RUNX1/ETO tetramerization-IN-1 is a small-molecule inhibitor targeting NHR2 of RUNX1/ETO, effectively inhibiting tetramerization with an EC50 value of 0.25 μM. This compound restores gene expression down-regulated by RUNX1/ETO and demonstrates anti-leukemic activity by inhibiting proliferation of RUNX1/ETO-dependent SKNO-1 cells and reducing tumor growth in a mouse model [1] [2] [3].

RUNX1/ETO, comprising the RUNX1 gene's DNA-binding Runt-domain5 and the ETO gene's four nervy homology regions (NHR1-4), is notable for its NHR2 domain, which facilitates tetramerization. RUNX1/ETO tetramerization-IN-1 (compound 7.44) at various concentrations (1 μM and 10 μM over 3, 5, 7 days; 25 μM and 50 μM over 5 days; and 100 μM over 7 days) has been shown to selectively diminish the viability and inhibit the growth, besides inducing differentiation, of RUNX1/ETO-dependent human leukemic cells (specifically SKNO-1, Kasumi-1, and K562 cells) and RUNX1/ETO^tr-expressing CD34+ progenitor cells while showing no significant effect on U937 cells. Furthermore, this compound, with favorable physicochemical and ADME (Absorption, Distribution, Metabolism, and Excretion) properties, including high aqueous solubility (60 μg/mL), stability in buffer and plasma, and low hepatic intrinsic clearance, not only demonstrates inhibition of critical cytochrome P450 enzymes (CYP2B6, 2C9, 2C19, and 3A4) but also inhibits the c-Jun N-terminal kinase (JNK) pathway at 50 μM over 16 hours. These findings underscore its potential as a targeted therapeutic agent in treating RUNX1/ETO-dependent leukemia, supported by cell viability assays and pharmacokinetic studies that reveal its significant stability, potent plasma protein binding, and negligible degradation in physiological conditions.

Compound 7.44 (RUNX1/ETO tetramerization-IN-1) administered at a dosage of 200-250 μg/kg via intraperitoneal injection five times weekly for 130 days significantly delays tumor growth in mice inoculated with RUNX1/ETO cells. This regimen resulted in a notable reduction in leukemic cell dissemination and ensured 75% survival in NSG immunodeficient mice (NOD.Cg-Prkdc scid Il2rg tm1WjI /SzJ) injected with Kasumi-1 cells at the end of the treatment period.