhydroquinoline derivatives, only 5r and 5v had good growth inhibitory effects against tested microorganisms while 5i and 5o showed no effect against tested microorganisms. [HSO4] (nanocatalyst), and seven of polyhydroquinoline derivatives (5c, 5i, 5j, 5r, 5s, 5o, and 5v) were investigated against Escherichia coli and Bacillus subtilis, and results shown in Table 6 and Figure 6. The results showed that the bare PpPDA and Fe3O4 NPs had good growth inhibitory effects against tested microorganisms, and among them, Fe3O4 NPs exhibited the highest antibacterial activity against both microorganisms while PpPDA@IL was effective against Bacillus subtilis. The nanocatalyst showed lower antimicrobial activity than that of bare minerals against tested bacteria. Moreover, among polyhydroquinoline derivatives, only 5r and 5v had good growth inhibitory effects against tested microorganisms while 5i and 5o showed no effect against tested microorganisms.     Benzyl2,7,7-trimethyl-5-oxo-4-(p-tolyl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (Table 5  added into the above solution under constant stirring for a half-hour, and the final pH was 10. Lastly, the black precipitate was isolated by a magnet and washed with distilled water and ethanol, and dried at 80 • C under vacuum for 2 h.

Pyridinium Hydrogen Sulfate [HPy][HSO4] Preparation
Pyridinium hydrogen sulfate [HPy][HSO4] was prepared as follows (Scheme 2a) [41]. 10 mL of pyridine was poured into a flask, then 6.76 mL of sulfuric acid solution was added slowly into pyridine for one hour under stirring at 0-5 °C. Afterward, the solution was maintained for 5 h at 0-5 °C to complete the reaction. Lastly, water was removed by a rotary evaporator to give a colorless liquid.

Overall Route for the Synthesis of Polyhydroquinoline Derivatives
One-pot synthesis of polyhydroquinoline compounds was carried out as follows: A mixture of dimedone (1.0 mmol), aldehyde (1.0 mmol), benzyl acetoacetate (1.0 mmol), ammonium acetate (1.0 mmol), and PpPDA@Fe 3 O 4 (0.04 g) in ethanol solvent (5 mL) was refluxed. Rection was traced by thin-layer chromatography (hexane/ethyl acetate 5:1). Once the reaction was completed the catalyst was separated easily by an external magnet. Afterward, the crude solid product was filtered and then purified by recrystallization from ethanol.

Antioxidant Activity
Antioxidant activity evaluation of prepared materials was studied in ethanolic DPPH solution (25 µM/L) by a UV-vis spectroscopy. The amount of each sample (10 mg) was added to tubes containing2 mL of ethanolic DPPH and then the tubes were kept in a dark place for 6 h. After that, DPPH inhibition (%) was measured by the following Equation: (1) In this equation, A b and A s are the absorption of DPPH solution and samples at 517 nm, respectively.

Antibacterial Activity
Kirby-Bauer disc diffusion technique was employed for antibacterial activities study of the prepared samples. Sample solutions (20 mg in 10 mL dimethyl sulfoxide, DMSO) were filtered by a Ministart (Sartorius). The antibacterial activity of the samples was evaluated against Bacillus subtilis PTCC 1023 (Gram-positive) and Escherichia coli PTCC 1330 (Gram-negative) bacterial species. The bacteria phase was prepared via inoculating of the cultures 1% (v/v) into the Muller-Hinton broth and incubating on a shaker at 37 • C for 24 h. Sterile paper discs were soaked with 10 µL of the sample solutions then allowed to dry. The soaked discs were placed on the agar plate and incubated at 37 • C for 24 h. The antibacterial activities of the compounds were compared with gentamicin and chloramphenicol antibiotics as positive control and DMSO as a negative control. Antibacterial activity was studied by evaluating the inhibition zone diameter (mm) of the surface of the plates and the results were reported as Mean ± SD after three repeats.

Conclusions
Antibacterial and antioxidant PpPDA@Fe 3 O 4 nanocomposite was successfully fabricated by in-situ oxidative polymerization in the presence of [HPy] [HSO 4 ] and iron oxide nanoparticles as a potential heterogeneous nanocatalyst for the synthesis of polyhydroquinolines derivatives. The nanocatalyst was characterized by different techniques and results displayed that the nanocatalyst showed superparamagnetic behavior with crystalline nature. The solubility test showed that prepared PpPDA in presence of [HPy][HSO 4 ] had better solubility than PpPDA. The PpPDA@Fe 3 O 4 nanocatalyst showed good antibacterial activity against Escherichia coli and Bacillus subtilis. The FESEM of nanocatalyst showed the hexagonal structure with a high agglomerate with a diameter of~100 nm. The PpPDA@Fe 3 O 4 nanocatalyst showed great catalytic performance in the synthesis of polyhydroquinolines derivatives and the corresponding products were synthesized with high yield (90-97%) without a difficult work-up procedure. Moreover, the PpPDA@Fe 3 O 4 nanocatalyst separated easily from the reaction media by a magnet. Reusability results showed that the nanocatalyst could use for at least five times without a significant decrease in catalytic activity. According to the proposed mechanistic scheme, the prepared PpPDA@Fe 3 O 4 nanocatalyst in [HPy] [HSO 4 ] played an important role in directing the synthesis reaction of polyhydroquinolines derivatives with favorable features, e.g., Brønsted acid, strong basic sites, and high surface area. It could be concluded the bioactive PpPDA@Fe 3 O 4 nanocomposite could be employed as an eco-friendly and high efficiency nanocatalyst for the synthesis of different organic reactions. PpPDA@Fe 3 O 4 nanocatalysts and 11 polyhydroquinolines derivatives showed antioxidant activity between 75% and 99%. Among polyhydroquinolines derivatives, only 5r and 5v had good growth inhibitory effects against tested microorganisms.