A solution of 4-nitrobenzyl chloroformate (9) (100 mg, 0.46 mmol, 1 eq.) in 3 mL of THF was added dropwise to a mixture of the appropriate aniline derivative (I-III) (0.46 mmol, 1 eq.) and K2CO3 (83 mg, 0.6 mmol, 1.3 eq.) in dry THF (10 mL) at 0º C. The reaction mixture was stirred at RT for 4 h. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure. The obtained residue was purified using MPLC (DCM: MeOH) to give the corresponding product. Recombinant human proteins HDAC1, HDAC2, and HDAC3/NCOR1 were purchased from ENZO Life Sciences AG (Lausen, CH). Recombinant human HDAC8 was produced by Romier et al. (IGBMC, Univ. Strasbourg), as described in . The in vitro testing on recombinant HDACs 1-3 was performed with a fluorogenic peptide derived from p53 (Ac-RHKK(Acetyl)-AMC) as reported . The measurements were performed in assay buffer (50 mM Hepes, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP, and 0.2 mg/mL BSA, pH 7.4 adjusted with NaOH) at 37 ˚C. All compounds at different concentrations were incubated with 10 nM HDAC1, 3 nM HDAC2, or 3 nM HDAC3 (final concentration) for at least 5 min. The reaction was first started with the addition of a fluorogenic peptide substrate (20 µM final concentration) and incubated for 30 min for HDAC2 and HDAC3 and 90 min for HDAC1. The reaction was then stopped with a solution of 1 mg/mL trypsin and 20 µM SAHA in 1 mM HCl and incubated for 1 h at 37 ˚C. The fluorescence intensity was measured with an Envision 2104 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA) with an excitation wavelength of 380 ± 8 nm and an emission wavelength of 430 ± 8 nm. The measured fluorescence intensities were normalized with uninhibited reaction as 100% and the reaction without enzyme as 0%. A nonlinear regression analysis was done to determine the IC50 value. The enzyme inhibition of HDAC8 was determined by using a reported homogenous fluorescence assay 2 . The enzyme was incubated for 90 min at 37 ˚C, with the fluorogenic substrate ZMAL (Z (Ac)Lys-AMC) in a concentration of 10.5 µM and increasing concentrations of tested compounds. Fluorescence intensity was reported at an excitation wavelength of 390 nm and an emission wavelength of 460 nm in a microtiter plate reader (BMG Polarstar). The NTR-expressing cell line THP1-NTR was generated in the group of Prof. Miething from the University of Freiburg Medical Center, by lentiviral transfection of wild-type THP1 cells (RRID:CVCL_0006), which was a kind gift of Prof. Lübbert from the University Hospital, Freiburg, with an nfsb gene construct. Both cell lines were cultivated in RPMI1640 medium supplemented with 10% (v/v) FCS, 2 mM L-glutamine and 1% Penicillin/Streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Cells were diluted to 5 x 10^4 cells•mL -1 , and mixed with compounds to a final DMSO concentration of 0.5% and seeded at 100 μL in 96well plates in triplicates. After 72 h incubation, the CellTiter 96 ® AQueous Non-Radioactive Cell Proliferation Assay from Promega was performed according to the manufacturer's instructions. Assay plates were measured at 492 nm on a POLARstar Optima microplate reader (BMG Labtech, Germany). Data was plotted as absorbance units against logarithm of compound concentration using OriginPro 9G (OriginLab, USA). 50% inhibition of viability (GI50) was determined as compound concentration required to reduce the number of metabolic active cells by 50% compared to DMSO control. The assay had already been described . 4.3.2.2. Cytotoxic activity against J774A.1 cell line Murine macrophage cell line J774A.1 was maintained under standard conditions at 37°C and 5% CO2 in DMEM (High glucose) supplemented with 10% fetal bovine serum.