Sustainable copper nanocomposite for multicomponent synthesis of triazolo quinolines and triazolyl benzamide derivatives and their bioactivity study†

Nandini R. ^a, Byresh B. Kempegowda ^b, Sudhanva M. Srinivasa ^b, Umesh A. Kshirsagar ^c, Jan Grzegorz Malecki ^d, Siddappa A. Patil ^a, Shoyebmohamad F. Shaikh ^e, Manoj V. Mane *^a and Ramesh B. Dateer *^a
aCentre for Nano and Material Sciences, Jain Global Campus, JAIN (Deemed-to-be University), Bangalore – 562112, Karnataka, India. E-mail: manoj.mane@jainuniversity.ac.in; d.ramesh@jainuniversity.ac.in
^bAdichunchanagiri Institute for Molecular Medicine, Adichunchanagiri Institute of Medical Sciences, Adichunchanagiri University, BG Nagara – 571448, Karnataka, India
^cIndian Institute of Technology, Indore – 453552, Madhya Pradesh, India
^dInstitute of Chemistry, University of Silesia, 9th Szkolna Street, Katowice 40-006, Poland
^eDepartment of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

First published on 26th March 2025

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1. Introduction

Heterocycles are found in a wide range of organic substances, medicines, natural products, and functional compounds as important building blocks having biologically active components/sites.¹ As a result, there has always been persistent interest in the synthetic community to discover novel diverse and greener approaches for the synthesis of heterocycles.^2,3 Nevertheless, the use of multicomponent reactions (MCRs) as a tool for the quick formation of small-molecule libraries has enormously grown in recent years due to the high level of reaction efficiency. Indeed, MCRs avoid the purification of the reaction intermediate and provide a way for the utilization of unstable reaction intermediates.^4–7 However, the main conceptual difficulty in developing novel forms of MCRs is learning unique combinations and sequences of elementary chemical reactions under comparable conditions.^8–11 In this regard, a specialized class of heterocyclic molecules known as 1,2,3-triazoles has gained a lot of consideration in synthetic,^12,13 pharmaceutical,⁷ and materials science.^12,14–16 Moreover, triazoles containing fused heterocycles have been proven to show a broad range of biological properties and hence they exhibit anti-inflammatory activity,¹⁷ excellent serine protease inhibition activity,^18,19 and anti-cancer^20–22 and anti-microbial properties^23,24 (Fig. 1A). Additionally, the free 1,2,3-triazole systems (Fig. 1B) show numerous activity, including anti-HIV, anti-plasmodial, and anti-cancerous qualities.²⁵ As a result, creating these structurally innovative fused N-heterocycles from basic materials using an elegant catalytic multi-component technique still presents an interesting and appealing challenge. One of the most effective synthetic methods to access triazoles is the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction via a combination of organic azides and terminal alkynes.^26–28 Nevertheless, a thoroughly favorable atom economy, tolerance of a wide substrate range, and straightforward reaction conditions make it an extremely effective tool.⁸ Among various approaches documented for triazole synthesis, a classical copper-catalyzed [3 + 2] cycloaddition involving aryl halide, sodium azide, and alkyne appears to be the most significant one.²⁹ In particular, Wang and his co-workers in early 2013 reported fused triazolo quinoline derivatives using a palladium catalyst assisted by TBAI (Scheme 1a).³⁰ Subsequently, Upadhyaya et al. demonstrated the synthesis of fused triazolo quinolines and their nucleoside analogs under a similar catalytic system (Scheme 1b).⁷ Nevertheless, a one-pot multicomponent reaction involving bromo benzaldehyde, aryl methyl ketones and NaN₃ to access 4-substituted triazolo quinolines was subsequently investigated (Scheme 1c).³¹ However, it has been identified that there are only a handful of reports for the synthesis of 1,4-disubstituted 1,2,3 triazole synthesis.⁸ For instance, in 2017, Hayeebueraheng and co-workers reported the synthesis of triazolyl benzamide derivatives via a one-step multicomponent reaction wherein the utility of precious metal precursors, copper salts, external ligands, and harsh reaction conditions, and the lack of recyclability were documented³² (Scheme 1d). Therefore, the development of sustainable heterogeneous nanocatalysts to access bioactive triazoles containing heterocycles under ligand-free and green reaction conditions is highly desirable from the perspective of pharmaceutical chemistry.

The smooth reversible redox cycle and effective particle stabilization of a complex catalyst are the major factors that determine a catalyst's performance. It has been anticipated that the redox interaction between a ligand and a metal cluster will significantly contribute to the catalytic system, if the ligand has a redox function.³³ In this context, the employment of appropriate conducting polymers like polyaniline, polypyrrole, and polythiophene helps to enhance the catalytic performance by forming heteroatom–metal bonds. Ultimately, this helps in the transfer of electrons more easily compared to normal copper salts due to the presence of functional groups in the polymer.^34–36 Additionally, in recent years, magnetic nanoparticles (MNPs) have generated a great deal of attention due to their ease of separation. Although Fe₃O₄ nanoparticles are frequently utilized as magnetic cores, their applications are severely hampered by anisotropic dipolar attraction, which causes aggregation.³⁷ Moreover, the colloidal instability of MNPs causes agglomeration via magnetic dipole–dipole interaction.³⁸ This can be overcome by surface functionalization of MNPs using a protective polymer coating.³⁹ Polymers containing amino functionalities act as linking agents via covalent interaction of organic molecules and the nanoparticles, which in turn captivates applications in heterogeneous catalysis, as drug carriers, in absorption techniques, etc.⁴⁰ In particular, among various polymers used as polymeric shells over the Fe₃O₄ core, polyaniline (PANI) can be easily used for the process of polymerization.^41,42 Next, it has been documented that copper is one of the most important earth-abundant transition metal based elements showcasing enormous catalytic activities for various organic transformations, especially in click reactions such as azide–alkyne cycloaddition.^43–45 Therefore, designing a sustainable, magnetically separable copper-based nanocomposite for the synthesis of triazoles containing heterocycles⁴⁶ has been highly necessary in recent times. In this line, we report the synthesis and characterisation of a magnetically separable Cu@PANI@Fe₃O₄ nanocomposite,⁴⁷ and its utility to access biologically important 4-substituted triazolo quinolines and triazolyl benzamide derivatives using a one-pot multicomponent reaction strategy is investigated. The stepwise mechanistic investigation of triazolo quinoline formation by the DFT study was performed. Moreover, the anti-cancer studies of triazolo quinoline derivatives were described in detail.

2. Experimental section

2.1 Synthesis of the Cu@PANI@Fe₃O₄ nanocomposite

The Cu@PANI@Fe₃O₄ nanocomposite has been synthesized following a recently reported procedure^48–51 (see the ESI† for details). The prepared Fe₃O₄@PANI (100 mg) was ultrasonically dispersed in a solution containing Cu(NO₃)₂ (2 mmol), hydrazine hydrate (1 mL) and ethanol (10 mL, Fig. 2). Later, the reaction mixture was sonicated for 3 hours followed by centrifugation (3 × 30 mL ethanol) and dried at 60 °C for 12 h to obtain the required nanocomposite. After the synthesis of the nanocomposite, it was thoroughly characterized using various techniques such as PXRD, FE-SEM, FTIR, UV-visible analysis, HR-TEM, XPS, TG-DTA, VSM, etc.

3. Results and discussion

3.1 Powder X-ray diffraction analysis

Powder X-ray diffraction (PXRD) measurements were carried out to understand the crystallinity of the synthesized nanocomposite (Fig. 3). The diffraction peaks at 2θ values of 30.11°, 35.46°, 43.10°, 53.47°, 57.00°, and 62.60° confirm the cubic phase of Fe₃O₄ corresponding to the (220), (311), (400), (422), (511), and (440) crystal planes, respectively (JCPDS card no. 89-0691, Fig. 3c). Similarly, the diffraction peaks at 2θ values of 43.29°, 50.43°, and 74.12° correspond to the (111), (200), and (220) crystal planes, respectively, of the Cu(0) cubic phase (JCPDS card no. 04-0836, Fig. 3e). The crystallite size of Cu(0) NPs was found to be 16.7 nm and it was calculated according to the Debye–Scherrer equation.

3.2 High-resolution transmission electron microscopy (HR-TEM) analysis

As demonstrated in Fig. 4, high-resolution transmission electron microscopy (HR-TEM) examination methodically depicts the form, structure, and surface morphology of the Cu@PANI@Fe₃O₄ nanocomposite. The nanocomposite was dispersed in ethanol by sonication, drop cast onto a copper grid, and dried in advance of the analysis. Accordingly, Fig. 4(a–e) confirm that the core spherical Fe₃O₄ NPs are surrounded by the polymer shell upon which the copper NPs (Cu) are dispersed on the surface. In the HR-TEM image (Fig. 4c), the (311) crystallographic plane of Fe₃O₄ NPs in the Cu@PANI@Fe₃O₄ nanocomposite is shown to have an interplanar spacing of 0.25 nm and the (111) crystallographic plane of Cu(0) NPs in the Cu@PANI@Fe₃O₄ nanocomposite is shown to have an interplanar spacing of 0.30 nm. The diffraction rings are visible in the SAED pattern which matches with the (422) and (440) planes of Fe₃O₄ NPs and the (111) and (220) planes of Cu(0) NPs as shown in Fig. 4(f) and is consistent with the PXRD data (Fig. 3).

3.3 Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis

From the ICP-OES analysis, the copper content in the prepared Cu@PANI@Fe₃O₄ nanocomposite was found to be 29.61% w/w.

3.4 X-ray photoelectron spectroscopy (XPS) analysis

The surface chemical composition and the chemical states of the synthesized Cu@PANI@Fe₃O₄ nanocomposite were analyzed using XPS analysis (Fig. 5). As shown in the wide survey spectrum (Fig. 5a), elements such as Cu, Fe, O, C, and N are present in the nanocomposite, which is also evident in the EDAX analysis (Fig. 4e and see the ESI†). Next, in the C 1s spectrum (Fig. 5b), the C–C, C–N and the shake-up satellite peaks have binding energies of 284.4, 288.1, and 292.0 eV, respectively. The high-resolution Cu 2p spectrum shows two characteristic peaks at the binding energies of 932.2 and 952.1 eV, corresponding to the deconvoluted spectra of Cu 2p_3/2 and Cu 2p_1/2 peaks, respectively (Fig. 5c). The binding energy at 932.5 eV corresponds to Cu(0) and Cu(I), which cannot be differentiated from each other. The binding energies of 934.2 and 954.0 eV correspond to Cu 2p_3/2 and Cu 2p_1/2 peaks, respectively, of Cu(II), which is most likely from oxides and hydroxides due to surface oxidation. Shake-up satellite peaks at 940.8, 943.9 and 962.2 eV, which match the distinctive d⁹ ground state structure, further support the significant presence of Cu²⁺ (ref. 52), whereas the peaks at 531.3 and 533.6 eV represent Cu–O and O–H from Cu(OH)₂ formed due to surface oxidation. The presence of different oxides/hydroxides of Cu²⁺ is not reflected in PXRD and is also not in accordance with the XPS data. However, the peak at 934.18 eV indicates the presence of Cu²⁺ species according to the literature.^53a Next, the peaks corresponding to Fe⁺³ and Fe⁺² of Fe₃O₄ match well with previous reports (Fig. 5d).⁵³ The prominent peaks at 709.8 and 711.1 eV validate the distinctive peaks originating from the Fe 2p_3/2 core level electrons, which are linked to the Fe³⁺ and Fe⁺² octahedral sites, respectively. Fe2p_1/2, the other spin–orbit component, is visible at 724.2 and 727.5 eV, which represent the +3 and +2 states, respectively. The two faint satellite peaks, located at 718.8 and 732.1 eV, are the Fe²⁺ shake-up peaks.⁵⁴ Thus, Fe³⁺ and Fe²⁺ are present in the high-resolution Fe 2p XPS spectrum. The additional peak at 718.7 eV may be the representation of a stoichiometric oxygen peak.⁵⁵ Three distinct peaks can be identified when the O 1s spectrum is deconvoluted (Fig. 5e). Next, the binding energy of 530.0 eV corresponds to the Fe–O peak of the Fe₃O₄ crystal lattice. Further, coming to the N 1s spectrum (Fig. 5f), the three divergent peaks having binding energies of 399.1, 402.3 and 406.3 eV correspond to the N–Cu bond, C–N bond and amine for the polymer (polyaniline) present in the nanocomposite.^56,57 Thus the XPS analysis gave us prominent evidence for the interaction of Cu NPs with polyaniline, which later helped us to propose the structure for the DFT study. Next, BET analysis was performed, which tells that the nanocomposite follows a type III isotherm and possesses the general characteristics of a mesoporous material with a surface area of 11.086 m² g⁻¹. Meanwhile from BJH analysis, the pore diameter was found to be 18.57 nm with a pore volume of 0.0836 cm³ g⁻¹ (see the ESI† for further details and other characterization results).

Application of the Cu@PANI@Fe₃O₄ nanocomposite for the synthesis of triazolo quinoline derivatives

The catalytic potential of the synthesized nanocomposite is tested for the synthesis of triazolo quinoline derivatives from 2-bromobenzaldehyde (1a) and acetophenone (2a) as model substrates using sodium azide (NaN₃) as an N-source and DMSO as a solvent. Delightfully, the formation of a new product (indicated by TLC) was observed at 100 °C after 24 h (entry 1). It is then subsequently isolated by column chromatography (eluent 15% ethyl acetate:hexane) and characterized by ¹H and ¹³C NMR. The appearance of quaternary carbon at 149.4 ppm and 142.8 ppm in ¹³C NMR indicates the desired triazole fused quinoline product 3a (Table 1).⁵⁸

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Encouraged by these early findings, we further tested several other parameters. Bases like K₃PO₄ furnished a poor yield while the desired product was not obtained with t-BuOK and NaHCO₃, and the reaction did not proceed in the absence of a base (entries 2–5). Further, the change in solvent to ethanol surprisingly enhanced the yield to 95% (entry 6) while DMF and acetonitrile provided moderate yields (entries 7 and 8). Other solvents such as toluene and PEG were ineffective (see the ESI† for details). The decrease in catalyst loading (5 mol%, entry 9 and 3 mol%, entry 10) diminished the reaction efficiency, while the reaction in the absence of a catalyst did not furnish the desired product, which indicates the involvement of the catalyst during the catalytic cycle (entry 11). Next, an increase in temperature did not alter the reaction efficiency, while a decrease in the product yield was recorded by decreasing the reaction time (entries 12 and 13). Next, the reaction with homogeneous CuI as a catalyst delivered a moderate yield, while the desired product was not obtained in the absence of the nanocomposite and only with PANI@Fe₃O₄ (entries 14 and 15). Finally, 10 mol% of catalyst, Cs₂CO₃ as a base and ethanol as a solvent at 80 °C for 12 h were found to be the optimized conditions (entry 6), which make it a green system. Furthermore, in line with our ongoing research for the development of N-heterocycles, we are curious to test the reactivity of 2-iodo-N-phenylbenzamide 4a and ethynylbenzene 5a under the optimized reaction conditions. Unfortunately, the expected triazolo quinazolinone 6a′ was not obtained presumably due to steric crowding that might prohibit the activation of the free triazole C–H bond. Instead, the pharmaceutically important triazolyl benzamide was effectively obtained in 96% even in the absence of a base at 80 °C in 2 h (eqn (1)).

	(1)

With the optimized conditions in hand, the generality of the reaction was initially tested using the Cu@PANI@Fe₃O₄ nanocomposite for the reaction with aromatic aldehydes and ketones. A series of triazolo quinoline derivatives were synthesized in good to excellent yields (Scheme 2). The aryl ketones with electron-donating 4-methyl (3b) and 4-methoxy (3c) and electron-withdrawing 4-chloro (3d) and 4-fluoro (3e) effectively furnished the desired product in good to excellent yields. Importantly, compounds 3b (CCDC: 2377550) and 3d (CCDC: 2377552) were unambiguously confirmed by X-ray crystallographic analysis indicating the formation of the product. In addition, sterically hindered 1-naphthyl ketone (3f) and heteroaryl thiophene-2-carbaldehyde (3g) were well tolerated and the expected compounds were accessed in moderate to good yields. On the other hand, 2-bromo benzaldehyde with 5-methyl (3h) and 5-chloro (3i) delivered the desired product without any issue, showcasing the generality of the developed protocol.

In the same line, the substrate scope for the synthesis of triazolyl benzamide derivatives was analyzed by subjecting 2-iodo aryl benzamides and aryl alkynes (Scheme 3). Aryl alkynes bearing both electron-donating 4-methyl (6b) and electron-withdrawing 4-chloro (6c) and 4-fluoro (6d) were well tolerated smoothly. Meanwhile, the structure of 6b (CCDC: 2377554) and 6d (CCDC: 2377553) was confirmed by X-ray crystallography.

Interestingly, 2-ethynylthiophene (6e) and 4-ethynyl-1,1′-biphenyl (6f) also reacted efficiently providing satisfactory results. Similarly, 2-iodo benzamides bearing N-benzyl amine (6g), benzyloxy (6h), N-naphthyl (6i), cyclohexyl (6j) and N-methoxy (6k) groups gave good to moderate yields. Meanwhile, the structure of 6h (CCDC: 2377555) was confirmed by X-ray crystallography. Astonishingly, smooth reactivity of phenylacetylene was observed with the substrate N-(2-bromophenyl)-2-iodobenzamide leading to the formation of benzamide with two substituted triazole moieties (6l).

Control experiments

A series of control experiments were conducted to support a mechanistic proposal (Scheme 4). Initially, the reaction between 2-bromo benzaldehyde (1a) and acetophenone (2b) with NaN₃ was performed under standard reaction conditions and terminated after 6 h, wherein the formation of the desired product 3a occurred up to 40% while the formation of 3a′ was up to 20%. Further, the reaction was allowed to continue for 20 h, wherein the complete formation of the expected 3a occurred smoothly. This observation indicates that the reaction proceeds through the chalcone intermediate and the chalcone is formed under catalyst free conditions (Scheme 4a). Alternatively, the reaction of isolated 3a′ with NaN₃ under standard conditions also furnished the expected compound 3a (Scheme 4b). Hence, we considered the catalytic cycle from 2-bromo chalcone 3a′ for DFT studies. Next, the reaction of (E)-chalcone 7 with NaN₃ under standard reaction conditions forms a triazole intermediate 8, while a separate reaction in the absence of the catalyst does not form compound 8. These results indicate the role of the catalyst in activating the chalcone for [3 + 2] cycloaddition with NaN₃ during triazole formation (Scheme 4c). This experiment is in agreement with transition state TS1 for the formation of intermediate IN2. Additionally, following a 20 hour reaction of azido intermediate 7a with NaN₃ under ideal circumstances, the desired product 3a was not detected. Instead, intermolecular annulation occurred to form quinoline 9 in 70% yield. This observation indicates that during the concerted reaction pathway, it first undergoes azide–alkyne cycloaddition followed by the SNAr reaction (Scheme 4d). This experiment is in good agreement with theoretical studies indicating the formation of a triazole ring prior to oxidative addition (Scheme 5-path B). Later, in terms of the industrial perspective, the practical usability of our developed protocol was successfully studied by scaling up to the gram scale without affecting the yield, and the desired product was obtained simply by washing with solvents (Scheme 4e). Further, a separate experiment was performed using a benzamide substrate, to confirm the in situ trapping of the azido intermediate by alkyne during [3 + 2] cycloaddition.⁵⁹ To prove that, a reaction was performed between 2-iodo-N-phenylbenzamide (4a) and NaN₃ in the absence of phenylacetylene under standard conditions, wherein the formation of 2-amino-phenylbenzamide^60–62 (6a′) was achieved predominantly in 2 h (indicated by GCMS). Later, phenylacetylene was added to the same reaction mixture but the formation of triazole 6a was not observed. This observation indicates that once the formation of the azido intermediate takes place it immediately undergoes [3 + 2] cycloaddition with phenyl acetylene and avoids the N₂ extrusion and thereby prohibits the 6a′ formation (Scheme 4f).

Computational studies

To shed light on the possible mechanism,⁶³ we performed density functional theory (DFT) calculations. The catalytic cycle begins after the formation of the 2-bromo chalcone intermediate (A) via base-catalyzed cross-aldol condensation, which is confirmed by gas chromatography-mass spectroscopy (GC-MS) analysis. We initiated our studies by two different approaches because there are two active sites for the copper to attack. However, the formation of a stable intermediate (IN2) proceeded after the addition of a nanocomposite (CuL) and NaN₃via transition state TS1 with a barrier of 20 kcal mol⁻¹ (path A), which was more feasible compared to path B where oxidative addition on copper occurs first before the triazole ring formation. In this step, the transition state barrier of path A (TS1) is more favorable than TS1′ by 1.7 kcal mol⁻¹. In addition, the intermediate IN2 is more stable than IN2′ by 21.7 kcal mol⁻¹. Next in path A, intermediate IN2 undergoes oxidative addition in the presence of the copper nanocomposite, leading to the formation of IN3 (−28.6 kcal mol⁻¹), through TS2, which has a barrier of 21 kcal mol⁻¹. Prior to addition of a second azide, formation of IN4 (−52.2 kcal mol⁻¹) takes place by leaving the NaBr, which is exergonic by 23.6 kcal mol⁻¹. Subsequent addition of azide across the C–Cu bond followed by liberation of dinitrogen leads to the generation of IN5, traversing the transition state TS4 with a barrier of 22.7 kcal mol⁻¹. In the next step, addition of dihydrogen gives IN7 with the regeneration of the active species (CuL) followed by intramolecular amine attack on the carbon center of the ketone group, giving stable complex IN8 (−83.6 kcal mol⁻¹). Finally, generation of the desired product with the liberation of water as the last step is associated with a barrier of 30.6 kcal mol⁻¹ with the completion of the catalytic cycle.

Catalyst recyclability

Next, considering the vital importance of catalyst recyclability in heterogeneous catalysis, the reaction of 2-bromobenzaldehyde (1a), acetophenone (2a) and NaN₃ under optimized conditions was performed to check the recyclability of the catalyst during the synthesis of 3a under standard reaction conditions. After the completion of the reaction, the nanocomposite was recovered from the reaction mixture by centrifugation, washed with ethyl acetate, water, and ethanol, and dried overnight in an oven, and was then used for the next cycle. Finally, results showed that the catalyst can be used up to 5 times without experiencing any significant loss in its activity. The FESEM of the recycled catalyst indicates that copper is stable for up to 5 cycles without much loss in the yield (see the ESI† for details).

Biological studies – anti-cancer activity

Cancer is a major global cause of death that has a significant negative impact on both the lives of individual people and society at large.^64,65 However, pharmaceutical chemists have found inspiration in 1,2,3-triazole moieties as a productive source for biological activities in the last few decades. Indeed, we have put a lot of emphasis on their synthetic accessibility using click chemistry and their varied inhibitory effects, such as anti-fungal, anti-bacterial, anti-allergic, anti-inflammatory, anti-cancer, etc.^66–68 Despite not being found in nature, the 1,2,3-triazole moiety has attracted a lot of attention due to its amazing pharmacophore.⁶¹ From this point of view, the synthetic library of compounds was screened against colon carcinoma cell lines (HCT116) and subjected to MTT assay. Compounds 3i and 3e displayed good activity against the cells with IC₅₀ values of 28.6 μM and 45.6 μM, respectively (see the ESI† for more details). Compound 3i displayed potent activity with lower IC₅₀ and was further used to evaluate anti-cancer activity against the HCT116 cells.

Compound 3i reduces viability and proliferation of cancer cells

Uncontrolled proliferation is a hallmark of cancer cells to propagate and form primary and secondary lesions. Cancer cells employ this ability to propagate and multiply, which results in the formation of tumours. Anticancer small molecules target various key pathway proteins to halt this process by triggering apoptotic cascade proteins. The ability of compound 3i to inhibit the proliferation was assessed by MTT and trypan analysis. The results demonstrated that compound 3i was very effective against the colon cancer cells, and inhibited the proliferation, reduced the viability of cells, and induced apoptosis in a concentration-dependent manner (Fig. S7a and b in the ESI†). The ability of the compound to induce cell death was analyzed by acridine orange/PI dual staining. The results demonstrated that compound 3i induces cell death of colon carcinoma cells in a concentration-dependent manner, which was unveiled by propidium iodide staining that permeates the cells lacking membrane integrity (Fig. S7c and d in the ESI†).

Effect of compound 3i on proliferation and cell viability

Cells were seeded and allowed to stand overnight, incubated with compound 3i in various concentrations and subjected to MTT and trypan analysis. In Fig. S7,† (a) presents bright field images of compound treated cells, (b) presents % of proliferation analysis, (c) presents acridine orange stained cells and (d) presents % of cell survival analysis.

Compound 3i inhibits proliferation and induces cell death

Cancer cells tend to proliferate abnormally by altering the expression of various pathway proteins. Anticancer small molecules target the abnormal proliferating cells to induce cell death by actuating various proapoptotic proteins involved in programmed cell death. To evaluate the effect of compound 3i on cell death, cells were treated with an increased concentration of compounds. The results demonstrated the potent anticancer effect of compound 3i to induce apoptotic cell death, which was demonstrated in Hoechst/PI dual staining. The compound-treated cells unveiled the nuclear deformation and penetration of propidium iodide into the cells, which permeates only into membrane integrity-compromised cells (Fig. S8a in the ESI†). The percentage of survival cells is represented in the bar graph (Fig. S8b in the ESI†).

Effect of compound 3i on cell death

Cells were seeded and allowed to stand overnight, incubated with compound 3i in various concentrations and subjected to Hoechst/PI dual staining. Hoechst and propidium iodide dual straining assay.

Conclusion

To summarize, we have synthesized a heterogeneous magnetically separable polymer-coated sustainable copper nanocomposite, and its formation was confirmed by various analytical and spectroscopic techniques. The synthesized nanocomposite was successfully utilized for the synthesis of a series of triazolo quinolines and triazolyl benzamide derivatives in good to excellent yields using greener reaction conditions, which stands as the 1st heterogeneous report. The mechanistic elucidation is well supported by key control experiments, literature precedents and DFT studies. Moreover, bioactivity studies were performed for selected triazole derivatives, wherein compound 3i displayed potent activity with lower IC₅₀ and it was subsequently used to evaluate anti-cancer activity against HCT116 cells. In addition, the catalyst recyclability studies and gram-scale reactions were also elaborated.

Data availability

The ESI† contains details of general and exact experimental procedures for the synthesis of triazolo quinolines and triazolyl benzamide derivatives, gram-scale reactions, hot filtration tests, catalyst recyclability, ¹H and ¹³C NMR & HR-MS of triazolo quinolines and triazolyl benzamide derivatives, and single crystal XRD data of compounds 3b, 3d, 6b, 6d and 6h.

The single crystal XRD data of compounds 3b, 3d, 6b, 6d and 6h and the .CIF file are submitted as part of the ESI.†

The experimental data of bioactivity studied have been provided in the ESI.†

All the coordinates of the compounds with respect to the DFT study have been provided in the ESI.†

Author contributions

Nandini R.: data curation, conceptualization, methodology, validation, software, and writing – original draft, review and editing. Byresh B. Kempegowda and Sudhanva M. Srinivasa: data curation, validation, and writing – original draft. Umesh A. Kshirsagar and Jan Grzegorz Malecki: data curation and resources. Siddappa A. Patil: validation, review, and editing. Manoj V. Mane, Ramesh B. Dateer and Shoyebmohamad F. Shaikh: supervision, funding acquisition, project administration, and review and editing.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

The authors thank the DST-SERB, Government of India for the financial support through the research grant no. SB/S2/RJN-042/2017 and ECR/2017/002207. The authors also thank the Jain University for the financial support through the Minor Research Project Grant (ref. JU/MRP/CNMS/17/2022). The authors extend their sincere appreciation to the Researchers Supporting Project number (RSP2025R370), King Saud University, Riyadh, Saudi Arabia. MVM thanks Prof. Luigi Cavallo from KAUST, Saudi Arabia, for providing access to a supercomputer (IBEX).

References

1. D. M. D'Souza and T. J. J. Müller, Chem. Soc. Rev., 2007, 36, 1095–1108 RSC .
2. E. Ruijter, R. Scheffelaar and R. V. A. Orru, Angew. Chem., Int. Ed., 2011, 50, 6234–6246 CrossRef CAS PubMed .
3. R. Keshri, D. Rana, A. Saha, S. A. Al-Thabaiti, A. A. Alshehri, S. M. Bawaked and D. Maiti, ACS Catal., 2023, 13, 4500–4516 CAS .
4. R. Nandini, R. Thrilokraj, U. A. Kshirsagar, R. V. Hegde, A. Ghosh, S. A. Patil, J. G. Malecki and R. B. Dateer, New J. Chem., 2024, 48, 1327–1335 Search PubMed .
5. U. K. Sharma, P. Ranjan, E. V. Van der Eycken and S.-L. You, Chem. Soc. Rev., 2020, 49, 8721–8748 CAS .
6. J. Peng, Y. Gao, W. Hu, Y. Gao, M. Hu, W. Wu, Y. Ren and H. Jiang, Org. Lett., 2016, 18, 5924–5927 CrossRef CAS PubMed .
7. K. Upadhyaya, A. Ajay, R. Mahar, R. Pandey, B. Kumar, S. K. Shukla and R. P. Tripathi, Tetrahedron, 2013, 69, 8547–8558 Search PubMed .
8. M. Selvaraju and C.-M. Sun, Adv. Synth. Catal., 2014, 356, 1329–1336 Search PubMed .
9. L. Biesen and T. J. J. Müller, Adv. Synth. Catal., 2021, 363, 980–1006 CAS .
10. S. Verma, S. Kumar, S. L. Jain and B. Sain, Org. Biomol. Chem., 2011, 9, 6943–6948 CAS .
11. R. C. Cioc, E. Ruijter and R. V. A. Orru, Green Chem., 2014, 16, 2958–2975 Search PubMed .
12. S. Rohilla, S. S. Patel and N. Jain, Eur. J. Org. Chem., 2016, 2016, 847–854 Search PubMed .
13. Y. Y. Xie, Y. C. Wang, Y. He, D. C. Hu, H. S. Wang and Y. M. Pan, Green Chem., 2017, 19, 656–659 Search PubMed .
14. D. Fournier, R. Hoogenboom and U. S. Schubert, Chem. Soc. Rev., 2007, 36, 1369–1380 CAS .
15. H. Nandivada, X. Jiang and J. Lahann, Adv. Mater., 2007, 19, 2197–2208 CAS .
16. S. G. Agalave, S. R. Maujan and V. S. Pore, Chem. – Asian J., 2011, 6, 2696–2718 CrossRef CAS PubMed .
17. V. Alagarsamy, V. R. Solomon and K. J. Dhanabal, Bioorg. Med. Chem., 2007, 15, 235–241 CrossRef CAS PubMed .
18. D. K. Mohapatra, P. K. Maity, M. Shabab and M. I. Khan, Bioorg. Med. Chem. Lett., 2009, 19, 5241–5245 CrossRef CAS PubMed .
19. A. Almulla, Der Pharma Chemica, 2017, 2017, 141–147 Search PubMed .
20. J. Qiao, G. Lin, A. Xia, Z. Xiang, P. Chen, G. Zhang, L. Li and S. Yang, Bioorg. Med. Chem. Lett., 2019, 29, 2595–2603 CrossRef CAS PubMed .
21. B. Banerji, S. K. Pramanik, P. Sanphui, S. Nikhar and S. C. Biswas, Chem. Biol. Drug Des., 2013, 82, 401–409 CrossRef CAS PubMed .
22. S. Pearce, Drug Discovery, 2017, 67 Search PubMed .
23. S. Chandrasekhar, M. Seenaiah, A. Kumar, C. R. Reddy, S. K. Mamidyala, C. G. Kumar and S. Balasubramanian, Tetrahedron Lett., 2011, 52, 806–808 CrossRef CAS .
24. N. Sudhapriya, A. Nandakumar, Y. Arun, P. Perumal, C. Balachandran and N. Emi, RSC Adv., 2015, 5, 66260–66270 RSC .
25. G. P. Costa, R. S. Baldinotti, M. G. Fronza, J. E. R. Nascimento, I. F. Dias, M. S. Sonego, F. K. Seixas, T. Collares, G. Perin, R. G. Jacob, L. Savegngo and D. Alves, ChemMedChem, 2020, 15, 610–622 CrossRef CAS PubMed .
26. J. P. Byrne, J. A. Kitchen and T. Gunnlaugsson, Chem. Soc. Rev., 2014, 43, 5302–5325 Search PubMed .
27. G. Rama-Martínez, M. Osorio-Celis, Y. Sabater-Algarra, D. Sánchez-Brunete, A. L. Llamas-Saiz, E. P. Quirós-Díez, M. E. Vázquez, M. V. López and M. D. López, Dalton Trans., 2024, 53, 18515–18527 RSC .
28. R. M. Meudtner, M. Ostermeier, R. Goddard, C. Limberg and S. Hecht, Chem. – Eur. J., 2007, 13, 9834–9840 CrossRef CAS PubMed .
29. K. Li, J. Chen, J. Li, Y. Chen, J. Qu, X. Guo, C. Chen and B. Chen, Eur. J. Org. Chem., 2013, 2013, 6246–6248 CrossRef CAS .
30. C. Y. Chen, C. H. Yang, W. P. Hu, J. K. Vandavasi, M. I. Chung and J. J. Wang, RSC Adv., 2013, 3, 2710–2719 Search PubMed .
31. C. Xu, S. F. Jiang, Y. D. Wu, F. C. Jia and A. X. Wu, J. Org. Chem., 2018, 83, 14802–14810 CrossRef CAS PubMed .
32. A. Hayeebueraheng, B. Kaewmee, V. Rukachaisirikul and J. Kaeobamrung, Eur. J. Org. Chem., 2017, 2017, 6714–6721 CrossRef CAS .
33. L. Fan, P. Sun, Y. Huang, Z. Xu, X. Lu, J. Xi, J. Han and R. Guo, ACS Appl. Bio Mater., 2020, 3, 1147–1157 CrossRef CAS PubMed .
34. E. Eskandari, M. Kosari, M. H. Davood Abadi Farahani, N. D. Khiavi, M. Saeedikhani, R. Katal and M. Zarinejad, Sep. Purif. Technol., 2020, 231, 115901 CrossRef CAS .
35. M. Butterworth, S. Bell, S. Armes and A. W. Simpson, J. Colloid Interface Sci., 1996, 183, 91–99 CrossRef CAS .
36. C. Zhou, J. Han, G. Song and R. Guo, J. Polym. Sci., Part A:Polym. Chem., 2008, 46, 3563–3572 CrossRef CAS .
37. L. Rodriguez-Arco, I. A. Rodriguez, V. Carriel, A. B. Bonhome-Espinosa, F. Campos, P. Kuzhir, J. D. Duran and M. T. Lopez, Nanoscale, 2016, 8, 8138–8150 RSC .
38. S. P. Yeap, J. Lim, B. S. Ooi and A. L. Ahmad, J. Nanopart. Res., 2007, 19, 1–15 Search PubMed .
39. T. E. Saraswati, A. Ogino and M. Nagatsu, Carbon, 2012, 50, 1253–1261 CrossRef CAS .
40. F. Sadegh, A. R. Modarresi-Alam, M. Noroozifar and K. Kerman, J. Environ. Chem. Eng., 2021, 9, 104942 CrossRef CAS .
41. M. S. Javed, A. J. Khan, M. Hanif, M. T. Nazir, S. Hussain, M. Saleem, R. Raza, S. Yun and Z. Liu, Int. J. Hydrogen Energy, 2021, 46, 9976–9987 CrossRef CAS .
42. J. Zhang, J. Han, M. Wang and R. Guo, J. Mater. Chem. A, 2017, 5, 4058–4066 RSC .
43. N. T. Patil, V. S. Shinde and B. Gajula, Org. Biomol. Chem., 2012, 10, 211–224 RSC .
44. R. Thrilokraj, J. G. Małecki, S. Budagumpi, U. A. Kshirsagar and R. B. Dateer, Green Chem., 2024, 26, 4723–4732 RSC .
45. H. Kim, J. Heo, J. Kim, M. H. Baik and S. Chang, J. Am. Chem. Soc., 2018, 140, 14350–14356 CrossRef CAS PubMed .
46. N. K. Nandwana, V. N. Shinde, H. K. Saini and A. Kumar, Eur. J. Org. Chem., 2017, 2017, 6445–6449 CrossRef CAS .
47. J. Fairoosa, M. Neetha and G. Anilkumar, RSC Adv., 2021, 11, 3452–3469 RSC .
48. M. Rawat, T. Taniike and D. S. Rawat, ChemCatChem, 2022, 14, e202101926 CrossRef CAS .
49. T. Wang, L. Zhang, H. Wang, W. Yang, Y. Fu, W. Zhou, W. Yu, K. Xiang, Z. Su, S. Dai and L. Chai, ACS Appl. Mater. Interfaces, 2013, 5, 12449–12459 CrossRef CAS PubMed .
50. C. Sun, R. Zhou, E. Jianan, J. Sun and H. Ren, RSC Adv., 2015, 5, 57058–57066 RSC .
51. T. Wang, L. Zhang, C. Li, W. Yang, T. Song, C. Tang, Y. Meng, S. Dai, H. Wang and L. Chai, Environ. Sci. Technol., 2015, 49, 5654–5662 CrossRef CAS PubMed .
52. R. Rajamohan, C. J. Raorane, S. C. Kim and Y. R. Lee, Materials, 2022, 16, 217 CrossRef PubMed .
53. (a) Q. Ai, Z. Yuan, R. Huang, C. Yang, G. Jiang, J. Xiong, Z. Huang and S. Yuan, J. Mater. Sci., 2019, 54, 4212–4224 CrossRef CAS ; (b) D. A. Bulushev, A. L. Chuvilin, V. I. Sobolev, S. G. Stolyarova, Y. V. Shubin, I. P. Asanov, A. V. Ishchenko, G. Magnani, M. Ricco and A. V. Okotrub, J. Mater. Chem. A, 2017, 5, 10574–10583 CAS .
54. M. Yuan, C. Nan, Y. Yang, G. Sun, H. Li and S. Ma, ACS Omega, 2017, 2, 4269–4277 CAS .
55. K. Mishra, H. Datta Khanal and Y. Lee, Eur. J. Org. Chem., 2021, 2021, 4477–4484 CAS .
56. M. Liu, W. Li, J. Rong and C. Zhou, Colloid Polym. Sci., 2012, 290, 895–905 CrossRef CAS .
57. Y. Y. Smolin, M. Soroush and K. K. Lau, Beilstein J. Nanotechnol., 2017, 8, 1266–1276 CAS .
58. K. Li, J. Chen, J. Li, Y. Chen, J. Qu, X. Guo, C. Chen and B. Chen, Eur. J. Org. Chem., 2013, 2013, 6246–6248 CAS .
59. N. A. Harry and R. V. Jagadeesh, Copper Catal. Org. Synth., 2020, 239–259 CAS .
60. C. Bouteiller, J. Becerril-Ortega, P. Marchand, O. Nicole, L. Barre, A. Buisson and C. Perrio, Org. Biomol. Chem., 2010, 8, 1111–1120 CAS .
61. Á. Georgiádes, S. B. Ötvös and F. Fülöp, Adv. Synth. Catal., 2018, 360, 1841–1849 Search PubMed .
62. C. Xu, S.-F. Jiang, Y.-D. Wu, F.-C. Jia and A.-X. Wu, J. Org. Chem., 2018, 83, 14802–14810 CAS .
63. A. Hayeebueraheng, B. Kaewmee, V. Rukachaisirikul and J. Kaeobamrung, Eur. J. Org. Chem., 2017, 2017, 6714–6721 CAS .
64. K. Lal and P. Yadav, Anti-Cancer Agents Med. Chem., 2018, 18, 21–37 CrossRef CAS PubMed .
65. L.-Y. Ma, L.-P. Pang, B. Wang, M. Zhang, B. Hu, D.-Q. Xue, K.-P. Shao, B.-L. Zhang, Y. Liu and E. J. E. Zhang, Eur. J. Med. Chem., 2014, 86, 368–380 CrossRef CAS PubMed .
66. H. A. M. El-Sherief, B. G. M. Youssif, S. N. Abbas Bukhari, A. H. Abdelazeem, M. Abdel-Aziz and H. M. Abdel-Rahman, Eur. J. Med. Chem., 2018, 156, 774–789 CrossRef CAS PubMed .
67. N. R. Penthala, L. Madhukuri, S. Thakkar, N. R. Madadi, G. Lamture, R. L. Eoff and P. A. Crooks, MedChemComm, 2015, 6, 1535–1543 RSC .
68. A. S. Limaye, P. Rananaware, A. Ghosh, T. Rajashekarreddy, N. Raghavendrarao, V. Brahmkhatri, R. V. Hegde and R. B. Dateer, ACS Appl. Bio Mater., 2024, 7, 1790–1800 CrossRef CAS PubMed .

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Footnote

† Electronic supplementary information (ESI) available. CCDC 2377550 and 2377552–2377555. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4cy01225a

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