Medicinal Chemistry and Use of Myosin II Inhibitor ( S)-Blebbistatin and Its Derivatives.

( S)-Blebbistatin, a chiral tetrahydropyrroloquinolinone, is a widely used and well-characterized ATPase inhibitor selective for myosin II. The central role of myosin II in many normal and pathological biological processes has been revealed with the aid of this small molecule. The first part of this manuscript provides a summary of myosin II and ( S)-blebbistatin literature from a medicinal chemist's perspective. The second part of this perspective deals with the physicochemical deficiencies that trouble the use of ( S)-blebbistatin in advanced biological settings: low potency and solubility, fluorescence interference, (photo)toxicity, and stability issues. A large toolbox of analogues has been developed in which particular shortcomings have been addressed. This perspective provides a necessary overview of these developments and presents guidelines for selecting the best available analogue for a given application. As the unmet need for high-potency analogues remains, we also propose starting points for medicinal chemists in search of nanomolar myosin II inhibitors.

Synthesis of C-ring-modified blebbistatin derivatives and evaluation of their myosin II ATPase inhibitory potency.

(S)-Blebbistatin is a micromolar myosin II ATPase inhibitor that is extensively used in research. In search of analogs with improved potency, we have synthesized for the first time C-ring modified analogs. We introduced hydroxymethyl or allyloxymethyl functionalities in search of additional favorable interactions and a more optimal filling of the binding pocket. Unfortunately, the resulting compounds did not significantly inhibit the ATPase activity of rabbit skeletal-muscle myosin II. This and earlier reports suggest that rational design of potent myosin II inhibitors based on the architecture of the blebbistatin binding pocket is an ineffective strategy.

Insights into the myosin II inhibitory potency of A-ring-modified (S)-blebbistatin analogs.

Myosin II is an interesting target for therapeutic intervention, as it is involved in a large number of motility-based diseases. (S)-Blebbistatin is a known micromolar inhibitor of this protein. A new series of (S)-blebbistatin derivatives with a modified A-ring was synthesized and the myosin II inhibitory properties were evaluated in vitro. In this way, we gained insight into the influence of structural modifications in this part of the scaffold on myosin II inhibitory potency. Our results indicate there are few possibilities for potency enhancement via ring A modification of the blebbistatin scaffold.

Improved synthesis and comparative analysis of the tool properties of new and existing D-ring modified (S)-blebbistatin analogs.

(S)-Blebbistatin is a widely used research tool to study myosin II, an important regulator of many motility based diseases. Its potency is too low to be of clinical relevance, but identification of analogs with enhanced potency could deliver leads for targeted pharmacotherapeutics. This, however, requires a profound insight into the structure-activity relationship of the (S)-blebbistatin scaffold. Therefore, new D-ring modified (S)-blebbistatin derivatives were prepared to extend the existing small library of analogs. These molecules were obtained via an improved synthesis pathway and their myosin II inhibitory properties were evaluated in vitro. Finally, all new and known D-ring modified (S)-blebbistatin analogs were compared and the most potent ones underwent a screening of their physicochemical properties.

Discovery of (S)-3'-hydroxyblebbistatin and (S)-3'-aminoblebbistatin: polar myosin II inhibitors with superior research tool properties.

In search of myosin II inhibitors with superior research tool properties, a chemical optimization campaign of the blebbistatin scaffold was conducted in this paper. (S)-Blebbistatin is the best known small-molecule inhibitor of myosin II ATPase activity. Unfortunately, as a research tool this compound has several deficiencies: it is photolabile and (photo)toxic, has low water solubility, and its (fluorescent) precipitates interfere in (fluorescence) readouts. In view of obtaining tool compounds with improved properties, both enantiomers of a series of D-ring modified polar analogs were prepared. We identified (S)-3'-hydroxyblebbistatin (S)-2 and (S)-3'-aminoblebbistatin (S)-3 as two myosin II inhibitors with a 30-fold higher water solubility than (S)-blebbistatin. These molecules furthermore do not cause interference in (fluorescence) readouts. (S)-2 and (S)-3 thus are superior alternatives to (S)-blebbistatin as research tools to study myosin II.

Further studies on anti-invasive chemotypes: An excursion from chalcones to curcuminoids.

In our ongoing search for new anti-invasive chemotypes, we have made an excursion from previously reported potent 1,3-diarylpropenones (chalcones) to congeners bearing longer linkers between the aromatic moieties. Nine 1,ω-diarylalkenones, including curcumin and bisdemethoxycurcumin, were evaluated in the chick heart invasion assay. Unfortunately, these compounds proved less potent and more toxic than earlier evaluated chemotypes. In the 1,3-diarylpenta-2,4-dien-1-one series, fluoro and/or trimethoxy substitution caused an increase in potency. This agrees with observations made earlier for the chalcone class.