Bioactive assessment of hexahydroquinoline derivatives prepared via a biochar/Fe₃O₄@APTMS magnetic catalyst: focus on antidiabetic and antibacterial properties

Abstract

This study aims to develop a green and effective magnetic catalyst, biochar/Fe₃O₄@APTMS, for the one-pot synthesis of bioactive hexahydroquinolines derivatives. Following synthesis, some biological activities were assessed including antibacterial activity and antidiabetic potential through polyol inhibition assays. The reaction involved four-component condensation of ammonium acetate, malononitrile or ethylcyanoacetate, dimedone (5,5-dimethyl-1,3-cyclohexanedione) and some aromatic aldehydes by refluxing in ethanol to afford products in high yields (91–97%) in a short time (10 minutes). Additionally, heterogeneous catalyst provides several advantages, including operational simplicity, rapid reaction times, easy product isolation, and recyclability of unreacted starting materials. The nano catalyst was fully characterized with Fourier Transform Infrared Spectroscopy (FT-IR), Raman, Field Emission Scanning Electron Microscopy (FE-SEM), and energy dispersive X-ray mapping (EDX-Map) while the characterization of the products with Nuclear magnetic resonance spectroscopy (¹³C NMR and ¹H NMR) confirmed their structure. Some of the compounds tested showed moderate but significant antidiabetic activity against aldose reductase (IC₅₀ values 4.03 to 18.29 μg mL⁻¹) and antibacterial activity against Gram-positive strains of bacteria, Staphylococcus aureus and Enterococcus faecalis, with inhibition zones up to 15.5 mm. These results showed promise for the compounds being used as dual-function therapeutic agents for diabetic complications and microbial infection.

An efficient and eco-friendly method was developed for the synthesis of hexahydroquinoline derivatives using biochar/Fe₃O₄@APTMS, and demonstrate the ability of these derivatives to inhibit the polyol pathway through aldose reductase inhibition.

Introduction

Human activities have led to unintentional contamination of the environment, prompting scientists to develop strategies for reducing pollution and designing processes that are both economically and environmentally friendly. Organic chemists have a crucial role in developing such strategies by systematically researching and designing processes that minimize environmental impacts.¹ Green Chemistry is a promising approach that utilizes synthetic techniques and avoids the use of hazardous materials. It also involves the development of more efficient methods for producing heterocyclic chemicals that have potential applications in various fields. Microwave irradiation, ultrasonication, mechanochemistry, UV irradiation, and other techniques have been used in the past to achieve this. However, the demand for skilled labor and specialized, expensive equipment is a serious concern for scale-up research and long-term viability. As a result, there is still a need for the development of a clean, green, and viable approach.^2–5

Heterogeneous catalysts are gaining increasing attention as they can substantially improve the organic reaction. Such approaches can have a significant impact on the development of efficient and environmentally friendly processes for producing heterocyclic chemicals and other important compounds. Green solvent and efficient heterogeneous catalysts can substantially improve the organic reaction.^6–10

Researchers have recently become interested in biochar. The thermochemical conversion of carbonaceous material using techniques such as pyrolysis, liquefaction, torrefaction, and gasification can produce biochar (solid product), bio-oil (liquid product), or bio-gas (gaseous product).¹¹ Biochar is distinguished by its high carbon content, high ion exchange capacity, stable and porous structure, large specific surface area, low raw material cost, eases of access, and numerous functional groups. It has been used for a variety of applications, including environmental remediation, energy storage, water and wastewater treatment, catalyst support, and so on, due to its significant characteristics, such as large surface area, abundant active functional groups, porosity, and pore volume.^12,13 Various processes, such as modification with acids, alkalis, oxidizing agents, and various activation methods, such as amination, hydrothermal synthesis, and magnetization, have been used to improve the physicochemical properties of biochar.^14,15

Magnetic catalysts are preferable for the synthesis of organic compounds. Due to synergistic effects, heat induction through the reaction mixture, simple and effective separation in the presence of an external magnetic field, easy catalyst recycling, high surface area that results in a high catalyst loading capacity and great dispersion. The magnetic catalyst can be recovered and reused with the help of an external magnetic field; magnetic separation is considered as a green technique. This can prevent the need of filtration or centrifugation steps in the separation process.^16,17 However, the hydrophobic naked nano-ferrites have a high surface area to volume ratio, strong magnetic dipole–dipole attraction, and always have issues like self-aggregation and a lack of functional groups.¹⁸ The aggregation of magnetic nanoparticles can be successfully avoided by using biochar as a carrier. Functionalization and surface modification with organic or inorganic supports are required to address these issues and boost their effectiveness for the specific application.^19–21

Heterocyclic compounds were shown to be a specific category of compounds with natural origin as well as chemical, medicinal, and industrial significance. They seem to be significant and useful agents against different types of medical disorders such as diabetes. Indeed, there is an urgent need for reliable and effective treatment strategies for retinopathy, neuropathy, and nephropathy (microvascular) and peripheral vascular, cerebrovascular and coronary heart diseases (macrovascular) diabetes complications.^22–25

The pharmaceutical and biological significance of hexahydroquinoline derivatives and their preparation is an ongoing research area for synthetic chemists. Several homogeneous and heterogeneous catalysts have been employed for the synthesis of substituted hexahydroquinolines derivatives. Among them, the synthesis using K₂CO₃,²⁶ triethylamine,²⁷ NH₄OAc,²⁸ [Dsim]HSO₄,²⁹ γ-Fe₂O₃ nanoparticles,³⁰ bioinspired AgNPs³¹ are noteworthy to mention. However, some of the described research may have drawbacks such as lengthy reaction periods, difficult catalyst separation, poor yields, and complicated purification techniques.

It's worth looking for new ways to produce hexahydroquinoline derivatives using heterogeneous nanocatalysts and multicomponent reactions that are low cost and environmentally friendly. In this study, we aimed to develop an eco-friendly and effective heterogeneous catalyst for a one-pot reaction to synthesize bioactive hexahydroquinoline (HHQ) derivatives through four component reaction of diverse aromatic aldehydes, malononitrile or ethylcyanoacetate, 5,5-dimethyl-1,3-cyclohexanedione, and ammonium acetate, the reaction was carried out at reflux in a green solvent of EtOH using biochar/Fe₃O₄@APTMS as magnetic nano-composite catalyst. The catalyst was synthesized using biochar obtained through pyrolysis of nerprun seed under low-oxygen conditions, and then a biochar/Fe₃O₄ nanocomposite was made in the presence of magnetic Fe₃O₄ nanoparticles. Finally, the biochar/Fe₃O₄ was functionalized with 3-aminopropyltrimethoxysilane (APTMS). The catalyst could be recycled at least six times without a significant decrease in catalytic activity. The structures of the hexahydroquinoline (HHQ) derivatives were characterized using FT-IR, ¹H NMR, and ¹³C NMR spectroscopy. A plausible reaction mechanism was also proposed. Then attempts are made to assess the antibacterial and antidiabetic properties of ten hexahydroquinoline (HHQ) derivatives and to elucidate the mode of action using selected biochemical targets relevant to diabetes.

Materials and methods

Materials

All reagents and chemicals used during the synthesis of final products were purchased from Sigma-Aldrich, and were used without further purification. Melting points (M.P.) were determined using open capillaries with Gallen Kamp's melting point apparatus and are uncorrected. The Perkin Elmer-Spectrum RX-IFTIR instrument was used to examine the FT-IR (Fourier transform infrared spectroscopy) spectra (ATR/KBr mode, cm⁻¹) of synthesized substances and catalyst in the 400–4000 cm⁻¹ range. The ¹H NMR and ¹³C NMR spectra (δ, ppm) in deuterated dimethyl sulphoxide (DMSO)-d₆ was recorded on a Bruker Avance NEO 500 (500 MHz) NMR spectrometer. SEM and EDX-Map figures were managed on a JSM 6100 (JEOL) emission scanning electron microscope.

Design of biochar/Fe₃O₄@APTMS

The synthesis of biochar/Fe₃O₄@APTMS goes through three stages:

First step biochar was prepared by using the nerprun seeds as a raw material. In the biochar preparation process, the nerprun seeds were first collected, ground, and powdered. Then the powdered nerprun seeds were pyrolyzed in a tube furnace at 700 °C for 2 h, using a heating rate of 10 °C min⁻¹. Second step 5 mL 5 M HCl, 40 mL water and 5 mL ethanol were mixed in a 100 mL flask. Then, 13.32 g FeCl₃·6H₂O and 19.88 g FeCl₂·4H₂O were added to the above solution and heated at 40 °C to complete dissolution of the salts. Then, 1 g biochar was added to 30 mL of the prepared solution and stirred for 2 h at room temperature. The prepared suspension was filtered and washed with distilled water and then immediately transfer into 1 M ammonia solution. After 2 h stirring at room temperature, the synthesized biochar/Fe₃O₄ was collected and washed with distilled water and dried. Third step 3-aminpropyltriethoxysilane (APTES) (1 ml) was added to a suspension of biochar/Fe₃O₄ (1 g) in dry toluene (25 ml) and refluxed for 24 h under N₂ atmosphere. Afterwards, the sample was collected by magnetic separation, washed with toluene and anhydrous ethanol several times, and then dried under vacuum in an oven at 60 °C overnight (Fig. 1).

Fig. 1. The process of biochar/Fe₃O₄@APTMS catalyst synthesis.

General procedure for the synthesis of hexahydroquinoline (HHQ) derivatives

In the one-pot synthesis of hexahydroquinoline (HHQ) derivatives, the four-component including diverse aromatic aldehydes 1 (1.0 mmol), malononitrile or ethylcyanoacetate 2 (1.0 mmol), 5,5-dimethyl-1,3-cyclohexanedione 3 (1.0 mmol), and ammonium acetate 4 (1.0 mmol) were mixed in round bottle flask at reflux with 10 mg of the biochar/Fe₃O₄@APTMS in the presence of EtOH (Scheme 1). TLC (thin layer chromatography) was used to monitor the progress of the reaction. After the reaction was completed, the catalyst was removed by an external magnet. To get pure hexahydroquinoline derivatives, the crude products were recrystallized from EtOH. The obtained biochar/Fe₃O₄@APTMS was then washed with EtOH, dried, and reuse in subsequent runs.

Scheme 1. Synthesis of hexahydroquinoline derivatives.

In silico study

AutoDock Tools (ADT) (version 1.5.6) was used to analyze the conformations of the 310 organic compounds (5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h, 5i and 5j). Ligand structures were edited using the Chemical Sketch Tool in PDB file. The crystal structure of human AR (accession id: 1IEI; resolution 2.5 Å) was downloaded from Protein Data Bank (PDB) in PDB format. The preparation of AR in ADT involved: i) removing non-protein atoms and water molecules from the PDB files to correct structural defects in the raw structure such as missing atoms and residues, and for energy minimization, ii) adding polar hydrogen atoms to the macromolecule and Kollman charges to compute pdbqt format, which is a step necessary for correct calculation of partial atomic charges. A grid map was prepared using a grid box, which size was set to 40 × 40 × 40 (x, y, and z points) with a grid spacing of 0.375 Å, and the grid center was designated at −3.194, 0.444 and 9.009 (x, y, and z). The docking program was analyzed by Lamarckian Genetic Algorithm to search the optimal conformation. The Discovery Studio 2021 program was used to visualize the protein–ligand interactions.

Experimental animals

The study was conducted on 40 adult, male Sprague Dawley (SD) rats (8 weeks old) with body weights ranging from 200 to 250 g. The rats were maintained in a single air-conditioned animal house at 25 °C and relative humidity of 55% with a 12 h light/12 h dark cycle. Food and water were provided ad libitum. Rats were acclimated to laboratory conditions during one month before starting the experiment. All the experiments were done in compliance with the guide for the care and use of laboratory animals.

Induction of diabetes and SA treatment

To induce diabetes, rats were injected intraperitoneally with a freshly prepared streptozotocin (STZ) solution in a citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 4.5) at a dose of 60 mg kg⁻¹. Animals displaying polydipsia and polyuria were further tested for hyperglycaemia using a blood glucose meter. Blood glucose levels larger than 20 mmol L⁻¹, after 24–48 h of STZ-injection, were considered diabetic.

Inhibition of polyol pathway enzymes

AR activity was assayed as described by ref. 32. The assay mixture of 1 mL consisted of 50 mM phosphate buffer (pH 6.2), 10 mM d-glyceraldehyde, 0.1 mM NADPH and rat kidney AR preparation. The reaction was initiated by adding NADPH and the change in the absorbance at 340 nm due to NADPH oxidation was measured. The SDH activity was assayed as described by ref. 32. The assay mixture of 1 mL consisted of 100 mM Tris-HCl buffer (pH 8.9), 40 mM sorbitol, 1.5 mM NAD⁺ and rat kidney preparation. The reaction was initiated by adding NAD⁺ and change in absorbance was measured at 340 nm.

Inhibition assay of AR activity was measured in the presence of different concentrations of organic compounds (10, 20, 40 μg mL⁻¹). The concentration of organic compounds resulting in 50% inhibition (IC₅₀) was determined by non-linear regression analysis of log concentration of compounds versus percentage inhibition.

Well diffusion assay

The bacterial growth inhibition of Escherichia. coli, Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecalis was determined by the well diffusion assay according to ref. 33. They were cultured in Mueller Hinton agar (MHA) at 37 °C for 24 hours. 20 mL of MHA was inoculated with 10⁸ CFU of indicator strains. Then, it was poured into a petri dish in which wells of 4 mm diameter were performed and filled with 10 μL of each molecule at a concentration of 50 μg mL⁻¹, dissolved in 10% dimethylsulfoxide (DMSO). The plates were then incubated for 24 hours at 37 °C and the inhibition diameters were measured.

Microdilution assay

The minimal inhibiting concentration (MIC) of the molecules was determined using the resazurin microtiter plate technique. 50 μL of MHA was distributed in all wells of the microtiter plates; then 10 μL of each molecule at a concentration of 50 μg mL⁻¹ dissolved in DMSO (10%) was added and serially diluted. 50 μL of 10⁸ CFU of the bacterial strains were added to all the wells and then incubated at 37 °C for 24 hours. A 30 μL of 0.01% (w/v) resazurin solution, sterilized by filtration through a 0.22 μm membrane, was added to each well. Then, the plates were incubated for 4 h at 37 °C. Three controls were used in the present assay: a column with all solutions without the bacterial culture, a column with DMSO (10%) solution, and a column with all solutions except the molecules.³³ Tetracycline was used as positive control. The MIC of each molecule corresponding to the absence of change in resazurin color was determined.^34,35

Statistical analysis

The experiments were carried out on triplicate and the results were given as means ± standard deviation (SD). The data were analyzed using GraphPad Prism 8.0 software using two-way ANOVA followed by Tukey's multiple comparisons test where a statistical significance difference was shown when P < 0.05. The differences between two groups were tested for statistical significance using Student t-test at a p < 0.05.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Department of Biology, Faculty of Science, Hassan II University, Morocco, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). In the absence of a formal Animal Ethics Committee at Hassan II University, the experimental protocol was reviewed and approved by the Department of Biology, Faculty of Science.

Results and discussion

Characterization of biochar/Fe₃O₄@APTMS

IR spectroscopy was utilized to qualitatively identify and confirm different functional groups of the current catalyst and the results are displayed in Fig. 2a biochar and biochar/Fe₃O₄@APTMS. Fig. 2 shows the distinctive bands of biochar, including the aromatic ring's C C stretching at 1497 cm⁻¹. A band at 1198 cm⁻¹ indicates O–H bending in polyphenols and the existence of aromatic groups.³⁶ The absorption band at 1043 cm⁻¹ is related to C–O–C and secondary O–H groups. A band around 775 cm⁻¹ indicates C C symmetric stretching, whereas an O–H stretching peak is seen at 3310 cm⁻¹.³⁷ The observed bands show that the biochar sample's surface has a significant number of functional groups, making it useful for modifying carbon-based nanomaterials. Fig. 2 shows a significant vibration band at 583 cm⁻¹, indicating the stretching vibration of the Fe–O link in Fe₃O₄ nanoparticles. A peak shows the existence of C–O–Fe bonds at 1059 cm⁻¹,³⁸ that's the outcome of the interaction between oxygen-containing functional groups on the surface of biochar and iron. The APTES-Fe₃O₄ (Fig. 2) has a distinct absorption peak at 1206 cm⁻¹ which is related to the silanol group. The bands at 2922 cm⁻¹ represent the stretching vibrations of CH₂ bonds in APTES molecules. The peak at 1529 cm⁻¹ indicates in-plane bending vibrations of CH₃. The peaks at 3452 cm⁻¹ and 1680 cm⁻¹ are mainly due to the stretching and bending vibrations of NH₂. The appearance of these different IR bands indicates that APTES was successfully integrated and deposited onto magnetite nanoparticles.³⁹

Fig. 2. IR of biochar/Fe₃O₄@APTMS.

Raman spectroscopy was used to investigate the carbon characteristics of the biochar, biochar/Fe₃O₄, and biochar/Fe₃O₄@APTMS samples (Fig. 3). The samples exhibited two distinct peaks at 1364–1370 cm⁻¹ and 1584–1600 cm⁻¹, representing the D band (sp³ vibrations of amorphous carbon) and the G band (sp² vibrations of graphite carbon), respectively. The I_D/I_G ratios for biochar, biochar/Fe₃O₄, and biochar/Fe₃O₄@APTMS were 0.931, 0.884, and 0.862, respectively. This shows that the composites have more graphite carbon.⁴⁰ These findings suggest that incorporating Fe₃O₄ and APTMS into the biochar structure lowers structural flaws and enhances their effective insertion, which is consistent with the outcomes of the FTIR study.

Fig. 3. Raman of biochar/Fe₃O₄@APTMS.

Fig. 4 shows SEM images analysis of biochar, biochar/Fe₃O₄, and biochar/Fe₃O₄@APTMS. The biochar has a very porous, heterogeneous surface with varied particle sizes. The biochar's pores and surface imperfections result from the emission of volatile molecules such hydrocarbons, CO, H₂O, and CO₂, along with the creation of a carbon–graphite structure. Adding Fe₃O₄ and APTMS nanoparticles to biochar resulted in the formation of tiny particles on the surface (Fig. 4), suggesting their assimilation.

Fig. 4. SEM of biochar/Fe₃O₄@APTMS.

The EDX-Map analysis revealed an abundance of Fe in the biochar/Fe₃O₄ magnetic nanocomposite, indicating that the Fe₃O₄ nanoparticles were properly incorporated into the biochar structure. Adding APTMS to the biochar/Fe₃O₄ magnetic nanocomposites affected the structure and porosity of the biochar/Fe₃O₄, as seen in Fig. 5. The picture shows irregular circular plate-like structures, magnetic particles coated with APTMS, and holes of varied sizes and EDX-Map examination revealed the existence of Si and N elements, showing that APTMS was successfully integrated into the biochar/Fe₃O₄ magnetic nanocomposite structure.^41,42

Fig. 5. EDX-map of biochar/Fe₃O₄@APTMS.

Optimal reaction conditions

After characterizing biochar/Fe₃O₄@APTMS, we adjusted reaction parameters such as solvent, temperature, and catalyst quantity for the synthesis of 2-amino-7,7-dimethyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carbonitrile. The model reaction consisted of a one-pot four-component condensation of benzaldehyde, malononitrile, 5,5-dimethyl-1,3-cyclohexanedione, and ammonium acetate. Initially, Table 1 summarizes the effect of different biochar/Fe₃O₄@APTMS masse on yield. It is important to note that without the catalyst, the reaction proceeds slowly; in fact, HHQ 5a was only found in small quantities by TLC after a lengthy reaction period, then we tested different catalyst levels and discovered that 10 mg produced the best results also Increasing the catalyst from 10 mg to 20 mg has no noticeable effect on yield. To estimate the minimum amount of time necessary for this synthesis while optimizing yield, we studied the kinetics of the model reaction through the previously adjusted parameters of 10 mg of biochar/Fe₃O₄@APTMS catalyst in ethanol. We changed the reaction duration from 3 to 20 min after determining the best quantity of catalyst and setting the synthesis temperature at the solvent's boiling point (78 °C). We noticed that the greatest yield of 97% was attained after just 10 min. Subsequently we investigated how different solvents affected the process. We evaluated the model reaction using the biochar/Fe₃O₄@APTMS catalyst under solvent-free conditions as well as a variety of polar and non-polar solvents such as water, ethanol, ethyl acetate, and hexane. According to the data, ethanol was the most efficient solvent for the model process, producing an 97% yield promptly at reflux. Non-polar solvent hexane produced lower yields of 48%. In the upcoming part of the survey, we investigated how temperature influenced reaction completion (entries 15–18). At room temperature, the reaction rate was low, and the yield was small (entry 15). However, the product yield rose with temperature, reaching up to reflux condition (entries 16–18). From Table 1 shows that 10 mg of biochar/Fe₃O₄@APTMS is needed as a catalyst for this reaction with ethanol as solvent at reflux.

Table 1. Molecular docking analysis of ten organic ligands.

	Entry	Solvent	Amount of catalyst (mg)	Temp (°C)	Time (min)	Yield (%)
Absence of catalyst	1	EtOH	—	Reflux	90	—
Effect of the amount of the catalyst	2	EtOH	5	Reflux	10	40
	3	EtOH	10	Reflux	10	97
	4	EtOH	25	Reflux	10	97
	5	EtOH	20	Reflux	10	97
Influence of reaction time	6	EtOH	10	Reflux	3	44
	7	EtOH	10	Reflux	6	68
	8	EtOH	10	Reflux	10	97
	9	EtOH	10	Reflux	20	97
Influence of the solvent	10	None	10	Reflux	10	42
	11	Hexane	10	Reflux	10	48
	12	AcOEt	10	Reflux	10	56
	13	H2O	10	Reflux	10	68
	14	EtOH	10	Reflux	10	97
Influence of temperature	15	EtOH	10	25	10	42
	16	EtOH	10	40	10	48
	17	EtOH	10	60	10	80
	18	EtOH	10	Reflux	10	97

Generalize study

The formerly established optimal conditions were successfully used to synthesis HHQ from a variety of substituted aromatic aldehydes, malononitrile or ethyl cyanoacetate, dimethyl-1,3-cyclohexanedione, and ammonium acetate, with the addition of 10 mg biochar/Fe₃O₄@APTMS (Fig. 6). The findings show that both electron-donating and electron-withdrawing substituted aromatic aldehydes generated HHQ derivatives in only 10 minutes, yielding 62% to 97%. Aldehydes containing electron-donating groups, such as the methyl (–CH₃) and methoxy (–OCH₃), produced products 5b and 5c in 95% and 94% yields, respectively (Fig. 6).

Fig. 6. Synthesis of hexahydroquinoline (HHQ) derivatives (5a–j) using biochar/Fe₃O₄@APTMS.

In contrast, aldehydes containing electron-drawing groups, such as chloro (–Cl) and nitro (–NO₂), yielded products 5d and 5e at 90% and 92%, respectively. When ethyl cyanoacetate was used instead of malononitrile, the yield decreased, which was most likely owing to ethyl cyanoacetate's reduced reactivity. Compared to earlier investigations,⁴³ we discovered that the reaction time was dramatically shortened in the presence of biochar/Fe₃O₄@APTMS, resulting in the synthesis of HHQ derivatives with no detectable side products. Recrystallization was used to purify all produced HHQ, which was then analyzed using IR, proton, and carbon-13 NMR spectroscopy (SI).

Suggested reaction mechanism

Scheme 2 depicts a potential method for producing HHQ using the biochar/Fe₃O₄@APTMS. The biochar/Fe₃O₄@APTMS act as a very effective magnetically solid acid catalyst. The nano catalyst's biochar component has a huge surface area, is charged, and contains a variety of functional groups. The presence of these functional groups enhances the polarization and electrophilicity of the carbonyl group.

Scheme 2. Suggested mechanism for hexahydroquinoline derivatives synthesis by biochar/Fe₃O₄@APTMS.

In this reaction biochar/Fe₃O₄@APTMS triggers the creation of two intermediates: β-enaminone 3′′, created by the condensation of 3 and 4, and intermediate 2′′, formed by the Knoevenagel condensation of 1 and 2. The interaction of the lone pair on the nitrile's nitrogen with the acidic sites of biochar/Fe₃O₄@APTMS enhances the Michael reaction between β-enaminone 3′′ and intermediate 2′′, resulting in the cyclization of intermediate 5′ into HHQ 5.

The reaction takes place on the catalyst's surface, which has two kinds of sites:

- Basic sites which are related with the nitrogen atoms in the NH₂ groups are expected to activate the reactants' acidic protons and electrophilic sites.

- Acidic sites such as the silicon in the Si(R)₃ groups most likely chelate the oxygen and nitrogen in the reactants and intermediates, promoting the reaction by breaking certain bonds and establishing new ones.

Catalyst recycling biochar/Fe₃O₄@APTMS

In the context of green chemistry, catalyst recovery and reusability are critical issues. Several reaction runs were performed to determine the reusability of the biochar/Fe₃O₄@APTMS in the production of hexahydroquinoline derivatives. After each reaction, the catalyst was separated using an external magnet, washed with ethanol, and dried in a 60 °C oven to determine its reusability. A consistent quantity of the recovered catalyst was then reused in future cycles. The findings in Fig. 7 indicate that the recycled catalyst may be used for at least 5 cycles without substantially reducing catalytic efficiency.

Fig. 7. Recyclability of biochar/Fe₃O₄@APTMS catalyst at optimum conditions of model reaction.

Inhibition of the polyol pathway in diabetes

The polyol pathway not only contributes significantly to diabetic complications by converting excess glucose into fructose, but also impacts cellular metabolism and signalling in ways that can promote disease progression.^44,45 Several natural and synthetic compounds have been reported as AR inhibitors. Among natural products, secondary plant metabolites such as phenolics, alkaloids, and terpenes have demonstrated promising health benefits, including antidiabetic, anticancer, antioxidant, and anti-inflammatory effects.⁴⁶ Meanwhile, synthetic compound development continues to focus on enhancing efficacy while minimizing adverse side effects. In this study, we hypothesized that the ten selected organic compounds would exhibit significant inhibitory effects on AR activity.

In silico docking studies showed that all the selected compounds have energy changes ranging between −9 to −10.6 kcal mol⁻¹ (Table 2) and revealed that all compounds fit in the active site of crystal structure of human AR. Five hydrophobic interactions mediated by the amino acids (TRP111, TRP79, VAL47, HIS110, and PRO217) tend to form contact with 5a with an energy change of −9.0 kcal mol⁻¹. Additionally, TRP219, LZU300, PHE122 and TYR48 were attached by van der Waals energy bonds, which are crucial for the formation of protein–ligand complex. Moreover, the interaction between 5a and AR resulted in one conventional hydrogen bond with TRP20. (Fig. 8A). 5b on the other hand shows an energy change of −9.6 kcal mol⁻¹. The interaction between 5b and AR is, however, involved in conventional hydrogen bonds with VAL47, and attached by van der Waals bond with TYR48. Hydrophobic interaction mediated by the amino acids (PHE122, LEU300, TRP217, HIS110, TRP20, TRP111, CYS298 and TYR205) were involved and surrounded 5b (Fig. 8B). 5c, however, shows an energy change of −9.4 kcal mol⁻¹. The interaction between 5c and AR resulted in one conventional hydrogen bond with TRP20. Moreover, three amino acids (LEU300, TRP219 and TYR48) were involved in van der Waals forces and surrounded 5c. The Pi–Pi stacked interaction was also observed with PHE122, and interactions with TRP111, TRP79, VAL47, HIS110 and PRO218 surrounded 5c (Fig. 8C). 5d shows an energy change of −9.3 kcal mol⁻¹. The antagonist ligand has interactions with TRP111, TRP219, TRP20 PHE122 and CYS298. Additionally, the interaction between 5c and AR is, however, involved in conventional hydrogen bonds with VAL47, and attached by one conventionnel hydrogen bond with LEU300 (Fig. 8D). 5e, the last compounds with carbonitrile group into the hexahydroquinoline, has interaction with five hydrophobic interactions mediated by the amino acids (PHE122, VAL47, TRP20, LEU300 and TRP219) and tend to form contact with 5e with an energy change of −9.4 kcal mol⁻¹ (Fig. 8E).

Table 2. Molecular docking analysis of ten organic ligands.

Enzyme	Ligand	Binding energy (kcal mol−1)
AR (PDB id: 1IEI)	5a	−9.0
	5b	−9.6
	5c	−9.4
	5d	−9.3
	5e	−9.4
	5f	−9.9
	5g	−10.6
	5h	−9.4
	5i	−9.9
	5j	−9.6

Fig. 8. Molecular interaction of compounds with AR protein. (A) Interaction of 5a, (B) interaction of 5b, (C) interaction of 5c, (D) interaction of 5d. (E) Interaction of 5e, (F) interaction of 5f, (G) interaction of 5g (H) interaction of 5h (I) interaction of 5i, (J) interaction of 5j with AR protein and its associated amino acid residues.

The simulated binding showed that the carboxylate group of 5f formed five hydrophobic interactions mediated by PHE122, VAL47, TRP20, TRP219 and LEU300, and TYR48 in van der Waals forces (Fig. 8F). 5g, however, showed an energy change of −10.6 kcal mol⁻¹, the best binding mode with the minimum binding energy. The results indicates that LEU300 and VAL47 were involved in van der Waals force and eight hydrophobic interactions mediated by the amino acids (TRP111, TRP219, PHE122, TRP20 and CYS228) tend to form contact with 5g (Fig. 8G). 5h made contact with the residues PHE122, LEU300, TRP219, TYR48, VAL47, GLN49, TRP111, CYS298 and TRP20 (Fig. 8H). 5i with AR is shown in Fig. 8I, the bonding energy was −9.9 kcal mol⁻¹, the docked results showed that 5i is attached by van der Waals bond with TYR48, TRP20, TRP219 and LEU300. Others important interactions such as Pi–Pi T-shaped were also reported in Fig. 8I. 5j high affinity was also associated with the presence of conventionnel hydrogen, van der Waals, Pi–Pi T-stacked bonds (Fig. 8J).

The above data supposes that all organic molecules tested can bind to the active centre of AR with a high affinity. Therefore, we tested each compound for an inhibitory activity on crude extract of diabetic rats. To confirm our findings, we realized a study in STZ-induced diabetes rats. We observed that the in vivo effect of the 5a, 5b, 5c, 5d and 5e on the activity of AR has shown inhibition in a dose-dependent manner and had an IC₅₀ ranging from 4.03 to 18.29 μg mL⁻¹ towards inhibition (Fig. 9A and B). Quercetin was used as a positive control and exhibited an IC₅₀ of 5.44 ± 0.07 μg mL⁻¹ under the same conditions. The introduction of the carbonitrile group into the hexahydroquinoline inhibit efficiency and sufficiently the activity of AR. Compounds 5a, 5b, 5c and 5d (bearing a –CN group) exhibited an IC₅₀, slightly more potent than quercetin (IC₅₀ = 5.44 ± 0.07 μg mL⁻¹), suggesting a potential interaction with the AR active site. This may be due to enhanced binding affinity conferred by the nitrile group's ability to participate in polar or hydrogen bonding interactions. Mild inhibition was observed for compounds 5f, 5g, 5h, 5i and 5j with the carboxylate substituent in the same position. Similarly, only mild activity was recorded for SDH inhibition related with carboxylate group (Fig. 9C and D).

Fig. 9. Organic compounds (A–D) IC₅₀ values. IC₅₀ values of compounds distinct R-carbonitrile (green square) and R-carboxylate (red triangle) were calculated using a non-linear regression model.

Several previous studies on medicinal chemistry of natural compounds were tested with the IC₅₀ values. Although, this later showed the potency of a natural compound, more valuable information was obtained by the kinetics of inhibition such as K_i. With that said, IC₅₀ values have two limitations, on one hand, it depends on the concentration of the substrate we used to observe the inhibition; however, K_i is sometimes perceived as a purer measurement of enzyme inhibition as the substrate concentration is not relevant. On the other hand, converting and comparing other research group's data would require the Cheng–Prusoff equation. For further use, we would need the substrate concentration that was used to generate the IC₅₀ and the K_m for the enzyme substrate complex. This would allow the deduction of K_i for that particular enzyme substrate. The ability to compare inhibition values of different inhibitors is very important as it allows to define which one is more potent.

The cellular damage in the kidney has been suggested to result from several mechanisms, including the stimulation of polyol pathway, activation of protein kinase C, production of advanced glycation end-products formation (AGEs), enhancement of hexosamine biosynthetic pathway (HBP).⁴⁷ Among this, the polyol pathway is an extremely attractive target for the treatment of diabetes nephropathy. Based on our results, we could speculate that i) in silico AR inhibition in humans is similar, with comparable IC₅₀ values, thereby allowing prediction of human drug activity from rat data.

Antibacterial assay

The phenomenon of antibiotic resistance is one of the major challenges that threaten the treatment of various infectious diseases. Bacteria can acquire resistance by horizontal gene transfer through exchange of plasmids, integrins, and transposons. They can inhibit the action of antibiotics through different ways, i) elimination by efflux pump that transports the targeted antibiotics to the extracellular environment, ii) altering the targets via genetic mutations or posttranslational modifications, iii) hydrolysis or modification of the antibiotic, iv) decrease the expression of the porin proteins in some bacteria.⁴⁸ Hence, it is crucial to design and synthesize new molecules that may provide potent biological activities to substitute antibiotics.

In this study, we evaluated the antibacterial activity of 10 synthesized hexahydroquinoline derivatives. Then, they were evaluated for their antibacterial activity using the well diffusion assay and the microdilution technique against four bacteria: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecalis.

Our results showed (Table 3) that the molecules 5a, 5b, 5c inhibited the growth of S. aureus, and E. faecalis, while 5l inhibited the growth of only S. aureus. The inhibition zone diameters were ranging from 9 to 15 mm, and the highest inhibition was obtained by the molecule 5c against E. faecalis and S. aureus with a diameter of 15.5 ± 0.707 mm and 14 ± 0.0 mm respectively. The agar well diffusion technique allowed a good diffusion of the antibacterial compounds into the medium, enhancing contact with the tested pathogens. On the other hand, the microdilution assay showed a MIC = 50 μg mL⁻¹ for all the molecules, compared to tetracycline, which exhibited MIC values of 128 μg mL⁻¹ against Escherichia coli, 15 μg mL⁻¹ against Staphylococcus aureus, 16 μg mL⁻¹ against Klebsiella pneumoniae, and 32 μg mL⁻¹ against Enterococcus faecalis.

Table 3. Antibacterial activity of synthesized molecules against Gram-positive and Gram-negative pathogenic strains.

Chemical compound	Escherichia coli	Staphylococcus aureus	Klebsiella pneumoniae	Enterococcus faecalis
5a	—	9 ± 0.0a	—	13 ± 1.414a
5b	—	10 ± 0.0a	—	13 ± 0.0a
5c	—	14 ± 0.0b	—	15.5 ± 0.707b
5d	—	—	—	—
5e	—	—	—	—
5f	—	—	—	—
5g	—	—	—	—
5h	—	—	—	—
5i	—	—	—	—
5j	—	—	—	13 ± 0.0a
Tetracycline	14 ± 0.0	10 ± 0.0a	10 ± 0.0	10 ± 0.0c

We note that the effective molecules showed an inhibition only against Gram-positive bacteria, which is possibly due to the difference in the composition of the bacterial cell walls, as Gram-negative bacteria possess a rigid outer membrane rich in lipopolysaccharides, complicating the penetration of the antibacterial compounds through bacterial membranes.

Tetracycline was used as a positive control, it inhibits the bacterial growth via the inhibition of the 30S ribosomal subunit, hindering the binding of the aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex. In this work, the active molecules have shown a potent inhibition of the bacterial growth of the Gram-positive bacteria (S. aureus, and E. faecalis), in our knowledge, their mechanism of action is not well elucidated. However, their effectiveness highly depends on the nature of the substituent, the structure of the tested molecules, and the sensitivity of the tested bacterial pathogens 45. Interestingly, the inhibition zones of the molecules 5a, 5b, 5c, and 5l against E. faecalis differ significantly than that gained by the reference drug. Moreover, 5c was more active than tetracycline, it inhibited the growth of S. aureus with an inhibition diameter of 14 mm over 10 mm for the positive control.

Our results go in line with the findings of ref. 49, who carried out an in vitro antibacterial activity assay against Escherichia coli, Staphylococcus aureus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. They showed that the heterocyclic derivatives of 8-hydroxyquinoline were more active against the Gram-positive than the Gram-negative bacteria because of their structure which was not suitable to raise the bacterial resistance determinants. They reported that the inhibition of Gram-negative bacterial growth requires more liposoluble compounds. Other researchers tested the antibacterial activity of some hexahydroquinoline derivatives against E. coli, P. aeruginosa, S. aureus, K. pneumoniae, and Bacillus subtilis. Among the five bacteria, only S. aureus and K. pneumoniae were inhibited, the authors suggested that the hexahydroquinoline derivatives containing halogen and methoxy groups act as potent antibacterial agent. The higher lipophilicity of these groups likely enhances cell membrane permeability, which is critical for overcoming bacterial resistance in Gram-positive organisms. Whereas, the hexahydroquinoline with 4-CN, 4-NO₂, and 4-Cl, were found to exhibit lower antibacterial activities, like the case of our molecules 5d, 5e, and 5i respectively.⁵⁰ Hence, the activity may involve disruption of the bacterial membrane or inhibition of essential enzymes, although further computational or experimental studies are needed to confirm this. Future work will focus on molecular docking and mechanistic validation of these proposed modes of action.

Furthermore, it has been reported that some hexahydroquinolines and their derivatives can inhibit S. aureus including methicillin-resistant S. aureus (MRSA), depending on its ability to form complexes with divalent metal ions through chelation, disrupting bacterial cell wall, thereby leading to cell lysis.⁵¹ Hence, a study evaluated the expression of norA efflux pump gene using real time PCR, and the results showed that the combination of hexahydroquinolines to some antibiotics that are no more effective because of bacterial resistance, such as the case of ciprofloxacin with MRSA, can abrogate the resistance mechanism in bacteria via the modulation of efflux systems, and thus, restore the effect of antimicrobial agents.⁵²

Regarding the structure–function relationship, the synthesis of hydroxyquinolines derivatives has shown a wide range of pharmacological properties related to the modifications realized on the original molecule, where the substitution of the phenyl ring or the introduction of the ester structure can increase the bioactivity of the molecule derivatives.

Conclusion

We established a more ecologically friendly and efficient process for producing pharmacologically active hydroxyquinolines derivatives (5a–j) with yields ranging from 88% to 97%. The reaction was catalyzed by the biochar/Fe₃O₄@APTMS in the presence of H₂O: EtOH at reflux. The benefits of this method involve biochar/Fe₃O₄@APTMS catalyst magnetic can be reused up to five times without losing catalytic activity, resulting in high yields. Furthermore, the catalyst may be readily isolated from the reaction mixture using an external magnet.

The in vitro antibacterial assay showed that the hexahydroquinoline derivatives 5a, 5b, 5c, 5j exhibit a good bacterial inhibitory activity against S. aureus, and E. faecalis. Thus, they offer a good potential to pharmaceutical industries to be exploited as antibacterial agents in order to substitute antibiotics that are increasingly becoming ineffective because of bacterial resistance. Moreover, 5a, 5b, 5c, 5d and 5e have shown a dose-dependent inhibition of aldose reductase activity involved in diabetes. Based on a bifunctional with joint polyol inhibitory pathway and antibacterial activity we showed that the synthesized molecules may also be beneficial for reducing diabetes complications. However, while the synthesized hexahydroquinoline derivatives demonstrated promising biological activity, this study is limited by the absence of comprehensive cytotoxicity data in order to assess the selectivity and safety of new antidiabetics candidates.

Author contributions

Abderrahmane Thoume: conceptualization, data curation, formal analysis, methodology, validation, visualization, supervision, writing – original draft, writing – review & editing. Imane Nait Irahal: conceptualization, data curation, formal analysis, methodology, validation, visualization, supervision, writing – original draft. Zineb Dahib: conceptualization, data curation, formal analysis, methodology, validation, visualization, writing – original draft. Asmaa Chbel: conceptualization, data curation, formal analysis, methodology, writing – original draft. Zineb Loukhmi: formal analysis, investigation, writing – review & editing. Fatima Abdou-Allah: data curation, formal analysis, investigation, methodology. Redouane Achagar: data curation, formal analysis, investigation, methodology. Mustapha Zertoubi: resources, supervision, validation, visualization, writing – original draft, writing – review & editing. Driss Benmessaoud Left: investigation, methodology, writing – original draft, writing – review & editing. Noureddine Bourhim: investigation, methodology, project administration, resources, supervision, validation, visualization, investigation, methodology, writing – original draft, writing – review & editing. Abdelhakim Elmakssoudi: investigation, methodology, project administration, resources, supervision, validation, visualization, writing – original draft, writing – review & editing.

Conflicts of interest

There is no conflict of interest to declare.

Data availability

Supplementary information: Including spectral data for all products and copies of the ¹H and ¹³C NMR spectra of compounds, is available in the SI file. You can consult these data for more details and information about the study. See DOI: https://doi.org/10.1039/D5MD00594A.

All data supporting the findings of this study are available within the article and its SI files.