*4.8. Molecular Docking*

The docking results of compound **11c** (ligand) and JAK proteins (receptor) were calculated by the MOE (version 2020) molecular docking program. Crystal structures of all JAK proteins were obtained from the RCSB Protein Data Bank (RCSB PDB). Firstly, compound **11c** was subjected to an energy minimization processing through Chemdraw 3D software (version, 14.0.0.17). Secondly, the water molecules of the receptor protein and the existing small molecule ligands in the crystal were removed. Finally, compound **11c** was introduced into the protein structure and docked in the pocket of the original ligand. Based on the scores calculated by GBVI/WSA combined with free energy, the top 5 results were analyzed using MOE.

#### *4.9. Flow cytometry Analysis of Apoptosis*

A549 and DU145 cells (5 × <sup>10</sup><sup>5</sup> cells/well) were bedded into 6-well plates and treated with **11c** at the indicated concentrations. After 24 h incubation, the cells were washed with phosphate-buffered saline (PBS) twice followed by incubating with the eBioscience™ Annexin VFITC Apoptosis Kit (Invitrogen, City, Carlsbad, California, USA) according to the manufacturer's instructions. Finally, cell apoptosis was analyzed by flow cytometry and the results were processed by flowjo software(version 10.6.2).

#### *4.10. Subcutaneous Tumor Xenograft Model and Antitumor Assay In Vivo*

Nude mice were randomly divided into 4 treatment groups (n = 5/group) before being implanted with subcutaneous tumor xenograft cells. DU145 cells were harvested, counted, and reserved on ice. About 15 × 106 cells were injected to establish a tumor transplantation nude mouse model. The four groups were treated as follows: vehicle group, Gefitinib (100 mg/kg) group, and **11c** (5 mg/kg or 10 mg/kg) group. Tumor size (1/2 × length × width2) and body weight were measured every 3 days for 21 days. On the last day the mice were sacrificed, and the tumor weight was determined [6].

#### *4.11. Immunohistochemical (IHC) Analysis*

The xenograft model tumor of mice was collected and fixed with 4% paraformaldehyde (PFA) for 72 h at 4 ◦C and then embedded in paraffin and cut into slices. After, the slices were baked, dewaxed in xylene, gradients ethanol, and boiled in a microwave to repair antigen [31]. The inactivation of endogenous peroxidase was blocked by incubating with fresh 3% H2O2. The slices were blocked with fat free milk and incubated with the primary antibody (1:3000) at 4 ◦C overnight. After that, they were washed with PBS followed by incubation with the HRP-conjugated secondary antibody at room temperature (Boster, Wuhan, China). Finally, slices were dyed by DAB/H2O2 reaction, brown color in the cell membrane indicated positive staining. Images were captured using an upright fluorescence microscope (Olympus BX53, Tokyo, Japan) [6,52].

#### *4.12. Statistical Analysis*

The data were presented as mean ± SD. GraphPad prism (version 8.0.2) was used for statistical analysis. *p*-value < 0.05 (\*) was considered a significant difference, which was calculated by *t*-test, one and two-way ANOVA (analysis of variance).

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/md21040218/s1, The general procedures for compounds **8a**, **8b**, **8c**, **9a**, **9b**, **9c**, **10a**, **10b**, **10c**, **11a**, **11b**, and **11c**. The data of 1H and 13C NMR of all the compounds are shown in Supplementary Materials. Table S1: The high-throughput-screen result of **aldisine** derivatives. Figure S1: The high-throughput screen of compounds graph.

**Author Contributions:** Conceptualization, C.-Y.Z. and T.J.; experimental studies performed by, D.-P.W., L.-H.W., R.L., N.H. and Q.-Y.Z.; writing–original draft preparation, L.-H.W., D.-P.W. and R.L.; writing–review and editing, C.-Y.Z. and T.J.; Our manuscript is original content and has not been submitted to other journals. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Natural Science Foundation of China: Grant No. 82073759; Shandong Provincial Key Laboratory Platform Project No. 2021ZDSYS11; Shandong Province Major Scientific and Technological Innovation Project: No. 2020CXGC010503; National Natural Science Foundation of China Major Project: No. 81991525.

**Institutional Review Board Statement:** The animal study protocol was approved by Committee of Experimental Animals of the Ocean University of China (protocol code: OUC-SMP-2020-11-01 and date of approval: 2 November 2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The authors declare that supporting data of this study are available within the article and the Supplementary Materials.

**Conflicts of Interest:** Authors have no conflict of interest.
