Design, synthesis, and biological evaluation of sphingosine kinase 2 inhibitors derived from K145

Sphingosine-1-phosphate (S1P) is a critical bioactive lipid mediator that regulates essential cellular processes-including proliferation, survival, migration, and inflammation-through binding to its cognate receptors (S1PRs) on the cell membrane or via direct intracellular actions. Sphingosine Kinase 2 (SphK2) has thus emerged as a promising therapeutic target. In this study, we designed and synthesized a novel series of SphK2 inhibitors (Q-series) based on the lead compound K145. Among these, compounds Q20 (IC₅₀ = 2.6 ± 0.46 μM), Q24 (IC₅₀ = 4.27 ± 1.51 μM), and Q25 (IC₅₀ = 6.25 ± 0.62 μM) displayed potent and selective inhibition of SphK2, while showing negligible activity against SphK1 (IC₅₀ > 50 μM). Notably, Q25 exhibited significant anti-proliferative effects against multiple colorectal cancer cell lines (LOVO, SW480, SW620, HT-29), with IC₅₀ values of 8.09 ± 4.36, 7.77 ± 2.48, 7.38 ± 3.41, and 6.41 ± 2.46 μM, respectively. Mechanistically, Q25 induced S-phase cell cycle arrest and apoptosis. The Q-series compounds (Q20, Q24, Q25) also demonstrated favorable metabolic stability in human liver microsomes, characterized by prolonged half-lives (T_1/2 > 90 min), low intrinsic clearance (CLint(mic) < 15 μL/min/mg), and high parent compound recovery (∼50% remaining after incubation). In vivo pharmacokinetic studies in mice indicated that Q25 is rapidly metabolized, classified as a high-clearance compound, and undergoes extrahepatic elimination. In a SW480 xenograft mouse model, Q25 effectively inhibited tumor growth without observable toxicity. Western blot analysis suggested that its anti-tumor effect is associated with reduced S1P production and subsequent suppression of the NF-κB pathway. In summary, these findings identify Q25 as a promising, selective SphK2 inhibitor worthy of further development as an anticancer agent.