Nm] resulting in no cost phenol indole 36 as a dark brown powder (0.02 g, 0.04 mmol, 27 , Rf = 0.14 (50:50 hexanes:EtOAc)). 1H NMR (CDCl3, 500 MHz): eight.57 (br s, 1H, NH), 7.71 (d, J = 9.0 Hz, 1H, ArH), six.972 (d, J = 9.0 Hz, 1H, ArH) 6.971 (d, J = 2.0 Hz, 1H, ArH), six.94 (s, 2H, ArH), six.79 (dd, J = 8.3 Hz, two.0 Hz, 1H, ArH), six.64 (d, J = eight.3 Hz, 1H, ArH), 5.63 (br s, 1H, OH), four.06 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), three.83 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.71 (s, 6H, OCH3). 13C NMR (CDCl3, 125 MHz): 192.0, 152.6, 148.1, 147.two, 145.7, 143.2, 141.1, 135.0, 134.0, 130.two, 125.four, 125.0, 122.0, 116.8, 114.eight, 113.four, 110.52, 110.45, 107.3, 61.three, 60.9, 57.4, 56.17, 56.15. HPLC: 11.45 min., purity at 254 nm 99 . HRMS (ESI): m/z calculated for C27H27NNaO8 [MNa] 516.1629, found 516.1626. four.2 Biological evaluation 4.two.1. SRB Assay52,53We assessed inhibition of human cancer cell growth making use of the National Cancer Institute’s regular sulforhodamine B assay, as previously described.52 Briefly, cancer cell lines inside a 5 fetal bovine serum/RPMI1640 medium, 1 gentamicin solution have been plated in 96well plates and incubated for 24 h. Serial dilutions of your compounds were then added. Immediately after 48 h, the cells had been fixed with trichloroacetic acid, stained with sulforhodamine B, and read with an automated Biotek plate reader. A development inhibition of 50 (GI50 or the drug concentration causing a 50 reduction inside the net protein improve) was calculated from optical density data. 4.two.2. Colchicine Binding AssayInhibition of [3H]colchicine binding was determined making use of 100 reaction mixtures containing 1.0 tubulin, 5.0 [3H]colchicine (from PerkinElmer), five (v/v) dimethyl sulfoxide, and possible inhibitors at 1.Methyl 5-bromo-1H-pyrazole-3-carboxylate Formula 0 or five.SulfoxFluor web 0 .PMID:24818938 Reaction mixtures also contained elements shown to potently stabilize the colchicine binding activity of tubulin:54 1.0 M monosodium glutamate (adjusted to pH six.6 with HCl in two.0 M stock solution), 0.five mg/mL bovine serum albumin, 0.1 M glucose1phosphate, 1.0 mM MgCl2, and 1.0 mM GTP. Incubation was for 10 min at 37 , a time point at which the reaction within the control is 400 full. Reactions were stopped by adding two.0 mL of icecold water and putting the samples on ice prior to filtration. Every single sample was poured onto a stack of two DEAEcellulose filters, followed right away by 6 mL of icecold water, as well as the water was aspirated under reduced vacuum. The filters have been washed with added water and placed into vials containing 5 mL of Biosafe II scintillation cocktail. The samples had been counted the following day within a Beckman scintillation counter. Samples with prospective inhibitors were in comparison with controls with no inhibitor to figure out percent inhibition. four.2.3. Inhibition of Tubulin PolymerizationTubulin assembly experiments were performed with 0.25 mL reaction mixtures (final volume). The mixtures contained 1 mg/mL (ten ) purified bovine brain tubulin, 0.eight M tubulin monosodium glutamate (adjusted to pHBioorg Med Chem. Author manuscript; obtainable in PMC 2014 November 01.NIHPA Author Manuscript NIHPA Author Manuscript NIHPA Author ManuscriptMacDonough et al.Page6.6 with HCl in two.0 M stock solution), 4 (v/v) dimethyl sulfoxide, 0.4 mM GTP, and varying concentrations of compound. Initially, all components except GTP have been preincubated for 15 min at 30 within a 0.24 mL volume. Following chilling the mixtures on ice, ten of ten mM GTP was added to each and every sample. The reaction mixtures were then transferred to cuvettes held at 0 in Beckman DU7400 and DU7500 spec.
