Absolute Stereochemical Assignment and Fluorescence Tuning of the Small Molecule Tool, (–)‐Blebbistatin

Abstract

(–)‐Blebbistatin (1), a recently discovered small molecule inhibitor of the ATPase activity of non‐muscle myosin II has been prepared from methyl 5‐methylanthranilate (6) in three steps. This flexible synthetic route has also been used to prepare a nitro group‐containing analogue 12 that has modified fluorescence properties and improved stability under microscope illumination. The key step in the synthesis of 1 and its analogues was the asymmetric hydroxylation of the quinolone intermediate 3 using the Davis oxaziridine methodology. The absolute stereochemistry of (–)‐blebbistatin (1) was shown to be S by X‐ray crystal structure analysis of a heavy atom (bromine) containing analogue 11, which was subsequently reduced and shown to be identical to 1. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The small molecule tool (S)-(−)-blebbistatin: novel insights of relevance to myosin inhibitor design

“…† An interesting variation in the degree of asymmetric induction in the quinolone hydroxylation reaction was also observed as a function of structure, although in all cases the absolute configuration of the resulting analogue was the same [(S)-(-)]. Quinolones 15-17 all reacted with (-)-[ (8,8-dichlorocamphoryl)sulfonyl]oxaziridine (18) 11 under optimised conditions to give the desired analogues 5-7 respectively in good yield and high enantiomeric excess (ee 86-90%) in line with our report 9 on the synthesis of 1 (Scheme 1, column 8). As expected, 9 the ee of the asymmetric hydroxylation reaction for these substrates was reduced significantly when the dihydro-oxaziridine 19 was used instead of 18 (Scheme 1, column 9).…”

“…Quinolones 15-17 all reacted with (-)-[ (8,8-dichlorocamphoryl)sulfonyl]oxaziridine (18) 11 under optimised conditions to give the desired analogues 5-7 respectively in good yield and high enantiomeric excess (ee 86-90%) in line with our report 9 on the synthesis of 1 (Scheme 1, column 8). As expected, 9 the ee of the asymmetric hydroxylation reaction for these substrates was reduced significantly when the dihydro-oxaziridine 19 was used instead of 18 (Scheme 1, column 9). Bach et al 12 have reported a detailed computational analysis of the transition state involved in lithium enolate hydroxylation reactions using an oxaziridine.…”

“…9 With the analogues of 1 in hand, assays against two myosin fragments (skeletal S1 and S1dC) were used to identify structure-activity relationships.…”

“…We have previously reported that a nitro-functional group can be incorporated into the core structure of 1 without significant loss of activity and that this leads to a dramatic improvement in the light stability of 1. 9 …”

Blebbistatin: use as inhibitor of muscle contraction

“…The drug exhibits stereo specificity, with the (−) isoform being the active compound as determined by in vitro motility assay and by its ability to prevent cell division [27,32].…”