DHW-221 is an orally active dual PI3K/mTOR inhibitor demonstrating potent inhibition at nanomolar levels across all four Class I PI3K isoforms and mTOR (PI3Kα, IC50= 0.50 nM; PI3Kβ, IC50= 1.9 nM; PI3Kγ, IC50= 1.8 nM; PI3Kδ, IC50= 0.74 nM; mTOR, IC50= 3.9 nM). By blocking the PI3K/Akt/mTOR pathway, DHW-221 induces mitochondrial apoptosis and paraptosis (through endoplasmic reticulum stress and MAPK signaling) and causes cell cycle arrest, thereby inhibiting cell migration, invasion, and angiogenesis to exert its antitumor effects. It suppresses tumor growth in A549/Taxol and HCC827 mouse models and is applicable in research related to non-small cell lung cancer (NSCLC), colon cancer, and breast cancer[1][2][3].

PI3Kα:0.50 nM

DHW-221 (compound 8i) demonstrates antiproliferative effects against standard human cancer cell lines with IC50 values of 0.36 μM for T47D, 0.14 μM for HCT116, and 0.31 μM for MCF-7. At concentrations ranging from 0.3 to 3 μM, DHW-221 decreases phosphorylated Akt (S473) levels in HCT116 cells in a dose-dependent manner without significantly affecting total Akt protein expression. It inhibits HCT116 cell proliferation dose-dependently over a range of 0 to 5 μM for two weeks. DHW-221 also impedes HCT116 cell migration and invasion in a dose-dependent fashion between 0.3 to 3 μM over 0 to 48 hours. HCT116 apoptosis is induced by DHW-221, accompanied by nuclear fragmentation and chromatin condensation. The compound can bind to the PI3K kinase site, forming hydrogen bonds with Val882, Lys833, and Thr887 and Pi-Pi interactions with numerous amino acid residues, similar to Omipalisib. In A549/Taxol and A549 cells, DHW-221 exhibits notable concentration and time-dependent cytotoxicity with IC50 values of 0.5274 μM and 0.4242 μM, respectively, after 72 hours. At 0 to 2.4 μM over 48 to 72 hours, it shows significant inhibitory activity against MDR cancer cells, causing severe cell damage compared to controls. DHW-221 enhances intracellular Rho-123 accumulation and significantly downregulates P-gp expression in A549/Taxol cells, indicating its role as a P-gp function and protein expression inhibitor. At 50 nM over 48 hours, it significantly increases the cytotoxic effects of Taxol, similar to P-gp inhibitor Verapamil, suggesting its potential as a P-gp inhibitor and MDR reversal agent. Through mitochondrial pathways, ER stress, and MAPK signaling, DHW-221 induces apoptosis and paraptosis in A549/Taxol cells. It arrests the cell cycle at G0/G1 phase via cyclin D1, CDK4, CDK6 downregulation, and p21 upregulation in A549/Taxol and A549 cells. Between 0.05 to 0.15 μM for 48 hours, it reverses EMT phenotypic changes, hindering A549/Taxol cell migration and invasion. It significantly blocks the PI3K/Akt signaling pathway by lowering PI3Kp110α and phosphorylated Akt levels in both A549 and A549/Taxol cells. In the range of 25 to 400 nM over 1 to 3 hours, it hampers endothelial tube formation in HUVEC by inhibiting the PI3K/HIF-1α/VEGF signaling axis. In vitro, DHW-221 inhibits microvessel sprouting in rat aortic ring assays at concentrations of 1.56 to 6.25 µM. Finally, it suppresses new blood vessel formation in the chick embryo chorioallantoic membrane at 1.25 to 5 µM without affecting embryo viability.

DHW-221 administered at doses of 10, 20, and 40 mg/kg (via gavage, once daily for two weeks) inhibits tumor growth in the A549/Taxol xenograft mouse model through the nuclear translocation of FOXO3a, without causing weight changes or toxicity. Additionally, DHW-221 (10, 20, and 40 mg/kg, orally, once daily for 21 days) suppresses tumor growth in the HCC827 xenograft mouse model.