Design, synthesis, and cytotoxic activities of isaindigotone derivatives as potential anti-gastric cancer agents

Figures & data

Figure 1. Isaindigotone and its derivatives. (A) General design strategy of the title compounds. (B) Chemical structures of Isaindigotone derivatives.

Scheme 1. Synthesis of isaindigotone derivatives. Reagents and conditions: (A) pyrrolidin-2-one or 2-piperidone, POCl₃, toluene, reflux; (B) 1-methylpiperazine, DMF, K₂CO₃, reflux; (C) different substituted benzaldehydes, AcOH, AcONa, reflux.

Figure 2. AGS and GES-1 cell viability after Compound 6 and cisplatin treatment. (A, B) Concentration and time dependent anti-proliferation effects of Compound 6 on AGS cells. (C, D) After 48 h treatment, the cytotoxic effects of the most active compounds, Isaindigotone and cisplatin on GES-1 cells. Values are shown as the means ± standard, n = 3. * p< 0.05, **p< 0.01, *** p< 0.001 compared with Pt.

Table 1. Anti-proliferative activities (IC₅₀, μM) of the derivatives for 48 h.

Compounds	PANC-1	HCT116	SMMC7721	AGS
1	>50.00	>50.00	>50.00	7.93 ± 0.97
2	>50.00	>50.00	>50.00	20.71 ± 0.93
3	>50.00	>50.00	>50.00	>50.00
4	19.15 ± 2.22	>50.00	>50.00	20.47 ± 2.65
5	>50.00	>50.00	>50.00	1.96 ± 0.84
6	9.94 ± 1.57	17.95 ± 0.00	>50.00	2.23 ± 0.01
7	>50.00	22.01 ± 1.25	>50.00	19.62 ± 0.31
8	>50.00	>50.00	>50.00	44.50 ± 2.10
9	>50.00	>50.00	>50.00	22.32 ± 1.05
10	>50.00	>50.00	>50.00	13.40 ± 0.11
11	>50.00	>50.00	>50.00	13.29 ± 1.90
12	28.12 ± 1.54	>50.00	>50.00	17.62 ± 2.54
13	>50.00	>50.00	>50.00	>50.00
14	37.32 ± 2.60	>50.00	27.31 ± 1.42	18.37 ± 3.06
15	>50.00	>50.00	>50.00	12.76 ± 3.13
16	31.93 ± 1.55	>50.00	40.42 ± 1.86	9.07 ± 1.19
17	21.37 ± 1.52	2.80 ± 0.90	>50.00	6.79 ± 1.03
18	14.87 ± 0.30	25.08 ± 2.22	49.05 ± 0.80	9.50 ± 1.43
19	37.12 ± 2.76	>50.00	>50.00	9.11 ± 0.22
20	>50.00	>50.00	>50.00	>50.00
21	>50.00	>50.00	>50.00	43.82 ± 1.00
22	>50.00	>50.00	>50.00	>50.00
23	23.94 ± 1.82	>50.00	>50.00	20.29 ± 0.44
24	>50.00	>50.00	>50.00	18.74 ± 2.07
25	>50.00	>50.00	>50.00	13.62 ± 1.23
26	>50.00	>50.00	>50.00	14.22 ± 1.90
Pt	>50.00	>50.00	>50.00	>50.00

Table 2. The structure of isaindigotone derivatives.

Figure 3. Cell cycle and apoptosis assays were tested with flow cytometry. (A, C) AGS cells were treated with Compound 6 (0.0, 2.0, 3.0, 4.0, and 5.0 μM) for 24 h, and analysed by flowed cytometry (B, D) Analysis of cell apoptosis induced by Compound 6 using Annexin V/PI assay. * p< 0.05, **p< 0.01, *** p< 0.001 compared with negative control.

Figure 4. Compound 6 induced apoptosis and mitochondrial depolarisation in AGS cells. (A) AGS cells were treated with Compound 6 (0.0, 2.0, 3.0, 4.0, and 5.0 μM) for 48 h and stained with Hoechst 33258. DMSO was used as negative control. (A) AGS cells were treated with Compound 6 (0.0, 2.0, 3.0, 4.0, and 5.0 μM), CCCP (5.0 μM) and DMSO for 48 h and MMP was determined using the JC-10 kit.

Figure 5. The result of molecular docking of Compound 6 and ipatasertib with AKT1 (PDB: 6HHF). (A, B) The binding conformation of ipatasertib in the active site of AKT1. (C, D) The binding conformation of Compound 6 in the active site of AKT1.

Figure 6. The effect of Compound 6 and the expression of mitochondrial-dependent pathway related proteins and PI3K/AKT/mTOR signal pathway related proteins and AKT1 inhibition effects of Compound 6 and positive control ipatasertib. (A, B) protein levels of PI3K, AKT, p-AKT, mTOR, Bcl-2, Bax, and cleaved-caspase 3. (C, D) Statistical analysis. (E) Inhibition effects of Compound 6. (F) Inhibition effects of Ipatasertib. * p< 0.05, **p< 0.01, *** p< 0.001 compared with control.

Figure 7. Effects of Compound 6 on tumour cell migration. (A) Transwell assay showed that cell migration of AGS were inhibited by Compound 6. (B) MTT assay showed that Compound 6 (0.0, 1.0, and 2.0 μM) inhibited AGS cell proliferation at 24, 48, and 72 h. (C) Statistic analysis. Values are shown as the means ± standard, n = 3. * p< 0.05, **p< 0.01, *** p< 0.001 compared with negative control (DMSO).