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Volumn 17, Issue 13, 2007, Pages 3562-3569

Synthesis and SAR of thiophene containing kinesin spindle protein (KSP) inhibitors

Author keywords

Anti mitotics; Eg5; Kinesin spindle protein; KSP; Thiophenes

Indexed keywords

KINESIN; PROTEIN INHIBITOR; THIAZOLE DERIVATIVE; THIOPHENE; THIOPHENE DERIVATIVE;

EID: 34250178457     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2007.04.076     Document Type: Article
Times cited : (52)

References (39)
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    • All final compounds displayed spectral data (NMR, MS) that were consistent with the assigned structure.
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    • note
    • 6-tagged) in 20 μl of assay buffer was dispensed to black 384-well assay plates. Compounds in DMSO and arrayed in 11 point 1/2 log dilution dose response (final top concentration of 96 μM, 1% DMSO in all assay wells) were added by passive pin transfer, followed by addition of 5 μl assay buffer containing taxol-stabilized bovine brain microtubules (Cytoskeleton) and ATP. The final concentrations of KSP, microtubules, and ATP were 15 nM, 500 nM, and 30 μM, respectively. After a 2-h room-temperature incubation, ADPQuest assay reagents were added according to the manufacturer's instructions, and the resulting fluorescence signal was read on a Molecular Devices Acquest plate reader. Raw fluorescence data were normalized to negative (DMSO) and positive (compound 2a, as a racemic mixture) controls. Data analysis was performed using Spotfire (v8.1, Spotfire, Inc.) and Kalypsys proprietary software.
  • 30
    • 34250194866 scopus 로고    scopus 로고
    • note
    • ′) horseradish peroxidase conjugate (Jackson ImmunoResearch), and a chemiluminescent substrate (POD, Roche Applied Science). Signal detection, data normalization, and data analysis were similar to that in Ref. 9.
  • 31
    • 34250163436 scopus 로고    scopus 로고
    • note
    • 5 cells/ml in medium with 1% fetal bovine serum. Compounds in DMSO and arrayed in 11 point 1/2 log dilution dose response (final top concentration of 96 μM, 1% DMSO in all assay wells) were added by passive pin transfer. After 60 h incubation, cell viability was measured by dispensing an equal volume of ATPLite reagent (Perkin-Elmer). Signal detection, data normalization, and data analysis were similar to that in Ref. 9.
  • 32
    • 34250169684 scopus 로고    scopus 로고
    • note
    • free = 0.208/0.272.
  • 35
    • 34250184045 scopus 로고    scopus 로고
    • note
    • Compound 33 was resolved using a Chiralpak IA column (4.6 mm × 250 mm) eluting 10% i-PrOH/90% heptane/0.1% TFA, 1 mL/min. The first eluting isomer showed ATPase activity of 0.48 μM versus 3.9 μM for the second eluting isomer. The absolute stereochemistry was not determined; the resolution of the crystal structure was not sufficient for definitive assignment.
  • 36
    • 34250222722 scopus 로고    scopus 로고
    • note
    • ′) Alexa 488 (Invitrogen). Chromosomal DNA was stained with 4′-6-diamidino-2-phenylindole (DAPI, Sigma). Cells were imaged on a Zeiss Axiovert 2 with an ORCA-ER CCD camera (Hamamatsu) and OpenLab software (Improvision). Images were imported into Adobe Photoshop for labeling and minor alterations in contrast.
  • 37
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    • note
    • A-549 cells in medium with 1% fetal bovine serum were treated with DMSO vehicle (<=0.15 % final) alone or containing compounds as specified for 18 h. To test reversibility of mitotic arrest, previously treated cells were washed three times with medium containing 1% fetal bovine serum and then replated in the same for an additional 24 h. Cells were harvested, washed twice with phosphate-buffered saline containing 2% fetal bovine serum, and then resuspended in 1 ml of the same wash buffer. For fixation, 3 ml of -20 °C absolute ethanol was added slowly, followed by a 1-h incubation on ice. Fixed cells were then washed twice with phosphate-buffered saline and resuspended in staining buffer (phosphate-buffered saline containing 3.8 mM sodium citrate, 50 μg/ml propidium iodide, and 100 μg/ml RNase A) for a minimum of 1 h at 4 °C. Cells were analyzed on a BD LSR flow cytometer.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.