1200 series device

The samples were withdrawn at various intervals and were analyzed with an Agilent 1200 series device equipped with a G1312BB inpump SL pump, G1367LHip‐ALS SL auto sampler, and a G1315A DAD detector (Agilent Technologies, Santa Clara, USA). The column employed was an Agilent ZORBAX Eclipse XDB‐C18 column (250 × 4.6 mm, 5 µm). The mobile phase consisted of a gradient of 60–100% methanol in water within 12 min at a flow rate of 0.5 mL/min and the sample injection volume was 5 µL. The column was maintained at 30°C and the detection wave length was 227 nm. The retention times of 2‐methyl‐3‐n‐butylaminoyl‐1,4‐benzoquinone (c) and 2‐methyl‐3,5‐n‐butylaminoyls‐1,4‐benzoquinone (d) were 4.13 and 4.74 min, respectively.

For stability tests, the carbamates were subjected to the described in vitro assay conditions (see above) without adding the substrate ZMAL and without the second incubation step with trypsin. Compounds were applied in a final concentration of 2.4 mM, and recombinant HDAC1 was used as the enzyme. After 90 min of incubation at 37 °C, the release of SAHA was detected via HPLC on an Agilent 1200 series device. The employed analytical column was a Kinetex 5u XB-C18, 100 Å, 250 × 4.6 mm (Phenomenex, Torrance, CA, USA) with the ReproSil 100 C18 5-µm precolumn (Jasco, Oklahoma City, OK, USA). The following conditions were applied: Eluent A: H₂O containing 0.05% TFA, Eluent B: acetonitrile containing 0.05% TFA, linear gradient conditions (0–4 min, A = 90%, B = 10%; 4–29 min, linear increase to 100% of B; 29–31 min, B = 100%; 31.5–40 min, A = 10%, B = 90%); injection volume: 30 µL, flow rate: 1 mL/min; detection wavelength: λ = 254 nm. For evaluation, the ChemStation software from Agilent was used. To quantify the released amount of SAHA and bufexamac, calibration curves were plotted by incubating DMSO stocks of these compounds under in vitro assay conditions with final compound concentrations ranging from 1 µM–200 µM. HPLC analysis with the conditions described above led to the following equations: y = 13.935x − 27.918 for SAHA and y = 0.6099x + 1.1301 for bufexamac.