Eltanexor br Conclusion br In summary we designed and
In summary, we designed and synthesized novel m-carborane-con-taining anti-cancer drug candidates based on the structure of screening-hit compound 1a. Among the synthesized compounds, 3,4,5-tri-methoxybenzyl derivative 2a and 3,4,5-trimethoxybenzoyl derivative 3a showed more potent cell growth inhibitory activity against the human breast cancer cell line MDA-MB-453 than lead compound 1a. As 3a showed tubulin polymerization inhibitory activity at 20 μM, we confirmed that tubulin is one of the target molecules of 3a. The iden-tification of other cancer-related target molecules of 2a and 3a is in progress, together with the investigation of the structure-activity re-lationships on the benzene ring of 3a. Bioorganic & Medicinal Chemistry 27 (2019) 1139–1144
Melting points were determined using a Yanaco micro melting point apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded on JEOL JNM-EX-270 and JNM-LA-400 spectrometers. Chemical shifts in the 1H NMR spectra are referenced to tetra-methylsilane (0.0 ppm) as the internal standard. Chemical shifts in the 13C NMR spectra are referenced to the signals of residual non-deuter-ated solvents within deuterated solvents. The splitting patterns are as-signed as follows: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad). Mass spectra were recorded on a JEOL JMS-DX-303 spec-trometer. Elemental analyses were performed on a Perkin Elmer 2400 CHN spectrometer. TLC was performed on F254 silica gel from Merck. Reagents were purchased from Wako Pure Chemical Industries, Ltd., Sigma-Aldrich Co., and Tokyo Chemical Industry, Ltd. (TCI). All sol-vents (reagent quality) were purchased from commercial sources and used without further purification. Yields of the test compounds in this section represent those after recrystallization.
methyl -1,7-dicarba-closo-dodecaborane (1d)
4.2. Biological assays
4.2.1. Cell growth inhibition assay
The human breast cancer cell line MDA-MB-453 was routinely cultivated in RPMI-1640 supplemented with 10% FBS, 100 IU/mL pe-nicillin, and 100 mg/mL streptomycin at 37 °C in a humidified in-cubator (5% CO2). Cells were trypsinized from the maintenance dish with trypsin-EDTA and seeded in a 96-well plate at the density of 4000 Eltanexor per 100 μ L (final volume) in the same media as that used for the cell cultures. After 1 day, the medium was removed and 100 μ L of the drug solution, supplemented with serial dilutions of test compounds (1–4) or DMSO (dilute control), was added to the microcultures in triplicate. MDA-MB-453 cells were incubated for 4 days. At the end of the incubation period, cell proliferation was evaluated by adding 5 μM WST-8 to the microcultures. The cells were incubated for 4 h, and then absorbance at 450 nm was measured. This parameter is related to the number of viable cells in the culture. The concentration required for 50% inhibition of cell growth (GI50) was calculated using Prism 4 software.
4.2.2. Tubulin polymerization inhibition assay
The time-dependent polymerization of tubulin into microtubules was monitored with a fluorescence-based tubulin polymerization assay kit (BK011P, Cytoskeleton, Inc.) in accordance with the manufacturer’s instructions. The reaction mixture with a final volume of 55 μ L in PEM buffer (80 mM PIPES, 0.5 mM ethylene glycol tetraacetic acid (EGTA), 2 mM MgCl2, pH = 6.9) contained 2 mg/mL bovine brain tubulin, 10 μM fluorescence reporter, 1 mM guanosine triphosphate (GTP), and 20% glycerol in the presence or absence of test compounds (final concentration: 20 mM) at 37 °C. Tubulin polymerization was detected by monitoring fluorescence enhancement due to the incorporation of a fluorescence reporter into microtubules upon polymerization. Fluorescence emission at 450 nm (λex = 360 nm) was measured for 1 h (1 min intervals) on a multimode plate reader (TECAN Infinite M1000). Tubulin polymerization assays were conducted with reagents as re-commended by the manufacturer (Cytoskeleton, Inc.). For that purpose, 2a and 3a (10–20 μM) were incubated with purified bovine tubulin and
This research was supported by a grant-in-aid from the Strategic Research Program for Private Universities (2015–2019) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT).
Appendix A. Supplementary data
Contents lists available at ScienceDirect
International Journal of Biochemistry
and Cell Biology
journal homepage: www.elsevier.com/locate/biocel
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