Polθ-IN-9 is an orally active inhibitor of Polθ polymerase with an IC50 of 9.6 nM and a Kd of 47.5 nM. It demonstrates remarkable selectivity, showing no inhibitory activity against other human DNA polymerases, such as Pol α, Pol ε, Pol γ, Pol λ, and Pol μ. In DLD1 BRCA2 KO cells, Polθ-IN-9 exhibits potent antiproliferative activity with an IC50 of 2.9 μM, and it is highly sensitive to MDA-MB-436 cells with an IC50 of 4.9 μM. In MDA-MB-436 xenograft tumor models, the combination of Polθ-IN-9 with Olaparib enhances DNA damage accumulation, increases γH2AX levels, and inhibits tumor growth. Polθ-IN-9 is useful in studying homologous recombination (HR)-deficient cancers such as breast cancer.

Polθ-IN-9 (Compound 33) enhances antiproliferative activity in DLD1 BRCA2 KO cells (IC 50 = 2.9 μM), MDA-MB-436 wild-type cells (IC 50 = 4.9 μM), MDA-MB-436 SHLD2 KO cells (IC 50 = 2.4 μM), and pancreatic cancer cells (Capan-1) (IC 50 = 13.8 μM). At concentrations of 0.097-25 μM over 14 days, Polθ-IN-9 boosts cell viability in MDA-MB-436 cells. It demonstrates enhanced enzymatic activity and significantly improved metabolic stability, with a half-life (T1/2) extended to 48.3 minutes in mouse liver microsomes and 30.5 minutes in human liver microsomes. Additionally, at 93.75-1500 nM over 14 days, Polθ-IN-9 shows synergistic effects with Olaparib, enhancing its antitumor activity in MDA-MB-436 cells.

Polθ-IN-9 (Compound 33) administered orally at 50 mg/kg daily for 25 consecutive days in combination with Olaparib increases γH2AX-positive cells, enhancing DNA damage and inhibiting tumor growth in the MDA-MB-436 xenograft model in NOD SCID mice, without causing significant tissue damage to major organs.