Mmol). The reaction mixture was stirred at space temperature for two h, quenched with distilled water, plus the aqueous layer was extracted with ethyl acetate. The combined organic layer was dried over Mg2 SO4 , along with the solvent was evaporated beneath lowered pressure. The product was isolated by preparative HPLC to acquire N-desisopropyl DN203368 (2.7 mg, 12 yield). MS (ESI+ ) m/z calculated for C27 H31 N2 O [M + H]+ 399.two; located 399.two. 1 H NMR (400 MHz, CD3 OD): 7.18 (t, J = eight.6 Hz, 3H), 7.11 (td, J = 1.2, eight.1 Hz, 3H), 6.80 (dd, J = 1.9, 6.eight Hz, 2H), 6.75 (d, J = 7.5 Hz, 1H), 6.71.69 (m, 2H), six.58 (d, J = eight.8 Hz, 2H), two.98.96 (m, 4H), two.90.88 (m, 4H), 0.95 (d, J = 6.9 Hz, 6H).Pharmaceutics 2021, 13,four of2.3. In Vitro Incubation of DN203368 in Liver Microsomes Liver microsomal incubation samples have been prepared as described previously [17]. DN203368 (one hundred ) was incubated with 1 mg/mL rat or human liver microsomal protein and 100 mM potassium phosphate buffer (pH 7.4) at 37 C for 5 min. Soon after preincubation, the reaction was initiated by Cereblon supplier adding an NADPH-generating system (three.3 mM G6P, 1 unit/mL G6PDH, 1.three mM -NADP+ , and three.three mM MgCl2 ). The reaction mixtures (final volume one hundred ) were further incubated for 120 min at 37 C inside a heated shaker (Eppendorf, Hamburg, Germany). Samples had been ready in triplicate, and controls comprised heatdenatured microsomal preparations (100 C for 30 min). The reaction was terminated by adding 100 cold acetonitrile followed by centrifugation at 14,000 rpm for ten min at four C. Lastly, the supernatants had been concentrated plus the residue was reconstituted in 100 acetonitrile. two.4. Liquid Chromatography andem Mass Spectrometry (LC-MS/MS) A Thermo Scientific Vanquish ultra-high-performance liquid chromatography method coupled to a Q Exactive focus orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was utilized to determine DN203368 and its putative metabolites. Chromatography was performed on a Caspase Formulation Phenomenex Kinetex C18 column (100 2.1 mm, 2.six , 100 . The mobile phase consisted of water with 0.1 formic acid (A) and acetonitrile with 0.1 formic acid (B). Gradient elution was conducted as follows: 0 min, 30 B; 15 min, 30 50 B; five min, 50 B; 7.1 min, 50 30 B; followed by 3 min re-equilibration (total run time: 10 min). The column oven temperature was maintained at 40 C. The flow rate was 0.two mL/min and also the injection volume was two . The electrospray ionization (ESI) parameters have been optimized as follows: heated capillary temperature: 320 C; spray voltage: 3.5 kV; sheath gas flow price: 40 arb; auxiliary gas flow rate: 10 arb; S-lens RF level: 50.0 V. Nitrogen was utilised for spray stabilization and because the collision gas within the C-trap. All data were acquired and analyzed utilizing the Thermo Xcalibur four.0 application (Thermo Fisher Scientific Inc., Waltham, MA, USA). 2.five. Metabolite Identification Using the Standard Approach For standard metabolite identification, information had been acquired in full scan and parallel reaction monitoring (PRM) mode with an inclusion list of predicted metabolites utilizing liquid chromatography igh-resolution mass spectrometry. The parameters for the complete scan mode had been as follows: resolution: 70,000; scan range: 30050; AGC target: 1 106 ; maximum injection time: one hundred ms. As for PRM mode: resolution: 17,500; normalized collision power: 30 eV; AGC target: 5 104 ; maximum injection time one hundred ms. An inclusion list contained the precursor ion mass of the predicted metabolic reaction (m/z.