Chitosan–Cu Catalyzed Novel Ferrocenated Spiropyrrolidines: Green Synthesis, Single Crystal X-ray Diffraction, Hirshfeld Surface and Antibacterial Studies

Chitosan-bounded copper (chitosan–Cu) was introduced for green synthesis of novel ferrocenated spiropyrrolidine hybrids, namely 3′-(4-.bromobenzoyl)-5′-(4-hydroxybenzyl)-4′-ferrocenylspiro[indoline-3,2′-pyrrolidin]-2-one and 3′-(4-bromobenzoyl)-4′-ferrocenylspiro[indoline-3,2′-pyrrolidin]-2-one, in good yield. A one-pot three-component 1,3-dipolar cycloaddition reaction was employed for the formation of spiropyrrolidines from 1-(4-bromophenyl)-ferrocene-prop-2-en-1-one and azomethine ylides, which were developed in situ from tyrosine, glycine, and isatin, respectively. Various spectroscopic methods were used to establish the structures of spiropyrrolidines, and a single crystal X-ray diffraction study of a spiropyrrolidine provided additional confirmation. The crystallographic study revealed that compound 3a has one independent molecule in its unit cell, which is correlated with Hirshfeld surface analysis, and describes intramolecular contacts adversely. The highly yielded products in green conditions were determined for their antibacterial significance and were found to have good activity against Gram-positive and Gram-negative bacterial strains.

In a continuation of our work, we are also focusing on preparing organometallicgrafted spiroheterocycles. Organometallic compounds usually hold significant efficacy due to their functioning in various commercial areas [14]. The synthesis of ferrocene-based heterocycles has attracted new attention because of their potential uses. Hence, as a simple and less costly organometallic material, ferrocene has received special attention in recent ongoing research for biologically active organic molecule synthesis.
One of the main objectives of "Green Chemistry" is the development of energy-efficient protocols. As such, catalysis receives much more attention in green chemistry research. The sustainability of a transition metal-catalyzed process depends on the catalyst's recovery

General
All the chemicals were used as acquired. All of the chemicals consumed were purchased from an international chemical supplier. The melting point was determined using a Stuart Scientific SMP3, version 5.0, melting point equipment (Bibby Scientific Limited, Staffordshire, UK), which was reported as uncorrected, on a clean Thermo Scientific NICOLE iS50 FT-IR spectrometer (Madison, WI, USA). Using TMS as an internal standard, the 1 H, and 13 C spectra were captured using Bruker 850 MHz equipment in a CDCl 3 . Chemical shifts are displayed in ppm, while coupling constants are given in hertz. Aluminum sheets covered with silica gel (Type 60 GF254, Merck, Darmstadt, Germany) were used for TLC, and the spots were found by exposing the sheets to UV light at either 254 or 360 nanometers. A PerkinElmer 2400 Series II Elemental CHN analyzer was used to perform elemental analyses. MoKa (k = 0.71073A) radiation was used to collect the single crystal X-ray data set for 2a on the Bruker Kappa X8 APPEX II diffractometer. The scan area was 2.22 • to 33.00 • .

Synthesis of Catalyst, Chitosan-Cu
Chitosan-Cu catalyst has been synthesized according to the reported procedure with minor modifications [22]. Here, Cu(CH 3 CO 2 ) 2 (200 mg) was dissolved in water and added to a suspension of chitosan (1 g) in 20 mL of water, which was then agitated for 3 h. After the copper had been adsorbed, the solid was filtered and extensively cleaned with water to remove lingering Cu compounds. It was then dried under vacuum for a whole night at 50 • C to produce the chitosan-Cu catalyst.

Synthesis of Spiropyyrolidines
Chalcone (1 mmol), isatin (0.147 g, 1 mmol), and amino acids (1 mmol) were mixed and dissolved in 10 mL of methanol. The mixture was then refluxed for 5 h. The reaction mixture was kept at room temperature for 30 min and poured in to 50 mL ice cold water. We filtered the precipitate, washed with water, and let it dry and recrystallize in chloroform-methanol to result in the pure 3a-b samples.

Green Method for Synthesis of Spiropyyrolidines (3a-b)
Isatin (0.147 g, 1 mmol), chalcone (1 mmol), and amino acids (1 mmol) were mixed well with the catalyst chitosan-Cu (50 mg) in a mortar. We transferred the reaction mixture into a 100 mL beaker, and it was heated at 60 • C. The reaction progress was checked by TLC. After the reaction completion, hot ethyl acetate was poured to recover the insoluble catalyst, which was then recovered by filtration. The filtrate was evaporated, and crude products 3a and 3b were recrystallized from the analytical grade chloroform-methanol mixture.

Synthesis of Spiropyyrolidines under Different Solvent Assisted Condition Using Chitosan-Cu Catalyst
Chalcone (1 mmol), isatin (0.147 g, 1 mmol), and tyrosine (1 mmol) mixture were dissolved in diverse solvents (chloroform, PEG-400, methanol, and H 2 O) (10 mL) and catalyst (50 mg) was added to it. The reaction mixture was refluxed for a particular time duration. The reaction progress was checked by TLC. After completion of the reaction the synthesized product was isolated and purified from the reaction medium by adding hot ethyl acetate. The catalyst was collected using filtration, and the remaining solvent was evaporated. A chloroform-methanol mixture was used to recrystallize the mixture to produce compound 3a.

Synthesis of Spiropyyrolidines under Solvent-Free Condition Using Different Catalytic System
A mixture of chalcone (1 mmol), isatin (0.147 g, 1 mmol), and tyrosine (1 mmol) were mixed well with different catalytic systems, namely sodium acetate, copper acetate, CuSO 4 , and chitosan, respectively, in a mortar, separately. We transferred the reaction mixture into a 100 mL beaker, and it was heated at 60 • C. Then, TLC was used to check the reaction progress After the reaction completion, ethyl acetate was added for the recovery of the insoluble catalyst. The filtrate was evaporated, and the chloroform-methanol was used to recrystallize the pure compound 3a.  13

Recycling and Reusage of Chitosan-Cu Catalyst
A mixture of chalcone (1 mmol), isatin (0.147 g, 1 mmol), and tyrosine (1 mmol) were mixed well with the catalyst chitosan-Cu (50 mg) in a mortar. We transferred the reaction mixture into a 100 mL beaker, and it was heated at 60 • C. The reaction progress was checked by TLC. Hot ethyl acetate added to recover the insoluble catalyst, and collected by filtration after the completion of reaction. We evaporated the filtrate, and the crude product 3a was recrystallized from chloroform-methanol mixture. The residual catalyst was washed with an isopropyl alcohol-ethyl acetate (1:9) solution, dried, and then used for the next cycles.

Single Crystal X-ray Crystallography
The prepared material was added to the suitable solvent until the preparation of the concentrated solution. The slow evaporation methodology was used to obtain the suitable single crystal. The selected fine and well-shaped single crystal was fixed onto the assembly and position-fitted with microfocus Cu/Mo Kα radiation for data collection via the Agilent SuperNova Dual Source Diffractometer (Agilent Technologies). The CrysAisPro software [29] was used for data collection, the system temperature was 296 K, and Mo Kα radiation was the source of radiation. The structure was solved and further refined with SHELXS-97 following the direct approach method [30], and improved by utilizing fullmatrix least-squares methods on F 2 , using SHELXL-97 [30]. The WinGX software [31] was used as a parent software to deal with structure solution and refinement. All non-hydrogen atoms were refined anisotropically using the full-matrix least squares techniques [27]. The required figures for understanding were generated with the use of well-acknowledged tools, such as PLATON [32] and ORTEP [33].
The U iso was fixed at 1.2 times for the C(H) groups (aromatic, cp-ferrocene, and methine), C (H, H) groups (methylene), and for N(H) groups (amide and secondary amine). The U iso was fixed at 1.5 times for the O(H) group (hydroxyl). All the available hydrogen atoms (C−H, N−H, O−H) were refined with riding coordinates. Crystal data were deposited at the Cambridge Crystallographic Data Centre; the following deposition number 2215469 was assigned to the compound's title CCDC number. Crystal data is available at no cost upon request to the CCDC, 12 Union Road, Cambridge, CB21 EZ, United Kingdom; (Fax-(+44) 1223 336-033; email-data_request@ccdc.cam.ac.uk).

Antibacterial Study
The synthesized compounds (spiropyrolidines) were tested for their capacity to avert the projected growth of several bacterial strains, including Pseudomonas aeruginosa (P. aeruginosa), Streptococcus pyogenes (S. pyogenes), Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Streptococcus aureus (S. aureus) using the disk diffusion method [34][35][36]. The standard inoculums (1 − 2 × 107 cfu/mL 0.5 McFarland standards) were obtained and dispersed on the sterile agar plate surface. After being dry heat sterilized for 1 h at 140 • C, the 6 mm-diameter microbial discs were prepared for soaked and put onto an inoculation plate. The previously soaked discs in DMSO and ciprofloxacin (30 g) were used as positive and negative controls, respectively. By flipping the plate and incubating it at 37 • C for 24 h, the mixture's impurities were removed. The antibacterial efficacy against several bacterial species was determined by the size of the inhibition zone using a broth microdilution technique. The tested compounds and standard drug were logarithmically and sequentially diluted two-fold in nutrient broth, and then growing bacterial cells at a concentration of about 5 × 10 5 cfu/mL were injected into the bacterial culture. After 24 h of incubation at 37 • C, bacterial growth was measured visually and spectrophotometrically. The MIC refers to the minimal concentration of a drug necessary to inhibit bacterial growth. The 100 L bacterial culture-inoculated sterile agar plates were used to calculate the minimal bactericidal concentration (MBC). The CFU counts were calculated after an incubation period of 18 to 24 h at 35 • C. The minimum concentration of a compound required to kill 99.9% of inoculum is the MBC value.

Results and Discussion
Spiroheterocyclic scaffolds are generally synthesized by the dipolarophiles reacting with azomethine ylides. Moreover, chalcones instead of simple dipolarophiles are used for the spiropyrrolidines formation mentioned by few authors [37][38][39]. Previously, we have reported a ferrocenated chalcone as 1-(4-bromophenyl)-ferrocene-prop-2-en-1-one 1, which was synthesized using the condensation of 4-bromoacetophenone and ferrocenecarboxaldehyde [40]. Herein, we report the novel spiropyrrolidine derivatives 3a-b synthesis using a 1,3-dipolar cycloaddition reaction of chalcone 1 with azomethine ylides formed in situ from tyrosine, glycine, and isatin, respectively. The synthetic strategy of 3a-b is depicted in Scheme 1. Initially the reaction was carried out under the usual solvent-assisted refluxing conditions, with methanol serving as the solvent. The reaction was found to be finished in 5 h; however, there was no appreciable product yield at any parameter, and, moreover, the method was not environmentally friendly. This inspired us to use solvent-free heating method for the same reaction, and the reaction was performed by heating the starting ingredients in the presence of a heterogeneous catalyst, chitosan-Cu, at 60 °C . The catalyst was synthesized according to the reported literature with minor modifications [25]. Chitosan, a bio-based polymer, is highly stable in acidic as well as basic medium and is, therefore, able to form a good heterogeneous support. The immobilization of Cu on chitosan helps to increase the surface area, stability, increased number of active sites, etc. Thus, the combined effect of both the support and acidic catalytic system afforded 3a-b in good Initially the reaction was carried out under the usual solvent-assisted refluxing conditions, with methanol serving as the solvent. The reaction was found to be finished in 5 h; however, there was no appreciable product yield at any parameter, and, moreover, the method was not environmentally friendly. This inspired us to use solvent-free heating method for the same reaction, and the reaction was performed by heating the starting ingredients in the presence of a heterogeneous catalyst, chitosan-Cu, at 60 • C. The catalyst was synthesized according to the reported literature with minor modifications [25].
Chitosan, a bio-based polymer, is highly stable in acidic as well as basic medium and is, therefore, able to form a good heterogeneous support. The immobilization of Cu on chitosan helps to increase the surface area, stability, increased number of active sites, etc. Thus, the combined effect of both the support and acidic catalytic system afforded 3a-b in good yields (88-91%) in a shorter time span (Table 1). The obtained products were purified using the appropriate solvents, and the IR and NMR spectra were used to determine the structures of compounds. The additional structural confirmation was followed by a single crystal X-ray diffraction study. The reaction condition was optimized with further studies using a model reaction. The model reaction was selected from chalcone, isatin, and tyrosine, which yields 3a. Initially, different kinds of solvents were selected for the examination of the solvent effect, namely methanol, PEG-400, chloroform, and water, along with the chitosan-Cu catalyst. A solventfree reaction was also compared along with it. The comparative study shows that a yield of 75% was produced after 50 min when using methanol as a solvent. Similarly, PEG-400 took 1 h for completion of its reaction, with a 65% of yield. The nonpolar solvent chloroform resulted in a 64% yield after 1.5 h. A much better result in terms of yield (78%) was obtained in water compared to other solvents. However, solvent-free heating condition afforded 3a a good yield (91%) in a shorter time span (15 min). Thus, the solvent-free condition became the efficient method in terms of relevant yield and time for this synthesis, as can be seen in Table 2. To determine the superiority of chitosan-Cu as a catalyst, a model reaction was studied with different catalysts in solvent-free condition. For this purpose, sodium acetate, copper acetate, CuSO 4 , and chitosan were selected. The result obtained with Na(CH 3 CO 2 ) was not satisfactory for the concerned reaction. Then, Cu(CH 3 CO 2 ) 2 and chitosan were evaluated separately. Cu(CH 3 CO 2 ) 2 resulted a 56% yield after 40 min. Similarly, the chitosan also resulted in same product in 1.5 h with a poor yield of 45%. Therefore, we expected that the combination of these two can produce much improved result in this reaction, and this become true, as shown in Entry 4 of Table 3. Chitosan-Cu can catalyze the said reaction much faster with an excellent yield of products. Immobilization of copper on chitosan increases the surface area of the catalyst and causes more active sites to become available for the interaction of reactants, enhancing the rate of reaction. Thus, the use of the chitosan-Cu catalyst in solvent-free condition was found to be the best condition for the synthesis of novel spiropyrrolidine derivatives 3a-b using a 1,3-dipolar cycloaddition reaction of chalcone 1 with azomethine ylides formed in situ from tyrosine, glycine, and isatin, respectively. Moreover, this combination has the advantage of the insolubility of the catalyst, which facilitates easy separation and simplifies the procedure.
The reusability is one of the green synthesis criteria to be proven by a heterogeneous catalyst. The investigation into catalyst recycling was carried out; ethyl acetate was added to the reaction mixture at the completion of the reaction (confirmed by TLC). It only took a simple filtration to separate the catalyst solution from the products due to the high solubility of the product and the insolubility of the catalyst in ethyl acetate. The catalyst, which was left over as residue, was washed with an isopropyl alcohol-ethyl acetate (1:9) solution, dried, and utilized for the subsequent cycles. The recorded catalyst was effective for roughly five consecutive runs without seeing a material drop in catalytic activity (Table 4). The plausible mechanism for spiropyrrolidine 3a formation has been given in Scheme 2. The reaction starts with the activation of the carbonyl oxygen of the isatin by coordination with the chitosan-Cu catalyst and further nucleophilic attack of tyrosine. The intermediate formed undergoes dehydration and decarboxylation to produce another nucleophile which attacks the β-carbon of chalcone, followed by cyclization to result in spiropyrrolidine 3a as the product.
In the 3a-b IR spectrum, the common absorption bands assigned at around 3310, 2923, 1718, and 1682 cm −1 correspond to NH, CH, and two C=O groups, respectively. The 1 H NMR spectrum of 3a exhibited two singlet peaks at 1.27 and 1.61 ppm assigned to tyrosine CH 2 and OH protons, respectively. The triplet signals at 4.21, 4.53, and 4.62 ppm were assigned to three CH protons. Due to the ferrocene moiety, the two protons appeared as singlets at 4.68 and 4.70 ppm, the six protons appeared as a multiplet at 4.72-4.76 ppm, and one proton appeared as a doublet at 4.80 ppm. The 12 aromatic protons were assigned as doublets, triplets, and a multiplet in the region 6.80−7.81 ppm. The rest of the oxindole NH and tyrosine OH protons appeared as singlet 7.96 and 8.10 ppm, respectively. In the 13 C spectrum, the two broad peaks at 188.5 and 194.2 ppm were assigned to two C=O carbons. The two CH and one CH 2 carbons appeared at 34.8, 40.2 and 29.7 ppm, respectively.
The plausible mechanism for spiropyrrolidine 3a formation has been given in Scheme 2. The reaction starts with the activation of the carbonyl oxygen of the isatin by coordination with the chitosan-Cu catalyst and further nucleophilic attack of tyrosine. The intermediate formed undergoes dehydration and decarboxylation to produce another nucleophile which attacks the β-carbon of chalcone, followed by cyclization to result in spiropyrrolidine 3a as the product. Scheme 2. Plausible mechanism for the formation of spiropyrrolidine 3a.
In the 3a-b IR spectrum, the common absorption bands assigned at around 3310, 2923, 1718, and 1682 cm −1 correspond to NH, CH, and two C=O groups, respectively. The 1 H NMR spectrum of 3a exhibited two singlet peaks at 1.27 and 1.61 ppm assigned to tyrosine CH2 and OH protons, respectively. The triplet signals at 4.21, 4.53, and 4.62 ppm were assigned to three CH protons. Due to the ferrocene moiety, the two protons appeared as singlets at 4.68 and 4.70 ppm, the six protons appeared as a multiplet at 4.72-4.76 ppm, and one proton appeared as a doublet at 4.80 ppm. The 12 aromatic protons were assigned as doublets, triplets, and a multiplet in the region 6.80−7.81 ppm. The rest of the oxindole NH and tyrosine OH protons appeared as singlet 7.96 and 8.10 ppm, respectively. In the Scheme 2. Plausible mechanism for the formation of spiropyrrolidine 3a.

Hirshfeld Surface Analysis
The crystallographic file of the compound was studied more on Crystal Explorer [46] as a theoretical investigation into some useful interconnections. This is an approach in which a particular study endorsed the interactions which were observed in experimental crystallographic analysis. Figure 3 shows the different maps indicating the nature of contacts (a-f). The variation in colors, such as red, blue, and white, indicates the intensity or type of contacts from strong to weak. Figure S1, added to the supplementary part, shows the hydrogen bonding interactions. The fingerprint plots of the compounds were generated and shown in Figure

Antibacterial Activity
The antibacterial efficacy of the spiropyrrolidines 3a-b against various Gram-positive and Gram-negative antibacterial pathogens, such as Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and E. coli was evaluated. The antibacterial activity findings of 3a-b demonstrated moderate to good activity (Table 7). Compared to the effectiveness of the standard antibiotic ciprofloxacin, 3a and 3b were found to be more active against S. pyogenes and methicillin-resistant Staphlococcus aureus (MRSA+Ve) (MIC−25 g/mL) (Table 8). Additionally, studied have indicated that these compounds are efficient against S. aureus, E. coli, and S. pyogenes. In actuality, Gram-positive bacterial strains were more effectively combated than Gram-negative bacterial strains by both compounds. In comparison to the equivalent MIC results of 3a and 3b revealed by the disk diffusion method, the MBC of compounds was found to be two, three, or four times greater. The results of the antibacterial study revealed that compounds have moderate to good antibacterial activity. The MBC values for both compounds were discovered to be two, three or four times greater than the corresponding MIC values (Table  8).

Antibacterial Activity
The antibacterial efficacy of the spiropyrrolidines 3a-b against various Gram-positive and Gram-negative antibacterial pathogens, such as Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and E. coli was evaluated. The antibacterial activity findings of 3a-b demonstrated moderate to good activity (Table 7). Compared to the effectiveness of the standard antibiotic ciprofloxacin, 3a and 3b were found to be more active against S. pyogenes and methicillin-resistant Staphlococcus aureus (MRSA+Ve) (MIC−25 g/mL) (Table 8). Additionally, studied have indicated that these compounds are efficient against S. aureus, E. coli, and S. pyogenes. In actuality, Gram-positive bacterial strains were more effectively combated than Gram-negative bacterial strains by both compounds. In comparison to the equivalent MIC results of 3a and 3b revealed by the disk diffusion method, the MBC of compounds was found to be two, three, or four times greater. The results of the antibacterial study revealed that compounds have moderate to good antibacterial activity. The MBC values for both compounds were discovered to be two, three or four times greater than the corresponding MIC values (Table 8).

Conclusions
In conclusion, the present investigation results showed that chitosan-Cu catalyzed the green synthesis of spiropyrrolidine derivatives which have advanced superiority in terms of improved product and short reaction times over spiropyrrolidines which were conventionally synthesized. The FTIR and NMR spectral analyses has been established to investigate the newly synthesized spiropyrrolidine structures, which were moreover corroborated by the diffraction study (single crystal X-ray). The crystallographic study confirmed that compound 3a has one independent molecule in its unit cell, which is correlated with Hirshfeld surface analysis, and described intramolecular contacts adversely. The good availability and dispersal of active chemical sites, which promote stronger interaction between substrate molecules and the catalyst, may be the cause of the key and maximum catalytic activity under solvent-free conditions. Additionally, because there is no solvent as a medium, there is no dilution effect, and the heat required for activation energy is directly available to the reactant molecules, which also help to improve yield as well as to reduce reaction time. Therefore, this methodology becomes greener, with attractive features, such as being eco-friendly, having a mild reaction condition, reduced period of reaction time, excellence in yields, recyclability of catalyst, etc. The target compound was crystalized in monoclinic crystal system with the space-group P2 1 /c. The unit cell dimensions are a = 16.7910(13) Å, b = 7.8298(6) Å, and c =23.1312(16) Å, while the beta angle is β = 106.316(8) • . The central pyrrolidine ring is substituted by various groups, and different functional groups are involved in hydrogen bonding interactions to provide stability to the structure. The spiropyrrolidines 3a-b were afforded in high yields in green conditions, and were determined for their antibacterial significance and found to show good activity against Gram-positive (G + ) and Gram-negative (G − ) bacterial strains.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/polym15020429/s1. Table S1: Bond Lengths for 3a; Table S2: Bond Angles for 3a; Table S3: Torsion angles for 3a; Figure S1: A view along the bc-plane showing the formation of a two-dimensional network.