Thiazolidinone-linked-1,2,3-triazoles with (R)-Carvone as new potential anticancer agents

Abstract

Aim: This study explores the cytotoxic and apoptotic effects of novel thiazolidinone-1,2,3-triazole hybrids on HT-1080, A-549, and MDA-MB-231 cancer cell lines.

Methods & results: The synthesized compounds underwent comprehensive characterization (NMR and HRMS) to confirm their structures and purity. Subsequent anticancer activity screening across diverse cancer cell lines revealed promising antitumor potential notably, compounds 6f and 6g. Mechanistic investigations unveiled that compound 6f triggers apoptosis through the caspase-3/7 pathway. In terms of in silico studies, the compound 6f was identified as a potent inhibitor of caspase-3 and caspase-7.

Conclusion: The present study underscores the therapeutic potential of thiazolidinone-1,2,3-triazole hybrids against certain cancer cells. These findings highlight a promising avenue for the development of cancer treatment strategies utilizing these (R)-Carvone-based derivatives.

Graphical Abstract

Plain language summary

Article highlights.

• Novel 1,2,3-triazole-thiazolidinone hybrids were synthesized and characterized.

• synthesized compounds were assessed for their cytotoxic activity.

• Compounds 6f–g exhibited the most potent cytotoxicity.

• Compound 6f triggers apoptosis through the caspase 3/7 pathway.

• Compound 6f exhibited superior binding affinity, selectivity and a well-defined binding mechanism toward caspase-3 and caspase-7.

1. Background

In the face of advancements against other illnesses, cancer persists as a significant adversary, with projections indicating it may soon surpass cardiovascular disease as the primary cause of death [1,2]. The cancer landscape is vast and varied, with breast cancer emerging prominently as one of the deadliest cancers worldwide, presenting a substantial public health challenge. Addressing this multifaceted threat requires tailored approaches, with treatment modalities such as surgery, chemotherapy, radiation, immunotherapy and hormone therapy carefully adapted to individual cases, considering factors like tumor characteristics, patient health and molecular profiling [3]. The escalating incidence of breast cancer, now the foremost cause of cancer-related mortality among women, underscores the pressing need for expedited discovery of effective therapeutics. Liver cancer also looms large on the global health stage, ranking sixth in incidence and fourth in mortality worldwide, with an annual toll of approximately 840,000 new cases and 782,000 lives lost [4,5].

Research on (R)-carvone, a natural compound, has shown promising anticancer properties against various cancers including lung, breast, gastric and prostate cancers, with mechanisms targeting apoptosis induction, cell cycle arrest, angiogenesis and autophagy [6–10]. Apoptosis, our body's intrinsic self-destruct mechanism, functions as a precise eliminator, removing unwanted cells such as tumor and pathogen-infected cells [11]. Enzymes like caspases-3 and -7, integral to the apoptosis process, sequentially cleave proteins, orchestrating controlled cellular demise [12]. Ongoing investigations offer prospects of personalized therapies leveraging apoptosis to selectively eradicate tumors [13,14]. The foundation of effective cancer drugs often lies in complex molecular structures known as heterocyclic pharmacophores.

These minute molecular frameworks serve as the foundational structure for potent drug compounds, holding a pivotal position in medicinal chemistry. Throughout history, plant extracts and their varied secondary metabolites have been utilized as therapeutic remedies for a multitude of ailments, including cancer [15]. Among the active constituents present in essential oils derived from medicinal plants, monoterpenes such as camphor, verbenone, carvone and limonene (Figure 1A) stand out as primary components [16]. In the vast array of chemical building blocks, 1,2,3-triazole compounds emerge as central players. Characterized by their distinctive ring structures, these compounds exhibit remarkable versatility, featuring prominently in both synthetic and medicinal chemistry, while also finding applications across various other domains. 1,2,3-Triazoles are increasingly recognised as frontrunners in the quest for novel drugs, owing to their involvement in the regulation of crucial biological processes.

Figure 1.

(A & B) Some 1,2,3-triazole and thiazolidine-containing drugs. (C) Potential of monoterpene-based inhibitors as cytotoxic agents.

Finding their way into established medicines like Carboxyamidotriazole, Rufinamide and Tazobactum strengthens this potential and paves the way for novel therapeutic designs (Figure 1B). In the biological world, these molecules wear many hats boasting antitubercular [17], antioxidant [18], antibacterial [19], anticancer [20], antiviral [21], anti-inflammatory [22] and antimicrobial [23] properties, showcasing their remarkable versatility.

However, the thiazolidinones have captivated researchers due to their presence as the cornerstone for a multitude of compounds boasting diverse biological activities. These versatile heterocycles offer a treasure trove of potential applications, from antiviral to antihyperglycemic, analgesic to antifungal and even anticancer [24–28]. Think of the thiazolidinone ring as a versatile molecular building block, seamlessly integrating into the structures of potent drugs across various therapeutic areas. Figure 1C showcases its presence in Ralitoline, enabling its anticonvulsant action, and in Etozoline, powering its diuretic properties. It even appears in Thiazolidomycin [29,30]. This range of applications highlights the remarkable potential of this tiny ring as a chemical scaffold. Because of the above facts and biological importance over moieties, while building on the promising properties of 1,2,3-triazole and thiazolidinone moieties, we have embarked on a mission to design exceptional therapeutic agents for cancer treatment. Motivated by the biological properties of both 1,2,3-triazoles and thiazolidinone, we strategically integrated them into a distinctive scaffold, as illustrated in Figure 2. This innovative hybrid structure holds the promise of harnessing potent biological effects for potential therapeutic use in the future. Building upon encouraging findings and ongoing investigations, we initiated the synthesis of a diverse array of novel derivatives tethering 1,2,3-triazole with thiazolidinone (6a–h). These newly synthesized compounds underwent rigorous evaluation for their cytotoxic efficacy both in vitro and via computational modelling (in silico), aimed at unlocking their potential as agents combating cancer.

Figure 2.

Design strategy of new thiazolidinone-1,2,3-triazole hybrids as potential anticancer agents.

2. Experimental section

2.1. Chemistry

2.1.1. Materials & methods

Chemicals and reagents: all chemicals and solvents required for the synthesis of compounds 6a–e were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (MI, USA). Purification: column chromatography was employed for the purification of isolated products throughout this work. Silica gel (230–400 mesh) served as the stationary phase, while a hexane/ethyl acetate eluent system facilitated the separation process. NMR Spectroscopy: each synthesized compound was analyzed using a Bruker Avance III NMR spectrometer (MA, USA; operating at 500 MHz for ¹H and 125 MHz for ¹³C) with CDCl₃ as the solvent. The ¹H and ¹³C NMR spectra were referenced to the residual solvent signal (CDCl₃ at δ H 7.26 ppm and δ C 77.16 ppm, respectively). High-resolution mass spectrometry (HRMS): the molecular weights of the synthesized compounds were determined using a Q-TOF micro mass spectrometer located within the Laboratory of Bioorganic Chemistry, Reactivity and Analysis at the University of Rouen Normandy, France.

Source of Starting Materials: It is noteworthy that the starting materials, compounds 2, 3a and 6a–e have been previously described in our earlier publication [31,32].

2.1.2. Preparation of thiazolidinone-N-etyle (3b & 4b)

Alkylation reaction: a solution of thiazolidinone–carvone derivative 2 or 4a (1 mmol) in anhydrous acetone was treated with potassium carbonate (1 eq.) and ethyl bromoacetate (1 equiv) at room temperature (25°C). The reaction mixture was stirred magnetically for 4–5 h. Workup and purification: after completion of the reaction, the solvent was removed under reduced pressure. The resulting residue was then quenched with water and extracted with ethyl acetate (EtOAc, 3 × 50 ml). The combined organic extracts were dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. Finally, the crude product was purified via silica gel column chromatography to afford the corresponding alkyne derivative 3b or 4b.

2.1.2.1. Ethyl 2-(2-(((R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene)hydrazono)-4-oxoth iazolidin-3-yl)acetate (3b)

Solid; yield 15%; m.p.: 126–128°C. ¹H NMR (500 MHz, CDCl₃) δ: 1.30 (3H, t, J = 7.1 Hz, CH₃); 1.78 (3H, s, CH₃); 1.91 (3H, s, CH₃); 2.00–3.40 (5H, m, CH and 2*CH₂); 3.81, 4.51 and 4.80 (6H, m, 3*CH₂); 4.21 (2H, q, J = 7.1 Hz, CH₂), 6.22 (1H, m, CH).¹³C NMR (125 MHz, CDCl₃) δ (ppm): 15.58 (CH₃); 18.38 (CH₃); 20.98 (CH₃); 31.07, 32.37, 41.34 and 44.21 (4*CH₂); 110.02 (CH₂); 135.83 (Cq); 136.35 (C); 149.23 (C); 160.20 (C); 165.49 (C); 167.29 (C); 173 (C). HRMS (TOF-MS ES+) (m/z) [M+H]⁺ calculated for C₁₇H₂₃N₃O₃S: 423.9583; found: 423.9732.

2.1.2.2. Ethyl 2-(5-(4-chlorobenzylidene)-2-(((R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylid ene)hydrazono)-4-oxothiazolidin-3-yl)acetate (4b)

Solid; yield 87%; m.p.: 123–125°C. ¹H NMR (500 MHz, CDCl₃) δ: 1.25 (3H, t, J = 7.1 Hz, CH₃); 1.26 (3H, s, CH₃); 1.87 (3H, s, CH₃); 2.00–3.50 (5H, m, CH and 2*CH₂); 4.75 and 5.08 (4H, m, 3*CH₂); 4.23 (2H, q, J = 7.1 Hz, CH₂), 6.24 (1H, m, CH), 7.25–7.00 (4H, m, CH_Ar); 7.23 (1H, s, CH). ¹³C NMR (125 MHz, CDCl₃) δ (ppm): 15.34 (CH₃); 18.23 (CH₃); 21.38 (CH₃); 31.23, 31.97, 38.54 and 42.38 (4*CH₂); 51.38 (CH); 110.23 (CH₂); 125.33 (Cq); 135.32 (C); 138.23 (C); 139.20 (C); 131.49 (C); 134.29 (C); 148 (C); 155.83 (Cq); 165.35 (C); 166.48 (C); 167.34 (CH). HRMS (TOF-MS ES+) (m/z) [M+H]⁺ calculated for C₂₄H₂₆N₃O₃SCl: 472.1582; found: 472.1582.

2.1.3. Preparation of thiazolidinone–benzylidene (4a)

2.1.3.1. 5-(4-chlorobenzylidene)-2-(((R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene)hyd razono)thiazolidin-4-one (4a)

Solid; yield 75%; m.p: 154–156°C. ¹H NMR (500 MHz, CDCl₃) δ: 1.75 (3H, s, CH₃); 1.85 (3H, s, CH₃); 2.00–2.50 and 3.00–3.25 (5H, m, CH, CH₂ and CH₂); 4.75 (2H, m, CH₂); 6.00 (1H, m, CH), 7.25–7.75 (4H, m, CH_Ar); 7.98 (1H, s, CH).¹³C NMR (125 MHz, CDCl₃) δ (ppm): 17.37 (CH₃); 20.3 (CH₃); 28.12 and 30.12 (CH₂ and CH₂); 40.32 (CH); 76.98 (C); 110.02 (CH₂); 122.83 (Cq); 129.35 (CH_Ar); 130.48 (CH); 131.21 (CH_Ar); 132.43 (CAr); 133.98 (Cq); 134.28 (CH); 136.50 (CAr); 147.27 (Cq); 157.71 (Cq); 167.03 (C=O). HRMS (TOF-MS ES+) (m/z) [M+H]⁺ calculated for C₂₃H₂₂ClN₃OS: 386.2020; found: 386.2085.

2.1.4. Preparation of thiazolidinone–alkyne (5)

To the solution of thiazolidinone–carvone 4 (385 mg; 1 mmol) in dry acetone, potassium carbonate (179 mg; 1.3 mmol) and 3-bromoprop1-yne (130 mg; 1.1 mmol) was added at 25°C. The reaction mixture was stirred at room temperature. After 4–5 h, the excess solvent in the reaction mixture was evaporated under reduced pressure; the resulting residue was diluted with water and extracted by using EtOAc (3 × 50 ml). The crude product was purified by silica gel column chromatography to give the corresponding alkyne 5.

2.1.4.1. 5-(4-chlorobenzylidene)-2-(((R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene)hyd razono)-3-(prop-2-yn-1-yl)thiazolidin-4-one (5)

Solid; yield 75%; m.p.: 174–176°C. ¹H NMR (500 MHz, CDCl₃) δ: 1.80 (3H, s, CH₃); 2.01 (3H, s, CH₃); 2.22 (1H, s,CH); 2.10–2.60 and 3.40–3.60 (5H, m, CH, 2*CH₂); 4.74 and 4.81 (4H, m, 2*CH₂); 6.28 (1H, m, CH), 7.40–7.60 (4H, m, CH_Ar); 7.7 (1H, s, CH). ¹³C NMR (125 MHz, CDCl₃) δ (ppm): 17.88 (CH₃); 20.71 (CH₃); 30.97, 31.22 and 32.53 (3*CH₂); 41.13 (CH); 71.66 (CH); 76.85 (Cq); 110.02 (CH₂); 122.83 (Cq); 129.35 (CH_Ar); 129.46 (CH); 131.20 (CH_Ar); 132.49 (CAr); 133.29 (Cq); 135.66 (CH); 136.50 (CAr); 147.95 (Cq); 154.71 (Cq); 165.77 (Cq); 167.03 (C=O). HRMS (TOF-MS ES+) (m/z) [M+H]⁺ calculated for C₂₃H₂₂ClN₃OS: 423.9583; found: 423.9732.

2.1.5. Preparation of (R)-carvone-thiazolidinone-1,2,3-triazole 6f–h

Cu-Catalyzed Azide-Alkyne Cycloaddition (CuAAC): a stirred solution of compound 5 (1 mmol) and appropriate azide (1 mmol) in a 1:5 (v/v) ethanol (EtOH)/water (H₂O) mixture (5 ml) was treated at room temperature (25°C) with sodium ascorbate (54.6 mg, 0.2 mmol, 20 mol%) and CuSO₄·5H₂O (12.4 mg, 0.06 mmol, 15 mol%) added sequentially. The reaction mixture was stirred for 5–6 h. Workup and purification: upon reaction completion, the reaction mixture was quenched with ice-cold water (100 ml) and extracted with ethyl acetate (EtOAc 3 × 50 ml). The combined organic extracts were dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. The crude product was then purified via silica gel column chromatography using a hexane/ethyl acetate eluent system (76:24 v/v) to afford the desired products 6f–h.

2.1.5.1. 5-(4-chlorobenzylidene)-2-(((R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene)hyd razono)-3-((1-(o-tolyl)-1H-1,2,3-triazol-4-yl)methyl)thiazolidin-4-one (6f)

Yield: 85%; solid; m.p.: 175–177°C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 1.63 (3H, s, CH₃); 2.15 (3H, s, CH₃); 2.32 (3H, s, CH₃); 2.00–3.65 (5H, m, CH, 2*CH₂); 4.75 (2H, s, =CH₂); 5.33 (2H, s, CH₂); 6.25 (1H, m, CH), 7.10–7.85 (8H, m, CH_Ar); 6.71 and 8.04 (2H, 2s, 2*CH). ¹³C NMR (125 MHz, CDCl₃) (ppm): 17.34 (CH₃); 20.54 (CH₃); 30.35 and 31.76 (2*CH₂ ); 38,51 (CH); 41.32 (N-CH₂); 110.45 (=CH₂); 120.45 (C=C); 121.32 (=C-N), 122.51 (CAr); 123.34 (CAr); 129.44 (CH_Ar); 130.76 (CH_Ar); 131.32 (CH_Ar), 130.87 (HC=); 131,55 (CAr); 136.56 (CH); 129.56 (CH_Ar); 132.73 (=C); 123.25 (CAr); 138.76 (CAr); 142.23 (C=C-N); 147.56 (C=C); 155,57 (N=C); 165,38 (C=N); 166.65 (C=O). HRMS (TOF-MS ES+) (m/z) [M+H]⁺ calculated for C₃₀H₂₉ClN₆OS: 557.1086; found: 557.1045.

2.1.5.2. 3-((1-(4-chloro-2-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-(4-chlorobenzylidene)-2-(2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene)hydrazono)thiazolidin-4-one (6g)

Yield: 87%; solid; m.p.: 181–183°C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 1.32 (3H, s, CH₃); 2.04 (3H, s, CH₃); 2.32 (3H, s, CH₃); 2.19–3.46 (5H, m, CH, 2*CH₂); 4.84 (2H, s, =CH₂); 5.23 (2H, s, CH₂); 6.31 (1H, m, CH), 7.20–7.85 (8H, m, CH_Ar); 7.64 and 8.23 (2H, 2s, 2*CH). ¹³C NMR (125 MHz, CDCl₃) (ppm): 17.58 (CH₃); 20.83 (CH₃); 30.45 and 31.29 (2*CH₂); 38,46 (N-CH₂); 41.31 (N-CH₂); 110.41 (=CH₂); 121.18 (C=C); 121.75 (CAr); 122.85 (CAr); 123.82 (N-CH); 129.45 (CAr); 129.59 (CAr); 129.87 (CH_Ar); 131.87 (CH_Ar); 132.05 (=C); 133,41 (HC=); 134.76 (=CH); 135.98 (CAr); 136.29 (CAr); 144.92 (CAr); 144.77 (C=C-N); 147.48 (=C); 155.96 (N=C); 165.63 (C=N); 166.71 (C=O). HRMS (TOF-MS ES+) (m/z) [M+H]⁺ calculated for C₃₀H₂₈Cl₂N₆OS: 591.5531; found: 591.5572.

2.1.5.3. 3-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-(4-chlorobenzylidene)-2-(((R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene)hydrazono)thiazolidin-4-one (6h)

Yield: 89%; solid; m.p.: 190–192°C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 1.67 (3H, s, CH₃); 1.95 (3H, s, CH₃); 2.20–3.60 (5H, m, CH, 2*CH₂); 4.78 (2H, 2s, H₂C=), 5.24 (2H, s, CH₂); 5.45 (2H, s, N-CH₂); 6.21 (1H, m, HC=), 6.80–7.50 (9H, m, HCAr); 7.72 (1H, s, =CH); 7.94 (1H, s, =CH). ¹³C NMR (125 MHz, CDCl₃) δ (ppm): 17.34 (CH₃); 21.56 (CH₃); 29.45 and 31.24 (2*CH₂); 40.56 (CH); 54.92 (CH₂); 61.72 (N-CH₂); 110.57 (H2C=); 120.47 (CH); 111.58, 123.87, 122.58, 124.98, 129.52 and 131.98 (HCAr); 123.89, 129.98, 129.29 and 135.98 (C-Ar); 133.21 (HC=); 134.39 (HC=); 143.98 (C); 149.39 (C); 157.92 (C); 158.35 (C); 167.83 (C); 169.32 (C). HRMS (TOF-MS ES+) (m/z) [M+H]⁺ calculated for C₃₀H₂₉ClN₆OS: 557.1087; found: 577.1080.

2.2. Anticancer activity

2.2.1. Cell culture

The cancer cells selected in this work (HT-1080 [CCL 121], A-549 [CCL-185], MCF-7 and MDA-MB-231) were purchased from Sigma Aldrich and American Type Culture Collection. The culture medium for these cancer cells contains both MEM for HT-1080, and DMEM for other cancer cells, as well as Earle salts and Glutamax I with 10% fetal bovine serum and, finally, 1% penicillin-streptomycin. Under conditions of 37°C and a humidified atmosphere containing 5% of CO₂, cancer cells and their culture medium were maintained. The treatment of cells after 24 h of incubation with the synthesis products was done using 0.05% trypsin, and 0.53 mM EDTA.

2.2.2. Cytotoxic activity

The experiment involved treating cells with various concentrations of the tested products, along with a cell density of 5 × 103 cells, followed by a 24-h incubation period. Concurrently, Doxorubicin from TEVA Pharma S.A. was selected as the positive control. The quantification of cancer cells treated with our products was conducted using the MTT assay, where cells were exposed to 5 mg/ml of MTT solution for 4 h at 37°C. Subsequently, 150 μl of DMSO was added to facilitate the formation of formazan. Absorbance analysis was performed at a wavelength of 570 nm. Cytotoxicity data, presented in the manuscript, were determined as the percentage of cell growth relative to untreated cells. The IC₅₀ value, expressed in micromoles (μM), indicates the concentration of the tested product required to inhibit cell growth by 50%.

2.2.3. Annexin-V binding assay

Cells were seeded at a density of 2 × 10⁵/wells and incubated overnight at 37°C. The cells were then treated with derivatives 6f and 6g at concentration of 20 μM for 24 h. Cells were then harvested and washed twice with phosphate-buffered saline (PBS) at 4°C. Then, the cells were centrifuged at 300 g for 5 min and washed twice with PBS at 4°C before adding 100 μl of Annexin V Binding Buffer staining (Annexin V Apoptosis Detection KIT with 7-AAD, Millipore-Merck, Fontenay sous Bois, France). The cells were then incubated for 20 min at room temperature in the dark. Apoptotic cells were evaluated using a Muse Cell Analyzer (Millipore-Merck, Darmstadt, Germany).

2.2.4. Caspase-3/7 activity

Cells were treated with 20 μM concentration of the derivatives 6f and 6g. After 24 h incubation, cells were then harvested and washed twice with PBS at 4°C. After staining with the Caspase assay kit (Millipore-Merck) and incubation for 30 min, caspase-3/7 activity was analyzed according to the manufacturer instructions and using the Muse Cell Analyzer (Millipore-Merck).

2.3. Molecular docking studies

2.3.1. Preparation of protein & ligand

In this study, the interactions between 6f and 6g derivatives and the proteins caspase-3 and caspase-7 were meticulously examined, highlighting these compounds' potential for inhibitory action against these enzymes. The structural information regarding caspase-3 and caspase-7 was retrieved from the RCSB Protein Data Bank (PDB; www.rcsb.org), specifically accessing PDB IDs 1NMS and 1SHJ. It's noteworthy that both caspase-3 and caspase-7 are composed of dimeric proteins, consisting of chains A and B with nearly identical sequences of amino acids. For this docking analysis, chain A was chosen for examination using the Discovery Studio 4.5 Client, and the corresponding PDB files were prepared accordingly [33]. The molecular structures of the ligands under study were designed and optimized using Chemsketch 2021 software MarvinSketch 21.19.0, ChemAxon (www.chemaxon.com). Initially, these structures were saved in the .mol file format and later converted to PDB format utilizing Open Babel 2.3.2 software, which is a prerequisite for conducting analyses with PyRx software. Before commencing the molecular docking process, the target enzymes were prepared by eliminating endogenous ligands and associated water molecules. Subsequently, the investigational ligands underwent processing, which included the addition of hydrogen atoms, assignment of torsions and identification of rotatable bonds, with the modified structures saved in the .pdbqt format. For detailed analysis, compounds demonstrating the lowest free binding energies were selected. The interactions between caspase-3 and caspase-7 with these compounds were examined using Discovery Studio 4.5 software, offering insights into the modes and dynamics of these molecular interactions [33,34].

2.3.2. Docking procedure analysis

In this phase of the study, the docking process was initiated by removing all un-associated water molecules and atoms from the protein structures using Auto Dock Tools. This was followed by adding polar hydrogen atoms and applying Kollman united atom type charges to both caspase-3 and caspase-7 proteins. The docking computations were carried out employing the Lamarckian Genetic Algorithm (LGA) as the primary method. The computational grid for caspase-3 docking was strategically centred with coordinates at X: -11.0038, Y: -4.4557, Z: 24.2692. The grid itself was defined with a midpoint spacing of 0.375 Å and covered a volume defined by 64*57*66 Å³ in the X, Y and Z dimensions, respectively. Similarly, for caspase-7, the docking grid was centred at X: 52.2913, Y: 16.9099, Z: 1.2239. The grid parameters were set with the same midpoint spacing of 0.375 Å, encompassing a volume of 71*57*69 Å³. All compound dockings were executed under consistent conditions, maintaining uniform Auto Dock parameters throughout the process. Following the completion of docking analyses, the conformation with the lowest binding energy was determined for each compound. Subsequently, a comparative evaluation was conducted to assess the affinity of all compounds in their interactions with caspase-3 and caspase-7. This comparison facilitated the identification of the most potent compound for inhibiting caspase-3 and caspase-7, based on the highest interaction affinity. Finally, the interaction dynamics of the ligand complexes with caspase-3 and caspase-7 were thoroughly examined using The Discovery Studio 4.5 software, offering detailed insights into the binding interactions and molecular conformations.

3. Results & discussion

3.1. Synthesis

The target hybrid compounds were synthesized from (R)-Carvone 1 using the synthetic protocol as depicted in Figure 3. The (R)-Carvone 1 was treated by thiosemicarbazide in the presence of a few drops of concentrated sulphuric acid, to obtain the (R)-Carvone-thiosemicarbazone 2 in 85% yield. Secondly, the reaction of the intermediate 2 with 1.5 equivalent of ethyl bromoacetate in the presence of anhydrous sodium acetate furnished (R)-carvone-thiazolidinone 3a and (R)-carvone-thiazolidinone-N-ethyl 3b which have been isolated with good yields of 75 and 15%, respectively. It is important to highlight that the low yield of compound 3b is due to the low amount of ethyl bromoacetate used (1.5 eq). Indeed, if an excess of ethyl bromoacetate is used (2.5 eq), the yield of compound 3b increases significantly, while that of compound 3a decreases. In a third step, this latter (3a) was reacted with para-chloro-benzaldehyde in a basic medium to afford the para-chloro-benzylidene-thiazolidinone 4a in 75% yield. Moreover, regarding the structure of compound 4a, we noticed on the ¹H-NMR spectrum the presence of the N–H group which can be involved in an N-alkylation reaction.

Figure 3.

Synthetic route for target compounds 6a–h. Operating conditions and reagents: (i) NH₂CSNHNH₂, three drops of H₂SO₄, EtOH with reflux for 3 h, (ii) BrCH₂COOEt, CH₃COONa, EtOH, reflux for 2 h, (iii) 4-chloro-benzaldehyde, KOH, Toluene, reflux for 2 h. (iv) Propargyl bromide, K₂CO₃, rt, 3 h, Acetone, (v) BrCH₂COOEt, K₂CO₃, Acetone, rt, 2 h, (vi) Aromatic azides, CuSO₄ 0.5 H₂O, sodium ascorbate, rt, 3 h, EtOH: H₂O (1:5).

rt: Room temperature.

From this finding, and as a first step to afford the targeted hybrids, compound 4a was treated with an equimolar quantity of propargyl bromide as an alkylating agent in the presence of K₂CO₃ as a base in acetone. After two h of agitation at room temperature, the reaction leads to the N-propargylated compound 5 in 89% yield. Under the same operating conditions, compound 4a was treated with an equimolar quantity of ethyl bromoacetate to give compound 4b with a yield of 87%. During the final step, the (R)-Carvone-thiazolidinone-1,2,3-triazole hybrids 6a–h were synthesized from compound 5 that is involved in a 1,3-dipolar cycloaddition reaction with aromatic azides in room temperature in ethanol/water (1:5), using CuSO₄·5H₂O and sodium ascorbate as a catalyst system (Figure 3). The targeted compounds 6a–h and their alkyne 5 and benzylidene 4 intermediates were purified using a silica gel column chromatography in good yields (Supplementary Table S1) and structurally identified using HRMS, ¹H- and ¹³C-NMR spectral data. As shown in Supplementary Table S1, the chemical structures of compounds 3b, 4a–b, 5 and 6a–h were fully elucidated by their HRMS spectra.

The propargyl unit within compound 5 proudly announced its presence in the ¹H NMR spectrum. Two characteriztic peaks – one at 4.56 ppm and another at 2.21 ppm, pinpointed the locations of its methylene and acetylene protons. While in the spectrum of the 1,2,3-triazole hybrid 6c (As a representative compound), we note the disappearance of the acetylenic proton and the appearance of the 1,2,3-triazole and aromatic protons which resonated respectively, as a singlet at 6.57 ppm and as two doublets centred at 7.42 and 7.72 ppm, each integrating for two protons. The methylene linker between the thiazolidinone and 1,2,3-triazole rings in 6c appears as a singlet at 5.19 ppm in the ¹H-NMR spectrum. Meanwhile, the 1,2,3-triazole-thiazolidinone skeleton of the same compound which is characterized by the methylene group of the thiazolidinone ring and the ethylenic proton of the (R)-Carvone core revealed respectively as a singlet at 3.87 ppm and a singlet at 5.38 ppm. On the other hand, the ¹³C-NMR spectrum of intermediate 5 exhibited characteriztic peaks at 76.97 and 71.53 ppm, which were assigned to the alkyne carbons. These peaks disappeared in the spectra of compound 6c, while other peaks appeared at 101.63 and 128.08–129.25 ppm, corresponding to the =CH carbons of the 1,2,3-triazole scaffold and the aromatic ring, respectively (Supplementary material). The following figure (Supplementary Figure S1) presents a comparative study of NMR (¹H and ¹³C) between the dipolarophile 5 and final compounds 6a–h, in particular, the effect of the different substitutions located in the para position of aryl of the triazole nucleus on the chemical shift of carbon =CH (sp2) of function 1,2,3-triazole. Here we can see that there's a significant 2.19 ppm resonance shift attributed to the alkyne (sp) proton to 7–8 ppm, and also a slight shift was detected in the case of substitution of H (more shielded) by NO₂ (more deshielded).

3.2. In vitro cytotoxic activity

We delved into the in vitro anticancer activity of both intermediate compounds (1–5) and the final hybrid molecules (6a–h) to uncover their potential as cancer-fighting agents. utilizing the MTT colorimetric assay, we put them to the test against four different human cancer cell lines: fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231) and lung carcinoma (A-549). Selected cells were treated with various concentrations (up to 100 μM) for 24 h to evaluate anticancer activity. Doxorubicin was used as a positive control. IC₅₀ values are shown in Table 1.

Table 1. .

Cytotoxic profile for the compounds of 1–5 and 6a–h against human cancer cells.

Compound	IC50 (μM)
	HT-1080	A-549	MCF-7	MDA-MB-231	Ref.
1	≥100	≥100	≥100	≥100
2	≥100	47.39 ± 1.23	≥100	72.87 ± 2.05
3a	48.30 ± 2.48	41.98 ± 2.93	78.87 ± 2.87	52.49 ± 0.89
3b	21.57 ± 1.29	19.30 ± 1.76	28.71 ± 1.97	25.47 ± 1.79
4a	≥100	52.21 ± 1.07	31.98 ± 1.34	44.29 ± 1.07
4b	47.48 ± 1.38	23.87 ± 1.81	19.36 ± 2.11	26.39 ± 1.62
5	78.21 ± 2.87	52.45 ± 0.73	82.76 ± 2.81	≥100
6a	≥100	≥100	≥100	≥100	[32]
6b	18.03 ± 1.07	26.22 ± 2.04	23.03 ± 2.07	31.65 ± 0.45	[32]
6c	25.19 ± 2.03	19.34 ± 1.12	27.34 ± 1.34	21.45 ± 1.78	[32]
6d	21.78 ± 0.56	18.32 ± 0.56	31.41 ± 0.45	27.71 ± 1.13	[32]
6e	31.87 ± 1.01	28.44 ± 2.87	37.88 ± 2.11	40.23 ± 2.32	[32]
6f	13.15 ± 1.82	17.33 ± 1.32	21.33 ± 1.63	22.34 ± 1.08
6g	16.62 ± 1.06	17.78 ± 1.92	15.94 ± 1.89	25.94 ± 1.47
6h	14.70 ± 2.43	20.33 ± 1.56	35.69 ± 1.77	18.41 ± 1.03
Dox	5.76 ± 1.98	6.38 ± 1.73	6.97 ± 1.38	5.82 ± 1.89

Dox: Doxorubicin.

Table 1 shows that intermediate compounds 1–5 exhibit weak to moderate cytotoxic activity against tumor cells, except for the ethoxy-substituted thiazolidinones (3b and 4b), which have important IC₅₀ values of 19–26 μM. Interestingly, the initial compound, (R)-carvone, exhibits significantly lower potency compared with the synthesized derivatives, as its cytotoxicity value reaches 100 μM. A deep dive into the structure–activity relationship (SAR) of hybrid compounds 6a–h yielded intriguing insights, guiding the path toward optimizing their impact on targeted pathways: among the compounds assessed, 6h demonstrated greater potency compared with the other triazole–thiazolidinone derivatives, exhibiting an IC₅₀ value of 14.70 ± 2.43 μM against HT-1080. In addition, the compounds 6f and 6g emerged as another potent warrior against all selected cells, boasting a strikingly similar cytotoxic profile to that of 6h. The stark decline in potency observed for 6a, whose plain aromatic ring lacks any substitutions, serves as a poignant reminder of the crucial role these modifications play in activity. Replacing the fluoro halogen group (6e) with a chloro type (6c) led to an increase in cytotoxic impact across all four selected cancer cell lines, notably in the MDA-MB-231 cell lines, decreasing from 40 to 21 μM. Similarly, substituting the CH₃ group from the para position of compound 6b with the ortho position (6f) demonstrated a positive effect on the cytotoxic profile, particularly in the A-549 cell lines, decreasing from 26 to 17 μM. Introducing an additional CH₃ group in the ortho position on the aromatic ring of compound 6c to synthesize a compound containing both groups in the ortho and para positions (6g) significantly enhanced the cytotoxic potential, indicating that occupying the ortho position strengthens interactions between the molecular candidates and the biological target. Furthermore, extending the aromatic ring of compound 6a by inserting a CH₂ group between the 1,2,3-triazole function and the phenyl ring (compound 6h) notably improved its cytotoxic potential compared with compound 6a. Based on the interpretation of these results and SAR analysis, substituting the phenyl group in the para position with a halogen atom, especially chlorine, is crucial for inducing cytotoxicity. Therefore, we suggest that a para-substituted aryl with a halogen atom and ortho-substituted with a CH₃ group are essential for enhancing cytotoxic activity. Moreover, extending the aromatic ring by inserting an n-CH₂ group is likely to provide significant anticancer activity (Figure 4). Considering all this information, we preliminarily preselect the chemical structure that is expected to be more active against cancer cells in our future research endeavours (Figure 4).

Figure 4.

Analysis of the structure–activity relationship.

SAR: Structure–activity relationship.

3.3. Analysis of results via structure–activity relationship with our previous work

To analyze the relationship between the anticancer activity and the modified structure of our starting material, (R)-carvone, particularly in comparison with the works conducted in our laboratory, we can conduct an SAR analysis. Our previous research, including the present study, focuses on evaluating the anticancer potential of compounds derived from (R)-carvone. Specifically, we have investigated the relationship between the chemical structures of these derivatives and their efficacy against cancer cells, with a particular emphasis on the same cancer cell lines utilized in this current study.

In our previous works, which span several years, we have synthesized a range of compounds derived from (R)-carvone. These compounds typically incorporate two heterocyclic nuclei, namely cyclopropane and thiadiazol types. Our investigation has centered on assessing the anticancer properties of these derivatives against the same set of cancer cells employed in the present study. By comparing the structure and activity of these compounds synthesized from (R)-carvone with those presented in this work, we aim to elucidate the SAR and understand how specific modifications to the chemical structure impact anticancer activity. Through this comparative analysis, we seek to identify structural features that contribute to enhanced anticancer efficacy, thereby informing future research directions and potentially facilitating the development of more potent anticancer agents derived from (R)-carvone (Derivatives of A, see Figure 5 [35]), these hybrid compounds showed a low cytotoxic profile with IC₅₀ values between 25 and 100 μM. The substitution of cyclopropane with isoxazoline (Derivatives of B, [36]) in another study revealed a slight decrease in cytotoxic values. This implies that the presence of various types of heteroatoms such as oxygen and nitrogen on the isoxazoline nucleus impacts the death of cancer cells more significantly.

Figure 5.

The analysis of structure–activity relationship issued from of the set of internal reactions on the (R)-Carvone monoterpene. Modified with permission from [32].

In the course of our structural modifications, we focused on omitting the thiadiazole nucleus and instead retained only the isoxazoline nucleus to evaluate the specific cytotoxic effect of isoxazole alone. This strategy led to the synthesis of derivatives of type C [37]. However, the results of our investigation revealed a low anticancer potential against HT-1080, A-549, MCF-7 and MDA-MB-231 cells when assessing these compounds. Subsequently, in the same study, we introduced the pyrazole function onto the double endo-cyclic bond to generate pyrazole–isoxazole type hybrids, referred to as derivatives of type D [38].

However, their in vitro cytotoxicity test revealed an inactive anticancer potential. The omission of the isoxazole and cyclopropane nucleus, replacing it with the pyrazole nucleus attached to the double endo-cyclic bond and introducing a new heterocycle nucleus of type 1,2,3-triazoles outside the cyclohexanone of (R)-Carvone, while maintaining the free double vinyl bond (Derivatives of E, [38]), has resulted in a significant enhancement in anticancer activity. This modification prompted us to ask a question about the grouping likely to be responsible for this activity, and according to the structural analysis of all these derivatives, it turned out that, the two double bonds are the elements responsible for this activity. That is why we decided in the other works to preserve the two double bonds of the monoterpene skeleton. The alteration of the position of the 1,2,3-triazole through a condensation reaction with oxime resulted in a profile of moderate anticancer activity, signifying a decline in anticancer efficacy (Derivatives of F [39], Figure 5). The incorporation of thiazolidinone as a bridging function for 1,2,3-triazole demonstrated notable anticancer activity, with IC₅₀ values reaching 10 μM (Derivatives of J, [40]). For the continuation, maintaining the thiazolidinone in its original position while altering the position of the 1,2,3-triazole yielded a highly significant cytotoxic profile in another study involving the same cancer cell lines, with IC₅₀ values reaching 15 μM (Derivatives of H, [41]). The comparison of the anticancer activity of the J and H derivatives synthesized previously with the derivatives presented in this work is very different because these 6a-h derivatives have been characterized by a very significant anticancer activity with IC₅₀ values around 13 and 25 μM in the majority of compounds. The free position of the vinylic double bond of (R)-Carvone is likely responsible for creating an environment rich in interactions with the biological target. Furthermore, this SAR study with our previous work have shown that the insertion of benzaldehyde also has a positive effect on biological activity.

3.4. Annexin-V/7-AAD staining to detect apoptosis

The process of controlled cell death, referred to as apoptosis, presents significant potential as a focal point for the development of innovative anticancer medications [42]. Apoptosis, or programmed cell death, hinges on a set of specialized enzymes such as cysteine-aspartic proteases, ultimately orchestrating the demise of cancer cells [43]. Caspases-3 and -7 serve as pivotal executors of apoptosis, with their precise enzymatic activities ensuring the irreversible destruction of cancerous cells. The identification of elevated expression levels of pro-caspase-3 in various cancers unveils new opportunities for therapeutic intervention, potentially through targeting its activation or downstream pathways [44]. Medications that activate this crucial enzyme deliver a targeted blow against cancer cells, prompting their programmed demise [45]. The hunt for the apoptotic trigger in compounds 6f and 6g led to a 24-h showdown in HT-1080 and A-549 cells, each challenged with 20 μM of the respective compound. Flow cytometry, equipped with annexin-V staining, meticulously analysed the cellular landscape, probing for the signs of programmed cell death. Compounds 6f and 6g emerged as the most potent inducers of apoptosis, exerting their effects on both HT-1080 and A-549 cells. Supplementary Figure S2 illustrates a notable increase in annexin-V staining, underscoring their efficacy. Compound 6f triggered a 16-fold increase in apoptotic cell numbers in HT-1080 cells and a sevenfold increase in A-549 cells compared with untreated cells (Supplementary Figure S2). Apoptotic cells surged from 1.35 to 21.90% in HT-1080 and from 4.02 to 27% in A-549 cells. Similarly, for HT-1080 cells, compound 6g demonstrated an approximately eightfold increase in total apoptosis (from 1.35 to 11.34%) compared with untreated cancer cells. Though slightly less pronounced in A-549 cells (from 4% to 21%), this still signifies a fivefold increase on average. Compounds 6f and 6g seem to target the early phase of apoptosis, as evidenced by the substantial rise in annexin-V staining, indicating early phosphatidylserine exposure and suggesting their potential to initiate programmed cell death.

3.5. Caspase-3/-7 activity assay

We turned to the spotlight of caspase-3/7 activation to dissect the machinery of cell death induced by compounds 6f and 6g in HT-1080 and A-549 cells. Could these executioner enzymes be the hidden weapons behind their anti-proliferative effect?

HT-1080 and A-549 cells, representing two distinct cancer cell lines, were exposed to a 20 μM dose of compounds 6f and 6g for 24 h so as to evaluate their caspase-3/-7 activation potential. Supplementary Figure S3 condenses the results of this experiment. The treatment with compounds 6f and 6g significantly heightened caspase-3/7 activation in both HT-1080 and A-549 cells compared with the control. While both cell lines responded positively to the caspase-3/7 activating prowess of compounds 6f and 6g, HT-1080 proved to be slightly more susceptible. Their control rate of 6.45% skyrocketed to 29.86 and 17.38% in the case of HT-1080, while A-549 cells have caused an increase from 5 to 23.73 and 18.68%.

3.6. Molecular docking studies

3.6.1. Molecular docking investigation of caspase inhibitors

To validate the efficacy of the molecular docking protocol, an exhaustive re-docking analysis was conducted on two specific inhibitors: 5-[4-(1-carboxymethyl-2-oxo-propylcarbamoyl)-benzylsulfamoyl]-2-hydroxy-benzoic acid (referred to as compound 161) and 2-(2,4-dichloro-phenoxy)-n-(2-mercapto-ethyl)-acetamide (denoted as NXN). These inhibitors were positioned within the active sites of the crystallised structures of caspase-3 and caspase-7. Upon re-docking into their respective binding sites, it was observed that both compound 161 and NXN formed molecular interactions with the same amino acid residues as delineated in the corresponding crystal structures. The root mean square deviation (RMSD) values, which quantify the spatial discrepancy between the crystallographic and re-docked conformations, were computed. The RMSD for all atoms in these comparative analyses were found to be 2.444 Å and 2.859 Å, with corresponding binding energies of -8.71 and -5.560 kcal/mol, respectively. These findings substantiate the precision and accuracy of the implemented molecular docking protocol, affirming its capability to reproduce the crystallographic complex with a remarkable degree of fidelity. Additionally, the spatial orientations of the inhibitors in both the crystallized and re-docked states are depicted in Supplementary Figure S4, providing visual confirmation of the congruence between the computationally predicted and experimentally determined molecular configurations. This comprehensive approach underscores the reliability of the docking simulations in replicating the actual biochemical interactions within the enzyme's active site [42,43].

3.7. In-depth molecular docking study of caspase-3 & caspase-7 inhibitors

This research employed molecular docking simulations to investigate the potential binding modes of selected compounds within the active sites of caspase-3 and caspase-7. The binding modes were identified and ranked based on their binding free energy, revealing that compounds 6f and 6g exhibited superior affinity and biological activity in both the binding and allosteric sites of caspase-3 and caspase-7, respectively [46,47]. These compounds demonstrated notable binding free energies within the active site of caspase-3 (-9.20 kcal/mol for 6f and -9.30 kcal/mol for 6g), surpassing the binding efficacy of the co-crystallised ligand inhibitor (161) of caspase-3, which exhibited a binding energy of -8.71 kcal/mol. The binding efficacies of compounds 6f and 6g were comparable to their co-crystalline ligand counterparts. Detailed binding scores against caspase-3 for all studied compounds are delineated in Table 2, highlighting primary interactions with nine amino acid residues, including His121, Arg207, Asn208, Trp214, Trp206, Phe256, Cys163 and Tyr204. Notably, the selectivity of these compounds appeared to be influenced by three hydrophobic residues in the pocket of caspases-3 and -7: Tyr204, Trp206 and Phe256. Arg207 and Trp204, prominent in the caspase-3 structure, played a significant role in positioning these compounds within the enzyme's active site Supplementary Figure S5. The interactions were predominantly characterized by hydrogen bonds and hydrophobic interactions, confirming the efficacy of compounds 6f and 6g as potent inhibitors of caspase-3 [48,49].

Table 2.

Summary of molecular docking outcomes with caspase-3 enzyme (Protein Data Bank ID: 1NMS).

Compound	Ligand	Receptor	Interaction	Distance	E (kcal/mol)
6f	N 2	O Arg207 (A)	H-donor	2.32	-8.12
	O	OH Tyr204 (A)	H-acceptor	2.99
	Cl 41	OH Ser120 (A)	Halogen acceptor	3.09
	C 17	O Arg207 (A)	H-donor	3.68
	6-ring	NH Arg207 (A)	Pi–cation	3.42
6g	S6	NH Arg207 (A)	Hydrogen bond	2.77	-9.30

Furthermore, in both compounds 6f and 6g, the thiazol-4(5H)-one ring was found to participate in hydrophobic interactions with two crucial residues, Tyr211 and Tyr223, within the active site of caspase-7. This study also encompassed the synthesis of novel reversible allosteric inhibitors targeting human caspase-3/7, with a particular emphasis on the allosteric exosite A at the dimerization interface to identify compounds capable of inhibiting enzyme function [50]. In vitro functional assays indicated that compounds 6f and 6g were notably potent among the synthesized molecules. These compounds share common structural features, including multiple aromatic rings, a carboxylate group and hydrogen bond donors, suggesting that π•••π stacking interactions with Tyr211 and Tyr223, alongside salt-bridge and polar interactions with Glu147, Glu146 and Cys290 at exosite A, are conducive to ligand binding [51]. The synthesized compounds with multiple aromatic rings, a carbonyl group of amide group and hydrogen bond donors share common structural features that contribute to their ligand binding capabilities. These features include π•••π stacking interactions with Tyr211 and Tyr223, as well as interactions with Glu147, Glu146 and Cys290 in active sites. The presence of multiple aromatic rings in these compounds allows for π•••π stacking interactions. This type of interaction occurs between the π–electron clouds of the aromatic rings, resulting in a stabilising force that enhances ligand binding Table 3 & Supplementary Figure S6 [51].

Table 3. .

Summary of molecular docking outcomes with caspase-7 enzyme (Protein Data Bank ID: 1SHJ).

Compound	Ligand	Receptor	Interaction	Distance	E (kcal/mol)
6f	O 7	SG Cys290 (A)	H-donor	3.33	-9.61
	N H 8	O Glu146 (A)	H-donor	2.692
	C 24	OE2 Glu147 (A)	H-donor	3.28
	N 8	OE2 Glu147 (A)	Ionic	3.86
	N H 3	OH Tyr211 (A)	1.77302	3.86
	N H 4	OH Tyr211 (A)	2.86552	3.99
	O-H	OG1 Thr225 (A)	2.85844	3.35
6g	N 8	SG Cys290 (A)	H-donor	2.392	-8.85
	N 14	OE2 Glu147 (A)	H-donor	1.889
	C 30	O Ile288 (A)	H-donor	3.22
	CL	CD Pro214 (A)	H-donor	3.055
	5-ring	OH Tyr211 (A)	pi-H	3.22

Overall, the combination of π•••π stacking interactions, interactions and polar interactions with specific amino acid residues creates a favourable environment for ligand binding in compounds with multiple aromatic rings, a carboxylate group and hydrogen bond donors. For compounds 6f and 6g, the inhibition mechanism was elucidated using the crystal structures of caspase-7 bound with NXN. These compounds exhibited well-ordered binding within the central cavity of caspase-7. Specifically, compound 6f engaged in hydrogen bonds with Cys290, Glu146, Glu147, Tyr211 and Thr225. In contrast, compound 6g adopted a planar, edge-to-edge arrangement within the cavity, forming four intermolecular hydrogen bonds with Cys290, Glu147, Ile288 and Pro214. Notably, the interaction of compound 6g was exclusive to chain A, primarily with Cys290 and Glu147. The distinct and well-defined binding within the central cavity, covered by the L2′ loops in contact with either compound, suggests a consistent and targeted inhibition mechanism, as evidenced by the protein conformations bound with both compounds 6g and 6f.

4. Conclusion

In the quest for biologically active heterocycles, we leveraged the Huisgen cycloaddition reaction to merge (R)-carvone, an easily accessible natural product, with thiazolidinones, yielding novel 1,2,3-triazole fused structures. Following meticulous purification, the products of the Huisgen cycloaddition underwent thorough analysis via NMR and HRMS. Among the synthesized derivatives (intermediaries 1–5 and hybrids 6a–h), compound 6g emerged as the most potent cytotoxic agent, displaying an IC₅₀ value of 16.62 ± 1.06 μM against HT-1080 cells. Although the average IC₅₀ for the derivatives against the four cancer cell lines reached 13.15 ± 1.82 μM, suggesting significant overall activity compared with the Doxorubicine reference. Notably, ortho-methyl-phenyl-1,2,3-triazole (6f) and ortho-methyl-para-chloro-phenyl-1,2,3-triazole (6g) exhibited the best performance against HT-1080 cells, with IC₅₀ values three-times higher than the positive control (5.76 ± 1.98 μM).

This molecular docking study delved into the binding modes and inhibitory potential of compounds within caspase-3 and caspase-7. Compounds 6f and 6g showcased robust affinity and biological activity in both binding and allosteric sites, surpassing the co-crystallised ligand inhibitor. Their interactions with specific amino acid residues involved hydrogen bonds and hydrophobic interactions, underscoring the potency of these compounds as caspase inhibitors. Common structural features, such as multiple aromatic rings and hydrogen bond donors, facilitated binding interactions. The investigation also unveiled well-defined inhibition mechanisms for compounds 6f and 6g within the central cavity of caspase-7.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2351287

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.