br antiproliferative effects against PC cancer cells In addi
antiproliferative effects against PC-3 cancer cells. In addition to its in-duction of HSP90 expression, 11 also downregulates the protein ex-pression levels of client proteins, such as EGFR, Src, FAK, and RB. The significant inhibition of the HSP90 chaperone by 11 was rationalized by its molecular docking into active site of HSP90. The outcome of the
present study is consistent with recent reports revealing the promising potential of HSP90 inhibitors against prostate cancer. These are ex-citing outcomes in light of the fact that prostate cancer is a leading cause of cancer-related mortality and the revelations could be useful for designing chemotherapeutic agents for prostate cancer. Further lead
Fig. 5. Molecular docking analysis of 11 in HSP90. (A) 11 (blue) is anchored within the HSP90 (gray) binding site. The three distinct sections of MPTG0G256 are colored as yellow, blue and red, which are located at sites S1, S2 and S3, respectively. Interacting residues are represented as sticks and labeled as shown. Hydrogen bonds are denoted by dotted green lines. (B) A 2D representation of 11 docked in HSP90. Hydrogen bonds are denoted by dashed lines. Green lines represent areas of hydrophobic interactions. Interacting residues are labeled as shown.
modifications on 11 including synthesis of compounds with diverse substitution on the quinoline rings along with positioning of other planar bicyclic heteroaryl rings in the hybrid structure design is under progress.
Nuclear magnetic resonance spectra were obtained with Bruker DRX-500 spectrometer operating at 300 MHz, with chemical shift re-corded in parts per million (ppm, d) downfield from TMS as an internal
standard. High-resolution mass spectra (HRMS) were measured with a JEOL (JMS-700) electron impact (EI) mass spectrometer. Flash column chromatography was accomplished with silica gel (Merck Kieselgel 60, No. 9385, 230e400 mesh ASTM). All reactions were carried out under an qPCR of dry N2.
Fig. 6. In vivo antitumor activity of 11 in PC3 xenograft model. (A) PC3-tumor-bearing nude mice were treated with vehicle or 11 (50, 100 and 200 mg/kg/d, by oral gavage qd). Tumor was excised when the tumor size reached 1200 mm3. (B) The body weight of the mice measured daily during the first week and then twice a week of administration. (MPT0G256 represents 11).
Sodium hydride (0.05 g, 2 mmol) was added to a solution of com-pound 28 (0.5 g, 1 mmol) in DMF (5 mL), and the reaction mixture was stirred for 20 min at °C. EtI (0.25 mL, 3.10 mmol) was then added and stirring was continued for 4 h at rt. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated. The residue obtained was used in a subsequent reaction without purification.
Human cancer cells were purchased from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in RPMI 1640 medium or DMEM supplemented with 10% FBS (v/v) and penicillin (100 U/mL)/streptomycin (100 μg/mL)/amphotericin B (0.25 μg/mL). Cultures were maintained at 37 °C in a humidified at-mosphere of 5% CO2/95% air.
Cells were seeded in 96-well plates in their cultured medium. After
24 h, cells were fixed with 10% trichloroacetic acid to represent cell population at the time of drug addition (T0). After incubation of DMSO or test compounds for 48 h, cells were fixed with 10% trichloroacetic acid and sulforhodamine B at 0.4% (w/v) in 1% AcOH was added to stain cells. Unbound sulforhodamine B was washed out with 1% AcOH and sulforhodamine B–bound cells were solubilized with 10 mmol/L Trizma base. The absorbance was read at a wavelength of 515 nm. Using the following absorbance measurements, such as time zero (T0), control growth (C), and cell growth in the presence of the drug (Tx), the percentage growth was calculated at each of the compound con-centrations levels. Percentage growth inhibition was calculated as 100 - [(Tx − T0)/(C − T0)] × 100. Growth inhibition of 50% (GI50) is de-termined at the drug concentration that results in 50% reduction of total protein increase in control cells during the compound incubation.
To assess the effects on HSP90 activity in vitro, Hsp90α Assay Kits (BPS Bioscience, San Diego, CA, USA) were used. Following the in-struction manual, master mixture (Hsp90 assay buffer, DTT, BSA and H2O) and FITC-labeled geldanamycin were added to all 96 wells. Then test compounds were added to each well designated “Test Inhibitor”. To the “Blank”, “Enzyme Positive Control” and “Enzyme Negative Control” wells, the same solution with inhibitor was added. To the “Enzyme Negative Control” and “Blank” wells, HSP90 assay buffer was added. Finally, the recombinant protein Hsp90α was incubated in every well designated “Enzyme Positive Control” and “Test Inhibitor” for 2–3 h at rt with slow shaking. The fluorescent polarization of the samples was determined by microtiter-plate reader, which can detect excitation ranging from 475 to 495 nm and emission ranging from 518 to 538 nm.