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50 values, consistent with an ATP-competitive mechanism of action.
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77953287249
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note
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All synthesized compounds displayed spectral data (MS, NMR) consistent with their assigned structures. Compounds were tested as racemic mixtures when present as such. Synthetic methods can be found in Kahraman, M. et al., U.S. patent application 20080021026.
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31
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77953286463
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note
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50 but less than 100% efficacy. In separate experiments, reference compounds dissolved in water at 100 mM allowed us to test concentrations up to 100 μM and confirm 100% efficacy compared to the most potent control, Y-39983. We tested reversibility by aspirating compound-containing media and replacing with compound-free media after multiple washes. Reversibility was tested up to 24 h after compound addition, and was complete about 3 h after media replacement. In parallel 24 h cytotoxicity assays, there was no significant effect of compounds up to at least 30 μM.
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77953288024
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note
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2 Alexa-488 secondary antibody (Invitrogen) under conventional conditions. Images were acquired on a Zeiss Axiovert 200 M equipped with a Hamamatsu ORCA ER cooled CCD camera and OpenLab software (Improvision). Images were imported into Adobe Photoshop (v.10) for labeling and minor alterations in brightness only.
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43
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77953286230
-
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note
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50 of 0.36 ± 0.08 μM against ROCK. The other compounds had dramatically different effects quantitatively, qualitatively, or both. For example, imatinib caused a rapid, small magnitude increase in impedance, followed by a slow decrease to baseline over 3 h. Other compounds with different effects than ROCK inhibitors included 6-anilinoquinoline-5,8-quinone (guanylate cyclase inhibitor), 2′,5′-dideoxyadenosine (adenylate cyclase inhibitor), and nocodazole (microtubule polymerization inhibitor, tested at 2 μM). Treatment with blebbistatin (myosin II inhibitor, 10 or 1 μM) showed a dose-dependent effect similar to ROCK inhibitors, but with one-third the magnitude, while taxol (microtubule stabilizer, 0.1 or 0.01 μM) showed a similar low magnitude, non dose-dependent effect. Only cytochalasin D (actin polymerization inhibitor) or an RGD containing peptide with sequence GRGDTP (integrin inhibitor; a control peptide with sequence GRADSP was inactive) showed dose dependent effects at 10 and 1 μM strikingly similar to that of ROCK inhibitors. The effects of the last four, and especially the last two, compounds are consistent with their targets and expected effects on cell adhesion. Sorafenib, PD-184352, gefitinib, imatinib, and VX-745 were synthesized by Kalypsys; all other compounds in impedance validation experiments were purchased from commercial vendors.
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44
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51
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77953287389
-
-
note
-
Prepared as described in Scheme 1; des-chloro 4 was used as an intermediate.
-
-
-
-
52
-
-
77953286275
-
-
note
-
3 in DCM.
-
-
-
-
53
-
-
77953290945
-
-
note
-
2, 3 h, 20%.
-
-
-
-
54
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77953287832
-
-
note
-
2O), THF/MeOH (1:1), rt, 43%.
-
-
-
-
55
-
-
77953288914
-
-
note
-
Compound 25 was tested at 10 μM for inhibition in functional assays of the following kinases (Invitrogen,% inhibition in parentheses if ≥50%): ALK4, AMPK, Aurora B (82), B-Raf, CaMK2α, CaMK4, CDK2/cyclinA, CDK5/p35, CK1α1, CK1ε1, CK2α1, DAPK3, EGFR, EphB4, Erk1, FGFR1, GSK3β, INSR, Jak2, Jnk1, Lck, Mek1, MKK6, MLK1, MAPKAPK2, MSK1 (94), p38α, p70S6K (55), Pak6, PDGFRα, PKA (67), PKCα, PKCε, PKN1 (53), RSK1, SGK1 (61), Src, TrkA, ZAP70.
-
-
-
-
56
-
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77953293842
-
-
note
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Solubility was assessed by conventional kinetic assays, measuring absorbance after precipitate filtration from compounds initially in 100% DMSO, with calculation from known standards.
-
-
-
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58
-
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77953292285
-
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note
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3 in DCM.
-
-
-
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60
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0038340963
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