Poly(aniline-co-melamine)@MnFe2O4 nanocatalyst for the synthesis of 4,4′-(arylmethylene) bis (1H-pyrazole-5-ol) derivatives, and 1,4- dihydropyrano[2,3-c]pyrazoles and evaluation of their antioxidant, and anticancer activities

In this work, magnetic poly(aniline-co-melamine) nanocomposite as an efficient heterogeneous polymer-based nanocatalyst was fabricated in two steps. First, poly(aniline-co-melamine) was synthesized through the chemical oxidation by ammonium persulfate, then the magnetic nanocatalyst was successfully prepared from the in-situ coprecipitation method in the presence of poly(aniline-co-melamine). The resulting poly(aniline-co-melamine)@MnFe2O4 was characterized by FTIR, FESEM, XRD, VSM, EDX, TGA, and UV-vis analyses. The catalytic activity of poly(aniline-co-melamine)@MnFe2O4 was investigated in the synthesis of 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives, and new alkylene bridging bis 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives in excellent yields. The yield of 1,4-dihydropyrano[2,3-c]pyrazoles, 4,4′-(arylmethylene)bis(1H-pyrazol-5-ol), yields, and new alkylene bridging bis 4,4′-(arylmethylene)bis(1H-pyrazol-5-ol) derivatives were obtained 89%–96%, 90%–96%, and 92%–96%, respectively. The poly(aniline-co-melamine)@MnFe2O4 nanocatalyst can be recycled without pre-activation and reloaded up to five consecutive runs without a significant decrease in its efficiency. In addition, the antioxidant activity of some derivatives was evaluated by DPPH assay. Results showed that the maximum antioxidant activity of 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives and 1,4-dihydropyrano[2,3-c]pyrazoles were 75% and 90%, respectively. Furthermore, 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives and 1,4-dihydropyrano[2,3-c]pyrazoles showed good potential for destroying colon cancer cell lines. Consequently, the poly(aniline-co-melamine)@MnFe2O4 nanocomposite is an excellent catalyst for green chemical processes owing to its high catalytic activity, stability, and reusability.


Introduction
Magnetic nanoparticles have been widely used as viable catalyst supports due to their high surface area, which results in high catalyst loading capacity, great dispersion, remarkable stability, and easy catalytic recycling (Zhu et al., 2010;Guo et al., 2021a;Lian et al., 2021;Soares et al., 2022). The recovery of catalysts from liquid-phase processes is significantly easier with magnetic separation than with cross-flow filtering and centrifugation (Gu et al., 2020;Dang et al., 2022;Xu et al., 2022). Additionally, except for those that are very corrosive or acidic, the particles' magnetic characteristics are persistent enough to withstand the majority of chemical conditions. Easy accessibility of reactants to the active sites is also possible since the catalysts are often bound to the surface of the magnetic nanoparticles (Stevens et al., 2005;Hassanzadeh-Afruzi et al., 2022;Movagharnezhad et al., 2022). Because of their electrical resistance, physical and magnetic characteristics, and great chemical stability, ferrites, a subclass of ferrimagnetic ceramics, are widely recognized (Alizadeh and Baseri, 2022). The spinel, hexagonal, and garnet structures of ferrites are determined by their original crystal lattice. The normal and inverse spinel ferrites are particularly appealing among these structures (Bououdina and Manoharan, 2020;Özdemir et al., 2020).
Superparamagnetism, single domain effects, high coercivity, moderate magnetization, and spin-filtering are just a few of the distinctive magnetic properties that make it possible for magnetic transition oxide nanostructures with spinel structure MFe 2 O 4 (M = Ni, Co, Fe, Mn, etc.) to have remarkable biological and industrial functionalities (Zipare et al., 2015). Due to its captivating electromagnetic and magnetic characteristics, as a significant member of the ferrite family, MnFe 2 O 4 (MFO) has garnered significant study attention. As MnFe 2 O 4 is partly inverse spinel, about 80% of Mn 2+ ions are found at the tetrahedral site, while just 20% are found at the octahedral site (Deraz and Alarifi, 2012). The features of MnFe 2 O 4 nanoparticles and thin films are varied, including high curie temperature, superparamagnetism, super spin glass state, sizedependent saturation magnetization, and high anisotropy constant (Carta et al., 2010). The magnetic nanoparticles such as MnFe 2 O 4 , MgFe 2 O 4 , CoFe 2 O 4 , ZnFe 2 O 4 , Fe 3 O 4 , and their nanocomposites due to their biocompatibility, good physicochemical properties and small particle size have several applications in targeted drug delivery, magnetic resonance imaging (MRI), mediators for hyperthermia applications, etc., (Islam et al., 2020).
The magnetic particles are rarely used in their unprocessed form; instead, they are often mixed with an organic polymer matrix or an inorganic silica carrier and placed in a liquid media to prevent aggregation (Guo et al., 2021b;Jazzar et al., 2021). The particles are endowed with organic polymers with the functional groups required for the desired uses (Kalia et al., 2014). This combination of organic polymers and magnetic nanoparticles provides a diverse range of opportunities for the creation of a new class of organic/inorganic hybrid materials that have dual properties. The resulting magnetic polymers have shown to be helpful for a range of purposes since they combine magnetic characteristics with high stability and reputable biocompatibility. The polymer nanocomposites due to high functionality, good phsicochmichal proprtes, and biocompatibility have received increasing attention in biomedical applications (Hule and Pochan, 2007). Thus, they've been used effectively in new applications including hyperthermia, bioseparation, medication delivery, targeted oil recovery, and so on (Philippova et al., 2011). Nevertheless, since magnetic nanoparticles possess a high surface-area-to-volume ratio and are scattered in a polymer matrix, it is difficult to create magnetic polymer nanocomposites that have a consistent magnetic field equivalent to that of an inorganic magnetic material. It is commonly known that a particle's magnetic properties depend on its chemical composition, shape, size, and surface characteristics. To determine the sorts of applications appropriate for polymer usage, precise control of the aforementioned factors is required. The creation of magnetic polymer nanocomposites has been investigated using a variety of methods, including simultaneous formation of polymers and particles, polymerization of monomers with the presence of particles, in-situ formation of particles into preformed polymers, and direct mixing (Massart, 1981;Jazzar et al., 2021).
Pyrazoles, commonly known as azoles, are five-membered ring heterocyclic compounds that include two nitrogen atoms in neighboring locations (Parajuli et al., 2013). Treatment of inflammation and illnesses linked to inflammation, such as arthritis, is the most well-known use of nearly every category of pyrazoles. Pyrazoles are the focus of numerous investigations due to their broad range of biological potential activities, including anticancer, anti-inflammatory (Amir et al., 2008), antihistaminic (Yildirim et al., 2005), antitumor (Park et al., 2005), antiviral (Larsen et al., 1999), anti-diabetic, anticonvulsant, andantimicrobial (Pimenova andVoronina, 2001) activities. Ethylacetoacetate and substituted hydrazine are typically used as the starting materials when synthesizing pyrazolone. With the aid of numerous catalysts, the production of pyrazoles has been reported to employ a variety of techniques and conditions. For example, pyrazolo [3,4-b]pyridine-5carboxamide derivatives were catalyzed by p-toluenesulfonic acid under mild reaction conditions and in good yields at ambient temperature (Shaabani et al., 2009), Poly(ethylene imine)-modified magnetic halloysite nanotubes was used as a catalyst for the synthesis of dihydropyrano[2,3-c] pyrazole derivatives (Hajizadeh and Maleki, 2018).

Preparation of poly(aniline-comelamine)@MnFe 2 O 4 nanocatalyst
To 40 ml of HCl (1 M), 1 ml aniline and 1.38 g melamine were added. The reaction mixture was stirred at 50°C until the melamine was entirely dissolved and was kept at 0°C-4°C under an inerth atmosphere. Then, 20 ml of ammonium persulfate solution (6.02 g) was added dropwise to the above solution. The reaction was maintained at 0°C-4°C for 12 h. The dark green poly(aniline-co-melamine) precipitate was then recovered by filtering and washed multiple times using water and methanol to eliminate the residual oxidizing agent and oligomers. The cleansed product was submerged in boiling water for 20 min and agitated to thoroughly dissolve the undissolved melamine. The in-situ co-precipitation method was used for the preparation of poly(aniline-co-melamine)@MnFe 2 O 4 nanocomposite as follows: 0.33 g of MnCl 2 .2H 2 O and 0.5 g of FeCl 3 .6H 2 O were dissolved in 25 ml of deionized water. The solution was then mixed with 1 g of poly(aniline-co-melamine) at 80°C for 20 min. The mixture was treated with a 1 M NaOH solution until the pH reached 12. After 3 h, the black precipitate was magnetically separated and repeatedly rinsed with deionized water and ethanol and drided at 40°C in the vacuum oven for 10 h. Figure 1A shows the reaction steps for the preparation of the nanocomposite.

Results and discussions
3.1 Characterization of polymer-based nanocatalyst

Fourier transform infrared spectroscopy
The FTIR absorption spectra of the prepared MnFe 2 O 4 (a), poly(aniline-co-melamine) (b), poly(aniline-co-melamine) @MnFe 2 O 4 (c) nanocomposite are shown in Figure 2. The MnFe 2 O 4 spectrum exhibits two prominent absorption bands below 1,000 cm −1 , which is a characteristic of ferrites. The distinctive bands at 449 and 573 cm −1 are caused by the metalinherent oxygen's stretching vibrations at the octahedral and tetrahedral sites, respectively (Zipare et al., 2015). The poly(aniline-co-melamine) (b) and poly(aniline-comelamine)@MnFe 2 O 4 (c) samples displayed very similar spectra. The stretching vibration of the NH 2 and N-H groups is observed at 3,390 cm −1 . The stretching modes of benzenoid amine and quinoid imine units, respectively, are associated with the two distinctive bands at around 1,500 and 1,600 cm −1 . The vibrations of the C-H and C-N stretching bonds in the benzene ring are responsible for the peaks at 1,106 and 1,296 cm −1 , respectively. The band corresponding to the bending vibrations of the C-H bond in the p-disubstituted benzene rings can be seen in the 797 cm −1 (Maroufi et al., 2020).  (Qin et al., 2021). The XRD patterns of the poly(aniline-co-melamine) (b) and (polyaniline-co-melamine)@MnFe 2 O 4 nanocomposite (c) showed a semicrystalline nature. The existence of corresponding peaks of MnFe 2 O 4 and poly(aniline-co-melamine) approved the successful preparation of the nanocomposite.

Thermogravimetric analysis
The TG/DTG curves of the MnFe 2 O 4 , poly(aniline-comelamine), and poly(aniline-co-melamine)@MnFe 2 O 4 nanocomposite are given in Figures 4A,B. The weight loss (below 150°C) observed in the TG curve of MnFe 2 O 4 is related to the evaporation of adsorbed water on the surface of the nanoparticles. Three weight losses are observed in the TG curve of poly(aniline-co-melamine). The first step of degradation between 80°C and 200°C corresponded to theremoval of water. The second and
Frontiers in Chemistry frontiersin.org 06 third weight losses at 210°C and up to 800°C are related to the melamine breakdown and minor degradation of polymeric chains, respectively (Maroufi et al., 2020). The comparison of TG curves of poly(aniline-co-melamine)and poly(aniline-comelamine)@MnFe 2 O 4 nanocomposite showed that the stability of poly(aniline-co-melamine)@MnFe 2 O 4 nanocomposite was higher than the poly(aniline-comelamine).

VSM
The VSM curves of the poly(aniline-co-melamine) @MnFe 2 O 4 nanocomposite and MnFe 2 O 4 are revealed in Figure 5. The VSM curves show that the magnetization saturation values of MnFe 2 O 4 nanoparticles and poly(aniline-co-melamine)@MnFe 2 O 4 were 21.28 and 41.81 emu/g, respectively. In addition, the magnetic property of the nanocomposite was lower than the MnFe 2 O 4 nanoparticles. This could be related to covering the magnetic nanoparticles with poly(aniline-co-melamine) copolymer.

UV-vis
The UV-vis absorption spectra of poly(aniline-comelamine), and poly(aniline-co-melamine)@MnFe 2 O 4 are presented in Figure 6. A specific DMSO concentration was used to evaluate both samples. The UV-vis spectrum of poly(aniline-co-melamine) shows two characteristic absorption peaks assigned to the n-π* and π-π* electron transition within the benzenoid segments in the range of 300-400 nm. A charge transfer from the benzenoid rings to the quinoid rings causes the Frontiers in Chemistry frontiersin.org 07 absorption peak at roughly 620 nm (Maroufi et al., 2020). The presence of MnFe 2 O 4 in the nanocomposite leads to reducing in the solubility of the poly(aniline-co-melamine)@MnFe 2 O 4. The UV-vis absorption spectrum of poly(aniline-co-melamine) @MnFe 2 O 4 is weaker than the UV-vis spectrum of poly(aniline-co-melamine). Figure 7A displays the FESEM images of MnFe 2 O 4 , poly(aniline-co-melamine), and poly(aniline-co-melamine) @MnFe 2 O 4 in magnifications of 200 nm. The average particle size for the of MnFe 2 O 4 was 60 nm, The FESEM image of poly(aniline-co-melamine) showed rod-like structure with a diameter of 44-183 nm. The FESEM image of poly(aniline-comelamine)@MnFe 2 O 4 show the particle size decreased after the composite was prepared. The average particle size diameter was 50 nm. These findings revealed that The FESEM image of the nanocomposite was successfully prepared.

Energy dispersive X-ray analysis
As shown in Figure 7B, the chemical composition of the produced MnFe 2 O 4 , poly(aniline-co-melamine), and poly(aniline-co-melamine) @MnFe 2 O 4 was also determined using the EDXanalysis. The presence of various quantities of O, C, N, Mn, and Fe elements was proven by comparing spectra and tabular data. The occurrence of S and Cl in poly(aniline-comelamine) is related to ammonium persulfate and HCl trapped in the polymer matrix.
The reaction conditions of benzaldehyde, 3-methyl-1phenyl-1H-pyrazole-5-ol, and malononitrile in the presence of poly(aniline-co-melamine)@MnFe 2 O 4 as a catalyst were optimized to get a higher yield. Following that, a solvent screening indicated that the reaction was more successful in water and ethanol. When benzaldehyde (1 mmol), and 3-methyl-1-phenyl-1H-pyrazole-5-ol (1 mmol) were reacted with malononitrile (1 mmol) in the presence of Poly(aniline-comelamine)@MnFe 2 O 4 (0.05 g) in Ethanol/H 2 O (1:1) at 40°C, the best results were attained (Table 1 entry 11). On the other hand, it is confirmed that the reaction requires a catalyst to continue and that the efficiency is not high in the absence of a catalyst (Table 1, entry 12). In this reaction model, the conversion rates were examined in the presence of poly(aniline-comelamine) and MnFe 2 O 4 ( Table 1, entries 13,14), the results showing that the best result has been achieved in poly(aniline-comelamine) but the major problem of poly(aniline-co-melamine) as a catalyst is easy solubility in organic solvents, and therefore its isolation is difficult. The poly(aniline-co-melamine)@MnFe 2 O 4   nanocomposite was consequently chosen as an efficient catalyst to carry out the reactions.
Frontiers in Chemistry frontiersin.org structures. This observation showed that the presence of MnFe 2 O 4 nanoparticles in the copolymer matrix led to decreasing the particle size and differencing in the morphology of the nanocomposite. It is well known that particle size and morphology are two important parameters that influence the catalytic performance of nanocatalysts (Kamalzare, 2022). Nanocatalysts with a high surface area could provide more active sites to less activation energy for catalytic reactions, and novel surface structures lead to product selectivity and catalytic efficacy. Thus, the higher catalytic activity of nanocomposite can be related to the high volume-to-surface ratio and different morphology of the nanocomposite. The reaction with several aromatic aldehydes to yield a wide range of 1, 4-dihydropyrano[2,3-c]pyrazoles were examined in the terms of substrate scope ( Table 2). The presence of poly(aniline-co-melamine)@MnFe 2 O 4 revealed that this reaction is generally tolerable to a wide range of benzaldehyde derivatives comprising electron-withdrawing and electrondonating groups (0.05 g). Calculation of turnover number (TON) and turnover frequencies (TOF) provides a comprehensive way to make comparisons based on a per active site basis. These values have been calculated from the EDX and TGA data. The results are shown in Table 2

Suggested mechanism
A possible mechanism (Figure 8) involves the initial activation of malononitrile using the catalyst and then attacking the carbonyl group of the aldehyde. By removing one water molecule, intermediate (I) is prepared (Knoevenagel condensation). The reaction is followed by deprotonation of 3-methyl-1-phenyl-1H-pyrazole-5-ol by a catalyst. Then, Michael's addition of 3-methyl-1-phenyl-
The poly(aniline-co-melamine)@MnFe 2 O 4 was employed also as a catalyst in the synthesis of bis pyrazolone. We investigated the synthesis of bispyrazolones using aromatic aldehyde (1 mmol) and 3-methyl-1-phenyl-1H-pyrazole-5-ol (2 mmol) ( Figure 1C). In order to optimize this reaction, benzaldehyde and 3-methyl-1-phenyl-1H-pyrazole-5-ol were selected as starting materials, and the effects of temperature, amount of catalyst, solvent, etc., were investigated. The influence of varied quantities of catalysts on the reaction result was examined. Among different catalyst loadings (e.g., 0.04, 0.05, and 0.06 g), the catalyst based on poly(aniline-co-melamine) @MnFe 2 O 4 (0.05 g) was selected as the most effective amount. Following that, it was investigated how the solvent affected the reaction's result. According to the data, ethanol was the best solvent for this kind of domino reaction. The findings of the subsequent investigation into the impact of temperature showed that room temperature conditions produced the product with the highest yield (Table 3). Different electron-withdrawing or electron-donating groups, such as OCH 3 , NO 2 , and CH 3 , were chosen to react with 3-Methyl-1H-phenyl-5-pyrazolone at the most favorable reaction conditions in order to examine the range and generality of this technique. Table 4 provides a summary of the findings. Different aromatic aldehydes containing both electron-withdrawing and electron-donating groups, including OH, OCH 3 , NO 2 , and CH 3 , generated their respective derivatives in good to exceptional yields, according to the experimental data. The calculation of turnover number (TON) and turnover frequencies (TOF) of 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives is shown in Table 4.

Suggested mechanism
In the comparison of catalytic activity of poly(aniline-comelamine) with nanoparticle MnFe 2 O 4 in Table 1, the result showed that poly(aniline-co-melamine) is more active than MnFe 2 O 4 . Therefore, it seems that the amine group on the polymer will be the active site of the catalyst. A possible mechanism showed in (Figure 9). The initial deprotonation of 3-methyl-1-phenyl-1H-pyrazole-5-ol by amino groups in catalyst and then Knoevenagel condensation on the carbonyl group of the aldehyde that activated by ammonium ion. Then by removing H 2 O, intermediate (I) is prepared. The reaction is followed by deprotonation of 3-methyl-1-phenyl-1Hpyrazole-5-ol, then Michael's addition of the resulted anion to intermediate 1 affords 2, and the product is obtained by tautomerization.
Frontiers in Chemistry frontiersin.org 1H-pyrazole-5-ol, and malononitrile at the conditions specified in the experimental section. The catalyst was separated by an external magnet after adding hot ethanol to the reaction mixture. The catalyst was rinsed in ethanol (5 ml) and dried at low pressure. The catalyst was then re-fed into the reaction, as seen in Figure 11, where it continued to display good activity for the fifth time.

In-vitro anticancer study
Anticancer study was evaluated against colon cancer (HCT116) and normal (L929) cell lines within 24-48 h and results were reported as the percent growth of the treated cells when compared to the untreated control cells ( Figure 13).
After 24 h of exposure to colon cancer cells ( Figure 13A) 6c and 4q samples showed significant toxicity in 25 µg/ml and higher doses. Other samples (5i, 5j, 5q, 4d, 4o, and 4l) had toxicity in 12.5 µg/ml and higher doses. After 48 h ( Figure 13B), the 4q sample showed significant toxicity in 50 µg/ml and higher doses. On the other hand, 6c, 5i, and 5j samples had significant toxicity in 25 µg/ml and higher doses. Other samples (5q, 4l, 4o, and 4d) showed considerable toxicity in 12.5 µg/ml and higher doses. These results show that the tested dervativies have good

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Frontiers in Chemistry frontiersin.org 19 potential for destroying cancer cells and can be used in drug compounds.
For better comparison, the normal cell line was also exposed to tested samples and results showed that after 24 h ( Figure 13C), no considerable toxicity was observed for 25 µg/ml and lower doses for all samples. 5j sample was toxic in 50 µg/ml and higher doses. In addition, 6c and 4l samples showed significant toxicity in 100 µg/ml and higher doses. 5q, 5i, and 4d samples were toxic only in 200 µg/ml concentration. 4q and 4o samples did not cause toxicity in any of the applied concentrations.
After 48 h ( Figure 13D), no considerable toxicity was observed in the 12.5 µg/ml concentration. 6c, 5i, 5j, and 4d samples showed significant toxicity in 25 µg/ml and higher doses. 5q and 4q samples showed significant toxicity in 50 µg/ ml and higher doses. 4l sample was toxic in 100 µg/ml and higher doses. 4o samples were toxic only in 200 µg/ml concentration.
Results showed that the most effective anticancer agents were 5q and 6c samples which lead to~30%-40% lethality in the highest applied dose (200 μg/ml). This concentration is relatively higher than conventional anticancer drugs. Two standard chemotherapeutic drugs commonly used in the treatment of colorectal cancer are 5-Fluorouracil (5-FU), and oxaliplatin (OXA) (Manoochehri et al., 2022). The IC50 for 5-FU in different colorectal cancer cell lines including HCT116 (used in this study) reported in a range between 0.6 and 4 μg/ml (Varghese et al., 2019). In a study performed by Yang et al. (2019), IC50 for 5-FU resistant HCT116 and HT29 colorectal cancer cell lines was 12.5 ± 1.4 μg/ml and 11.5 ± 1.5 μg/ml, respectively. They showed that IC50 of oxaliplatin in parental HCT-116 and OXAresistant HCT116 cells was 9.18 and 70.01 μg/ml, respectively (Yang et al., 2019). However, chemical anticancer drugs have considerable toxicity against body normal cells owing to a lack of specificity (Sarfjoo et al., 2022), but 5q sample dose does not have considerable toxicity against normal cells in lower than 200 μg/ml doses in time points of 24 h.