Axl-IN-21 is an orally bioavailable selective AXL inhibitor with a Kd of 2.7 nM and an IC50 of 4.0 nM. It demonstrates strong inhibitory activity against various cancer-related kinases while maintaining kinase selectivity, including Mer (Kd = 1.4 nM), DDR1 (IC50 = 22.2 nM), HIPK4 (Kd = 11.0 nM), and LOK (Kd = 10 nM). By blocking the AXL/STAT3/ABCG1 signaling pathway induced by GAS6 from tumor-associated fibroblasts, Axl-IN-21 can overcome tumor microenvironment-driven resistance, restore chemotherapy sensitivity, and inhibit drug efflux. In MDA-MB-231 cells, it inhibits TGF-β1-induced epithelial-mesenchymal transition, cell migration, and invasion. Axl-IN-21 exhibits no significant toxicity to non-cancer cells and is relevant for research in triple-negative breast cancer and gastric cancer.

DDR1:22.2 nM

Axl-IN-21 (compound 9im) effectively inhibits AXL phosphorylation in a dose-dependent manner within SNU668 and MKN1 gastric cancer cells, even when co-cultured with cancer-associated fibroblasts (CAF). This compound reverses epithelial-mesenchymal transition by restoring E-cadherin and reducing N-cadherin expression, and it enhances chemosensitivity by blocking CAF-induced AXL activation and downstream signaling, despite CAF co-cultivation normally reducing apoptosis markers (cleaved PARP and cleaved Caspase-3) after 5-Fluorouracil (5-FU) and cisplatin chemotherapy. Axl-IN-21 (0.5-32 μM) shows lower cytotoxicity in non-cancerous cells compared to BGB324. At concentrations of 0.1-0.5 μM, Axl-IN-21 suppresses the JAK1/STAT3, PI3K/AKT, and MEK/ERK signaling pathways and reduces CAF-induced migration in SNU668 cells, even in the presence of CAF-conditioned medium or direct co-culture. In SNU668 cells co-cultured with CAF and/or treated with chemotherapeutic agents, Axl-IN-21 (0.5-2 μM) inhibits CAF-induced AXL activation, downstream signaling, gastric cancer cell migration, and chemoresistance. When treated with recombinant GAS6, Axl-IN-21 (0.1-0.5 μM) prevents upregulation of ABCG1 expression and phosphorylation of AXL, JAK1/STAT3, PI3K/AKT, and MEK/ERK in AXL-activated gastric cancer cells. The compound reduces ABCG1 expression and increases cleaved Caspase-3 positive cells in combination with CAF and 5-FU, indicating enhanced apoptosis. In MDA-MB-231 breast cancer cells, Axl-IN-21 (0.03-3 μM, 6 h) serves as a potent Axl kinase inhibitor. It also dose-dependently restrains TGF-β1-induced Axl activation (0.04-5 μM, 96-144 h) and inhibits cell migration and invasion (0.04-5 μM, 24 h).

Axl-IN-21 (compound 9im), administered orally at 90 mg/kg once daily for three weeks, enhances the expression of E-cadherin in CAF-mixed SNU668 xenograft tumors, suggesting its ability to inhibit CAF-induced AXL activation and downstream signaling pathways. Additionally, Axl-IN-21 at 30 or 90 mg/kg, given orally once daily for 21 days, is used in a xenograft model of highly metastatic 4T1 mouse breast cancer cells.