Solubility assays were carried out on a Biomek FX lab automation workstation (Beckman Coulter Inc.) using μSOL Evolution software (pION Inc.). Compound stock (16.7 μM) (in DMSO) was used to make the reference plate. Compound stock (0.1 mM) was used in storage plate and incubated at room temperature for 18 hours. The suspension was then filtered through a 96-well filter plate (pION Inc.), diluted with 1-propanol to make the sample plate, and the ultraviolet (UV) spectrum of the reference and sample plate was read. Calculations were carried out with μSOL Evolution software based on the area under the curve (AUC) of the UV spectrum of the sample and reference plates. All compounds were tested in triplicate. For analysis of permeability, a parallel artificial membrane permeability assay (PAMPA) was conducted with the same instrument using PAMPA Evolution 96 Command software (pION Inc.). Compound stock (50 μM) was used to make the reference plate, and the UV spectrum of this plate was read. Gastrointestinal tract (GIT) lipid (pION Inc.) was used as the membrane on the preloaded PAMPA Sandwich (pION Inc.). The acceptor and donor chamber of the sandwich was then filled with 200 μl of acceptor solution buffer (pION Inc.) and test compound. The PAMPA Sandwich was assembled, placed on the Gut-Box, and stirred for 30 min. Then, the UV spectrum of the donor and the acceptor was read. The permeability coefficient was calculated on the basis of the AUC of the reference plate, the donor plate, and the acceptor plate. All compounds were tested in triplicate. Cytotoxicity studies were based on BJ (human foreskin fibroblasts), HEK293 (HEK cells), HepG2 (human hepatocellular carcinoma cells), and Raji (human lymphoblast cells; Burkitt’s lymphoma) cell lines [American Type Culture Collection (ATCC)], which were cultured according to recommendations and cell culture media from ATCC. Exponentially growing cells were plated in 384-well white custom assay plates (Corning) and incubated overnight at 37°C in a humidified 5% CO2 incubator. DMSO inhibitor stock solutions were added the following day to a top final concentration of 25 μM of 0.25% DMSO and then diluted 1:3 for a total of 10 testing concentrations. Cytotoxicity was determined after 72 hours of incubation using Cell Titer Glo Reagent (Promega), according to the manufacturer’s recommendation. Luminescence was measured on an Envision plate reader (PerkinElmer).

Compound stability was assessed using mouse liver microsomes (microsomes, 20 mg/ml) in potassium phosphate buffer (0.1 M, pH 7.4). Compound stocks (10 mM) in DMSO were diluted with DMSO and acetonitrile to three different intermediate concentrations. Only one concentration of controls (diphenhydramine HCl, verapamil HCl, and ketoprofen) was used. Each diluted compound stock (37.83 μl) was added to an aliquot of the liver microsomal solution (3 ml), vortexed, and transferred to five 96-well assay plates. A single assay plate was tested at each time point: 0, 0.5, 1, 2, and 4 hours. For the 0-hour time point, precooled (4°C) internal standard (2 μM warfarin in methanol) was added to the plate before the reaction starts. For other time points, NADPH solution (Thermo Fisher Scientific) was added first. The plates were sealed, and all plates except the 0-hour plate were incubated at 37°C, shaken at a speed of 60 rpm. At each time point, 437.5 μl of precooled internal standard was added to quench the reaction. The final compound concentrations were 20, 4, and 0. 8 μM. The centration of controls was 4 μM. The quenched plate was then centrifuged at 4000 rpm for 15 min at 4°C. The resulting supernatant was transferred to a 96-well plate and analyzed by UPLC-MS (Waters Inc.). The compounds and internal standard were detected by selected ion recording. The amount of material was measured as a ratio of peak area to the internal standard and graphed. Using the slope from the most linear portion of this curve, the degradation rate constant is calculated. The rate constant was then used to calculate the compounds half-life in plasma. Intrinsic clearance was calculated as CLint′ = (0.693/(t1/2)) × (1/microsomal concentration in the reaction solution) × (45 mg of microsome/gram liver) × (gram liver/kg body weight), where microsomal concentration in the reaction solution is 0.5 mg/ml and gram liver/kg body weight of mouse is 52.

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