Immunoproteasome-IN-2 is a selective immunoproteasome inhibitor with an IC50 of 232.3 nM for the LMP7 subunit and 9384.9 nM for the β5 subunit. It additionally displays inhibitory activity against LMP2 and MECL-1 subunits. In an arthritis mouse model, Immunoproteasome-IN-2 demonstrates anti-inflammatory effects and shows low hepatotoxicity and hematotoxicity in toxicology studies, linking its LMP7/β5 selectivity to reduced systemic toxicity. Immunoproteasome-IN-2 can be utilized in arthritis-related research.

Immunoproteasome-IN-2 (compound A33) exhibits inhibitory activity against immunoproteasome subunits in Molt-4 cells (human leukemia T cells) with an IC₅₀ of 12.5 nM for LMP7, 2022.3 nM for β5, 1043.7 nM for LMP2, and 570.2 nM for MECL-1. In mouse B lymphoma cells, Immunoproteasome-IN-2 (1.37-3000 nM) demonstrates exceptional binding efficiency with LMP7, forming a covalent bond faster than with β5 (LMP7 Kᵢ = 1.10 × 10⁰ μM, β5 Kᵢ = 2.07 × 10¹ μM). Additionally, it has a low cardiotoxic risk, showing an IC₅₀ greater than 30 μM on HERG channels in HEK293 hERG cells. It has outstanding selectivity for immunoproteasome subunits and shows no inhibitory activity against β1 (IC₅₀ > 30 μM).

Immunoproteasome-IN-2 (compound A33) administered intravenously at 5 mg/kg three times a week for two weeks exhibits potent anti-inflammatory activity in the BALB/c mouse collagen antibody-induced arthritis (CAIA) model. At 20 mg/kg, it did not result in any mortality in SD rats and maintained lymphocyte, platelet, and reticulocyte levels within normal fluctuation ranges during hematological analysis. In the spleen of BALB/c mice, Immunoproteasome-IN-2 (5 mg/kg, intravenously administered) inhibits LMP7 activity (relative activity of 1.5%) and moderately inhibits LMP2 and MECL-1 (relative activities of 44.5% and 49.2%, respectively), while in the kidney, it shows lower inhibition of the β5 subunit (relative activity of 76.9%). Moreover, Immunoproteasome-IN-2 (5-20 mg/kg, administered intravenously twice a week for four weeks) demonstrates a favorable safety profile in SD rats.