Insights on Antitumor Activity and Mechanism of Natural Benzophenanthridine Alkaloids

Benzophenanthridine alkaloids are a class of isoquinoline compounds, which are widely found in the plants of papaveraceae, corydalis, and rutaceae. Biological activities and clinical studies have shown that benzophenanthridine alkaloids have inhibitory effects on many cancers. Considering that the anticancer activities and mechanisms of many natural benzophenanthridine alkaloids have been discovered in succession, the purpose of this paper is to review the anticancer effects of benzophenanthridine alkaloids and explore the application potential of these natural products in the development of antitumor drugs. A literature survey was carried out using Scopus, Pubmed, Reaxys, and Google Scholar databases. This review summarizes and analyzes the current status of research on the antitumor activity and antitumor mechanism of natural products of benzophenanthridine from different sources. The research progress of the antitumor activity of natural products of benzophenanthridine from 1983 to 2023 was reviewed. The antitumor activities of 90 natural products of benzophenanthridine and their related analogues were summarized, and the results directly or indirectly showed that natural products of benzophenanthridine had the effects of antidrug-resistant tumor cell lines, antitumor stem cells, and inducing ferroptosis. In conclusion, benzophenanthridine alkaloids have inhibitory effects on a variety of cancers and have the potential to counteract tumor resistance, and they have great application potential in the development of antitumor drugs.


Introduction
Tumor refers to a disease with the highest mortality rate.Under the action of a wide variety of factors, cells in local tissues lose their normal regulation of growth at the gene level, thus causing abnormal cell proliferation [1].Over 60% of anticancer drugs have been derived from natural products and natural product derivatives [2].
Figure 1 presents the structure of the benzophenanthridine compound, containing a non-aromatic heterocycle (B ring).It is primarily distributed in papaveraceae and rutaceae [3]; pertains to isoquinoline alkaloids; and exhibits antitumor, antifungal, antiviral, anti-inflammatory, immune regulation, and other pharmacological activities [4].Indeed, benzophenanthridine alkaloids play an anticancer role via different mechanisms.Benzophenanthridine alkaloids are capable of affecting the activity of DNA topoisomerase I and topoisomerase II, suppressing the rapid proliferation of tumor cells [5], inducing cancer cells ferroptosis [6], inhibiting the growth of tumor stem cells [7], and so forth.Benzophenanthridine natural products combine with negatively charged membrane surfaces and proteins, and react with sulfhydryl residues of amino acids, thus interfering with collagenase, tubulin assembly, Na + /K + ATP ase, as well as other functions [8].The antitumor activities of benzophenanthridine alkaloids are reviewed to lay a theoretical basis for the development of novel antitumor drugs with natural benzophenanthridine alkaloids as the lead compounds.

Antitumor Activities of Benzophenanthridine Alkaloids from Papaver SPP
Quaternary benzophenanthridine alkaloids (QBAs) primarily originate from plants of the genus Papaver.Representative compounds comprise sanguinarine (1), chelerythrine (2), sanguilutine (3), sanguirubine (4), chelirubine (5), chelilutine (6), and macarpine (7) (Figure 2).The presence of moderate or high levels of ROS in tumor cells affects the initiation and proliferation of cancer to a certain extent.Benzophenanthridine natural products are capable of inducing, or activating different molecular signal transduction, activating proteins of related pathways, or making them apoptosis by interfering with the signal pathway of ROS, and treating and eliminating tumor cells by regulating dysfunctional proteins [9].Sanguinarine (1), one of the most famous benzophenanthridine alkaloids, can act on ROS-dependent mitochondria to induce autophagy and apoptosis, or inhibit the mitosis of cancer cells by changing the acidic conditions of lysosomes and interfering with the formation of autophagosomes lysosomes, such that liver cancer [10] and MDA-MB-231 Indeed, benzophenanthridine alkaloids play an anticancer role via different mechanisms.Benzophenanthridine alkaloids are capable of affecting the activity of DNA topoisomerase I and topoisomerase II, suppressing the rapid proliferation of tumor cells [5], inducing cancer cells ferroptosis [6], inhibiting the growth of tumor stem cells [7], and so forth.Benzophenanthridine natural products combine with negatively charged membrane surfaces and proteins, and react with sulfhydryl residues of amino acids, thus interfering with collagenase, tubulin assembly, Na + /K + ATP ase, as well as other functions [8].The antitumor activities of benzophenanthridine alkaloids are reviewed to lay a theoretical basis for the development of novel antitumor drugs with natural benzophenanthridine alkaloids as the lead compounds.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 26 [3]; pertains to isoquinoline alkaloids; and exhibits antitumor, antifungal, antiviral, antiinflammatory, immune regulation, and other pharmacological activities [4].Indeed, benzophenanthridine alkaloids play an anticancer role via different mechanisms.Benzophenanthridine alkaloids are capable of affecting the activity of DNA topoisomerase I and topoisomerase II, suppressing the rapid proliferation of tumor cells [5], inducing cancer cells ferroptosis [6], inhibiting the growth of tumor stem cells [7], and so forth.Benzophenanthridine natural products combine with negatively charged membrane surfaces and proteins, and react with sulfhydryl residues of amino acids, thus interfering with collagenase, tubulin assembly, Na + /K + ATP ase, as well as other functions [8].The antitumor activities of benzophenanthridine alkaloids are reviewed to lay a theoretical basis for the development of novel antitumor drugs with natural benzophenanthridine alkaloids as the lead compounds.

Antitumor Activities of Benzophenanthridine Alkaloids from Papaver SPP
Quaternary benzophenanthridine alkaloids (QBAs) primarily originate from plants of the genus Papaver.Representative compounds comprise sanguinarine (1), chelerythrine (2), sanguilutine (3), sanguirubine (4), chelirubine (5), chelilutine (6), and macarpine (7) (Figure 2).The presence of moderate or high levels of ROS in tumor cells affects the initiation and proliferation of cancer to a certain extent.Benzophenanthridine natural products are capable of inducing, or activating different molecular signal transduction, activating proteins of related pathways, or making them apoptosis by interfering with the signal pathway of ROS, and treating and eliminating tumor cells by regulating dysfunctional proteins [9].Sanguinarine (1), one of the most famous benzophenanthridine alkaloids, can act on ROS-dependent mitochondria to induce autophagy and apoptosis, or inhibit the mitosis of cancer cells by changing the acidic conditions of lysosomes and interfering with the formation of autophagosomes lysosomes, such that liver cancer [10] and MDA-MB-231 The presence of moderate or high levels of ROS in tumor cells affects the initiation and proliferation of cancer to a certain extent.Benzophenanthridine natural products are capable of inducing, or activating different molecular signal transduction, activating proteins of related pathways, or making them apoptosis by interfering with the signal pathway of ROS, and treating and eliminating tumor cells by regulating dysfunctional proteins [9].Sanguinarine (1), one of the most famous benzophenanthridine alkaloids, can act on ROS-dependent mitochondria to induce autophagy and apoptosis, or inhibit the mitosis of cancer cells by changing the acidic conditions of lysosomes and interfering with the formation of autophagosomes lysosomes, such that liver cancer [10] and MDA-MB-231 human breast cancer [11] can be inhibited.Sanguinarine (1) is capable of significantly targeting ephrin type-B receptor 4 (EphB4) and hypoxia inducible factor-1α (HIF-1α) in breast cancer, inhibiting the activation of the downstream protein signal transducer and activator of transcription 3 (STAT3) in cells, blocking hypoxia-induced HIF-1α or STAT3 interacts, and downregulating the mRNA levels of its target genes, thus inhibiting breast cancer cell hyperplasia [12].In addition, in the breast cancer model, sanguinarine (1) has been proven to have the effects of inhibiting the metastasis of breast cancer and anti epithelial mesenchymal transformation (EMT) [13].A recent review article also showed that sanguinarine (1) is a very promising therapeutic option for breast cancer [14].Sanguinarine (1) facilitates apoptosis in HeLa cells as a treatment for cervical cancer with an IC 50 value of 3.5 µM [15].Sanguinarine (1) exhibits anti-microtubule activity while inhibiting the binding of colchicine and podophyllotoxin to tubulin with IC 50 values of 32 µM and 46 µM.The IC 50 values for chelerythrine (2) have been obtained as 55 µM and 60 µM [16].Sanguinarine (1) induces apoptosis in HeLa cells by upregulating the expression of the proapoptotic protein Bax and inhibiting the antiapoptotic protein BcI-2, and 0.5 µM sanguinarine treatment leads to a significantly reduced number of colonies formed by HeLa cells [15].Sanguinarine (1) is cytotoxic to different resistant cancer cell lines, and the main mechanisms of action are the inhibition of P-glycoprotein transporters, NF-KB activation, and so forth.For CCRF-CEM, CEM/ADR5000, U87MG, U87∆EGFR, MDA231, MDA-BCRP, p53 +/+ , p53 −/− , HEK293, and HEK293/ABCB5 cell lines are significantly inhibited with IC 50 values of 0.3-4.1 µM [17].
Moreover, sanguinarine (1) exhibits strong cytotoxicity against non-small cell lung cancer (NSCLC) with an IC 50 value of 2.19 µM.Its mechanism of action is likely to be correlated with blocking NF-κB, and Akt and ERK1 signaling pathways have a correlation with the inhibition of cancer cell migration [18].Sanguinarine (1) inhibits the proliferation of BGC-823 gastric cancer cells by downregulating the expression of miR-96-5p and miR-29c-3p and upregulating the expression of MAP4K4, pMEK4, and pJNK1 protein in gastric cancer cells BGC-823 [19].Sanguinarine (1) inhibits human prostate cancer cells by inducing ROS-dependent Par-4 cleavage and increasing ROS concentrations in cancer cells.Moreover, it induces growth arrest and apoptosis of human prostate cancer cells PC3 and DU145 with active caspases, which are activated at 2 µM concentration, such that their colony formation can be inhibited [20].The existing research has also suggested that long-term treatment with sanguinarine (1) causes telomere attrition and cell growth retardation, such that cancer cells become senescent.The main mechanisms are associated with the downregulation of the reverse transcriptase hTERT gene expression and inhibition of telomerase activity [21].Sanguinarine (1) induces apoptosis in human HT-29 cells, demonstrating potential therapeutic applications in the treatment of colon cancer [22].
Moreover, recent research has confirmed that sanguinarine (1) is capable of inducing H 2 O 2 -dependent cellular ferroptosis in human cervical cancer (Hela) based on a major mechanism that is correlated with the downregulation of SLC7A11 and the depletion of GSH [6].Besides the above cancer cells, existing studies have found that sanguinarine (1), which specifically targets lung cancer stem cells, is a natural anti-lung cancer drug-resistant compound.Furthermore, sanguinarine (1) inhibits pancreatic cancer stem cells by inhibiting the sonic hedgehog signaling pathway [27].The therapeutic effect of sanguinarine on various tumors has been verified at the animal level [28][29][30].Overall, sanguinarine (1) has anticancer potential and is expected to become a leading compound of anticancer natural products [31].
In human hepatocellular carcinoma (HCC), chelerythrine (2) can inhibit human hepatocellular carcinoma Hep3B cells by downregulating the expression of p-FAK and MMP-2/9.Moreover, the main mechanism of action is correlated with the alteration of phosphoinositide 3-kinase (PI3K), Akt, and the mammalian target of rapamycin (mTOR) signaling pathways [35].Chelerythrine (2) has exhibited anticancer activity in vivo and in vitro, and considerable existing research has confirmed that chelerythrine can act on different pathways (e.g., DNA, MAPK, apoptosis, ROS, cell cycle, autophagy, tumor metastasis, and PKC) to inhibit or facilitate apoptosis in a variety of cancer cells (e.g., non-small cell lung cancer [36], prostate cancer [37], lung adenocarcinoma [38], renal cancer [39], and melanoma cells [40], colorectal cancer [41]), thus suggesting that the benzophenanthridine alkaloids exhibit high anticancer activity.Previous studies have proven that chelerythrine (2) exhibits antitumor stem cell properties, which are mediated by the downregulation of β-Catenin expression, thus inhibiting non-small cell lung cancer stem cells [42].Both sanguinarine (1) and chelerythrine (2) have anticancer activities on human breast cancer cells, but sanguinarine (1) has more potential [43].However, chelerythrine (2) has been reported as a promoter that can regulate c-MYC oncogenes, which has become a new strategy to develop anticancer molecules [44].
Existing research has confirmed that the hydroxymethyl group at the C-6 position of benzophenanthridine alkaloids takes on a critical significance to cellular activity, and the introduction of different groups at the C-6 position can change their activity (Figures 3 and 4) [45].For instance, the introduction of malonate, dialkylphosphite, and nitroalkanes significantly enhances their cytotoxicity.Furthermore, derivatives (8-12) (Table 1) obtained after the insertion of the electron-donating group at the C-6 position by sanguinarine exhibit higher cellular activity than those of chelerythrine (13-16) (Table 2).
In human hepatocellular carcinoma (HCC), chelerythrine (2) can inhibit human hepatocellular carcinoma Hep3B cells by downregulating the expression of p-FAK and MMP-2/9.Moreover, the main mechanism of action is correlated with the alteration of phosphoinositide 3-kinase (PI3K), Akt, and the mammalian target of rapamycin (mTOR) signaling pathways [35].Chelerythrine (2) has exhibited anticancer activity in vivo and in vitro, and considerable existing research has confirmed that chelerythrine can act on different pathways (e.g., DNA, MAPK, apoptosis, ROS, cell cycle, autophagy, tumor metastasis, and PKC) to inhibit or facilitate apoptosis in a variety of cancer cells (e.g., non-small cell lung cancer [36], prostate cancer [37], lung adenocarcinoma [38], renal cancer [39], and melanoma cells [40], colorectal cancer [41]), thus suggesting that the benzophenanthridine alkaloids exhibit high anticancer activity.Previous studies have proven that chelerythrine (2) exhibits antitumor stem cell properties, which are mediated by the downregulation of β-Catenin expression, thus inhibiting non-small cell lung cancer stem cells [42].Both sanguinarine (1) and chelerythrine (2) have anticancer activities on human breast cancer cells, but sanguinarine (1) has more potential [43].However, chelerythrine (2) has been reported as a promoter that can regulate c-MYC oncogenes, which has become a new strategy to develop anticancer molecules [44].
Existing research has confirmed that the hydroxymethyl group at the C-6 position of benzophenanthridine alkaloids takes on a critical significance to cellular activity, and the introduction of different groups at the C-6 position can change their activity (Figures 3  and 4) [45].For instance, the introduction of malonate, dialkylphosphite, and nitroalkanes significantly enhances their cytotoxicity.Furthermore, derivatives (8-12) (Table 1) obtained after the insertion of the electron-donating group at the C-6 position by sanguinarine exhibit higher cellular activity than those of chelerythrine (13-16) (Table 2).
Sanguinaria canadensis L. can extract and purify sanguilutine (3).Some research has suggested that the antiproliferative activities of sanguilutine (3) and chelilutine ( 6) are related to the induction of oxidative stress.As indicated by the results, against three different cancer cell lines, HeLa, A2780, and HL-60, the IC 50 values of sanguilutine range from 0.04 to 0.46 (µg/mL), and the IC 50 values of chelilutine (µg/mL) range from 0.16 to 0.84 [49].
Hammerová, Jindřiš Ka et al. [50] further elucidated the mechanism of sanguilutine (3) in inducing apoptosis in melanoma cells.As a result, sanguinarine caused a decrease in the mitochondrial membrane potential and levels of antiapoptotic proteins of the bcl-2 protein family, BCL XL, and myeloid cell leukemia protein 1 (Mcl-1), as well as downregulated levels of the X-linked inhibitor of apoptosis protein (XIAP) to facilitate melanoma cell apoptosis.

Antitumor Activities of Benzophenanthridine Alkaloids from Corydalis saxicola Bunting
The 6-acetyl-5,6-dihydrosanguinarine ( 17), 8-acetyldihydrochelerythrine (18), and dihydrochelerythrine (19) (Figure 5) are isolated from Corydalis saxicola Bunting, and the above alkaloids have some antitumor effects on squamous cell carcinoma, lung cancer, and liver cancer of the tongue.To be specific, the mechanism of action against human tongue squamous cell carcinoma may be inhibiting NF-κB activation, downregulating BcI-2 protein expression on mRNA, and reducing telomerase activity; inhibiting non-small cell lung cancer A549 cell proliferation, migration, and inducing apoptosis; inhibiting proliferation and migration and upregulating the intracellular NF-κB p65 expression of the subunit [51].
Sanguinaria canadensis L. can extract and purify sanguilutine (3).Some research has suggested that the antiproliferative activities of sanguilutine (3) and chelilutine ( 6) are related to the induction of oxidative stress.As indicated by the results, against three different cancer cell lines, HeLa, A2780, and HL-60, the IC50 values of sanguilutine range from 0.04 to 0.46 (µg/mL), and the IC50 values of chelilutine (µg/mL) range from 0.16 to 0.84 [49].
Hammerová, Jindřiš Ka et al. [50] further elucidated the mechanism of sanguilutine (3) in inducing apoptosis in melanoma cells.As a result, sanguinarine caused a decrease in the mitochondrial membrane potential and levels of antiapoptotic proteins of the bcl-2 protein family, BCL XL, and myeloid cell leukemia protein 1 (Mcl-1), as well as downregulated levels of the X-linked inhibitor of apoptosis protein (XIAP) to facilitate melanoma cell apoptosis.

Antitumor Activities of Benzophenanthridine Alkaloids from Corydalis saxicola Bunting
The 6-acetyl-5,6-dihydrosanguinarine ( 17), 8-acetyldihydrochelerythrine ( 18), and dihydrochelerythrine (19) (Figure 5) are isolated from Corydalis saxicola bunting, and the above alkaloids have some antitumor effects on squamous cell carcinoma, lung cancer, and liver cancer of the tongue.To be specific, the mechanism of action against human tongue squamous cell carcinoma may be inhibiting NF-κB activation, downregulating BcI-2 protein expression on mRNA, and reducing telomerase activity; inhibiting non-small cell lung cancer A549 cell proliferation, migration, and inducing apoptosis; inhibiting proliferation and migration and upregulating the intracellular NF-κB p65 expression of the subunit [51].

Antitumor Activities of Benzophenanthridine Alkaloids from Chelidonium
Sakineh Kazemi noureini et al. [53] have indicated that chelidonine ( 22) (Figure 7) can inhibit MCF-7 in a dose-dependent manner, including cell senescence, apoptosis of autophagic syncytial cells by inhibiting telomerase activity, and chelidonine ( 22) at a 0.05 µM concentration, thus inducing senescence in MCF-7 cells with an LD50 value of 8 µM.Chelidonine ( 22) is capable of inhibiting the pharmacological activity of the NRAS activa-

Antitumor Activities of Benzophenanthridine Alkaloids from Chelidonium
Sakineh Kazemi noureini et al. [53] have indicated that chelidonine (22) (Figure 7) can inhibit MCF-7 in a dose-dependent manner, including cell senescence, apoptosis of autophagic syncytial cells by inhibiting telomerase activity, and chelidonine ( 22) at a 0.05 µM concentration, thus inducing senescence in MCF-7 cells with an LD 50 value of 8 µM.Chelidonine ( 22) is capable of inhibiting the pharmacological activity of the NRAS activator stk19 kinase.It inhibits arginine, lysine, and leucine (Q61R, Q61K, and Q61L) in NRAS mutant melanoma at glutamate 61 (Q61), thus inhibiting the downstream signaling pathways of RAS proteins (e.g., Raf/MEK and P13K-AKT).As a result, cellular senescence and apoptosis are caused.Chelidonine inhibits STK19 kinase with an IC 50 value of 123.5 ± 19.3 Nm [54].Csomós, I., et al. [55] have indicated that chelidonine (22) arrests the G2/S phase of melanoma cells by inhibiting the phosphorylation of complexine and serine in the STAT3 signaling pathway in human melanoma cells, thus inhibiting melanoma.The inhibition effect is significant at 1 µg/mL concentration.It has also been documented that chelidonine (22) can inhibit growth, invasion, angiogenesis, and suppress gene expression in head and neck cancer cell lines.At 10 µg/mL, it significantly inhibits FADU, HLaC78, HlaC79, and HLaC79-Tax cell lines [56].Radim havelek et al. [57] indicated that chelidonine (22) can inhibit the cell cycle of leukemic T cells in different p53 states while suppressing tubulin polymerization in A549 cells.The IC 50 value of different tumor suppressor proteins of the p53 gene for MOLT-4, HL-60, U-937, Raji, Jurkat, and others ranges from 2.2 to 5.0 µM.For non-small cell lung cancer cells (NSCLC), chelidonine (22) has strong inhibitory effects, which are achieved primarily through the ability to selectively inhibit the EGFR phosphorylation and inhibit mitochondrial function in EGFR double mutant cells.The IC 50 of chelidonine after 72 h treatment of various NSCLC cell lines (e.g., H1975, PC9, H460, and H358) ranges from 2.58 to 12.77 µM.Against A549, CCD19 is less active with IC 50 value > 20 µM [58].Chelidonine (22) can induce cell death in T98G cells through two apoptotic pathways: caspase-dependent and caspase-independent.As a result, cell mitosis is arrested, thus causing cell death, and inhibiting human glioblastoma.0.6 µM of chelidonine (22) can significantly inhibit the G2/M phase of mitosis in T98G cells [59].Lenvatinib is capable of enhancing the apoptosis of HCC cells by chelidonine (22), thus inhibiting the epithelial mesenchymal transition (EMT)-related factor of HCC cells based on the possible mechanism.Moreover, chelidonine inhibits HCC cells MHCC97-H and LM-3 with IC 50 values of 7.72 µM and 6.34 µM [60].Chelidonine (22)    Havelek R. et al. [61] have indicated that homochelidonine (23) (Figure 8) induces apoptosis and arrests the G2 phase mitotic cell cycle in cancer cells, and 20 µM homochelidonine (23) inhibits the cell growth of SK-BR-3, HepG2, and MCF-7 by over 50%.

Antitumor Activities of Benzophenanthridine Alkaloids from Corydalis
Corynoline (27) (Figure 9) is a natural product derived from the traditional Chinese medicine Corydalis.It significantly inhibits the cell cycle and induces apoptosis in melanoma cells B16F10 and A375 in vivo, with an IC50 value of 6.16 µM.The IC50 value of A375 reaches 5.56 µM.The mechanism between them is correlated with the upregulated gene expression of Bax and cleavage of Caspase-3 [63].
Corygaline A (28) (Figure 9), isolated from Corydalis bungeana Turcz, refers to a hexahydrobenzophenanthridine alkaloid with an unusual carbon skeleton.Corygaline A ( 28) is capable of inhibiting the NO production in LPS-activated RAW264.7 macrophages with an IC50 value of 2.9 µM.Moreover, it is independent of dose [64].
Acetylcorynoline (29) (Figure 9), originating from the rhizome of the natural plant Corydalis incisa, inhibits the mitotic process of cancer cells by affecting chromosomes, spindles, and the cytoplasm during mitosis, which eventually arrests the mitotic process and induces apoptosis.The mitotic process of the cells is significantly inhibited by 10 µM acetylcorynoline.Acetylcorynoline (29) potently inhibits human colon carcinoma HCT-116,    Corygaline A (28) (Figure 9), isolated from Corydalis bungeana Turcz, refers to a hexahydrobenzophenanthridine alkaloid with an unusual carbon skeleton.Corygaline A ( 28) is capable of inhibiting the NO production in LPS-activated RAW264.7 macrophages with an IC 50 value of 2.9 µM.Moreover, it is independent of dose [64].
Acetylcorynoline (29) (Figure 9), originating from the rhizome of the natural plant Corydalis incisa, inhibits the mitotic process of cancer cells by affecting chromosomes, spindles, and the cytoplasm during mitosis, which eventually arrests the mitotic process and induces apoptosis.The mitotic process of the cells is significantly inhibited by 10 µM acetylcorynoline.Acetylcorynoline (29) potently inhibits human colon carcinoma HCT-116, lung adenocarcinoma cell NCI-H23, lung carcinoma H460, as well as cervical carcinoma TuWi with EC 50 values < 20 µg/mL [65].

Antitumor Activities of Benzophenanthridine Alkaloids from Macleaya cordata
The benzophenanthridine alkaloids cordatine (32) and 6-methoxyldihydrochelerythrine (33) in Figure 11 are extracted from the fruits of Macleaya cordata.Hui Liang Zou et al. [67] determined the cytotoxic activity of the above two benzophenanthridine alkaloids against MCF-7 and SF-268 through the MTT assay.As revealed by the results, the IC50 value of cordatine (32) against MCF-7 cells is 34.78 mM, and that against SF-268 cells reaches 11.79 mM.Furthermore, the IC50 value of 6-methoxydihydrochelerythrine (33) reaches 21.45 mM and 4.28 mM, respectively.

Antitumor Activities of Benzophenanthridine Alkaloids from Macleaya cordata
The benzophenanthridine alkaloids cordatine (32) and 6-methoxyldihydrochelerythrine (33) in Figure 11 are extracted from the fruits of Macleaya cordata.Hui Liang Zou et al. [67] determined the cytotoxic activity of the above two benzophenanthridine alkaloids against MCF-7 and SF-268 through the MTT assay.As revealed by the results, the IC 50 value of cordatine (32) against MCF-7 cells is 34.78 mM, and that against SF-268 cells reaches 11.79 mM.Furthermore, the IC 50 value of 6-methoxydihydrochelerythrine (33) reaches 21.45 mM and 4.28 mM, respectively.

Antitumor Activities of Benzophenanthridine Alkaloids from Zanthoxylum rhoifolium
Nitidine chloride (54), a herb derived from the root of Zanthoxylum avicennae, exhibits anti-inflammatory, antifungal, anti-HIV, and antimalarial biological activities and shows high activity against tumor cancer cells [77], as indicated by the IC50 values (µM) against four cancer cell lines (including HepG2, A549, NCI-H460, and CNE1) that reach 1.40 ± 0.16, 1.88 ± 0.24, 2.35 ± 0.35, and 1.85 ± 0.08, respectively.Pan X et al. [78] have confirmed that nitidine chloride (54) can inhibit the protrusion formation and partial proteolytic activity of MMP-9 and MMP-2 in a dose-dependent manner; reduce the PDGF-induced phosphorylation of c-Src, FAK, and MAPKs; and decrease AP-1 transcriptional activity to inhibit the human breast cancer MDA-MB-231 cell line.Nitidine chloride (54) can inhibit HepG2, HCCLM3, and Huh7 growth, arrest G1/s cell cycle, inhibit proliferation, induce apoptosis, and suppress the expression of cegf-a and VEGFR2 in HCC cells in vitro and in vivo by activating the mitochondria-dependent pathway [79].Huaping Mou et al. [80] have suggested that nitidine chloride (54) can inactivate S-phase kinase-associated protein 2 (Skp2) to inhibit ovarian cancer, mainly downregulating Skp2 expression and enhancing the sensitivity of ovarian cancer cells to nitidine chloride, in which p < 0.05.Nitidine chloride ( 54) is capable of inhibiting renal cancer cell 786-O and A498 cells, as confirmed by cell viability and flow cytometric apoptosis analysis.Its mechanism is primarily induced by downregulating the signaling process of p53, BcI-2, caspase-3, and inhibiting ERK [81].Nitidine chloride (54) can induce autophagy and apoptosis of ovarian cancer cells through various signaling pathways, such as Akt/mTOR.It may become a potential target for ovarian cancer chemotherapy [82].
In cancer cell proliferation, nitidine chloride (54) can serve as a potent inhibitor while inhibiting cell growth, apoptosis, migration, and invasion in glioblastoma cell lines by downregulating the expression of calmodulin-dependent protein kinase III, which may be correlated with the activation of Cdc20 oncoprotein expression [83].Moreover, osteosarcoma cells can be inhibited by nitidine chloride (54), and Hui Xu et al. [84] have indicated that nitidine chloride (54) inhibits the growth, migration, and invasion and induces the apoptosis of osteosarcoma cells through an MTT assay and flow cytometric analysis.The mechanism may be correlated with the inhibition of sin1 expression in osteosarcoma cells.As revealed by the result, nitidine chloride (54) inhibits rectal cancer HCT-116 cells  [80] have suggested that nitidine chloride (54) can inactivate S-phase kinase-associated protein 2 (Skp2) to inhibit ovarian cancer, mainly downregulating Skp2 expression and enhancing the sensitivity of ovarian cancer cells to nitidine chloride, in which p < 0.05.Nitidine chloride ( 54) is capable of inhibiting renal cancer cell 786-O and A498 cells, as confirmed by cell viability and flow cytometric apoptosis analysis.Its mechanism is primarily induced by downregulating the signaling process of p53, BcI-2, caspase-3, and inhibiting ERK [81].Nitidine chloride (54) can induce autophagy and apoptosis of ovarian cancer cells through various signaling pathways, such as Akt/mTOR.It may become a potential target for ovarian cancer chemotherapy [82].
In cancer cell proliferation, nitidine chloride (54) can serve as a potent inhibitor while inhibiting cell growth, apoptosis, migration, and invasion in glioblastoma cell lines by downregulating the expression of calmodulin-dependent protein kinase III, which may be correlated with the activation of Cdc20 oncoprotein expression [83].Moreover, osteosarcoma cells can be inhibited by nitidine chloride (54), and Hui Xu et al. [84] have indicated that nitidine chloride (54) inhibits the growth, migration, and invasion and induces the apoptosis of osteosarcoma cells through an MTT assay and flow cytometric analysis.The mechanism may be correlated with the inhibition of sin1 expression in osteosarcoma cells.As revealed by the result, nitidine chloride (54) inhibits rectal cancer HCT-116 cells by inhibiting the phosphorylation pathway of ERK, and the mechanism is the upregulation of the expression of Bax, p53, and the downregulation of the expression of caspase-3, caspase-9, and BcI-2 [85].Hyoung Yang et al. [86] have found that nitidine chloride (54) inhibits human oral squamous cell carcinoma (OSCC) by inhibiting the signal transducer and activator of transcription 3 (STAT3), and the main mechanism is the downregulation of myeloid cell leukemia-1 (MCl-1) protein in HSC-3 and HSC-4 by inhibiting the STAT3 pathway.Furthermore, acute myeloid leukemia (AML) can be inhibited by nitidine chloride (54) by inhibiting the phosphorylation of Akt and ERK.Its main mechanism is to downregulate cyclin B1, CDK1, and BCl; upregulate p27 and Bax in AML cells; inactivate PARP; and activate caspase-3-related signaling pathways [87].Existing research on the anticancer effect of nitidine chloride (54) has suggested that it is capable of inhibiting leukemia (CML) by downregulating the expression of proto oncogene c-myc, and the possible mechanism of action of nitidine chloride ( 54) is to induce apoptosis and upregulate caspase-3 and PARP-1 in K562 cells, thus enhancing the effect of imatinib on K562 cells [88].Nitidine chloride ( 54) is also active against BRCA1-deficient cancer cells.Existing research has suggested that bicarbonate chloride (DSBs) is also active at 0.2 µM, thus significantly inhibiting MDA-436BRCA1-KO cells (p ≤ 0.001), and 0.4 µ m is significantly (p ≤ 0.001) resistant to HCC1937-BRCA1 −/− inhibition [89].
Some studies have indicated that bicarbonate chloride ( 54) exhibits antitumor stem cell properties (e.g., inhibiting the epithelial mesenchymal transition (EMT) and inhibiting glioma stem cell properties through the JAK2/STAT3 signaling pathway [90]).Bicarbonate chloride ( 54) also exhibited dose-dependent anti-liver cancer stem cell activity, which was confirmed in nude mice experiments [91].Furthermore, nitidine chloride (54) inhibits the epithelial mesenchymal transition process while suppressing tumor stem cell properties in breast cancer cells through the hedgehog signaling pathway [92].In the in vitro activity study, nitidine chloride (54) inhibited bladder cancer cells by downregulating the expression of Lymphocyte antigen 75 (LY75) [93].
Avicine (55) and nitidine chloride (54) (Figure 17) are two types of benzophenanthridine alkaloids derived from the roots of Zanthoxylum avicennae that serve as cholinesterases (AChE), monoamine oxidases A (Maos), and A β 1-42 targeted inhibitors.Erika Plaza et al.  54) by inhibiting the phosphorylation of Akt and ERK.Its main mechanism is to downregulate cyclin B1, CDK1, and BCl; upregulate p27 and Bax in AML cells; inactivate PARP; and activate caspase-3-related signaling pathways [87].Existing research on the anticancer effect of nitidine chloride ( 54) has suggested that it is capable of inhibiting leukemia (CML) by downregulating the expression of proto oncogene c-myc, and the possible mechanism of action of nitidine chloride ( 54) is to induce apoptosis and upregulate caspase-3 and PARP-1 in K562 cells, thus enhancing the effect of imatinib on K562 cells [88].Nitidine chloride ( 54) is also active against BRCA1-deficient cancer cells.Existing research has suggested that bicarbonate chloride (DSBs) is also active at 0.2 µM, thus significantly inhibiting MDA-436BRCA1-KO cells (p ≤ 0.001), and 0.4 µ m is significantly (p ≤ 0.001) resistant to HCC1937-BRCA1 −/− inhibition [89].Some studies have indicated that bicarbonate chloride ( 54) exhibits antitumor stem cell properties (e.g., inhibiting the epithelial mesenchymal transition (EMT) and inhibiting glioma stem cell properties through the JAK2/STAT3 signaling pathway [90]).Bicarbonate chloride ( 54) also exhibited dose-dependent anti-liver cancer stem cell activity, which was confirmed in nude mice experiments [91].Furthermore, nitidine chloride (54) inhibits the epithelial mesenchymal transition process while suppressing tumor stem cell properties in breast cancer cells through the hedgehog signaling pathway [92].In the in vitro activity study, nitidine chloride (54) inhibited bladder cancer cells by downregulating the expression of Lymphocyte antigen 75 (LY75) [93].
Natural benzophenanthridine alkaloids have promising applications in the fight against the drug resistance of tumors.For instance, NK-109 (85) exhibits strong cytotoxicity to drug-resistant cell lines K562/ADM, AdrR MCF7, PC-9/CDDP, and SKOV3/V, with IC 50 values of 0.045, 0.42, 0.19, and 0.21 µg/mL, respectively, and the drug molecule has entered the clinical research stage.In addition, the natural products of benzophenanthridine exhibit significant cell activity against cancer stem cells.Sanguinarine (1) exerts a specific targeting effect on lung cancer stem cells, and it is a natural drug-resistant compound against lung cancer.In addition, sanguinarine (1) inhibits pancreatic cancer stem cells by inhibiting the sonic hedgehog signaling pathway.Chelerythrine (2) downregulates β-Catenin, thus inhibiting non-small cell lung cancer stem cells.Nitidine chloride (54) inhibits epithelial mesenchymal transformation (EMT) and glioma stem cell characteristics by the JAK2/STAT3 signaling pathway.Furthermore, it exhibits a dose-dependent anti-hepatoma stem cell activity, as confirmed in nude mouse experiments.Moreover, it can inhibit the epithelial mesenchymal transformation of breast cancer cells and suppress the characteristics of tumor stem cells through the hedgehog signaling pathway.Ferroptosis takes on critical significance in the treatment of cancer stem cells [111].Recent research has revealed that sanguinarine (1) can induce H 2 O 2 -dependent cell ferroptosis in human cervical cancer (HeLa) by downregulating SLC7A11 and GSH [6].

Figure 3 .
Figure 3. Study on structure-activity relationship of sanguinarine.

Figure 3 .
Figure 3. Study on structure-activity relationship of sanguinarine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.

Figure 4 .
Figure 4. Study on structure-activity relationship of chelerythrine.
can inhibit the cell cycle of leukemic T cells in different p53 states while suppressing tubulin polymerization in A549 cells, and the IC 50 values of different tumor suppressor proteins' p53 gene for MOLT-4, HL-60, U-937, and Raji range from 4.8 to 8.3 µM [51].Molecules 2023, 28, x FOR PEER REVIEW 8 of 26

Figure 16 .
Figure 16.Benzophenanthridine alkaloids from Thalictrum microgynum Lecoy ex Oliv.2.3.Antitumor Activities of Benzophenanthridine Alkaloids from Rutaceae 2.3.1.Antitumor Activities of Benzophenanthridine Alkaloids from Zanthoxylum rhoifolium Nitidine chloride (54), a herb derived from the root of Zanthoxylum avicennae, exhibits anti-inflammatory, antifungal, anti-HIV, and antimalarial biological activities and shows high activity against tumor cancer cells [77], as indicated by the IC 50 values (µM) against four cancer cell lines (including HepG2, A549, NCI-H460, and CNE1) that reach 1.40 ± 0.16, 1.88 ± 0.24, 2.35 ± 0.35, and 1.85 ± 0.08, respectively.Pan X et al. [78] have confirmed that nitidine chloride (54) can inhibit the protrusion formation and partial proteolytic activity of MMP-9 and MMP-2 in a dose-dependent manner; reduce the PDGF-induced phosphorylation of c-Src, FAK, and MAPKs; and decrease AP-1 transcriptional activity to inhibit the human breast cancer MDA-MB-231 cell line.Nitidine chloride (54) can inhibit HepG2, HCCLM3, and Huh7 growth, arrest G1/s cell cycle, inhibit proliferation, induce apoptosis, and suppress the expression of cegf-a and VEGFR2 in HCC cells in vitro and in vivo by activating the mitochondria-dependent pathway [79].Huaping Mou et al.[80] have suggested that nitidine chloride (54) can inactivate S-phase kinase-associated protein 2 (Skp2) to inhibit ovarian cancer, mainly downregulating Skp2 expression and enhancing the sensitivity of ovarian cancer cells to nitidine chloride, in which p < 0.05.Nitidine chloride (54) is capable of inhibiting renal cancer cell 786-O and A498 cells, as confirmed by cell viability and flow cytometric apoptosis analysis.Its mechanism is primarily induced by downregulating the signaling process of p53, BcI-2, caspase-3, and inhibiting ERK[81].Nitidine chloride (54) can induce autophagy and apoptosis of ovarian cancer cells through various signaling pathways, such as Akt/mTOR.It may become a potential target for ovarian cancer chemotherapy[82].In cancer cell proliferation, nitidine chloride (54) can serve as a potent inhibitor while inhibiting cell growth, apoptosis, migration, and invasion in glioblastoma cell lines by downregulating the expression of calmodulin-dependent protein kinase III, which may be correlated with the activation of Cdc20 oncoprotein expression[83].Moreover, osteosarcoma cells can be inhibited by nitidine chloride(54), and Hui Xu et al.[84] have indicated that nitidine chloride (54) inhibits the growth, migration, and invasion and induces the apoptosis of osteosarcoma cells through an MTT assay and flow cytometric analysis.The mechanism may be correlated with the inhibition of sin1 expression in osteosarcoma cells.As revealed by the result, nitidine chloride (54) inhibits rectal cancer HCT-116 cells by inhibiting the phosphorylation pathway of ERK, and the mechanism is the upregulation [79]  analyzed the IC 50 values for inhibiting EeAChEH, rAChE, EqAChE, MAO A, and MAO B using avicine(55) and nitidine chloride(54).The IC 50 values (µM) of avicineinhibited AChEW as 0.15 ± 0.01, 0.52 ± 0.05, and 0.88 ± 0.08 reach 0.41 ± 0.02, and the inhibition Maos IC 50 values (>100 µM).However, the IC 50 values (µM) of nitidine chlorideinhibited AChE reach 0.65 ± 0.09, 1.25 ± 0.09, and 5.73 ± 0.60, and those of inhibition MAOs IC 50 values reach 1.89 ± 0.17 and >300 µM.Molecules 2023, 28, x FOR PEER REVIEW 14 of 26 by inhibiting the phosphorylation pathway of ERK, and the mechanism is the upregulation of the expression of Bax, p53, and the downregulation of the expression of caspase-3, caspase-9, and BcI-2 [85].Hyoung Yang et al. [86] have found that nitidine chloride (54) inhibits human oral squamous cell carcinoma (OSCC) by inhibiting the signal transducer and activator of transcription 3 (STAT3), and the main mechanism is the downregulation of myeloid cell leukemia-1 (MCl-1) protein in HSC-3 and HSC-4 by inhibiting the STAT3 pathway.Furthermore, acute myeloid leukemia (AML) can be inhibited by nitidine chloride (

Figure 20 .
Figure 20.Study on structure-activity relationship of rhoifoline B.

Figure 20 .
Figure 20.Study on structure-activity relationship of rhoifoline B.

Figure 20 .
Figure 20.Study on structure-activity relationship of rhoifoline B.

Figure 29 .
Figure 29.Study on structure-activity relationship of oxynitidine.

Figure 29 .
Figure 29.Study on structure-activity relationship of oxynitidine.

Table 1 .
The IC50 value of compounds 8

Table 2 .
The IC50 values of compounds

Table 1 .
The IC50 value of compounds 8

Table 1 .
The IC50 value of compounds 8

Table 1 .
The IC50 value of compounds 8

Table 1 .
The IC50 value of compounds 8

Table 1 .
The IC50 value of compounds 8

Table 2 .
The IC 50 values of compounds