Synthesis and Thrombin, Factor Xa and U46619 Inhibitory Effects of Non-Amidino and Amidino N²-Thiophenecarbonyl- and N²-Tosylanthranilamides.

Thrombin (factor IIa) and factor Xa (FXa) are key enzymes at the junction of the intrinsic and extrinsic coagulation pathways and are the most attractive pharmacological targets for the development of novel anticoagulants. Twenty non-amidino N²-thiophencarbonyl- and N²-tosyl anthranilamides 1-20 and six amidino N²-thiophencarbonyl- and N²-tosylanthranilamides 21-26 were synthesized to evaluate their activated partial thromboplastin time (aPTT) and prothrombin time (PT) using human plasma at a concentration of 30 µg/mL in vitro. As a result, compounds 5, 9, and 21-23 were selected to study the further antithrombotic activity. The anticoagulant properties of 5, 9, and 21-23 significantly exhibited a concentration-dependent prolongation of in vitro PT and aPTT, in vivo bleeding time, and ex vivo clotting time. These compounds concentration-dependently inhibited the activities of thrombin and FXa and inhibited the generation of thrombin and FXa in human endothelial cells. In addition, data showed that 5, 9, and 21-23 significantly inhibited thrombin catalyzed fibrin polymerization and mouse platelet aggregation and inhibited platelet aggregation induced by U46619 in vitro and ex vivo. Among the derivatives evaluated, N-(3'-amidinophenyl)-2-((thiophen-2''-yl)carbonylamino)benzamide (21) was the most active compound.

Myocardin marks the earliest cardiac gene expression and plays an important role in heart development.

Myocardin belongs to the SAP domain family of transcription factors and is expressed specifically in cardiac and smooth muscle during embryogenesis and in adulthood. Myocardin functions as a transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. However, the in vivo function of myocardin during cardiogenesis is not completely understood. Here we clone myocardin from chick embryonic hearts and show that myocardin protein sequences are highly conserved cross species. Detailed studies of chick myocardin expression reveal that myocardin is expressed in cardiac and smooth muscle lineage during early embryogenesis, similar to that found in mouse. Interestingly, the expression of myocardin in the heart was found enriched in the outflow tract and the sinoatrial segments shortly after the formation of linear heart tube. Such expression pattern is also maintained in later developing embryos, suggesting that myocardin may play a unique role in the formation of those cardiac modules. Similar to its mouse counterpart, chick myocardin is able to activate cardiac and smooth muscle promoter reporter genes and induce smooth muscle gene expression in nonmuscle cells. Ectopic overexpression of myocardin enlarged the embryonic chick heart. Conversely, repression of the endogenous chick myocardin using antisense oligonucleotides or a dominant negative mutant form of myocardin inhibited cardiogenesis. Together, our data place myocardin as one of the earliest cardiac marker genes for cardiogenesis and support the idea that myocardin plays an essential role in cardiac gene expression and cardiogenesis.