*3.11. Statistical Analysis*

All the data were made with SPSS 22.0 (IBM, Armonk, NY, USA), GraphPad PriM 8.0 (Insightful Science, Boston, MA, USA), and AI 2020 (Adobe Systems, San Jose, CA, USA), and the error bars of all data results were represented by mean ± standard deviation. One-way ANOVA and *t*-test were used to test the differences between the analyzed data.

#### **4. Conclusions**

In summary, we designed and synthesized 36 neocryptolepine derivatives, based on 8-chloroneocryptolepine-based, and performed an in-depth structure-activity relationship study on five human gastric cancer cells, as well as evaluated toxicity on human normal gastric mucosa cells. Compounds **C5** and **C8** showed strong cytotoxicity to AGS and HGC27 cells. Moreover, compound **C8** inhibited the proliferation of AGS and HGC27 cells in a concentration-dependent and time-dependent manner. Cell colony formation and cell migration experiments showed that compound **C8** could inhibit the proliferation and migration of AGS and HGC27 cells, and compound **C5** could also inhibit the proliferation of AGS and HGC27 cells. Compound **C5** had a significant inhibitory effect on HGC cell migration. However, compound **C5** did not significantly affect the migration of AGS cells. In cell cycle and apoptosis experiments, the results showed that compounds **C5** and **C8** did not induce apoptosis of AGS and HGC27 cells but, mainly, caused cell necrosis. Compound **C5** had no significant effect on AGS and HGC27 cell cycle at low concentration. After treatment with 5 μM compound **C5** for 24 h, the AGS cell cycle could be significantly arrested in the G2/M phase. Compound **C8** had no significant effect on AGS and HGC27 cell cycles at 2.5 μM and 5 μM concentrations. The results of molecular docking and Western blot showed that compounds **C5** and **C8** might produce cytotoxic effects by the PI3K/AKT signaling pathway. Compounds **C5** and **C8** showed the best scores with AKT protein in molecular docking results. The experimental results showed that AKT might be the target of the compounds **C5** and **C8** acting on gastric cancer cells. In conclusion, by structural modification of neocryptolepine, two neocryptolepine derivatives with good activity were obtained, compounds **C5** and **C8**, which provided a lead compound for the development of drugs for the clinical treatment of gastric cancer.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijms231911924/s1.

**Author Contributions:** P.C. conceived the project and contributed to the experimental designs. Conceptualization, Y.M. and Y.T.; methodology, Z.Z.; software, Y.T., H.L. and S.C.; validation, K.D., H.Z. (Hao Zhang) and X.J.; formal analysis, J.L.; investigation, Y.N.; resources, Y.L.; data curation, L.T.; writing—original draft preparation, Y.M.; writing—review and editing, Y.M. and P.C.; visualization, J.W. and H.Z. (Hongmei Zhu); supervision, P.C. and Y.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Key Research and Development Program of Gansu Province (Grant No. 21YF5FA112), the Technological Innovation Guidance Program of Gansu Province (Grant No. 21CX6QA127), the Key Program for International S&T Cooperation Projects of China Gansu Province (18YF1WA115), National College Students' Innovation and Entrepreneurship Training Program (Grant No. 202210730011), the College Students' Innovation and Entrepreneurship Training Program of Lanzhou University, China (Grant No. 20220260010) and Innovation and Entrepreneurship Training Program of Lanzhou University, China (Grant No. cxcy202207).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to all lab members for their useful suggestions, support and encouragement.

**Conflicts of Interest:** The authors declare no conflict of interest.
