G9a-IN-4 is a specific inhibitor of G9a with an IC50 of 32 nM. It exhibits high selectivity against other tested lysine/arginine methyltransferases. G9a-IN-4 shows enhanced enzymatic activity and demonstrates stronger antiproliferative effects on all tested cancer cell lines. It significantly inhibits H3K9me2 levels and induces autophagy via ROS generation, leading to apoptosis and G0/G1 cell cycle arrest in CT26 colon cancer cells. G9a-IN-4 is applicable for colon cancer research.

G9a-IN-4 (Compound 31) is a selective inhibitor of G9a/GLP, with IC50 values of 0.2 nM and 0.43 nM, respectively. It exhibits high specificity towards other epigenetic targets tested in AlphaLISA. This compound demonstrates potent inhibitory effects on all tested cancer cell lines, including hematological tumor cells like RPMI8226 (IC50 = 0.85 μM), MV-4-11 (IC50 = 0.49 μM), Jeko-1 (IC50 = 0.7 μM), and Molt4 (IC50 = 1.34 μM), as well as solid tumor cells such as HeLa (IC50 = 1.55 μM), U2OS (IC50 = 3 μM), and CT26 (IC50 = 3.31 μM). At concentrations of 1.25–2.5 μM over 72 hours, G9a-IN-4 significantly reduces the dimethylation levels of H3K9 in a dose-dependent manner in CT26 cells. Additionally, at 2.5–5 μM for 48 hours, it induces the accumulation of reactive oxygen species (ROS) dose-dependently in the same cells. When applied at 2.5–10 μM for 12 hours, it significantly decreases LC3-I expression at 10 μM and increases LC3-II levels at all tested concentrations, thereby enhancing the LC3-II/LC3-I ratio in a dose-dependent manner, inducing autophagy in CT26 cells. Furthermore, G9a-IN-4 (2.5–10 μM, 24 hours) increases the apoptosis rate in CT26 cells from 5.48% to 91.37% in a dose-dependent fashion. It also causes G0/G1 phase arrest in CT26 cells at 2.5–5 μM over 24 hours.

G9a-IN-4, administered at 10 mg/kg via intraperitoneal injection every other day for two weeks, effectively suppresses tumor growth in a mouse model of colon cancer without causing significant weight loss or mortality.