Regular Article
Design, Synthesis, and Antitumor Activity of Novel Eupatilin Derivatives Based on the Mannich Reaction
2025 Volume 73 Issue 2 Pages 112-120
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2025 Volume 73 Issue 2 Pages 112-120
Eupatilin, a natural bioactive flavone, is the active ingredient in traditional Chinese medicine Artemisia argyi Levl. et Vant. To enhance the antitumor effect of eupatilin, we designed a series of novel eupatilin–Mannich derivatives and investigated antitumor activity against several human cancer cell lines, including gastric cancer cells (AGS), esophageal cancer cells (Eca-109), and breast cancer cells (MDA-MB-231). Among all derivatives, the majority demonstrated superior antitumor activity compared to eupatilin, with compound 3d exhibiting the most effective antitumor activity against AGS cells. Furthermore, compound 3d effectively inhibited colony formation and migration of AGS cells. Network pharmacology combined with molecular docking studies indicated that compound 3d exerts antitumor activity by targeting the Hsp90AA1 and multiple signaling pathways. In addition, the Western blot experiment results showed that compound 3d reduced the expression of Hsp90AA1 in AGS cells, indicating that Hsp90AA1 may be the potential target of compound 3d. In summary, several novel eupatilin derivatives were prepared via the Mannich reaction, representing the first structure modification study of eupatilin. The mechanism of action of compound 3d was estimated through cell experiments, network pharmacology, molecular docking, and Western blot experiments, to provide lead compounds for the discovery of natural product-based antitumor candidates.
Cancer, a complex multifactorial disease, is the second leading cause of death after cardiovascular diseases and threatens human life and health. According to authoritative global statistics, there were 19.3 million new cases of cancer and almost 10 million deaths worldwide in 2020.1) Despite various cancer treatment methods that have been developed over the years, chemotherapy is the priority means of effectively treating cancer. However, serious adverse reactions and drug resistance make it difficult to meet clinical needs.2–4) The discovery and development of new antitumor drugs with high efficiency and low side effects have high research value and practical significance. Natural products possess diverse pharmacological activities and have a significant impact on the world, especially in the development of pharmaceuticals and agricultural chemicals.5) Several natural products are approved as antitumor drugs, such as paclitaxel, camptothecin, vinblastine, etc. In addition, natural product derivatives account for a larger proportion of innovative drugs that are being studied. According to the statistics of new chemical entities from 1981 to 2019, nearly 75% of antitumor drugs are not pure synthetic chemicals, and 47% are natural products including derivatives or imitation natural products.6–10) Therefore, natural products and their scaffold types have become indispensable sources for pharmaceutical chemistry and drug discovery.
Eupatilin is a pharmacologically active flavone mainly isolated from Artemisia argyi Levl. et Vant of traditional Chinese medicine and has various pharmacological properties encompassing antitumor, anti-inflammatory, antiallergic, antioxidation, etc. In addition, eupatilin was approved for clinical use in the treatment of gastritis and peptic ulcer in Korea in 2005.11–13) Several studies have shown that eupatilin possesses potent antitumor properties against multiple cancer cell lines including gastric cancer, leukemia cancer cells, renal carcinoma, glioma cancerous cells, and other cancer types, by acting on cell cycle arrest, apoptosis, and numerous signaling pathways.11,14–17) Although eupatilin is a promising antitumor compound, its antitumor activity is far from the required concentration of clinical antitumor drugs. At present, research on the structural modification of eupatilin is relatively poor. It is of great significance to further enhance the antitumor activity by modifying the structure of eupatilin to find candidate drugs with good antitumor activity. Therefore, for the first time, we designed and synthesized a series of eupatilin derivatives and evaluated their antitumor activities on several cancer cell lines. The structure–activity relationships, preliminary antitumor mechanism, network pharmacology, and molecular docking research were carried out sequentially in this research, hoping to discover natural product-based antitumor candidates.
The general synthesis route of the target compounds is shown in Fig. 1. Eupatilin as raw material was modified by the Mannich reaction. Eupatilin reacted with formaldehyde and various substituted piperazines in N,N-dimethylformamide (DMF) produced eupatilin–Mannich derivatives with a preferable yield. The structures of the targeted compounds were characterized by spectral analyses of 1H-NMR, 13C-NMR, and high-resolution mass spectrometry (HR-MS).
The antiproliferation activity of the eupatilin–Mannich derivatives was evaluated using several human cancer cell lines, including gastric cancer cells (AGS), esophageal cancer cells (Eca-109), and breast cancer cells (MDA-MB-231) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for 72 h and 5-fluorouracil (5-Fu) as positive control. The results of antiproliferation activity are summarized in Table 1. As a result, most eupatilin derivatives exerted moderate to excellent cytotoxic activity against 3 human cancer cells, which was superior to that of eupatilin. The majority of compounds had better antitumor activity against AGS cells and Eca-109 cells than MDA-MB-231 cells. Comparing the antitumor results of compounds 3a, 3b, and 3d, it was found that the antitumor activity of alkane substituents was superior to that of aromatic substituents. Comparing the antitumor results of 3b, 3c, 3e, and 3g, it was found that the antitumor activity of phenyl substituents was superior to that of heterocyclic substituents. Among all target compounds, compound 3d exhibited the most excellent antitumor activity against AGS cells with an IC50 value of 20.25 μM, compared to eupatilin with an IC50 value of 100 μM.17) Therefore, to investigate the preliminary mechanism of antitumor effect, compound 3d and AGS cells were selected for further investigation.
| Compound | R | AGS | Eca-109 | MDA-MB-231 |
|---|---|---|---|---|
| 3a | CH3 | 55.25 ± 0.97 | 46.25 ± 1.11 | 98.58 ± 1.21 |
| 3b | Ph | 58.58 ± 1.01 | 62.35 ± 0.88 | 86.35 ± 1.24 |
| 3c | 2-Pyrimidine | 90.85 ± 1.02 | 51.69 ± 1.31 | 68.24 ± 1.38 |
| 3d | Cyclohexyl | 20.25 ± 0.24 | 20.28 ± 1.02 | 40.55 ± 1.57 |
| 3e | 2-Pyrazine | 80.25 ± 0.78 | 26.98 ± 0.78 | 74.28 ± 0.96 |
| 3f | Cyclohexane methyl | 95.36 ± 1.07 | 49.55 ± 1.05 | 88.59 ± 0.88 |
| 3g | 4-Pyridinomethyl | 60.14 ± 1.34 | 38.59 ± 1.14 | 45.36 ± 0.37 |
| Eupatilin | — | 100.25 ± 1.02 | 106.16 ± 1.25 | 110.27 ± 0.64 |
| 5-Fub) | — | 12.15 ± 1.15 | 10.38 ± 1.04 | 17.22 ± 1.21 |
a) Antitumor activity was assayed by exposure for 72 h to substances and expressed as the concentration required to inhibit tumor cell proliferation by 50% (IC50). Dates are presented as the mean ± S.D. of 3 independent experiments. b) Positive control.
The excellent antiproliferation activity of compound 3d against AGS cells prompted us to investigate the effect on colony formation and migration. The clone formation of cancer cells, on the one hand, can reflect the effect of drugs on tumor cell proliferation, on the other hand, represents an indirect estimate of tumor transformation. As shown in Fig. 2, the presence of compound 3d at different concentrations resulted in a decrease in the number of colonies formed by AGS cells, indicating that compound 3d effectively inhibited the proliferation of AGS cells. In addition, the cell migration ability was assessed by the wound healing assay. As shown in Fig. 3, treatment of AGS cells with compound 3d at the indicated concentration suppressed wound healing in a time- and dose-dependent manner.
(A) Clone formation of AGS cells with different concentrations of compound 3d. (B) Quantitative analysis of clone formation. Data are the mean ± S.D. All experiments were carried out at least 3 times. (* p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group). AGS, gastric cancer cell; S.D., standard deviation.
(A) Wound healing assay of AGS cells with different concentrations of compound 3d. (B) Quantitative analysis of wound healing assay. Data are the mean ± S.D. All experiments were carried out at least 3 times. (* p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group). AGS, gastric cancer cell; S.D., standard deviation.
Recently, network pharmacology based on interdisciplinary disciplines has emerged worldwide, which has accelerated the process of drug discovery.18,19) Therefore, the use of network pharmacology to study the targets and mechanisms of traditional Chinese medicine and its natural products has become an emerging and widespread research method in recent years.20–22) Molecular docking involves placing ligand molecules at the active sites of receptor molecules, searching for different molecular conformations, and achieving the optimal state of chemical structure matching and spatial complementarity of ligand-receptor binding. As one of the most important technical means, it has been widely used in drug development.23,24) The combination of network pharmacology and molecular docking technology provides new research ideas for the mechanism of action of natural product derivatives with novel structures, significant activity, and diverse targets, and has been widely used in the modernization research of traditional Chinese medicine.
Targets Screening of Compound 3dTo further explore the mechanism of compound 3d against gastric cancer, we predicted potential targets of compound 3d against gastric cancer using network pharmacology technology. The Swiss Target Prediction and Targetnet databases were used to obtain compound 3d therapeutic targets, and “Homo sapiens” was selected as the prediction condition. The Swiss Target Prediction obtained 105 targets, while the Targetnet obtained 235 targets. After removing duplicate targets, they were imported into the UniProt database for standardization using Retrieve/ID mapping, resulting in a total of 292 targets for compound 3d.
Screening of Gastric Cancer TargetsUsed the keyword “gastric cancer” to search for targets in the GeneCards and OMIM databases, filtered search results based on relevance score, and only accepted targets larger than the median correlation score. Duplicate targets were deleted, and 1106 gastric cancer targets were obtained.
Construction of Protein–Protein Interaction Network and Screening of Core TargetsCompound 3d and gastric cancer targets were separately uploaded to the Venny platform. A total of 104 intersection targets of compound 3d with gastric cancer were obtained, which were the potential anti-gastric cancer targets of compound 3d (Fig. 4). Intersection targets were imported into the STRING database, using high confidence 0.700 as the criterion for screening, and other settings were set as default. After removing free nodes, protein–protein interaction (PPI) analysis was completed. The PPI network analysis was imported into Cytoscape for further data mining (Fig. 5). The network topology eigenvalues of Degree, Betweenness, and Closeness were analyzed to determine the core targets using the media value exceeding the above 3 topology feature values as the screening criterion.
Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to identify the core mechanism and pathway of compound 3d anti-gastric cancer in the DAVID database. The results were imported into bioinformatics to visualize the biological functions and associated signaling pathways of compound 3d in the treatment of gastric cancer. A total of 688 GO items were obtained from the David database (p < 0.01), including 518 entries of biological process (BP), 71 entries of cell composition (CC), and 99 entries of molecular function (MF). We further selected the top 10 BP, CC, and MF catalogs for visualization (Fig. 6). According to the p-value, the main terms in the BP category were phosphorylation and protein phosphorylation. The CC category was mainly distributed in the receptor complex, cytoplasm, and plasma membrane. Furthermore, these MFs were mainly associated with protein kinase activity, ATP binding, and transmembrane receptor protein tyrosine kinase activity.
The results of KEGG pathway enrichment analysis showed that 104 targets were enriched in 152 signaling pathways (p < 0.01), mainly involving pathways in cancer, phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway. We selected the top 20 results to draw a bubble diagram (Fig. 7). The larger the bubble, the more genes are enriched along the pathway. The redder the color, the smaller the p-value and the more meaningful the result.
The PDB IDs were obtained from the PDB database and molecular docking was conducted with Autodock. Among them, Hsp90AA1 had the highest absolute affinity with 11.54 kcal/mol and encodes a PDB ID of 1UY6. Pymol was used for visual analysis to demonstrate the binding mode between proteins and ligands to better demonstrate the interaction forces. The interaction forces between proteins and ligands were distinguished by different colors, with hydrogen bonds represented by yellow lines, hydrophobic forces represented by blue lines, and π-cation forces represented by gray lines. The main interaction between compound 3d and 1UY6 protein includes hydrophobic interaction with amino acids ASN51, ALA55, ILE96, LEU103, LEU107, PHE138, TRP162, hydrogen bonds with amino acids ASN51, LYS58, ASP93, and π–cation interaction with amino acid PHE138 (Fig. 8). The molecular docking result suggested that the key gene Hsp90AA1 may be the potential target of compound 3d against gastric cancer.
The interaction forces between proteins and ligands were distinguished by different colors, with hydrogen bonds represented by yellow lines, hydrophobic forces by blue lines, and π–cation forces by gray lines.
Heat shock protein 90 (Hsp90) is a highly conserved member of the heat shock protein family, with several Hsp90 subtypes in humans including Hsp90α and Hsp90β, as well as endoplasmic reticulum and mitochondrial subtypes Grp94 and TRAP1. Hsp90 is one of the most abundant chaperone proteins in cells, accounting for 1–2% of cytoplasmic proteins. It is composed of multiple chaperone proteins and participates in the folding, activation, and assembly of various proteins. In addition to participating in various physiological functions, Hsp90 is overexpressed in various tumor cells, leading to increased expression of many client proteins that typically drive tumor occurrence and progression. Hsp90 is involved in regulating various proteins and signaling pathways in cancer, making it a promising target for cancer treatment.25) Among them, Hsp90AA1, as an important member of the family, encodes heat shock protein 90α (Hsp90α), which has been proven to be highly expressed in ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, and other tumors, and can promote proliferation, reduce prognosis, and produce drug resistance. Currently, multiple Hsp90 inhibitors have been developed.26,27) Therefore, inhibiting the expression of Hsp90AA1 can suppress tumor progression and improve prognosis.
Western blot experiment was conducted to investigate the effect of compound 3d on the expression of Hsp90AA1 protein in gastric cancer cells. As shown in Fig. 9, treatment of AGS cells with compound 3d resulted in a decreased expression of Hsp90AA1 in a concentration-dependent manner, indicating that compound 3d may act on the target of Hsp90AA1 proteins to exert an antitumor effect.
(A) Compound 3d induced expression changes of Hsp90AA1 in AGS cells. (B) Statistical analysis of expression levels of Hsp90AA1. Results were presented as mean ± S.D. for 3 independent experiments. (nsp > 0.05, ** p < 0.01, *** p < 0.001 compared with the control group). AGS, gastric cancer cell; S.D., standard deviation.
A series of novel eupatilin derivatives were synthesized and the antitumor activity of the target compounds on human cancer cell lines AGS, Eca-109, and MDA-MB-231 were evaluated. Most compounds exhibited moderate to good antitumor activity against 3 selected cancer cell lines, of which compound 3d exerted the best antitumor activity against AGS cells, and further antitumor mechanisms suggested that compound 3d can inhibit colony formation and migration of AGS cells. The anti-gastric cancer mechanism of compound 3d was preliminarily investigated employing network pharmacology, molecular docking, and Western blot experiments. Compound 3d reduced the expression of Hsp90AA1, indicating that compound 3d may exert antitumor activity by acting on the target Hsp90AA1. The structural modification and antitumor mechanism of eupatilin were studied for the first time, which laid the foundation for further development and mechanism study of more efficient and less toxic eupatilin derivatives.
Reagents and solvents were purchased from commercial sources and were used without further purification. 1H-NMR and 13C-NMR spectra were recorded on a Bruker 300 or 400 MHz. Mass spectra were recorded on a Waters of ultra-performance LC-electron spray ionization-quadrupole-time of flight-mass spectrometry. Melting points were determined on an RD-1 melting point apparatus. Reactions were monitored by TLC.
General Procedure for the Synthesis of Compounds 3a–3gEupitilin (1 equivalent (equiv.)) was dissolved in an appropriate amount of N,N-dimethyl formamide, followed by the addition of 5 equiv. of formaldehyde and 1.5 equiv. of substituents and reaction at 70 °C for 6–10 h. The reaction process was monitored by TLC. After the reaction, ethyl acetate was extracted anhydrous sodium sulfate was dried, and column chromatography was performed to purify the target compounds 3a–3g.
2-(3,4-Dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-8-((4-methylpiperazin-1-yl)methyl)-4H-chromen-4-one (3a)Yellow solid, 78.9% yield, Mp 196.5–197.2 °C. 1H-NMR (400 MHz, Chloroform-d) δ: 12.88 (s, 1H), 7.49–7.45 (m, 1H), 7.28 (t, J = 1.8 Hz, 1H), 6.99 (s, 1H), 6.55 (d, J = 1.5 Hz, 1H), 4.04 (d, J = 1.5 Hz, 2H), 3.97 (s, 3H), 3.96 (d, J = 1.4 Hz, 6H), 2.76 (s, 4H), 2.59 (s, 4H), 2.35 (d, J = 1.5 Hz, 3H). 13C-NMR (101 MHz, Chloroform-d) δ: 183.54, 163.09, 158.72, 153.13, 152.31, 150.25, 149.40, 131.88, 123.99, 111.74, 110.75, 108.98, 108.51, 104.63, 104.03, 98.44, 60.17, 54.43, 53.77, 52.36, 45.63, 29.66. HR-MS Calcd for C24H29N2O7 [M + H]+: 457.1975, Found: 457.1988.
2-(3,4-Dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-8-((4-phenylpiperazin-1-yl)methyl)-4H-chromen-4-one (3b)Yellow solid, 69.3% yield, Mp 139.5–140.3 °C. 1H-NMR (400 MHz, Chloroform-d) δ: 12.89 (s, 1H), 7.47 (dd, J = 8.5, 2.1 Hz, 1H), 7.26 (d, J = 7.4 Hz, 3H), 6.99 (d, J = 8.5 Hz, 1H), 6.92 (d, J = 8.2 Hz, 3H), 6.55 (s, 1H), 4.09 (s, 2H), 3.95 (s, 9H), 3.29 (d, J = 5.5 Hz, 4H), 2.86 (d, J = 5.4 Hz, 4H). 13C-NMR (101 MHz, Chloroform-d) δ: 181.67, 162.10, 159.76, 157.57, 152.19, 151.30, 149.64, 149.27, 148.37, 128.27, 128.13, 122.96, 119.59, 118.78, 115.81, 115.56, 115.27, 110.26, 107.70, 103.23, 97.40, 59.74, 55.12, 52.27, 48.17. HR-MS Calcd for C29H31N2O7[M + H]+: 519.2131, Found: 519.2154.
2-(3,4-Dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-8-((4-(pyrimidin-2-yl)piperazin-1-yl)methyl)-4H-chromen-4-one (3c)Yellow solid, 77.9% yield, Mp 240.5–241.4 °C. 1H-NMR (400 MHz, Chloroform-d) δ: 12.95 (s, 1H), 8.32 (d, J = 4.7 Hz, 2H), 7.48 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 6.99 (d, J = 8.6 Hz, 1H), 6.59–6.51 (m, 3H), 4.12 (s, 2H), 4.02–3.93 (m, 5H), 3.95 (s, 8H), 2.78 (d, J = 5.3 Hz, 4H). 13C-NMR (151 MHz, Chloroform-d) δ: 182.66, 163.19, 161.33, 157.82, 153.30, 152.33, 150.45, 149.39, 131.81, 124.46, 123.90, 119.81, 111.29, 110.58, 108.72, 104.81, 104.29, 60.79, 56.13, 56.09, 52.52, 43.12, 31.44, 30.19. HR-MS Calcd for C27H29N4O7[M + H]+: 521.2036, Found 521.2035.
8-((4-Cyclohexylpiperazin-1-yl)methyl)-2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-chromen-4-one (3d)Yellow solid, 88.9% yield, Mp 156.3–156.7 °C. 1H-NMR (400 MHz, Chloroform-d) δ: 12.90 (s, 1H), 7.47 (dd, J = 8.5, 2.1 Hz, 1H), 7.28 (s, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.55 (s, 1H), 4.04 (s, 2H), 3.97 (s, 3H), 3.96 (d, J = 1.7 Hz, 6H), 2.78 (s, 8H), 2.39 (s, 1H), 1.90 (s, 2H), 1.82 (s, 2H), 1.31 (d, J = 19.6 Hz, 2H), 1.25 (s, 4H). 13C-NMR (101 MHz, Chloroform-d) δ: 181.82, 163.08, 159.56, 153.09, 152.14, 150.27, 148.87, 131.75, 124.89, 120.11, 113.96, 111.15, 108.91, 108.37, 104.60, 100.18, 71.97, 63.77, 61.16, 60.31, 55.71, 54.27, 52.83, 48.85, 27.86. HR-MS Calcd for C29H37N2O7[M + H]+: 525.2601, Found: 525.2625.
2-(3,4-Dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-8-((4-(pyrazin-2-yl)piperazin-1-yl)methyl)-4H-chromen-4-one (3e)Yellow solid, 84.3% yield, Mp 200.1–200.5 °C. 1H-NMR (300 MHz, Chloroform-d) δ: 12.55 (s, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.70 (q, J = 1.9 Hz, 1H), 7.53 (t, J = 2.2 Hz, 1H), 7.09 (dt, J = 8.5, 2.0 Hz, 1H), 6.89 (d, J = 1.4 Hz, 1H), 6.61 (dd, J = 8.6, 1.7 Hz, 1H), 6.18 (d, J = 1.7 Hz, 1H), 3.72 (d, J = 1.7 Hz, 2H), 3.61-3.55 (m, 9H), 3.42–3.27 (m, 4H), 2.44 (t, J = 4.9 Hz, 4H). 13C-NMR (101 MHz, Chloroform-d) δ: 196.34, 186.00, 182.31, 176.57, 170.65, 169.41, 163.37, 154.77, 146.92, 141.79, 129.62, 128.10, 124.21, 121.83, 118.70, 114.18, 111.15, 103.04, 97.80, 85.42, 62.66, 61.19, 54.83, 40.56, 30.12. HR-MS Calcd for C27H29N4O7[M + H]+: 521.2036, Found: 521.2054.
Yellow solid, 66.9% yield, Mp 148.1–148.8 °C. 1H-NMR (400 MHz, Chloroform-d) δ: 12.88 (s, 1H), 7.47 (dd, J = 8.4, 2.1 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.55 (s, 1H), 4.04 (s, 2H), 3.97 (s, 6H), 3.96 (s, 3H), 2.64 (d, J = 76.1 Hz, 8H), 2.18 (d, J = 7.1 Hz, 2H), 1.76 (d, J = 14.8 Hz, 2H), 1.72-1.66 (m, 2H), 1.35 (d, J = 15.0 Hz, 1H), 1.31–1.23 (m, 4H), 1.24-1.15 (m, 2H). 13C-NMR (101 MHz, Chloroform-d) δ: 182.66, 163.01, 158.99, 153.08, 152.26, 150.24, 149.37, 131.88, 124.03, 119.77, 111.26, 108.68, 104.55, 104.18, 98.50, 65.23, 60.72, 56.14, 56.08, 53.91, 53.10, 52.69, 34.95, 31.82, 26.71, 26.07. HR-MS Calcd for C30H39N2O7[M + H]+: 539.2679, Found: 539.2698.
2-(3,4-Dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-8-((4-(pyridin-4-ylmethyl)piperazin-1-yl)methyl)-4H-chromen-4-one (3g)Yellow solid, 54.8% yield, Mp 177.6–178.5 °C. 1H-NMR (400 MHz, Chloroform-d) δ: 12.73 (s, 1H), 8.39 (s, 2H), 7.44-7.27 (m, 2H), 7.20 (s, 1H), 6.96 (dt, J = 9.1, 2.6 Hz, 1H), 6.84 (dd, J = 8.7, 2.6 Hz, 1H), 6.39 (d, J = 2.7 Hz, 1H), 3.91 (d, J = 2.7 Hz, 2H), 3.82 (d, J = 2.6 Hz, 6H), 3.80 (d, J = 2.7 Hz, 3H), 3.39 (d, J = 2.6 Hz, 2H), 2.60 (s, 4H), 2.44 (s, 4H). 13C-NMR (151 MHz, Chloroform-d) δ: 182.66, 160.77, 150.61, 149.82, 149.43, 147.12, 124.47, 123.76, 123.48, 121.78, 119.84, 119.52, 118.88, 111.32, 110.80, 108.73, 108.60, 105.04, 104.76, 104.30, 61.57, 56.15, 53.50, 52.44, 45.57. HR-MS Calcd for C29H32N3O7[M + H]+: 534.2240, Found:534.2224.
All compounds were dissolved in dimethyl sulfoxide (DMSO) to make a 10 mM stock solution, and culture medium RPMI-1640 and MTT were purchased from Solarbio. Fetal bovine serum (FBS) was obtained from every green. Crystal violet staining solution purchased from Beyotime (Shanghai, China). All the other reagents used were of analytical grade.
Cell Culture and TreatmentHuman cancer cells AGS, MDA-MB-231, and Eca-109 were purchased from the American Type Culture Collection (ATCC, Shanghai, China). All of them were cultured in RPMI-1640 with 10% FBS. Cells were cultivated at 37 °C in a 5% carbon dioxide incubator and stimulated with different concentrations of eupatilin derivatives. Cells were fed every 2–3 d and subculture when they reached 70–80% confluence.
MTT AssayCells in the logarithmic growth phase were seeded in 96-well plates at 3000–5000 cells per well. After the cells were cultured for 24 h, different concentrations of compound 3d were treated for 72 h. MTT was added to each well at a final concentration of 0.5 mg/mL. After 4 h in a 37 °C incubator, the medium was aspirated. 150 mL DMSO was then added to each well to dissolve the formazan, and the plate was shaken on a shaker for 10 min. The absorbance was measured by Full wavelength enzyme-linked immunosorbent assay (Epoch2) at the wavelength of 490 nm. Cell viability rate = Abs 490 treated cells/Abs 490 control cells 100%. The SPSS 26.0 software was used to determine the IC50 value. The results were mean ± standard deviation (S.D.) of 3 independent experiments.
Colony Formation AssayAGS cells in the logarithmic growth phase were plated in a 6-well plate at a density of 1000 cells per well, and then the cells were cultured at 37 °C 5% carbon dioxide incubator. After the cells were cultured for 24 h, different concentrations of compound 3d were treated for 9 d. The cells were incubated with 4% paraformaldehyde fixing solution for 15 min and stained with crystal violet solution. After the plate was naturally dried, images were photographed, and the results using Image J. The experiment was repeated 3 times.
Wound Healing AssayAGS cells in the logarithmic growth phase were collected and seeded in 6-well plates at 1.5 × 105 cells per well. After the cells were cultured for 24 h, a line was drawn at the bottom of the plate using the 200 μL gun head. Cells were cultured in RPMI-1640 medium containing 5% FBS. Different concentrations of compound 3d were treated for 72 h and take photos. Images were processed with Photoshop software. The experiment was repeated 3 times.
Western BlotThe cells were treated with different concentrations of compound 3d. The intracellular proteins were extracted from the RIPA lysate buffer (Beyotime) and the protein contents were determined by the BCA protein quantification kit (Beyotime). The experiments of protein extraction, electrophoresis, and transfer were performed as per standard methods. Total proteins were separated by the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) based on the molecular weight of the protein. Blots were blocked at room temperature for 2 h in 5% nonfat dry milk–PBS–0.05% Tween 20. Incubate the primary antibody (corresponding dilution ratio) overnight at 4 °C, then wash 3 times with PBS–Tween20 (0.05%) for 10 min each time. Incubate the horseradish peroxidase-conjugated secondary antibody (corresponding dilution ratio) at room temperature for 2 h, and then wash 3 times with PBS–Tween20 (0.05%) for 10 min each time. Blot exposure was performed with an ECL chemiluminescence solution. The experiment was repeated 3 times.
Statistical AnalysisAll experimental data are the results of the experiment repeated 3 times. Statistical analysis operations and mapping were done using Photoshop, ImageJ, SPSS, GraphPad, etc. All experimental results are expressed as mean ± S.D.
This work was supported by Henan Province Science and Technology Research Project (No.242102310562), the Major Science and Technology Projects of Nanyang (No. 2023ZDZX105), the Program for Science & Technology Innovation Talents in Universities of Henan Province (No. 19HASTIT007), the Nanyang Medical College Science Research Fund Project (No. 2022ZRKX023), Nanyang Science and Technology Research Project (Nos. 23KJGG180, 23KJGG169, 22KJGG106), the Key Research Project of Henan College and University (No. 23B350007), Special Program of Traditional Chinese Medicine Scientific Research of Henan Province (No. 2015ZY02005), and Horizontal Research Project of Nanyang Traditional Chinese Medicine Development Service Center (HX-001).
The authors declare no conflict of interest.
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