*2.3. Preliminary Cytotoxic Mechanism of Compounds C5 and C8 against AGS and HGC27 Cells* 2.3.1. The Cytotoxic Effect of Compounds **C5** and **C8** on Gastric Cancer Cells

The cytotoxicity of compounds **C5** and **C8** were systematically evaluated by an MTT assay. As shown in Figure 2, we performed cytotoxicity experiments on five gastric cancer cell lines—AGS, HGC27, MKN45, MGC803, and SGC7901—using neocryptolepine and cisplatin as the control. The results showed that the IC50 values of neocryptolepine were 20, 18, 19, 40, and 37 μM on AGS, HGC27, MKN45, MGC803, and SGC7901 cells, after 48 h, respectively. Compared with the cytotoxicity of the parent nucleus of neocryptolepine, we found that compounds **C5** and **C8** had stronger cytotoxicity by structural modification. The IC50 values of compound **C5** on AGS, HGC27, MKN45, MGC803, and SGC7901 cells, for 48 h, were 9.2, 6.6, 5.9, 13, and 8.7 μM, respectively. The IC50 values of compound **C8** on AGS, HGC27, MKN45, MGC803, and SGC7901 cells, after 48 h treatment, were 6.9, 4.3, 3.5, 10, and 10 μM, respectively. Compared with the positive drugs, compounds **C5** and **C8** showed significantly stronger cytotoxicity than cisplatin. In Figure 2G, it was found that the cytotoxic effects of compounds **C5** and **C8** to normal cells (IC50 = 12.8 and 12.6 μM, respectively) were relatively weaker than gastric cancer cells. We also performed concentration-dependent and time-dependent experiments on the cytotoxicity of compound **C8** and cisplatin to AGS, and the results showed (Figure 2) that compound **C8** and cisplatin could inhibit the growth of gastric cancer cell lines AGS, HGC27, and MKN45 in a concentration-dependent and time-dependent manner. Moreover, compound **C8** showed strong cytotoxicity to gastric cancer cells at 5 μM, while cisplatin was weak. Therefore, the results of the MTT assay showed that we improved the cytotoxicity of the parent nuclear structure of neocryptolepine by structural modification, and compounds **C5** and **C8** showed stronger cytotoxicity effects compared with the positive drug cisplatin.

**Figure 2.** Cytotoxicity evaluation of neocryptolepine (NC), compounds **C5**, **C8,** and CIS on gastric cancer cell lines. (**A**–**F**) The cytotoxic effects of NC, compound **C5**, compound **C8,** and CIS at different concentrations on AGS, HGC27, MKN45, MGC803, GES-1, and SGC7901 cells, at 48 h, respectively. (**G**) IC50 comparison of NC, compound **C5**, compound **C8,** and CIS on gastric cancer cell lines at 48 h. (**H**) Chemical structure of compounds **C5** and **C8**. (**I**–**K**) The cytotoxic effects of compound **C8** with different concentrations on AGS, HGC27, and MKN45 cells at 24 h, 48 h, and 72 h, respectively. (**L**–**N**) Cytotoxic effects of CIS at different concentrations on AGS, HGC27, and MKN45 cells at 24 h, 48 h, and 72 h. Values are shown as the means ± standard, n = 3. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to negative DMSO control group.

2.3.2. Compounds **C5** and **C8** Inhibited the Proliferation and Migration of AGS and HGC27 Cells

To study the effects of compounds **C5** and **C8,** on the proliferation of AGS and HGC27 cells, the colony formation assays were performed. The results showed that, as shown in Figure 3A, compound **C8** at 1 μM and 2 μM, as well as compound **C5** at 2 μM and 4 μM, could inhibit the proliferation of AGS HGC27 cells. When the concentration of compound **C8** was 4 μM and compound **C5 was** at 6 μM, the proliferation of AGS and HGC27 cells was completely inhibited, and no cell clones were formed. Therefore, our results showed that compounds **C5** and **C8** had a certain inhibitory effect on the proliferation of AGS and HGC27 cells, and the proliferation of AGS and HGC27 cells could be completely inhibited when compounds **C5** and **C8** reached a certain concentration.

**Figure 3.** The proliferation and migration of AGS and HGC27 cells were inhibited after treatment compounds **C5** or **C8** for 48 h. (**A**) The proliferation of AGS and HGC27 cells were inhibited for treatment of different concentrations of compounds **C5** or **C8** for 48 h. (**B**) Statistical analysis of AGS and HGC27 cell migration numbers. (**C**) The proliferation of AGS and HGC27 cells was inhibited after the treatment of compounds **C5** and **C8** for 8–10 days. (**D**) Statistical analysis of AGS and HGC27 cell proliferation numbers. Values are shown as the means ± standard. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to negative DMSO control group.

The migration of tumor cells is an important embodiment of the lethal effect of tumor. Therefore, cell migration experiments were performed to study the effects of compounds **C5** and **C8** on the migration ability of AGS and HGC27 cells. Results are shown in Figure 3C, after treatment with compound **C8** at 1.25 μM for 48 h, and the migrating ability of AGS and HGC27 cells was significantly inhibited. As the concentration of compound **C8** increased, the number of AGS cells decreased from 243 to 28, and the number of HGC27 cells decreased from 170 to 47. Similarly, compound **C5** inhibited the migration of HGC27 cells in a concentration-dependent manner. However, our results showed that compound **C5** did not have an obvious effect on the migration of AGS cells. It may be that the tumor specificity caused the different inhibitory effect of compounds on AGS and HGC27 cells. In conclusion, different concentrations of compound **C8** inhibited the migration of AGS and HGC27 cells, and compound **C5** also inhibited the migration of HGC27 cells at certain concentrations.

#### 2.3.3. The Effects of Compounds **C5** and **C8** on AGS and HGC27 Cell Cycle

Cell cycle regulation plays an important role in anti-tumor drugs. Therefore, to evaluate the effect of neocryptolepine derivatives on cell cycle, flow cytometry was used to test the changes of AGS and HGC27 cell cycle after neocryptolepine derivatives treatment. Results are shown in Figure 4A, AGS cells were treated with 2.5 μM and 5 μM compound **C8** for 24 h, and there was no significant change in AGS and HGC27 cell cycles. After the AGS and HGC27 cells were treated with different concentrations of compound **C5** for 24 h, it was found that HGC27 cells died when treated with 10 μM of compound **C5**. Therefore, we reduced the concentration of compound **C5**. The results showed that compound **C5** had no significant change in the HGC27 cell cycle when treated with 2.5 μM and 5 μM. However, the AGS cells were treated with 10 μM compound **C5** for 24 h, the AGS cells were mainly arrested in the G2/M phase. In the apoptosis experiment, although compound **C5** caused the necrosis of most AGS cells after treatment for 48 h, AGS cells did not die completely after treatment with compound **C5** for 24 h in the cell cycle. The cell cycle experiments suggested that compounds **C5** and **C8** had no significant effect on the cell cycles of AGS and HGC27 cells at low concentration, while compound **C5** could significantly block AGS cells in G2/M phase at high concentration.

**Figure 4.** *Cont*.

**Figure 4.** Effects of different concentrations of compounds **C5** or **C8** on AGS cell cycle and cell apoptosis after treatment for 24 h or 48 h. (**A**) Effects of different concentrations of compounds (**C5**) and **C8** on AGS and HGC27 cell cycle after 24 h treatment. (**B**) Analysis of the proportion of AGS and HGC27 cells, at different cell stages, before and after treatment with compounds **C5** or **C8**. (**C**) Statistical analysis of apoptosis percentage of AGS and HGC27 cells. (**D**) Treatment of AGS or HGC27 cells, with different concentrations of compound **C5** or **C8** for 48 h, caused cell necrosis.

#### 2.3.4. The Effects of Compounds **C5** and **C8** on Apoptosis of AGS and HGC27 Cells

Apoptosis is a common characteristic of most chemotherapeutic drugs that can exert antitumor effects. To investigate whether the neocryptolepine derivatives exert cytotoxic effects by cell apoptosis, we detected the effects of compounds **C5** and **C8** on the apoptosis of AGS and HGC27 cells by flow cytometry. The results (Figure 4C) showed that, when the concentration of compound **C8** was 2.5 μM, it did not cause the apoptosis or necrosis of AGS and HGC27 cells, but when the concentration reached 5 μM, compound **C8** had caused the necrosis of AGS and HGC27 cells, and when the concentration reached 10 μM, all AGS and HGC27 cells had apoptosis or necrosis. Similarly, compound **C5** showed the same effect in AGS and HGC27 cells, but it should be noted that, after treatment with 10 μM compound **C5** for 48 h, most AGS cells died. Moreover, cell cycle results also showed that compound **C5** treatment of AGS cells, at a concentration of 10 μM for 24 h, did not completely cause the death of AGS. Therefore, it can be concluded that compound **C5** and **C8** do not exert cytotoxic effects, mainly, through apoptosis but directly lead to cell necrosis.
