Cysteic acid grafted to magnetic graphene oxide as a promising recoverable solid acid catalyst for the synthesis of diverse 4H-chromene

4H-chromenes play a significant role in natural and pharmacological products. Despite continuous advances in the synthesis methodology of these compounds, there is still a lack of a green and efficient method. In this study, we have designed cysteic acid chemically attached to magnetic graphene oxide (MNPs·GO-CysA) as an efficient and reusable solid acid catalyst to synthesize 4H-chromene skeletons via a one-pot three components reaction of an enolizable compound, malononitrile, an aldehyde or isatin, and a mixture of water–ethanol as a green solvent. This new heterogeneous catalyst provides desired products with a good to excellent yield, short time, and mild condition. This procedure presents an environmentally friendly approach for the synthesis of a great number of 4H-chromene derivatives.

Cysteic acid grafted to magnetic graphene oxide as a promising recoverable solid acid catalyst for the synthesis of diverse 4H-chromene Firouz Matloubi Moghaddam * , Mohammad Eslami & Golfamsadat Hoda 4H-chromenes play a significant role in natural and pharmacological products. Despite continuous advances in the synthesis methodology of these compounds, there is still a lack of a green and efficient method. In this study, we have designed cysteic acid chemically attached to magnetic graphene oxide (MNPs·GO-CysA) as an efficient and reusable solid acid catalyst to synthesize 4H-chromene skeletons via a one-pot three components reaction of an enolizable compound, malononitrile, an aldehyde or isatin, and a mixture of water-ethanol as a green solvent. This new heterogeneous catalyst provides desired products with a good to excellent yield, short time, and mild condition. This procedure presents an environmentally friendly approach for the synthesis of a great number of 4H-chromene derivatives.
4H-chromenes represent an important class of oxygen-containing heterocycles and a key building block of many natural products. They are also widely found in nature, in some edible fruits and vegetables, to name but a few, and these compounds also are broadly used as cosmetics and pigments [1][2][3] . 4H-chromene scaffolds exhibit pharmacological properties and biological activities such as antioxidant 4 , antimicrobial 5,6 , antiviral, antibacterial, pro-apoptotic 7-11 , anticancer 12 , antifungal, anticoagulant, antinociceptive 13 , antiproliferative 14 , antitubercular, antiallergic, antibiotic, hypolipidemic, and immunomodulating activities. They also are used as cognitive enhancers, in order to treat neurodegenerative disease 15,16 such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, AIDS, Parkinson's disease, Down's syndrome, and also myoclonus and schizophrenia 17 . For example, 4H-chromene derivatives such as A, B, C, D, and E are known as apoptosis inducer, insulin-regulated aminopeptidase for enhancing memory, learning functions inhibitor, and anticancer therapeutic agents respectively, which are shown in Fig. 1 11,18-20 . In this regard, multi-component reactions (MCRs) have gained considerable attention for constructing a broad range of complex molecules in a highly efficient, rapid, step-economic, low-cost, and eco-friendly manner [21][22][23][24][25][26][27] . Because of the benefits of MCRs and the great importance of 4H-chromenes, syntheses of these compounds have been developed using different catalysts in the multicomponent reaction between malononitrile (or ethyl cyanoacetate), a diverse electron-rich phenol or enolizable carbonyl compound, and an aldehyde 1 . Recent catalytic systems for the synthesis of 4H-chromene derivatives consist of Fe(HSO 4 ) 3 28 , nickel nanoparticles 29

Results and discussion
The MNPs·GO-CysA catalyst was synthesized using a few steps presented in Fig. 3. Details of the preparation method are described in the experimental section.
The FTIR spectrum is shown in Fig. 4. demonstrates the formation of desired bonds and the presence of new functional groups of MNPs·GO-CysA. The band at 640 cm −1 is attributed to the Fe-O bond vibration as proof of the existence of Fe 3 O 4 in MNPs·GO-CysA 107 . The intense broad bands at ̴ 3400 can be attributed to stretching of    108 . The peaks that appeared at 1137 cm −1 and 1026 cm −1 are due to the SO 2 asymmetrical and symmetrical stretching vibrations 109 . In addition to the previous note, the absorption peak at 854 cm −1 correspondings to bonded N-H stretching, confirmed the formation of a chemical bond between cysteic acid and the magnetic GO sheets (Fig. 4). XRD analysis of MNPs·GO-CysA clearly indicates the Fe 3 O 4 spinel structure (Fig. 5 In order to evaluate the structure, morphology, and size of the catalyst, SEM, TEM and EDX analysis were collected. As can be seen in Fig. 6a. the microstructure of MNPs·GO-CysA presented the average size of 16 nm. The TEM micrograph of MNPs·GO-CysA catalyst is shown in Fig. 6b. as shown in Fig. 6b, the sizes of the magnetic Fe 3 O 4 nanoparticles with tiny particles possessing the spherical morphology were obtained from 10 to 15 nm on a lighter shaded substrate corresponding to the GO sheet. The TEM image of the catalyst (MNPs·GO-CysA) also confirmed that the Fe 3 O 4 nanoparticles were attached to the surface of graphene oxide free from aggregation. Furthermore, the EDX pattern of the catalyst (Fig. 6c) turned out that MNPs·GO-CysA contains Fe, O, C, N, and S. CHNS elemental analysis was performed and the results proved the presence of Sulfur atoms in the structure with a scale of 6.68%. The percentage of Sulfur atom in the sample of magnetic GO before the addition of Cysteic acid was determined to be 0.009%. It also appeared that MNPs·GO-CysA contains 1.56% Nitrogen. These results confirm that cysteic acid is successfully attached to the magnetic GO (~ 1.37 mmol g −1 ).  www.nature.com/scientificreports/ TG-DTG thermograms explain the thermal stability of the MNPs·GO-CysA nanocomposite. All the results derived from TGA analysis are shown in Fig. 7. The first stage of decomposition observed below 180 °C is attributed to physically and chemically trapped water between magnetic GO nanosheets. The second stage of the weight loss, which counted 11.24% between 180 and 220 °C, can be ascribed to the attached organic groups (Cysteic acid) on the surface of magnetic GO nanosheets. Such a relatively high grafting yield suggests successful  www.nature.com/scientificreports/ attachment of Cysteic acid to Fe 3 O 4 /GO surface. The last stage weight loss between 220-600 °C related to the decomposition of graphene oxide nanosheets (the removal of epoxide, hydroxyl, and carboxylic acid surface groups of GO were performed at the beginning of this stage).
In the next section, the magnetic behavior of the Magnetic graphene oxide functionalized with cysteic acid nanocomposite has been investigated. In this respect, vibrating sample magnetometer (VSM) measurements were carried out at room temperature for both the Finally, the activity of the magnetically separable acid catalyst was consequently investigated upon characterization in the diver's derivatives of 4H-chromen synthesis. In this regard, to obtain the optimum condition, the three-component reaction between benzaldehyde (1), malononitrile (2), and 4-hydroxy-6-methyl-2H-pyran-2one (3) (molar ratio 1.0:1.1:1.0) was selected as the model reaction and studied in different conditions. The results are summarized in Table 1. In the first step, the reaction was carried out in different solvents including ethanol, water, water-ethanol (3:1), acetonitrile, THF, and solvent-free without any catalysts. The best yield (29%) was obtained in water-ethanol (3:1) in 3 h at room temperature ( Table 1, entries 1-6). The Knoevenagel condensation product was formed in quantitative yield as a result of the reaction between benzaldehyde and malononitrile. This result shows that the presence of a catalyst is necessary to improve the desired reaction rate and yield.
In the studies of different green catalysts such as ethylenediaminetetraacetic acid on (EDTA), cysteic acid, graphene oxide, and Fe 3 O 4 nanoparticles on the model reaction, it was found that the best yield of desired product 5e (43%) was obtained when the cysteic acid was used as the catalyst in ethanol-water under reflux conditions (Table 1, entry 7-10).
Studies showed that acidic reagent plays the main role in the catalytic cycle in these reactions. Therefore, cysteic acid was attached as a green biodegradable amino acid to the magnetic graphene oxide to increase the  www.nature.com/scientificreports/ acidic sites of graphene oxide and to obtain a suitable catalytic activity. Then, the prepared catalyst was used in the model reaction. The reaction was studied under different conditions including running the model reaction at r.t., at 80 °C and using the grinding technique under the solvent-free condition. The desired product was not formed in a suitable yield at any of the mentioned conditions. Interestingly, the product was obtained properly under reflux in water-ethanol (1:3) in the presence of 5 mg of MNPs·Go-CysA nano-catalyst with a yield of 68% after 3 h (Table 1, entries [11][12][13][14]. Therefore, the effect of catalyst loading on the completion of the reaction was examined in the next experiments (Table 1, entries [15][16][17][18]. By increasing the catalyst amount from 5 to 15 mg, the reaction yield improved from 68 to 92% at 30 min. This result clearly shows that the catalyst is effective enough to improve the reaction yield. The active sites of the magnetic nano-catalyst, as a solid acid, can be the acidic functional groups, including the graphene oxide's carboxylic acid group, and the cysteic acid's carboxylic and sulphonic acid groups. These active sites as Bronsted acids improve the reaction yield. The Fe 3 O 4 nano-particles also can race the reaction up as Lewis acids. However, the optimization results indicate the major active site of the nano-particles to be cysteic acid's functional groups. Consequently, we developed the optimized reaction condition (15 mg of MNPs·GO-CysA in 2 ml of water-ethanol (3:1) under reflux conditions) for other derivatives of aromatic aldehydes (1) and enolizable compound (3, 4, 7, 10) for the synthesis of the various derivatives of 2-amino-3-cyano-4H-chromenes (5a-j, 6a-j, 8a-j, 11a-l). The results have been presented in Tables 2, 3, 4.
It is noteworthy that in all processes of 2-amino-3-cyano-4H-chromene derivatives (5,6,8) syntheses, the reaction of aromatic aldehydes which possessed electron-withdrawing groups are shown to be faster than the reaction of aromatic aldehydes with electron-donating groups. Additionally, the reaction was proceeding with heterocyclic aldehydes, and the desired products were obtained in good yields.  www.nature.com/scientificreports/ Finally, to demonstrate the effectiveness and efficiency of this new method, the optimized conditions were developed for four-component one-pot condensation of ethyl acetate, hydrazine hydrate/or phenyl hydrazine, malononitrile, and isatin. It should be pointed out that the reaction proceeded at reflux conditions to give the purely expected 6′-amino-3′-methyl-2-oxo-1′H-spiro[indoline-3,4′-pyrano[2,3-c]pyrazole]-5′-carbonitrile (11a-l) in quantitative yields in very short reaction time (Table 4).
In all studies, the reaction catalyst was simply separated from the main product employing an external magnet after cooling the mixture to r.t. and the precipitated product was filtered out of the reaction mixture.
Due to the importance of using heterogeneous catalysts in industrial processes, we studied the recyclability of the MNPs·GO-CysA nano-catalyst by using it in repeatedly five runs reactions for product 11a under the optimized reaction conditions. After each reaction cycle, the superparamagnetic catalyst was separated by an external magnetic field and washed twice with hot deionized water (5 mL), once with 5 mL ethanol, dried in an oven at 70 °C, and reused in the model reaction. Summarized results in Fig. 9. implied that a significant reduction in catalytic efficiency was not observed after 5 runs. The strong covalence interaction of cysteic with the GO surface could be the reason for the repetitive use of the catalyst in a greater number of catalytic runs with high efficiency. Eventually, the comparison of the results of FT-IR, EDX, and VSM analysis of the recycled catalyst after five cycles revealed that there are no significant structural changes occurred at the nanoparticle surface (Fig. 10).
In order to evaluate the catalytic efficiency of MNPs·Go-CysA nano-catalyst, we compared the advantages of this catalyst with some other previously reported catalysts for the synthesis of 4H-chromenes. The comparison proved that the catalyst possesses higher activity compared to other catalysts (Table 5).

Conclusions
In summary, we have successfully developed a solid acid supported magnetic graphene oxide catalyzed reaction of an enolizable compound, active methylene nitriles, and aldehydes, which provides rapid and efficient access to different 4H-chromene derivatives. Cysteic acid has been incorporated on the magnetic GO successfully and properly dispersed between GO sheets. We have demonstrated that MNPs·GO-CysA acts as a non-hazardous, efficient, reusable, and convenient catalytic system for the synthesis of a wide range of 4H-chromenes in water/ ethanol as a green solvent. Additionally, the presented catalyst was easily removed from the reaction mixture by means of an external magnet and reused several times with little loss of activity. It can be seen that simplicity in preparation of the present catalyst, efficient recyclability and reusability of the catalyst, short reaction times, and high yields of the products can be considered as outstanding characteristics of the present protocol.

Experimental
Materials. All the materials which were used in our experiments including reagents and solvents were purchased from Merck or Sigma Aldrich. The materials were used without further purification, while benzaldehyde was used freshly after distillation. We used our University's Bruker (Avance DRX-500) spectrometer in order to have our products been checked via 1 H NMR and 13 C NMR spectrometry using CDCl 3 as solvent at room temperature. Chemical shifts from the initial standard tetramethylsilane are reported in parts per million (ppm). Using an ABB Bomem MB100 FTIR spectrophotometer, the samples were tested, the results of which are reviewed. CHNS analysis was done by LECO Truspec. Scanning electron microscopy (SEM) was performed on   Tables 2, 3, and 4. After reaction completion, which was controlled by Thin Layer Chromatography (TLC) test (using EtOAc/ n-Hexane, 1:3 as solvent), the catalyst was separated by a magnet, and the obtained solid product was filtered. In the case of impurities, the obtained product was recrystallized from ethanol.

Synthesis of Spiro 4H-chromene derivatives.
A glass vial was successively charged with hydrazine monohydrate (1.1 mmol), ethyl acetoacetate (1 mmol), MNPs·GO-CysA (15 mg), water-ethanol (3:1, 2 mL) and stirred at reflux conditions for 5 min. Then isatin derivatives (1 mmol), methylene reagent (malononitrile, 1.1 mmol) were added to reaction mixture and stirred for the appropriate time brought in Table 4. After reaction completion, which was controlled by Thin Layer Chromatography (TLC) test (using EtOAc/ n-Hexane, 1:3 as solvent) and the reaction color change from red to white, the catalyst was separated by a magnet, and the obtained solid product was filtered. In the case of impurities, the obtained product was recrystallized from ethanol.