HDAC6-IN-13 (Compound 35m) is a potent and highly selective orally active inhibitor of HDAC6, with an IC50 of 0.019 μM. It also demonstrates inhibitory activity against HDAC1, HDAC2, and HDAC3, with IC50 values of 1.53 μM, 2.06 μM, and 1.03 μM, respectively. HDAC6-IN-13 exhibits substantial blood-brain barrier permeability and displays anti-inflammatory properties [1].

HDAC6-IN-13 (Compound 35m), at concentrations ranging from 0.1 to 1 μM over 24 hours, demonstrates high selectivity for HDAC6 over class I HDAC enzymes. This compound is characterized by its slow-on and slow-off tight-binding inhibition of HDAC6, contrasting with its rapid binding affinity for HDAC1, 2, and 3. Furthermore, HDAC6-IN-13 exhibits anti-inflammatory effects in vitro at higher concentrations of 5 to 20 μM after an 8-hour exposure. Western Blot Analysis on the MV4 11 and J774A.1 cell lines, using 0.1, 0.2, 0.5, and 1 μM concentrations for 24 hours, revealed a concentration-dependent increase in acetylated tubulin (Ac-Tubulin), with no significant changes in acetylated histone H3 (AcHH3) and H4 (AcHH4) at the 1 μM level. Another analysis on J774A.1 cells, at concentrations of 5, 10, and 20 μM for 8 hours, demonstrated dose-dependent inhibition of pro-caspase 1 cleavage to p20 and prevented the interaction between HDAC6 and dynein, highlighting its potential for therapeutic applications in inflammation and cancer.

HDAC6-IN-13 (Compound 35m) at a dosage of 20 mg/kg, administered both orally (p.o.) and intraperitoneally (i.p.), once, significantly reduces LPS-induced inflammation in mice, demonstrating its potent anti-inflammatory effects. Additionally, when given orally at the same dosage, HDAC6-IN-13 exhibits high oral bioavailability (93.4%) and substantial blood-brain barrier (BBB) permeability. This compound was tested in male C57BL/6 WT mice using an LPS-induced endotoxic shock model and male CD-1 mice for pharmacokinetic studies, revealing a notable decrease in serum IL-1β levels post-administration. The pharmacokinetic analysis showed a maximum plasma concentration (Cmax) of 4604 ± 551 ng/mL and 5570 ± 551 ng/mL, half-life (t 1/2) of 7.95 ± 0.370 h and 6.80 ± 0.145 h, and area under the curve (AUC) of 2755 ± 395 ng h/mL and 10292 ± 1385 ng h/mL for intravenous (5 mg/kg) and oral (20 mg/kg) administration, respectively. These results, backed by blood samples analyzed through LC-MS/MS, underscore the effective delivery and promising pharmacodynamic profile of HDAC6-IN-13 for combating inflammation.