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  • Eltanexor br Conclusion br In summary we designed and

    2020-08-12


    3. Conclusion
    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
    4. Experimental
    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
    Acknowledgments
    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
    References
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