2.3.5. Molecular Docking of Compounds **C5** and **C8** with AKT Protein

The activation of carcinogenic signaling pathway is very frequent in the occurrence of cancer, and multiple signaling pathways are involved in the development of gastric cancer [25,26]. To find the cause of cytotoxicity of neocryptolepine derivatives to gastric cancer cell lines, that is, the intracellular targets of compounds **C5** and **C8**, a series of proteins related to cancer cell signaling pathways were docked with compounds **C5** and **C8** by molecular docking experiments. The results (Figure 5) showed that both compounds **C5** and **C8** had the best scores with the AKT protein. Its scores with the AKT protein were −8.529 and −8.359 kcal/mol, respectively. The side of the benzene ring in the small molecule is close to the hydrophobic region consisting of LEU210, TYR263, LEU264, VAL270, and TYR272, forming hydrophobic interactions with the receptor. The benzene ring of the small molecule forms a Π–Π interaction with the pyrrole of tryptophan 80. Therefore, the

molecular docking results suggested that AKT may be a potential intracellular target of compounds **C5** and **C8**.

**Figure 5.** Molecular docking between compounds **C5** or **C8** and AKT proteins. Different colours in **A** and **D** indicated different types of amino acids, and different colours in B, C, E and F indicated different structures of the protein. (**A**) 2D diagram of AKT interacting with small molecule ligand compound **C5**. (**B**) Diagram of compound **C5** binding interaction with AKT protein. (**C**) Overall view of compound **C5** binding to the AKT protein. (**D**) A 2D diagram of AKT interacting with small molecule ligand compound **C8**. (**E**) Diagram of compound **C8** binding interaction with AKT protein. (**F**) Overall view of compound **C8** binding to the AKT protein.

2.3.6. Compounds **C5** and **C8** Inhibited AGS and HGC27 Cell Growth by Regulating PI3K/AKT Signaling Pathway

A variety of cell signaling pathways are involved in the occurrence of gastric cancer [26]. PI3K/AKT is an important signaling pathway, and when this pathway is abnormally activated, it affects the expression of downstream related proteins, thus affecting the occurrence and development of gastric cancer [39]. To further verify the results of molecular docking, Western blot experiments were performed to study the effects of compounds **C5** and **C8** on PI3K/AKT signaling pathway-related proteins in AGS and HGC27 cells. Results are shown in Figure 6, after AGS and HGC27 cells were treated with 5 μM compounds **C5** and **C8** for 48 h, and the expression levels of PI3KCA, AKT, and p-Akt proteins in the PI3K/AKT signaling pathway were down-regulated. The results further confirmed the molecular docking results: compounds **C5** and **C8** may act as AKT inhibitors to play an anti-proliferation role in gastric cancer. In conclusion, compounds **C5** and **C8** may inhibit the proliferation of gastric cancer AGS and HGC27 cells through the PI3K/AKT signaling pathway.

**Figure 6.** Changes of PI3K/AKT signaling pathway related proteins in AGS and HGC27 cells treated with compounds **C5** or **C8** for 48 h. (**A**) The expression of PI3KCA, AKT, and p-AKT of AGS or HGC27 cells was inhibited after treatment with compound **C5** or **C8** for 48 h at 5 μM. (**B**) Changes of PI3KCA, AKT, and p-AKT protein expression levels on AGS and HGC27 cells, after treatment with 5 μM of compounds **C5** and **C8,** for 48 h. \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to negative DMSO control group.
