Design and synthesis of chitosan-gelatin modified magnetic composite capped Palladium nanoparticles for the C-N Ullmann coupling reactions and study of its anti-prostate cancer effects

2.1. Materials

Chitosan (98%), gelatin, PdCl₄, haloarenes, imidazole, indole, and solvents were obtained from Sigma-Aldrich and Merck. The crystal structure of the materials was characterized using a Bruker D8 Advanced X-ray diffractometer (XRD) (Germany), operating at a scan rate of 4° per minute with a step size of 0.02 degrees. The X-ray source was Cu Kα, at 40 kV and 35 mA. The morphology of the samples was analyzed using Field Emission Scanning Electron Microscopy (FESEM), utilizing the TESCAN Mira3 model (Czech Republic) and the ZEISS SIGMA VP model.

2.2. Fe₃O₄@CS-Gl synthesis

The Fe₃O₄ nanoparticles (NPs) were synthesised following methods described in the literature [32]. To prepare a CS-Gl solution, 0.1 g chitosan and 0.1 g gelatin were dissolved in 50 mL of water with 0.5 mL acetic acid and subjected to ultrasound irradiation for 30 min. Next, 0.5 g of as-prepared Fe₃O₄ nanoparticles were scattered in the solution by subjecting it to ultrasound irradiation for 20 min. This was then mixed with the CS-Gl filtrate. The mixture was then stirred for 6 h at room temperature. The Fe₃O₄@CS-Gl composite was separated with a magnet, thoroughly rinsed with pure water, and dried at 50°C.

2.3. Preparation of Fe₃O₄@CS-Gl/Pd NPs

In this stage, 0.5 g of the Fe₃O₄@CS-Gl compound was sonicated for 20 min in 100 mL of water to ensure uniform dispersion. A diluted solution of Na₂PdCl₄ (30 mg in 10 mL) was added to the suspension and stirred at 80°C for 5 h. Following this, the Fe₃O₄@CS-Gl/Pd NPs nanocomposite was isolated using a magnetic field. It was then washed thoroughly with a water/acetone mixture and left to air-dry. The palladium (Pd) loading of the composite was quantified by inductively coupled plasma optical emission spectrometer (ICP-OES), which revealed a concentration of 0.065 mmol/g.

2.4. Ullmann-type coupling reactions catalyzed by Fe₃O₄@CS-Gl/Pd NPs

We set up a mixture in a 25 mL flask consisting of haloarenes (1 mmol), dimethylformamide (DMF) (3 mL), either imidazole or indole (1 mmol), and Et₃N (2 mmol), along with 0.020 g of Fe₃O₄@CS-Gl/Pd NPs, which include 0.01 mmol of Pd. The blend was stirred to 80°C and for a suitable duration, while monitoring the progress of reaction by thin layer chromatography (TLC) (n-hexane/ethyl acetate 5:1). At end of reaction, we allowed the mixture to cool, and the catalyst was subsequently separated by a magnet. The catalyst was washed with ethyl acetate and EtOH. The reaction mixture was then dried using dry Na₂SO₄ and concentrated under reduced pressure. Finally, the target product was purified through TLC on silica gel.

2.5. Inquiring into the antioxidant consequences

The antioxidant activity of Fe₃O₄@CS-Gl/Pd NPs was evaluated using the conventional 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. For the experiment, 2 mg DPPH powder was dissolved in 33.8 mL of 96% EtOH and stored in the dark at 37°C for 30 min. Various sample mixtures with different concentrations of nanoparticles (ranging from 1 to 1000 μg/mL) were then added to the DPPH solution. The mixtures were kept in the dark for an additional hour before measuring absorbance. The DPPH solution, in its ethanolic form, exhibits a purple color with a peak absorption at 517 nm. Following the reaction between the solution and the nanoparticles, a decrease in absorbance was observed, indicating the scavenging of free radicals or protons by the sample. DPPH stabilizes by accepting an electron or a hydrogen atom. As a positive control, butylated hydroxytoluene (BHT), a common antioxidant, was used, and the radical scavenging activity was calculated using the following formula. $\text{DPPH scavenging effect}\left(\%\right)=\left[\left(\text{A}0-\text{A1}\right)/\text{A}0\right]\times \text{1}00$

2.6. Anti-prostate cytotoxic action of Fe₃O₄@CS-Gl/Pd NPs nanoparticles

To assess the antioxidant potential of the synthesized material, we investigated the cytotoxicity of the Fe₃O₄@CS-Gl/Pd NPs nanocomposite against PC cell lines (LNCaP and PC3) using the 3-(4,5-di methyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The cells were initially cultured under standard conditions and then treated with various concentrations of the Fe₃O₄@CS-Gl/Pd NPs nanocomposite (ranging from 1 to 1000 μg/mL) for 24 h. After incubation, the culture medium was removed from the wells of a 96-well plate, and MTT reagent was added to each well. The resulting formazan crystals were then dissolved in dimethyl sulfoxide (DMSO), and the absorbance of the purple solution was measured using an enzyme-linked immunosorbent assay (ELISA) reader (Organon Teknika, Netherlands) at 570 nm. The percentage of cell viability was calculated using the following formula: $\text{Cell viability }\left(\text{\%}\right)=\frac{\text{Sample A}.}{\text{Control A. }}\times 100$

2.7. Statistical analysis

We performed the statistical analysis in triplicate and evaluated the results using one-way analysis of variance (ANOVA) and the liquid crystal display (LCD) post-hoc tests.

3.2. Catalytic application of Fe₃O₄@CS-Gl/Pd NPs in the C-N coupling

After characterizing the Fe₃O₄@CS-Gl/Pd NPs structure, we assessed its catalytic activity in the C(aryl)-N bond formation through Ullmann-type coupling (Scheme 1). In the preliminary phase of the study, the N-arylation of indole with iodo-benzene was chosen as a model reaction to optimize various reaction parameters, including catalyst loading, solvent selection, and temperature, without the use of an inert atmosphere (Table 1). Initially, we tested a range of solvents, including DMF, EtOH, CH₃CN, CH₂Cl₂, among others, using 15 mg of catalyst and 2 equivalents of Et₃N at 80°C (Table 1, entries 1-7). As shown in Table 1, the best results were achieved with DMF (Entry 7). Following this, we examined several bases, such as Et₃N, Na₂CO₃, KOH, NaHCO₃, K₃PO₄, and K₂CO₃, with Et₃N yielding the highest reaction efficiency (Table 1, entries 1, 12-16). In contrast, the use of K₃PO₄ resulted in poor outcomes (Table 1, entry 10).

Table 1. Optimization condition for N-arylation of indole with iodo-benzene.^a

Entry	Catalyst (g)	Solvent	Base	t (h)	T (°C)	Yield (%)b
1	0.015	Aetonitrile	Et3N	12	82	30
2	0.015	Ethanol	Et3N	12	78	40
3	0.015	Toluene	Et3N	12	110	60
4	0.015	Water	Et3N	12	100	20
5	0.015	Dicholromethane	Et3N	12	40	25
6	0.015	Dimethylsulfoxide	Et3N	12	80	80
7	0.015	Dimethylformamide	Et3N	6	80	90
8	0.010	Dimethylformamide	Et3N	12	80	60
9	0.020	Dimethylformamide	Et3N	5	80	96
10	0.020	Dimethylformamide	-	12	80	Trace
11	0.020	Dimethylformamide	Et3N	12	80	0
12	0.020	Dimethylformamide	K2CO3	5	80	70
13	0.020	Dimethylformamide	Na2CO3	5	80	50
14	0.020	Dimethylformamide	K3PO4	5	80	55
15	0.020	Dimethylformamide	KOH	5	80	70
16	0.020	Dimethylformamide	NaHCO3	5	80	40
17	0.020	Dimethylformamide	Et3N	5	25	20
18	0.020	Dimethylformamide	Et3N	5	60	55

^a Reaction conditions: Iodo-benzene (1.1 mmol), indole (1.0 mmol), catalyst, base (2.0 mmol), solvent (5.0 mL). ^bIsolated yield.

We observed that the temperature had a significant effect on the outcome (Table 1, entries 9, 17, 18). The highest yield (96%) was achieved at 80°C (Table 1, entry 9). Moreover, increasing the catalyst quantity from 0.015 g to 0.020 mg (0.01 mmol Pd) enhanced the reaction efficiency. However, reducing the catalyst to 10 mg led to a decrease in yield to 60% (Table 1, entries 7-9). It is important to note that no reaction took place in the absence of the Pd catalyst (Table 1, entry 11). In summary, the optimal conditions for the N-arylation of indole with iodo-benzene were identified as 0.020 g of catalyst (0. 01 mmol Pd), 2 equivalents of Et₃N, in DMF at 80°C for 2 h (Table 1, Entry 9).

After determining the optimal reaction conditions, we proceeded with the N-arylation of indole by utilizing aromatic halides, including I^-, Br^-, and Cl^-. The reactions with iodides and bromides went forward quickly and productively, yielding good results (Table 2, entries 1, 2, 4, 5, 7, 8). However, when the halides were switched to chlorides, the yields were significantly lower (Table 2, entries 3, 6, 9). These observations led to the conclusion that the reaction order follows: R-I > R-Br > R-Cl. This approach not only enhanced the yields but reduced the reaction time as well, achieving results under milder conditions compared to traditional methods.

To explore the broader applicability of the protocol, we next studied the coupling of imidazole with haloarenes (Table 2, entries 10-12). The outcomes were promising, fluctuating between moderate and excellent yields. Notably, this reaction is highly selective, providing only N-arylated products, in contrast to many other methods that typically result in C-arylation of the amine.

To investigate whether the observed catalysis was due to the Fe₃O₄@CS-Gl/Pd NPs or to homogeneous Pd NPs that might leach from the support and later reattach, we performed a hot filtration test [35]. This test was conducted by employing the N-arylation reaction of indole with iodo-benzene. Following 2.5 h reaction, the Fe₃O₄@CS-Gl/Pd NPs were filtered out, and the filtrate continued with the same reaction conditions for an additional 2.5 h. The purification was carried out at the reaction temperature of 80°C to avoid any precipitation or re-coordination of leached Pd NPs upon cooling.

Initially, the reaction of indole with iodobenzene at 80°C for 2.5 h yielded 62% of N-phenylindole. Following hot purification, no further reaction took place, indicating that the Pd NPs remained on the Fe₃O₄@CS-Gl support at elevated temperatures during the reaction, thereby confirming the heterogeneous nature of the catalysis.

For heterogeneous change metal catalysts, evaluating their isolation, capability of improving, and reusability can be essential. In this context, we tested the reusability of Fe₃O₄@CS-Gl/Pd NPs in the reaction between iodobenzene and indole. After each reaction, the Fe₃O₄@CS-Gl/Pd NPs were separated from the mixture using an external magnet, rinsed in hot ethanol and water, and recycled in subsequent reactions. In the Figure 7, subpart Figure 7(c) the catalyst could be recycled up to six times with just a slight decrease in catalytic power. The good reusability of the catalyst are likely due to the chelating interaction between the guar gum groups and the Palladium, which contributes to stabilizing the catalyst. The stability of the catalyst used six times was further validated through FE-SEM and TEM analysis, demonstrating that the texture and pattern are nearly identical to those of the fresh catalyst (Figure 7a and b).

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Figure 7.

(a) FE-SEM, (b) TEM images of recovered catalyst after 6^th run and (c) the recycling of the Fe₃O₄@CS-Gl/Pd NPs catalyst.

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3.3. The biological application of Fe₃O₄@CS-Gl/Pd NPs nanocomposite

At low concentrations, antioxidant substances work by inhibiting and preventing the oxidation of specific biochemical compounds, protecting them from damage caused by free radicals. Antioxidants are crucial in mitigating various health risks in living organisms, and they play a significant role in extending lifespan. They are widely used in industries such as food production, fuels, lubricants, and polymers. In addition to their industrial applications, antioxidants are highly effective in managing health by reducing oxidative stress. Pd NPs in particular, have garnered considerable attention for their antioxidant properties. To assess the antioxidant potential of the Fe₃O₄@CS-Gl/Pd NPs nanocomposite, we employed the widely recognized DPPH assay. Six different concentrations of the nanocomposite (31.25, 62.5, 125, 250, 500, and 1000 µg/mL) were tested by adding 150 µL of each concentration (at 0.04 mg/mL) to the DPPH solution in EtOH. The DPPH scavenging activity was quantified by spectroscopic analysis, using the equation outlined in section 2.3. The results, shown in Figure 8, demonstrated a dose-dependent increase in antioxidant activity, reaching a maximum of 87.25% at 1000 µg/mL. The IC₅₀ value for the antioxidant activity was calculated to be 127 µg/mL.

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Figure 8.

Antioxidant activity of Fe₃O₄@CS-Gl/Pd NPs nanocomposite.

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The Fe₃O₄@CS-Gl/Pd NPs nanocomposite demonstrates promising potential for anti-cancer applications, as evidenced by its significant antioxidant activity. Reactive oxygen species (ROS), which are typically generated as free radicals, can be produced through the action of the antioxidant material within living organisms. Both in vitro and in vivo studies have shown that ROS play a critical role in inhibiting the growth, senescence, and apoptosis of cancer cells, potentially interfering with their proliferation. Another mechanism involves the nanomaterial’s unique interface, which may cause cancer cells to degrade their own cell membranes, leading to DNA damage. Given these noteworthy properties, we aimed to investigate the effectiveness of Fe₃O₄@CS-Gl/Pd NPs nanocomposite against two PC cell lines, LNCaP and PC3, using the MTT assay. This colorimetric test was used to evaluate cell viability at varying concentrations of the nanomaterial (5, 50, 500, 1000, and 2000 µg/mL), with the toxicity percentage calculated according to the equation described in section 2.4. As shown in Figure 9, for both cell lines, the toxicity percentage increased with higher concentrations of the nanocomposite, meaning that cell viability decreased in a dose-dependent manner. This observation indicates a direct correlation between the concentration of the nanomaterial and the death of cancer cells. The corresponding IC₅₀ values for the two cell lines were determined to be 219 µg/mL and 336 µg/mL. Additionally, cytotoxicity tests on the normal human endothelial cell line (HUVEC) showed minimal toxicity, suggesting that Fe₃O₄@CS-Gl/Pd NPs present a relatively low risk to healthy human cells (Figure 10).

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Figure 9.

Study of cytotoxicity of Fe₃O₄@CS-Gl/Pd NPs against (a) LNCaP and (b) PC3.

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Figure 10.

Study of cytotoxicity of Fe₃O₄@CS-Gl/Pd NPs against HUVEC cell.

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