4-(tert-butyl)-N-(8-chloro-5-methyl-5H-indolo[2,3-b]quinolin-11-yl)benzenesulfonamide (**D6**)

Yield, 61%; white solid, m.p. > 400 ◦C (DMSO); 1H NMR (400 MHz, DMSO-*d*6) *δ* 12.73 (s, 1H), 9.01 (d, *J* = 9.9 Hz, 1H), 8.06 (d, *J* = 8.3 Hz, 1H), 8.02 (d, *J* = 8.7 Hz, 1H), 7.91 (t, *J* = 7.8 Hz, 1H), 7.80 (d, *J* = 8.1 Hz, 2H), 7.57–7.53 (m, 3H), 7.51 (s, 1H), 7.09 (d, *J* = 8.4 Hz, 1H), 4.16 (s, 3H), 1.33 (s, 9H). 13C NMR (100 MHz, DMSO-*d*6) *δ* 156.71, 148.30, 144.72, 141.53, 138.53, 137.83, 132.58, 130.04, 129.60, 125.74 (2C), 125.53 (2C), 124.22, 123.05, 122.44, 121.60, 118.08, 116.34, 110.53, 99.61, 35.14, 34.73, 31.46 (3C). MS-ESI *m*/*z*: calcd for C26H24ClN3O2S[M + H]+: 478.1278; found: 478.2500.

#### *3.3. Cell Culture*

The human gastric cell lines AGS was obtained from the American Type Culture Collection (ATCC), AGS(LOT:70012225). The human gastric cancer cell lines HGC27, MKN45, SGC7901, MGC803, liver cancer cell SMMC7721, and gastric mucosa GES-1 were obtained from the genetic resource reserve of our laboratory. The AGS, HGC27, MKN45, and SMMC7721 cells were cultured in RPMI medium (Solarbio Invitrogen Corp., Beijing, China), and the SGC7901, MGC803, and GES-1 cells were cultured in a DMEM (high glucose) medium (Solarbio Invitrogen Corp., Beijing, China) containing 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at 37 ◦C in humidified atmosphere of 5% CO2 in air.

#### *3.4. MTT Assay*

The cells were seeded in 96 well plates at the density of 8000 per well. Then, the cells were incubated in the cell incubator for about 24 h. The experiment included the treatment group, negative DMSO group, and positive group. Each group was set with 4–8 repeated wells. After the cells were incubated in the cell incubator for the corresponding time, 10 μL MTT solution of 5 mg/mL was added. The plates were incubated in the cell incubator for another 4 h. The formazan was solved in DMSO, and the absorbance value at 490 nm was measured with a microplate reader.

### *3.5. Colony Formation Assay*

When AGS and HGC27 cells were in the logarithmic growth phase, AGS and HGC27 cells were inoculated into 24-well plates at a density of 1000 cells/well, and incubated in a cell incubator for 24 h. Cells were treated with compound **C5** or **C8** for 8–10 days. The cells were washed twice with PBS buffer, fixed in 4% paraformaldehyde solution for 40 min, and then stained with 1% crystal violet solution for 20 min. After staining, the cells

were washed twice with fresh PBS buffer solution. The photos were taken, and Image J (National Institutes of Health, Bethesda, MD, USA) was used for counting analysis.

#### *3.6. Migration Assay*

A 600 μL complete medium, containing 20% fetal bovine serum, was added to the lower chamber of the transwell chamber. The cells were counted, and compounds with different concentrations were prepared using 1% complete medium. There was 150 μL cells suspension added into the upper chamber of transwell with a cell density of 50,000 cells/well. The suspension was placed in a cell incubator for further incubation for 48 h. The cells were washed twice with PBS and were fixed with 4% paraformaldehyde solution for 40 min, followed by staining with 1% crystal violet solution for 20 min. The cells in the upper transwell cells were carefully erased with cotton swabs. The number of migrating cells of the lower compartment were observed under a microscope and analyzed by Image J counting.

#### *3.7. Cell Cycle Analysis*

The AGS or HGC27 cells were seeded in six-well plates at a density of 800,000 cells/well. The plates were incubated in a cell incubator for about 24 h and treated with different concentrations of compounds. After 24 h, cells were collected and fixed with 1 mL of precooled 70% anhydrous ethanol for overnight. Additionally, 100 μL RNase A were added and incubated at 37 ◦C for 30 min. Then, the cells suspension was transferred to flow tube, and 400 μL PI solution was added and incubated at 4 ◦C for 30 min. The cells proportion of different cell cycles were detected by A BD FACSCanto II (BD LSRFortessa, USA). Modfit is used to process and analyze data.

#### *3.8. Cell Apoptosis Analysis*

AGS and HGC27 cells were seeded out in six-well plates with a cell density of 700,000 cells/well and incubated in a cell incubator for 24 h. Then, the cells were treated with different concentrations for 48 h. The supernatant was collected, the cells were washed with PBS twice, and the washing solution was collected. Then, the cells were digested and collected with trypsin without EDTA, centrifuged at 1000 r/min for 5 min, the cells were washed with PBS twice, and the cells were collected. The cell density was adjusted to <sup>1</sup> − <sup>5</sup> × <sup>10</sup><sup>6</sup> cells/mL. Then, 100 <sup>μ</sup>L cell suspension was added into the flow tube, and 5 <sup>μ</sup><sup>L</sup> Annexin V/Alexa Fluor 488 solution was added. After incubation for 5 min, 10 μL PI and 400 μL PBS solution were added. A BD FACSCanto II (BD Biosciences, USA) was used for detection, and Flowjo was used for statistical analysis.

### *3.9. Molecular Docking Analysis*

Schrödinger 10.1 software (Schrödinger, USA) was used for molecular docking correlation analysis. The crystal structure of AKT protein binding to the molecule during docking was obtained from the PDB database (PDB: 6hhf). In this experiment, we used the Prime module in the Schrödinger software to fill in the missing side chains and loops in the protein, processed the protein by protonation, dehydration, hydrogenation, etc., and then minimized and initially optimized the protein under the OPLS2005 force field. The compound was then docked with the protein AKT appropriately. During the docking process, the small molecular center bound to the protein was used as the docking site of the compound to complete the whole docking process. The crystal structures of all proteins bound to molecules in docking were obtained from the PDB database, and their PDB ID, resolution, and sources could be obtained in Supplementary Information Tables S1 and S2.

#### *3.10. Western Blotting Assay*

When AGS or HGC27 cells grew to about 80–90%, they were inoculated in six-well plates at a density of 500,000 cells/well and, then, incubated in cell incubator for 24 h. The cells were treated with a certain concentration of compounds for 48 h and DMSO was used as a negative control group. Cells were lysed with a high-potency tissue cell lysate containing 1% protease inhibitor PMSF and phosphatase inhibitor RIPA on ice for 30 min. Cell suspension was suspended every 10 min. After centrifugation, the supernatant was collected, and 5× protein denaturation buffer was added. The supernatant was placed in a constant temperature dry metal bath at 100 ◦C for denaturation for 10 min. The supernatant was stored at −20 ◦C. The protein was isolated with 10% separation gel and 5% concentrate gel. The total protein content was 25 μg per well. After electrophoresis, the protein was transferred to 0.22 μm PVDF membrane and blocked on a 5% skim milk shaker for 2 h. Then, the PVDF membrane was washed three times on the shaker with TBST buffer solution, 10 min each time, and blocked with primary antibody overnight at a ratio of 1:2000 at 4 ◦C. The PVDF membrane was washed with TBST buffer solution and incubated with a second antibody, at the ratio of 1:10,000, at room temperature for about 1 h. BCL luminescence system was used to record the photo, and the recorded protein bands were saved. Image J was used for statistical analysis of gray values.
