Design and synthesis of 1,2,4-oxadiazole-functionalized funtumine derivatives as potent anticancer agents

2.1. Instruments and chemicals

All reagents and solvents were commercially obtained and purified as needed. NMR spectra were recorded on a Bruker Avance Neo 400 MHz spectrometer (Bruker, Bremerhaven, Germany) using CDCl₃ and TMS. High-resolution mass spectrometry were obtained on a Waters Xevo G2-S QTOF mass spectrometer (Waters, Milford, MA, USA). Melting points were measured with a ZGX-4 micro melting point apparatus (Shanghai Zhuoguang Instrument Co., Ltd.).

2.2. General synthetic procedure of title compounds 3(a-p) and 4(a-p)

2.3. Biology assays

2.4. Transcriptome analysis

2.5. Molecular docking

The interaction between compound 4m and the target proteins cytochrome P450 1A2 (CYP1A2) and UDP glucuronosyltransferase family 2 member B11 (UGT2B11) in HepG2 cells was explored through a molecular docking study. The 3D structure of CYP1A2 was retrieved from the Protein Data Bank (https://www.rcsb.org, PDB: 2HI4), and the 3D structure of UGT2B11 was obtained from the AlphaFold protein structure database (https://alphafold.com/). Prior to docking, native ligands and water molecules were eliminated from the protein structure. Compound 4m was prepared by hydrogenation and energy minimization. Using AutoDock Tools 1.5.7, the proteins were processed by adding hydrogen atoms, calculating partial charges, and merging non-polar hydrogens, and the output was saved in PDBQT format. The active site of CYP1A2 and UGT2B11 were defined with the center coordinates (x, y, z) set to (5.436, 20.254, 21.733) and (-5.92, 0.089, -0.578). AutoDock Tools 1.5.7 facilitated the molecular docking process, and the resulting binding modes were visualized with Discovery Studio 2021.

2.6. Density functional theory calculations

Following the results of the biological activity screening, compound 4m, identified as the most potent, was chosen for theoretical calculations in accordance with established methodologies [24]. Gaussian 09 was used for density functional theory (DFT) calculations, utilizing the becke, 3-parameter, lee-yang-parr (B3LYP ) functional for geometry optimization and electronic property analysis. The highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), and MEP maps were derived and visualized using GaussView 6.0.

2.7. Data analyses

The compounds’ effects on cell proliferation were assessed with the formula: Inhibition rate (%) = (OD_negative − OD_experiment)/(OD_negative − OD_blank) × 100. Cell viability was calculated as: Viability (%) = (OD_experiment − OD_blank)/(OD_negative − OD_blank) × 100. Experiments were done in triplicate, with results shown as mean ± SD. Data were analyzed using SPSS version 21.0.

3.1. Chemistry

Compounds 3(a–p) and 4(a–p) were synthesized following the method outlined in Scheme 1. The key intermediates 2a–p were obtained in a two-step procedure starting from commercially available aromatic nitriles. First, various aromatic nitriles were reacted with hydroxylamine hydrochloride and sodium hydroxide to give the corresponding aromatic amidoximes (1a–p). Subsequent cyclization of 1(a–p) yielded 2-(chloromethyl)-5-substituted-1,2,4-oxadiazoles (2a–p). In the next step, funtumine was coupled with intermediates 2(a–p) using potassium carbonate and potassium iodide to give the target derivatives 3(a–p). Finally, reduction of compounds 3(a–p) with NaBH₄ afforded the title compounds 4(a–p). The synthesized derivatives underwent complete characterization through ¹H NMR, ¹³C NMR, and HRMS. Moreover, single-crystal X-ray diffraction was utilized for compounds 2j, 3a, and 4k to confirm their molecular structures (Figure 2 and Table S1). Spectral crystallographic and data are found in the Supporting Information.

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Scheme 1. Synthesis route of target compounds 3(a-p) and 4(a-p).

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Figure 2. X-ray crystal structure of compounds 3n, 4i and 4j.

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3.2. Anticancer activities and structure−activity relationships

The anticancer properties of the thirty-two target compounds were evaluated using the CCK-8 assay against HCT116, HeLa, and HepG2, with sorafenib and the lead compound funtumine as positive controls. As summarized in Table 1, the majority of synthesized derivatives exhibited excellent antiproliferative activities across the tested cancer cell lines. Notably, fifteen compounds demonstrated inhibition rates exceeding 70% against all three cancer lines, with derivatives 4g, 4l, and 4o showing particularly potent activity—close to or above 90% inhibition. In addition to broad-spectrum efficacy, several compounds displayed distinct selectivity profiles. For instance, 3p showed preferential cytotoxicity toward HeLa and HepG2 cells, while 3j was more active against HCT116 and HeLa. Similarly, 4c and 4h exhibited enhanced inhibitory effects on HCT116 and HepG2. An initial structure-activity relationship (SAR) analysis was performed to pinpoint key structural components influencing antitumor efficacy. Reduction of the C-20 carbonyl group in funtumine to a hydroxyl group significantly enhanced antiproliferative activity, as evidenced by the consistently superior performance of derivatives 4(a–p) over their carbonyl-containing counterparts 3(a–p). Furthermore, for the 4(a–p) series, incorporating electron-withdrawing groups into the 1,2,4-oxadiazole ring enhanced its potency. For instance, compounds 4(e–g), featuring electron-withdrawing groups, exhibited inhibition rates exceeding 80% across all three cancer cell lines, whereas derivatives 4(b–d), which contain electron-donating groups, showed rates below 80% against both HCT116 and HeLa cells. Interestingly, compared with our earlier studies on the antitumor activity of Funtumine acyl/sulfonyl derivatives, the 1,2,4-oxadiazole derivatives evaluated here generally exhibited significantly higher antitumor activity. This indicated that 1,2,4-oxadiazole, serving as a bioisostere of acyl/sulfonyl groups, represented an effective strategy for enhancing the biological activity of drug candidates. Notably, the incorporation of specific heterocyclic systems, such as furan and chloropyridine rings, yielded the most active analogues—compounds 4m and 4o demonstrated inhibition rates approaching or exceeding 90%. These findings underscore that strategic structural modification of the 1,2,4-oxadiazole moiety can markedly modulate anticancer activity.

Based on the promising results from initial screening, the compounds with the highest activity were chosen for additional assessment of their antiproliferative strength by measuring IC₅₀ values, as summarized in Figure 3. Among the tested derivatives, twenty-five compounds exhibited IC₅₀ values of 11.30 ∼ 53.91 μM against HCT116 cells. Against HeLa cells, twenty-four compounds showed IC₅₀ values between 11.27 and 31.58 μM. Twenty-six compounds showed increased effectiveness against HepG2 cells, with IC₅₀ values between 3.15 and 51.86 μM. In particular, compound 4m displayed the strongest inhibitory activities against all three cell lines, with IC₅₀ values of 25.33 μM (HCT116), 17.56 μM (HeLa), and 3.15 μM (HepG2). These values were lower than those of the lead compound funtumine (30.28, 36.06, and 33.45 μM, respectively) and comparable to those of the reference drug sorafenib (5.39, 7.34, and 11.55 μM, respectively). To investigate the potential safety aspects of the compounds, the cytotoxicity of the most potent derivative, 4m, was tested against the human normal cells (BEAS-2B). The results demonstrated low cytotoxicity with IC₅₀ values of 65.15 μM toward BEAS-2B cells (Figure S1). This suggested that compound 4m exhibited favorable selectivity toward cancer cells compared to normal cells.

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Figure 3. IC₅₀ values of target compounds against three human cancer cells.

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3.3. Cell apoptosis analysis

Building on the promising in vitro antiproliferative results, we further investigated the potential anticancer mechanism of this compound series. The derivative 4m, which showed the highest activity, was chosen to test its potential to trigger apoptosis in HepG2 cells through a TUNEL staining assay. Cells underwent treatment with either dimethyl sulfoxide (DMSO) as a control or 4m at 2, 4, and 8 μM for 48 h. The treated group exhibited a concentration-dependent increase in total apoptosis rates, reaching 45.2%, 75.0%, and 92.4%, respectively, compared to only 11.2% in the control group (Figure 4). These results demonstrated that the antiproliferative impact of compound 4m was facilitated by dose-dependent apoptosis induction.

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Figure 4. Effect of compound 4m on cell apoptosis in HepG2 cells. (*** p < 0.001).

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3.4. Transcriptome analysis

Transcriptomics provides a powerful approach for assessing global gene expression patterns in response to specific conditions or treatments over time, enabling the identification of differentially expressed genes associated with pathological changes, and has become an indispensable tool for elucidating drug mechanisms of action [25]. This research utilized transcriptome analysis to explore how compound 4m inhibits proliferation in HepG2 cells. Sequencing of six biological samples generated a total of 45.56 Gb of high-quality clean data after rigorous filtering. Each sample produced at least 6.77 Gb of data, with Q30 scores exceeding 94.10% (Table S2). The alignment rates of clean reads to the reference genome ranged from 95.77% to 97.23% (Table S3). PCA showed a distinct separation between the group treated with compound 4m and the control group, confirming the high quality and biological reproducibility of the transcriptome data and supporting its suitability for subsequent in-depth analyses (Figure 5a).

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Figure 5. Transcriptome and PPI network analysis in HepG2 cells treated by 4m. (a) PCA figure, (b) DEGs volcanic figure, (c) GO enrichment, (d) KEGG enrichment, (e) Topological network schematic of proteins targeted by 4m and associated with HepG2, (f) Constituent-target-pathway network of top 20 pathways.

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As depicted in Figure 5(b), analysis of DEGs identified 87 genes that were significantly altered in HepG2 cells following treatment with compound 4m, including 40 upregulated and 47 downregulated genes. These changes confirmed that 4m substantially influences the transcriptional profile of HepG2 cells. GO and KEGG pathway enrichment analyses were performed to further interpret the biological implications of these DEGs. GO analysis (Figure 5c) indicated that 4m affected biological processes like xenobiotic metabolic process, cellular response to xenobiotic stimulus, and oxidative demethylation, while molecular functions, including aromatase activity, steroid hydroxylase activity, and monooxygenase activity, were also disrupted. KEGG pathway enrichment analysis indicated that compound 4m influenced HepG2 cells through 110 significantly enriched pathways. The top 20 pathways, ranked by enrichment significance and gene count, are displayed in a bubble chart (Figure 5d). Among these, several pathways such as chemical carcinogenesis-DNA adducts, steroid hormone biosynthesis, and metabolism of xenobiotics by cytochrome P450, cytochrome P450-mediated xenobiotic metabolism were notably prominent. These results collectively suggested that compound 4m likely exerted its antiproliferative effects through multiple pathways, including interfering with the metabolic activation of carcinogens, disrupting hormonal homeostasis, and modulating cellular differentiation and proliferation signaling, as well as potentially reversing drug metabolism-related resistance.

3.5. PPI network analysis

To identify specific potential targets of compound 4m, high-confidence PPI data were retrieved using the STRING database based on the DEGs and their encoded proteins. After filtering out unconnected proteins, 27 overlapping targets common to both 4m and HepG2 cells were obtained. The PPI data obtained were loaded into Cytoscape to build an interaction network (Figure 5e). Among the nodes, five targets—CYP1A2, SERPINE1, early growth response 1(EGR1), CYP3A5, and UGT2B11—exhibited the highest number of interactions, suggesting their central role in the network. A comprehensive ingredient-target-pathway network was further established to visualize the relationships between 4m, the potential targets, and the top 20 enriched pathways (Figure 5f). Notably, highly connected targets such as UGT2B11, CYP1A2, and glutathione S-transferase alpha 2 (GSTA2) are likely to contribute significantly to the anti-HepG2 activity of 4m.

CYP1A2, part of the cytochrome P450 family, is crucial for metabolizing drugs, toxins, and hormones [26]. It suppresses proliferation and tumorigenicity in hepatocellular carcinoma (HCC) but is often downregulated in HCC tissues [27]. Its upregulation enhances the conversion of 17β-estradiol to cytotoxic 2-methoxyestradiol, inhibiting HCC cells. This highlights CYP1A2’s tumor-suppressive role in liver cancer [28]. Conversely, UGT2B11, a UDP-glucuronosyltransferase, aids in substrate elimination through glucuronidation and is linked to increased cancer cell proliferation, migration, and invasion [29-31]. Transcriptomic analysis showed that compound 4m treatment upregulated CYP1A2 and downregulated UGT2B11 in HepG2 cells. Overall, these results indicate that CYP1A2 and UGT2B11 represented the most promising candidate targets for the mechanism of action of compound 4m.

3.6. Molecular docking

Molecular docking, a computational method for predicting the binding interactions and affinities between small molecules and protein targets, is crucial in drug discovery. It is essential for target validation and aids in developing new anticancer agents [32,33]. This research involved molecular docking to confirm possible interactions between compound 4m and two major nodes from the PPI network, CYP1A2 and UGT2B11. The results demonstrated strong binding affinity between 4m and both targets, with binding energies of –11.87 kcal/mol for CYP1A2 and –11.14 kcal/mol for UGT2B11, supported by diverse interaction modes. As illustrated in Figure 6(a), 4m formed multiple stabilizing contacts with CYP1A2, including a hydrogen bond between its hydroxyl group and residue Hem 900, π–π stacking of the furan and oxadiazole rings with Phe226, and alkyl interactions involving the steroid nucleus and residues Ala317, Phe125, Leu382, Ile386, and Leu497. Meanwhile, as shown in Figure 6(b), 4m interacted with UGT2B11 via a hydrogen bond between its hydroxyl group and Ser311. The furan ring exhibited π–π T-shaped stacking with Phe397 and π-alkyl contacts with Leu116, Val153, Pro176, and Met226. The steroid nucleus also engaged in alkyl interactions with Val312, Val94, His374, Ile90, and Phe396. These results indicated that compound 4m bound to both CYP1A2 and UGT2B11 through multiple sites and various types of interactions, suggesting that their might serve as potential functional targets.

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Figure 6. Molecular models of 4m binding to (a) CYP1A2 and (b) UGT2B1.

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3.7. Density functional theory (DFT) calculations

HOMO and LUMO, derived from frontier molecular orbital theory, serve as fundamental quantum-chemical descriptors that provide valuable insights into a compound’s potential biological interactions [24]. The frontier orbital properties of compound 4m, including total energy, HOMO/LUMO energies, and the energy gap, were calculated using the B3LYP method. As summarized in Table 2 and visualized in Figure 7, the HOMO of 4m was mainly localized on the double bond of the furan and oxadiazole rings to provide electrons, whereas the LUMO was primarily distributed around the oxygen atom and the junction between the two ring systems to accept electrons. The computed energy gap was 0.18188 eV, indicating high chemical reactivity. Compounds with aromatic rings could be stabilized within the protein’s active pocket through weak interactions and hydrophobic interactions with specific residues. These weak interactions were essentially orbital interactions between the HOMO or LUMO of the substrate molecule and the HOMO or LUMO of the residues. The computed low energy gap of compound 4m indicates that the heterocyclic aromatic ring portion of the compound was prone to forming weak interactions with residues in the protein pocket, such as π- π stacked and π-π T-shaped interactions. This further corroborated the results obtained from molecular docking. The molecular electrostatic potential (MEP) map offers critical information on molecular charge distribution and is particularly useful for understanding recognition processes, such as those between a drug and its biological target [34]. As shown in Figure 7, regions of negative electrostatic potential (red) in 4m are concentrated on the oxygen atoms of the heterocyclic rings, while positive regions (blue) are mainly located around hydroxyl and nitrogen atoms in the structure. These electronic features support the ability of compound 4m to form strong and specific interactions with amino acid residues of target proteins.

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Figure 7. The HOMO and LUMO, MEP map of compound 4m.

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