Abstract As a top-three cancer in global incidence and mortality, colorectal cancer (CRC) urgently demands novel treatments. β-Caryophyllene (β-CP) and its derivatives, a class of sesquiterpenoids with broad anticancer potential, were structurally optimized in this study to enhance efficacy against CRC. Among the synthesized derivatives, AC-7 exhibited potent cytotoxicity and selectivity in HT-29 cells (IC[50] = 3.09 μM, SI = 6.1), comparable to 5-fluorouracil (5-FU, IC[50] = 3.63 μM, SI = 0.4). Network pharmacology and gene enrichment analyses indicated that apoptosis, autophagy, ROS, and NF-κB were key downstream pathways of AC-7, which were later validated experimentally. AC-7 arrested the cell cycle in the G0/G1 phase, promoted autophagy and apoptosis. ROS were identified as having a central role in regulating these related pathways. In vivo studies revealed the significant antitumor and DNA damage activity of AC-7 in a nude mouse model. These findings suggest that AC-7 is a promising candidate for anti-CRC therapy, acting through the ROS-mediated apoptosis pathway. __________________________________________________________________ The β-CP derivative AC-7 exerted potent anti-tumor activity both in vitro and in vivo, suggesting that AC-7 is a promising candidate for anti-CRC therapy, acting through the ROS-mediated apoptosis pathway.[43] graphic file with name d4md00951g-ga.jpg Introduction Colorectal cancer (CRC) is a malignant tumor caused by the abnormal proliferation and malignant transformation of cells in the lining of the colon, ranking among the top three cancers worldwide in both incidence and mortality rates.^[44]1 Treatment options for CRC have traditionally included 5-fluorouracil (5-FU) based chemotherapy, and targeted therapies.^[45]2 However, the limited efficacy of chemotherapy and its adverse side effect result in cancer recurrence and poor outcomes. Moreover, only a small fraction of CRC patients benefit from targeted therapies due to the necessity of mutation matching and high costs. According to the World Health Organization, the incidence of CRC is expected to continue rising in the coming years, reflecting the urgent need for new anti-CRC drugs to achieve more effective treatment outcomes. Medicinal plants and their active compounds have long been recognized as productive sources for drug development, especially for anti-cancer drugs.^[46]3 β-Caryophyllene (β-CP), a natural bicyclic sesquiterpene found in the essential oils of plants like basil, cinnamon, black pepper and rosemary,^[47]4 has been approved as a food additive and flavoring agent.^[48]5 Numerous studies have demonstrated that β-CP and its natural analogues have antiproliferative effects on various human cancer cell lines, particularly colorectal cancer cells.^[49]6,7 Furthermore, β-CP has been found to potentiate the anticancer effects of paclitaxel in CRC and breast cancer cells.^[50]8 Recently, it was reported that β-CP induces apoptosis and inhibits angiogenesis in CRC and holds promise as a treatment option for tumors.^[51]9,10 However, β-CP's hydrophobic nature and relatively weak anticancer activity, with IC[50] values in the high micromolar range, limit its further development as a drug. Structural optimization is a crucial strategy to improve druggability, but few synthesized β-CP derivatives have been reported. Recently, β-caryolanol, a natural product derived from transannular cyclization of β-CP,^[52]11 has garnered our attention due to its structural novelty and stronger antiproliferative activity against CRC cells compared to β-CP. This discovery prompted further investigation into the anticancer effects of β-CP derivatives with a β-caryolanol scaffold. In this work, we designed and synthesized a series of novel β-CP derivatives, and studied their anti-CRC effects in vitro and in vivo and their molecular mechanism. This work provides new insights into the potential of β-CP derivatives as treatments for CRC, highlighting their novel role in modulating key cancer-related pathways. Results and discussion Chemistry It was previously reported that under acid-catalyzed conditions, β-CP can undergo transannular cyclization with nucleophiles, such as water, alcohol or phenol, to form β-caryolanol or its ether derivatives.^[53]12–15 The unique stereoscopic structure of the product was confirmed by various spectral and single crystal X-ray analyses, and the stereoscopic transformation mechanism of the transannular cyclization reaction was also proposed as shown in [54]Scheme 1.^[55]16–18 However, the bioactivity studies of β-caryolanol and its ether derivatives have rarely been reported.^[56]19 In addition, it is well known that the introduction of nitrogen-containing groups into drug design can be a useful strategy that has the potential to introduce new interactions with target proteins, such as hydrogen bonding or electrostatic interactions, thereby improving their pharmacological properties. Therefore, we designed and prepared a series of novel β-caryolanol ether derivatives, including phenol ethers AC-1–4, alkyl ethers AC-5–6 and aminoalkyl ethers AC-7–23 ([57]Fig. 1). After trying a variety of acids and reaction solvents, trifluoroacetic acid and dichloromethane were found to be the most ideal combination of reaction conditions. At 0–10 °C, most substrates can be converted into target products with good to excellent yields. Products AC-7–23 containing amino groups were obtained and used in the form of trifluoroacetate. Scheme 1. Acid-catalyzed reaction mechanism of β-CP to β-caryolanol ether. [58]Scheme 1 [59]Open in a new tab Fig. 1. Transannular cyclization reaction of β-CP and alcohol or phenol under acid-catalyzed conditions, and structures of β-CP derivatives (β-caryolanol and AC-1 to AC-23). Reaction conditions: TfOH (0.1 eq., 1.1 eq. if substrates contain an amino group), anhydrous DCM, 0–10 °C, 43–98%. AC-7–23 are trifluoromethanesulfonates, and TfOH and TfO^− have been omitted for clarity. [60]Fig. 1 [61]Open in a new tab Pharmacology β-CP derivatives inhibit cancer cell growth The antiproliferative effects of compounds AC-1–AC-23 and β-caryolanol were initially assessed on the human colon cancer cell HT-29 using CCK assay, and β-CP and 5-FU were used as control compounds. As shown in [62]Table 1, β-CP showed low antiproliferative activity with an IC[50] of 295.67 ± 17.93 μM, while β-caryolanol increased its activity by 3.8 times with an IC[50] of 77.49 ± 2.51 μM. In the absence of nitrogen-containing substituents, phenol ethers AC-1–AC-4 and alkyl ethers AC-5–AC-6 showed no significant antiproliferative activity at 100 μM. However, compounds AC-7–AC-23 with nitrogen-containing substituents inhibited cell growth significantly, and their IC[50] values were almost all less than 10 μM. Among these, AC-7 is the most potent, with an IC[50] of 3.09 ± 0.09 μM. Table 1. IC[50] values of AC-1–AC-23 and β-caryolanol determined in four human cancer cell lines (HT-29, HepG2, MCF-7 and A549) and the normal human colon mucosal epithelial cell line (NCM-460), with β-CP and 5-FU as the positive control. The selectivity index (SI) was calculated as the IC[50] ratio (SI = IC[50] (NCM-460)/IC[50] (HT-29)). IC[50] values listed as >20 indicate that no 50% inhibition was observed at the 20 μM dose, nor was maximum inhibition. Cmpd IC[50] (μM) SI HT-29 HepG2 MCF-7 A549 NCM-460 AC-7 3.09 ± 0.09 13.60 ± 0.69 >20 14.77 ± 3.21 18.85 ± 1.40 6.1 AC-8 4.42 ± 0.194 14.91 ± 1.21 >20 >20 22.57 ± 1.17 5.1 AC-9 4.12 ± 0.23 6.13 ± 0.27 9.56 ± 0.39 10.30 ± 0.69 12.53 ± 0.18 3.0 AC-10 6.23 ± 0.31 >20 >20 >20 24.23 ± 1.06 3.9 AC-11 4.94 ± 0.23 6.57 ± 0.54 8.15 ± 0.37 7.82 ± 0.15 10.62 ± 1.42 2.1 AC-12 6.19 ± 0.29 5.68 ± 0.16 9.52 ± 1.49 9.85 ± 0.44 11.25 ± 0.35 1.8 AC-13 4.23 ± 0.34 2.87 ± 0.04 4.44 ± 0.31 7.35 ± 0.33 3.28 ± 0.05 0.8 AC-14 3.20 ± 0.20 2.19 ± 0.04 13.48 ± 11.79 6.21 ± 0.24 3.92 ± 0.29 1.2 AC-15 5.04 ± 0.20 2.14 ± 0.07 4.14 ± 0.20 4.46 ± 0.16 2.04 ± 0.08 0.4 AC-16 4.31 ± 0.25 4.03 ± 0.08 4.91 ± 1.61 6.91 ± 0.35 1.68 ± 0.07 0.4 AC-17 4.54 ± 0.20 4.71 ± 0.43 14.27 ± 9.80 13.40 ± 1.35 5.73 ± 0.22 1.3 AC-18 10.33 ± 0.26 17.09 ± 2.24 >20 >20 15.09 ± 0.88 1.5 AC-19 5.16 ± 0.42 7.33 ± 0.23 12.90 ± 1.05 11.96 ± 0.76 20.59 ± 1.15 4.0 AC-20 6.47 ± 0.17 5.33 ± 0.72 7.19 ± 0.46 13.72 ± 1.08 10.01 ± 0.87 1.4 AC-21 5.30 ± 0.16 7.29 ± 0.44 11.57 ± 0.40 >20 13.85 ± 0.55 2.6 AC-22 5.84 ± 0.60 9.65 ± 0.76 10.70 ± 0.39 >20 17.07 ± 0.41 2.9 AC-23 >20 >20 >20 >20 14.45 ± 0.88 — β-CP 295.67 ± 17.93 292.71 ± 36.65 276.83 ± 46.50 205.08 ± 13.38 188.2 ± 2.6 0.6 β-Caryolanol 77.49 ± 2.51 100.03 ± 9.85 91.75 ± 4.83 110.53 ± 5.15 82.52 ± 4.11 1.1 5-FU 3.63 ± 0.61 1.12 ± 0.06 0.21 ± 0.02 2.24 ± 0.10 1.57 ± 0.07 0.4 [63]Open in a new tab These active compounds were also tested on three additional human cancer cell lines including HepG2, MCF-7 and A549, as well as the corresponding normal cell lines (normal human colon cell NCM-460, human hepatocytes LO2, human mammary epithelial cells MCF-10A and human bronchial epithelial cells BEAS-2B) to assess their selectivity. In each cancer cell line, compounds AC-7–AC-23 demonstrated markedly enhanced anti-proliferative activity compared to the parent compounds β-CP and β-caryolanol. Moreover, it's obvious that these compounds were generally more sensitive to HT-29 cells than to the other cells. The selectivity index (SI) was calculated as the IC[50] ratio (SI = IC[50] (normal)/IC[50] (cancer)), and the results are shown in [64]Tables 1 and S1.[65]† Compound AC-7 exhibited the highest SI value of 6.1, indicating its superior selectivity. Considering both activity and selectivity, AC-7 was selected for further pharmacological studies. Potential targets of AC-7 in treating CRC To predict the potential targets of AC-7 in treating CRC, the SuperPred database was used, identifying 213 targets. Additional CRC-related targets were collected from databases such as DisGeNET (758 targets), GeneCards (3857 targets), OMIM (497 targets), and PharmGKB (285 targets). After removing duplicates, 4698 unique CRC-related targets were obtained. Among these, 124 overlapping targets between AC-7 and CRC were identified as potential therapeutic targets for AC-7 in treating colorectal cancer ([66]Fig. 2A–C). Fig. 2. Identification of AC-7 targets in colorectal cancer and pathway enrichment analysis. (A) Venn diagram of AC-7 targets. (B) Venn diagram of colorectal cancer targets. (C) Venn diagram of potential targets for AC-7 for treating colorectal cancer. (D) Top 10 significantly enriched items in GO analysis. (E) Top 20 significantly enriched pathways in KEGG pathway enrichment analysis. [67]Fig. 2 [68]Open in a new tab GO enrichment analysis revealed that the 124 targets were primarily involved in biological processes such as phosphorylation, regulation of NF-κB transcription factor activity, ROS production, and apoptosis. In terms of cellular components, the targets were mainly associated with the formation of structures such as ficolin-1-rich granule lumen, the cell surface, chromosomes, telomeric regions, and membrane rafts. Molecular functions were mainly related to ATP binding and RNA polymerase II activity. KEGG pathway enrichment analysis suggested that AC-7 may interfere with CRC through pathways such as PI3K-Akt, mTOR and NF-κB ([69]Fig. 2D and E). Protein–protein interaction (PPI) network analysis revealed a significant correlation between the predicted targets of AC-7 and the ROS, NF-κB, PI3K-Akt, mTOR, apoptosis, and autophagy pathways. The PPI network comprised 123 nodes and 1019 edges, with an average degree value of 16.6. Targets with values greater than the median for degree centrality (DC > 12), betweenness centrality (BC > 0.0025), and closeness centrality (CC > 0.4792) were identified as the top 25 core targets. These mainly include NF-κB, PARP1, PIK3R1, KEAP1, CDK2 and CDK1, which were considered as the primary functional targets ([70]Fig. 3A–C). Fig. 3. Key target screening and molecular docking with AC-7. (A) PPI network of potential targets for AC-7 in treating CRC. (B) PPI network of AC-7-target-pathway interactions. (C) Key genes based on topological parameters. (D) Molecular docking of AC-7 and potential targets. [71]Fig. 3 [72]Open in a new tab In molecular docking, the more negative the binding energy of a receptor molecule, the more stable the docking conformation. Molecular docking was performed between the core target proteins NF-κB, KEAP1, PARP1 and PIK3R1 with the protonated form of AC-7. The results showed that the binding energies were −7.34 kJ mol^−1, −9.44 kJ mol^−1, −7.06 kJ mol^−1 and −8.67 kJ mol^−1 respectively, indicating good binding affinity between the target proteins and AC-7 ([73]Fig. 3D). AC-7 induced apoptosis in HT-29 cells via the extrinsic apoptosis signaling pathway Following 24 hours of treatment with AC-7, the morphology of HT-29 cells was significantly changed. Several typical features, such as shrinkage of cells and fragmentation into apoptotic bodies ([74]Fig. 4A), suggest that AC-7 induced apoptosis in HT-29 cells. To confirm this, we performed flow cytometry assays and found an increase in apoptosis rates from 14.41% to 38.93% with 0 μM to 10 μM of AC-7 ([75]Fig. 4B and C). Consistent with this, western blot analysis indicated a significant increase in apoptosis-related proteins, including cleaved caspase-3 and cleaved PARP1 ([76]Fig. 4D and E). Additionally, the effect of caspase inhibitor Z-VAD-FMK on preventing AC-7-induced cell death was examined. Pretreating with 50 μM Z-VAD-FMK for 8 hours, followed by treatment with 1 μM AC-7 for 72 hours, increased HT-29 cell viability from 42.98% to 53.13% ([77]Fig. 4F). Fig. 4. AC-7 induced apoptosis in HT-29 cells via the extrinsic apoptosis signaling pathway. (A) Morphology changes of HT-29 cells after treatment with AC-7 (5–100 μM) for 24 hours. (B and C) Flow cytometry analysis of apoptosis in HT-29 cells after treatment with AC-7 (0, 5, 10 μM) for 24 hours. (D and E) Western blot analysis of Bax, Bcl-2, FADD, cleaved caspase-8, caspase8, PARP1, cleaved PARP1, caspase-3 and cleaved caspase-3 protein expression in HT-29 cells after treatment with AC-7 (0, 5, 20, 50 μM) for 24 hours. (F) Cell viability of HT-29 cells pretreated with Z-VAD-FMK (50 μM) for 8 hours, followed by washing with PBS and subsequent incubation with or without 1 μM AC-7 for 72 hours. [78]Fig. 4 [79]Open in a new tab The mechanism by which AC-7 induced apoptosis in HT-29 cells was further investigated using western blot to analyze proteins involved in the intrinsic apoptosis and extrinsic apoptosis pathway. Mitochondrial proteins Bax and Bcl-2 showed no changes in expression after 24 hours of incubation with AC-7 ([80]Fig. 4D). However, the expressions of FADD, cleaved caspase-8 and cleaved caspase-3 were upregulated in a dose-dependent manner ([81]Fig. 4D and E), suggesting that AC-7 activates the extrinsic apoptosis signaling pathway in HT-29 cells. AC-7 induced cell cycle arrest at the G0/G1 phase and ROS production in HT-29 cells The effect of AC-7 on the cell cycle was analyzed using flow cytometry analysis and western blotting. The percentage of cells in the G0/G1 phase increased in a concentration-dependent manner after 24 hours of AC-7 treatment ([82]Fig. 5A and B), while the expression levels of CDK2 and cyclin E were downregulated ([83]Fig. 5C and D). Fig. 5. AC-7 induced HT-29 cell cycle arrest at the G0/G1 phase and ROS generation. (A and B) Flow cytometry analysis of HT-29 cell cycle distribution after treatment with AC-7 (0, 5, 10 μM) for 24 h. (C and D) Western blot analysis of CDK2 and cyclin E protein expression in HT-29 after treatment with AC-7 (0, 5, 20, 50 μM) for 24 h. (E and F) Detection of ROS using the DCFH-DA probe in HT-29 cells after incubation with AC-7 (0, 2.5, 10 μM) and NAC (0.25, 0.5 mM) for 24 h. (G) Cell viability of HT-29 cells pretreated with NAC (0.4 mM) for 8 h, followed by incubation with or without 1 μM AC-7 for 72 h. [84]Fig. 5 [85]Open in a new tab Excessive ROS can cause cell damage, leading to cell death, including apoptosis. To assess AC-7 induced oxidative stress in HT-29 cells, ROS generation was measured by confocal microscopy and flow cytometry with DCFH-DA as the probe. AC-7 significantly increased ROS generation by more than two times, which was quenched by ROS scavenger NAC ([86]Fig. 5E and F). Furthermore, the protective effect of NAC on AC-7 induced cell death was investigated. After 8 hours of pretreatment with 0.4 mM NAC, the viability of HT-29 cells treated with 1 μM AC-7 for 72 hours increased ([87]Fig. 5G). AC-7 induced apoptosis in HT-29 cells via the ROS-mediated NF-κB signaling pathway GO analysis indicated that NF-κB was involved in the signaling pathways induced by AC-7, which was confirmed by western blot analysis. The results demonstrated that the p-p65 levels significantly increased ([88]Fig. 6A and C). To determine whether the activation of apoptosis and NF-κB signaling pathways was ROS-dependent, HT-29 cells were treated with ROS scavenger NAC. NAC significantly reduced the levels of cleaved caspase-8, cleaved caspase-3 and p-p65, all of which were elevated by AC-7 ([89]Fig. 6B and C). Fig. 6. AC-7 induced apoptosis in HT-29 cells via ROS-mediated caspase-8/caspase-3 and NF-κB signaling pathways. (A and C) Western blot analysis of p-p65 expression in HT-29 cells after treatment with AC-7 (0, 5, 20, 50 μM) for 24 h. (B and C) Western blot analysis of cleaved caspase-8, cleaved caspase-3 and p-p65 protein expression in HT-29 cells after treatment with AC-7 (0, 50 μM) and NAC (0, 2 mM) for 24 h. [90]Fig. 6 [91]Open in a new tab AC-7 induced autophagy in HT-29 cells via the PI3K/Akt/mTOR signaling pathway The relationship between autophagy and cancer is complex, as autophagy can both promote tumor growth and metastasis, as well as inhibit tumorigenesis. To investigate the effect of AC-7 on autophagy, the expression of the autophagy-related protein p62 was analyzed. The results showed that p62 was significantly upregulated ([92]Fig. 7A and B). In cancer cells, autophagy is regulated by several signaling pathways, with the PI3K/Akt/mTOR pathway being the most classical, known for its ability to inhibit autophagy. The PI3K inhibitor, for example, LY294002,^[93]20 induces cell death with IC[50] = 25.92 ± 5.68 μM (Fig. S1[94]†). Western blot results demonstrated that AC-7 downregulated the expression of p-PI3K, p-Akt, and p-mTOR ([95]Fig. 7C and D). These data indicated that AC-7 induces autophagy in HT-29 cells via the PI3K/Akt/mTOR signaling pathway, consistent with the findings from network pharmacology analysis. Fig. 7. AC-7 inhibited autophagy in HT-29 cells via the PI3K/Akt/mTOR signaling pathway. (A and B) Western blot analysis of p62 protein expression in HT-29 after treatment with AC-7 (0, 5, 20, 50 μM) for 24 h. (C and D) Western blot analysis of p-PI3k, p-Akt, and p-mTOR protein expression in HT-29 after treatment with AC-7 (0, 5, 20, 50 μM) for 24 h. [96]Fig. 7 [97]Open in a new tab AC-7 suppressed HT-29 tumor growth in vivo To assess the in vivo antitumor effect of AC-7, a BALB/C nude mouse xenograft tumor model was established by subcutaneous injection of HT-29 cells. The mice were divided into four groups, with six mice in each group. Mice were administered vehicle, AC-7 (5 and 15 mg kg^−1), or 5-FU (10 mg kg^−1) by intraperitoneal injection daily. After 14 days of treatment, tumor volumes and weights in all AC-7-treated groups were significantly reduced compared to the vehicle-treated group, while the mice in all groups maintained normal weights throughout the treatment period ([98]Fig. 8A–D). Additionally, the TUNEL staining revealed that AC-7 induced significant DNA damage and apoptosis in tumor tissues ([99]Fig. 8E). Fig. 8. In vivo anti-tumor activity of AC-7 on the HT-29 xenograft mouse model. (A) Body weight changes and (B) tumor volume measurements following administration of AC-7. (C) Photographs of isolated tumors at the endpoint. (D) Tumor weights after treatment with AC-7 and 5-FU. (E) Representative TUNEL staining images after treatment with vehicle, 5-FU (10 mg kg^−1), and AC-7 (5, 15 mg kg^−1). (F) Representative H&E staining of HT-29 xenograft BALB/C nude mouse major organs. Images depict tumors from mice treated with vehicle, 5-FU (10 mg kg^−1), and AC-7 (5, 15 mg kg^−1), highlighting histopathological alterations following treatment. [100]Fig. 8 [101]Open in a new tab Further H&E analysis was performed to investigate the antitumor effect and potential pathological alterations in the heart, liver, spleen, lung, and kidney tissues of AC-7-treated mice. Major organs and tumor samples were collected from all groups after 14 days of treatment. H&E staining showed no significant pathological alterations compared to the control group, indicating that AC-7 did not cause detectable toxicity in these organs ([102]Fig. 8F). In contrast, H&E staining revealed significant necrosis in tumor tissues following AC-7 treatment. Discussion Many studies have demonstrated the anti-proliferative activity of β-CP toward numerous cancer cell lines.^[103]21 Dahham et al. found that β-CP appears to have a stronger inhibitory effect on colon cancer cells than other cancer cell lines.^[104]22 They also demonstrated the efficacy of β-CP against colon cancer in animal models.^[105]9 However, relatively few studies have focused on β-CP derivatives and their anti-cancer studies. As a widely used natural derivative of β-CP, it was found for the first time that β-caryolanol has stronger anti-CRC activity than β-CP in this study. We further developed a series of derivatives (AC-1–AC-23) based on the β-caryolanol skeleton. The structure–activity relationship (SAR) study showed that derivatives AC-7–AC-22 with amino substituents exhibited much stronger antiproliferative activity than AC-1–AC-6 without amino substituents, and appeared to be more sensitive to the CRC cell line HT-29 compared to other cancer cell lines. In addition, compounds containing primary, secondary or tertiary amine groups have similar high activity, only the quaternary ammonium compound AC-23 is almost inactive. These results suggested that the active β-CP derivatives are highly tolerant to the amino groups. Among these, AC-7 displayed the strongest inhibition of HT-29 cell proliferation, with an IC[50] of 3.09 ± 0.09 μM, comparable to the first-line CRC drug 5-FU (IC[50] 3.63 ± 0.61 μM). Additionally, AC-7 showed the highest selectivity (SI = 6.1), much higher than that of 5-FU (SI = 0.4), suggesting a potential safety profile. To explore the underlying mechanisms of AC-7 mediated CRC inhibition, we performed target and pathway enrichment analysis in silico. Network pharmacology analysis indicated that the potential targets were primarily involved in apoptosis, ROS, NF-κB, PI3K/Akt and mTOR pathways. Molecular docking results further demonstrated the potential interaction between AC-7 and NF-κB, KEAP1, PARP1, and PIK3R1. Apoptosis and cell cycle arrest are common mechanisms in tumor suppression. Apoptosis is a tightly regulated process of programmed cell death, and defects in this process can promote tumorigenesis. Morphological changes and flow cytometry results confirmed that AC-7 induced apoptosis in HT-29 cells. This apoptosis could be partially reversed by the caspase inhibitor Z-VAD-FMK, suggesting caspase involvement. Additionally, the upregulation of apoptosis-related proteins such as FADD, cleaved caspase-8, cleaved caspase-3, and cleaved PARP1, along with unaltered levels of mitochondrial proteins Bax and Bcl-2, indicated that AC-7 induces apoptosis via the extrinsic apoptotic pathway. Cell cycle arrest is another promising therapeutic strategy for cancer suppression. CDK2 is a key regulator of the cell cycle, controlling both G1/S and G2/M transitions, while cyclin E interacts with CDK2 to promote the G1/S transition. AC-7 significantly downregulated CDK2 and cyclin E, suggesting G0/G1 cell cycle arrest. Our findings showed that AC-7 suppresses HT-29 cell proliferation through G0/G1 phase arrest. Excessive ROS accumulation is well-known for inhibiting tumor growth and serving as a key mediator of apoptosis and cell cycle arrest. Our results demonstrated that intracellular ROS levels were significantly increased by AC-7. The ROS scavenger NAC quenched this increase and partially reversed AC-7-induced cell death. Additionally, TUNEL staining revealed DNA damage in tumor tissue after AC-7 treatment, which is consistent with the role of ROS in causing DNA damage,^[106]23 triggering cell cycle arrest, and ultimately leading to apoptosis when damage is irreparable.^[107]24 NF-κB is a critical transcription factor involved in tumor initiation, promotion, and progression. RelA (p65), the most important member of the NF-κB transcription factor family, regulates genes associated with inflammation, immune response, cell survival, and apoptosis. ROS can induce P65 phosphorylation through PKAc and promote the proteasomal degradation of IκBα. Although NF-κB is generally considered anti-apoptotic, it can promote apoptosis in response to cellular stress, such as hypoxia or DNA damage.^[108]25,26 Our results showed that AC-7 significantly upregulated p65 phosphorylation in HT-29 cells, and this effect was reversed by NAC, indicating that AC-7 activates the NF-κB pathway via ROS, leading to apoptosis. Autophagy, which provides biological materials and energy during cellular stress, plays a crucial role in tumorigenesis and tumor progression. ROS can inactivate the upstream tyrosine phosphatase of PI3K and PTEN, thus promoting autophagy. Our findings demonstrated that AC-7 suppressed the PI3K/Akt/mTOR pathway^[109]27 and upregulated p62 expression in CRC cells. During autophagy, p62 targets pro-caspase-8 oligomers to the autophagosomal surface, leading to caspase-8 activation,^[110]28 which is consistent with the observed upregulation of caspase-8 by AC-7. Therefore, AC-7 promotes autophagy in HT-29 cells via the PI3K/Akt/mTOR pathway. Using a BALB/C nude mouse xenograft tumor model, we confirmed that AC-7 significantly reduces CRC tumor growth in vivo. Tumor volume and weight were suppressed by AC-7, while body weight remained stable, and H&E staining revealed no pathological changes in major organs. In contrast, AC-7 treatment caused significant necrosis and DNA damage in tumor tissues. These results suggest that AC-7 may act as a novel ROS inducer for anti-CRC treatment. Conclusion In conclusion, our study focused on developing a novel, highly efficient anti-CRC compound derived from β-CP. SAR studies revealed that β-CP derivatives with amino substituents generally exhibit potent anticancer activity, with AC-7 showing the highest activity and selectivity against HT-29 cells. Additionally, this study provided insight into AC-7s mode of action using network pharmacology analysis, which revealed that AC-7 may bind to multiple cancer-associated targets and affect various signaling pathways. AC-7 induced G0/G1 cell cycle arrest of HT-29 cells, and significantly enhanced ROS accumulation, which modulated several cancer-inhibitory events, including DNA damage, apoptosis, autophagy, NF-κB pathway activation, and PI3K/Akt/mTOR pathway suppression, ultimately leading to CRC cell death ([111]Fig. 9). It is also worth noting that both the ROS scavenger NAC and the apoptosis inhibitor Z-VAD-FMK only partially reversed AC-7-induced cell death, suggesting additional underlying mechanisms. Altogether, these findings demonstrate that AC-7 may act as a promising anti-CRC agent through multiple mechanisms. Fig. 9. Summary of the anticancer mechanism of compound AC-7. This figure illustrates the multifaceted anticancer effects of AC-7, including induction of apoptosis, ROS accumulation, G0/G1 cell cycle arrest, activation of the NF-κB pathway, and suppression of the PI3K/Akt/mTOR signaling pathway, leading to enhanced cell death in CRC cells. [112]Fig. 9 [113]Open in a new tab Experimental Chemistry β-CP was purchased from Shanghai Bidepharm Technology Co., Ltd, China. All other reagents and solvents, including trifluoromethanesulfonic acid (TfOH), alcohols, phenols and dichloromethane (DCM) are commercially available and used without further purification. The transannular cyclization reaction of β-CP with nucleophilic substrates is as follows: β-CP (2.0–10.0 equivalents) and nucleophilic substrates (water, alcohols or phenols, 1.0 equivalent) were dissolved in anhydrous DCM and cooled to 0–10 °C, and TfOH (0.1 equivalent, 1.1 equivalents when substrates contain an amino group) was added as a catalyst. The resulting mixture was stirred at room temperature for 12 hours, then quenched with water and extracted with DCM. The organic solution was dried with anhydrous sodium sulfate. After removing the solvent, the crude product was purified by column chromatography on silica gel to provide the product AC. ^1H-/^13C-NMR and HRMS spectra of compound AC were detected. The purity of the compounds was more than 95% by HPLC. (1R,2S,5R,8S)-1-(4-Methoxyphenoxy)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodeca ne (AC-1) According to the general procedure, compound AC-1 could be obtained with 70% yield as a white solid using β-caryophyllene and 4-methoxyphenol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 6.88–6.81 (m, 2H), 6.81–6.74 (m, 2H), 3.78 (s, 3H), 2.46–2.34 (m, 1H), 2.07–1.97 (m, 1H), 1.89–1.74 (m, 4H), 1.73–1.65 (m, 2H), 1.58 (m, 2H), 1.38 (m, 3H), 1.21–1.15 (m, 1H), 1.13(d, J = 9.6 Hz, 1H), 1.05(s, 3H), 1.04 (s, 3H), 1.02–0.95 (m, 1H), 0.89 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 153.76, 147.91, 122.81, 112.64, 79.20, 54.49, 45.77, 44.20, 39.60, 36.19, 36.11, 35.64, 33.94, 33.76, 33.70, 32.40, 29.38, 21.12, 19.95, 19.86. 4-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)oxy)phenol (AC-2) According to the general procedure, compound AC-2 could be obtained with 70% yield as a white solid using β-caryophyllene and hydroquinone as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 6.77–6.72 (m, 2H), 6.71–6.65 (m, 2H), 2.42–2.32 (m, 1H), 2.04–1.95 (m, 1H), 1.84–1.76 (m, 2H), 1.72–1.65 (m, 3H), 1.54–1.48 (m, 3H), 1.36–1.32 (m, 3H), 1.17–1.12 (m, 1H), 1.12–1.07 (m, 2H), 1.02 (s, 3H), 1.01 (s, 3H), 0.86 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 151.18, 148.43, 124.16, 115.32, 80.41, 46.72, 45.16, 40.49, 37.22, 37.15, 36.58, 35.00, 34.76, 34.74, 33.44, 30.42, 22.09, 21.01, 20.91. (3-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)oxy)pheny l)methanol (AC-3) According to the general procedure, compound AC-3 could be obtained with 70% yield as a white solid using β-caryophyllene and 3-hydroxybenzyl alcohol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 7.16 (t, J = 7.9 Hz, 1H), 6.87 (m, 2H), 6.74–6.63 (m, 1H), 5.08 (s,1H), 4.44 (d, J = 12.1 Hz, 1H), 4.32 (d, J = 12.1 Hz, 1H), 2.28–2.18 (m, 1H), 1.91 (m, 1H), 1.87–1.77 (m, 2H), 1.75–1.71 (m, 2H), 1.64 (d, J = 9.6 Hz, 1H), 1.52 (ddd, J = 10.3, 6.1, 2.5 Hz, 2H), 1.45–1.37 (m, 2H), 1.37–1.25 (m, 2H), 1.21 (d, J = 12.8 Hz, 1H), 1.16–1.05 (m, 2H), 1.00 (s, 6H), 0.89 (d, J = 2.8 Hz, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 155.64, 142.21, 129.39, 119.15, 113.93, 113.85, 63.82, 46.93, 46.00, 40.41, 37.75, 37.69, 36.78, 35.28, 34.69, 34.60, 33.92, 30.50, 22.76, 20.95, 20.64. (1R,2S,5R,8S)-4,4,8-Trimethyl-1-(4-(trifluoromethyl)phenoxy)tricyclo[6.3.1.0^ 2,5]dodecane (AC-4) According to the general procedure, compound AC-4 could be obtained with 91% yield as a white solid using β-caryophyllene and 4-(trifluoromethyl)phenol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 8.00 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H), 4.55 (s, 1H), 2.40–2.24 (m, 1H), 1.78–1.68 (m, 4H), 1.63–1.58 (m, 1H), 1.58–1.53 (m, 2H), 1.49–1.40 (m, 2H), 1.38–1.26 (m, 4H), 1.18 (d, J = 12.8 Hz, 1H), 1.12–1.08(m, 1H), 1.02 (s, 3H), 0.99 (s, 3H), 0.80 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 147.80, 133.75 (d), 133.57, 127.22, 126.05 (q, J = 3.7 Hz), 59.66, 46.11, 45.16, 40.11, 37.33, 37.23, 36.94, 35.20, 34.45, 34.32, 33.56, 30.51, 21.92, 20.71, 20.02. (1R,2S,5R,8S)-4,4,8-Trimethyl-1-(2-(prop-2-yn-1-yloxy)ethoxy)tricyclo[6.3.1.0 ^2,5] dodecane (AC-5) According to the general procedure, compound AC-5 could be obtained with 70% yield as a white solid using β-caryophyllene and propynol ethoxylate as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 4.22 (d, J = 2.4 Hz, 2H), 3.64 (dd, J = 8.0, 3.2 Hz, 2H), 3.61–3.54 (m, 1H), 3.40 (dt, J = 10.2, 5.1 Hz, 1H), 2.41 (t, J = 2.4 Hz, 1H), 2.19–2.09 (m, 1H), 1.88–1.81 (m, 1H), 1.66–1.57 (m, 3H), 1.56–1.47 (m, 3H), 1.47–1.41 (m, 2H), 1.34–1.28 (m, 3H), 1.15–1.08 (m, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.87 (s, 3H).^13C NMR (101 MHz, CDCl[3]) δ 80.00, 76.00, 74.22, 69.87, 61.33, 58.54, 46.92, 45.89, 40.19, 37.69, 36.65, 35.19, 34.61, 34.35, 33.86, 30.42, 22.72, 20.90, 20.62. (1R,2S,5R,8S)-4,4,8-Trimethyl-1-(pent-4-yn-1-yloxy)tricyclo[6.3.1.0^2,5]dodec ane (AC-6) According to the general procedure, compound AC-6 could be obtained with 70% yield as a white solid using β-caryophyllene and 4-pentyn-1-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.39 (dt, J = 9.0, 6.6 Hz, 1H), 3.22 (dt, J = 9.0, 6.4 Hz, 1H), 2.19 (td, J = 7.1, 2.6 Hz, 2H), 2.14–2.02 (m, 1H), 1.85 (t, J = 2.7 Hz, 1H), 1.79–1.71 (m, 1H), 1.69–1.57 (m, 6H), 1.51–1.36 (m, 3H), 1.32–1.18 (m, 4H), 1.09–0.96 (m, 3H), 0.92 (s, 3H), 0.91 (s, 3H), 0.80 (s, 3H).^13C NMR (101 MHz, CDCl[3]) δ 84.46, 75.46, 68.17, 60.22, 46.73, 45.89, 40.31, 37.74, 36.50, 35.13, 34.63, 34.36, 33.95, 30.47, 29.61, 22.74, 20.92, 20.64, 15.38. (R)-1-Methyl-2-((((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1 -yl)oxy)methyl)pyrrolidine trifluoromethanesulfonate (AC-7) According to the general procedure, compound AC-7 could be obtained with 70% yield as a white solid using β-caryophyllene and (R)-(1-methylpyrrolidin-2-yl)methanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.98 (m, 1H), 3.80 (dd, J = 10.8, 9.2 Hz, 1H), 3.49 (dd, J = 10.8, 3.2 Hz, 1H), 3.40–3.28 (m, 1H), 3.09 (d, J = 5.0 Hz, 3H), 3.00–2.86 (m, 1H), 2.28–2.12 (m, 3H), 2.12–2.01 (m, 1H), 1.83–1.75 (m, 3H), 1.74–1.69 (m, 2H), 1.65 (d, J = 9.1 Hz, 1H), 1.63–1.59 (m, 1H), 1.53–1.43 (m, 3H), 1.41–1.34 (m, 2H), 1.32–1.28 (m, 1H), 1.26 (s, 1H), 1.17 (d, J = 12.9 Hz, 1H), 1.10–1.03 (m, 1H), 0.99 (s, 6H), 0.89 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 77.20, 69.90, 61.27, 58.05, 45.93, 45.91, 42.59, 40.06, 37.66, 37.40, 36.53, 35.38, 34.74, 33.80, 33.32, 30.53, 27.01, 22.63, 22.28, 20.80, 20.52. HRMS (ESI): m/z calculated for [M + H]^+: 320.2948, found 320.2740. (S)-1-Methyl-2-((((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1 -yl)oxy)methyl)pyrrolidine trifluoromethanesulfonate (AC-8) According to the general procedure, compound AC-8 could be obtained with 60% yield as a white solid using β-caryophyllene and (S)-(1-methylpyrrolidin-2-yl)methanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.85–3.72 (m, 1H), 3.72–3.61 (m, 2H), 3.42–3.27 (m, 1H), 3.17–2.99 (m, 1H), 2.98 (s, 3H), 2.27–2.11 (m, 3H), 2.12–1.94 (m, 2H), 1.82–1.69 (m, 3H), 1.67–1.55 (m, 3H), 1.51–1.43 (m, 3H), 1.43–1.28 (m, 4H), 1.18–1.10 (m, 2H), 1.09–1.02 (m, 1H), 0.99 (s, 3H), 0.98 (s, 3H), 0.89 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 76.92, 60.39, 57.65, 45.85, 44.95, 40.26, 37.56, 37.39, 36.07, 35.23, 34.85, 33.87, 33.22, 30.62, 29.71, 26.91, 22.50, 22.12, 20.82, 20.52. HRMS (ESI): m/z calculated for [M + H]^+: 320.2948, found 320.2743. 1-Methyl-3-((((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl) oxy)methyl)pyrrolidine trifluoromethanesulfonate (AC-9) According to the general procedure, compound AC-9 could be obtained with 68% yield as a white solid using β-caryophyllene and (1-methylpyrrolidin-3-yl)methanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.57–3.21 (m, 4H), 3.06 (brs, 1H), 2.89 (s, 3H), 2.70–2.59 (m, 1H), 2.23–2.05 (m, 2H), 2.00–1.83 (m, 1H), 1.75–1.66 (m, 1H), 1.66–1.58 (m, 2H), 1.57–1.50 (m, 2H), 1.45–1.36 (m, 2H), 1.35–1.27 (m, 2H), 1.28–1.19 (m, 3H), 1.10–0.95 (m, 3H), 0.92 (s, 3H), 0.91 (s, 3H), 0.81 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.31 (q, J = 318 Hz), 75.76, 75.72, 62.00, 61.81, 58.53, 58.36, 56.60, 56.46, 45.98, 45.88, 41.47, 41.43, 40.32, 40.30, 37.83, 37.73, 37.65, 37.60, 36.39, 36.35, 35.22, 34.73, 34.71, 33.95, 33.85, 30.63, 30.61, 29.67, 27.16, 27.05, 22.62, 20.84, 20.49. HRMS (ESI): m/z calculated for [M + H]^+ 320.2953, found 320.2813. 1-Methyl-2-((((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl) oxy)methyl)piperidine trifluoromethanesulfonate (AC-10) According to the general procedure, compound AC-10 could be obtained with 98% yield as a white solid using β-caryophyllene and (1-methylpiperidin-2-yl)methanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.76–3.68 (m, 1H), 3.56 (dd, J = 10.2, 4.9 Hz, 0.6H), 3.42 (dd, J = 10.2, 4.8 Hz, 0.4H), 3.25 (dd, J = 10.7, 3.3 Hz, 1H), 2.70 (s, 1.8H), 2.62 (s, 1.2H), 2.59–2.48 (m, 2H), 2.23–2.09 (m, 1H), 1.92–1.73 (m, 5H), 1.72–1.65 (m, 3H), 1.66–1.57 (m, 3H), 1.55–1.49 (m, 1H), 1.49–1.43 (m, 2H), 1.42–1.34 (m, 3H), 1.34–1.28 (m, 2H), 1.15–1.07 (m, 2H), 1.07–1.01 (m, 1H), 1.00–0.95 (m, 6H), 0.89–0.87 (m, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 76.49, 76.23, 65.74, 65.29, 63.57, 62.59, 57.06, 46.22, 46.22, 45.94, 45.87, 45.79, 40.34, 40.17, 37.74, 37.57, 36.51, 36.34, 35.25, 35.21, 34.74, 33.95, 33.82, 30.56, 30.53, 29.71, 28.12, 23.95, 23.75, 22.87, 22.70, 22.89, 20.90, 20.86, 20.58. HRMS (ESI): m/z calculated for [M + H]^+: 334.3110, found 334.2894. 1-Methyl-3-((((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl) oxy)methyl)piperidine trifluoromethanesulfonate (AC-11) According to the general procedure, compound AC-11 could be obtained with 55% yield as a white solid using β-caryophyllene and (1-methylpiperidin-3-yl)methanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 7.71 (s, 1H), 7.44 (s, 1H), 3.43–3.33 (m, 2H), 3.29 (dd, J = 9.3, 4.8 Hz, 0.5H), 3.21–3.13 (m, 1H), 3.08 (dd, J = 9.2, 5.8 Hz, 0.5H), 2.77 (dq, J = 43.8, 10.7 Hz, 2H), 2.07 (dd, J = 19.1, 11.0 Hz, 1H), 2.01–1.91 (m, 1H), 1.88–1.81 (m, 1H), 1.79–1.67 (m, 3H), 1.64–1.57 (m, 2H), 1.56–1.50 (m, 2H), 1.45–1.35 (m, 3H), 1.34–1.27 (m, 2H), 1.26–1.18 (m, 3H), 1.09–0.95 (m, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.80 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.31 (q, J = 318 Hz), 75.60, 75.52, 63.39, 63.32, 58.22, 47.87, 47.85, 45.93, 45.84, 45.04, 40.32, 40.26, 37.73, 37.69, 37.63, 36.22, 36.16, 35.16, 34.66, 34.58, 34.47, 33.98, 33.94, 33.75, 30.45, 30.42, 25.68, 22.69, 22.64, 21.78, 21.74, 20.84, 20.55, 18.23. HRMS (ESI): m/z calculated for [M + H]^+: 334.3110, found 334. 2943. 1-Methyl-4-((((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl) oxy)methyl)piperidine trifluoromethanesulfonate (AC-12) According to the general procedure, compound AC-12 could be obtained with 63% yield as a white solid using β-caryophyllene and (1-methylpiperidin-4-yl)methanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.47 (d, J = 10.1 Hz, 2H), 3.22–3.14 (m, 1H), 3.12–3.03 (m, 1H), 2.79 (s, 3H), 2.78–2.60 (m, 2H), 2.08 (td, J = 11.0, 7.9 Hz, 1H), 2.02–1.87 (m, 3H), 1.75–1.69 (m, 1H), 1.65–1.59 (m, 4H), 1.57–1.51(m, 3H), 1.45–1.36 (m, 3H), 1.26–1.23 (m, 2H), 1.08–1.03 (m, 1H), 1.02–0.96 (m, 2H), 0.91 (s, 3H), 0.91 (s, 3H), 0.81 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.20(q, J = 375.2 Hz), 75.53, 64.90, 46.28, 45.99, 40.35, 37.74, 37.65, 36.37, 35.19, 34.70, 34.05, 34.00, 30.56, 29.70, 22.71, 20.87, 20.56. HRMS (ESI): m/z calculated for [M + H]^+: 334.3110, found 334.2943. 3-((((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)oxy)methy l)piperidine trifluoromethanesulfonate (AC-13) According to the general procedure, compound AC-13 could be obtained with 55% yield as a white solid using β-caryophyllene and piperidin-3-ylmethanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 7.71 (s, 1H), 7.44 (s, 1H), 3.43–3.33 (m, 2H), 3.29 (dd, J = 9.3, 4.8 Hz, 0.5H), 3.21–3.13 (m, 1H), 3.08 (dd, J = 9.2, 5.8 Hz, 0.5H), 2.77 (dq, J = 43.8, 10.7 Hz, 2H), 2.07 (dd, J = 19.1, 11.0 Hz, 1H), 2.01–1.91 (m, 1H), 1.88–1.81 (m, 1H), 1.79–1.67 (m, 3H), 1.64–1.57 (m, 2H), 1.56–1.50 (m, 2H), 1.45–1.35 (m, 3H), 1.34–1.27 (m, 2H), 1.26–1.18 (m, 3H), 1.09–0.95 (m, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.80 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.31 (q, J = 318 Hz), 75.60, 75.52, 63.39, 63.32, 58.22, 47.87, 47.85, 45.93, 45.84, 45.04, 40.32, 40.26, 37.73, 37.69, 37.63, 36.22, 36.16, 35.16, 34.66, 34.58, 34.47, 33.98, 33.94, 33.75, 30.45, 30.42, 25.68, 22.69, 22.64, 21.78, 21.74, 20.84, 20.55, 18.23. HRMS (ESI): m/z calculated for [M + H]^+: 320.2953, found 320.2793. 4-((((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)oxy)methy l)piperidine trifluoromethanesulfonate (AC-14) According to the general procedure, compound AC-14 could be obtained with 56% yield as a white solid using β-caryophyllene and piperidin-4-ylmethanol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 7.46 (d, J = 60.0 Hz, 2H), 3.78 (s, 1H), 3.31 (s, 2H), 3.15 (s, 2H), 2.22–2.08 (m, 1H), 1.90 (s, 2H), 1.75–1.56 (m, 5H), 1.56–1.46 (m, 3H), 1.46–1.35 (m, 3H), 1.35–1.20 (m, 3H), 1.10–1.01 (m, 2H), 1.01–0.95 (m, 1H), 0.92 (s, 3H), 0.91 (s, 3H), 0.81 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.20(q, J = 375.2 Hz), 76.95, 60.61, 45.49, 45.41, 41.28, 41. 24, 40.27, 37.76, 37.45, 35.45, 35.03, 34.97, 34.55, 34.08, 30.63, 30.54, 30.36, 22.35, 21.06, 20.65. HRMS (ESI): m/z calculated for [M + H]^+: 320.2953, found 320.2796. (1S,2R)-2-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)ox y)cyclohexan-1-amine trifluoromethanesulfonate (AC-15) According to the general procedure, compound AC-15 could be obtained with 71% yield as a white solid using β-caryophyllene and (1R,2S)-2-aminocyclohexan-1-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 4.90 (brs, 3H), 3.85–3.75 (m, 1H), 3.30 (td, J = 6.8, 3.1 Hz, 1H), 2.19 (dd, J = 20.1, 10.6 Hz, 1H), 1.80–1.70 (m, 3H), 1.69–1.58 (m, 4H), 1.54 (d, J = 10.1 Hz, 1H), 1.49–1.36 (m, 7H), 1.32–1.21 (m, 5H), 1.19–1.16 (m, 2H), 1.07 (dd, J = 10.0, 7.1 Hz, 1H), 1.04–0.97 (m, 1H), 0.93 (s, 3H), 0.92 (s, 3H), 0.83 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 119.67 (q, J = 375.2 Hz), 77.27, 67.10, 65.17, 53.59, 45.29, 44.63, 40.52, 37.68, 37.41, 35.29, 35.12, 35.05, 34.59, 33.71, 30.88, 30.44, 29.71, 25.96, 22.21, 21.27, 20.64. HRMS (ESI): m/z calculated for [M + H]^+: 320.2953, found 320.2798. (1S,2S)-2-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)ox y)cyclohexan-1-amine trifluoromethanesulfonate (AC-16) According to the general procedure, compound AC-16 could be obtained with 67% yield as a white solid using β-caryophyllene and (1S,2S)-2-aminocyclohexan-1-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 5.40 (s, 3H), 3.46–3.31 (m, 1H), 2.82–2.71 (m, 1H), 2.19 (dd, J = 20.1, 10.3 Hz, 1H), 2.06 (d, J = 11.2 Hz, 1H), 1.96 (d, J = 9.6 Hz, 1H), 1.78–1.59 (m, 5H), 1.56–1.46 (m, 4H), 1.45–1.33 (m, 3H), 1.31–1.20 (m, 7H), 1.03 (d, J = 12.2 Hz, 2H), 0.91 (s, 6H), 0.82 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 119.67 (q, J = 375.2 Hz), 70.23, 67.08, 56.43, 45.55, 44.45, 39.45, 37.31, 36.49, 35.32, 34.90, 34.70, 34.06, 33.55, 33.00, 30.58, 29.70, 28.44, 23.93, 23.86, 21.41, 20.81. HRMS (ESI): m/z calculated for [M + H]^+: 320.2953, found 320.2812. (S)-3-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)oxy)py rrolidine trifluoromethanesulfonate (AC-17) According to the general procedure, compound AC-17 could be obtained with 63% yield as a white solid using β-caryophyllene and (S)-pyrrolidin-3-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 7.00 (brs, 2H), 3.58–3.39 (m, 1H), 3.37–3.21 (m, 2H), 2.71 (d, J = 9.0 Hz, 3H), 2.15–2.02 (m, 1H), 1.79–1.70 (m, 1H), 1.65 (d, J = 7.9 Hz, 1H), 1.59–1.52 (m, 2H), 1.46–1.36 (m, 3H), 1.34–1.19 (m, 7H), 1.10–1.02 (m, 2H), 1.02–0.95 (m, 1H), 0.92 (s, 3H), 0.90 (s, 3H), 0.81 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.31 (q, J = 318 Hz), 76.80, 76.72, 61.42, 60.78, 56.15, 46.02–45.83, 45.82, 45.70, 45.44, 40.28, 40.22, 37.78, 37.50, 36.28, 36.14, 35.25, 35.19, 34.71, 34.67, 33.91, 33.87, 33.69, 33.68, 31.12, 31.11, 30.38, 30.35, 22.70, 20.87, 22.83, 20.45, 13.82, 13.52. HRMS (ESI): m/z calculated for [M + H]^+: 292.2640, found 292.2494. (R)-3-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)oxy)py rrolidine trifluoromethanesulfonate (AC-18) According to the general procedure, compound AC-18 could be obtained with 56% yield as a white solid using β-caryophyllene and (R)-pyrrolidin-3-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 7.92 (d, J = 21.7 Hz, 2H), 4.19 (s, 1H), 3.35 (s, 3H), 3.13 (s, 1H), 2.16–2.01 (m, 2H), 1.95 (s, 1H), 1.74–1.68 (m, 1H), 1.62–1.50 (m, 4H), 1.44–1.35 (m, 3H), 1.33–1.23 (m, 4H), 1.08–1.02 (m, 1H), 0.99 (d, J = 12.9 Hz, 2H), 0.92 (s, 3H), 0.91 (s, 3H), 0.80 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.31 (q, J = 318 Hz), 77.14, 68.62, 52.22, 45.50, 44.87, 43.62, 39.05, 36.93, 36.77, 35.46, 34.53, 34.13, 33.80, 33.17, 31.80, 29.37, 28.68, 21.80, 20.16, 19.47. HRMS (ESI): m/z calculated for [M + H]^+: 292.2640, found 292.2497. (S)-1-methyl-3-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1- yl)oxy)pyrrolidine trifluoromethanesulfonate (AC-19) According to the general procedure, compound AC-19 could be obtained with 72% yield as a white solid using β-caryophyllene and (S)-1-methylpyrrolidin-3-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 4.32–4.23 (m, 1H), 3.74 (dd, J = 12.1, 5.3 Hz, 1H), 3.64 (t, J = 8.0 Hz, 1H), 3.23–3.10 (m, 1H), 2.91 (s, 3H), 2.91–2.83(s, 1H), 2.27–2.16 (m, 1H), 2.11 (td, J = 11.1, 7.8 Hz, 1H), 2.03–1.93 (m, 1H), 1.70–1.60 (m, 4H), 1.59–1.51 (m, 2H), 1.47–1.37 (m, 2H), 1.36–1.31 (m, 1H), 1.27–1.21 (m, 3H), 1.10–1.04 (m, 1H), 1.02–0.96 (m, 2H), 0.92 (s, 3H), 0.92 (s, 3H), 0.82 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.20 (q, J = 375.2 Hz), 78.52, 69.89, 63.13, 55.51, 46.49, 46.23, 42.96, 40.26, 38.08, 37.75, 36.72, 35.70, 35.21, 34.94, 34.51, 34.37, 30.58, 22.95, 21.13, 20.41. HRMS (ESI): m/z calculated for [M + H]^+: 306.2797, found 306.2641. 2-(((1R,2S,5R,8S)-4,4,8-Trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)oxy)ethan- 1-amine trifluoromethanesulfonate (AC-20) According to the general procedure, compound AC-20 could be obtained with 51% yield as a white solid using β-caryophyllene and 2-aminoethan-1-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 6.19 (s, 3H), 3.54 (dt, J = 10.8, 5.4 Hz, 1H), 3.46–3.30 (m, 1H), 3.16–3.02 (m, 2H), 2.08 (td, J = 11.1, 7.9 Hz, 1H), 1.79–1.69 (m, 1H), 1.65–1.54 (m, 4H), 1.47–1.34 (m, 3H), 1.34–1.26 (m, 2H), 1.26–1.19 (m, 2H), 1.10–0.95 (m, 3H), 0.92 (s, 3H), 0.91 (s, 3H), 0.80 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.20(q, J = 375.2 Hz), 76.85, 56.77, 45.84, 45.81, 41.03, 40.19, 37.78, 37.52, 36.24, 35.26, 34.68, 33.90, 33.86, 30.27, 22.72, 20.87, 20.52. HRMS (ESI): m/z calculated for [M + H]^+: 266.2484, found 266.2363. N-Methyl-1-(((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1-yl)o xy)propan-2-amine trifluoromethanesulfonate (AC-21) According to the general procedure, compound AC-21 could be obtained with 49% yield as a white solid using β-caryophyllene and 2-(methylamino)propan-1-ol as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 7.00 (brs, 2H), 3.58–3.39 (m, 1H), 3.37–3.21 (m, 2H), 2.71 (d, J = 9.0 Hz, 3H), 2.15–2.02 (m, 1H), 1.79–1.70 (m, 1H), 1.65 (d, J = 7.9 Hz, 1H), 1.59–1.52 (m, 2H), 1.46–1.36 (m, 3H), 1.34–1.19 (m, 7H), 1.10–1.02 (m, 2H), 1.02–0.95 (m, 1H), 0.92 (s, 3H), 0.90 (s, 3H), 0.81 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.31 (q, J = 318 Hz), 76.80, 76.72, 61.42, 60.78, 56.15, 45.83, 45.82, 45.70, 45.44, 40.28, 40.22, 37.78, 37.50, 36.28, 36.14, 35.25, 35.19, 34.71, 34.67, 33.91, 33.87, 33.69, 33.68, 31.12, 31.11, 30.38, 30.35, 22.70, 20.87, 22.83, 20.45, 13.82, 13.52. HRMS (ESI): m/z calculated for [M]^+: 294.2797, found 294.2646. N,N-Dimethyl-1-(((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan-1- yl)oxy) propan-2-amine trifluoromethanesulfonate (AC-22) According to the general procedure, compound AC-22 could be obtained with 49% yield as a white solid using β-caryophyllene and choline hydroxide as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.70–3.63 (m, 0.5H), 3.51–3.35 (m, 2.5H), 2.85 (s, 6H), 2.11 (tt, J = 11.0, 7.4 Hz, 1H), 1.77–1.69 (m, 1H), 1.67–1.63 (m, 2H), 1.60–1.53 (m, 2H), 1.45–1.38 (m, 2H), 1.38–1.33 (m, 2H), 1.33–1.26 (m, 5H), 1.08–1.03 (m, 2H), 1.01–0.96 (m, 1H), 0.92 (m, 6H), 0.82 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.31 (q, J = 318 Hz), 62.18, 62.03,60.80, 60.62, 46.00, 45.72, 45.47, 40.21, 40.10, 37.70, 37.67, 37.48, 36.47, 36.36, 35.39, 35.30, 34.84, 34.79, 33.94, 33.92, 33.65, 33.50, 30.61, 30.54, 29.71, 22.65, 22.60, 20.86, 20.83, 20.45, 11.99, 11.84. HRMS (ESI): m/z calculated for [M + H]^+: 308.2953, found 308.2798. N,N,N-Trimethyl-2-(((1R,2S,5R,8S)-4,4,8-trimethyltricyclo[6.3.1.0^2,5]dodecan -1-yl)oxy)ethan-1-aminium trifluoromethanesulfonate (AC-23) According to the general procedure, compound AC-23 could be obtained with 43% yield as a white solid using β-caryophyllene and choline hydroxide as substrates. ^1H NMR (400 MHz, CDCl[3]) δ 3.76 (d, J = 7.1 Hz, 1H), 3.62–3.53 (m, 1H), 3.53–3.43 (m, 2H), 3.19 (s, 9H), 2.10 (td, J = 11.1, 7.9 Hz, 1H), 1.74–1.61 (m, 3H), 1.61–1.53 (m, 3H), 1.48–1.37 (m, 2H), 1.36–1.28 (m, 3H), 1.27–1.22 (m, 1H), 1.09–1.01 (m, 2H), 0.98 (dd, J = 8.7, 4.9 Hz, 1H), 0.92(s, 3H), 0.91(s, 3H), 0.81 (s, 3H). ^13C NMR (101 MHz, CDCl[3]) δ 120.60 (q, J = 318 Hz), 77.49, 66.65, 56.20, 54.45, 54.42, 54.39, 45.93, 45.64, 39.97, 37.61, 37.44, 36.38, 35.28, 34.80, 33.91, 33.46, 30.49, 22.56, 20.79, 20.47. HRMS (ESI): m/z calculated for [M + H]^+: 308.2948, found 308.2799. Biology Reagents and cell lines HT-29 (SCSP-5032), HepG2 (SCSP-510), MCF-7 (SCSP-531) and A549 (SCSP-503) were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). Normal cell line NCM-460 (iCell-h373) was obtained from Cellverse Co., Ltd (Shanghai, China). CCK-8 and Z-VAD-FMK were purchased from Meilunbio (Dalian, China). 5-FU was purchased from Yuanye Biotechnology (Shanghai, China). NAC, DCFH-DA, RIPA lysis buffer, protease inhibitors, phosphatase inhibitors, 4% paraformaldehyde fix solution, hematoxylin and eosin staining kit, western blocking buffer, enhanced BCA protein assay kit, SDS-PAGE gel kit, BeyoECL Plus kit, annexin V-FITC cell apoptosis detection kit and PI cell cycle detection kit were purchased from Beyotime Biotechnology (Shanghai, China). TBST buffer, glycine, Tris and SDS were purchased from Solarbio (Beijing, China). The primary antibodies were as follows: Bax (5023T, CST, American), Bcl-2 (4223T, CST, American), p-p65 (3033T, CST, American), p-65 (8242T, CST, American), FADD (2782T, CST, American), caspase-8 (4790T, CST, American), Cleaved caspase-8 (9496T, CST, American), caspase-3 (14220T, CST, American), Cleaved caspase-3 (9664T, CST, American), PARP1 (9532T, CST, American), Cleaved PARP1 (5625T, CST, American), p62 (5114T, CST, American), PI3K (4249T, CST, American), p-Akt (4060T, CST, American), Akt (4691T, CST, American), p-mTOR (5536T, CST, American), mTOR (2983T, CST, American), GAPDH (2118T, CST, American), CDK2 (10122-1-Ap, Proteintech, China), cyclin E (11554-1-Ap, Proteintech, China), calnexin (10427-1-Ap, Proteintech, China), tubulin (10094-1-Ap, Proteintech, China) and p-PI3K (YP0224, ImmunoWay, American). The secondary antibodies were as follows: HRP-labeled goat anti-rabbit and anti-mouse IgG (SA00001-2 and SA00001-1, Proteintech, China). Cell culture and anti-proliferation assay NCM-460 and A549 cells were cultured in RPMI 1640 medium, HT-29 cells were cultured in McCoy's 5A medium, HepG2 and MCF-7 cells were cultured in DMEM medium. All media were supplemented with 10% FBS and 1% penicillin–streptomycin, and cells were maintained in a 5% CO[2] incubator at 37 °C. HT-29 cells were digested and plated into 6-well plates (1 × 10^6 cells per well) and incubated overnight. Different concentrations of AC-7 were added to each well, and after 24 hours, cell morphology was observed using an inverted microscope (Leica, German). HT-29, HepG2, MCF-7, A549 and NCM-460 cells were plated into 96-well plates (4 × 10^3 cells per well) and incubated overnight. Different concentrations of 5-FU and β-CP derivatives were added to measure the antiproliferation effects. After 72 hours, old media were replaced with fresh media containing 10% CCK-8 (Meilunbio, China). After incubation for 1 hour, absorbance at 450 nm was measured. Cell survival rate (%) = OD of the dosing group/OD of the control group × 100%. IC[50]: the concentration of compounds that can cause 50% inhibition of cell viability. Determination of intracellular reactive oxygen species (ROS) HT-29 cells were seeded in 96-well plates (1 × 10^4 cells per well), treated with AC-7 and NAC (Beyotime, China) for 24 hours, and stained with DCFH-DA (Beyotime, China) to detect ROS, with green fluorescence observed using an inverted fluorescence microscope (Leica, Germany). HT-29 cells were seeded in 6-well plates (2 × 10^5 cells per well), then treated with AC-7 and NAC for 24 hours, and stained with DCFH-DA. After staining, the cells were detected and analyzed using flow cytometry. Cell apoptosis assay HT-29 cells were seeded in 6-well plates (2 × 10^5 cells per well) and incubated overnight. After treatment with AC-7 for 24 hours, cells were stained with annexin V-FITC and PI (Beyotime, China) for 20 min. After staining, the cells were detected and analyzed using flow cytometry. Cell cycle assay HT-29 cells were plated into 6-well plates (2 × 10^5 cells per well), treated with AC-7 for 24 hours, and fixed in 70% ethanol at 4 °C for 1 hour. Cells were then stained with PI and RNase (Beyotime, China) for 0.5 hours. After staining, the cells were detected and analyzed using flow cytometry. Z-VAD-FMK and NAC inhibition assay HT-29 cells were seeded into 96-well plates (4 × 10^3 cells per well) and pretreated with Z-VAD-FMK (Meilunbio, China) and NAC for 8 hours. Cells were then treated with AC-7, and viability was measured using the CCK-8 assay. Absorbance at 450 nm was recorded after 1 hour. Cell survival rate (%) = OD of the dosing group/OD of the control group × 100%. Western blot assay HT-29 cells were seeded into 6-well plates (1 × 10^6 cells per well) and incubated overnight. Cells were treated with AC-7 for 24 hours, lysed using RIPA lysis buffer (Beyotime, China) with protease and phosphatase inhibitors (Beyotime, China), and the supernatants were collected after centrifugation. Total protein was measured using the enhanced BCA protein assay kit (Beyotime, China). Equal amounts of protein were loaded onto SDS-PAGE gels, transferred to PVDF membranes, and blocked for 1 hour. Membranes were incubated with primary antibodies overnight at 4 °C, washed, and then incubated with secondary antibodies for 1 hour. Protein bands were visualized using the BeyoECL Plus kit (Beyotime, China) on a ChemiDoc XRS+ system (Bio-Rad, USA) and quantified using ImageJ (NIH, USA). Potential AC-7 targets in CRC treatment Potential targets of AC-7 were identified using the SuperPred database ([114]https://prediction.charite.de/), and standardized with corresponding gene names from the UniProt database ([115]https://www.uniprot.org/). CRC-related targets were gathered from DisGeNET ([116]https://www.disgenet.org/), GeneCards ([117]https://www.genecards.org/), OMIM ([118]https://omim.org/), and PharmGKB databases ([119]https://www.pharmgkb.org/). A Venn diagram was created using Venny 2.0 to identify overlapping genes between datasets, which were selected for further study. GO functional annotation and KEGG pathway enrichment analysis The DAVID database ([120]https://david.ncifcrf.gov/) was used to enrich the biological processes (BP), cellular components (CC), molecular functions (MF), and KEGG pathways of the intersecting targets. The top 10 significant BP, CC, and MF terms were visualized, along with the top 20 KEGG pathways. PPI network and screening of core targets Cytoscape (version 3.10.1) was employed to construct the interaction network of active compounds, target, pathway, and disease. The STRING database ([121]https://cn.string-db.org/) was used for PPI analysis with an interaction score above 0.4. Core targets were selected based on degree centrality (DC), closeness centrality (CC), and betweenness centrality (BC). Molecular docking 3D structures of core target proteins were downloaded from the PDB database ([122]https://www.rcsb.org/) and prepared using PyMOL and AutoDockTools. Ligand structures were obtained from PubChem and converted into mol2 format, and molecular docking was performed using AutoDock. The docking results were visualized using PyMOL and Discovery Studio 2019. Animals Balb/c mice (4–6 weeks old, female, 18–20 g) (Gempharmatech, China) were raised at constant temperature (23 ± 2 °C) on a 12-hour light–dark cycle, with free access to diet and water. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jiangsu Hanjiang Biotechnology Co., Ltd and approved by the Animal Ethics Committee of Jiangsu Hanjiang Biotechnology Co., Ltd, Ethical Approval No HJSW-22111501. Establishment and treatment of mouse tumor models 0.1 mL of HT-29 cell suspension in PBS was subcutaneously injected at a density of 5 × 10^7 cells per mL into the back of the left forelimb of mice. The positive control group was treated with 10 mg kg^−1 5-FU (Yuanye, China). The two drug treatment groups were treated with AC-7 at doses of 5 and 15 mg kg^−1, respectively. 5-FU or AC-7 solution (10% ethanol + 40% PEG400 + 50% normal saline) was administered intraperitoneally daily for 14 days. In addition, 6 normal mice were randomly selected as the normal group. Histopathology analysis The tumor, heart, liver, spleen, lung, and kidney tissues were fixed with 4% paraformaldehyde fix solution (Beyotime, China), embedded in paraffin and sliced. Slices were stained with the hematoxylin and eosin staining kit (HE) (Beyotime, China), and photographed using an Olympus microscope. Statistical analysis Statistical analysis was analyzed by one-way analysis of variance followed by the Dunnett test using GraphPad Prism software. Data were shown as average ± standard deviation. Independent experiments were performed at least three times. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 compared with the control (comparing all columns vs. control column). Abbreviations CRC Colorectal cancer β-CP β-Caryophyllene ROS Reactive oxygen species 5-FU 5-Fluorouracil NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells PI3K Phosphoinositide 3-kinase Akt Serine/threonine kinase mTOR Mammalian target of rapamycin DNA Deoxyribonucleic acid TfOH Trifluoromethanesulfonic acid LC-MS Liquid chromatograph-mass spectrometer FBS Fetal bovine serum CCK-8 Cell Counting Kit-8 FITC Fluorescein isothiocyanate PI Propidium iodide PARP1 Poly(ADP-ribose) polymerase 1 CDK2 Cyclin dependent kinase 2 HRP Horseradish peroxidase FACS Fluorescence activated cell sorter RIPA Radio immunoprecipitation assay BCA Bicinchoninic acid SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride DCFH-DA 2′,7′-Dichlorodihydrofluorescein diacetate GO Gene ontology KEGG Kyoto encyclopedia of genes and genomes H&E Hematoxylin and eosin TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling SI Selectivity index PPI Protein–protein interaction PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1 KEAP1 Kelch-like ECH-associated protein 1, CDK1, cyclin dependent kinase 1 Data availability All relevant data are within the manuscript and its additional files. Conflicts of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary Material MD-OLF-D4MD00951G-s001 [123]MD-OLF-D4MD00951G-s001.pdf^ (4.5MB, pdf) MD-OLF-D4MD00951G-s002 [124]MD-OLF-D4MD00951G-s002.pdf^ (7.5MB, pdf) ^† Electronic supplementary information (ESI) available. See DOI: [125]https://doi.org/10.1039/d4md00951g References